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Archive for the ‘Methylation’ Category


Cancer Genomics: Multiomic Analysis of Single Cells and Tumor Heterogeneity

Curator: Stephen J. Williams, PhD

 

scTrio-seq identifies colon cancer lineages

Single-cell multiomics sequencing and analyses of human colorectal cancer. Shuhui Bian et al. Science  30 Nov 2018:Vol. 362, Issue 6418, pp. 1060-1063

To better design treatments for cancer, it is important to understand the heterogeneity in tumors and how this contributes to metastasis. To examine this process, Bian et al. used a single-cell triple omics sequencing (scTrio-seq) technique to examine the mutations, transcriptome, and methylome within colorectal cancer tumors and metastases from 10 individual patients. The analysis provided insights into tumor evolution, linked DNA methylation to genetic lineages, and showed that DNA methylation levels are consistent within lineages but can differ substantially among clones.

Science, this issue p. 1060

Abstract

Although genomic instability, epigenetic abnormality, and gene expression dysregulation are hallmarks of colorectal cancer, these features have not been simultaneously analyzed at single-cell resolution. Using optimized single-cell multiomics sequencing together with multiregional sampling of the primary tumor and lymphatic and distant metastases, we developed insights beyond intratumoral heterogeneity. Genome-wide DNA methylation levels were relatively consistent within a single genetic sublineage. The genome-wide DNA demethylation patterns of cancer cells were consistent in all 10 patients whose DNA we sequenced. The cancer cells’ DNA demethylation degrees clearly correlated with the densities of the heterochromatin-associated histone modification H3K9me3 of normal tissue and those of repetitive element long interspersed nuclear element 1. Our work demonstrates the feasibility of reconstructing genetic lineages and tracing their epigenomic and transcriptomic dynamics with single-cell multiomics sequencing.

Fig. 1 Reconstruction of genetic lineages with scTrio-seq2.

Global SCNA patterns (250-kb resolution) of CRC01. Each row represents an individual cell. The subclonal SCNAs used for identifying genetic sublineages were marked and indexed; for details, see fig. S6B. On the top of the heatmap, the amplification or deletion frequency of each genomic bin (250 kb) of the non-hypermutated CRC samples from the TCGA Project and patient CRC01’s cancer cells are shown.

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Fig. 1 Reconstruction of genetic lineages with scTrio-seq2.

Global SCNA patterns (250-kb resolution) of CRC01. Each row represents an individual cell. The subclonal SCNAs used for identifying genetic sublineages were marked and indexed; for details, see fig. S6B. On the top of the heatmap, the amplification or deletion frequency of each genomic bin (250 kb) of the non-hypermutated CRC samples

 

 

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Reporter and Curator: Dr. Sudipta Saha, Ph.D.

 

The trillions of microbes in the human gut are known to aid the body in synthesizing key vitamins and other nutrients. But this new study suggests that things can sometimes be more adversarial.

 

Choline is a key nutrient in a range of metabolic processes, as well as the production of cell membranes. Researchers identified a strain of choline-metabolizing E. coli that, when transplanted into the guts of germ-free mice, consumed enough of the nutrient to create a choline deficiency in them, even when the animals consumed a choline-rich diet.

 

This new study indicate that choline-utilizing bacteria compete with the host for this nutrient, significantly impacting plasma and hepatic levels of methyl-donor metabolites and recapitulating biochemical signatures of choline deficiency. Mice harboring high levels of choline-consuming bacteria showed increased susceptibility to metabolic disease in the context of a high-fat diet.

 

DNA methylation is essential for normal development and has been linked to everything from aging to carcinogenesis. This study showed changes in DNA methylation across multiple tissues, not just in adult mice with a choline-consuming gut microbiota, but also in the pups of those animals while they developed in utero.

 

Bacterially induced reduction of methyl-donor availability influenced global DNA methylation patterns in both adult mice and their offspring and engendered behavioral alterations. This study reveal an underappreciated effect of bacterial choline metabolism on host metabolism, epigenetics, and behavior.

 

The choline-deficient mice with choline-consuming gut microbes also showed much higher rates of infanticide, and exhibited signs of anxiety, with some mice over-grooming themselves and their cage-mates, sometimes to the point of baldness.

 

Tests have also shown as many as 65 percent of healthy individuals carry genes that encode for the enzyme that metabolizes choline in their gut microbiomes. This work suggests that interpersonal differences in microbial metabolism should be considered when determining optimal nutrient intake requirements.

 

References:

 

https://news.harvard.edu/gazette/story/2017/11/harvard-research-suggests-microbial-menace/

 

http://www.cell.com/cell-host-microbe/fulltext/S1931-3128(17)30304-9

 

https://www.ncbi.nlm.nih.gov/pubmed/23151509

 

https://www.ncbi.nlm.nih.gov/pubmed/25677519

 

http://mbio.asm.org/content/6/2/e02481-14

 

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Pancreatic Cancer Targeted Treatment?

Curator: Larry H. Bernstein, MD, FCAP

 

 

MGH study identifies potential treatment target for pancreatic cancer

Molecular signature found in 30 percent of PDAC tumors, associated with more aggressive cancer

http://www.massgeneral.org/about/pressrelease.aspx?id=1933&

 

Massachusetts General Hospital (MGH) investigators have identified the first potential molecular treatment target for the most common form of pancreatic cancer, which kills more than 90 percent of patients. Along with finding that the tumor suppressor protein SIRT6 is inactive in around 30 percent of cases of pancreatic ductal adenocarcinoma (PDAC), the team identified the precise pathway by which SIRT6 suppresses PDAC development, a mechanism different from the way it suppresses colorectal cancer. The paper will appear in the June 2 issue of Cell and have been published online.

“With the advance of cancer genomics, it has become evident that alterations in epigenetic factors – those that control whether and when other genes are expressed – represent some of the most frequent alterations in cancer,” says Raul Mostoslavsky, MD, PhD, of the MGH Cancer Center, senior author of the report.  “Yet, not many of those factors have been described before, and those that have been identified have not been linked to specific downstream targets.  Not only did more than a third of analyzed PDAC patient samples exhibit the molecular signature we identified, those patients also turned out to have very poor prognoses.”

Among its other functions, SIRT6 is known to control how cells process glucose, and a 2012 study by Mostoslavsky’s team found that its ability to suppress colorectal cancer involves control of a process called glycolysis.  But while that study also found reduced SIRT6 expression in PDAC tumor cells, the current investigation indicated that SIRT6 deficiency promotes PDAC through a different mechanism. Experiments in cell lines and animal models revealed that low SIRT6 levels in PDAC were correlated with increased expression of Lin28b, an oncoprotein normally expressed during fetal development.

Lin28b expression proved to be essential to the growth and survival of SIRT6-deficient PDAC cells and acted by preventing a family of tumor-suppressing mRNAs called let-7 from blocking expression of three genes previously associated with increased aggressiveness and metastasis in pancreatic cancers.  All of these hallmarks – reduced SIRT6, increased Lin28b and reduced let-7 expression – were found in tumor samples from patients who died more quickly.

“A general message from these studies is that cancer cells benefit from modulating epigenetic factors like SIRT6 by acquiring the ability to override normal cellular growth control patterns,” says Mostoslavsky, an associate professor of Medicine at Harvard Medical School and an associate member at the Broad Institute.  “Each tumor type may acquire a unique set of capabilities that may provide tumor-specific growth and survival advantages, which may need to be determined for each kind of cancer.  In terms of our findings regarding PDAC, we are intrigued by the downstream pathways controlled by Lin28b and how they increase aggressiveness and metastasis, and we are hopeful that developing in the future Lin28b inhibitors could benefit this subset of PDAC patients, who currently have very few treatment options.”

 

SIRT6 Suppresses Pancreatic Cancer through Control of Lin28b

Sita Kugel, Carlos Sebastián, Julien Fitamant,…., Alon Goren, Vikram Deshpande, Nabeel Bardeesy, Raul Mostoslavsky

Figure thumbnail fx1
Highlights
  • Loss of SIRT6 cooperates with oncogenic Kras to drive pancreatic cancer
  • SIRT6 regulates the oncofetal protein Lin28b through promoter histone deacetylation
  • Lin28b drives the growth and survival of SIRT6-deficient pancreatic cancer
  • SIRT6 and Lin28b expression define prognosis in specific pancreatic cancer subsets

Chromatin remodeling proteins are frequently dysregulated in human cancer, yet little is known about how they control tumorigenesis. Here, we uncover an epigenetic program mediated by the NAD+-dependent histone deacetylase Sirtuin 6 (SIRT6) that is critical for suppression of pancreatic ductal adenocarcinoma (PDAC), one of the most lethal malignancies. SIRT6 inactivation accelerates PDAC progression and metastasis via upregulation of Lin28b, a negative regulator of the let-7 microRNA. SIRT6 loss results in histone hyperacetylation at theLin28b promoter, Myc recruitment, and pronounced induction of Lin28b and downstream let-7 target genes, HMGA2, IGF2BP1, and IGF2BP3. This epigenetic program defines a distinct subset with a poor prognosis, representing 30%–40% of human PDAC, characterized by reduced SIRT6 expression and an exquisite dependence on Lin28b for tumor growth. Thus, we identify SIRT6 as an important PDAC tumor suppressor and uncover the Lin28b pathway as a potential therapeutic target in a molecularly defined PDAC subset.

 

The multifaceted functions of sirtuins in cancer

Angeliki Chalkiadaki & Leonard Guarente  Affiliations  Corresponding author
Nature Reviews Cancer (2015); 15:608–624     http://dx.doi.org:/10.1038/nrc3985

The sirtuins (SIRTs; of which there are seven in mammals) are NAD+-dependent enzymes that regulate a large number of cellular pathways and forestall the progression of ageing and age-associated diseases. In recent years, the role of sirtuins in cancer biology has become increasingly apparent, and growing evidence demonstrates that sirtuins regulate many processes that go awry in cancer cells, such as cellular metabolism, the regulation of chromatin structure and the maintenance of genomic stability. In this article, we review recent advances in our understanding of how sirtuins affect cancer metabolism, DNA repair and the tumour microenvironment and how activating or inhibiting sirtuins may be important in preventing or treating cancer.

 

Figure 1: Overview of the role of sirtuins in the regulation of cancer metabolism

The inhibitory effects of sirtuin 3 (SIRT3), SIRT4 and SIRT6 on metabolic pathways that drive cancer cells are depicted. In normal cells, SIRT6 functions as a co-repressor for the transcription factors hypoxia-inducible factor 1α (HIF1α…

 

Tumor suppressor p53 cooperates with SIRT6 to regulate gluconeogenesis by promoting FoxO1 nuclear exclusion

Ping Zhanga,1, Bo Tua,1, Hua Wangb , Ziyang Caoa , Ming Tanga , … , Bin Gaob , Robert G. Roederd,2, and Wei-Guo Zhua,e,2
PNAS | July 22, 2014;111(29): 10684–10689 |   http://www.pnas.org/content/111/29/10684.full.pdf  http://www.pnas.org/lookup/suppl/doi:10. 1073/pnas.1411026111/-/DCSupplemental.

In mammalian cells, tumor suppressor p53 plays critical roles in the regulation of glucose metabolism, including glycolysis and oxidative phosphorylation, but whether and how p53 also regulates gluconeogenesis is less clear. Here, we report that p53 efficiently down-regulates the expression of phosphoenolpyruvate carboxykinase (PCK1) and glucose-6-phosphatase (G6PC), which encode rate-limiting enzymes in gluconeogenesis. Cell-based assays demonstrate the p53-dependent nuclear exclusion of forkhead box protein O1 (FoxO1), a key transcription factor that mediates activation of PCK1 and G6PC, with consequent alleviation of FoxO1- dependent gluconeogenesis. Further mechanistic studies show that p53 directly activates expression of the NAD+-dependent histone deacetylase sirtuin 6 (SIRT6), whose interaction with FoxO1 leads to FoxO1 deacetylation and export to the cytoplasm. In support of these observations, p53-mediated FoxO1 nuclear exclusion, down-regulation of PCK1 and G6PC expression, and regulation of glucose levels were confirmed in C57BL/J6 mice and in liver-specific Sirt6 conditional knockout mice. Our results provide insights into mechanisms of metabolism-related p53 functions that may be relevant to tumor suppression.

As the “guardian of the genome,” tumor suppressor p53 has been reported to coordinate diverse cellular responses to a broad range of environment stresses (1) and to play antineoplastic roles by activating downstream target genes involved in DNA damage repair, apoptosis, and cell-cycle arrest (2). Recent studies have indicated broader roles for p53 in mediating metabolic changes in cells under various physiological and pathological conditions (3–7). For example, p53 was reported to influence the balance between glycolysis and oxidative phosphorylation by inducing the p53-induced glycolysis and apoptosis regulator (TIGAR) and by regulating the synthesis of cytochrome c oxidase 2 (SCO2) (3), respectively, thus promoting the switch from glycolysis to oxidative phosphorylation. p53 also may impede metabolism by reducing glucose import (4) or by inhibiting the pentose phosphate pathway (PPP) (5). More recently, context-dependent inhibitory (6) or stimulatory (7, 8) effects of p53 on gluconeogenesis have been reported. It thus is clear that p53 plays important roles in glucose regulation in mammalian cells. Glucose homeostasis is maintained by a delicate balance between intestinal absorption of sugar, gluconeogenesis, and the utilization of glucose by the peripheral tissues, irrespective of feeding or fasting (9). The gluconeogenesis pathway is catalyzed by several key enzymes that include the first and last rate-limiting enzymes of the process, phosphoenolpyruvate carboxykinase (PCK1) and glucose-6-phosphatase (G6PC), respectively. The expression of both PCK1 and G6PC is controlled mainly at the transcription level. For example, the transcription factor forkhead box protein O1 (FoxO1) activates gluconeogenesis through direct binding to the promoters of G6PC and PCK1 (10). A dominant negative FoxO1 lacking its transactivation domain significantly decreases gluconeogenesis (11) whereas FoxO1 ablation impairs fasting- and cAMP-induced PCK1 and G6PC expression (12). Therefore, factors influencing expression of FoxO1 or its binding activity to the PCK1 and G6PC promoters are potential targets for gluconeogenesis regulation. The transcription activity of FoxO family members is regulated by a sophisticated signaling network. Various environmental stimuli cause different posttranslational modifications of FoxO proteins, including phosphorylation, acetylation, ubiquitination, and methylation (13–15). The phosphorylation of FoxO proteins is known to be essential for their shuttling between the nucleus and cytoplasm. For example, kinase Akt/PKB phosphorylates FoxO1 at threonine 24 and at serines 256 and 319, which in turn leads to 14-3-3 binding and subsequent cytoplasmic sequestration. The acetylation of FoxO proteins also affects their trafficking and DNA-binding activities (15–17). Sirtuin (SIRT)1, a homolog of the yeast silent information regulator-2 (Sir2), has been identified as a deacetylase for FoxO proteins (15, 17, 18). Of the seven mammalian sirtuins, SIRT1, SIRT6, and SIRT7 are localized to the nucleus (19), and SIRT6 was recently reported to act as a central player in regulating the DNA damage response, glucose metabolism, and aging (20–26). Using a knockout mouse model, it was found that SIRT6 functions as a corepressor of the transcription factor Hif1α to suppress glucose uptake and glycolysis.

Significance: Beyond its canonical functions in processes such as cell-cycle arrest, apoptosis, and senescence, the tumor suppressor p53 has been increasingly implicated in metabolism. Here, in vitro and in vivo studies establish a role for p53 in gluconeogenesis through a previously unidentified mechanism involving (i) direct activation of the gene encoding the NAD-dependent deacetylase sirtuin 6 (SIRT6), (ii) SIRT6-dependent deacetylation and nuclear exclusion of forkhead box protein O1 (FoxO1), and (iii) downregulation of FoxO1-activated genes (G6PC and PCK1) that are rate-limiting for gluconeogenesis. These results have implications for proposed tumor-suppressor functions of p53 through regulation of metabolic pathways.

Among a variety of other functions, SIRT6 was previously connected to glucose metabolism. For example, SIRT6 acts as a corepressor of the transcription factor Hif1-α to suppress glycolysis (23). Conversely, the deletion of Sirt6 in mice results in severe hypoglycemia (33) whereas the liver-specific deletion of Sirt6 leads to increased glycolysis and triglyceride synthesis (23, 34). Our study adds further evidence that SIRT6 plays an important role in glucose metabolism by connecting p53 transcription activity and gluconeogenesis. Our data also reemphasize a previously established role for SIRT6 in regulating the acetylation state and nuclear localization of FoxO proteins, albeit in a divergent manner. Thus, the Caenorhabditis elegans SIRT6/7 homolog SIR-2.4 was implicated in DAF-16 deacetylation and consequent nuclear localization and function in stress responses (35); and the effect was reported to be indirect and to involve a stress-induced inhibition by SIR-2.4 of CBP-mediated acetylation of DAF-16 that is independent of its deacetylase activity. These results, emphasizing context-dependent SIRT6 mechanisms, contrast with the SIRT6 deacetylase activity requirement for FoxO1 nuclear exclusion in the present study and a likely direct effect of SIRT6 on FoxO1 deacetylation based on their direct interaction, the SIRT6 deacetylase activity requirement, and precedent (15, 17, 18) from direct SIRT1-mediated deacetylation of FoxO proteins.

Despite a high genetic diversity, cancer cells exhibit a common set of functional characteristics, one being the “Warburg effect”: i.e., continuous high glucose uptake and a higher rate of glycolysis than that in normal cells (36). To favor the rapid proliferation requirement for high ATP/ADP and ATP/AMP ratios, cancer cells use large amounts of glucose. p53, as one of the most important tumor suppressors, exerts its antineoplastic function through diverse pathways that include the regulation of glucose metabolism. Thus, p53 regulates glucose metabolism by activation of TIGAR (3), which lowers the intracellular concentrations of fructose-2,6-bisphosphate and decreases glycolysis. On the other hand, p53 activation causes down-regulation of several glycolysisrelated factors such as phosphoglycerate mutase (PGM) (37) and the glucose transporters (4). Expression of p53 also can limit the activity of IκBα and IκBβ, thereby restricting the activation of NFκB and dampening the expression of glycolysis-promoting genes such as GLUT3 (38). As a reverse glycolysis pathway, gluconeogenesis generates glucose from noncarbohydrate precursors and is conceivably essential for tumor cell growth. However, the current study further supports the notion (6) that p53 is also involved in a gluconeogenesis inhibition pathway, which in this case is executed by enhanced SIRT6 expression and subsequent FoxO1 nuclear exclusion. These results raise the interesting possibility that an inhibition of gluconeogenesis may contribute to the tumorsuppressive function of p53.

 

http://www.frontiersin.org/Journal/DownloadFile/1/2496/20065/1/21/fphar-03-00022_pdf

http://www.jcpjournal.org/journal/DOIx.php?id=10.15430/JCP.2013.18.3.221
Keywords : Oncogenes, Tumor suppressors, Glutamine metabolism, Cancer cells … p53 is a well-known protein which is involved in many cellular functionsincluding cell … deprivation activates p53 by regulating protein phosphatase 2A ( PP2A). …. and tumor suppressors may affect glutamine metabolism in cancercells

 

Protein controlling glucose metabolism also a tumor suppressor

Finding supports metabolic strategies to control tumor growth

http://www.massgeneral.org/about/pressrelease.aspx?id=1530    December 6, 2012

A protein known to regulate how cells process glucose also appears to be a tumor suppressor, adding to the potential that therapies directed at cellular metabolism may help suppress tumor growth.  In their report in the Dec. 7 issue of Cell, a multi-institutional research team describes finding that cells lacking the enzyme SIRT6, which controls how cells process glucose, quickly become cancerous.  They also found evidence that uncontrolled glycolysis, a stage in normal glucose metabolism, may drive tumor formation in the absence of SIRT6 and that suppressing glycolysis can halt tumor formation.

“Our study provides solid evidence that SIRT6 may function as a tumor suppressor by regulating glycolytic metabolism in cancer cells,” says Raul Mostoslavsky, MD, PhD, of the Massachusetts General Hospital (MGH) Cancer Center, senior author of the report.  “Critically, our findings indicate that, in tumors driven by low SIRT6 levels, drugs that may inhibit glycolysis – currently a hot research topic among biotechnology companies – could have therapeutic benefits.”

The hypothesis that a switch in the way cells process glucose could set off tumor formation was first proposed in the 1920s by German researcher Otto Warburg, who later received the Nobel Prize for discoveries in cellular respiration.  He observed that, while glucose metabolism is normally a two-step process involving glycolysis in the cellular cytoplasm followed by cellular respiration in the mitochondria, in cancer cells rates of glycolysis are up to 200 times higher.  Warburg’s proposition that this switch in glucose processing was a primary cause of cancer did not hold up, as subsequent research supported the role of mutations in oncogenes, which can spur tumor growth if overexpressed, and tumor suppressors, which keep cell proliferation under control.  But recent studies have suggested that alterations in cellular metabolism may be part of the process through which activated oncogenes or inactivated tumor suppressors stimulate cancer formation.

A 2010 study led by Mostoslavsky found that the absence of SIRT6 – one of a family of proteins called sirtuins that regulate many important biological pathways – appears to “flip the switch” from normal glucose processing to the excess rates of glycolysis seen in cancer cells. The current study was specifically designed to investigate whether SIRT6’s control of glucose metabolism also suppresses tumor formation.  The research team first showed that cultured skin cells from embryonic mice lacking SIRT6 proliferated rapidly and quickly formed tumors when injected into adult mice.  They also confirmed elevated glycolysis levels in both cells lacking SIRT6 and tumor cells and found that formation of tumors through SIRT6 deficiency did not appear to involve oncogene activation.

Analysis of tumor samples from patients found reduced SIRT6 expression in many – particularly in colorectal and pancreatic tumors.  Even among patients whose tumors appeared to be more aggressive, higher levels of SIRT6 expression may have delayed or, for some, prevented relapse.   In a mouse model programmed to develop numerous colon polyps, the researchers showed that lack of intestinal SIRT6 expression tripled the formation of polyps, many of which became invasive tumors.  Treating the animals with a glycolytic inhibitor significantly reduced tumor formation, even in the absence of SIRT6.

“Our results indicate that, at least in certain cancers, inhibiting glycolytic metabolism could provide a strong alternative way to halt cancer growth, possibly acting synergistically with current anti-tumor therapies,” says Mostoslavsky, an assistant professor of Medicine at Harvard Medical School.  “Cancer metabolism has only recently emerged as a hallmark of tumorigenesis, and the field is rapidly expanding.  With the current pace of research and the speed at which some basic discoveries are moving into translational studies, it is likely that drugs targeting cancer metabolism may be available to patients in the near future.”

 

THE HISTONE DEACETYLASE SIRT6 IS A NOVEL TUMOR SUPPRESSOR THAT CONTROLS CANCER METABOLISM

Reprogramming of cellular metabolism is a key event during tumorigenesis. Despite being known for decades (Warburg effect), the molecular mechanisms regulating this switch remained unexplored. Here, we identify SIRT6 as a novel tumor suppressor that regulates aerobic glycolysis in cancer cells. Importantly, loss of SIRT6 leads to tumor formation without activation of known oncogenes, while transformed SIRT6-deficient cells display increased glycolysis and tumor growth, suggesting that SIRT6 plays a role in both establishment and maintenance of cancer. Using a conditional SIRT6 allele, we show that SIRT6 deletion in vivoincreases the number, size and aggressiveness of tumors. SIRT6 also functions as a novel regulator of ribosome metabolism by co-repressing MYC transcriptional activity. Lastly, SIRT6 is selectively downregulated in several human cancers, and expression levels of SIRT6 predict prognosis and tumor-free survival rates, highlighting SIRT6 as a critical modulator of cancer metabolism. Our studies reveal SIRT6 to be a potent tumor suppressor acting to suppress cancer metabolism.

Cancer cells are characterized by the acquisition of several characteristics that enable them to become tumorigenic (Hanahan and Weinberg, 2000). Among them, the ability to sustain uncontrolled proliferation represents the most fundamental trait of cancer cells. This hyperproliferative state involves the deregulation of proliferative signaling pathways as well as loss of cell cycle regulation. In addition, tumor cells need to readjust their energy metabolism to fuel cell growth and division. This metabolic adaptation is directly regulated by many oncogenes and tumor suppressors, and is required to support the energetic and anabolic demands associated with cell growth and proliferation (Lunt and Vander Heiden, 2011).

Alteration in glucose metabolism is the best-known example of metabolic reprogramming in cancer cells. Under aerobic conditions, normal cells convert glucose to pyruvate through glycolysis, which enters the mitochondria to be further catabolized in the tricarboxylic acid cycle (TCA) to generate adenosine-5’-triphosphate (ATP). Under anaerobic conditions, mitochondrial respiration is abated; glucose metabolism is shifted towards glycolytic conversion of pyruvate into lactate. This metabolic reprogramming is also observed in cancer cells even in the presence of oxygen and was first described by Otto Warburg several decades ago (Warburg, 1956; Warburg et al., 1927). By switching their glucose metabolism towards “aerobic glycolysis”, cancer cells accumulate glycolytic intermediates that will be used as building blocks for macromolecular synthesis (Vander Heiden et al., 2009). Most cancer cells exhibit increased glucose uptake, which is due, in part, to the upregulation of glucose transporters, mainly GLUT1 (Yamamoto et al., 1990; Younes et al., 1996). Moreover, cancer cells display a high expression and activity of several glycolytic enzymes, including phospho-fructose kinase (PFK)-1, pyruvate kinase M2, lactate dehydrogenase (LDH)-A and pyruvate dehydrogenase kinase (PDK)-1 (Lunt and Vander Heiden, 2011), leading to the high rate of glucose catabolism and lactate production characteristic of these cells. Importantly, downregulation of either LDH-A or PDK1 decreases tumor growth (Bonnet et al., 2007; Fantin et al., 2006; Le et al., 2010) suggesting an important role for these proteins in the metabolic reprogramming of cancer cells.

Traditionally, cancer-associated alterations in metabolism have been considered a secondary response to cell proliferation signals. However, growing evidence has demonstrated that metabolic reprogramming of cancer cells is a primary function of activated oncogenes and inactivated tumor suppressors (Dang et al., 2012;DeBerardinis et al., 2008; Ward and Thompson, 2012). Despite this evidence, whether the metabolic reprogramming observed in cancer cells is a driving force for tumorigenesis remains as yet poorly understood.

Sirtuins are a family of NAD+-dependent protein deacetylases involved in stress resistance and metabolic homeostasis (Finkel et al., 2009). In mammals, there are seven members of this family (SIRT1-7). SIRT6 is a chromatin-bound factor that was first described as a suppressor of genomic instability (Mostoslavsky et al., 2006). SIRT6 also localizes to telomeres in human cells and controls cellular senescence and telomere structure by deacetylating histone H3 lysine 9 (H3K9) (Michishita et al., 2008). However, the main phenotype SIRT6 deficient mice display is an acute and severe metabolic abnormality. At 20 days of age, they develop a degenerative phenotype that includes complete loss of subcutaneous fat, lymphopenia, osteopenia, and acute onset of hypoglycemia, leading to death in less than ten days (Mostoslavsky et al., 2006). Recently, we have demonstrated that the lethal hypoglycemia exhibited by SIRT6 deficient mice is caused by an increased glucose uptake in muscle and brown adipose tissue (Zhong et al., 2010). Specifically, SIRT6 co-represses HIF-1α by deacetylating H3K9 at the promoters of several glycolytic genes and, consequently, SIRT6 deficient cells exhibit increased glucose uptake and upregulated glycolysis even under normoxic conditions (Zhong et al., 2010). Such a phenotype, reminiscent of the “Warburg Effect” in tumor cells, prompted us to investigate whether SIRT6 may protect against tumorigenesis by inhibiting glycolytic metabolism.

Here, we demonstrate that SIRT6 is a novel tumor suppressor that regulates aerobic glycolysis in cancer cells. Strikingly, SIRT6 acts as a first hit tumor suppressor and lack of this chromatin factor leads to tumor formation even in non-transformed cells. Notably, inhibition of glycolysis in SIRT6 deficient cells completely rescues their tumorigenic potential, suggesting that enhanced glycolysis is the driving force for tumorigenesis in these cells. Furthermore, we provide new data demonstrating that SIRT6 regulates cell proliferation by acting as a co-repressor of c-Myc, inhibiting the expression of ribosomal genes. Finally, SIRT6 expression is downregulated in human cancers, strongly reinforcing the idea that SIRT6 is a novel tumor suppressor.

…..

In addition to controlling glucose metabolism in cancer cells, our current work unravels a novel function of SIRT6 as a regulator of ribosomal gene expression. One of the main features of cancer cells is their high proliferative potential. In order to proliferate, cancer cells readjust their metabolism to generate biosynthetic precursors for macromolecular synthesis (Deberardinis et al., 2008). However, protein synthesis also requires the activation of a transcriptional program leading to ribosome biogenesis and mRNA translation (van Riggelen et al., 2010). As a master regulator of cell proliferation, MYC regulates ribosome biogenesis and protein synthesis by controlling the transcription and assembly of ribosome components as well as translation initiation (Dang et al., 2012; van Riggelen et al., 2010). Our results show that SIRT6 specifically regulates the expression of ribosomal genes. In keeping with this, SIRT6-deficient tumor cells exhibit high levels of ribosomal protein gene expression. Beyond ribosome biosynthesis, MYC regulates glucose and glutamine metabolism (Dang et al., 2012). Our results show that glutamine – but not glucose – metabolism is rescued in SIRT6-deficient/MYC knockdown cells, suggesting that SIRT6 and MYC might have redundant roles in regulating glucose metabolism.

Overall, our results indicate that SIRT6 represses tumorigenesis by inhibiting a glycolytic switch required for cancer cell proliferation. Inhibition of glycolysis in SIRT6-deficient cells abrogates tumor formation, providing proof of concept that inhibition of glycolytic metabolism in tumors with low SIRT6 levels could provide putative alternative approaches to modulate cancer growth. Furthermore, we uncover a new role for SIRT6 as a regulator of ribosome biosynthesis by co-repressing MYC transcriptional activity. Our results indicate that SIRT6 sits at a critical metabolic node, modulating both glycolytic metabolism and ribosome biosynthesis (Figure 7L). SIRT6 deficiency deregulates both pathways, leading to robust metabolic reprogramming that is sufficient to promote tumorigenesis bypassing major oncogenic signaling pathway activation.

 

Lack of cellular enzyme triggers switch in glucose processing

Understanding mechanism underlying SIRT6 activity may help treat diabetes, cancer

http://www.massgeneral.org/about/pressrelease.aspx?id=1196   January 21, 2010

A study investigating how a cellular enzyme affects blood glucose levels in mice provides clues to pathways that may be involved in processes including the regulation of longevity and the proliferation of tumor cells. In their report in the January 22 issue of Cell, a Massachusetts General Hospital (MGH)-based team of researchers describes the mechanism by which absence of the enzyme SIRT6 induces a fatal drop in blood sugar in mice by triggering a switch between two critical cellular processes.

“We found that SIRT6 functions as a master regulator of glucose levels by maintaining the normal processes by which cells convert glucose into energy,” says Raul Mostoslavsky, MD, PhD, of the MGH Cancer Center, who led the study. “Learning more about how this protein controls the way cells handle glucose could lead to new approaches to treating type 2 diabetes and even cancer.”

SIRT6 belongs to a family of proteins called sirtuins, which regulate important biological pathways in organisms from bacteria to humans. Originally discovered in yeast, sirtuins in mammals have been shown to have important roles in metabolic regulation, programmed cell death and adaptation to stress. SIRT6 is one of seven mammalian sirtuins, and Mostoslavsky’s team previously showed that mice lacking the protein die in the first month of life from acute hypoglycemia. The current study was designed to investigate exactly how lack of SIRT6 causes this radical drop in blood sugar.

Normally cells convert glucose into energy through a two-step process. The first stage called glycolysis takes place in the cytoplasm, where glucose is broken down into an acid called pyruvate and a few molecules of ATP, the enzyme that provides the energy to power most biological processes. Pyruvate is taken into cellular structures called mitochondria, where it is further processed to release much greater amounts of ATP through a process called cellular respiration.

In a series of experiments in mouse cells, the researchers showed that SIRT6-deficiency hypoglycemia is caused by increased cellular uptake of glucose and not by elevated insulin levels or defects in the absorption of glucose from food. They then found increased levels of glycolysis and reduced mitochondrial respiration in SIRT6-knockout cells, something usually seen when cells are starved for oxygen or glucose, and showed that activation of the switch from cellular respiration to glycolysis is controlled through SIRT6’s regulation of a protein called HIF1alpha. Normally, SIRT6 represses glycolytic genes through its role as a compactor of chromatin – the tightly wound combination of DNA and a protein backbone that makes up chromosomes. In the absence of SIRT6, this structure is opened, causing activation of these glycolytic genes. The investigators’ finding increased expression of glycolytic genes in living SIRT6-knockout mice – which also had elevated levels of lactic acid, characteristic of a switch to glycolytic glucose processing – supported their cellular findings.

Studies in yeast, worms and flies have suggested a role for sirtuins in aging and longevity, and while much of the enzymes’ activity in mammals is unclear, SIRT6’s control of critical glucose-metabolic pathways could signify a contribution to lifespan regulation. Elevated glycolysis also is commonly found in tumor cells, suggesting that a lack of SIRT6 could contribute to tumor growth. Conversely, since knocking out SIRT6 causes blood sugar to drop, limited SIRT6 inhibition could be a novel strategy for treating type 2 diabetes.

“There’s a lot we still don’t know about SIRT6,” adds Mostoslavsky, who is an assistant professor of Medicine at Harvard Medical School. “We need to identify the factors that interact with SIRT6 and determine how it is regulated; investigate whether it acts as a tumor suppressor and how it might help lower glucose levels in diabetes; and determine its target organs in living animals, all of which we are investigating.”

 

A tale of metabolites: the crosstalk between chromatin and energy metabolism

Mitochondrial metabolism influences histone and DNA modifications by retrograde signaling and activation of transcriptional programs. Considering the high number of putative sites for acetylation and methylation in chromatin, we propose in this Perspective that epigenetic modifications might impinge on cellular metabolism by affecting the pool of acetyl-CoA and SAM.

Metabolism can be defined as the sum of chemical reactions that occur within a cell to sustain life. It is also the way that a cell interacts with energy sources: in other words, it is the coordination of energy intake, its utilization and storage that ultimately allows growth and cell division. In animal cells, mitochondria have evolved to become the most efficient system to generate energy. This organelle consumes carbon sources via oxidative phosphorylation to produce ATP, the energy currency of the cell. Additionally, the mitochondria produces intermediate metabolites for the biosynthesis of DNA, proteins and lipids.

Under basic dividing conditions, uptake of nutrients is tightly regulated through growth signaling pathways, thus differentiated cells engage in oxidative metabolism, the most efficient mechanism to produce energy from nutrients. Cells metabolize glucose to pyruvate through glycolysis in the cytoplasm, and this pyruvate is then oxidized into CO2 through the mitochondrial TCA cycle. The electrochemical gradient generated across the inner mitochondrial membrane facilitates ATP production in a highly efficient manner. Studies in recent years indicate that under conditions of nutrient excess, cells increase their nutrient uptake, adopting instead what is known as aerobic glycolysis, an adaptation that convert pyruvate into lactate, enabling cells to produce intermediate metabolites to sustain growth (anabolic metabolism) (1). Interestingly, most cancer cells undergo the same metabolic switch (Warburg Effect), a unique evolutionary trait that allows them to grow unabated. Although aerobic glycolysis generates much less ATP from glucose compared to oxidative phosphorylation, it provides critical intermediate metabolites that are used for anaplerotic reactions, and therefore is an obligatory adaptation among highly proliferative cells. In response to variations in nutrient availability, cells regulate their metabolic output, coordinating biochemical reactions and mitochondrial activity by altering transcription of mitochondrial genes through both activation of transcription factors, such as PGC1α, and chromatin modulators that exert epigenetic changes on metabolic genes.

Mitochondrial dysfunction has been implicated in aging, degenerative diseases and cancer. Proper mitochondrial function can be compromised by the accumulation of mutations in mitochondrial DNA that occur during aging. In addition, reactive oxygen species (ROS) produced during oxidative phosphorylation can promote oxidative damage to DNA, protein and lipids, in turn adversely affecting global cellular functions. In recent years, several studies have illustrated a novel unexpected link between metabolism and gene activity: fluctuations in mitochondrial and cytoplasmic metabolic reactions can reprogram global metabolism by means of impacting epigenetic dynamics. These studies will be briefly summarized in the first part of this article. In the second part, we will propose a provocative novel hypothesis: the crosstalk between metabolism and epigenetics is a two-way street, and defects in chromatin modulators may affect availability of intermediate metabolites, in turn influencing energy metabolism.

Metabolism impacts epigenetics

A regulated crosstalk between metabolic pathways in the mitochondria and epigenetic mechanisms in the nucleus allows cellular adaptations to new environmental conditions. Fine-tuning of gene expression is achieved by changes in chromatin dynamics, including methylation of DNA and posttranslational modifications of histones: acetyl, methyl and phosphate groups can be added by acetyltransferases, methyltransferases and kinases, respectively, to different residues on histones. Given the number of residues that can potentially undergo modifications in histone tails and in the DNA, it is reasonable to consider that metabolic changes affecting the availability of these metabolites will impact epigenetics (as discussed below).

Recently, acetylation of proteins was revealed to be as abundant as phosphorylation (2). This posttranslational modification involves the covalent binding of an acetyl group obtained from acetyl-CoA to a lysine. In histones, acetylation can modify higher order chromatin structure and serve as a docking site for histone code readers. Recent mass spectrometry studies have uncovered the complete acetylome in human cells and revealed that protein acetylation occurs broadly in the nucleus, cytoplasm and mitochondria, affecting more than 1700 proteins (2). Acetylation of proteins depends on the availability of acetyl-CoA in each cellular compartment, but this metabolite is produced in the mitochondria and cannot cross the mitochondrial membrane. In single cell eukaryotes, the pool of acetyl groups required for histone acetylation comes from the production of acetyl-CoA by the enzyme acetyl-CoA synthetase (Acs2p), which is responsible of converting acetate into acetyl-CoA. In mammalian cells, although they have a homolog enzyme to Acs2p, AceCS1, the majority of acetyl-CoA is produced from mitochondrion-derived citrate by the enzyme adenosine triphosphate (ATP)-citrate lyase (ACL) (3). ACL is present in the cytoplasm and in the nucleus, and is responsible for the production of acetyl-CoA from citrate in both compartments. Citrate is generated in the metabolism of glucose and glutamine in the TCA cycle. In contrast to acetyl-CoA, citrate can cross the mitochondrial membrane and diffuse through the nuclear pores into the nucleus, where it can be converted into acetyl-CoA by ACL. Wellen and colleagues found that ACL is required for acetylation of histones under normal growth conditions; knockdown of ACL decreases the pool of acetyl-CoA in the nucleus and reduces the level of histone acetylation (3). Strikingly, reduction in histone acetylation occurs preferentially around glycolytic genes, leading to downregulation of their transcription and therefore inhibition of glycolysis. These observations reveal a process where glucose metabolism dictates histone acetylation that in a feedback mechanism controls the rate of glycolysis.

Notably, deacetylation of histones also exhibits a metabolic influence. Deacetylation of histones is achieved by class I and class II histone deacetylases (HDACs) and by a separate class (class III), also known as sirtuins. Sirtuins use NAD+ as a cofactor for deacetylation, and the ratio of NAD+/NADH regulates their activity. In diets rich in carbohydrates, growth factors stimulate cellular glucose uptake and the production of energy is carried out through glycolysis. In this context, the NAD+/NADH ratio decreases, in turn inhibiting, in theory, sirtuins in the cytoplasm (Sirt2) and nucleus (Sirt1, Sirt6 and Sirt7). In fact, low Sirt1 and Sirt6 activity generates a global increase in protein acetylation. Interestingly, Sirt6, which is exclusively nuclear, deacetylates H3K9 Hif1α target genes, repressing their transcription. Since most of these genes are glycolytic, deacetylation of histones by Sirt6 modulates glycolysis. Indeed, SIRT6-deficient mice experience a dramatic increase in glucose uptake for glycolysis, triggering a fatal hypoglycemia in few weeks (4).

In animal cells, both histone acetylation and deacetylation are under the control of glucose metabolism through the availability of acetyl-CoA and NAD+, respectively. However, is this metabolic control restricted to acetylation, or can other reactions in the nucleus be influenced by the energy status of the cell?

Histone methyltransferases (HMTs) use S-adenosylmethionine (SAM) to transfer a methyl group onto lysine and arginine residues on histone tails. SAM is produced from methionine by the enzyme S-adenosyl methionine transferase (MAT) in a reaction that uses ATP. The recent finding of MAT in the nucleus suggests that the SAM pool could also be controlled locally in this compartment (5). The reverse reaction catalyzed by histone demethylases (HDMs) uses flavin adenine dinucleotide (FAD+) and α-ketoglutarate as coenzymes. FAD is a common redox coenzyme that exists in two different redox states. In its reduced state, FADH2 is a carrier of energy and when oxidized, FAD+ is consumed in the oxidation of succinate to fumarate by the enzyme succinate dehydrogenase (complex II) in one of the last steps of the TCA cycle. On the other hand, α-ketoglutarate is an intermediate in the TCA cycle. It is generated from isocitrate by the enzymes isocitrate dehydrogenase 1 and 2 (IDH1-cytosolic and IDH2-mitochondrial) (Figure 1A–B). Based on these findings, it is easy to infer that the amount of coenzymes used for histone methylation and demethylation could also be controlled by metabolic reactions. Moreover, the different cellular compartments compete for the same metabolites. Indeed, changes in diet that affect the biosynthesis of SAM, FAD and α-ketoglutarate in the mitochondria and cytoplasm have been shown to impact histone methylation (6).

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Figure 1   A) Diagram depicting two-way crosstalk between metabolites in cytoplasm/mitochondria and chromatin.

More recently, some of the metabolic enzymes responsible for producing cofactors for nuclear biochemical reactions have been found mutated in cancer. For instance, IDH1 and IDH2 somatic mutations are recurrent in gliomas and acute myeloid leukemias (AML). These mutations lead not only to a decreased production of α-ketoglutarate but also to a new activity: α-ketoglutarate is in fact converted into 2-hydroxyglutarate (2-HG), a metabolite rarely found in normal cells. The new metabolite is a competitive inhibitor of α-ketoglutarate-dependent dioxygenase enzymes, including the Jumonji C (JmjC) domain containing histone demethylases and the recently discovered TET family of 5-methylcytosine (5mC) hydroxylases involved in DNA demethylation (7). By inhibiting JmjC and TET enzymes, the aberrant production of 2-HG generates a genome-wide histone and DNA hypermethylation phenotype. This is considered to be, at least in part, at the origin of tumorigenesis in IDH1 and IDH2 mutated cells and for this reason, 2-HG may earn its place as an oncometabolite. The discovery that mutations in metabolic enzymes may influence tumorigenesis by means of controlling genome-wide epigenetic changes caused a paradigm shift, indicating that such metabolic abnormalities may affect cancer beyond the Warburg Effect.  ….

Chromatin modifications and cellular metabolism are tightly connected. Thus far the only aspects that have been considered are the retrograde signaling, with mitochondrial metabolites affecting histone modifications, and the anterograde transcriptional regulation of metabolism. A third aspect of the link between nucleus and metabolism has been, in our opinion, omitted so far: a direct influence of chromatin on acetyl-CoA and SAM availability, which may have an essential role also in cancer establishment and development (Figure 1A–B). Notably, a shift towards glycolytic metabolism is now considered a hallmark of cancer cells. It is also true that multiple tumors carry mutations in chromatin modifiers. However, new studies suggest that those two processes may be much more intertwined that previously appreciated, further blurring the limits on their respective roles in tumorigenesis. There is no doubt that changes in metabolite availability can drastically impact chromatin modifications. We believe that the opposite may be true as well. At least in mouse models, deficiency in two chromatin modifiers, SIRT6 and Jhdm2, causes drastic metabolic abnormalities. Even though some of those phenotypes depend on changes in gene-expression, we would like to propose that severe attrition of metabolite pools might as well play a role, a possibility that awaits experimental proof.

……

 

Investigators at UC San Diego say that when they blocked a well known signaling molecule that plays a major role in driving colorectal cancer, an escape pathway emerged that allowed tumors to continue to grow.

The pathway they explored, ERK1/2, is a problem for about a third of all colorectal cancer patients, says Petrus R. de Jong, MD, PhD, a co-first author on the paper.

“Since we were genetically deleting the ERK1/2 pathway, we expected to see less cell proliferation,” said de Jong. “Instead, the opposite occurred. There was more cell growth and loss of organization within the cells.”

The problem was ERK5, the investigators add. And when that was blocked as well in animal models and cell lines for the disease, the combination approach proved more effective in halting cancer growth.

“If you block one pathway, cancer cells usually mutate and find another pathway that ultimately allows for a recurrence of cancer growth,” said co-first author Koji Taniguchi. “Usually, mutations occur over weeks or months. But other times, as in this case, the tumor does not need to develop mutations to find an escape route from targeted therapy. When you find the compensatory pathway and block both, there is no more escape.”

 

GEN News Highlights    May 18, 2016   http://www.genengnews.com/gen-news-highlights/blocking-cancer-signaling-leads-to-discovery-of-new-tumor-promoting-pathway/81252738/
Blocking Cancer Signaling Leads to Discovery of New Tumor-Promoting Pathway

 Immunofluorescent staining of intestinal epithelium tissue shows cell growth (green). In a normal mouse model (left), cell growth is controlled, but in a mouse model with the ERK1/2 pathway blocked (right) increased cell proliferation and loss of organization occurred. [UC San Diego Health]

An international research team lead by scientists at the University of California San Diego School of Medicine uncovered some surprising results while investigating a potential therapeutic target for the ERK1 and two pathways. These signaling pathways are widely expressed and known to drive cancer growth in one-third of patients with colorectal cancer (CRC). The UCSD team found that an alternative pathway immediately emerges when ERK1/2 is halted, thus allowing tumor cell proliferation to continue.

“Since we were genetically deleting the ERK1/2 pathway, we expected to see less cell proliferation,” explained co-lead study author Petrus R. de Jong, M.D., Ph.D., translational scientist at Sanford Burnham Prebys Medical Discovery Institute. “Instead, the opposite occurred. There was more cell growth and loss of organization within the cells.”

The exciting part of this new study is investigators found that treating both ERK1/2 and the compensatory pathway ERK5 concomitantly with a combination of drug inhibitors halted CRC growth more effectively in both mouse models and human CRC cell lines.

“We show that loss of Erk1/2 in intestinal epithelial cells results in defects in nutrient absorption, epithelial cell migration, and secretory cell differentiation,” the authors wrote. “However, intestinal epithelial cell proliferation is not impeded, implying compensatory mechanisms. Genetic deletion ofErk1/2 or pharmacological targeting of MEK1/2 results in supraphysiological activity of the ERK5 pathway. Furthermore, targeting both pathways causes a more effective suppression of cell proliferation in murine intestinal organoids and human CRC lines.”

The findings from this study were published recently in Nature Communications in an article entitled “ERK5 Signalling Rescues Intestinal Epithelial Turnover and Tumour Cell Proliferation upon ERK1/2 Abrogation.”

The ERK pathway plays a critical role in embryonic development and tissue repair because it instructs cells to multiply and start dividing, but when overactivated cancer growth often occurs.

“Therapies aimed at targeting ERK1/2 likely fail because this mechanism is allowing proliferation through a different pathway,” noted senior study author Eyal Raz, M.D., professor of medicine at UC San Diego School of Medicine. “Previously, ERK5 didn’t seem important in colorectal cancer. This is an underappreciated escape pathway for tumor cells. Hence, the combination of ERK1/2 and ERK5 inhibitors may lead to more effective treatments for colorectal cancer patients.”

Currently, there are 1.2 million people living with CRC in the United States, making it the third most common cancer among men and women. In 2016 alone, an estimated 134,490 new cases are expected to be diagnosed, so understanding the molecular mechanisms that drive tumor promotion are paramount to treating this disease effectively.

“If you block one pathway, cancer cells usually mutate and find another pathway that ultimately allows for a recurrence of cancer growth,” remarked co-lead study author Koji Taniguchi, M.D., Ph.D., senior researcher at the Keio University School of Medicine in Tokyo. “Usually, mutations occur over weeks or months. But other times, as in this case, the tumor does not need to develop mutations to find an escape route from targeted therapy. When you find the compensatory pathway and block both, there is no more escape.”

The researchers were excited by their findings but urged caution at over interpretation of their initial findings and suggested that other classes of inhibitors be tested in combination with ERK5 inhibitors in human CRC cells in preclinical mouse models before any patient trial can begin.

 

ERK5 signalling rescues intestinal epithelial turnover and tumour cell proliferation upon ERK1/2 abrogation

Petrus R. de JongKoji TaniguchiAlexandra R. HarrisSamuel BertinNaoki TakahashiJen DuongAlejandro D. CamposGarth PowisMaripat CorrMichael Karin & Eyal Raz
Nature Communications 7, Article number:11551  doi:10.1038/ncomms11551

The ERK1/2 MAPK signalling module integrates extracellular cues that induce proliferation and differentiation of epithelial lineages, and is an established oncogenic driver, particularly in the intestine. However, the interrelation of the ERK1/2 module relative to other signalling pathways in intestinal epithelial cells and colorectal cancer (CRC) is unclear. Here we show that loss of Erk1/2in intestinal epithelial cells results in defects in nutrient absorption, epithelial cell migration and secretory cell differentiation. However, intestinal epithelial cell proliferation is not impeded, implying compensatory mechanisms. Genetic deletion of Erk1/2 or pharmacological targeting of MEK1/2 results in supraphysiological activity of the ERK5 pathway. Furthermore, targeting both pathways causes a more effective suppression of cell proliferation in murine intestinal organoids and human CRC lines. These results suggest that ERK5 provides a common bypass route in intestinal epithelial cells, which rescues cell proliferation upon abrogation of ERK1/2 signalling, with therapeutic implications in CRC.

The extracellular signal-regulated kinases 1 and 2 (ERK1/2) are part of the classical family of mammalian mitogen-activated protein kinases (MAPKs), which also include three c-Jun amino-terminal kinases (JNK1/2/3), four p38 isoforms and its lesser-known counterpart, ERK5. The serine/threonine kinases ERK1 (MAPK3, also known as p44 MAPK) and ERK2 (MAPK1, also known as p42 MAPK) show 83% amino acid identity, are ubiquitously expressed and typically activated by growth factors and phorbol esters, whereas the p38 and JNK pathways are mainly activated by inflammatory cytokines and stress1. MAPKs are involved in regulation of mitosis, gene expression, cell metabolism, cell motility and apoptosis. ERK1/2 are activated by MEK1 and MEK2, which themselves are activated by Raf-1, A-Raf or B-Raf1, 2. Ras proteins (K-Ras, H-Ras or N-Ras) are small GTPases that can be activated by receptor tyrosine kinases (RTKs) or G-protein coupled receptors (GPCRs), which recruit Raf proteins to the plasma membrane where they are activated. Together, these modules constitute the Ras–Raf–MEK–ERK pathway3.

The activation of ERK1/2 results in their nuclear translocation where they can phosphorylate a variety of nuclear targets such as Elk-1, c-Fos and c-Myc1, in addition to p90 ribosomal S6 kinases (p90RSKs) and mitogen- and stress-activated protein kinases, MSK1/2. The full repertoire of substrates for ERK1/2 consists of at least 160 cellular proteins4. These proteins are typically involved in the regulation of cell proliferation—more specifically, G1/S-phase cell cycle progression—and differentiation. However, their cellular effects are context-dependent and determined by the spatial and temporal dynamics of ERK1/2 activity5, which are highly regulated by scaffolding proteins and phosphatases3, 6, 7.

Despite vast literature on the role of ERK1/2 in cell proliferation, the absolute requirement of this signalling module in rapidly dividing tissues relative to other signalling pathways is unknown. The small intestinal epithelium is particularly suitable to address this question given the short (4–8 days) and dynamic life cycle of intestinal epithelial cells (IECs). Lgr5+ intestinal stem cells at the intestinal crypt base produce transit-amplifying cells, which then undergo a number of proliferative cycles before terminal differentiation into absorptive enterocytes at the crypt–villus border. Enterocytes then migrate to the villus tip where they undergo anoikis and are shed into the gut lumen8. All of these cellular events are tightly coordinated by the Wnt, Notch, bone morphogenetic protein (BMP) and Hedgehog pathways9, whereas the roles of ERK1/2 remain to be charted. In the intestines, the ERK1/2 pathway is likely activated by autocrine and paracrine factors downstream of RTKs, such as epidermal growth factor receptor (EGFR)10, and by exogenous microbial-derived substrates that signal through the Toll-like receptor (TLR)/MyD88 pathway11.

To study the effects of ERK1/2 in the adult intestinal epithelium, we generated mice with a conditional (IEC-specific) and tamoxifen-inducible deletion of Erk2 on the Erk1−/− background, which completely abrogates this pathway. We show that the ERK1/2 signalling module, surprisingly, is dispensable for IEC proliferation. Genetic deletion of Erk1/2 in primary IEC or treatment of colorectal cancer (CRC) cell lines with MEK1/2 inhibitors results in compensatory activation of the ERK5 pathway. Moreover, the treatment of human CRC lines with a combination of MEK1/2 and ERK5 inhibitors is more efficacious in the inhibition of cancer cell growth. Thus, compensatory signalling by ERK5 suggests a potential rescue pathway that has clinical implications for targeted therapy in colorectal cancer.

….

Figure 1: Wasting disease associated with malabsorption in Erk1/2ΔIEC mice.

ERK1/2ΔIEC causes wasting and enterocyte dysfunction

Here we show that ERK1/2 signalling in mouse intestinal epithelium is dispensable for cell proliferation, while it resulted in abnormal differentiation of enterocytes, wasting disease and ultimately lethality (Fig. 1). Consistent with these findings, ERK1/2 MAPKs were shown to be associated with the enterocyte brush border and activated upon RTK stimulation or feeding27 or electrical field stimulation in polarized epithelium28. This seems at odds with literature that suggest that maintained ERK1/2 signalling precludes enterocyte differentiation29, 30. A possible explanation for this discrepancy could be that cycling IEC in the transit amplifying zone of the crypt require relatively high levels of active ERK1/2, which is readily blocked by pharmacological intervention, whereas a transition to low level ERK1/2 activity in IEC migrating into the villus compartment promotes the absorptive enterocyte differentiation program that is only perturbed upon complete genetic deletion of Erk1/2. Little is known about the role of ERK1/2 signalling in the life cycle of secretory cells in the gut. A recent report by Heuberger et al.15 described that IEC-specific deletion of non-receptor tyrosine phosphatase, Shp2, resulted in the loss of p-ERK1/2 levels in the small intestine. This coincided with an increased number of Paneth cells at the expense of goblet cells in the small intestine, as well as shortening of villi. They also observed the strongest staining for epithelial p-ERK1/2 in the TA zone. This p-ERK1/2+ staining pattern and the architectural organization of the TA zone were lost in Shp2 knockout mice. Interestingly, the deleterious effects of Shp2 deficiency were rescued by expression of constitutively active MEK1. A model was proposed in which the balance between Wnt/β-catenin and MAPK signalling determines Paneth cell versus goblet cell differentiation, respectively15. This proposed crucial role for ERK1/2 MAPK signalling in intestinal secretory cell differentiation is consistent with our observations inERK1/2ΔIECmice.

Migration and differentiation are functionally intertwined in the intestines, as demonstrated by the immature phenotype of mislocalized Paneth cells observed in ΔIEC mice (Fig. 2). Critical to epithelial cell migration is proper cytoskeleton reorganization mediated by the small GTPases of the Rho family, cell polarization regulated by Cdc42 and dynamic adhesion through cell–matrix and cell–cell interaction via integrin/FAK/Src signalling31. The ERK1/2 module is used as a downstream effector of many of these pathways in the intestine, including Rho GTPases32, FAK33and Src34, and has been suggested to promote cell motility33, 35. RTK signalling also contributes to cell migration, for example, Eph–Ephrin receptor interactions are crucial for correct positioning of Paneth cells36. Ephrin receptor-induced epithelial cell migration has been shown to be mediated by Src and ERK1/2 activation37, 38, which may explain the Paneth cell mislocalization observed in ΔIEC mice. In summary, the ERK1/2 module is indispensable for full maturation of both absorptive enterocytes and the secretory lineage (Fig. 7a), confirming its crucial role in the integration of cellular cues required for determination of epithelial cell fate.

Figure 7: Roles of ERK1/2 and ERK5 in intestinal homeostasis and tumorigenesis.

Roles of ERK1/2 and ERK5 in intestinal homeostasis and tumorigenesis.

(a) When the ERK1/2 pathway is intact, extracellular cues that are transduced via RTKs or GPCRs activate Ras under physiological conditions, or alternatively, Ras is constitutively active in colorectal cancer (RasΔ*), which preferentially activates the Raf–MEK1/2–ERK1/2 module. The nuclear and transcriptional targets of ERK1/2 are crucial for enterocyte and secretory cell differentiation, IEC migration, as well as cell proliferation under homeostatic and oncogenic conditions. Importantly, ERK1/2 activation also results in the activation of negative feedback mechanisms that suppress its upstream kinases (for example, RTKs, son of sevenless, Raf) and activate dual specificity phosphatases (DUSPs), resulting in the silencing of the ERK5 module. (b) Upon abrogation of MEK1/2 or genetic knockout ofErk1/2, the lack of negative feedback mechanisms (that is, feedback activation) results in upregulation of the Ras–Raf–MEK5–ERK5 module, which maintains cell proliferation under physiological conditions, or results in continued tumour cell proliferation in colorectal cancer, respectively. However, the lack of activation of ERK1/2-specific targets results in differentiation and migration defects of intestinal epithelial cells culminating in malabsorption, wasting disease and mortality. Compensatory upregulation of the ERK5 pathway in CRC can be reversed by targeted treatment with its specific inhibitor, XMD8-92.

An unexpected finding was the redundancy of ERK1/2 in the gut with regard to cell proliferation.Erk1/2 deletion was compensated by upregulated ERK5 signalling. Genetic targeting of ERK1/2 in vitro previously showed that Erk2 knockdown is more effective than Erk1 knockdown in suppressing cell proliferation, although this may be related to higher expression levels of the former39. The effect of gene dosage was demonstrated in vivo by the observations that whileErk1−/− mice are viable12 and Erk2−/− mice die in utero13, Erk2+/− mice are only viable when at least one copy of Erk1 is present. However, mice heterozygous (+/−) for both Erk1 and Erk2 alleles were born at lower than Mendelian ratio39. More recently, it was reported that transgenic expression of ERK1 can compensate for Erk2 deletion40, demonstrating functional redundancy between both family members. Deletion of Erk1/2 in adult skin tissue resulted in hypoplasia, which was associated with G2/M cell cycle arrest, without notable differentiation defects of keratinocytes41. These data differ from our observations in the intestines, which might be explained by incomplete and transient siRNA-mediated knockdown of ERK1/2 in primary keratinocyte cultures41, compared with more efficient genomic deletion of Erk1 and Erk2 that is typically achieved by the Villin-Cre-ERT2 system14, possibly resulting in different outcomes.

Both ERK1/2 and ERK5 have been described to promote cell cycle progression, although they have different upstream signalling partners, MEK1/2 and MEK5, respectively1. Furthermore, ERK2 and ERK5 proteins share only about 66% sequence identity, and MEK5 is phosphorylated by MEKK2/3, which can also activate the p38 and JNK pathways42. The ERK5 pathway is classically activated by stress stimuli, in addition to mitogens; thus, it shares features of both the ERK1/2, and p38 and JNK pathways, respectively43. ERK5 induces expression of cyclin D1 (refs 44, 45), and suppresses expression of cyclin dependent kinase inhibitors46, thereby promoting G1/S-phase cell cycle progression. Importantly, the role of ERK5 in IEC differentiation and intestinal homeostasis is currently unknown. Knockout of Erk1/2 in IEC induced activity of ERK5, which was not detectable in naive mice (Fig. 3). These data suggest that the ERK1/2 and ERK5 modules may share proximal signalling components. Although EGFR is a likely candidate in this context19, 20, we found that abrogation of EGFR signalling did not prevent enhanced ERK5 activity upon MEK1/2 inhibition. Although it was originally suggested that ERK5 signalling is independent of Ras20, other groups established that Ras, either through physiological signalling47, or by its oncogenic activity48,49, activates the MEK5–ERK5 signalling axis. Thus, rewiring of signalling networks downstream of Ras could explain the supraphysiological activity of ERK5 upon conditional deletion of Erk1/2 in the intestines. In fact, it has been shown that ERK1/2 signalling mediates negative feedback on ERK5 activity50, possibly through transcriptional activation of dual specificity phosphatases (DUSPs)51. Alternatively, ERK1/2-induced FOS-like antigen 1 (Fra-1) may negatively regulate MEK5 (ref. 52). These data suggest that ERK5 is a default bypass route downstream of RTK-Ras and activated upon loss of ERK1/2-mediated repression, thereby ensuring the transduction of mitogenic signals to the nucleus (Fig. 7b). Consistent with this concept, we found that ERK5 inhibition induces atrophy of ΔIEC intestinal organoids (Fig. 4). In addition, important downstream transcriptional targets of ERK5 and ERK1/2 overlap, such as immediate-early gene Fra1 and oncogene c-Myc, whereas c-Fos and Egr1 were specifically induced by ERK1/2 (Fig. 6 and Supplementary Fig. 7). Specificity of ERK1/2 over ERK5 and other MAPK family members for the activation of c-Fos has been previously described53, demonstrating their differential biological output despite the shared ability to transduce potent mitogenic signals.

Our findings may be relevant for the use of MAPK inhibitors in the treatment of colorectal cancer. Although there was only a mild phenotype in the colons of ΔIEC mice under homeostatic conditions, the Ras–RAF–MEK–ERK pathway is generally upregulated in malignant cells including CRC54. Targeted therapy typically results in feedback activation of upstream players of the targeted kinase, which are then able to reactivate the same pathway or utilize bypass signalling routes55. For example, on activation, ERK1/2 phosphorylates EGFR, son of sevenless56, and Raf57, thereby terminating upstream signalling activity. Knockout of Erk1/2 eliminates this negative feedback. Our data suggest that ERK5 is a putative resistance pathway in the context of targeted treatment with MEK1/2 or ERK1/2 inhibitors (Fig. 7b). Different classes of MEK1/2 inhibitors display various modes of resistance to therapy (innate, adaptive and acquired)58. Since we have only used one MEK1/2 inhibitor (PD0325901) in our studies, it will be necessary to evaluate other classes of inhibitors in combination with ERK5 inhibitors. Importantly, while treatment with either the MEK1/2 or ERK5 inhibitor suppressed tumour growth in murine Apc−/− organoids, only the latter was able to inhibit the proliferation of Apc−/−;KRASG12V organoids (Fig. 6), which are more representative of human CRC. In line with this, suppression of ERK5 expression by forced expression of miR-143/145 inhibited intestinal adenoma formation in the ApcMin/+ model59, and activated MEK5 correlated with more invasive CRC in human60. ERK5 has been previously reported to mediate resistance to cytotoxic chemotherapy-induced apoptosis61. The highly specific and bioavailable ERK5 inhibitor, XMD8-92, has shown antitumour effects in multiple preclinical cancer models by inhibiting tumour angiogenesis, metastasis and chemo-resistance62. Furthermore, ERK5 inhibition does not induce feedback activation of upstream or parallel signalling pathways62. In conclusion, the ERK1/2 and ERK5 MAPK modules display a high degree of signalling plasticity in the intestinal epithelium, which has implications for targeted treatment of colorectal cancer.

 

Researchers Reveal Role of Transcription Factor Isoforms in Colon Diseases

http://www.genengnews.com/gen-news-highlights/researchers-reveal-role-of-transcription-factor-isoforms-in-colon-diseases/81252735/


 

 

 

 

 

 

 

 

Balance between the two isoforms, P1 and P2, of nuclear receptor HNF4a in the colonic crypt influences susceptibility to colitis and colon cancer. P1 is seen here in green. P2 is seen in red. [Poonamjot Deol, Sladek lab, UC Riverside]

Scientists at the University of California, Riverside have determined the distribution of the P1 and P2 isoforms of hepatocyte nuclear factor 4α (HNF4α) in the colons of mice. They report (“Opposing Roles of Nuclear Receptor HNF4α Isoforms in Colitis and Colitis-Associated Colon Cancer”) in eLife that maintaining a balance of P1 and P2 is crucial for reducing risk of contracting colon cancer and colitis.

What is already known in the field of cell biology is that the HNF4α transcription factor plays a key role in both diseases. HNF4α comes in two major isoforms, P1-HNF4α and P2-HNF4α (P1 and P2), but just how each isoform is involved in colitis and colon cancer is not understood.

“P1 and P2 have been conserved between mice and humans for 70 million years,” said Frances M. Sladek, Ph.D., professor of cell biology, who led the research project. “Both isoforms are important and we want to keep an appropriate balance between them in our gut by avoiding foods that would disrupt this balance and consuming foods that help preserve it. What these foods are is our next focus in the lab.”

The intestine is the only adult tissue in the body that expresses both P1 and P2. Dr. Sladek and her team have shown for the first time that these isoforms perform nonredundant functions in the intestine and are relevant to colitis and colitis-associated colon cancer.

“Our study also suggests that finding a drug to stabilize one isoform should be more effective than targeting both isoforms for treating colitis and colon cancer,” said Karthikeyani Chellappa, Ph.D., the first author of the research paper and a former postdoctoral researcher in Sladek’s lab.

Dr. Sladek explained that the colonic epithelial surface has finger-like invaginations (into the colonic wall) called colonic crypts that house stem cells at their base. These stem cells help regenerate new epithelial cells that continuously migrate up toward the surface, thus ensuring complete renewal of the intestinal lining every 3–5 days.

The researchers observed that the P1-positive cells were found in the surface lining and the top portion of the crypt (green in the accompanying image) whereas P2-positive cells were mostly in the proliferative compartment in the lower half of the crypt (the proliferation marker is red in the image.) Furthermore, when transgenic mice genetically engineered to have only either P1 or P2 were subjected to a carcinogen and, subsequently, to an irritant to stress the epithelial lining of the colon, the researchers found that the P1 mice showed fewer tumors than wild-type control mice. When treated with irritant alone, these mice were resistant to colitis. In sharp contrast, mice with only P2 showed more tumors and were much more susceptible to colitis.

The researchers explain these findings by invoking the “barrier function,”  a mucosal barrier generated by the colon’s epithelial cells that prevents bacteria in the gut from entering the body. In the case of P1 mice, this barrier function was enhanced. The P2 mice, on the other hand, showed a compromised barrier function, presumably allowing bacteria to pass through.

Next, the researchers examined genes expressed in the P1 and P2 mice. They found that resistin-like molecule (RELM)-beta, a cytokine (a signaling molecule of the immune system) expressed in the gastrointestinal tract and implicated in colitis, was expressed far more in the P2 mice than the P1 mice.

“This makes sense since a reduced barrier function means bacteria can go across the barrier, which activates RELM-beta,” Dr. Sladek said. “We also found that the P2 protein transcribes RELM-beta more effectively than the P1 protein.”

Next, Poonamjot Deol, Ph.D.,  an assistant project scientist in Dr. Slaked’s lab and the second author of the eLife study, will lead a project aimed at understanding how diet affects the distribution of P1 and P2 in the gut. She and others in the lab also plan to investigate how obesity and colitis may be linked. (Diet studies performed in Dr. Sladek’s lab in the past illustrated soybean oil’s adverse effect on obesity.)

“In the case of colitis, could soybean oil be playing a part in allowing bacteria to get across the barrier function?” Dr. Deol said. “We do not know. We know its detrimental effect on obesity. But more research needs to be done where colitis is concerned.”

Opposing roles of nuclear receptor HNF4α isoforms in colitis and colitis-associated colon cancer

 Karthikeyani Chellappa, 

HNF4α has been implicated in colitis and colon cancer in humans but the role of the different HNF4α isoforms expressed from the two different promoters (P1 and P2) active in the colon is not clear. Here, we show that P1-HNF4α is expressed primarily in the differentiated compartment of the mouse colonic crypt and P2-HNF4α in the proliferative compartment. Exon swap mice that express only P1- or only P2-HNF4α have different colonic gene expression profiles, interacting proteins, cellular migration, ion transport and epithelial barrier function. The mice also exhibit altered susceptibilities to experimental colitis (DSS) and colitis-associated colon cancer (AOM+DSS). When P2-HNF4α-only mice (which have elevated levels of the cytokine resistin-like β, RELMβ, and are extremely sensitive to DSS) are crossed with Retnlb-/- mice, they are rescued from mortality. Furthermore, P2-HNF4α binds and preferentially activates the RELMβ promoter. In summary, HNF4α isoforms perform non-redundant functions in the colon under conditions of stress, underscoring the importance of tracking them both in colitis and colon cancer.

 

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Somatic Mutation Theory – Why it’s Wrong for Most Cancers

Reporter: Aviva Lev-Ari, PhD, RN

 

 

Somatic Mutation Theory – Why it’s Wrong for Most Cancers.

Cell Physiol Biochem 2016;38:1663-1680. http://www.karger.com/Article/FullText/443106

Björn L.D.M. Brücher and Ijaz S. Jamall

Brucher [a to d] and Jamall [a,b,e]

a Theodor-Billroth-Academy®, Munich, Germany – Sacramento, California, USA;

b INCORE, International Consortium of Research Excellence of the Theodor-Billroth-Academy®, Munich, Germany – Sacramento, California, USA;

c Bon Secours Cancer Institute, Richmond, VA, USA;

d Dep. of Surgery, Carl- Thiem-Klinikum, Cottbus, Germany;

e Risk-Based Decisions Inc., Sacramento, CA, USA

 

Key Words

Carcinogenesis • Somatic mutation theory • Microenvironment • Cell communication • Signaling • Inflammation • Chronic inflammation • Fibrosis • Cell transition • Precancerous niche

Abstract

Hysteron proteron reverses both temporal and logical order and this syllogism occurs in carcinogenesis and the somatic mutation theory (SMT): the first (somatic mutation) occurs only after the second (onset of cancer) and, therefore, observed somatic mutations in most cancers appear well after the early cues of carcinogenesis are in place. It is no accident that mutations are increasingly being questioned as the causal event in the origin of the vast majority of cancers as clinical data show little support for this theory when compared against the metrics of patient outcomes. Ever since the discovery of the double helical structure of DNA, virtually all chronic diseases came to be viewed as causally linked to one degree or another to mutations, even though we now know that genes are not simply blueprints, but rather an assemblage of alphabets that can, under non-genetic influences, be used to assemble a business letter or a work of Shakespearean literature. A minority of all cancers is indeed caused by mutations but the SMT has been applied to all cancers, and even to chemical carcinogenesis, in the absence of hard evidence of causality. Herein, we review the 100 year story of SMT and aspects that show why genes are not just blueprints, how radiation and mutation are associated in a more nuanced view, the proposed risk of cancer and bad luck, and the in vitro and in vivo evidence for a new cancer paradigm. This paradigm is scientifically applicable for the majority of non-heritable cancers and consists of a six-step sequence for the origin of cancer. This new cancer paradigm proclaims that somatic mutations are epiphenomena or later events occurring after carcinogenesis is already underway. This serves not just as a plausible alternative to SMT and explains the origin of the majority of cancers, but also provides opportunities for early interventions and prevention of the onset of cancer as a disease.

  

Conclusions

The incorrect interpretation of data can sometimes appear to be the more parsimonious explanation especially when it has acquired the mantle of a paradigm, as in the case of the SMT. Summa Cancerologica is not hypothetical or ontological. Its syllogism of carcinogenesis needs the consideration of all reasonable perspectives such as whether somatic mutations are later events or epiphenomena occurring at the end of the sequence of events in carcinogenesis. This mutatio praemissarum leads to a reflection of reasoned judgments of correct findings in cancer (mutations within tumors) together with clinical observations (relevance of such mutations to cancer therapy). An overemphasis of the SMT as the sole reason of the origin of carcinogenesis elevated it to the status of a dogma which downplays significant findings of mutations and genetics in different fields of nature, biology and science. However, there is hope that hereditary cancers can be treated in the near future as new technologies make it possible to manipulate proteins packaging DNA to turn on specific gene promoters and enhancers [164]. If this were applicable to the mass of non-hereditary cancers this approach would still be only symptomatic as the genesis of non-hereditary cancers is not caused by somatic mutations though somatic mutations occur within tumors. Focusing on the tumor cell without its origin including the microenvironment won’t be enough [165]. The reasoning on the origin of carcinogenesis, including different step-wise sequences, may help unmask mechanisms of the transition of a normal into a cancer cell (cancer genesis) as well as its different primary pathogenic stimulus, which can serve to prevent or retard cancers instead of concentrating on symptomatic strategies or for a cure for all cancers. It is scientifically valid based on in vitro and in vivo genetic findings that carcinogenesis consists of a six-step multi sequence process [17, 18]. This serves not just as a plausible alternative to the SMT to explain the origin of the majority of cancers, but could also suggest early interventions and thereby prevent the onset of cancer as a disease.

FULL ARTICLE

2016-CELL-PHYSIOL-BIOCHEM-Somatic-Mutation-Theory

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Microbe meets cancer

Larry H. Bernstein, MD, FCAP, Curator

LPBI

 

Microbes Meet Cancer

Understanding cancer’s relationship with the human microbiome could transform immune-modulating therapies.

By Kate Yandell | April 1, 2016  http://www.the-scientist.com/?articles.view/articleNo/45616/title/Microbes-Meet-Cancer

 © ISTOCK.COM/KATEJA_FN; © ISTOCK.COM/FRANK RAMSPOTT  http://www.the-scientist.com/images/April2016/feature1.jpg

In 2013, two independent teams of scientists, one in Maryland and one in France, made a surprising observation: both germ-free mice and mice treated with a heavy dose of antibiotics responded poorly to a variety of cancer therapies typically effective in rodents. The Maryland team, led by Romina Goldszmidand Giorgio Trinchieri of the National Cancer Institute, showed that both an investigational immunotherapy and an approved platinum chemotherapy shrank a variety of implanted tumor types and improved survival to a far greater extent in mice with intact microbiomes.1 The French group, led by INSERM’s Laurence Zitvogel, got similar results when testing the long-standing chemotherapeutic agent cyclophosphamide in cancer-implanted mice, as well as in mice genetically engineered to develop tumors of the lung.2

The findings incited a flurry of research and speculation about how gut microbes contribute to cancer cell death, even in tumors far from the gastrointestinal tract. The most logical link between the microbiome and cancer is the immune system. Resident microbes can either dial up inflammation or tamp it down, and can modulate immune cells’ vigilance for invaders. Not only does the immune system appear to be at the root of how the microbiome interacts with cancer therapies, it also appears to mediate how our bacteria, fungi, and viruses influence cancer development in the first place.

“We clearly see shifts in the [microbial] community that precede development of tumors,” says microbiologist and immunologist Patrick Schloss, who studies the influence of the microbiome on colon cancer at the University of Michigan.

But the relationship between the microbiome and cancer is complex: while some microbes promote cell proliferation, others appear to protect us against cancerous growth. And in some cases, the conditions that spur one cancer may have the opposite effect in another. “It’s become pretty obvious that the commensal microbiota affect inflammation and, through that or through other mechanisms, affect carcinogenesis,” says Trinchieri. “What we really need is to have a much better understanding of which species, which type of bug, is doing what and try to change the balance.”

Gut feeling

In the late 1970s, pathologist J. Robin Warren of Royal Perth Hospital in Western Australia began to notice that curved bacteria often appeared in stomach tissue biopsies taken from patients with chronic gastritis, an inflammation of the stomach lining that often precedes the development of stomach cancer. He and Barry J. Marshall, a trainee in internal medicine at the hospital, speculated that the bacterium, now called Helicobacter pylori, was somehow causing the gastritis.3 So committed was Marshall to demonstrating the microbe’s causal relationship to the inflammatory condition that he had his own stomach biopsied to show that it contained no H. pylori, then infected himself with the bacterium and documented his subsequent experience of gastritis.4 Scientists now accept that H. pylori, a common gut microbe that is present in about 50 percent of the world’s population, is responsible for many cases of gastritis and most stomach ulcers, and is a strong risk factor for stomach cancer.5 Marshall and Warren earned the 2005 Nobel Prize in Physiology or Medicine for their work.

H. pylori may be the most clear-cut example of a gut bacterium that influences cancer development, but it is likely not the only one. Researchers who study cancer in mice have long had anecdotal evidence that shifts in the microbiome influence the development of diverse tumor types. “You have a mouse model of carcinogenesis. It works beautifully,” says Trinchieri. “You move to another institution. It works completely differently,” likely because the animals’ microbiomes vary with environment.

IMMUNE INFLUENCE: In recent years, research has demonstrated that microbes living in and on the mammalian body can affect cancer risk, as well as responses to cancer treatment. Although the details of this microbe-cancer link remain unclear, investigators suspect that the microbiome’s ability to modulate inflammation and train immune cells to react to tumors is to blame.
See full infographic: WEB | PDF
© AL GRANBERG

Around the turn of the 21st century, cancer researchers began to systematically experiment with the rodent microbiome, and soon had several lines of evidence linking certain gut microbes with a mouse’s risk of colon cancer. In 2001, for example, Shoichi Kado of the Yakult Central Institute for Microbiological Research in Japan and colleagues found that a strain of immunocompromised mice rapidly developed colon tumors, but that germ-free versions of these mice did not.6 That same year, an MIT-based group led by the late David Schauer demonstrated that infecting mice with the bacterium Citrobacter rodentium spurred colon tumor development.7 And in 2003, MIT’s Susan Erdman and her colleagues found that they could induce colon cancer in immunocompromised mice by infecting them with Helicobacter hepaticus, a relative of? H. pylori that commonly exists within the murine gut microbiome.8

More recent work has documented a similar link between colon cancer and the gut microbiome in humans. In 2014, a team led by Schloss sequenced 16S rRNA genes isolated from the stool of 90 people, some with colon cancer, some with precancerous adenomas, and still others with no disease.9 The researchers found that the feces of people with cancer tended to have an altered composition of bacteria, with an excess of the common mouth microbes Fusobacterium or Porphyromonas. A few months later, Peer Bork of the European Molecular Biology Laboratory performed metagenomic sequencing of stool samples from 156 people with or without colorectal cancer. Bork and his colleagues found they could predict the presence or absence of cancer using the relative abundance of 22 bacterial species, including Porphyromonas andFusobacterium.10 They could also use the method to predict colorectal cancer with about the same accuracy as a blood test, correctly identifying about 50 percent of cancers while yielding false positives less than 10 percent of the time. When the two tests were combined, they caught more than 70 percent of cancers.

Whether changes in the microbiota in colon cancer patients are harbingers of the disease or a consequence of tumor development remained unclear. “What comes first, the change in the microbiome or tumor development?” asks Schloss. To investigate this question, he and his colleagues treated mice with microbiome-altering antibiotics before administering a carcinogen and an inflammatory agent, then compared the outcomes in those animals and in mice that had received only the carcinogenic and inflammatory treatments, no antibiotics. The antibiotic-treated animals had significantly fewer and smaller colon tumors than the animals with an undisturbed microbiome, suggesting that resident bacteria were in some way promoting cancer development. And when the researchers transferred microbiota from healthy mice to antibiotic-treated or germ-free mice, the animals developed more tumors following carcinogen exposure. Sterile mice that received microbiota from mice already bearing malignancies developed the most tumors of all.11

Most recently, Schloss and his colleagues showed that treating mice with seven unique combinations of antibiotics prior to exposing them to carcinogens yielded variable but predictable levels of tumor formation. The researchers determined that the number of tumors corresponded to the unique ways that each antibiotic cocktail modulated the microbiome.12

“We’ve kind of proven to ourselves, at least, that the microbiome is involved in colon cancer,” says Schloss, who hypothesizes that gut bacteria–driven inflammation is to blame for creating an environment that is hospitable to tumor development and growth. Gain or loss of certain components of the resident bacterial community could lead to the release of reactive oxygen species, damaging cells and their genetic material. Inflammation also involves increased release of growth factors and blood vessel proliferation, potentially supporting the growth of tumors. (See illustration above.)

Recent research has also yielded evidence that the gut microbiota impact the development of cancer in sites far removed from the intestinal tract, likely through similar immune-modulating mechanisms.

Systemic effects

In the mid-2000s, MIT’s Erdman began infecting a strain of mice predisposed to intestinal tumors withH. hepaticus and observing the subsequent development of colon cancer in some of the animals. To her surprise, one of the mice developed a mammary tumor. Then, more of the mice went on to develop mammary tumors. “This told us that something really interesting was going on,” Erdman recalls. Sure enough, she and her colleagues found that mice infected with H. hepaticus were more likely to develop mammary tumors than mice not exposed to the bacterium.13The researchers showed that systemic immune activation and inflammation could contribute to mammary tumors in other, less cancer-prone mouse models, as well as to the development of prostate cancer.

MICROBIAL STOWAWAYS: Bacteria of the human gut microbiome are intimately involved in cancer development and progression, thanks to their interactions with the immune system. Some microbes, such as Helicobacter pylori, increase the risk of cancer in their immediate vicinity (stomach), while others, such as some Bacteroides species, help protect against tumors by boosting T-cell infiltration.© EYE OF SCIENCE/SCIENCE SOURCE
http://www.the-scientist.com/images/April2016/immune_2.jpg

 

 

© DR. GARY GAUGLER/SCIENCE SOURCE  http://www.the-scientist.com/images/April2016/immune3.jpg

At the University of Chicago, Thomas Gajewski and his colleagues have taken a slightly different approach to studying the role of the microbiome in cancer development. By comparing Black 6 mice coming from different vendors—Taconic Biosciences (formerly Taconic Farms) and the Jackson Laboratory—Gajewski takes advantage of the fact that the animals’ different origins result in different gut microbiomes. “We deliberately stayed away from antibiotics, because we had a desire to model how intersubject heterogeneity [in cancer development] might be impacted by the commensals they happen to be colonized with,” says Gajewski in an email to The Scientist.

Last year, the researchers published the results of a study comparing the progression of melanoma tumors implanted under the mice’s skin, finding that tumors in the Taconic mice grew more aggressively than those in the Jackson mice. When the researchers housed the different types of mice together before their tumors were implanted, however, these differences disappeared. And transferring fecal material from the Jackson mice into the Taconic mice altered the latter’s tumor progression.14

Instead of promoting cancer, in these experiments the gut microbiome appeared to slow tumor growth. Specifically, the reduced tumor growth in the Jackson mice correlated with the presence of Bifidobacterium, which led to the greater buildup of T?cells in the Jackson mice’s tumors. Bifidobacteriaactivate dendritic cells, which present antigens from bacteria or cancer cells to T?cells, training them to hunt down and kill these invaders. Feeding Taconic mice bifidobacteria improved their response to the implanted melanoma cells.

“One hypothesis going into the experiments was that we might identify immune-suppressive bacteria, or commensals that shift the immune response towards a character that was unfavorable for tumor control,” says Gajewski.  “But in fact, we found that even a single type of bacteria could boost the antitumor immune response.”

http://www.the-scientist.com/images/April2016/immune4.jpg

 

Drug interactions

Ideally, the immune system should recognize cancer as invasive and nip tumor growth in the bud. But cancer cells display “self” molecules that can inhibit immune attack. A new type of immunotherapy, dubbed checkpoint inhibition or blockade, spurs the immune system to attack cancer by blocking either the tumor cells’ surface molecules or the receptors on T?cells that bind to them.

CANCER THERAPY AND THE MICROBIOME

In addition to influencing the development and progression of cancer by regulating inflammation and other immune pathways, resident gut bacteria appear to influence the effectiveness of many cancer therapies that are intended to work in concert with host immunity to eliminate tumors.

  • Some cancer drugs, such as oxaliplatin chemotherapy and CpG-oligonucleotide immunotherapy, work by boosting inflammation. If the microbiome is altered in such a way that inflammation is reduced, these therapeutic agents are less effective.
  • Cancer-cell surface proteins bind to receptors on T cells to prevent them from killing cancer cells. Checkpoint inhibitors that block this binding of activated T cells to cancer cells are influenced by members of the microbiota that mediate these same cell interactions.
  • Cyclophosphamide chemotherapy disrupts the gut epithelial barrier, causing the gut to leak certain bacteria. Bacteria gather in lymphoid tissue just outside the gut and spur generation of T helper 1 and T helper 17 cells that migrate to the tumor and kill it.

As part of their comparison of Jackson and Taconic mice, Gajewski and his colleagues decided to test a type of investigational checkpoint inhibitor that targets PD-L1, a ligand found in high quantities on the surface of multiple types of cancer cells. Monoclonal antibodies that bind to PD-L1 block the PD-1 receptors on T?cells from doing so, allowing an immune response to proceed against the tumor cells. While treating Taconic mice with PD-L1–targeting antibodies did improve their tumor responses, they did even better when that treatment was combined with fecal transfers from Jackson mice, indicating that the microbiome and the immunotherapy can work together to take down cancer. And when the researchers combined the anti-PD-L1 therapy with a bifidobacteria-enriched diet, the mice’s tumors virtually disappeared.14

Gajewski’s group is now surveying the gut microbiota in humans undergoing therapy with checkpoint inhibitors to better understand which bacterial species are linked to positive outcomes. The researchers are also devising a clinical trial in which they will give Bifidobacterium supplements to cancer patients being treated with the approved anti-PD-1 therapy pembrolizumab (Keytruda), which targets the immune receptor PD-1 on T?cells, instead of the cancer-cell ligand PD-L1.

Meanwhile, Zitvogel’s group at INSERM is investigating interactions between the microbiome and another class of checkpoint inhibitors called CTLA-4 inhibitors, which includes the breakthrough melanoma treatment ipilimumab (Yervoy). The researchers found that tumors in antibiotic-treated and germ-free mice had poorer responses to a CTLA-4–targeting antibody compared with mice harboring unaltered microbiomes.15 Particular Bacteroides species were associated with T-cell infiltration of tumors, and feedingBacteroides fragilis to antibiotic-treated or germ-free mice improved the animals’ responses to the immunotherapy. As an added bonus, treatment with these “immunogenic” Bacteroides species decreased signs of colitis, an intestinal inflammatory condition that is a dangerous side effect in patients using checkpoint inhibitors. Moreover, Zitvogel and her colleagues showed that human metastatic melanoma patients treated with ipilimumab tended to have elevated levels of B. fragilis in their microbiomes. Mice transplanted with feces from patients who showed particularly strong B. fragilis gains did better on anti-CTLA-4 treatment than did mice transplanted with feces from patients with normal levels of B. fragilis.

“There are bugs that allow the therapy to work, and at the same time, they protect against colitis,” says Trinchieri. “That is very exciting, because not only [can] we do something to improve the therapy, but we can also, at the same time, try to reduce the side effect.”

And these checkpoint inhibitors aren’t the only cancer therapies whose effects are modulated by the microbiome. Trinchieri has also found that an immunotherapy that combines antibodies against interleukin-10 receptors with CpG oligonucleotides is more effective in mice with unaltered microbiomes.1He and his NCI colleague Goldszmid further found that the platinum chemotherapy oxaliplatin (Eloxatin) was more effective in mice with intact microbiomes, and Zitvogel’s group has shown that the chemotherapeutic agent cyclophosphamide is dependent on the microbiota for its proper function.

Although the mechanisms by which the microbiome influences the effectiveness of such therapies remains incompletely understood, researchers once again speculate that the immune system is the key link. Cyclophosphamide, for example, spurs the body to generate two types of T?helper cells, T?helper 1 cells and a subtype of T?helper 17 cells referred to as “pathogenic,” both of which destroy tumor cells. Zitvogel and her colleagues found that, in mice with unaltered microbiomes, treatment with cyclophosphamide works by disrupting the intestinal mucosa, allowing bacteria to escape into the lymphoid tissues just outside the gut. There, the bacteria spur the body to generate T?helper 1 and T?helper 17 cells, which translocate to the tumor. When the researchers transferred the “pathogenic” T?helper 17 cells into antibiotic-treated mice, the mice’s response to chemotherapy was partly restored.

Microbiome modification

As the link between the microbiome and cancer becomes clearer, researchers are thinking about how they can manipulate a patient’s resident microbial communities to improve their prognosis and treatment outcomes. “Once you figure out exactly what is happening at the molecular level, if there is something promising there, I would be shocked if people don’t then go in and try to modulate the microbiome, either by using pharmaceuticals or using probiotics,” says Michael Burns, a postdoc in the lab of University of Minnesota genomicist Ran Blekhman.

Even if researchers succeed in identifying specific, beneficial alterations to the microbiome, however, molding the microbiome is not simple. “It’s a messy, complicated system that we don’t understand,” says Schloss.

So far, studies of the gut microbiome and colon cancer have turned up few consistent differences between cancer patients and healthy controls. And the few bacterial groups that have repeatedly shown up are not present in every cancer patient. “We should move away from saying, ‘This is a causal species of bacteria,’” says Blekhman. “It’s more the function of a community instead of just a single bacterium.”

But the study of the microbiome in cancer is young. If simply adding one type of microbe into a person’s gut is not enough, researchers may learn how to dose people with patient-specific combinations of microbes or antibiotics. In February 2016, a team based in Finland and China showed that a probiotic mixture dubbed Prohep could reduce liver tumor size by 40 percent in mice, likely by promoting an anti-inflammatory environment in the gut.16

“If it is true that, in humans, we can alter the course of the disease by modulating the composition of the microbiota,” says José Conejo-Garcia of the Wistar Institute in Philadelphia, “that’s going to be very impactful.”

Kate Yandell has been a freelance writer living Philadelphia, Pennsylvania. In February she became an associate editor at Cancer Today.

GENETIC CONNECTION

The microbiome doesn’t act in isolation; a patient’s genetic background can also greatly influence response to therapy. Last year, for example, the Wistar Institute’s José Garcia-Conejo and Melanie Rutkowski, now an assistant professor at the University of Virginia, showed that a dominant polymorphism of the gene for the innate immune protein toll-like receptor 5 (TLR5) influences clinical outcomes in cancer patients by changing how the patients’ immune cells interact with their gut microbes (Cancer Cell, 27:27-40, 2015).

More than 7 percent of people carry a specific mutation in TLR5 that prevents them from mounting a full immune response when exposed to bacterial flagellin. Analyzing both genetic and survival data from the Cancer Genome Atlas, Conejo-Garcia, Rutkowski, and their colleagues found that estrogen receptor–positive breast cancer patients who carry the TLR5 mutation, called the R392X polymorphism, have worse outcomes than patients without the mutation. Among patients with ovarian cancer, on the other hand, those with the TLR5 mutation were more likely to live at least six years after diagnosis than patients who don’t carry the mutation.

Investigating the mutation’s contradictory effects, the researchers found that mice with normal TLR5produce higher levels of the cytokine interleukin 6 (IL-6) than those carrying the mutant version, which have higher levels of a different cytokine called interleukin 17 (IL-17). But when the researchers knocked out the animals’ microbiomes, these differences in cytokine production disappeared, as did the differences in cancer progression between mutant and wild-type animals.

“The effectiveness of depleting specific populations or modulating the composition of the microbiome is going to affect very differently people who are TLR5-positive or TLR5-negative,” says Conejo-Garcia. And Rutkowski speculates that many more polymorphisms linked to cancer prognosis may act via microbiome–immune system interactions. “I think that our paper is just the tip of the iceberg.”

References

  1. N. Iida et al., “Commensal bacteria control cancer response to therapy by modulating the tumor microenvironment,” Science, 342:967-70, 2013.
  2. S. Viaud et al., “The intestinal microbiota modulates the anticancer immune effects of cyclophosphamide,” Science, 342:971-76, 2013.
  3. J.R. Warren, B. Marshall, “Unidentified curved bacilli on gastric epithelium in active chronic gastritis,”Lancet, 321:1273-75, 1983.
  4. B.J. Marshall et al., “Attempt to fulfil Koch’s postulates for pyloric Campylobacter,” Med J Aust, 142:436-39, 1985.
  5. J. Parsonnet et al., “Helicobacter pylori infection and the risk of gastric carcinoma,” N Engl J Med, 325:1127-31, 1991.
  6. S. Kado et al., “Intestinal microflora are necessary for development of spontaneous adenocarcinoma of the large intestine in T-cell receptor β chain and p53 double-knockout mice,” Cancer Res, 61:2395-98, 2001.
  7. J.V. Newman et al., “Bacterial infection promotes colon tumorigenesis in ApcMin/+ mice,” J Infect Dis, 184:227-30, 2001.
  8. S.E. Erdman et al., “CD4+ CD25+ regulatory T lymphocytes inhibit microbially induced colon cancer in Rag2-deficient mice,” Am J Pathol, 162:691-702, 2003.
  9. J.P. Zackular et al., “The human gut microbiome as a screening tool for colorectal cancer,” Cancer Prev Res, 7:1112-21, 2014.
  10. G. Zeller et al., “Potential of fecal microbiota for early-stage detection of colorectal cancer,” Mol Syst Biol, 10:766, 2014.
  11. J.P. Zackular et al., “The gut microbiome modulates colon tumorigenesis,” mBio, 4:e00692-13, 2013.
  12. J.P. Zackular et al., “Manipulation of the gut microbiota reveals role in colon tumorigenesis,”mSphere, doi:10.1128/mSphere.00001-15, 2015.
  13. V.P. Rao et al., “Innate immune inflammatory response against enteric bacteria Helicobacter hepaticus induces mammary adenocarcinoma in mice,” Cancer Res, 66:7395, 2006.
  14. A. Sivan et al., “Commensal Bifidobacterium promotes antitumor immunity and facilitates anti-PD-L1 efficacy,” Science, 350:1084-89, 2015.
  15. M. Vétizou et al., “Anticancer immunotherapy by CTLA-4 blockade relies on the gut microbiota,”Science, 350:1079-84, 2015.

……..

 

Microbially Driven TLR5-Dependent Signaling Governs Distal Malignant Progression through Tumor-Promoting Inflammation

Melanie R. Rutkowski, Tom L. Stephen, Nikolaos Svoronos, …., Julia Tchou,  Gabriel A. Rabinovich, Jose R. Conejo-Garcia
Cancer cell    12 Jan 2015; Volume 27, Issue 1, p27–40  http://dx.doi.org/10.1016/j.ccell.2014.11.009
Figure thumbnail fx1
  • TLR5-dependent IL-6 mobilizes MDSCs that drive galectin-1 production by γδ T cells
  • IL-17 drives malignant progression in IL-6-unresponsive tumors
  • TLR5-dependent differences in tumor growth are abrogated upon microbiota depletion
  • A common dominant TLR5 polymorphism influences the outcome of human cancers

The dominant TLR5R392X polymorphism abrogates flagellin responses in >7% of humans. We report that TLR5-dependent commensal bacteria drive malignant progression at extramucosal locations by increasing systemic IL-6, which drives mobilization of myeloid-derived suppressor cells (MDSCs). Mechanistically, expanded granulocytic MDSCs cause γδ lymphocytes in TLR5-responsive tumors to secrete galectin-1, dampening antitumor immunity and accelerating malignant progression. In contrast, IL-17 is consistently upregulated in TLR5-unresponsive tumor-bearing mice but only accelerates malignant progression in IL-6-unresponsive tumors. Importantly, depletion of commensal bacteria abrogates TLR5-dependent differences in tumor growth. Contrasting differences in inflammatory cytokines and malignant evolution are recapitulated in TLR5-responsive/unresponsive ovarian and breast cancer patients. Therefore, inflammation, antitumor immunity, and the clinical outcome of cancer patients are influenced by a common TLR5 polymorphism.

see also… Immune Influence

In recent years, research has demonstrated that microbes living in and on the mammalian body can affect cancer risk, as well as responses to cancer treatment.

By Kate Yandell | April 1, 2016

http://www.the-scientist.com/?articles.view/articleNo/45644/title/Immune-Influence

Although the details of this microbe-cancer link remain unclear, investigators suspect that the microbiome’s ability to modulate inflammation and train immune cells to react to tumors is to blame. Here are some of the hypotheses that have come out of recent research in rodents for how gut bacteria shape immunity and influence cancer.

HOW THE MICROBIOME PROMOTES CANCER

Gut bacteria can dial up inflammation locally in the colon, as well as in other parts of the body, leading to the release of reactive oxygen species, which damage cells and DNA, and of growth factors that spur tumor growth and blood vessel formation.

http://www.the-scientist.com/images/April2016/ImmuneInfluence1_640px.jpg

http://www.the-scientist.com/images/April2016/ImmuneInfluence2_310px1.jpg

Helicobacter pylori can cause inflammation and high cell turnover in the stomach wall, which may lead to cancerous growth.

HOW THE MICROBIOME STEMS CANCER

Gut bacteria can also produce factors that lower inflammation and slow tumor growth. Some gut bacteria (e.g., Bifidobacterium)
appear to activate dendritic cells,
which present cancer-cell antigens to T cells that in turn kill the cancer cells.

http://www.the-scientist.com/images/April2016/ImmuneInfluence3_310px1.jpg

http://www.the-scientist.com/images/April2016/ImmuneInfluence4_310px1.jpg

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ZNF154 hypermethylation signature

Larry H. Bernstein, MD, FCAP, Curator

LPBI

 

Pan-Cancer Epigenetic Signature Readable in Circulating Tumor DNA   

GEN News  http://www.genengnews.com/gen-news-highlights/pan-cancer-epigenetic-signature-readable-in-circulating-tumor-dna/81252334/

If cancer has a signature, the dotted line may be a gene called ZNF154, say scientists at the National Institutes of Health. Although the scientists don’t know exactly what the gene does, they do know that it may carry distinctive methylation marks, and that these marks have been associated with multiple types of cancer. In their most recent work, the scientists have evaluated whether these marks might serve as a universal cancer biomarker.

The team, led by Laura Elnitski, Ph.D., a computational biologist at the NIH’s National Human Genome Research Institute, previously identified hypermethylation around the ZNF154 gene in 15 solid epithelial tumor types from 13 different organs. In their current work, which was described February 5 in the Journal of Molecular Diagnostics, these scientists are going further. They are testing whether ZNF154 hypermethylation can distinguish tumor samples from normal tissue samples. What’s more, the scientists are running computer simulations to evaluate whether the tiny amounts of tumor DNA that end up circulating in blood can be detected on the basis of ZNF hypermethylation.

The results of this work led the scientists to conclude that ZNF hypermethylation is a relevant biomarker for identifying solid tumor DNA. Moreover, the scientists say that it “may have utility as a generalizable biomarker for circulating tumor DNA.”

Details appeared in the Journal of Molecular Diagnostics article (“Robust Detection of DNA Hypermethylation of ZNF154 as a Pan-Cancer Locus with in Silico Modeling for Blood-Based Diagnostic Development”), which described how the magnitude and pattern of ZNF hypermethylation across colon, lung, breast, stomach, and endometrial tumor samples were measured using next-generation bisulfite amplicon sequencing.

“To evaluate this site as a possible pan-cancer marker, we compare the ability of several sequence analysis methods to distinguish the five tumor types (184 tumor samples) from normal tissue samples (n = 34),” wrote the authors. “The classification performance for the strongest method, measured by the area under (the receiver operating characteristic) curve (AUC), is 0.96, close to a perfect value of 1. Furthermore, in a computational simulation of circulating tumor DNA, we were able to detect limited amounts of tumor DNA diluted with normal DNA.”

Even when the scientists reduced the amount of methylated molecules by 99%, the computer could still detect the cancer-related methylation marks in the mixture. Knowing that tumors often shed DNA into the bloodstream, the scientists calculated the proportions of circulating tumor DNA that could be found in the blood.

Going forward, Dr. Elnitski’s group will begin screening blood samples from patients with bladder, breast, colon, pancreatic, and prostate cancers to determine the accuracy of detection at low levels of circulating DNA. Tumor DNA in a person with cancer typically constitutes 1–10% of all DNA circulating in the bloodstream. The group noted that when 10% of the circulating DNA contains the tumor signature, their detection rate is quite good. Because the methylation could be detected at such low levels, it should be adequate to detect advanced cancer as well as some intermediate and early tumors, depending on the type.

Dr. Elnitski’s group will also collaborate with Christina Annunziata, M.D., Ph.D., an investigator at the National Cancer Institute (NCI). The scientists will test blood samples from women with ovarian cancer to validate the process over the course of treatment and to determine if this type of analysis leads to improved detection of a recurrence and, ultimately, improved outcomes.

Current blood tests are specific to a known tumor type. In other words, clinicians must first find the tumor, remove a sample of it and determine its genome sequence. Once the tumor-specific mutations are known, they can be tracked for appearance in the blood. The potential of the new approach is that no prior knowledge of cancer is required, it would be less intrusive than other screening approaches like colonoscopies and mammograms and it could be used to follow individuals at high risk for cancer or to monitor the activity of a tumor during treatment.

“Finding a distinctive methylation-based signature is…a technical challenge, but we found an elevated methylation signature around the gene known as ZNF154 that is unique to tumors,” declared Dr. Elnitski. “We have laid the groundwork for developing a diagnostic test, which offers the hope of catching cancer earlier and dramatically improving the survival rate of people with many types of cancer.”

NIH Team IDs Recurrent Methylation Site in Five Cancer Types

NEW YORK (GenomeWeb) – Multiple tumor types are recurrently methylated at a specific CpG island, according to researchers from the National Institutes of Health. In addition, the biomarker could potentially be analyzed noninvasively to detect late stage and potentially intermediate and early stage cancers.

In a study published today in the Journal of Molecular Diagnostics, the researchers described a specific site, ZNF154, that is methylated across five different tumor types — lung, stomach, colon, breast, and gynecologic — and can be identified through a targeted bisulfite sequencing strategy. In addition, the researchers performed a computational simulation of circulating tumor DNA, demonstrating that they could detect the methylation biomarker on tumor DNA in a solution composed of 99 percent normal DNA.

The work builds on a previous study in which the team discovered that the epigenetic marker was found across a wide variety of samples in the Cancer Genome Atlas. In the more recent study, the researchers sought to test the reproducibility of the marker as well as whether it could be identified from blood.

The researchers looked at 184 tumor samples across the five different cancer types as well as genomic DNA extracted from 34 normal tissue samples. First, they validated the idea that the targeted bisulfite sequencing protocol would be robust even on small amounts of DNA, since bisulfite conversion can fragment and damage DNA. After verifying that the methylation signal was “robust” with “minimal variation” they moved on to testing the 184 tumor samples.

The DNA from the samples was bisulfite converted and the researchers designed a 302-base PCR product covering the ZNF154 CpG island, including the transcriptional start site, which they had previously found to be hypermethylated in the TCGA data. They then sequenced the amplicons on the Illumina MiSeq instrument.

………

“The large magnitude of this hypermethylation bodes well for a strong discriminant in each tissue type,” the authors wrote.

Nonetheless, they noted that there were still a few tumor samples that had lower levels of methylation than the average methylation in the normal samples, including one sample each from lung, stomach, and colon, and three breast samples.

Within each tumor type, the researchers noted a couple of additional interesting findings. For instance, within the lung cancer samples, small cell carcinomas and squamous cell carcinomas had average methylation levels around 15 percent higher than adenocarcinomas and bronchioalveolar carcinomas.

The group next wanted to assess whether ZNF154 methylation could be used as a pan-cancer marker in blood-based screening. The researchers randomly matched one of the 34 normal samples to each of the 184 tumor samples and created mixtures of various dilutions ranging from 10 percent normal to 99 percent normal.

They were able to reliably identify tumor DNA in mixtures containing up to 90 percent normal tissue. Using the area under a curve measurement (AUC), which plots the false positive rate and true positive rate, the team achieved an AUC of .89. When the percentage of normal data was increased to 99 percent, AUC dropped to .74.

 

Recurrent patterns of DNA methylation in the ZNF154, CASP8, and VHL promoters across a wide spectrum of human solid epithelial tumors and cancer cell lines.

Epigenetics. 2013 Dec;8(12):1355-72.   http://dx. doi.org:/10.4161/epi.26701. Epub 2013 Oct 22.
The study of aberrant DNA methylation in cancer holds the key to the discovery of novel biological markers for diagnostics and can help to delineate important mechanisms of disease. We have identified 12 loci that are differentially methylated in serous ovarian cancers and endometrioid ovarian and endometrial cancers with respect to normal control samples. The strongest signal showed hypermethylation in tumors at a CpG island within the ZNF154 promoter. We show that hypermethylation of this locus is recurrent across solid human epithelial tumor samples for 15 of 16 distinct cancer types from TCGA. Furthermore, ZNF154 hypermethylation is strikingly present across a diverse panel of ENCODE cell lines, but only in those derived from tumor cells. By extending our analysis from the Illumina 27K Infinium platform to the 450K platform, to sequencing of PCR amplicons from bisulfite treated DNA, we demonstrate that hypermethylation extends across the breadth of the ZNF154 CpG island. We have also identified recurrent hypomethylation in two genomic regions associated with CASP8 and VHL. These three genes exhibit significant negative correlation between methylation and gene expression across many cancer types, as well as patterns of DNaseI hypersensitivity and histone marks that reflect different chromatin accessibility in cancer vs. normal cell lines. Our findings emphasize hypermethylation of ZNF154 as a biological marker of relevance for tumor identification. Epigenetic modifications affecting the promoters of ZNF154, CASP8, and VHL are shared across a vast array of tumor types and may therefore be important for understanding the genomic landscape of cancer.
Robust Detection of DNA Hypermethylation of ZNF154 as a Pan-Cancer Locus with in Silico Modeling for Blood-Based Diagnostic Development
Gennady Margolin, Hanna M. Petrykowska, Nader Jameel, Daphne W. Bell, Alice C. Young, Laura Elnitski   http://dx.doi.org/10.1016/j.jmoldx.2015.11.004
Sites that display recurrent, aberrant DNA methylation in cancer represent potential biomarkers for screening and diagnostics. Previously, we identified hypermethylation at the ZNF154 CpG island in 15 solid epithelial tumor types from 13 different organs. In this study, we measure the magnitude and pattern of differential methylation of this region across colon, lung, breast, stomach, and endometrial tumor samples using next-generation bisulfite amplicon sequencing. We found that all tumor types and subtypes are hypermethylated at this locus compared with normal tissue. To evaluate this site as a possible pan-cancer marker, we compare the ability of several sequence analysis methods to distinguish the five tumor types (184 tumor samples) from normal tissue samples (n = 34). The classification performance for the strongest method, measured by the area under (the receiver operating characteristic) curve (AUC), is 0.96, close to a perfect value of 1. Furthermore, in a computational simulation of circulating tumor DNA, we were able to detect limited amounts of tumor DNA diluted with normal DNA: 1% tumor DNA in 99% normal DNA yields AUCs of up to 0.79. Our findings suggest that hypermethylation of the ZNF154 CpG island is a relevant biomarker for identifying solid tumor DNA and may have utility as a generalizable biomarker for circulating tumor DNA.

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Deciphering the Epigenome

Curator: Larry H. Bernstein, MD, FCAP

 

UPDATED on 1/29/2016

 

RNA Epigenetics

DNA isn’t the only decorated nucleic acid in the cell. Modifications to RNA molecules are much more common and are critical for regulating diverse biological processes.

By Dan Dominissini, Chuan He and Gidi Rechavi | January 1, 2016

 

RNA SOUP: Newly transcribed messenger RNA exiting the nucleus via nuclear pores
© BENJAMIN CAMPILLO/SCIENCE SOURCE
http://www.the-scientist.com/January2016/feature2.jpg

For years, researchers described DNA and RNA as linear chains of four building blocks—the nucleotides A, G, C, and T for DNA; and A, G, C, and U for RNA. But these information molecules are much more than their core sequences. A variety of chemical modifications decorate the nucleic acids, increasing the alphabet of DNA to about a dozen known nucleotide variants. The alphabet of RNA is even more impressive, consisting of at least 140 alternative nucleotide forms. The different building blocks can affect the complementarity of the RNA molecules, alter their structure, and enable the binding of specific proteins that mediate various biochemical and cellular outcomes.

The large size of RNA’s vocabulary relative to that of DNA’s is not surprising. DNA is involved mainly with genetic information storage, while RNA molecules—mRNA, rRNA, tRNA, miRNA, and others—are engaged in diverse structural, catalytic, and regulatory activities, in addition to translating genes into proteins. RNA’s multitasking prowess, at the heart of the RNA World hypothesis implicating RNA as the first molecule of life, likely spurred the evolution of numerous modified nucleotides. This enabled the diversified complementarity and secondary structures that allow RNA species to specifically interact with other components of the cellular machinery such as DNA and proteins.

Methylating RNA

The nucleotide building blocks of RNA contain pyrimidine or purine rings, and each position of these rings can be chemically altered by the addition of various chemical groups. Most frequently, a methyl (–CH3) group is tacked on to the outside of the ring. Other chemical additions such as acetyl, isopentenyl, and threonylcarbamoyl are also found added to RNA bases.

Among the 140 modified RNA nucleotide variants identified, methylation of adenosine at the N6 position (m6A) is the most prevalent epigenetic mark in eukaryotic mRNA. Identified in bacterial rRNAs and tRNAs as early as the 1950s, this type of methylation was subsequently found in other RNA molecules, including mRNA, in animal and plant cells as well. In 1984, researchers identified a site that was specifically methylated—the 3′ untranslated region (UTR) of bovine prolactin mRNA.1 As more sites of m6A modification were identified, a consistent pattern emerged: the methylated A is preceded by A or G and followed by C (A/G—methylated A—C).

The alphabet of RNA consists of at least 140 alternative nucleotide forms.

Although the identification of m6A in RNA is 40 years old, until recently researchers lacked efficient molecular mapping and quantification methods to fully understand the functional implications of the modification. In 2012, we (D.D. and G.R.) combined the power of next-generation sequencing (NGS) with traditional antibody-mediated capture techniques to perform high-resolution transcriptome-wide mapping of m6A, an approach we termed m6A-seq.2 Briefly, the transcriptome is randomly fragmented and an anti-m6A antibody is used to fish out the methylated RNA fragments; the m6A-containing fragments are then sequenced and aligned to the genome, thus allowing us to locate the positions of methylation marks.

Analyzing the human transcriptome in this way, we identified more than 12,000 methylated sites in mRNA molecules derived from approximately 7,000 protein-coding genes. The transcripts of most expressed genes, in a variety of cell types, were shown to be methylated, indicating that m6A modifications are widespread. In addition, about 250 noncoding RNA sequences—including well-characterized long noncoding RNAs (lncRNAs), such as the XIST transcripts that have a key role in X-chromosome inactivation—are decorated by m6A. In almost all cases, the epigenetic mark was found on adenosines embedded in the predicted A/G—methylated A—C sequence. We found that this pattern was consistently preceded by an additional purine (A or G) and followed by a uracil (U), extending the known consensus sequence to A/G—A/G—methylated A—C—U.2

At the macro level, we found that m6A methylation sites were enriched at two distinct landmarks. The highest relative representation of m6A was found in the stop codon–3′ UTR segment of the RNA, with nearly a third of such methylation found in this sequence just beyond a gene’s coding region. Within the coding regions of the RNA molecules, m6A enrichment mapped to unusually long internal exons; 87 percent of the exonic methylation peaks were found in exons longer than 400 nucleotides. (The average human exon is only 145 nucleotides in length). This pattern of decoration of transcribed RNA suggests that m6A is involved in the mediation of splicing of long-exon transcripts. RNAs transcribed from single­-isoform genes were found to be relatively undermethylated, while transcripts that are known to have multiple isoforms, determined by alternative splicing patterns, were hypermethylated.2 Moreover, specific alternative splicing types, such as intron retention, exon skipping, and alternative first or last exon usage, were highly correlated with m6A decoration. And silencing the m6A methylating protein METTL3 affected global gene expression and alternative splicing patterns in both human and mouse cells.2

These findings clearly indicate the importance of m6A decoration in regulating the expression of diverse transcripts. Moreover, our parallel study of the human and mouse methylome by m6A-seq has uncovered a remarkable degree of conservation in both consensus sequence and areas of enrichment, further supporting the importance of m6A function.2 But research into understanding how m6A marks themselves are regulated, and how this affects various cellular processes, is only just beginning.

Writers, erasers, and readers

The accumulating findings regarding the cellular consequences of m6A transcriptome decoration led to the search for the mediators that enable m6A to exert its influence. Epigenetic marks are introduced by enzymes and cofactors known as “writers,” and m6A is no exception. This mark is added to RNA by a large protein complex that includes three well-characterized components: METTL3, METTL14, and WTAP.3,4 (See illustration on opposite page.)

The transcripts of most expressed genes, in a variety of cell types, were shown to be methylated.

The reverse process of RNA demethylation is performed by “erasers.” In 2011, one of us (C.H.) and an international group of colleagues identified the first m6A eraser: the fat mass and obesity–associated protein (FTO).5 Four years earlier, three independent studies had discovered that a single-nucleotide polymorphism in the first intron of Fto was strongly associated with body mass index and obesity risk, and studies of mouse models where Fto was deleted or overexpressed further demonstrated its link with altered body weight. The research from the C.H. group showed that silencing the Fto gene or protein increased total m6A levels, while overexpression decreased levels of the epigenetic mark.5 C.H.’s group later discovered that another protein from the same protein family as FTO, ALKBH5, behaves as an active m6A demethylase.6 In contrast to the ubiquitous expression of Fto in all tissues, the highest expression level of Alkbh5 was demonstrated in mouse testes. Indeed, Alkbh5-null male mice exhibit aberrant spermatogenesis, probably a result of m6A-mediated altered expression of spermatogenesis-related genes.6

 

RNA METHYLATION DYNAMICS: At least 140 alternative RNA nucleotide forms exist. On mRNA, the most common is the methylation of adenosine on the N6 position (m6A). This epigenetic mark is laid down by a “writer” protein complex that includes three well-characterized components: METTL3, METTL14, and WTAP. The reverse process of RNA demethylation is performed by “erasers,” such as the enzymes FTO and ALKBH5.

http://www.the-scientist.com/January2016/methylation2.jpg

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These writers and erasers facilitate the dynamic nature of m6A methylation, which was shown when we (D.D. and G.R.) demonstrated changes in response to environmental stimuli, such as UV irradiation, heat shock, and exposure to interferon gamma or hepatocyte growth factor.2 Once RNA epigenetic modifications are laid down, they are recognized by specific “reader” proteins that bind to the modified nucleotide and mediate enhancement or inhibition of gene expression. In 2012, the G.R. group used methylated and nonmethylated versions of synthetic RNA baits that include the m6A consensus sequence to identify such readers of m6A.2 By preferential binding to the methylated bait, we isolated several specific m6A-binding proteins, including members of the RNA-binding YTH domain family, whose function was previously unknown.2

The finding of the first m6A-binding reader proteins has accelerated the deciphering of the various molecular and cellular processes mediated by m6A marking. In 2014, for example, we (C.H. and colleagues) showed that the human YTH domain family 2 (YTHDF2) reader protein selectively recognizes m6A and mediates mRNA degradation.7 We identified more than 3,000 cellular RNA targets of YTHDF2, most of which are mRNAs. Binding of YTHDF2 to m6A in mRNA results in the translocation of bound mRNA from the translatable pool to mRNA decay sites, such as processing bodies in the cytoplasm where mRNA turnover is regulated.

Recently, C.H. and colleagues identified another m6A reader protein, YTHDF1, with a very different function—stimulating protein synthesis by ramping up the efficiency of translation machinery.8 The dueling functions of YTHDF2 and YTHDF1 provide a mechanism by which cells can adjust gene expression promptly and precisely to environmental stimuli. Finally, G.R. and his group have identified an additional reader protein, the RNA-binding protein heterogeneous nuclear ribonucleoprotein A2B1 (HNRNPA2B1),2 which directly binds a set of m6A decorated transcripts and mediates alternative splicing.9

Clearly, m6A plays diverse roles in regulating cellular function, starting with basic processes such as gene expression, translation, and alternative splicing. As work on this epigenetic mark continues, we will undoubtedly link m6A to numerous phenotypes, and its dysregulation may undergird various diseases and syndromes.

RNA epigenetics in action

Understanding the molecular mechanisms by which m6A regulation controls RNA stability, translation efficiency, and alternative splicing is helping researchers decipher the importance of this new epigenetic mark in physiological and pathological processes. For example, researchers recently showed that translation increases in stressed mice thanks to m6A decoration. In 2015, two studies from Cornell University and Weill Cornell Medical College found increased m6A methylation of specific 5′ UTR adenosines in newly transcribed mRNAs as a result of stress-induced nuclear localization of the m6A YTHDF2 reader. The researchers suggested that the nuclear YTHDF2 preserves the unique 5′ UTR m6A methylation of stress-induced transcripts by limiting the demethylation activity of the FTO eraser. Increased 5′ UTR m6A methylation in turn promotes translation of specific transcripts, such as those for the heat shock protein Hsp70. While conventional mRNA translation starts by binding of the ribosome components to a region of the 5′ UTR marked by the unusual nucleotide 7meG (the “cap”), under stress conditions initiation of translation can start farther downstream.10

DECIDING CELL FATE: Among its many roles in the cell, m6A methylation helps regulate the expression of RNA transcripts that mediate the transition from pluripotency to differentiation. The presence of m6A appears to decrease the stability of transcripts important for maintaining pluripotency, priming the cells for future differentiation. The loss of METTL3, an m6A methlyase component, in mouse embryonic stem cells leads to the cells’ inability to exit the pluripotent state, a lethal outcome in the early embryos.
http://www.the-scientist.com/January2016/feature2_21.jpg

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In a second study, Weill Cornell Medical College’s Samie Jaffrey, who collaborated on the previous study, led a team that showed m6A-methylated mRNAs can be translated in a cap-independent manner. The researchers showed that a specific 5′ UTR m6A binds the eukaryotic initiation factor 3 (eIF3), which recruits the ribosomal 43S complex and initiates cap-independent translation. This study also demonstrated increased m6A levels in the Hsp70 mRNA that enhanced its cap-independent translation following heat-shock stress.11

Other work has hinted at m6A’s role in the regulation of circadian rhythms. Researchers identified m6A sites on many transcripts of genes involved in the regulation of daily cycles. Inhibition of m6A methylation by silencing of the METTL3 writer led to circadian period elongation, with altered distribution and processing of the transcripts of the clock genes Per2 and Arntl.12

It’s quickly becoming clear that m6A decoration has diverse cellular and physiological functions. But perhaps the best illustration of its critical ability to precisely control processes at the cellular level is its involvement in early embryogenesis. Cell-fate decisions are coordinated by alterations in global gene expression, which are orchestrated by epigenetic regulation. Well-established epigenetic marks, such as DNA methylation and histone modifications, are known to mediate embryonic stem cell (ESC) cell-fate decisions, and it turns out that m6A modification is no different.

Dynamic m6A RNA markings, the new kid on the epigenetic block, herald the era of tripartite epigenetics where modifications of DNA, RNA, and proteins join hands to fine-tune gene expression and to execute prompt and precise responses to environmental stimuli and stresses.

We (G.R. and collaborators) and other groups recently demonstrated that the m6A writer METTL3 is also an essential regulator for termination of mouse embryonic stem cell pluripotency. Knocking out Mettl3 in preimplantation murine epiblasts and in undifferentiated ESCs led to depletion of m6A in mRNAs. Cell viability was not affected, suggesting that m6A decoration is not essential for the maintenance of the ESC naive state, but m6A marks were critical for early differentiation. The loss of this modification led to aberrant and restricted lineage priming at the post-implantation stage, resulting in early embryonic lethality.13 The presence of m6A also decreased mRNA stability, including in those transcripts important for maintaining pluripotency. These findings demonstrated, for the first time, an essential function for an mRNA modification in vivo.14

Beyond mRNA

While m6A methylation is most prevalent on mRNAs, this mark also decorates other RNA species. It is well established, for example, that m6A is abundant on rRNAs, tRNAs, and small nuclear RNAs (snRNAs), which mediate splicing and other RNA processing and protein synthesis reactions.

More recently, researchers found that the reader protein HNRNPA2B1 binds to m6A marks in a subset of primary microRNA (miRNA) transcripts, recruiting the miRNA-microprocessor complex and promoting primary miRNA processing that is essential for mature miRNA biogenesis.9 Not only is the biogenesis of miRNA regulated by m6A marking and recruitment of HNRNPA2B1, miRNAs themselves appear to play a role in the placement of the m6A epigenetic marks. MiRNAs regulate m6A modification in specific transcript sites using a sequence-pairing mechanism where the “seed” sequence of a specific miRNA binds a complementary target sequence in the 3′ UTR of mRNA and directs methylation.15 The interaction is bidirectional: manipulation of miRNA sequence or expression affects m6A modification also by reducing binding of the METTL3 writer to the target mRNA sites.

Similarly, m6A appears to be involved in structural alterations of mRNAs and lncRNAs to facilitate binding of heterogeneous nuclear ribonucleoprotein C (HNRNPC), an abundant RNA-binding protein responsible for mRNA processing. This novel mechanism, termed m6A-switch, was shown to affect alternative splicing and abundance of multiple target mRNAs.16 Taken together, these results demonstrate that m6A is an important mark on diverse RNA species.

Dynamic m6A RNA markings, the new kid on the epigenetic block, herald the era of tripartite epigenetics where modifications of DNA, RNA, and proteins join hands to fine-tune gene expression and to execute prompt and precise responses to environmental stimuli and stresses. Indeed, m6A is just one of 140 modified RNA nucleotides that likely affect the function of the nucleic acid messenger and key cellular actor in diverse ways. Molecular approaches are paving the way for the study of additional RNA modifications.

As the list of RNA epigenetic marks continues to expand, researchers will gain a clearer picture of how diverse cellular processes are regulated. The extremely large repertoire of such modifications is expected to reveal various RNA marks analogous to the known DNA and histone epigenetic marks, and the various modifications of DNA, RNA, and proteins can enrich the language that allows the development, adaptation, and diversity of complex organisms.

Dan Dominissini is a postdoctoral fellow in Chuan He’s group at the University of Chicago. Gidi Rechavi is a pediatric hematologist-oncologist and a researcher in genetics and genomics at the Chaim Sheba Medical Center in Tel Hashomer, Israel, and a Professor of Hematology at the Sackler School of Medicine at Tel Aviv University. Sharon Moshitch-Moshkovitz, a senior researcher in RNA biology at the Chaim Sheba Medical Center, also contributed to this article.

References

  1. S. Horowitz et al., “Mapping of N6-methyladenosine residues in bovine prolactin mRNA,” PNAS, 81:5667-71, 1984.
  2. D. Dominissini et al., “Topology of the human and mouse m6A RNA methylomes revealed by m6A-seq,” Nature, 485:201-06, 2012.
  3. Y. Fu et al., “Gene expression regulation mediated through reversible m6A RNA methylation,” Nat Rev Genet, 15:293-306, 2014.
  4. K.D. Meyer, S.R. Jaffrey, “The dynamic epitranscriptome: N6-methyladenosine and gene expression control,” Nat Rev Mol Cell Biol, 15:313-26, 2014.
  5. G. Jia et al., “N6-methyladenosine in nuclear RNA is a major substrate of the obesity-associated FTO,” Nat Chem Biol, 7:885-87, 2011.
  6. G. Zheng et al., “ALKBH5 is a mammalian RNA demethylase that impacts RNA metabolism and mouse fertility,” Mol Cell, 49:18-29, 2013.
  7. X. Wang et al., “N6-methyladenosine-dependent regulation of messenger RNA stability,” Nature, 505:117-20, 2014.
  8. X. Wang et al., “N6-methyladenosine modulates messenger RNA translation efficiency,” Cell, 161:1388-99, 2015.
  9. C.R. Alarcón et al., “HNRNPA2B1 is a mediator of m6A-dependent nuclear RNA processing events,”Cell, 162:1299-308, 2015.
  10. J. Zhou et al., “Dynamic m6A mRNA methylation directs translational control of heat shock response,” Nature, 526:591-94, 2015.
  11. K.D. Meyer et al. “5′ UTR m6A promotes cap-independent translation,” Cell, 163:999-1010, 2015.
  12. J.-M. Fustin et al., “RNA-methylation-dependent RNA processing controls the speed of the circadian clock,” Cell, 155:793-806, 2013.
  13. S. Geula et al., “m6A mRNA methylation facilitates resolution of naive pluripotency toward differentiation,” Science, 347:1002-06, 2015.
  14. P.J. Batista et al., “m6A RNA modification controls cell fate transition in mammalian embryonic stem cells,” Cell Stem Cell, 15:707-19, 2014.
  15. T. Chen et al., “m6A RNA methylation is regulated by microRNAs and promotes reprogramming to pluripotency,” Cell Stem Cell, 16:289-301, 2015.
  16. N. Liu et al., “N6-methyladenosine-dependent RNA structural switches regulate RNA-protein interactions,” Nature, 518:560-64, 2015.

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RNA methylationRNA epigeneticsrnamethylationepigenetics and epigenetic regulation

 

Telomerase Overdrive

Two mutations in a gene involved in telomere extension reverse the gene’s epigenetic silencing.

By Ashley P. Taylor | January 1, 2016

http://www.the-scientist.com//?articles.view/articleNo/44768/title/Telomerase-Overdrive/

EPIGENETIC ACTIVATION: A single base-pair mutation (lower allele) leads to epigenetic changes that promote expression of a telomerase gene.COURTESY OF JOSH STERN
http://www.the-scientist.com/January2016/shortlit2.jpg

EDITOR’S CHOICE IN GENETICS & GENOMICS

The paper
J.L. Stern et al., “Mutation of the TERT promoter, switch to active chromatin, and monoallelic TERTexpression in multiple cancers,” Genes Dev, doi:10.1101/gad.269498, 2015.

The foundation
Chromosome ends are slightly shortened with each DNA replication. Terminal repetitive sequences called telomeres buffer coding DNA from this fate. In stem cells, telomerase extends the telomeres so that cell division can continue, perhaps indefinitely. In somatic cells, telomerase is inactive in part because the gene encoding telomerase’s catalytic sub­unit, telomerase reverse transcriptase (TERT), is epigenetically silenced. In most cancers, however, telomerase is again turned on and aids proliferation.

The mutations
In 2013, researchers found two mutations in the TERT promoter that occur frequently in cancer cell lines and are tied with TERT expression.

Regulation
To probe the mechanism of TERT activation, Josh Stern, a postdoctoral fellow in the lab of Thomas Cech at the University of Colorado Boulder, studied cancer cell lines that were heterozygous for one of these TERTmutations. Stern and his colleagues determined that the mutant TERT allele had histone methylation marks associated with gene activation and was transcribed, whereas the wild-type allele bore other histone methylation marks characteristic of gene silencing and was not transcribed.
“It’s very nice biochemical work to show that a single-base-pair mutation in the cancer genome activates the expression of the telomerase gene,” says Dana-Farber Cancer Institute’s Franklin Huang.

Application
“Telomerase is a fantastic therapeutic target for cancers because so many cancers are absolutely reliant on telomerase,” says Stern. “These TERT promoter mutations only occur in cancer, so if we can understand the mechanism, then we can potentially develop a highly specific cancer therapeutic.”

Tags

transcriptiontelomerestelomerasemutationliteraturegenetics & genomicsepigenetics and cancer

 

CRISPR Fixes Stem Cells Harboring Blindness-Causing Defect

http://www.genengnews.com/gen-news-highlights/crispr-fixes-stem-cells-harboring-blindness-causing-defect/81252293/

Marking yet another CRISPR-related first, scientists have replaced a defective gene associated with a sensory disease in stem cells that were derived from a patient’s tissue. The disease, retinitis pigmentosa (RP), is an inherited condition that degrades the retina and leads to blindness. A patient with the disease supplied a skin sample that was used to generate the stem cells, which were manipulated by means of the CRISPR/Cas9 gene-editing system.

CRISPR/Cas9, which zeroed in on a single disease-causing mutation in the RGPR gene, was able to make the necessary correction in 13% of the stem cells. This correction rate, according to the Columbia University and University of Iowa scientists who announced the results, is indicative of a practical approach—albeit one that still needs work. The Columbia/Iowa team added that they are working to show that their technique does not introduce any unintended genetic modifications in human cells, and that the corrected cells are safe for transplantation.

While the scientists freely acknowledge that their technique needs additional development before any cures are possible, they basked in the success of having accomplished a difficult genetic fix. The RGPR mutation that needed to be repaired sits in a highly repetitive sequence of the gene where it can be tricky to discriminate one region from another. In fact, it was not clear that CRISPR/Cas9 would be able to home in on and correct the point mutation.

The scientists described their work January 27 in the journal Scientific Reports in an article entitled “Precision Medicine: Genetic Repair of Retinitis Pigmentosa in Patient-Derived Stem Cells.”

“Fibroblasts cultured from a skin-punch biopsy of an XLRP patient were transduced to produce [induced pluripotent stem cells (iPSCs)] carrying the patient’s c.3070G > T mutation,” the authors wrote. “The iPSCs were transduced with CRISPR guide RNAs, Cas9 endonuclease, and a donor homology template. Despite the gene’s repetitive and GC-rich sequences, 13% of RPGR gene copies showed mutation correction and conversion to the wild-type allele.”

The authors asserted that theirs was the first report of CRISPR/Cas9 being used to correct a pathogenic mutation in iPSCs derived from a patient with photoreceptor degeneration. This proof-of-concept finding, they added, supports the development of personalized iPSC-based transplantation therapies for retinal disease.

The authors also emphasized that because the corrections are made in cells derived from the patient’s own tissue, doctors can retransplant the cells with fewer fears of rejection by the immune system. Previous clinical trials have shown that generating retinal cells from embryonic stem cells and using them for transplantation is a safe and potentially effective procedure.

Recently, another group has used CRISPR to ablate a disease-causing mutation in rats with retinitis pigmentosa. Going forward, the first clinical use of CRISPR could be for treating an eye disease because compared to other body parts, the eye is easy to access for surgery, readily accepts new tissue, and can be noninvasively monitored.

 

 

Edited stem cells offer hope of precision therapy for blindness

http://www.rdmag.com/news/2016/01/edited-stem-cells-offer-hope-precision-therapy-blindness

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Skin cells from a patient with X-linked Retinitis Pigmentosa were transformed into induced pluripotent stem cells and the blindness-causing point mutation in the RPGR gene was corrected using CRISPR/Cas9. Credit: Vinit Mahajan, Univ.of Iowa Health Care

Using a new technology for repairing disease genes–the much-talked-about CRISPR/Cas9 gene editing–Univ. of Iowa researchers working together with Columbia Univ. Medical Center ophthalmologists have corrected a blindness-causing gene mutation in stem cells derived from a patient. The result offers hope that eye diseases might one day be treated by personalized, precision medicine in which patients’ own cells are used to grow replacement tissue.

With the aim of repairing the deteriorating retina in patients with an inherited blinding disease, X-linked Retinitis Pigmentosa (XLRP), Alexander Bassuk, MD, PhD, and Vinit Mahajan, MD, PhD, led a team of researchers who generated stem cells from patient skin cells and then repaired the damaged gene. The editing technique is so precise it corrected a single DNA change that had damaged the RPGR gene. More importantly, the corrected tissue had been derived from the patient’s own stem cells, and so could potentially be transplanted without the need for harmful drugs to prevent tissue rejection. The research was published Jan. 27 in the journal Scientific Reports.

“With CRISPR gene editing of human stem cells, we can theoretically transplant healthy new cells that come from the patient after having fixed their specific gene mutation, ” says Mahajan, clinical assistant professor of ophthalmology and visual sciences in the UI Carver College of Medicine. “And retinal diseases are a perfect model for stem cell therapy, because we have the advanced surgical techniques to implant cells exactly where they are needed.”

The study was a “proof-of-concept” experiment showing it is possible not only to repair a rare gene mutation, but that it can be done in patient stem cells. Use of stem cells is key because they can be re-programmed into retinal cells.

The CRISPR technology was able to correct the RPGR mutation in 13 percent of the stem cells, which is a practically workable correction rate.

Bassuk notes this result is particularly encouraging because the gene mutation sits in a highly repetitive sequence of the RPGR gene where it can be tricky to discriminate one region from another. In fact, initially determining the DNA sequence in this part of the gene was challenging. It was not clear that CRISPR/Cas9 would be able to home in on and correct the “point mutation.”

“We didn’t know before we started if we were going to be able to fix the mutation,” says Bassuk, associate professor in the Stead Family Department of Pediatrics at University of Iowa Children’s Hospital.

 

Epigenetics Research Reveals a Range of Clinical Possibilities

Advantageously Epigenetic Analyses Can Capture both Genetic Factors and Environmental Exposures

Richard A. Stein, M.D., Ph.D.

http://www.genengnews.com/gen-articles/epigenetics-research-reveals-a-range-of-clinical-possibilities/5650/

  • Over half a century ago, Conrad Hal Waddington introduced his model of the epigenetic landscape. He depicted a differentiating cell as a ball rolling down a landscape of bifurcating valleys and ridges, with each valley representing an alternative developmental path. Just as a ball may roll from valley to valley until it reaches the bottom of the landscape, a cell may progress from one developmental alternative to another until it reaches its fully differentiated state.

The model’s original purpose was to integrate concepts from genetics and developmental biology and to describe mechanisms that connect the genotype to the phenotype. Today, the model remains a compelling metaphor for epigenetics, which has developed into one of the most vibrant biomedical fields. Epigenetics has become indispensable for exploring development, differentiation, homeostasis, and diseases that span virtually every clinical discipline.

  • Analyzing Methylation Patterns

“Modern efforts toward explaining human disease purely based upon sequencing cannot possibly succeed in isolation,” says Andrew P. Feinberg, M.D., professor of medicine and director of the Center for Epigenetics at Johns Hopkins University School of Medicine. “At least half of human disease is caused by exposure to the environment.”

While the contribution of genetic factors to disease is more predictable and easier to study in the case of highly penetrant Mendelian disorders, most medical conditions involve multiple genes that may interact with one another and with environmental factors. Particularly for these conditions, capturing epigenetic changes becomes a crucial aspect of understanding pathogenesis and designing prophylactic and therapeutic interventions.

“In these cases,” notes Dr. Feinberg, “an approach not including epigenetics will be severely limited in what it can accomplish.”

In a recent study, Dr. Feinberg and colleagues reported that large blocks of the human genome are hypomethylated in the epidermis as a result of sun exposure, which together with aging represents a known risk factor for skin cancer. These hypomethylated regions overlap with regions that have methylation changes in patients with squamous cell carcinoma.

This overlap could explain the causal link between sun exposure and the increased risk of malignancy found in many epidemiological studies. Most of the methylation changes were observed in the epidermis, not in the dermis, pointing toward the combination between the genotype and exposure, acting on specific cell types, as a key factor in shaping disease.

“One of the advantages of epigenetic analyses is that they capture both genetic factors and environmental exposures,” explains Dr. Feinberg. In the study of complex diseases, the existence of many distinct genetic variants identified in different individuals makes it challenging to understand their roles in pathogenesis. “But if genetic variants converge on gene regulatory loci, then measuring methylation can still be informative about these variants,” continues Dr. Feinberg, “even if genetic changes are inconsistent across the patients.”

In combining data from genome-wide association analysis and epigenome-wide analysis, Dr. Feinberg and colleagues revealed that two single-nucleotide polymorphisms on human chromosome 11, located 100 kb apart and involved in different aspects of lipid metabolism, controlled DNA methylation at two CpG sites in a bidirectional promoter situated between two genes encoding the fatty acid desaturases FADS1 and FADS2. Genome-wide association studies alone would not capture the convergence of these two single-nucleotide polymorphisms as they regulate DNA methylation in the shared promoter region.

“Measuring DNA methylation,” concludes Dr. Feinberg, “can pick up the fact that these single nucleotide polymorphisms act through DNA methylation to regulate the genes.”

The image shows a cleavage-stage human embryo. This is around the same stage that DNA methylation is ‘set’ at metastable epialleles. [Instituto Bernabeu]

http://www.genengnews.com/Media/images/Article/Jan15_2016_RobertWaterland_CleavageStageHumanEmbryo3202193100.jpg

Identifying Metastable Epialleles

Over the years, genome-wide association studies provided opportunities to establish links between genetic variation and phenotypic changes. For these analyses, genetic material from any of an individual’s cells, such as a peripheral white blood cell, is informative about the individual’s genotype. However, for epigenetic changes, which vary across tissues and within the same tissue among different cells, it is much more challenging to examine associations with disease.

Robert A. Waterland, Ph.D., associate professor of pediatrics and molecular and human genetics at Baylor College of Medicine, thinks that identifying human metastable epialleles will help circumvent some of these challenges. “Getting investigators and the field interested in metastable epialleles is going to be an important first step in helping us understand how epigenetic dysregulation contributes to human disease,” says Dr. Waterland.

The term metastable epialleles refers to genomic loci with differential epigenetic regulation that are variably expressed in genetically identical individuals, and where the epigenetic state is established stochastically in the very early embryo, before gastrulation, and subsequently maintained. This leads to systemic (non-tissue-specific) interindividual epigenetic differences that are not genetically mediated.

The fact that DNA methylation at metastable epialleles is particularly sensitive to environmental influences makes these loci valuable in mechanistically exploring the developmental origins hypothesis, the concept that environmental exposures during critical periods of prenatal and early postnatal development can have long-term implications in the risk of disease. Previous studies have implicated epigenetic modifications as a mechanism by which environmental changes during pregnancy may lead to epigenetic changes that influence health later in life.

In the most recent genome-wide screen meant to identify metastable epialleles in humans, Dr. Waterland teamed up with Dr. Andrew Prentice and colleagues at the London School of Hygiene and Tropical Medicine and used two independent and complementary experimental approaches to identify DNA methylation changes that occur in the cleavage-stage embryo (shortly after the time of conception). The first approach involved a genome-wide screen for DNA methylation in multiple tissues from two healthy Caucasian adults. In parallel, genome-wide DNA methylation profiling was performed in a rural population from The Gambia to examine the link between the season of conception (a proxy for maternal nutritional status) and DNA methylation in the offspring and sought to capture the effect of maternal nutritional status on the epigenetic profile of the offspring.

“We identified the same genomic locus as the top hit in both screens, suggesting that this is likely to be a key indicator of early environmental influences on the epigenome,” explains Dr. Waterland. Both approaches identified VTRNA2-1 as the lead candidate for an environmentally-responsive epiallele.

VTRNA2-1, a genomically imprinted small noncoding RNA and a putative tumor suppressor gene, is preferentially methylated on the maternally inherited allele, and loss of imprinting at this locus promises to link the early embryonic environment to epigenetic changes that shape disease risk later in life. Besides VTRNA2-1, over 100 metastable epialleles were identified in the study.

“At metastable epialleles such as VTRNA2-1, DNA methylation in peripheral blood or in any easily accessible tissue can give an indication about the epigenetic regulation throughout the body,” concludes Dr. Waterland. “That is what is really different.”

http://www.genengnews.com/Media/images/Article/thumb_Jan15_2016_DavidBazettJones_ElectronSpectroscopic1381311078.jpg

Electron spectroscopic image of a region of the nucleus of a mouse embryonic fibroblast. Phosphorus and nitrogen maps allow chromatin (yellow) to be distinguished from protein-based structures (cyan). The arrow indicates the nuclear envelope. The large structure in the middle of the field, a chromocentre, is an accumulation of pericentric heterochromatin. It is surrounded by dispersed chromatin fibers. The heterochromatin mark, trimethlated H3K9, is immunolabelled and visualized with gold tags (white foci). [David Bazett-Jones]

Mapping Heterochromatin Domains

Electron spectroscopic image of a region of the nucleus of a mouse embryonic fibroblast. Phosphorus and nitrogen maps allow chromatin (yellow) to be distinguished from protein-based structures (cyan). The arrow indicates the nuclear envelope. The large structure in the middle of the field, a chromocentre, is an accumulation of pericentric heterochromatin. It is surrounded by dispersed chromatin fibers. The heterochromatin mark, trimethlated H3K9, is immunolabelled and visualized with gold tags (white foci). [David Bazett-Jones]

“For the first time, we found that a histone chaperone is implicated in organizing chromatin at a large scale,” says David Bazett-Jones, Ph.D., professor of biochemistry at the University of Toronto and senior scientist at the Hospital for Sick Children. The discovery and characterization of histone variants has been a vital facet of understanding chromatin organization and dynamics.

One of the most extensively studied histone variants is H3.3. Although H3.3 is 96% identical at the amino acid level to histone H3.1, histones H3.3 and H3.1 are functionally distinct. Histone H3.3 is expressed throughout the cell cycle, and it is enriched in transcriptionally active chromatin and in certain types of post-translational modifications. The death domain-associated protein DAXX, one of the proteins associated with histone H3.3 deposition, was recently identified as its chaperone.

Dr. Bazett-Jones and colleagues, including his graduate student Lindsy Rapkin, revealed that the loss of DAXX led to a global structural change in the chromatin landscape, characterized by genomic regions enriched in the trimethylated H3K9 epigenetic mark that were juxtaposed to large chromatin domains devoid of this modification.

“These major changes probably occur because the boundaries between heterochromatin domains and other regions were not being respected, leading to the inappropriate insertions of histone H3.3, and this exerted quite profound effects,” explains Dr. Bazett-Jones. The loss of DAXX led to the uncoupling of the epigenetic marks from the global chromatin architecture. “This shows that a major global reorganization of the chromatin was taking place,” Dr. Bazett-Jones continues.

To visualize chromatin changes that result from the loss of DAXX, Dr. Bazett-Jones and colleagues used electron spectroscopic imaging, an experimental approach that is based on the principle of electron energy loss spectroscopy. When a biological specimen is targeted with electrons and its atoms become ionized, the ionization energy is equal to the energy that is lost by the incident electrons that generated the event.

The electron microscope technique generates nitrogen and phosphorus maps, which are used to discriminate between nucleic-acid-rich and protein-rich cellular structures. These maps offer high-contrast images of chromatin and its three-dimensional organization in intact cells.

Another component of the DAXX deletion phenotype included the loss of nucleolar structural integrity, resulting in an increased number of cells containing mini-nucleoli, and the dispersal of ribosomal DNA genes outside the nucleolus. Collectively, these findings pointed toward a novel role that DAXX plays in the subnuclear organization of chromatin and in maintaining nucleolar structural integrity.

“Historically, we thought that the well-known epigenetic modifications dictate the compact character of heterochromatin,” notes Dr. Bazett-Jones. “But our findings, and those from other groups, reveal that a heterochromatin domain epigenetically marked with H3K9 trimethylation, for example, can be found in a structurally ‘open’ state, similar to euchromatin.”

This indicates that the boundaries between heterochromatin and euchromatin are much more fluid than previously envisioned, a concept that is crucial for understanding factors that dynamically shape the three-dimensional interaction between epigenetic changes. A key implication of these findings is that the epigenetic marks at a specific genomic locus depend on both the local environment and the three-dimensional context.

“We need to look at what loci come together in specific regions of the nucleus in three dimensions and how they affect each other,” concludes Dr. Bazett-Jones. “This is on top of capturing epigenetic marks, which are on top of the genomic sequences that we need to explore.”

Identifying Druggable Epigenetic Processes

“There is a big gap in understanding the biology of epigenetics,” says Chris J. Burns, Ph.D., laboratory head, Division of Chemical Biology, Walter and Eliza Hall Institute of Medical Research, Melbourne. “And this goes hand in hand with the need to learn how to generate small molecule probes or drugs.”

When interrogating epigenetic processes, researchers find it useful to integrate biological and chemical perspectives. For example, researchers have generated a large body of literature demonstrating that many epigenetic processes involve highly complicated protein complexes.

Historically, genetics studies have typically relied on knocking down or knocking out a gene and its protein product to examine the resulting phenotype. “In contrast, knocking down a protein that is part of a protein complex fundamentally alters that complex, and the phenotype could be quite different from the one that can be seen with a small molecule inhibition of a catalytic component of the protein complex,” notes Dr. Burns. This opens an acute need to identify small molecules that can selectively impact just one particular aspect of these protein complexes.

A major effort in Dr. Burns’ lab is focusing on identifying therapeutic agents that could target epigenetic processes. “Epigenetics in terms of drug discovery and development is still in an early stage,” explains Dr. Burns. While several drugs that target epigenetic processes have become available in recent years—drugs such as HDAC inhibitors and DNA methyl transferase inhibitors—many other drugs are still at early stages of development.

“Some epigenetic processes have not yet been drugged,” Dr. Burns points out. “For some of them, there may not be any therapeutic agents that are particularly good.”

Dr. Burns’ lab has collaborated with investigators led by Carl Walkley, Ph.D., joint head, Stem Cell Regulation Unit, St. Vincent’s Institute of Medical Research, Melbourne. Together, the research teams revealed that several bromodomain inhibitors exert powerful antitumor activity in human osteosarcoma cell lines and in osteosarcoma primary cells from mouse models of the disease.

The researchers’ findings were surprising. JQ1, one the bromodomain inhibitors tested, exerted its antiproliferative activity by inducing apoptosis, and not by mediating cell cycle arrest, as expected. Moreover, even though previous studies identified MYC as an oncogenic driver in osteosarcoma, the activity of JQ1 was exerted independently of MYC downregulation.

At the same time, this work revealed that downregulation of FOSL1, a gene previously implicated in osteoblast differentiation, is an important contributor to the effects of JQ1, marking the first time when this gene was implicated in osteosarcoma.

“Because we used primary cell from animals, these findings reflect the disease process better than cell lines, which may take on a number of other mutations,” concludes Dr. Burns. “This explains why our findings are contrary to previous reports in the literature.”

“We have shown that epigenetic drugs may work not only on protein-coding genes but also on the noncoding part of the genome,” says Claes Wahlestedt, M.D., Ph.D., professor and associate dean for therapeutic innovation at the University of Miami Miller School of Medicine.

A therapeutically promising class of epigenetic compounds consists of bromodomain inhibitors. These compounds have received increasing attention in recent years, and several leads have entered clinical trials for malignancies, atherosclerosis, and type 2 diabetes.

”One of our interests is to see if bromodomain inhibitors could be used for diseases of the nervous system,” notes Dr. Wahlestedt.

Using in vitro and in vivo approaches, investigators in Dr. Wahlestedt’s group, in collaboration with investigators led by Nagi Ayad, Ph.D., found that BET bromodomain inhibitors can inhibit glioblastoma cell proliferation by inducing a cyclin-dependent kinase inhibitor. These findings set the stage for subsequent experiments that used single molecule sequencing to profile long noncoding RNAs (lncRNAs) differentially expressed in glioblastoma multiforme. This helped identify a set of transcripts that are specific for this malignancy and could be regulated by bromodomain inhibitors.

In glioblastoma multiforme cells, the I-BET151 bromodomain inhibitor localized to the promoter of HOTAIR, a tumor-promoting lncRNA that acts as an epigenetic silencer and has been implicated in several cancers, decreased its expression, and restored the expression of several lncRNA species that are downregulated in this malignancy.

In another collaborative endeavor, Dr. Wahlestedt and colleagues conducted a semi-high-throughput gene-expression-based screen to identify small molecules that could increase the expression of C9ORF72. A GGGGCC hexanucleotide repeat expansion in the noncoding region of the C9ORF72 gene is the most common genetic cause for amyotrophic lateral sclerosis. Individuals without this condition harbor 2 to 25 of these repeats, but their number can reach up to several hundreds in ALS patients, reducing C9ORF72 expression, which has been implicated in the pathogenesis of this condition.

The gene-expression-based screen identified, in fibroblasts from affected and unaffected individuals, small interfering RNAs against the BRD3 bromodomain protein and several small molecule bromodomain inhibitors that were able to increase C9ORF72 expression. This effect occurred without changes in promoter CpG hypermethylation and trimethylated H3K9 marks, which are heterochromatin markers of the expanded C9ORF72 alleles.

“The mechanism of action of these compounds is probably broader than we thought before,” concludes Dr. Wahlestedt.

 

CRISPR Works Well but Needs Upgrades

More Effective and Reliable CRISPR Tools Will Have To Be Developed

MaryAnn Labant

http://www.genengnews.com/gen-articles/crispr-works-well-but-needs-upgrades/5652/

http://www.genengnews.com/Media/images/Article/thumb_UnivIllinois_Cas9DSB1921455173.jpg

In this image, which comes from the University of Illinois at Urbana-Champaign, Cas9 (green) is shown cutting DNA (white and brown) at the target sequence specified by the single guide RNA (red). The image was created from the Protein Data Bank file 4un3.pdb using Pymol, and it was enhanced using Photoshop.

The gene-editing technology known as CRISPR-Cas9 went through a disruptive phase when it first took the research world by storm.

Now, thousands of research articles later, it is starting to raise expectations in the therapeutic realm. In fact, CRISPR-Cas9 and other CRISPR systems are moving so close to therapeutic uses that the technology’s ethical implications are starting to attract notice. For example, people worry that CRISPR could be used to alter human germline cells, introducing genomic changes that could impact future generations.

Before any of that can happen, however, CRISPR will have to overcome a number of practical obstacles. If CRISPR is to be harnessed effectively and leveraged to its full potential, it will have to be better understood. Also, more effective and reliable CRISPR tools will have to be developed.

For example, little progress has been made in the area of targeted integration. “We effectively have the tools to cut, yet we lack efficient tools to paste. How the cells repair the double-strand break created by the RNA-guided nucleases, or RGNs, depends almost exclusively on the cells themselves in that there is no control over the repair mechanism. In addition to the RGNs, we deliver a vector that can function as a repair template, and hope the cells will use it,” explained Pablo Perez-Pinera, M.D., Ph.D., assistant professor, department of bioengineering, University of Illinois at Urbana-Champaign.

 

 

Fun with Lego (molecules)

http://www.rdmag.com/news/2016/01/fun-lego-molecules

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Depending on the relative amounts of different building-block molecules, it is possible to create different sandwich and wheel topologies (shown above in micrographs and below as models). Credit: American Chemical Society. Copyright 2016

A great childhood pleasure is playing with Legos and marveling at the variety of structures you can create from a small number of basic elements. Such control and variety of superstructures is a goal of polymer chemists, but it is hard to regulate their specific size and how the pieces fit together. This week in ACS Central Science, researchers report a simple system to make different nano-architectures with precision.

Using a variety of highly efficient chemical transformations and other techniques to ensure high yields and purity, Stephen Z. D. Cheng, Yiwen Li, Wen-Bin Zhang and coworkers designed systems to create giant molecules with ‘orthogonal’ ends, meaning that they only fit together with a specific partner just like Legos. Depending on the relative amounts of different building-block molecules, these molecules come together in different superstructures — ranging from cubes to wheels and sandwiches. Eventually, they could be employed in device-creation, where it is crucial to have precise control over the positions of the components.

 

Protein Expression Systems Proliferate

Bioprocessing Assembly Lines Are Being Retooled, Often At the Genomic Scale

Angelo DePalma, Ph.D.

http://www.genengnews.com/Media/images/Article/thumb_iStock_32784234_eColi1930160883.jpg

Despite some bells and whistles, most E. coli production systems have been the same. Now, new systems are being introduced that purport to express proteins more efficiently. [iStock/Scharvik]

Biomanufacturers enjoy a host of tools to optimize the production of therapeutic proteins, including expression systems, media, feeds, and gene-editing tools. Suffice it to say that protein expression is a growth industry.

Industry research firm Future Market Insights (FMI) breaks down the protein expression market into four product areas: competent cells, expression vectors, instruments, and reagents serving demand for research-grade and therapeutic proteins.

FMI has identified noteworthy growth drivers: the rising significance of biologics; innovations in proteomics; and patent expirations among small-molecule drugs. “These demands will boost the overall protein expression market in the coming future,” FMI literature states. “However, [attempts to contain rising costs] in various R&D activities in the fields of biotechnology and pharmaceutical industry as well as market consolidation of a high degree are some restraining factors for this market.”

The largest market for protein expression is expected to emerge in North America, given this region’s “well-established healthcare infrastructure.” North America is followed by Europe, and the Asia-Pacific region shows the highest growth. This information was derived from an FMI report (“Protein Expression Market: Global Industry Analysis and Opportunity Assessment 2015–2025”) that was issued last December.

 

 

Landmark Year

Through the efforts of scientists at Thermo Fisher Scientific, 2015 was a landmark year for transient protein production in CHO cells. The company’s ExpiCHO™ transient expression system achieved multiple g/L levels of protein expression previously thought possible only in stable cell lines, according to Jonathan Zmuda, Ph.D., associate director of cell biology at Thermo Fisher Scientific’s Gibco business unit.

“ExpiCHO allows drug developers to obtain meaningful quantities of protein from CHO cells at the very earliest stages of biologics development,” Dr. Zmuda asserts. “It allows CHO-derived protein to be used from discovery day one through the transition to stable cell lines, bioproduction, clinical trials, and product licensing.”

This has had the effect of streamlining drug development by eliminating the risk of starting a program with HEK 293-derived drug candidates, while also providing an alternative high-expressing system for proteins that are difficult to express in HEK 293.

New E. Coli Expression System

http://www.genengnews.com/Media/images/Article/NewEnglandBiolabsFigure28614117612.jpg

New England BioLabs says that its SHuffle T7 E. coli expression system is able to express non-di-sulfide bonded proteins more efficiently than wild-type E. coli. The actual SHuffle strain expressing GFP is shown here.

Since E. coli was recruited for service around 1950, hundreds of thousands of publications have sung the praises of this bedrock expression system. But Mehmet Berkmen, Ph.D., staff scientist at New England BioLabs, notes that no more than a dozen distinct protein production strains exist. When production strains are examined closely, all are found to belong to just two basic strains, E. coli K-12 and E. coli B.

“Some strains have ‘bells and whistles,’ but the basic platform is the same,” Dr. Berkmen points out. “People are still looking for engineered lines that express protein more efficiently.”

Most expression systems are based on E. coli B, but that strain is not engineered specifically for protein production. The B strain is somewhat less domesticated than K-12, which has gone through numerous generations of selection for DNA manipulation. “E. coli B is more wild and tends to make protein better,” Dr. Berkmen notes. “But if you ask people why that is the case, they can’t provide an answer.”

New England BioLabs claims that its SHuffle® T7 E. coli expression system represents a breakthrough for microbial fermentation. The bacteria, which are chemically competent E. coli K-12 cells engineered to form proteins containing disulfide bonds in the cytoplasm, are suitable for T7-promoter-driven protein expression. The company has recently produced full-length antibodies, complete with disulfide bonds, in SHuffle organisms, which Dr. Berkmen calls “a significant step toward engineering and developing novel antibody formats and tools.”

New England BioLabs manufactures more than 500 proteins, 98% of them in E. coli. Perhaps even more interesting is the SHuffle system’s ability to express non-disulfide-bonded proteins more efficiently than wild-type E. coli. “SHuffle,” insists Dr. Berkmen, “represents a new chassis for protein production.”

The E. coli bacterium does not form disulfide bonds in its cytoplasm because two reducing pathways maintain the cytoplasmic proteome in its reduced state. Dr. Berkmen’s group knocked out those pathways and inserted a gene for a disulfide bond isomerase that increases fidelity of disulfide bond formation.

In addition to benefits already mentioned, SHuffle has a greatly diminished reducing capacity, permitting the formation of disulfide bonds for proteins that require it for folding and activity. Additionally, the cells, which are under oxidative stress, produce chaperones that also improve folding. For example, the activity of green fluorescent protein (GFP) expressed in SHuffle is much higher than protein produced in wild-type E. coli B.

It should be noted that a lack of glycosylation machinery persists in SHuffle cells. This problem, however, can be circumvented, as demonstrated in a seminal study carried out by Dr. Berkmen and colleagues. This study, which appeared last year in Nature Communications, described how IgG could be produced in SHuffle cells. Specifically, the investigators introduced mutations into the Fc portion of IgG. This resulted in efficient binding of aglycosylated IgG to its cognate receptor FcγRI.

Even in the absence of such ingenuity, E. coli remains a valuable expression system. It can be used to produce diagnostic and reagent proteins, or proteins for which glycosylation is noncritical.

“A Matter of Trying”

The principal advantages of using E. coli. are time and cost. “It takes basically one day, more or less, to obtain enough protein to suit many applications,” says David Chereau, Ph.D., CSO at Biozilla, a biotechnology contract research organization. As previously noted, the main disadvantages are lack of glycosylation apparatus and inability to support disulfide bond formation.

Workaround strategies can achieve stable disulfide bonds for some proteins. One strategy involves the following steps: Express the protein as an inclusion body, in insoluble form. Isolate the insoluble fraction. Solubilize this fraction with urea or some other suitable agent. Refold the protein.

“The process is relatively straightforward,” observes Dr. Chereau. “It’s much more difficult to find refolding conditions, which are normally determined empirically.” Refolding requires just the right buffer, salt concentrations, and additives. Also, refolding must be done in an oxidizing environment if disulfide bonds are to be achieved or maintained.

Dr. Chereau is philosophical about CHO cells’ inability to glycosylate: “Lack of glycosylation can be seen as an advantage or an inconvenience, depending.” E. coli is definitely out where glycosylation is a sine qua non. “But for the many applications where glycosylation isn’t needed, E. coli can be advantageous,” comments Dr. Chereau. Diagnostics and reagents are two such products. Additionally, obtaining a crystal structure during protein characterization is easier with glycans absent.

As part of its proof-of-concept services, Biozilla performs rapid screens to determine if E. coli is the right expression system for a particular product. Screening resembles design-of-experiment for mammalian cells, varying plasmids and vectors, as well as expression conditions.

Due to the success of CHO cells, bioprocessors tend to dismiss microbial fermentation, particularly for large proteins. “A lot of people think that expressing large proteins in E coli is difficult,” Dr. Chereau states, “but it’s often just a matter of trying. We have recently expressed a protein of 215 kDa in E. coli, which most people will tell you cannot be done. And we achieved it in very high yield.”

Rapid Prototyping

In June 2015, Invenra, a preclinical stage biotech company specializing in next-generation antibodies and antibody derivatives, entered an agreement with Oxford BioTherapeutics (OBT) to identify and characterize fully human therapeutic monoclonal antibodies (mAbs) against a novel cancer target that OBT has identified.

Invenra’s protein expression platform, through which it is capable of producing hundreds of thousands of full-length antibodies, uses cell-free expression to multiplex up to 10,000 protein variants simultaneously.

“We think of our technology as a rapid prototyping tool for proteins,” says Bryan Glaser, Ph.D., Invenra’s R&D director. “Once we have DNA, we can get protein in less than a day.” Invenra’s expression platform is suitable mainly for discovery and rapid protein prototyping. Yields are quite good: up to 500 μg/mL.

Other firms, such as Sutro Biopharma, are working on cell-free expression at much larger scales. Sutro claims that its Express CF™ technology can produce g/L yields in eight hours.

Cell-free expression involves E. coli extracts, typically S30 (used by most cell-free expression systems) and S12. The numbers reflect centrifugation speed. “Our system is based on S12, which is spun at lower speed than S30,” informs Dr. Glaser. “Our extract also does not undergo dialysis. We think of it as a ‘whole grain’ version.”

In addition to E. coli extracts, additives contain varying quantities of supplemental energy sources, nucleotides, and other small molecules that facilitate in vitro transcription and translation. Every vendor has its own unique blend.

Invenra’s standard mix, which is similar to off-the-shelf products from most commercial sources, is optimized for less complex molecules that don’t require disulfide bonds. Another mix has been optimized to include chaperones formulated to help expression and folding of IgGs and IgG-like molecules.

The upshot: fully functional, correctly folded IgGs and some bispecific antibodies, scFvs, and Fabs. More complex molecules are also possible, but each must be investigated independently. It is possible those could be made, but they would need to be optimized structure by structure. Dr. Glaser says expression capability depends to a large extent on amino acid sequence.

“We can fine-tune expression and folding conditions better than is possible in E. coli,” Dr. Glaser asserts. “We have better control over redox environment to facilitate disulfide bond formation, and we can add chaperones that are not present in E. coli organisms.” Still, the more disulfides the more complex the structure, and the lower the yield.

Dr. Glaser adds that antibody frameworks that express well in E. coli express well in cell-free systems, and ones that don’t express well in bacteria or mammalian cells tend not to express well cell-free. “It could be a framework sequence dependency,” he speculates. “It could be how well that framework folds. Perhaps the best-expressing molecules are those that do not require as much assistance from various chaperones and isomerases.”

Invenra’s expression system lends itself well to large-scale parallelism. The company has developed a credit-card-sized nanowell platform that expresses up to 10,000 unique antibodies per nanowell array. Cell-free expression of IgG using the Invenra nanowell platform system enables the incorporation of functional screening very early into the discovery process.

The ability to screen in excess of 100,000 IgG molecules can reduce the antibody display selection steps and preserve a larger diversity of epitope coverage. In addition, large combinations of binding partners can be empirically tested in various bispecific formats with relevant functional assays to identify the best pair and format for activity.

Getting the Bugs Out

Interest is growing for insect cell expression systems transiently transfected through the baculovirus expression vector system (BEVS). More and more clinical candidates are being generated in insect cells, including development-stage products for respiratory syncytial virus, Ebola virus, and norovirus.

 

A good deal of BEVS’ success is the ability of insect cells to produce multivalent, multisubunit vaccines through virus-like particles. These proteins can be made at large scale with BEVS for structural studies or to elucidate protein function.

Additionally, insect cells are ideal for making proteins that are toxic to mammalian or E. coli expression systems. BEVS shows its flexibility by providing rapid development cycles for treatments like seasonal influenza or pandemic infection vaccines. Because it is a transient system, BEVS allows for rapid turnaround times compared with mammalian cells, from identification of vaccine candidates to production.

 

Progress toward Therapeutic Epigenetics    

Epigenetic Targets Are Plentiful but Well Camouflaged

Angelo DePalma, Ph.D.

GEN  Jan 15, 2016 (Vol. 36, No. 2)   http://www.genengnews.com/gen-articles/progress-toward-therapeutic-epigenetics/5664/

  • Epigenetics is poised to become a cornerstone of drug development in oncology, diabetes, inflammation, developmental and metabolic disorders, cardiovascular and autoimmune diseases, pain, and neurological disorders.

    According to citations from PubMed Epigenetics, 40% year-on-year increases in epigenetics-related scientific publications occurred during the last decade, accompanied by a substantial increase in research funding. Data from ClinicalTrials.gov indicate that more than 40 different epigenetics-related drugs are undergoing clinical trials. Epigenetics will also likely affect developments in animal, plant, and environmental health.

    Jim Corbett, president of the human health business at PerkinElmer, notes that epigenetics research is currently limited by the number and availability of fully validated targets and preclinical disease models. “Another limitation stems from the relative dearth of fully selective antibodies for some of the writer and eraser targets to elucidate these complex signaling and modification events,” he points out. “Epigenetics research also suffers from a lack of a translational continuum for specific applications and for solutions from bench to bedside.”

    Nevertheless, the field is characterized by a high level of optimism. Research by Mordor Intelligence (“North America and Europe Epigenetics Market Growth, Trends and Forecasts, 2014–2020”) estimates that epigenetics will grow in market reach from approximately $2.9 billion in 2012 to about $12 billion in 2018.

    “I anticipate the development of second-generation epigenetic inhibitors with increased selectivity and targeting potential, standardization of epigenetic assays, and the validation of preclinical disease models leading to an improved understanding of epigenetic targets and mechanisms,” Corbett ventures. “The emergence of selective genome-editing technologies such as CRISPR will also apply in epigenetics and epigenome editing. I envision the future the emergence of personalized epigenetic profiles in patients.”

  • Computer Analogy

    Randy L. Jirtle, Ph.D., professor of epigenetics at North Carolina State University, describes epigenetics as a type of biological software. He explains an embryo’s combination of paternal and maternal genetic information, and eventual differentiation into 200–300 cell types, on the basis of cells running different programs.

    “The cell can be thought of as a programmable computer where the hardware is DNA and the software is the epigenome,” says Dr. Jirtle. “Very shortly after fertilization, this computer tells the cell how to work. And as with actual computers, things can go wrong because of viruses or—in the case of cells—mutations.”

    Dr. Jirtle demonstrated in 2003 that epigenetic modifications in utero may determine adult disease susceptibility, a notion that was not welcomed enthusiastically. “If you think of life as hardware, no known mechanism would explain this [connection],” asserts Dr. Jirtle. “But when you consider the ‘software,’ it becomes understandable.”

    Epigenetics can bring about positive effects as well. Through a process known as hormesis, low doses of a toxic agent or low doses of radiation can be administered strategically to improve an organism’s subsequent health. For example, mice exposed to low levels of ionizing radiation experienced a positive adaptive effect, which flies in the face of prevailing “no safe dosage” logic.

    In one strain of an experimental mouse bred to develop human-like diseases, 1 cGy of exposure—about the dose received from five X-rays—resulted in a decidedly positive hypermethylation of the epigenome. Exposed mice developed obesity, diabetes, and cancer at significantly lower rates than nonexposed mice. Negative effects occur at significantly higher doses as expected.

    Similarly positive epigenetic effects have been observed in plants exposed to very low doses of herbicides.

    Dr. Jirtle believes that the characterization of the repertoire of genes imprinted in humans, and their regulatory elements, the imprintome, will guide epigenome-based therapies. Imprinting is the process by which one parental copy of a gene is silenced. Thus, depending on the effectiveness of silencing, one could have two copies of a gene or none, either of which could potentially be deadly. In some cancers, for example, the inability to silence one parental gene for IGF2, which influences apoptosis, allows cancer cells to grow out of control.

    “There are probably around 150–500 disease-influencing genes that are regulated this way,” Dr. Jirtle points out.

  • Implementation Hurdles

    The connection between dysregulated DNA methylation and cancer is well established. Keith Booher, Ph.D., epigenetic service projects manager at Zymo Research, believes that modifying how methylation patterns change could allow a reset. Essentially, cells destined to become cancerous could be returned to a normal state.

    But significant hurdles block straightforward implementation. For example, getting drugs into cells, particularly solid tumors, is not easy. “It’s no coincidence that DNA methylation inhibitors have proved most successful for blood-based cancers, which are easier to target,” Dr. Booher tells GEN.

    Another hurdle is drug resistance, an issue with nearly all oncology agents. Moreover, drugs that alter the activity of the ubiquitous DNA methyl transferase will have broad activity on normal as well as abnormal cell processes.

    “Normal cells show low and high methylation levels,” Dr. Booher explains. “DNA methylation tends to limit gene expression, so you want to shut down those genes. And where DNA methylation is absent, genes tend to be expressed.

    “Methylation will change across the genome at different development stages, but in adult cells or developing blood cell you want methylation patterns to change in a regulated way. It’s difficult to limit the effect to diseased cells.”

    Finally, the way methylation inhibitors interact with DNA is in itself harmful. The original epigenetics-based drugs tested as broad chemotherapeutics, but their toxicology was high. It was only later, after the understanding the relationship between DNA methylation and carcinogenesis was better established, that the potential to use these agents at much lower doses became possible.

     

    Diagnostic Relevance

     

     

    This Circos plot from Swift Biosciences represents the methylation status of 1 Mb bins across chromo­somes 1–22 for Sample 8 (Metastatic colorectal adenocarcinoma with liver metastasis, 2 cm primary).

    One of the most important advances in epigenetic research is the ability to obtain comprehensive, per-base methylation status of the methylome using next-generation sequencing (NGS). The significant drop in sequencing costs enables both whole-genome bisulfite sequencing and hybridization capture for targeted enrichment of the methylome.

    Initially, notes Laurie Kurihara, Ph.D., director of R&D at Swift Biosciences, these techniques were developed for microgram inputs of genomic DNA that undergo standard NGS library preparation followed by bisulfite conversion, a chemical process that converts nonmethylated cytosines to uracil. Subsequently, the polymerase chain reaction (PCR) process can be used to convert uracil to thymidine. “But the methylated cytosines are protected, thus demarcating methylation status when the DNA sequence is determined,” Dr. Kurihara observes. “The drawback is that bisulfite-induced DNA fragmentation destroys the bulk of the prepared NGS library. Hence the requirement for microgram DNA inputs.”

    To enable lower DNA inputs and improved methylome coverage and uniformity, Swift Biosciences has developed an NGS library preparation performed on bisulfite-converted DNA fragments. The underlying technology, Adaptase, is a proprietary NGS adapter attachment chemistry for single-stranded DNA.

    “By significantly improving sample recovery from bisulfite-converted DNA,” explains Dr. Kurihara, “more complete analysis of clinical samples is possible, particularly cell-free DNA from plasma that is limited to low-nanogram quantities of DNA.”

    Dr. Kurihara cites an example provided by Dennis Lo, M.D., Ph.D., professor of chemical pathology at the Chinese University of Hong Kong. Dr. Lo developed a noninvasive test for cancer by detection of genome-wide hypomethylation of cell-free DNA from patient plasma. Although this “liquid biopsy” does not uncover actionable cancer mutations, it may prove to be a sensitive blood test for early cancer detection as well as treatment monitoring.

    More recently, Dr. Lo’s group mapped the tissue of origin for cell-free plasma DNA using genome-wide bisulfite sequencing after mapping tissue-specific methylation patterns. Such noninvasive testing from blood may identify tissue- or organ-specific pathologies, including cancer, stroke, myocardial infarction, autoimmune disorders, and transplant rejection.

    “Given that advances in epigenetic technologies have enabled per-base methylation status from low DNA input clinical samples, proof of concept has been established that ‘liquid biopsy’ testing of patient blood may be a universal screen for a variety of diseases that may be pinpointed to individual organs or tissues,” Dr. Kurihara tells GEN. “Such universal testing could be particularly advantageous for early detection of cancer and other diseases where noninvasive screening has not previously been possible.”

  • NGS: An Enabling Technology

    The widespread adoption of next-generation genomic sequencing means that for the first time scientists can sequence large numbers of cancer patient genomes. Thus far, these studies have demonstrated that a large proportion of mutated cancer genes may be classified as epigenetic modifying factors.

    “Chromatin remodeling and modifying factors are involved in the regulation of gene expression,” says Ali Shilatifard, Ph.D., chairman of the department of biochemistry and molecular genetics at Northwestern University’s Feinberg School of Medicine. “The DNA methylation factors are highly mutated in most cancers characterized thus far.”

    Dr. Shilatifard provides the example of a family of mixed-lineage leukemia genes within the complexes known as COMPASS (complex proteins associated with Set1), which are highly mutated in a large number of cancers. “We’ve shown that MLL3/4, two members of the COMPASS family, modify regulatory elements known as enhancers,” notes Dr. Shilatifard. “The job of this COMPASS family member is to regulate these cis-regulatory elements during development.”

    It has been shown that MLL3/4 and another component of COMPASS, UTX, are some of the most mutated genes in cancer. “We propose that perhaps these mutations function through enhancer malfunction,” Dr. Shilatifard continues. “And enhancer malfunction through these family members could result in miscommunication of the regulatory elements and promoters and mis-regulation of the expression pattern, resulting in tissue-specific cancers. It’s now very clear that epigenetic regulation and enhancer malfunction are key events in cancer pathogenesis.”

    Dr. Shilatifard believes that over the next several years, academic labs and pharmaceutical companies will increasingly rely on agents that intervene epigenetically. For example, a recent study indicated that approximately 75% of patients with diffuse intrinsic pontine glioma (DIPG), a rare brain cancer in children, carried a single point mutation on histone H3, transforming lysine 27 into methionine. Many copies of histone H3 exist in these patients, but mutation in just one copy is sufficient to cause DIPG.

    After modeling this mutation in Drosophila, Dr. Shilatifard’s laboratory discovered that a single point mutation on one histone was associated with a global loss of histone methylation and an increase in histone acetylation.

    “Epigenetic regulators could be central for treating this disease,” comments Dr. Shilatifard. “Numerous examples in the literature suggest that inhibition of epigenetic regulators and interactors could be very important for treating cancer, and this may work for the treatment of DIPG through the inhibition of factors that bind to hyper-acetylated histones.”

 

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Metabolic Genomics and Pharmaceutics, Vol. 1 of BioMed Series D available on Amazon Kindle


Metabolic Genomics and Pharmaceutics, Vol. 1 of BioMed Series D available on Amazon Kindle

Reporter: Stephen S Williams, PhD

 

Leaders in Pharmaceutical Business Intelligence would like to announce the First volume of their BioMedical E-Book Series D:

Metabolic Genomics & Pharmaceutics, Vol. I

SACHS FLYER 2014 Metabolomics SeriesDindividualred-page2

which is now available on Amazon Kindle at

http://www.amazon.com/dp/B012BB0ZF0.

This e-Book is a comprehensive review of recent Original Research on  METABOLOMICS and related opportunities for Targeted Therapy written by Experts, Authors, Writers. This is the first volume of the Series D: e-Books on BioMedicine – Metabolomics, Immunology, Infectious Diseases.  It is written for comprehension at the third year medical student level, or as a reference for licensing board exams, but it is also written for the education of a first time baccalaureate degree reader in the biological sciences.  Hopefully, it can be read with great interest by the undergraduate student who is undecided in the choice of a career. The results of Original Research are gaining value added for the e-Reader by the Methodology of Curation. The e-Book’s articles have been published on the Open Access Online Scientific Journal, since April 2012.  All new articles on this subject, will continue to be incorporated, as published with periodical updates.

We invite e-Readers to write an Article Reviews on Amazon for this e-Book on Amazon.

All forthcoming BioMed e-Book Titles can be viewed at:

https://pharmaceuticalintelligence.com/biomed-e-books/

Leaders in Pharmaceutical Business Intelligence, launched in April 2012 an Open Access Online Scientific Journal is a scientific, medical and business multi expert authoring environment in several domains of  life sciences, pharmaceutical, healthcare & medicine industries. The venture operates as an online scientific intellectual exchange at their website http://pharmaceuticalintelligence.com and for curation and reporting on frontiers in biomedical, biological sciences, healthcare economics, pharmacology, pharmaceuticals & medicine. In addition the venture publishes a Medical E-book Series available on Amazon’s Kindle platform.

Analyzing and sharing the vast and rapidly expanding volume of scientific knowledge has never been so crucial to innovation in the medical field. WE are addressing need of overcoming this scientific information overload by:

  • delivering curation and summary interpretations of latest findings and innovations on an open-access, Web 2.0 platform with future goals of providing primarily concept-driven search in the near future
  • providing a social platform for scientists and clinicians to enter into discussion using social media
  • compiling recent discoveries and issues in yearly-updated Medical E-book Series on Amazon’s mobile Kindle platform

This curation offers better organization and visibility to the critical information useful for the next innovations in academic, clinical, and industrial research by providing these hybrid networks.

Table of Contents for Metabolic Genomics & Pharmaceutics, Vol. I

Chapter 1: Metabolic Pathways

Chapter 2: Lipid Metabolism

Chapter 3: Cell Signaling

Chapter 4: Protein Synthesis and Degradation

Chapter 5: Sub-cellular Structure

Chapter 6: Proteomics

Chapter 7: Metabolomics

Chapter 8:  Impairments in Pathological States: Endocrine Disorders; Stress

                   Hypermetabolism and Cancer

Chapter 9: Genomic Expression in Health and Disease 

 

Summary 

Epilogue

 

 

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Nonhematologic Cancer Stem Cells [11.2.3]

Writer and Curator: Larry H. Bernstein, MD, FCAP 

Nonhematologic Stem Cells

11.2.3.1 C8orf4 negatively regulates self-renewal of liver cancer stem cells via suppression of NOTCH2 signalling

Pingping Zhu, Yanying Wang, Ying Du, Lei He, Guanling Huang, et al.
Nature Communications May 2015; 6(7122). http://dx.doi.org:/10.1038/ncomms8122

Liver cancer stem cells (CSCs) harbor self-renewal and differentiation properties, accounting for chemotherapy resistance and recurrence. However, the molecular mechanisms to sustain liver CSCs remain largely unknown. In this study, based on analysis of several hepatocellular carcinoma (HCC) transcriptome datasets and our experimental data, we find that C8orf4 is weakly expressed in HCC tumors and liver CSCs. C8orf4 attenuates the self-renewal capacity of liver CSCs and tumor propagation. We show that NOTCH2 is activated in liver CSCs. C8orf4 is located in the cytoplasm of HCC tumor cells and associates with the NOTCH2 intracellular domain, which impedes the nuclear translocation of N2ICD. C8orf4 deletion causes the nuclear translocation of N2ICD that triggers the NOTCH2 signaling, which sustains the stemness of liver CSCs. Finally, NOTCH2 activation levels are consistent with clinical severity and prognosis of HCC patients. Altogether, C8orf4 negatively regulates the self-renewal of liver CSCs via suppression of NOTCH2 signaling.

Like stem cells, CSCs are characterized by self-renewal and differentiation simultaneously9. Not surprisingly, CSCs share core regulatory genes and developmental pathways with normal tissue stem cells. Accumulating evidence shows that NOTCH, Hedgehog and Wnt signaling pathways are implicated in the regulation of CSC self-renewal4. NOTCH signaling modulates many aspects of metazoan development and tissue stemness1011. NOTCH receptors contain four members (NOTCH1–4) in mammals, which are activated by engagement with various ligands. The aberrant NOTCH signaling was first reported to be involved in the tumorigenesis of human T-cell leukaemia1213. Recently, a number of studies have reported that the NOTCH signaling pathway is implicated in regulating self-renewal of breast stem cells and mammary CSCs1415. However, how the NOTCH signaling regulates the liver CSC self-renewal remains largely unknown.

C8orf4, also called thyroid cancer 1 (TC1), was originally cloned from a papillary thyroid carcinoma and its surrounding normal thyroid tissue16. C8orf4 is ubiquitously expressed across a wide range of vertebrates with the sequence conservation across species. A number of studies have reported that C8orf4 is highly expressed in several tumors and implicated in tumorigenesis171819. In addition, C8orf4 augments Wnt/β-catenin signaling in some cancer cells2021, suggesting it may be involved in the regulation of self-renewal of CSCs. However, the biological function of C8orf4 in the modulation of liver CSC self-renewal is still unknown. Here we show that C8orf4 is weakly expressed in HCC and liver CSCs. NOTCH2 signaling is highly activated in HCC tumors and liver CSCs. C8orf4 negatively regulates the self-renewal of liver CSCs via suppression of NOTCH2 signaling.

C8orf4 is weakly expressed in HCC tissues and liver CSCs

To search for driver genes in the oncogenesis of HCC, we performed genome-wide analyses using several online-available HCC transcriptome datasets by R language and Bioconductor approaches. After analysing gene expression profiles of HCC tumor and peri-tumor tissues, we identified >360 differentially expressed genes from both Park’s cohort (GSE36376; ref. 22) and Wang’s cohort (GSE14520; refs 2324). Of these changed genes, we focused on C8orf4, which was weakly expressed in HCC tumors derived from both Park’s cohort (GSE36376) and Wang’s cohort (GSE14520) (Fig. 1a). Lower expression of C8orf4 was further confirmed in HCC samples by quantitative reverse transcription–PCR (qRT–PCR) and immunoblotting (Fig. 1b,c). In this study, HCC patient samples we used included all subtypes of HCC. In addition, these observations were further validated by immunohistochemical (IHC) staining (Fig. 1d). These data indicate that C8orf4 is weakly expressed in HCC tumor tissues.

C8orf4 is weakly expressed in HCC tumours and liver CSCs

C8orf4 is weakly expressed in HCC tumours and liver CSCs

Figure 1. C8orf4 is weakly expressed in HCC tumours and liver CSCs

http://www.nature.com/ncomms/2015/150519/ncomms8122/images_article/ncomms8122-f1.jpg

(a)C8orf4 is weakly expressed in HCC patients. Using R language and Bioconductor methods, we analyzed C8orf4 expression in HCC tumor and peri-tumor tissues provided by Park’s cohort (GSE36376) and Wang’s cohort (GSE14520) datasets. (b,c) C8orf4 expression levels were verified in HCC patient samples by quantitative RT–PCR (qRT–PCR) (b) and immunoblotting (c). β-actin served as a loading control. 18S: 18S rRNA. (d) HCC samples were assayed by immunohistochemical staining. Scale bar—left: 50 μm; right: 20 μm. (eC8orf4 is weakly expressed in CD13+CD133+ cells sorted from Huh7 cells and primary HCC samples. C8orf4 messenger RNA (mRNA) was measured by qRT–PCR. Six HCC samples got similar results. (fC8orf4 is much more weakly expressed in oncospheres than non-sphere tumor cells. Non-sphere: Huh7 or HCC primary cells that failed to form spheres. (g) HCC sample tissues were co-stained with anti-C8orf4 and anti-CD13 or anti-CD133 antibodies, then counterstained with DAPI for confocal microscopy. White arrows indicate CD13+ or CD133+ cells. Scale bars: 20 μm. For a,b, data are shown as box and whisker plot. Boxes represent interquartile range (IQR); upper and lower edge corresponds to the 75th and 25th percentiles, respectively. Horizontal lines within boxes represent median levels of gene intensity. Whiskers below and above boxes extend to the 5th and 95th percentiles, respectively. For e and f, Student’s t-test was used for statistical analysis, *P<0.05;**P<0.01, data are shown as mean ± standard deviation. Data are representative of at least three independent experiments. P, peri-tumor; T, tumor.

 

Notably, C8orf4 was also weakly expressed in embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) by analysis of its expression profiles derived from online datasets (GSE14897; ref. 25 and GSE25417; ref. 26) (Supplementary Fig. 1a,b). C8orf4 was also lowly expressed in normal liver stem cells (Supplementary Fig. 1c,d), suggesting that C8orf4 may be involved in the regulation of self-renewal of liver stem cells. Thus, we propose that C8orf4 might play a role in the maintenance of liver CSCs. Since CD13 and CD133 were widely used as liver CSC surface markers, we sorted CD13+CD133+ cells from Huh7 and Hep3B HCC cell lines as well as HCC samples, serving as liver CSCs. We observed that C8orf4 was weakly expressed in liver CSCs enriched from both HCC cell lines and patient samples (Fig. 1e). Six HCC samples were analyzed for these experiments. Similar results were obtained in CD13+CD133+ cells from Hep3B cells. Furthermore, we performed sphere formation experiments using Huh7 cells and HCC primary sample cells, and detected expression levels of C8orf4. We observed that C8orf4 was dramatically reduced in the oncospheres generated by both HCC cell lines and patient samples (Fig. 1f). In addition, we noticed that C8orf4 expression was negatively correlated with liver CSC markers such as CD13 and CD133 in HCC samples (Fig. 1g), suggesting lower expression of C8orf4 in liver CSCs. Moreover, C8orf4 was mainly located in the cytoplasm of tumour cells. Altogether, C8orf4 is weakly expressed in HCC tumor tissues and liver CSCs.

C8orf4 negatively regulates self-renewal of liver CSCs

We then wanted to look at whether C8orf4 plays a critical role in the self-renewal maintenance of liver CSCs. C8orf4 was knocked out in Huh7 cells through a CRISPR/Cas9 system (Fig. 2a). TwoC8orf4-knockout (KO) cell strains were established and C8orf4 was completely deleted in these two strains. C8orf4 deletion dramatically enhanced oncosphere formation (Fig. 2b). We co-stained SOX9, a widely used progenitor marker, and Ki67, a well-known proliferation marker, in C8orf4 KO sphere cells. We found that SOX9 was strongly stained in C8orf4 KO sphere cells (Supplementary Fig. 2a). In contrast, Ki67 staining was not significantly altered in C8orf4 KO sphere cells versus WT sphere cells. We also digested sphere cells and examined the SOX9 and Ki67 expression by flow cytometry. Similar results were achieved (Supplementary Fig. 2b). Importantly, through serial passage of CSC sphere cells, similar observations were obtained in the fourth generation oncosphere assay (Supplementary Fig. 2c,d). These data suggest that C8orf4 is involved in the regulation of liver CSC self-renewal.

(not shown)

Figure 2: C8orf4 knockout enhances self-renewal of liver CSCs.

http://www.nature.com/ncomms/2015/150519/ncomms8122/images_article/ncomms8122-f2.jpg

  • C8orf4-deficient Huh7 cells were established using a CRISPR/Cas9 system. T7 endonuclease I cleavage confirmed the efficiency of sgRNA (left panel, white arrowheads), and C8orf4-knockout efficiency was confirmed by western blot (right panel). Two knockout cell lines were used.  C8KO#1:C8orf4KO#1;  C8KO#2C8orf4KO#2. (bC8orf4-deficient cells enhanced sphere formation activity. Calculated ratios are shown in the right panel. (cC8orf4-deficient or WT Huh7 cells (1 × 106) were injected into BALB/c nude mice. Tumor sizes were observed every 5 days. (dC8orf4 deficiency enhances tumor-initiating capacity. Diluted cell numbers of Huh7 cells were implanted into BALB/c nude mice for tumor initiation. Percentages of tumor-formation mice were calculated (left panel), and frequency of tumor-initiating cells was calculated using extreme limiting dilution analysis (right panel). Error bars represent the 95% confidence intervals of the estimation. (e) Expression levels of CD13 andCD133 were analyzed in C8orf4-knockout Huh7 cells. (f) C8orf4 was silenced in HCC primary cells and C8orf4 depletion enhanced sphere formation activity. Calculated ratios are shown at the right panel. Three HCC specimens obtained similar results. (g) C8orf4-overexpressing Huh7 cells were established (left panel). C8orf4-overexpressing Huh7 cells and control Huh7 cells were cultured for sphere formation. (h,i) Xenograft tumor growth (h) and frequency of tumor-initiating cells (i) for C8orf4-overexpressing Huh7 cells were analyzed as c,d. (j) C8orf4 overexpression reduces expression of CD133 and CD13 in Huh7 cells. (k) C8orf4 was transfected in HCC primary cells and cultured for sphere formation. Three HCC patient samples obtained similar results. Scale bars: b,f,g,k, 500 μm. Student’s t-test was used for statistical analysis,    *P<0.05; **P<0.01; ***P<0.001, data are shown as mean ± standard deviation. Data represent at least three independent experiments. oeC8orf4, overexpression of C8orf4; oeVec, overexpression vector.

In addition, C8orf4-deficient Huh7 cells overtly increased xenograft tumour growth (Fig. 2c). We then performed sphere formation and digested oncospheres formed by C8orf4-deficient or WT cells into single-cell suspension, then subcutaneously implanted 1 × 104, 1 × 103, 1 × 102 and 10 cells into BALB/c nude mice. Tumour formation was examined for tumour-initiating capacity at the third month. C8orf4 deficiency remarkably enhanced tumour-initiating capacity and liver CSC ratios (Fig. 2d). In addition, C8orf4 deletion significantly enhanced expression levels of the liver CSC markers such as CD13 and CD133 (Fig. 2e). We also silenced C8orf4 in HCC primary cells using a lentivirus infection system and established C8orf4-silenced cells. Two pairs of short hairpin RNA (shRNA) sequences obtained similar knockdown efficiency. C8orf4 knockdown remarkably promoted sphere formation and xenograft tumour growth (Fig. 2f and Supplementary Fig. 2e). These data indicate that C8orf4 deletion potentiates the self-renewal of liver CSCs.
We next overexpressed C8orf4 in Huh7 cells and HCC primary cells using lentivirus infection. We observed that C8orf4 overexpression in Huh7 cells remarkably reduced sphere formation and xenograft tumour growth (Fig. 2g,h). In addition, C8orf4 overexpression remarkably reduced tumour-initiating capacity and expression of liver CSC markers (Fig. 2i,j). Similar results were observed by C8orf4 overexpression in HCC primary cells (Fig. 2k). We tested three HCC samples with similar results. Overall, C8orf4 negatively regulates the maintenance of liver CSC self-renewal and tumour propagation.

C8orf4 suppresses NOTCH2 signaling in liver CSCs

To further determine the underlying mechanism of C8orf4 in the regulation of liver CSCs, we analyzed three major self-renewal signaling pathways, including Wnt/β-catenin, Hedgehog and NOTCH pathways, in C8orf4-deleted Huh7 cells and HCC primary cells. We found that only NOTCH target genes were remarkably upregulated in C8orf4-deficient cells (Fig. 3a), whereasC8orf4 deficiency did not significantly affect the Wnt/β-catenin or the Hedgehog pathway. Given that the NOTCH family receptors have four members, we wanted to determine which NOTCH member was involved in the C8orf4-mediated suppression of liver CSC stemness. We noticed that only NOTCH2 was highly expressed in both Huh7 cells and HCC samples (Fig. 3b). In addition, this result was also confirmed by analysis of NOTCH expression levels derived from Wang’s cohort (GSE14520) and Petel’s cohort (E-TABM-36; ref. 27) (Fig. 3c). Moreover, we analysed expression profiles of C8orf4 and NOTCH target genes using Park’s cohort (GSE36376) and Wurmbach’s cohort (GSE6764; ref. 28). These cohort datasets provided several Notch signaling and its target genes. HEY1NRARP and HES6 genes were highly expressed in HCC tumour tissues (GSE6764; ref. 28), which were further confirmed in HCC samples by real-time PCR (Supplementary Fig. 3a,b). Furthermore, HEY1NRARP and HES6 genes have been reported to be relatively specific NOTCH target genes. We then examined these three genes as the NOTCH2 target genes throughout this study. We found that the C8orf4 expression level was negatively correlated with the expression levels of HEY1 and HES6, suggesting that C8orf4 inhibited NOTCH signaling in HCC patients (Fig. 3d). Finally these results were further confirmed in HCC samples by qRT-PCR (Fig. 3e). To further explore the activation status of NOTCH2 signaling in liver CSCs, we examined the expression levels of NOTCH downstream target genes in oncospheres and CD13+CD133+ cells derived from both Huh7 cells and HCC cells. We observed that NOTCH target genes were highly expressed in liver CSCs (Fig. 3f,g). These observations were verified by immunoblotting (Fig. 3h). In addition, the expression levels of NRARPHES6 and HEY1 were positively related to the expression levels of EpCAM and CD133 derived from Zhang’s cohort (GSE25097; ref. 29) and Wang’s cohort (GSE14520; Supplementary Fig. 3c,d). These data suggest that the NOTCH2 signaling plays a critical role in the maintenance of self-renewal of liver CSCs.

(not shown)

Figure 3: C8orf4 suppresses NOTCH2 signaling in liver CSCs.

http://www.nature.com/ncomms/2015/150519/ncomms8122/images_article/ncomms8122-f3.jpg

(aC8orf4 deficiency or depletion activates NOTCH signaling. The indicated major stemness signalling pathways were analysed in C8orf4-knockout Huh7 cells (left panel) and C8orf4-silenced primary cells of HCC samples (right panel). (b) Four receptor members of NOTCH family were examined in both Huh7 cells (left panel) and 29 pairs of HCC samples (right panel). (cNOTCH receptors were analyzed from Wang’s cohort (left panel) and Petel’s cohort (right panel) datasets. (dHEY1 and HES6 were highly expressed in C8orf4low samples by analysis of Park’s cohort (upper panel) and Wurmbach’s cohort (lower panel). (e) Expression levels of HEY1 and HES6 along with C8orf4 were analysed in HCC samples by qRT–PCR. (f,g) Expression levels of NRARPHEY1 and HES6 in spheres generated by Huh7 cells and HCC primary cells (f) and in CD13+CD133+ cells sorted from Huh7 cells and HCC primary cells (g). Non-sphere: Huh7 cells or HCC cells that failed to form spheres. (h) HEY1, HES6 and NRARP expression in sphere and non-sphere cells was detected by immunoblotting. β-actin was used as a loading control. For c,d, data are shown as box and whisker plot. Box: interquartile range (IQR); horizontal line within box: median; whiskers: 5–95 percentile. For a,b,f,g, Student’s t-test was used for statistical analysis, *P<0.05;**P<0.01; ***P<0.001, data are shown as mean ± standard deviation. Data are representative of at least three independent experiments.

C8orf4 interacts with NOTCH2 that is critical for liver CSCs

On ligand–receptor binding, the NOTCH receptor experiences a proteolytic cleavage by metalloprotease and γ-secretase, releasing a NOTCH extracellular domain (NECD) and a NOTCH intracellular domain (NICD), respectively30. Then the active NICD undergoes nuclear translocation and activates the expression of NOTCH downstream target genes31.Then we constructed the NOTCH2 extracellular domain (N2ECD) and intracellular domain (N2ICD) and examined the interaction with C8orf4 via a yeast two-hybrid approach. Interestingly, we found that C8orf4 interacted with N2ICD, but not N2ECD (Fig. 4a). The interaction was validated by co-immunoprecipitation (Fig. 4b). Through domain mapping, the ankyrin repeat domain of NOTCH2 was essential and sufficient for its association with C8orf4 (Fig. 4c). Taken together, C8orf4 interacts with the N2ICD domain of NOTCH2.

Figure 4: C8orf4 interacts with NOTCH2 that is required for the self-renewal maintenance of liver CSCs.

C8orf4 interacts with NOTCH2 that is required for the self-renewal maintenance of liver CSCs

C8orf4 interacts with NOTCH2 that is required for the self-renewal maintenance of liver CSCs

http://www.nature.com/ncomms/2015/150519/ncomms8122/images_article/ncomms8122-f4.jpg

(a) C8orf4 interacts with N2ICD. Yeast strain AH109 was co-transfected with Gal4 DNA-binding domain (BD) fused C8orf4 and Gal4-activating domain (AD) fused N2ICD. p53 and large T antigen were used as a positive control. (b) Recombinant Flag-N2ICD and GFP–C8orf4 were incubated for co-immunoprecipitation. (c) The ankyrin repeat AR domain is essential and sufficient for the interaction of C8orf4 with N2ICD. Various N2ICD truncation constructs were co-transfected with GFP–C8orf4 for domain mapping. NLS: nuclear location signal. (d) NOTCH2 was knocked down in Huh7 cells and detected by qRT–PCR and immunoblotting. (e) NOTCH2-silenced Huh7 cells were cultured for sphere formation assays. Two pairs of shRNAs against NOTCH2 obtained similar results. (f,g) Xenograft tumor growth (f) and frequency of tumor-initiating cells (g) for NOTCH2-silenced Huh7 cells were analyzed. (h) NOTCH2 was silenced in HCC primary cells and NOTCH2 depletion declined sphere formation activity. Three HCC specimens obtained similar results. (i) Sphere formation capacity was examined in differently treated HCC primary cells. (j) HCC primary cells were treated with indicated lentivirus and implanted into BALB/c nude mice for xenograft tumor growth assays. Scale bars: e,h,i, 500 μm, Student’s t-test was used for statistical analysis, *P<0.05; **P<0.01; ***P<0.001, data are shown as mean ± standard deviation. Data are representative of at least three independent experiments. IB, immunoblotting; IP, immunoprecipitation; NS, not significant.

To further verify the role of NOTCH2 in the maintenance of liver CSC self-renewal, we knocked down NOTCH2 in Huh7 cells and established stably depleted cell lines by two pairs of NOTCH2 shRNAs (Fig. 4d). NOTCH2 knockdown dramatically reduced sphere formation (Fig. 4e), as well as attenuated xenograft tumor growth and tumor-initiating capacity (Fig. 4f,g). Similar observations were achieved in NOTCH2-depleted HCC primary cells (Fig. 4h). In addition, we found that simultaneous knockdown of NOTCH2 and overexpression of C8orf4 failed to reduce sphere formation capacity compared with individual knockdown of NOTCH2 (Fig. 4i), suggesting that NOTCH2 and C8orf4 affected sphere formation through the same pathway. Meanwhile, C8orf4 knockdown failed to rescue the sphere formation ability of NOTCH2-depleted HCC primary cells (Fig. 4i). Similar observations were obtained in Huh7 cells (Supplementary Fig. 4). Finally, NOTCH2 depletion in C8orf4-silenced Huh7 cells or HCC primary cells also abrogated the C8orf4 depletion-mediated enhancement of xenograft tumor growth (Fig. 4j), suggesting that C8orf4 acted as upstream of NOTCH2 signaling. These data suggest that C8orf4 suppresses the liver CSC stemness through inhibiting the NOTCH2 signaling pathway.

C8orf4 blocks nuclear translocation of N2ICD

As shown in Fig. 1g, C8orf4 was mainly localized in the cytoplasm in tumor cells of HCC samples. To confirm these observations, we stained C8orf4 in several HCC cell lines and noticed that C8orf4 also resided in the cytoplasm of Huh7 cells and Hep3B cells (Fig. 5a and Supplementary Fig. 5a). These results were further validated by cellular fractionation (Fig. 5b). Importantly, C8orf4 KO led to nuclear translocation of N2ICD (Fig. 5c). In addition, we also examined the intracellular location of N2ICD in Huh7 spheres. We found that C8orf4 deletion caused complete nuclear translocation of N2ICD in oncosphere cells (Fig. 5d,e), while N2ICD was mainly located in the cytoplasm of WT oncosphere cells. However, we found that C8orf4 KO did not affect subcellular localization of β-catenin (Supplementary Fig. 5b,c). Through luciferase assays, C8orf4 transfection did not significantly influence promoter transcription activity of Wnt target genes such as TCF1, LEF and SOX4 (Supplementary Fig. 5d). These data indicate that C8orf4 resides in the cytoplasm of HCC cells and inhibits nuclear translocation of N2ICD.

C8orf4 deletion causes the nuclear translocation of N2ICD

C8orf4 deletion causes the nuclear translocation of N2ICD

Figure 5: C8orf4 deletion causes the nuclear translocation of N2ICD.

http://www.nature.com/ncomms/2015/150519/ncomms8122/images_article/ncomms8122-f5.jpg

(a) C8orf4 resides in the cytoplasm of Huh7 cells. Huh7 cells were permeabilized and stained with anti-C8orf4 antibody, then counterstained with PI for confocal microscopy. (b) Cellular fractionation was performed and detected by immunoblotting. (c,d) C8orf4 knockout causes the nuclear translocation of N2ICD. C8orf4-deficient Huh7 cells (c) and sphere cells (d) were permeabilized and stained with anti-C8orf4 and anti-N2ICD antibodies, then counterstained with DAPI followed by confocal microscopy. (e) Cellular fractionation was performed in C8orf4-deficient sphere and WT sphere cells followed by immunoblotting. (f) C8orf4-deficient Huh7 cells were implanted into BALB/c nude mice. Xenograft tumors were analyzed by immunohistochemical staining. Red arrowheads denote nuclear translocation of N2ICD. (g) C8orf4-overexpressing Huh7 cells were permeabilized for immunofluorescence staining. (h) Cellular fractionation was performed in C8orf4-overexpressing Huh7 cells for immunoblotting. (i,j) C8orf4 was overexpressed in N2ICD-overexpressing Huh7 cells followed by immunofluorescence staining (i) and immunoblotting (j). (k) NOTCH target genes were measured in cells treated as in i by real-time PCR. Scale bars: a,c,d,g,i, 10 μm; f, 40 μm. Student’s t-test was used for statistical analysis, **P<0.01;***P<0.001, data are shown as mean±s.d.. Data represent at least three independent experiments.

To further determine whether C8orf4 inhibits the NOTCH2 signaling in the propagation of xenograft tumors, we examined the distribution of N2ICD and NOTCH2 target gene activation inC8orf4-deficient xenograft tumor tissues. We found that C8orf4-deficient tumors displayed much more nuclear translocation of N2ICD compared with WT tumors (Fig. 5f). Expectedly, C8orf4-deficient tumors showed elevated expression levels of NOTCH2 target genes such as HEY1, HES6 and NRARP (Supplementary Fig. 5e). Furthermore, C8orf4 overexpression blocked the nuclear translocation of N2ICD (Fig. 5g,h). Consequently, C8orf4-overexpressing tumors showed much less N2ICD nuclear translocation and reduced expression levels of NOTCH2 target genes compared with control tumors (Supplementary Fig. 5f,g). Of note, C8orf4 overexpression in N2ICD-overexpressing Huh7 cells still blocked nuclear translocation of N2ICD (Fig. 5i,j). Consequently, C8orf4 overexpression abolished the activation of Notch2 signaling (Fig. 5k). These results suggest that C8orf4 deletion causes the nuclear translocation of N2ICD leading to activation of NOTCH2 signaling.

NOTCH2 signalling is required for the stemness of liver CSCs

To further verify the role of NRARP and HEY1 in the maintenance of liver CSC self-renewal, we knocked down these two genes in Huh7 cells and established stably depleted cell lines by two pairs of shRNAs. As expected, NRARP knockdown dramatically reduced sphere formation (Fig. 6a,b). NRARP knockdown also attenuated tumor-initiating capacity and liver CSC ratios (Fig. 6c). Similar results were achieved in NRARP-silenced HCC primary cells (Fig. 6d,e). Similarly, HEY1 silencing remarkably reduced sphere formation derived from Huh7 and HCC primary cells (Fig. 6f–i), as well as declined xenograft tumor growth and tumor-initiating capacity (Supplementary Fig. 6a,b). In sum, NOTCH2 signaling is required for the maintenance of liver CSC self-renewal.

(not shown)

Figure 6: Depletion of NRARP and HEY1 impairs stemness of liver CSCs.

http://www.nature.com/ncomms/2015/150519/ncomms8122/images_article/ncomms8122-f6.jpg

(a,b) NRARP-silenced Huh7 cells were established (a) and showed reduced sphere formation capacity (b). Two pairs of shRNAs against NRARP obtained similar results. (c) NRARP-silenced Huh7 cells decline tumour-initiating capacity (left panel) and reduce liver CSC frequency (right panel). Error bars represent the 95% confidence intervals of the estimation. (d,e) NRARP was knocked down in HCC primary cells (d) and sphere formation was detected (e). Three HCC samples were tested with similar results. (f,g) HEY1-silenced Huh7 cells were established (f) and sphere formation was assayed (g). Two pairs of shRNAs against HEY1 obtained similar results. (h,i) HEY1 was knocked down in HCC primary cells (h) and HEY1 depletion impaired sphere formation capacity (i). Three HCC samples were tested with similar results. Scale bars: b,e,g,i, 500 μm. For a,b,di, Student’s t-test was used for statistical analysis, *P<0.05; **P<0.01;  ***P<0.001, data are shown as mean ± standard deviation. Data are representative of at least three independent experiments.

NOTCH2 signaling is correlated with HCC severity

As shown above, the NOTCH2 signaling was highly activated in liver CSCs and involved in the regulation of liver CSC stemness. We further examined the relationship of NOTCH2 signaling with the progression of HCC. First, we analyzed NOTCH2 activation levels in HCC tumor tissues and peri-tumor tissues derived from Park’s cohort (GSE36376). We observed that HEY1HES6 and NRARP were highly expressed in the tumor tissues of HCC patients (Fig. 7a). Consistently, high expression levels of HEY1HES6 and NRARP in HCC tumors were validated by Zhang’s cohort (GSE25097) (Fig. 7b). Importantly, high expression of these three genes was confirmed in HCC samples through quantitative RT–PCR (Fig. 7c), as well as immunoblotting (Fig. 7d). To confirm a causative link between low C8orf4 expression level and nuclear N2ICD, we examined 93 HCC samples (31 peri-tumor, 37 early stage of HCC patients and 25 advanced stage of HCC patients) with immunohistochemistry staining. We observed that nuclear staining of N2ICD appeared in ~10% tumor cells in the majority of early HCC patients we tested (Fig. 7e,f). In advanced HCC patients, nuclear staining of N2ICD in tumor cells increased to ~30% in almost all the advanced HCC patients we examined. Consequently, HEY1 staining existed in ~10% tumor cells with scattered distribution and increased to 30% tumor cells in the advanced HCC patients (Fig. 7e). Consistently, low expression of C8orf4 was well correlated with activation of NOTCH2 signaling (Fig. 7e,f).

NOTCH2 activation levels are consistent with clinical severity and prognosis of HCC patients

NOTCH2 activation levels are consistent with clinical severity and prognosis of HCC patients

Figure 7: NOTCH2 activation levels are consistent with clinical severity and prognosis of HCC patients.

http://www.nature.com/ncomms/2015/150519/ncomms8122/images_article/ncomms8122-f7.jpg

(a,b) NOTCH target genes were highly expressed in HCC tumour tissues derived from Park’s cohort (a) and Zhang’s cohort (b). (c) High expression levels of NOTCH target genes in HCC tumor tissues were verified by qRT–PCR. (d) HEY1 expression in HCC tumor tissues was detected by western blot. (e) IHC staining for N2ICD, C8orf4 and HEY1. These images represent 93 HCC samples. Scale bars, 50 μm. (f) IHC images were calculated using Image-Pro Plus 6. (g) Expression levels of NOTCH target genes were elevated in HCC tumors and advanced HCC patients derived from Wang’s cohort. (hHEY1 expression level was positively correlated with prognosis prediction of HCC patients analyzed by Petel’s cohort and Wang’s cohort. HCC samples were divided into two groups according to HEY1 expression levels followed by Kaplan–Meier survival analysis. For ac, data are shown as box and whisker plot, Box: interquartile range (IQR); horizontal line within box: median; whiskers: 5–95 percentile. For f,g, Student’s t-test was used for statistical analysis, *P<0.05; **P<0.01; ***P<0.001; data are shown as mean ± standard deviation. Experiments were repeated at least three times. aHCC, advanced HCC; CL, cirrhosis liver; eHCC, early HCC; IL, inflammatory liver; NL, normal liver; NS, not significant.

Serial passages of colonies or sphere formation in vitro, as well as transplantation of tumor cells, are frequently used to assess the long-term self-renewal capacities of CSCs32. We used HCC primary cells for serial passage growth in vitro and tested the expression levels of C8orf4HEY1 and SOX9. We found that C8orf4 expression was gradually reduced over serial passages in oncosphere cells (Supplementary Fig. 7a). Consequently, the expression of NOTCH2 targets such as HEY1 and SOX9 was gradually increased in oncosphere cells during serial passages (Supplementary Fig. 7b). In addition, N2ICD nuclear translocation appeared in oncosphere cells with high expression of HEY1 plus low expression of C8orf4 (termed as C8orf4/N2ICDnuc/HEY1+cells) (Supplementary Fig. 7c). These data suggest that the C8orf4/N2ICDnuc/HEY1+ fraction cells represent a subset of liver CSCs.

Through analyzing Wang’s cohort (GSE54238), we noticed that the NOTCH2 activation levels were positively correlated with the development and progression of HCC (Fig. 7g). By contrast, the NOTCH2 pathway was not activated in inflammation liver, cirrhosis liver and normal liver (Fig. 7f). Consistently, similar observations were achieved by analysis of Zhang’s cohort (GSE25097) (Supplementary Fig. 7d). In addition, the NOTCH2 activation levels were consistent with clinicopathological stages of HCC patients derived from Wang’s cohort (GSE14520) (Supplementary Fig. 7e). Finally, HCC patients with higher expression of HEY1 displayed worse prognosis derived from Petel’s cohort (E-TABM-36) and Wang’s cohort (GSE14520) (Fig. 7h). These two cohorts (E-TABM-36 and GSE14520) have survival information of HCC patients. Taken together, the NOTCH2 activation levels in tumor tissues are consistent with clinical severity and prognosis of HCC patients.

Discussion

CSC have been identified in many solid tumors, including breast, lung, brain, liver, colon, prostate and bladder cancers4633. CSCs have similar characteristics associated with normal tissue stem cells, including self-renewal, differentiation and the ability to form new tumors. CSCs may be responsible for cancer relapse and metastasis due to their invasive and drug-resistant capacities34. Thus, targeting CSCs may become a promising therapeutic strategy to deadly malignancies3536. However, it remains largely unknown about hepatic CSC biology. In this study, we used CD13 and CD133 to enrich CD13+CD133+
subpopulation cells as liver CSCs. Based on analysis of several online-available HCC transcriptome datasets, we found that C8orf4 is weakly expressed in HCC tumors as well as in CD13+CD133+ liver CSCs. NOTCH2 signaling is required for the maintenance of liver CSC self-renewal. C8orf4 resides in the cytoplasm of tumor cells and interacts with N2ICD, blocking the nuclear translocation of N2ICD. Lower expression of C8orf4 causes nuclear translocation of N2ICD that activates NOTCH2 signaling in liver CSCs. NOTCH2 activation levels are consistent with clinical severity and prognosis of HCC patients. Therefore, C8orf4 negatively regulates self-renewal of liver CSCs via suppression of NOTCH2 signaling.

Elucidating signaling pathways that maintains self-renewal of liver CSCs is pivotal for the understanding of hepatic CSC biology and the development of novel therapies against HCC. Several signaling pathways, such as Wnt/β-catenin, transforming growth factor-beta, AKT and STAT3 pathways, have been defined to be implicated in the regulation of liver CSCs37. Not surprisingly, some liver CSC subsets and normal tissue stem cells may share core regulatory genes and common signaling pathways. The NOTCH signaling pathway plays an important role in development via cell-fate determination, proliferation and cell survival3839. The NOTCH family receptors contain four members in mammals (NOTCH1–4), which are activated by binding to their corresponding ligands. A large body of evidence provides that NOTCH signaling is implicated in carcinogenesis40. However, the role of NOTCH signaling in liver cancer is controversial. A previous study reported that NOTCH1 signaling suppresses tumor growth of HCC41. Recently, several reports showed that NOTCH signaling enhances liver tumor initiation424344. Importantly, a recent study showed that various NOTCH receptors have differential functions in the development of liver cancer45. Here we demonstrate that NOTCH2 signaling is activated in HCC tumor tissues and liver CSCs, which is required for the maintenance of liver CSC self-renewal.

C8orf4, also known as TC1, was originally cloned from a papillary thyroid cancer16, 46. The copy number variations of C8orf4 are associated with acute myeloid leukemia and other hematological malignancies19, 47. C8orf4 has been reported to be implicated in various cancers. C8orf4 was highly expressed in thyroid cancer, gastric cancer and breast cancer16, 20, 46. C8orf4 has been reported to enhance Wnt/β-catenin signaling in cancer cells that is associated with poor prognosis20, 21. However, C8orf4 is downregulated in colon cancer48. In this study, we show that C8orf4 is weakly expressed in HCC tumor tissues and liver CSCs. Our observations were confirmed by two HCC cohort datasets. Importantly, C8orf4 negatively regulates the NOTCH2 signaling to suppress the self-renewal of liver CSCs. Therefore, C8orf4 may exert distinct functions in the regulation of various malignancies.

NOTCH receptors consist of noncovalently bound extracellular and transmembrane domains. Once binding with membrane-bound Delta or Jagged ligands, the NOTCH receptors undergoes a proteolytic step by metalloprotease and γ-secretase, generating NECD and NICD fragments11, 31. The NICD, a soluble fragment, is released in the cytoplasm on proteolysis. Then the NICD translocates to the nucleus and binds to the transcription initiation complex, leading to activation of NOTCH-associated target genes49. However, it is largely unclear how the NICD is regulated during NOTCH signaling activation. Here we show that N2ICD binds to C8orf4 in the cytoplasm of liver non-CSC tumor cells, which impedes the nuclear translocation of N2ICD. By contrast, in liver CSCs, lower expression of C8orf4 causes the nuclear translocation of N2ICD, leading to activation of NOTCH signaling.

CSCs or tumour-initiating cells, behave like tissue stem cells in that they are capable of self-renewal and of giving rise to hierarchical organization of heterogeneous cancer cells4. Thus, CSCs harbour the stem cell properties of self-renewal and differentiation. Actually, the CSC model cannot account for tumorigenesis in all tumours. CSCs could undergo genetic evolution, and the non-CSCs might switch to CSC-like cells4. These results highlight the dynamic nature of CSCs, suggesting that the clonal evolution and CSC models can act in concert for tumorigenesis. Furthermore, low C8orf4 expression in tumor cells results in overall Notch2 activation, which then may have more of a progenitor signature and be more aggressive. These cells would likely have a growth advantage in non-adherent conditions and express many of the stemness markers. The dynamic nature of CSCs or persistent NOTCH2 activation may contribute to the high number of C8orf4/N2ICDnuc/HEY1+ cells in advanced HCC tumors and correlation in the patient cohort.

A recent study showed that NOTCH2 and its ligand Jag1 are highly expressed in human HCC tumors, suggesting activation of NOTCH2 signaling in HCC45. In addition, inhibiting NOTCH2 or Jag1 dramatically reduces tumor burden and growth. However, suppression of NOTCH3 has no effect on tumor growth. Dill et al.43 reported that Notch2 is an oncogene in HCC. Notch2-driven HCC are poorly differentiated with a high expression level of the progenitor marker Sox9, indicating a critical role of Notch2 signaling in liver CSCs. Here we found that NOTCH2 and its target genes such as NRARP, HEY1 and HES6 are highly expressed in HCC samples. In addition, depletion of NRARP and HEY1 impairs the stemness maintenance of liver CSCs and tumor propagation. Moreover, the expression levels of NRARP, HEY1 and HES6 in tumors are positively correlated with clinical severity and prognosis of HCC patients. Finally, the NOTCH2 activation status is positively related to the clinicopathological stages of HCC patients. Altogether, C8orf4 and NOTCH2 signaling can be detected for the diagnosis and prognosis prediction of HCC patients, as well as used as targets for eradicating liver CSCs for future therapy.

11.2.3.2 Quantifying the Landscape for Development and Cancer from a Core Cancer Stem Cell Circuit

The authors developed a landscape and path theoretical framework to investigate the global natures and dynamics for a core cancer stem cell gene network. The landscape exhibits four basins of attraction, representing cancer stem cell, stem cell, cancer and normal cell states. They also uncovered certain key genes and regulations responsible for determining the switching between different states. [Cancer Res]

Chunhe Li and Jin Wang
Cancer Res May 13, 2015; 75(10).
http://dx.doi.org:/10.1158/0008-5472.CAN-15-0079

Cancer presents a serious threat to human health. The understanding of the cell fate determination during development and tumor genesis remains challenging in current cancer biology. It was suggested that cancer stem cell (CSC) may arise from normal stem cells, or be transformed from normal differentiated cells. This gives hints on the connection between cancer and development. However, the molecular mechanisms of these cell type transitions and the CSC formation remain elusive. We quantified landscape, dominant paths and switching rates between cell types from a core gene regulatory network for cancer and development. Stem cell, CSC, cancer, and normal cell types emerge as basins of attraction on associated landscape. The dominant paths quantify the transition processes among CSC, stem cell, normal cell and cancer cell attractors. Transition actions of the dominant paths are shown to be closely related to switching rates between cell types, but not always to the barriers in between, due to the presence of the curl flux. During the process of P53 gene activation, landscape topography changes gradually from a CSC attractor to a normal cell attractor. This confirms the roles of P53 of preventing the formation of CSC, through suppressing self-renewal and inducing differentiation. By global sensitivity analysis according to landscape topography and action, we identified key regulations determining cell type switchings and suggested testable predictions. From landscape view, the emergence of the CSCs and the associated switching to other cell types are the results of underlying interactions among cancer and developmental marker genes. This indicates that the cancer and development are intimately connected. This landscape and flux theoretical framework provides a quantitative way to understand the underlying mechanisms of CSC formation and interplay between cancer and development. Major Findings: We developed a landscape and path theoretical framework to investigate the global natures and dynamics for a core cancer stem cell gene network. Landscape exhibits four basins of attraction, representing CSC, stem cell, cancer and normal cell states. We quantified the kinetic rate and paths between different attractor states. We uncovered certain key genes and regulations responsible for determining the switching between different states.

11.2.3.3 IMP3 Promotes Stem-Like Properties in Triple-Negative Breast Cancer by Regulating SLUG

Scientists observed that insulin-like growth factor-2 mRNA binding protein 3 (IMP3) expression is significantly higher in tumor initiating than in non-tumor initiating breast cancer cells and demonstrated that IMP3 contributes to self-renewal and tumor initiation, properties associated with cancer stem cells. [Oncogene]

S Samanta, H Sun, H L Goel, B Pursell, C Chang, A Khan, et al.
Oncogene
 , (18 May 2015) |
http://dx.doi.org:/10.1038/onc.2015.164

IMP3 (insulin-like growth factor-2 mRNA binding protein 3) is an oncofetal protein whose expression is prognostic for poor outcome in several cancers. Although IMP3 is expressed preferentially in triple-negative breast cancer (TNBC), its function is poorly understood. We observed that IMP3 expression is significantly higher in tumor initiating than in non-tumor initiating breast cancer cells and we demonstrate that IMP3 contributes to self-renewal and tumor initiation, properties associated with cancer stem cells (CSCs). The mechanism by which IMP3 contributes to this phenotype involves its ability to induce the stem cell factor SOX2. IMP3 does not interact with SOX2 mRNA significantly or regulate SOX2 expression directly. We discovered that IMP3 binds avidly to SNAI2 (SLUG) mRNA and regulates its expression by binding to the 5′ UTR. This finding is significant because SLUG has been implicated in breast CSCs and TNBC. Moreover, we show that SOX2 is a transcriptional target of SLUG. These data establish a novel mechanism of breast tumor initiation involving IMP3 and they provide a rationale for its association with aggressive disease and poor outcome.

11.2.3.4 Type II Transglutaminase Stimulates Epidermal Cancer Stem Cell Epithelial-Mesenchymal Transition

Researchers investigated the role of type II transglutaminase (TG2) in regulating epithelial mesenchymal transition (EMT) in epidermal cancer stem cells. They showed that TG2 knockdown or treatment with TG2 inhibitor, resulted in a reduced EMT marker expression, and reduced cell migration and invasion. [Oncotarget]

ML Fisher, G Adhikary, W Xu, C Kerr, JW Keillor, RL Ecker
Oncotarget May 08, 2015;

Type II transglutaminase (TG2) is a multifunctional protein that has recently been implicated as having a role in ECS cell survival. In the present study we investigate the role of TG2 in regulating epithelial mesenchymal transition (EMT) in ECS cells. Our studies show that TG2 knockdown or treatment with TG2 inhibitor, results in a reduced EMT marker expression, and reduced cell migration and invasion. TG2 has several activities, but the most prominent are its transamidase and GTP binding activity. Analysis of a series of TG2 mutants reveals that TG2 GTP binding activity, but not the transamidase activity, is required for expression of EMT markers (Twist, Snail, Slug, vimentin, fibronectin, N-cadherin and HIF-1α), and increased ECS cell invasion and migration. This coupled with reduced expression of E-cadherin. Additional studies indicate that NFϰB signaling, which has been implicated as mediating TG2 impact on EMT in breast cancer cells, is not involved in TG2 regulation of EMT in skin cancer. These studies suggest that TG2 is required for maintenance of ECS cell EMT, invasion and migration, and suggests that inhibiting TG2 GTP binding/G-protein related activity may reduce skin cancer tumor survival.

Epidermal squamous cell carcinoma (SCC) is among the most common cancers and the frequency is increasing at a rapid rate [1,2]. SCC is treated by surgical excision, but the rate of recurrence approaches 10% and the recurrent tumors are aggressive and difficult to treat [2]. We propose that human epidermal cancer stem (ECS) cells survive at the site of tumor excision, that these cells give rise to tumor regrowth, and that therapies targeted to kill ECS cells constitute a viable anti-cancer strategy. An important goal in this context is identifying and inhibiting activity of key proteins that are essential for ECS cell survival. Working towards this goal, we have developed systems for propagation of human ECS cells [3]. These cells display properties of cancer stem cells including self-renew and high level expression of stem cell marker proteins [3].

In the present study we demonstrate that ECS cells express proteins characteristic of cells undergoing EMT (epithelial-mesenchymal transition). EMT is a morphogenetic process whereby epithelial cells lose epithelial properties and assume mesenchymal characteristics [4]. The epithelial cells lose cell-cell contact and polarity, and assume a mesenchymal migratory phenotype. There are three types of EMT. This first is an embryonic process, during gastrulation, when the epithelial sheet gives rise to the mesoderm [5]. The second is a growth factor and cytokine-stimulated EMT that occurs at sites of tissue injury to facilitate wound repair [6]. The third is associated with epithelial cancer cell acquisition of a mesenchymal migratory/invasive phenotype. This process mimics normal EMT, but is not as well controlled and coordinated [478]. A number of transcription factors (ZEB1, ZEB2, snail, slug, and twist) that are expressed during EMT suppress expression of epithelial makers, including E-cadherin, desmoplakin and claudins [4]. Snail proteins also activate expression of vimentin, fibronectin and metalloproteinases [4]. Snail factors are not present in normal epithelial cells, but are present in the tumor cells and are prognostic factors for poor survival [4].

An important goal is identifying factors that provide overarching control of EMT in cancer stem cells. In this context, several recent papers implicate type II transglutaminase (TG2) as a regulator of EMT [912]. TG2, the best studied transglutaminase, was isolated in 1957 from guinea pig liver extract as an enzyme involved in the covalent crosslinks proteins via formation of isopeptide bonds [13]. However, subsequent studies reveal that TG2 also serves as a scaffolding protein, regulates cell adhesion, and modulates signal transduction as a GTP binding protein that participates in G protein signaling [14]. TG2 is markedly overexpressed in cancer cells, is involved in cancer development [1518], and has been implicated in maintaining and enhancing EMT in breast and ovarian cancer [10121920]. The G protein function may have an important role in these processes [102123].

In the present manuscript we study the role of TG2 in regulating EMT in human ECS cells. Our studies show that TG2 is highly enriched in ECS cells. We further show that these cells express EMT markers and that TG2 is required to maintain EMT protein expression. TG2 knockdown, or treatment with TG2 inhibitor, reduces EMT marker expression and ECS cell survival, invasion and migration. TG2 GTP binding activity is absolutely required for maintenance of EMT protein expression and EMT-related responses. However, in contrast to breast cancer [910], we show that TG2 regulation of EMT is not mediated via NFκB signaling.

TG2 is required for expression of EMT markers

EMT is a property of tumor stem cells that confers an ability to migrate and invade surrounding tissue [2426]. We first examined whether ECS cells express EMT markers. Non-stem cancer cells and ECS cells, derived from the SCC-13 cancer cell line, were analyzed for expression of EMT markers. Fig. 1A shows that a host of EMT transcriptional regulators, including Twist, Snail and Slug, are increased in ECS cells (spheroid) as compared to non-stem cancer cells (monolayer). This is associated with increased levels of vimentin, fibronectin and N-cadherin, which are mesenchymal proteins, and reduced expression of E-cadherin, an epithelial marker. HIF-1α, an additional marker frequently associated with EMT, is also elevated. We next examined whether TG2 is required to maintain EMT marker expression. SCC-13 cell-derived ECS cells were grown in the presence of control- or TG2-siRNA, to reduce TG2, and the impact on EMT marker level was measured. Fig. 1B shows that loss of TG2 is associated with reduced expression of Twist, Snail, vimentin and HIF-1α. To further assess the role of TG2, we utilized SCC13-Control-shRNA and SCC13-TG2-shRNA2 cell lines. These lines were produced by infection of SCC-13 cells with lentiviruses encoding control- or TG2-specific shRNA. Fig. 1C shows that SCC13-TG2-shRNA2 cells express markedly reduced levels of TG2 and that this is associated with reduced expression of EMT associated transcription factors and target proteins, and increased expression of E-cadherin. To confirm this, we grew SCC13-Control-shRNA and SCC13-TG2-shRNA2 cells as monolayer cultures for immunostain detection of EMT markers. As shown in Fig. 2A, TG2 levels are reduced in TG2-shRNA expressing cells, and this is associated with the anticipated changes in epithelial and mesenchymal marker expression.

Tumor cells that express EMT markers display enhanced migration and invasion ability [2426]. We therefore examined the impact of TG2 reduction on these responses. To measure invasion, control-shRNA and TG2-shRNA cells were monitored for ability to move through matrigel. Fig. 2B shows that loss of TG2 reduces movement through matrigel by 50%. We further show that this is associated with a reduction in cell migration using a monolayer culture wound closure assay. The control cells close the wound completely within 14 h, while TG2 knockdown reduces closure rate (Fig. 2C).

TG2 inhibitor reduces EMT marker expression and EMT functional responses

NC9 is a recently developed TG2-specific inhibitor [2728]. We therefore asked whether pharmacologic inhibition of TG2 suppresses EMT. SCC-13 cells were treated with 0 or 20 μM NC9. Fig. 3A shows that NC9 treatment reduces EMT transcription factor (Twist, Snail, Slug) and EMT marker (vimentin, fibronectin, N-cadherin, HIF-1α) levels. Consistent with these changes, the level of the epithelial marker, E-cadherin, is elevated. Fig. 3B and 3C show that pharmacologic inhibition of TG2 activity also reduces EMT biological response. Invasion (Fig. 3B) and cell migration (Fig. 3C) are also reduced.

Identification of TG2 functional domain required for EMT

We next performed studies to identify the functional domains and activities required for TG2 regulation of EMT. TG2 is a multifunctional enzyme that serves as a scaffolding protein, as a transamidase, as a kinase, and as a GTP binding protein [21]. The two best studied functions are the transamidase and GTP binding/G-protein related activities [21]. Transamidase activity is observed in the presence of elevated intracellular calcium, while GTP binding-related signaling is favored by low calcium conditions (reviewed in [21]). To identify the TG2 activity required for EMT, we measured the ability of wild-type and mutant TG2 to restore EMT in SCC13-TG2-shRNA2 cells, which have reduced TG2 expression (Fig. 4A). SCC13-TG2-shRNA2 cells display reduced expression of EMT markers including Twist, Snail, Slug, vimentin, fibronectin, N-cadherin and HIF-1α, and increased expression of the epithelial maker, E-cadherin, as compared to SCC13-Control-shRNA cells. Expression of wild-type TG2, or the TG2-C277S or TG2-W241A mutants, restores marker expression in SCC13-TG2-shRNA2 cells (Fig. 4A). TG2-C277S and TG2-W241A lack transamidase activity [10,2931]. In contrast, TG2-R580A, which lacks G-protein activity [2931], and TG2-Y516F, which retains only partial G-protein activity [30], do not efficiently restore marker expression. These findings suggest that the TG2 GTP binding function is required for EMT.

We next assayed the ability of the TG2 mutants to restore EMT functional responses-invasion and migration. Fig. 4B4C shows that wild-type TG2, TG2-C277S and TG2-W241A restore the ability of SCC13-TG2-shRNA2 cells to invade matrigel, but TG2-R580A and Y516F are less active. Fig. 4D shows a similar finding for cell migration, in that the TG2-R580A and Y517F mutant are only partially able to restore SCC13-TG2-shRNA2 cell migration. These findings suggest that TG2 GTP binding/G-protein related activity is required for EMT-related migration and invasion by skin cancer cells.

Role of TG2 in regulating EMT in A431 cells

The number of available epidermis-derived squamous cell carcinoma cell lines is limited, and so we compared our findings with A431 cells. A431 cells are squamous cell carcinoma cells established from human vulvar skin. A431 cells were grown as monolayer (non-stem cancer cells) and spheroids (ECS cells) and after 10 d the cells were harvested and assayed for expression of TG2 and EMT makers. Fig. 5A shows that TG2 levels are elevated in ECS cells and that this is associated with increased levels of mesenchymal markers, including Twist, Snail, Slug, vimentin, fibronectin, N-cadherin and HIF-1α. In contrast, E-cadherin levels are reduced. We next examined the impact of TG2 knockdown on EMT marker expression. Fig. 5B shows that mesenchymal markers are globally reduced and E-cadherin level is increased. As a biological endpoint of EMT, we examine the impact of TG2 knockdown on spheroid formation and found that TG2 loss leads to reduced spheroid formation (Fig. 5C). We next examined the impact of NC9 treatment on EMT and found a reduction in EMT markers expression associated with an increase in epithelial (E-cadherin) marker level (Fig. 5D). This loss of EMT marker expression is associated with reduced matrigel invasion (Fig. 5E), reduced spheroid formation (Fig. 5F) and reduced cell migration (Fig. 5G).

Role of NFκB

Previous studies in breast [183236], ovarian cancer [123738], and epidermoid carcinoma [11] indicate that NFκB signaling mediates TG2 impact on EMT. We therefore assessed the role of NFκB in skin cancer cells. As shown in Fig. 6A, the increase in TG2 level observed in ECS cells (spheroids) is associated with reduced NFκB level. In addition, NFκB level is increased in TG2 knockdown cells (Fig. 6B). Thus, increased NFκB is not associated with increased TG2. We next assessed the impact of NFκB knockdown on TG2 control of EMT marker expression. Fig. 6C shows that TG2 is required for increased expression of EMT markers (HIF-1α, snail, twist, N-cadherin, vimentin and fibronectin) and reduced expression of the E-cadherin epithelial marker; however, knockdown of NFκB expression does not interfere with TG2 regulation of these endpoints. We next examined the effect of TG2 knockdown on NFκB and IκBα localization. The fluorescence images in Fig. 6D suggest that TG2 knockdown with TG2-siRNA does not alter the intracellular localization of NFκB or IκBα. This is confirmed by subcellular fractionation assay (Fig. 6E) which compares NFκB level in SCC13-TG2-Control and SCC13-TG2-shRNA2 (TG2 knockdown) cells. We also monitored NFκB subcellular distribution following treatment with NC9, the TG2 inhibitor. Fig. 6F shows that cytoplasmic/nuclear distribution of NFκB is not altered by NC9. Finally, we monitored the impact of TG2 expression on NFκB binding to a canonical NFκB-response element. Increased NFκB binding to the response element is a direct measure of NFκB activity [10]. Fig. 6G shows that overall binding is reduced in nuclear (N) extract prepared from ECS cells (spheroids) as compared to non-stem cancer cells (monolayer), and that NFkB binding, as indicated by gel supershift assay, is also slightly reduced in ECS cell extracts. These findings indicate that NFkB binding is slightly reduced in ECS cells, which are TG2-enriched (Fig. 1A).

We next monitored the role of NFκB on biological endpoints of EMT. Fig. 7A and 7B show that TG2 knockdown reduces migration through matrigel, but NFκB knockdown has no impact. Likewise, TG2 knockdown reduces wound closure, but NFκB knockdown does not. These findings suggest that NFκB does not mediate the pro-EMT actions of TG2 in epidermal squamous cell carcinoma.

The metastatic cascade, from primary tumor to metastasis, is a complex process involving multiple pathways and signaling cascades [3941]. Cells that complete the metastatic cascade migrate away from the primary tumor through the blood to a distant site and there form a secondary tumor. Identifying the mechanisms that allow cells to survive this journey and form secondary tumors is an important goal. The processes involved in epithelial-mesenchymal transition (EMT) are important cancer therapy targets, as EMT is associated with enhanced cancer cell migration and stem cell self-renewal. EMT regulators, including Snail, Twist, Slug, are increased in expression in EMT and control expression of genes associated with the EMT phenotype [42].

TG2 is required for EMT

We have characterized a population of ECS cells derived from epidermal squamous cell carcinoma [3]. The present studies show that these cells, which display enhanced migration and invasion, possess elevated levels of TG2. Moreover, these cells are enriched in expression of transcription factors associated with EMT (Snail, Slug, and Twist, HIF-1α) as well as mesenchymal structural proteins including vimentin, fibronectin and N-cadherin. Consistent with a shift to mesenchymal phenotype, E-cadherin, an epithelial marker, is reduced in level. Additional studies show that TG2 knockdown results in a marked reduction in EMT marker expression and that this is associated with reduced ability of the cells to migrate to close a scratch wound and reduced movement in matrigel invasion assays. We also examined the impact of treatment with a TG2 inhibitor. NC9 is an irreversible active site inhibitor of TG2, that locks the enzyme in an open conformation [284345]. NC9 treatment of ECS cells results in decreased levels of Snail, Slug and Twist. These transcription factors suppress E-cadherin expression [46] and their decline in level is associated with increased levels of E-cadherin. NC9 inhibition of TG2 also reduces expression of vimentin, fibronectin and N-cadherin, and these changes are associated with reduced cell migration and reduced invasion through matrigel.

(Figures are not shown)

We also examined the role of TG2 in A431 squamous cell carcinoma cells derived from the vulva epithelium. TG2 is elevated in A431-derived ECS cells, as are EMT markers, and knockdown of TG2, with TG2-siRNA, reduces EMT marker expression and spheroid formation. Studies with NC9 indicate that NC9 inhibits A431 spheroid formation, EMT, migration and invasion. These studies indicate that TG2 is also required for EMT and migration and invasion in A431 cells. Based on these findings we conclude that TG2 is essential for EMT, migration and invasion, and is likely to contribute to metastasis in squamous cell carcinoma.

TG2 GTP binding activity is required for EMT

TG2 is a multifunctional enzyme that can act as a transamidase, GTP binding protein, protein disulfide isomerase, protein kinase, protein scaffold, and DNA hydrolase [21294447]. The two most studied functions are the transamidase and GTP binding functions [294447]. To identify the TG2 activity responsible for induction of EMT, we studied the ability of TG2 mutants to restore EMT in SCC13-TG2-shRNA2 cells, which express low levels of TG2 and do not express elevated levels of EMT markers or display EMT-related biological responses. These studies show that wild-type TG2 restores EMT marker expression and the ability of the cells to migrate on plastic and invade matrigel. TG2 mutants that retain GTP binding activity (TG2-C277S and TG2-W241A) also restore EMT. In contrast, TG2-R580A, which lacks GTP binding function, does not restore EMT. This evidence suggests that the GTP binding function is essential for TG2 induction of the EMT phenotype in ECS cells. Recent reports suggest that the TG2 is important for maintenance of stem cell survival in breast [91017] and ovarian [123848] cancer cells. Moreover, our findings are in agreement of those of Mehta and colleagues who reported that the TG2 GTP binding function, but not the crosslinking function, is required for TG2 induction of EMT in breast cancer cells [10].

TG2, NFκB signaling and EMT

To gain further insight into the mechanism of TG2 mediated EMT, we examined the role of NFκB. NFκB has been implicated as mediating EMT in breast, ovarian, and pancreatic cancer; however, NFκB may have a unique role in epidermal squamous cell carcinoma. In keratinocytes, NFκB has been implicated in keratinocyte dysplasia and hyperproliferation [49]. However, inhibition of NFκB function has also been shown to predispose murine epidermis to cancer [50]. Here we show that TG2 levels are elevated and NFκB levels are reduced in ECS cells as compared to non-stem cancer cells, and that TG2 knockdown is associated with increased NFκB level. In addition, TG2 knockdown, or inhibition of TG2 by treatment with NC9, does not altered the nuclear/cytoplasmic distribution of NFκB. We further show that elevated levels of TG2 in spheroid culture results in a slight reduction in NFκB binding to the NFκB response element, as measured by gel mobility supershift assay. These molecular assays strongly suggest that NFκB does not mediate the action of TG2 in epidermal cancer stem cells. Moreover, knockdown of NFκB-p65 in TG2 positive cells does not result in a reduction in Snail, Slug and Twist, or mesenchymal marker proteins expression, and concurrent knockdown of TG2 and NFκB does not reduce EMT marker protein levels beyond that of TG2 knockdown alone. These findings suggest that NFκB is not an intermediary in TG2-stimulated EMT in ECS cells. This is in contrast to the required role of NFκB in mediating TG2 induction of cell survival and EMT in breast cancer cells [183233] and ovarian cancer [123738] and epidermoid carcinoma [11].

11.2.3.5 CD24+ Ovarian Cancer Cells are Enriched for Cancer Initiating Cells and Dependent on JAK2 Signaling for Growth and Metastasis

Investigators showed that CD24+ and CD133+ cells have increased tumorsphere forming capacity. CD133+ cells demonstrated a trend for increased tumor initiation while CD24+ cells vs CD24– cells, had significantly greater tumor initiation and tumor growth capacity. [Mol Cancer Ther]

D Burgos-OjedaR Wu, K McLean, Yu-Chih Chen, M Talpaz, et al.
Molec Cancer Ther May 12, 2015; 14(5)
http://dx.doi.org:/10.1158/1535-7163.MCT-14-0607

Ovarian cancer is known to be composed of distinct populations of cancer cells, some of which demonstrate increased capacity for cancer initiation and/or metastasis. The study of human cancer cell populations is difficult due to long requirements for tumor growth, inter-patient variability and the need for tumor growth in immune-deficient mice. We therefore characterized the cancer initiation capacity of distinct cancer cell populations in a transgenic murine model of ovarian cancer. In this model, conditional deletion of Apc, Pten, and Trp53 in the ovarian surface epithelium (OSE) results in the generation of high grade metastatic ovarian carcinomas. Cell lines derived from these murine tumors express numerous putative stem cell markers including CD24, CD44, CD90, CD117, CD133 and ALDH. We show that CD24+ and CD133+ cells have increased tumor sphere forming capacity. CD133+ cells demonstrated a trend for increased tumor initiation while CD24+ cells vs CD24- cells, had significantly greater tumor initiation and tumor growth capacity. No preferential tumor initiating or growth capacity was observed for CD44+, CD90+, CD117+, or ALDH+ versus their negative counterparts. We have found that CD24+ cells, compared to CD24- cells, have increased phosphorylation of STAT3 and increased expression of STAT3 target Nanog and c-myc. JAK2 inhibition of STAT3 phosphorylation preferentially induced cytotoxicity in CD24+ cells. In vivo JAK2 inhibitor therapy dramatically reduced tumor metastases, and prolonged overall survival. These findings indicate that CD24+ cells play a role in tumor migration and metastasis and support JAK2 as a therapeutic target in ovarian cancer.

11.2.3.6 EpCAM-Antibody-Labeled Noncytotoxic Polymer Vesicles for Cancer Stem Cells-Targeted Delivery of Anticancer Drug and siRNA

Researchers designed and synthesized a novel anti-epithelial cell adhesion molecule (EpCAM)-monoclonal-antibody-labeled cancer stem cells (CSCs)-targeting, noncytotoxic and pH-sensitive block copolymer vesicle as a nano-carrier of anticancer drug and siRNA. [Biomacromolecules]

Jing Chen , Qiuming Liu , Jiangang Xiao , and Jianzhong Du
Biomacromolecules May 19, 2015. (just published)
http://dx.doi.org:/10.1021/acs.biomac.5b00551

Cancer stem cells (CSCs) have the capability to initiate tumor, to sustain tumor growth, to maintain the heterogeneity of tumor, and are closely linked to the failure of chemotherapy due to their self-renewal and multilineage differentiation capability with an innate resistance to cytotoxic agents. Herein, we designed and synthesized a novel anti-EpCAM (epithelial cell adhesion molecule)-monoclonal-antibody-labeled CSCs-targeting, noncytotoxic and pH-sensitive block copolymer vesicle as a nano-carrier of anticancer drug and siRNA (to overcome CSCs drug resistance by silencing the expression of oncogenes). This vesicle shows high delivery efficacy of both anticancer drug doxorubicin hydrochloride (DOX∙HCl) and siRNA to the CSCs because it is labeled by the monoclonal antibodies to the CSCs-surface-specific marker. Compared to non-CSCs-targeting vesicles, the DOX∙HCl or siRNA loaded CSCs-targeting vesicles exhibited much better CSCs killing and tumor growth inhibition capabilities with lower toxicity to normal cells (IC50,DOX decreased by 80%), demonstrating promising potential applications in nanomedicine.

11.2.3.7 Survival of Skin Cancer Stem Cells Requires the Ezh2 Polycomb Group Protein

Investigators showed that Ezh2 is required for epidermal cancer stem (ECS) cell survival, migration, invasion and tumor formation, and that this is associated with increased histone H3 trimethylation on lysine 27, a mark of Ezh2 action. They also showed that Ezh2 knockdown or treatment with Ezh2 inhibitors, GSK126 or EPZ-6438, reduced Ezh2 level and activity, leading to reduced ECS cell spheroid formation, migration, invasion and tumor growth. [Carcinogenesis]

G Adhikary, D Grun, S Balasubramanian, C Kerr, J Huang and RL Eckert
Carcinogenesis (2015)
http://dx.doi.org:/10.1093/carcin/bgv064

Polycomb group (PcG) proteins, including Ezh2, are important candidate stem cell maintenance proteins in epidermal squamous cell carcinoma. We previously showed that epidermal cancer stem cells (ECS cells) represent a minority of cells in tumors, are highly enriched in Ezh2 and drive aggressive tumor formation. We now show that Ezh2 is required for ECS cell survival, migration, invasion and tumor formation, and that this is associated with increased histone H3 trimethylation on lysine 27, a mark of Ezh2 action. We also show that Ezh2 knockdown or treatment with Ezh2 inhibitors, GSK126 or EPZ-6438, reduces Ezh2 level and activity, leading to reduced ECS cell spheroid formation, migration, invasion and tumor growth. These studies indicate that epidermal squamous cell carcinoma cells contain a subpopulation of cancer stem (tumor-initiating) cells that are enriched in Ezh2, that Ezh2 is required for optimal ECS cell survival and tumor formation, and that treatment with Ezh2 inhibitors may be a strategy for reducing epidermal cancer stem cell survival and suppressing tumor formation.

11.2.3.8 Inhibition of STAT3, FAK and Src mediated signaling reduces cancer stem cell load, tumorigenic potential and metastasis in breast cancer

R Thakur, R Trivedi, N Rastogi, M Singh & DP Mishra
Scientific Reports May 14, 2015; 5(10194)
http://dx.doi.org:/10.1038/srep10194

Cancer stem cells (CSCs) are responsible for aggressive tumor growth, metastasis and therapy resistance. In this study, we evaluated the effects of Shikonin (Shk) on breast cancer and found its anti-CSC potential. Shk treatment decreased the expression of various epithelial to mesenchymal transition (EMT) and CSC associated markers. Kinase profiling array and western blot analysis indicated that Shk inhibits STAT3, FAK and Src activation. Inhibition of these signaling proteins using standard inhibitors revealed that STAT3 inhibition affected CSCs properties more significantly than FAK or Src inhibition. We observed a significant decrease in cell migration upon FAK and Src inhibition and decrease in invasion upon inhibition of STAT3, FAK and Src. Combined inhibition of STAT3 with Src or FAK reduced the mammosphere formation, migration and invasion more significantly than the individual inhibitions. These observations indicated that the anti-breast cancer properties of Shk are due to its potential to inhibit multiple signaling proteins. Shk also reduced the activation and expression of STAT3, FAK and Src in vivo and reduced tumorigenicity, growth and metastasis of 4T1 cells. Collectively, this study underscores the translational relevance of using a single inhibitor (Shk) for compromising multiple tumor-associated signaling pathways to check cancer metastasis and stem cell load.

Breast cancer is the most common endocrine cancer and the second leading cause of cancer-related deaths in women. In spite of the diverse therapeutic regimens available for breast cancer treatment, development of chemo-resistance and disease relapse is constantly on the rise. The most common cause of disease relapse and chemo-resistance is attributed to the presence of stem cell like cells (or CSCs) in tumor tissues12. CSCs represent a small population within the tumor mass, capable of inducing independent tumors in vivo and are hard to eradicate2. Multiple signaling pathways including Receptor Tyrosine Kinase (RTKs), Wnt/β-catenin, TGF-β, STAT3, Integrin/FAK, Notch and Hedgehog signaling pathway helps in maintaining the stem cell programs in normal as well as in cancer cells3456. These pathways also support the epithelial-mesenchymal transition (EMT) and expression of various drug transporters in cancer cells. Cells undergoing EMT are known to acquire stem cell and chemo-resistant traits7. Thus, the induction of EMT programs, drug resistance and stem cell like properties are interlinked7. Commonly used anti-cancer drugs eradicate most of the tumor cells, but CSCs due to their robust survival mechanisms remain viable and lead to disease relapse8. Studies carried out on patient derived tumor samples and in vivo mouse models have demonstrated that the CSCs metastasize very efficiently than non-CSCs91011. Therefore, drugs capable of compromising CSCs proliferation and self-renewal are urgently required as the inhibition of CSC will induce the inhibition of tumor growth, chemo-resistance, metastasis and metastatic colonization in breast cancer.

Shikonin, a natural dietary component is a potent anti-cancer compound1213. Previous studies have shown that Shk inhibits the cancer cell growth, migration, invasion and tumorigenic potential12. Shk has good bioavailability, less toxicity and favorable pharmacokinetic and pharmacodynamic profiles in vivo12. In a recent report, it was shown that the prolonged exposure of Shk to cancer cells does not cause chemo-resistance13.Other studies have shown that it inhibits the expression of various key inflammatory cytokines and associated signaling pathways1214. It decreases the expression of TNFα, IL12, IL6, IL1β, IL2, IFNγ, inhibits ERK1/2 and JNK signaling and reduces the expression of NFκB and STAT3 transcription factors1415. It inhibits proteasome and also modulates the cancer cell metabolism by inhibiting tumor specific pyurvate kinase-M214,1516. Skh causes cell cycle arrest and induces necroptosis in various cancer types14. Shk also inhibits the expression of MMP9, integrin β1 and decreases invasive potential of cancer cells1417. Collectively, Shk modulates various signaling pathways and elicits anti-cancer responses in a variety of cancer types.

In breast cancer, Shk has been reported to induce the cell death and inhibit cell migration, but the mechanisms responsible for its effect are not well studied1819. Signaling pathways modulated by Shk in cancerous and non-cancerous models have previously been shown important for breast cancer growth, metastasis and tumorigenicity20. Therefore in the current study, we investigated the effect of Shk on various hallmark associated properties of breast cancer cells, including migration, invasion, clonogenicity, cancer stem cell load and in vivo tumor growth and metastasis.

Shk inhibits cancer hallmarks in breast cancer cell lines and primary cells

We first examined the effect of Shk on various cancer hallmark capabilities (proliferation, invasion, migration, colony and mammosphere forming potential) in breast cancer cells. MTT assay was used to find out effect of Shk on viability of breast cancer cells. Semi-confluent cultures were exposed to various concentrations of Shk for 24 h. Shk showed specific anti-breast cancer activity with IC50 values ranging from 1.38 μM to 8.3 μM in MDA-MB 231, MDA-MB 468, BT-20, MCF7, T47D, SK-BR-3 and 4T1 cells (Fig. 1A). Whereas the IC50 values in non-cancerous HEK-293 and human PBMCs were significantly higher indicating that it is relatively safe for normal cells (Fig. S1A). Shk was found to induce necroptotic cell death consistent with previous reports (Fig. S1B). Treatment of breast cancer cells for 24 h with 1.25 μM, 2.5 μM and 5.0 μM of Shk significantly reduced their colony forming potential (Fig. 1B). To check the effect of Shk on the heterogeneous cancer cell population, we tested it on patient derived primary breast cancer cells. Shk reduced the viability and colony forming potential of primary breast cancer cells in dose dependent manner (Fig. 1C,D). Further we checked its effects on migration and invasion of breast cancer cells. Shk (2.5 μM) significantly inhibited the migration of MDA-MB 231, MDA-MB 468, MCF7 and 4T1 cells (Fig. 1E). It also inhibited the cell invasion in dose dependent manner (Fig. 1F and S1CS1DS1E,S1F). We further examined its effect on mammosphere formation. MDA-MB 231, MDA-MB 468, MCF7 and 4T1 cell mammosphere cultures were grown in presence or absence of 1.25 μM, 2.5 μM and 5.0 μM Shk for 24 h. After 8 days of culture, a dose dependent decrease in the mammosphere forming potential of these cells was observed (Figs. 1G,H). Collectively, these results indicated that Shk effectively inhibits the various hallmarks associated with aggressive breast cancer.

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Figure 1: Shk inhibits multiple cancer hallmarks

Shk reduces cancer stem cell load in breast cancer

As Shk exhibited strong anti-mammosphere forming potential; therefore it was further examined for its anti-cancer stem cell (CSC) properties. Cancer stem cell loads in breast cancer cells were assessed using Aldefluor assay which measures ALDH1 expression. MDA-MB 231 cells with the highest number of ALDH1+ cells were selected for further studies (Fig. S2A). We also checked the correlation between ALDH1 expression and mammosphere formation. Sorted ALDH1+ cells were subjected to mammosphere cultures. ALDH1+ cells formed highest number of mammospheres compared to ALDH1-/low and parent cell population, indicating that ALDH1+ cells are enriched in CSCs (Fig. S2B). Shk reduced the Aldefluor positive cells in MDA-MB 231 cells after 24 h of treatment (Fig. 2A,B). Next, we examined the effect of Shk on the expression of stem cell (Sox2, Oct3/4, Nanog, AldhA1 and c-Myc) and EMT (Snail, Slug, ZEB1, Twist, β-Catenin) markers, associated with the sustenance of breast CSCs. Shk (2.5 μM) treatment for 24 h reduced the expression of these markers (Fig. 2C and S2D). Shk also reduced protein expression of these markers in dose dependent manner (Fig. 2D,E and S2C).

(not shown)

Figure 2: Shk decreases stem cell load in breast cancer cells and enriched CD44+,CD24−/low breast cancer stem cells.

To further confirm anti-CSC properties of Shk, we checked the effect of shikonin on the load of CD44+ CD24− breast CSCs in MCF7 cells grown on matrigel. Shikonin reduced CD44+ CD24− cell load in dose dependent manner after 24 h of treatment (Fig S2E). We also tested its effects on the enriched CSC population. CD44+ CD24− cells were enriched from MCF7 cells using MagCellect CD24− CD44+ Breast CSC Isolation Kit (Fig. S2F). Enriched CSCs formed highest number of mammosphere in comparison to parent MCF7 cell population or negatively selected CD24+ cells (Fig. S2G). Enriched CSCs were treated with indicated doses of Shk (0.625 μM, 1.25 μM and 2.5 μM) for 24 h and were either analyzed for ALDH1 positivity or subjected to colony or mammosphere formation. 2.5 μM dose of Shk reduced ALDH1+ cells by 50% and inhibited colony and mammosphere formation (Fig. S2H2F2G and 2H). Shk also reduced the mRNA expression of CSC markers in CD44+ CD24− cells and patient derived primary cancer cells (Fig. 2I,J). These results collectively indicated that Shk inhibits CSC load and associated programs in breast cancer.

Shk is a potent inhibitor of STAT3 and poorly inhibits FAK and Src

To identify the molecular mechanism responsible for anti-cancer properties of Shk, we used a human phospho-kinase antibody array to study a subset of phosphorylation events in MDA-MB 231 cells after 6h of treatment with 2.5 μM Shk. Amongst the 46 phospho-antibodies spotted on the array, the relative extent of phosphorylation of three proteins decreased to about ≳ 2 fold (STAT3, 3.3 fold; FAK, 2.5 fold and Src, 1.8 fold) upon Shk treatment (Fig. 3A,B). These proteins (STAT3, FAK and Src) are known to regulate CSC proliferation and self renewal212223. Therefore, we focused on these proteins and the result of kinase-array was confirmed by western blotting. Shk effectively inhibits STAT3 at early time point (1 h) while activation of FAK and Src decreased on or after 3 h (Fig. 3C) confirming Shk as a potent inhibitor of STAT3. Shk also reduced the protein expression of STAT3, FAK and Src at 24 h (Fig. 3C).

(not shown)

Figure 3. Shk inhibits STAT3, FAK and Src signaling pathways.

We also observed that Shk does not inhibit JAK2 at initial time-points (Fig. 3C). This raised a possibility that Shk either regulates STAT3 independent of JAK2 or it binds directly to STAT3. To check the first probability, we activated STAT3 by treating the cells with IL6 (100 ng ml−1) for 1 h followed by treatment with Shk (2.5 μM) for 1 h. Both immunofluorescence and western-blotting results showed that Shk inhibited activated STAT3 without inhibiting JAK2 (Fig. S3AS3B) confirming that Shk inhibits JAK2 mediated activation of STAT3 possibly by binding directly to STAT3. For further confirmation, we performed an in silico molecular docking analysis to examine binding of Shk with the STAT3 SH2 domain. In a major conformational cluster, Shk occupied Lys-707, Lys-709 and Phe-710 binding sites in the STAT3 SH2 domain similar to the STAT3 standard inhibitor S3I-201 (Fig. S3C and S3D). The binding energy of Shk to STAT3 was −4.20 kcal mol−1. Collectively, these results showed that Shk potently inhibits STAT3 activation and also attenuates FAK and Src activation.

STAT3, Src and FAK are differentially expressed and activated in breast CSCs (BCSCs)

STAT3 and FAK are known to play an important role in proliferation and self-renewal of CSCs in various cancer types including breast cancer212224. Src also support CSC phenotype in some cancer types, but there are limited reports of its involvement in breast cancer25. Therefore, we checked the expression and activation of STAT3, FAK and Src in CSCs and non-CSCs. Here we used two methods to enrich the CSCs and non-CSCs. In the first method, the MDA-MB 231 cells were subjected to mammosphere formation for 96 h. After 96 h, mammosphere and non-mammosphere forming cells were clearly visible (Fig. 4A). These mammosphere and non-mammosphere forming cells were separated by using a 70 micron cell strainer. Mammospheres were subjected to two subculture cycles to enrich CSCs. With each passage, the viable single cells (non-mammosphere forming cells) and mammospheres were collected in RIPA lysis buffer and western blotting was done (Fig. 4B). We found that the activation and expression of the STAT3, FAK and Src is higher in enriched mammosphere cultures (Fig. 4C). In the second method, CD44+ CD24− cells were isolated from MCF7 cultures using MagCellect Breast CSC Isolation Kit. STAT3, FAK and Src activation and their mRNA and protein expression were assessed in enriched CSCs and were compared to parent MCF7 cell population. STAT3, FAK and Src all were differentially activated in CSCs (Fig. 4E). High mRNA as well as protein expressions of all the three genes was also observed in CSCs (Fig. 4D,E). Collectively, these results indicate that STAT3, FAK and Src are over expressed and activated in BCSCs.

Figure 4: STAT3, FAK and Src are differentially activated and expressed in breast cancer cells.

  • Representative picture indicating mammosphere and single suspended cells. (B) Schematic outline of mammosphere enrichment. (C) Protein expression and activation of STAT3, FAK and Src was determined in single suspended cells (non-mammosphere forming cells) and mammospheres by western blot. The full size blots corresponding to the cropped blot images are given in  S10. (D) Gene expression of STAT3, FAK and Src was determined in MCF7 parent population and CD44+ CD24−/low MCF7 cells using PCR. The full agarose gel images corresponding to the cropped images are given in Fig. S10. (E) Protein expression and activation of STAT3, FAK and Src was in CD44+ 24− cells and parent population.
STAT3, FAK and Src are differentially activated and expressed in breast cancer cells.

STAT3, FAK and Src are differentially activated and expressed in breast cancer cells.

http://www.nature.com/srep/2015/150514/srep10194/images_article/srep10194-f4.jpg

STAT3 is important for mammosphere formation and CSC programs in breast cancer

As our results indicated that the expression and activation of STAT3, FAK and Src is high in BCSCs and Shk is capable of inhibiting these signaling proteins; therefore to find out functional relevance of each protein and associated effects on their pharmacological inhibition by Shk, we used specific inhibitors against these three. Effect of these inhibitors was first tested on the mammosphere forming potential of MDA-MB 231, MDA-MB 468 and MCF7 cells. A drastic reduction in the mammosphere formation was observed upon STAT3 inhibition. FAK and Src inhibition also reduced the primary and secondary mammosphere formation but STAT3 inhibition showed most potent effect (Fig. 5A and S4). Further, we also checked the effect of these inhibitors on the expression of various CSC and EMT related markers in MDA-MB 231 cells. STAT3 inhibition decreased the expression of most of the CSC and EMT markers (Fig. 5B). These two findings indicated that STAT3 inhibition is more effective in reducing mammosphere forming potential and weakens major CSC programs and the anti-CSC potential of Shk is possibly due to its strong STAT3 inhibitory effect.
(not shown)

STAT3, FAK and Src activation status correlates with mammosphere forming potential in breast cancer

STAT3, FAK and Src activation status correlates with mammosphere forming potential in breast cancer

Figure 5: STAT3, FAK and Src activation status correlates with mammosphere forming potential in breast cancer.

http://www.nature.com/srep/2015/150514/srep10194/carousel/srep10194-f5.jpg

(A) Bar graph represents number of mammospheres formed from 2500 cells in presence and absence of indicated treatments. MDA-MB 231, MDA-MB 468 and MCF7 24 h mammosphere cultures were treated with Shk (2.5 μM), FAK inhibitor (FAK inhibitor 14; 2.5 μM), Src inhibitor (AZM 475271; 10 μM) and STAT3 inhibitor (WP1066; 10 μM). After 24 h, treatments were removed and cells were allowed to grow in fresh mammosphere culture media for 8 days. (B) Expression of various stem cell and EMT related transcription factors and markers were detected using western blotting in MDA-MB 231 cells with or without indicated treatments. The full size blots corresponding to the cropped blot images are given in Fig. S10. (C) MDA-MB 231, MDA-MB 468 and MCF7 cells were pre-treated with either IL6 (100 ng ml−1), Fibronectin (1 μg ml−1) or EGF (25 ng ml−1) for two population doublings and subjected to mammosphere formation. Bar graph represents average of three independent experiments. (D) MCF7 cells were pre-treated with either IL6 (100 ng ml−1), Fibronectin (1 μg ml−1) or EGF (25 ng ml−1) for two population doublings and subjected to mammosphere formation. After 24 h, cells were treated with DMSO (untreated) or Shk (treated) as indicated in the bar graph. Data are shown as the mean ±SD. (*) p < 0.05 and (**) p < 0.01.

To further check the involvement of these pathways in CSCs, we cultured MDA-MB 231, MDA-MB 468 and MCF7 cells in the presence of either IL6 (100ng ml−1), EGF (25 ng ml−1) or Fibronectin (1 μg ml−1) coated surface for two population doublings. Cells were then subjected to mammosphere formation. In IL6 pre-treated cultures, there was a sharp rise in mammosphere formation, indicating that the STAT3 activation shifts CSC and non-CSC dynamics towards CSCs (Fig. 5C). IL6 is previously known to induce the conversion of non-CSC to CSC via STAT3 activation26. In MCF7 cells, mammosphere forming potential after IL6 pre-treatment increased nearly by three fold. Therefore, we further checked the effectiveness of Shk on mammosphere forming potential in pre-treated MCF7 cells. It was found that Shk inhibits mammosphere formation most effectively in IL6 pre-treated cultures (Fig. 5D). However, in EGF and Fibronectin pre-treated cultures, Shk was relatively less effective. This was possibly due to its weak FAK and Src inhibitory potential. Collectively, these results illustrated that STAT3 activation is significantly correlated with the mammosphere forming potential of breast cancer cells and its inhibition by a standard inhibitor or Shk potently reduce the mammosphere formation.

Shk inhibit CSCs load by disrupting the STAT3-Oct3/4 axis

In breast cancer, STAT3 mediated expression of Oct3/4 is a major regulator of CSC self-renewal2627. As we observed that both Shk and STAT3 inhibitors decreased the Oct3/4 expression (Figs. 2C and 5B), we further checked the effect of STAT3 activation on ALDH1+ CSCs and Oct3/4 expression. On IL6 pre-treatment, number of ALDH1+ cells increased in all three (MDA-MB 231, MDA-MB 468 and MCF7) cancer cells (Fig. 6A). MCF7 cells showed highest increase. Therefore, to check the effect of STAT3 inhibition on CSC load, we incubated IL6 pre-treated MCF7 cells with Shk and STAT3 inhibitor for 24 h and analyzed for ALDH1 positivity. It was observed that both Shk and STAT3 inhibitor reduced the IL6 induced ALDH1 positivity from 10% to < 2% (Fig. 6B). These results suggested that Shk induced inhibition of STAT3 and decrease in BCSC load is interlinked. We further checked the effect of STAT3 activation status on Oct3/4 expression in MDA-MB 231, MDA-MB 468 and MCF7 cells. We observed that expression of Oct3/4 increases with the increase in STAT3 activation (Fig. 6C–E).

(not shown)

Figure 6: STAT3 activation status and its effect on cancer stem cell load

STAT3 transcriptional activity is important in maintaining CSC programs2829. Therefore, we also examined the effect of Shk on STAT3 promoter activity. STAT3 reporter assay was performed in presence of IL6 and Shk; it was found that Shk reduced the promoter activity of STAT3 in a dose dependent manner (Fig. S5). Collectively, these results showed that Shk mediated STAT3 inhibition are responsible for decrease in CSC load and Oct3/4 associated stem cell programs.

Shk inhibits mammosphere formation, migration and invasion through inhibition of STAT3, FAK and Src in breast cancer cells

As the earlier results (Fig. 1) showed that Shk inhibits cell migration and invasion in breast cancer cells, we further examined the effect of STAT3, FAK and Src inhibitors on cell migration and invasion in MDA-MB 231 cells. It was found that STAT3 inhibitor poorly inhibits cell migration while both Src and FAK inhibitors were effective in reducing cell migration (Fig. 7A). All the three inhibitors decreased the cell invasion and MMP9 expression significantly (Fig. 7B and S6). It was also observed that effect of all these inhibitors, except STAT3 inhibitor on mammosphere formation and FAK inhibitor on cell migration, were not comparable to that of Shk. Shk inhibited all these properties more effectively than individual inhibition of STAT3, FAK and Src. This made us to assume that the ability of Shk to inhibit multiple signaling molecules simultaneously is the reason behind its potent anti-cancer effect. To check this notion, we combined STAT3, FAK and Src inhibitors with each other and examined the effect of combinations on invasion, migration and mammosphere forming potential in MDA-MB 231 cells. We observed further decrease in cell migration and invasion on combining STAT3 and FAK, STAT3 and Src, or FAK and Src (Figs. 7A,B). Combination of FAK and Src was not very effective in inhibiting mammosphere formation in MDA-MB 231 cells and CD44+ CD24− MCF7 CSCs. However, their combination with STAT3 decreased the mammosphere forming potential equivalent to that of Shk (Fig. 7C,D). We also compared the mammosphere forming potential of Shk with Salinomycin (another anti-CSC agent) and found that at 2.5 μM dose of Shk was almost two times more potent than Salinomycin (Fig. S7). Collectively, these results indicated that Shk inhibits multiple signaling proteins (STAT3, FAK and Src) to compromise various aggressive breast cancer hallmarks.

Figure 7: Combination of FAK, Src and STAT3 inhibitors is more potent than individual inhibition against various cancer hallmarks.

combination-of-fak-src-and-stat3-inhibitors-is-more-potent-than-individual-inhibition-against-various-cancer-hallmarks

combination-of-fak-src-and-stat3-inhibitors-is-more-potent-than-individual-inhibition-against-various-cancer-hallmarks

http://www.nature.com/srep/2015/150514/srep10194/images_article/srep10194-f7.jpg

  • Cell migration and (B) cell invasion potential of MDA-MB 231 cells was assessed in the presence of Shk (2.5 μM), FAK inhibitor (FAK inhibitor 14; 2.5 μM), Src inhibitor (AZM 475271; 10 μM) and STAT3 inhibitor (WP1066; 10 μM). Various combinations of these inhibitors were also used STAT3+FAK inhibitor (WP1066; 10 μM + FAK inhibitor 14; 2.5 μM), STAT3 + Src Inhibitor (WP1066; 10 μM + AZM 475271; 10 μM) and FAK+Src Inhibitor (FAK inhibitor 14; 2.5 μM + AZM 475271; 10 μM). Cell migration and cell invasion was assessed through scratch cell migration assay and transwell invasion after 24 h of treatments. (C,D) Mammosphere forming potential of MDA-MB 231 cells and CD44+ CD24−/low enriched MCF7 cells was assessed in presence of similar combination of STAT3+FAK inhibitor (WP1066; 10 μM + FAK inhibitor 14; 2.5 μM), STAT3 + Src Inhibitor (WP1066; 10 μM+ AZM 475271; 10 μM) and FAK + Src Inhibitor (FAK inhibitor 14; 2.5 μM + AZM 475271; 10 μM). Cells were subjected to mammosphere cultures for 24 h and treated with the indicated inhibitors for next 24 h, followed by media change and growth of mammospheres were monitored for next 8 days. Data are shown as the mean ±SD. (**) p < 0.01.

Shk inhibits breast cancer growth, metastasis and decreases tumorigenicity

To explore whether Shk may have therapeutic potential for breast cancer treatment in vivo, we tested Shk against 4T1-induced breast cancer syngenic mouse model. 4T1 cells (mouse breast cancer cells) are capable of growing fast and metastasize efficiently in vivo30. Prior to the in vivo experiments, we checked the effect of Shk on ALDH1 positivity and on activation of STAT3, FAK and Src in 4T1 cells in vitro. Shk effectively decreased the ALDH1+ cells and inhibited STAT3, FAK and Src in 4T1 cells in vitro (Fig. S8A and S8B). For in vivo tumor generation, 1 × 106 cells were injected subcutaneously in the fourth nipple mammary fat pad of BALB/c mice. When the average size of tumors reached around 50 mm3, mice were divided into three groups, vehicle and two Shk treated groups each received either 2.5 mg Kg−1 or 5.0 mg Kg−1 Shk. Shk was administered via the intraperitoneal injection on every alternate day. It significantly suppressed the tumor growth in 4T1 induced syngenic mouse model (Fig. 8A). The average reduction in 4T1 tumor growth was 49.78% and 89.73% in 2.5 mg Kg−1 and 5.0 mg Kg−1 groups respectively compared with the vehicle treated group (Fig. 8A). No considerable change in body weight of the treated group animals was observed (Fig. S9A). We further examined the effect of Shk on the tumor initiating potential of breast cancer cells. 4T1 induced tumors were excised from the control and treatment groups on the second day after 4th dose of Shk was administered. Tumors were dissociated; cells were allowed to adhere and then re-injected into new animals for secondary tumor formation. Growth of secondary tumors was monitored till day 15 post-reinjection. Shk treated groups showed a marked decrease in secondary tumor formation (Fig. 8D). We also observed a drastic reduction in the number of metastatic nodules in the lungs of treatment group animals (Fig. 8F). The reduction in the metastatic load was not proportional to the decrease in tumor sizes; however within the treatment group, some animals with small tumors were carrying higher number of metastatic nodules. As FAK is an important mediator of cancer metastasis and metastatic colonization, we further examined the effects of Shk on metastatic colonization. For this, 1 × 105 4T1 cells were injected to BALB/c mice through tail vein. Animals were divided into three groups, as indicated above. Shk and vehicle were administered through intraperitoneal injections at alternate days starting from the 2nd day post tail vein injections till 33rd day. The average reduction in total number of metastatic nodules was 88.6% – 90.5% in Shk treated mice compared to vehicle control (Fig. 8F). An inset picture (Fig. 8A lower panel) represents lung morphology of vehicle control and treated groups. We further examined the activation and expression status of STAT3, FAK and Src between vehicle control and treated group tumors. There were low expression and activation of STAT3, FAK and Src in treated tumors as compared to the vehicle control (Fig. 8B,C). Similar trend was observed in ALDH1 expressions (Fig. 8B). Further, the mice tumor sections were subjected to immunohistochemistry, immunofluorescence and hematoxylin and eosin (H&E) staining to study histology and expression of key proteins being examined in this study. Fig. 8G shows representative images of H&E staining, proliferating cell nuclear antigen (PCNA), terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), STAT3 and Oct3/4 immunostaining. PCNA expression was low while TUNEL positive cells were high in tumor tissues of Shk treated groups. STAT3 and Oct3/4 expression was low in Shk treated groups. These results collectively demonstrated that Shk modulates the expression and activation of STAT3, FAK and Src in vivo and is effective in suppressing tumorigenic potential and metastasis in syngenic mouse model.

Figure 8: Shk inhibits breast cancer growth, tumorigenicity and metastasis in vivo.

Shk inhibits breast cancer growth, tumorigenicity and metastasis in vivo

Shk inhibits breast cancer growth, tumorigenicity and metastasis in vivo

http://www.nature.com/srep/2015/150514/srep10194/images_article/srep10194-f8.jpg

  • Shk inhibited 4T1 tumor growth. Bar graph represents the average tumor volumes in vehicle control and Shk treated tumor bearing mice (n = 6). (*) p < 0.05 and (**) p < 0.01. Inset picture of upper panel represents tumor sizes and lower pane represents lung morphology in vehicle control and Shk treatment groups. (B) Western blot examination of indicated proteins for their expression and activation in vehicle control and treated tumor groups. The full size blots corresponding to the cropped blot images are given in Fig. S10. (C) Gene expression of stem cell and EMT markers in tumor tissues excised from the vehicle control and Shk treated groups (n = 3). (D) Number of secondary tumors formed after injecting indicated cell dilutions from Vehicle treated and Shk treated 4T1 tumors. (E) Number of lung nodules formed in mice injected with 4T1 mouse mammary tumor cells in the mammary fat pad and administered with 2.5 mg Kg−1 Shk or vehicle control on every alternate day for 3 weeks (n = 6). (F) Number of lung nodules in mice injected with 4T1 mouse mammary tumor cells through tail vein and administered with 2.5 mg Kg−1 Shk or vehicle control on every alternate day for 3 weeks. (n = 8) (G) Representative panel of the histological H&E staining, immunofluorescence staining for the STAT3, Oct3/4, cell proliferation marker PCNA and DNA damage indicator-TUNEL staining of tumor sections from vehicle and treatment groups.

Recent studies have shown that aggressiveness, therapy resistance and disease relapse in breast cancer is attributed to a small population of CSCs involved in continuous self-renewal and differentiation through signaling pathways similar to that of the normal stem cells31. Therapeutic targeting of CSCs therefore, has profound clinical implications for cancer treatment31. Recent studies indicated that therapies / agents targeting both differentiated cancer cells and CSCs may possibly have significant therapeutic advantages32. Therefore, it is imperative to look for novel therapeutic agents with lesser side effects urgently for effective targeting of CSCs. In search of novel, nontoxic anti-CSC agents, attention has been focused on natural agents in recent times33,34. In this study, we have used a natural napthoquinone compound, Shk with established antitumorigenic, favorable pharmacokinetic and toxicity profiles and report for the first time its potent anti-CSC properties. Shk significantly inhibits breast cancer cell proliferation in vitroex vivoand in vivo. It decreases the cell migration and invasion of breast cancer cells in vivo, as well as inhibits tumorigenicity, metastasis and metastatic colonization in a syngenic mouse model of breast cancer in vivo. These finding suggest a strong potential of Shk in breast cancer therapy.

We assessed the effect of Shk on the CSC load in breast cancer cells through various functional assays (tumorsphere in vitro and syngenic mouse model of breast cancer in vivo) and quantification of specific stem cell markers. In breast cancer, CD44+ CD24− cells and ALDH1+ cells are considered to be BCSCs2125. Shk significantly decreased the mammosphere formation (Fig. 1HS1G and 2H), ALDH1+ cell and CD44+ CD24− cell loads in vitro (Fig. 2BS2E and S2H). It also reduced the expression of CSC markers (Oct3/4, Sox2, Nanog, c-Myc and Aldh1) in vivo andin vitro (Fig. 2C,DS2C and S2D). These genes are known to regulate stem cell programs and in cancer, they are established promoters and regulators of CSC phenotype353637383940. Decrease in the expression of these genes on Shk treatment indicates its potential to suppress CSC programs. Tumor initiating potential (tumorigenicity) is the bona fide measure of CSCs. Reduction in the tumorigenic potential of cells isolated form Shk treated tumors indicates in vivoanti-CSC effects of Shk.

We further demonstrated that Shk is a potent inhibitor of STAT3 and it also inhibits FAK and Src (Fig. 3A–C). Its STAT3 inhibitory property was found to be responsible for its anti-CSC effects (Figs. 6B and 7B). STAT3 and FAK inhibitors are previously known to compromise CSC growth41,42. Here, we found that pharmacological inhibition of STAT3 was more effective in compromising CSC load than FAK and Src inhibitions (Fig. 5A). STAT3 activation through IL6 increases mammosphere formation more significantly than Src and FAK activation through EGF and Fibronectin (Fig. 5C). This indicates that IL6-STAT3 axis is a key regulator of BCSC dynamics.

11.2.3.9 Ovatodiolide Sensitizes Aggressive Breast Cancer Cells to Doxorubicin Anticancer Activity, Eliminates Their Cancer Stem Cell-Like Phenotype, and Reduces Doxorubicin-Associated Toxicity

Investigators evaluated the usability of ovatodiolide (Ova) in sensitizing triple negative breast cancer (TNBC) cells to doxorubicin (Doxo), cytotoxicity, so as to reduce Doxo effective dose and consequently its adverse effects. Ova-sensitized TNBC cells also lost their cancer stem cell-like phenotype evidenced by significant dissolution and necrosis of formed mammospheres, as well as their terminal differentiation. [Cancer Lett]

11.2.3.10 Glabridin Inhibits Cancer Stem Cell-Like Properties of Human Breast Cancer Cells: An Epigenetic Regulation of miR-148a/SMAd2 Signaling

The authors report that glabridin (GLA) attenuated the cancer stem cell (CSC)-like properties through microRNA-148a (miR-148a)/transforming growth factor beta-SMAD2 signal pathway in vitro and in vivo. In MDA-MB-231 and Hs-578T breast cancer cell lines, GLA enhanced the expression of miR-148a through DNA demethylation. [Mol Carcinog]

11.2.3.11 Ginsenoside Rh2 Inhibits Cancer Stem-Like Cells in Skin Squamous Cell Carcinoma

The effects of ginsenoside Rh2 (GRh2) on Lgr5-positive cancer stem cells (CSCs) were determined by flow cytometry and by tumor sphere formation. Scientists found that GRh2 dose-dependently reduced skin squamous cell carcinoma viability, possibly through reduced the number of Lgr5-positive CSCs. [Cell Physiol Biochem]

Liu S. Chen M. Li P. Wu Y. Chang C. Qiu Y. Cao L. Liu Z. Jia C.
Cell Physiol Biochem 2015;36:499-508
http://dx.doi.org:/10.1159/000430115

Background/Aims: Treatments targeting cancer stem cells (CSCs) are most effective cancer therapy, whereas determination of CSCs is challenging. We have recently reported that Lgr5-positive cells are cancer stem cells (CSCs) in human skin squamous cell carcinoma (SCC). Ginsenoside Rh2 (GRh2) has been shown to significantly inhibit growth of some types of cancers, whereas its effects on the SCC have not been examined. Methods: Here, we transduced human SCC cells with lentivirus carrying GFP reporter under Lgr5 promoter. The transduced SCC cells were treated with different doses of GRh2, and then analyzed cell viability by CCK-8 assay and MTT assay. The effects of GRh2 on Lgr5-positive CSCs were determined by fow cytometry and by tumor sphere formation. Autophagy-associated protein and β-catenin were measured by Western blot. Expression of short hairpin small interfering RNA (shRNA) for Atg7 and β-catenin were used to inhibit autophagy and β-catenin signaling pathway, respectively, as loss-of-function experiments. Results: We found that GRh2 dose-dependently reduced SCC viability, possibly through reduced the number of Lgr5-positive CSCs. GRh2 increased autophagy and reduced β-catenin signaling in SCC cells. Inhibition of autophagy abolished the effects of GRh2 on β-catenin and cell viability, while increasing β-catenin abolished the effects of GRh2 on autophagy and cell viability. Conclusion: Taken together, our data suggest that GRh2 inhibited SCC growth, possibly through reduced the number of Lgr5-positive CSCs. This may be conducted through an interaction Carcinoma account for more than 80% of all types of cancer worldwide, and squamous cell carcinoma (SCC) is the most frequent carcinoma. Skin SCC causes a lot of mortality yearly, which requires a better understanding of the molecular carcinogesis of skin SCC for developing efficient therapy [1,2]. Ginsenoside Rh2 (GRh2) is a characterized component in red ginseng, and has proven therapeutic effects on inflammation [3] and a number of cancers [4,5,6,7,8,9,10,11,12,13,14], whereas its effects on the skin SCC have not been examined.

Cancer stem cells (CSCs) are cancer cells with great similarity to normal stem cells, e.g., the ability to give rise to various cell types in a particular cancer [15,16]. CSCs are highly tumorigenic, compared to other non-CSCs. CSCs appear to persist in tumors as a distinct population and CSCs are believed to be responsible for cancer relapse and metastasis after primary tumor resection [15,16,17,18]. Recently, the appreciation of the critical roles of CSCs in cancer therapy have been continuously increasing, although the identification of CSCs in a particular cancer is still challenging.

To date, different cell surface proteins have been used to isolate CSCs from a variety of cancers by flow cytometry. Among these markers for identification of CSCs, the most popular ones are prominin-1 (CD133), side population (SP) and increased activity of aldehyde dehydrogenase (ALDH). CD133 is originally detected in hematopoietic stem cells, endothelial progenitor cells and neuronal and glial stem cells. Later on, CD133 has been shown to be expressed in the CSCs from some tumors [19,20,21,22,23], but with exceptions [24]. SP is a sub-population of cells that efflux chemotherapy drugs, which accounts for the resistance of cancer to chemotherapy. Hoechst (HO) has been experimentally used for isolation of SP cells, while the enrichment of CSCs by SP appears to be limited [25]. Increased activity of ALDH, a detoxifying enzyme responsible for the oxidation of intracellular aldehydes [26,27], has also been used to identify CSCs, using aldefluor assay [28,29]. However, ALDH has also been detected in other cell types, which creates doubts on the purity of CSCs using ALDH method [30,31]. Moreover, all these methods appear to be lack of cancer specificity.

The Wnt target gene Lgr5 has been recently identified as a stem cell marker of the intestinal epithelium, and of the hair follicle [32,33]. Recently, we reported that Lgr5 may be a potential CSC marker for skin SCC [34]. We detected extremely high Lgr5 levels in the resected skin SCC specimen from the patients. In vitro, Lgr5-positive SCC cells grew significantly faster than Lgr5-negative cells, and the fold increase in growth of Lgr5-positive vs Lgr5-negative cells is significantly higher than SP vs non-SP, or ALDH-high vs ALDH-low, or CD133-positive vs CD133-negative cells. Elimination of Lgr5-positive SCC cells completely inhibited cancer cell growth in vitro.

Here, we transduced human SCC cells with lentivirus carrying GFP reporter under Lgr5 promoter. The transduced SCC cells were treated with different doses of GRh2, and then analyzed cell viability by CCK-8 assay and MTT assay. The effects of GRh2 on Lgr5-positive CSCs were determined by flow cytometry and by tumor sphere formation. Autophagy-associated protein and β-catenin were measured by Western blot. Expression of short hairpin small interfering RNA (shRNA) for autophagy-related protein 7 (Atg7) and β-catenin were used to inhibit autophagy and β-catenin signaling pathway, respectively, as loss-of-function experiments. Atg7 was identified based on homology to Pichia pastoris GSA7 and Saccharomyces cerevisiae APG7. In the yeast, the protein appears to be required for fusion of peroxisomal and vacuolar membranes. The protein shows homology to the ATP-binding and catalytic sites of the E1 ubiquitin activating enzymes. Atg7 is a mediator of autophagosomal biogenesis, and is a putative regulator of autophagic function [35,36,37,38]. We found that GRh2 dose-dependently reduced SCC viability, possibly through reduced the number of Lgr5-positive CSCs. GRh2 increased autophagy and reduced β-catenin signaling in SCC cells. Inhibition of autophagy abolished the effects of GRh2 on β-catenin and cell viability, while increasing β-catenin abolished the effects of GRh2 on autophagy and cell viability.

Transduction of SCC cells with GFP under Lgr5 promoter

We have recently shown that Lgr5 is CSC marker for skin SCC [34]. In order to examine the role of GRh2 on SCC cells, as well as a possible effect on CSCs, we transduced human skin SCC cells A431 [34] with a lentivirus carrying GFP reporter under Lgr5 promoter (Fig. 1A). The Lgr5-positive cells were green fluorescent in culture (Fig. 1B), and could be analyzed or isolated by flow cytometry, based on GFP (Fig. 1C).

(not shown)

Fig. 1. Transduction of SCC cells with GFP under Lgr5 promoter. (A) The structure of lentivirus carrying GFP reporter under Lgr5 promoter. (B) The pLgr5-GFP-transduced A431 cells in culture. Lgr5-positive cells were green fluorescent. Nuclear staining was done by DAPI. (C) Representative flow chart for analyzing pLgr5-GFP-transduced A431 cells by flow cytometry based on GFP. Gated cells were Lgr5-positive cells. Scar bar is 20µm.

GRh2 dose-dependently inhibits SCC cell growth

Then, we examined the effect of GRh2 on the viability of SCC cells. We gave GRh2 at different doses (0.01mg/ml, 0.1mg/ml and 1mg/ml) to the cultured pLgr5-GFP-transduced A431 cells. We found that from 0.01mg/ml to 1mg/ml, GRh2 dose-dependently deceased the cell viability in either a CCK-8 assay (Fig. 2A), or a MTT assay (Fig. 2B). Next, we questioned whether GRh2 may have a specific effect on CSCs in SCC cells. Thus, we analyzed GFP+ cells, which represent Lgr5-positive CSCs in pLgr5-GFP-transduced A431 cells after GRh2 treatment. We found that GRh2 dose-dependently deceased the percentage of GFP+ cells, by representative flow charts (Fig. 2C), and by quantification (Fig. 2D). We also examined the capability of the GRh2-treated cells in the formation of tumor sphere. We found that GRh2 dose-dependently deceased the formation of tumor sphere-like structure, by quantification (Fig. 2E), and by representative images (Fig. 2F). Together, these data suggest that GRh2 dose-dependently inhibited SCC cell growth, possibly through inhibition of CSCs.

Fig. 2. GRh2 dose-dependently inhibits SCC cell growth. We gave GRh2 at different doses (0.01mg/ml, 0.1mg/ml and 1mg/ml) to the cultured pLgr5-GFP-transduced A431 cells. (A-B) GRh2 dose-dependently deceased the cell viability in either a CCK-8 assay (A), or a MTT assay (B). (C-D) GFP+ cells after GRh2 treatment were analyzed by flow cytometry, showing that GRh2 dose-dependently deceased the percentage of GFP+ cells, by representative flow charts (C), and by quantification (D). The capability of the GRh2-treated cells to form tumor sphere-like structures was examined, shown by quantification (E), and by representative images (F). *p

http://www.karger.com/Article/ShowPic/430115?image=000430115_f02.JPG

GRh2 treatment decreases β-catenin and increases autophagy in SCC cells

We analyzed the molecular mechanisms underlying the cancer inhibitory effects of GRh2 on SCC cells. We thus examined the growth-regulatory proteins in SCC. From a variety of proteins, we found that GRh2 treatment dose-dependently decreases β-catenin, and dose-dependently upregulated autophagy-related proteins Beclin, Atg7 and increased the ratio of LC3 II to LC3 I, by quantification (Fig. 3A), and by representative Western blots (Fig.3B). Since β-catenin signaling is a strong cell-growth stimulator and autophagy can usually lead to stop of cell-growth and cell death, we feel that the alteration in these pathways may be responsible for the GRh2-mediated suppression of SCC growth.

(not shown)

Figure 3. GRh2 treatment decreases β-catenin and increases autophagy in SCC cells.

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Inhibition of autophagy abolishes the effects of GRh2 on β-catenin

In order to find out the relationship between β-catenin and autophagy in this model, we inhibited autophagy using a shRNA for Atg7, and examined its effect on the changes of β-catenin by GRh2. First, the inhibition of Atg7 in A431 cells by shAtg7 was confirmed by RT-qPCR (Fig. 4A), and by Western blot (Fig. 4B). Inhibition of Atg7 resulted in abolishment of the effects of GRh2 on other autophagy-associated proteins (Fig. 4B), and resulted in abolishment of the inhibitory effect of GRh2 on β-catenin (Fig. 4B). Moreover, the effects of GRh2 on cell viability were completely inhibited (Fig. 4C). Together, inhibition of autophagy abolishes the effects of GRh2 on β-catenin. Thus, the regulation of GRh2 on β-catenin needs autophagy-associated proteins.

Fig. 4. Inhibition of autophagy abolishes the effects of GRh2 on β-catenin.

A431 cells were transfected with shRNA for Atg7, or scrambled sequence (scr) as a control. (A) RT-qPCR for Atg7. (B) Quantification of β-catenin, Beclin, Atg7 and LC3 by Western blot. (C) Cell viability by CCK-8 assay. *p

http://www.karger.com/Article/ShowPic/430115?image=000430115_f04.JPG

Overexpression of β-catenin abolishes the effects of GRh2 on autophagy

Next, we inhibited the effects of GRh2 on β-catenin by overexpression of β-catenin in A431 cells. First, the overexpression of β-catenin in A431 cells was confirmed by RT-qPCR (Fig. 5A), and by Western blot (Fig. 5B). Overexpression of β-catenin resulted in abolishment of the effects of GRh2 on autophagy-associated proteins (Fig. 5B). Moreover, the effects of GRh2 on cell viability were completely inhibited (Fig. 5C). Together, inhibition of β-catenin signaling abolishes the effects of GRh2 on autophagy. Thus, the regulation of GRh2 on autophagy needs β-catenin signaling. This model is thus summarized in a schematic (Fig. 6), suggesting that GRh2 may target both β-catenin signaling and autophagy, which interacts with each other in the regulation of SCC cell viability and growth.

http://www.karger.com/Article/ShowPic/430115?image=000430115_f05.JPG

Fig. 5. Overexpression of β-catenin abolishes the effects of GRh2 on autophagy. A431 cells were transfected with β-catenin, or scrambled sequence (scr) as a control. (A) RT-qPCR for β-catenin. (B) Quantification of β-catenin, Beclin, Atg7 and LC3 by Western blot. (C) Cell viability by CCK-8 assay. *p

http://www.karger.com/Article/ShowPic/430115?image=000430115_f06.JPG

Fig. 6. Schematic of the model. GRh2 may target both β-catenin signaling and autophagy, which interacts with each other in the regulation of SCC cell viability and growth.

Understanding the cancer molecular biology of skin SCC and identification of an effective treatment are both critical for improving the current therapy [1]. Lgr5 has been recently identified as a novel stem cell marker of the intestinal epithelium and the hair follicle, in which Lgr5 is expressed in actively cycling cells [32,33]. Moreover, we recently showed that Lgr5-positive are CSCs in skin SCC [34]. Thus, specific targeting Lgr5-positive cells may be a promising therapy for skin SCC.

In the current study, we analyzed the effects of GRh2 on the viability of SCC. Importantly, we not only found that GRh2 dose-dependently decreases SCC cell viability, but also dose-dependently decreased the number of Lgr5-positive CSCs in SCC cells. These data suggest that the CSCs in SCC may be more susceptible for the GRh2 treatment, and the decreases in CSCs may result in the decreased viability in total SCC cells. This point was supported by following mechanism studies. Activated β-catenin signaling by WNT/GSK3β prevents degradation of β-catenin and induces its nuclear translocation [39]. Nuclear β-catenin thus activates c-myc, cyclinD1 and c-jun to promote cell proliferation, and activates Bcl-2 to inhibit apoptosis [39]. High β-catenin levels thus are a signature of CSCs. Therefore, it is not surprising that CSCs are more affected than other cells when GRh2 targets β-catenin signaling.

In addition, GRh2 appears to target autophagy. Although altered metabolism may be beneficial to the cancer cells, it can create an increased demand for nutrients to support cell growth and proliferation, which creates metabolic stress and subsequently induces autophagy, a catabolic process leading to degradation of cellular components through the lysosomal system [40]. Cancer cells use autophagy as a survival strategy to provide essential biomolecules that are required for cell viability under metabolic stress [40]. However, autophagy not only results in a staring in cell growth, but also may result in cell death [40]. Increases in autophagy may substantially decrease cancer cell growth. Thus, GRh2 has its inhibitory effect on skin SCC cells through a combined effect on cell proliferation (by decreasing β-catenin) and autophagy [40].

Interestingly, our data suggest an interaction between β-catenin and autophagy. This finding is consistent with previous reports showing that autophagy negatively modulates Wnt/β-catenin signaling by promoting Dvl instability [41,42], and with other studies showing that β-catenin regulates autophagy [38,43,44].

Of note, we have checked other SCC lines and essentially got same results. Together with our previous reports showing that Lgr5-positive cells are CSCs in skin SCC [34], these findings thus highlight a future engagement of Lgr5-directed GRh2 therapy, which could be performed in a sufficiently frequent manner, to substantially improve the current treatment for skin SCC.

Normal vs Cancer Thyroid Stem Cells: The Road to Transformation
The authors discuss new insights into thyroid stem cells as a potential source of cancer formation in light of the available information on the oncogenic role of genetic modifications that occur during thyroid cancer development. Understanding the fine mechanisms that regulate tumor transformation may provide new ground for clinical intervention in terms of prevention, diagnosis and therapy. [Oncogene] Abstract
Cancer Stem Cells: A Potential Target for Cancer Therapy
The identification of cancer stem cells (CSCs) and a better understanding of the complex characteristics of CSCs will provide invaluable diagnostic, therapeutic and prognostic targets for clinical application. The authors introduce the dysregulated properties of CSCs in cancers and discuss the possible challenges in targeting CSCs for cancer treatment. [Cell Mol Life Sci] Abstract
Targeting Cancer Stem Cells Using Immunologic Approaches
Wicha, M; Chang, A; Yingxin, X; Xiaolian, Z; Ning, N; Liu, Shuang, Q, L; Pan, Q
Stem Cells 2015-04-15 4.15 | Apr 22
Targeting Notch, Hedgehog, and Wnt Pathways in Cancer Stem Cells: Clinical Update
Ivy, P; Takebe, N
Nat Rev Clin Oncol 2015-04-07 4.14 | Apr 15
Hypoxia-Inducible Factors in Cancer Stem Cells and Inflammation
Liu, Y; Peng, G
Trends Pharmacol Sci 2015-04-06 4.14 | Apr 15
NANOG in Cancer Stem Cells and Tumor Development: An Update and Outstanding Questions
Tang, D; Chao, HP; Wang, J; Yang, Tao; Jeter, C
Stem Cells 2015-03-26 4.12 | Apr 1

Two Genes Control Breast Cancer Stem Cell Proliferation and Tumor Properties

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Cancer Biomarkers [11.3.2.3]

Writer and Curator: Larry H. Bernstein, MD, FCAP

Cancer Biomarkers

This discussion is extracted from the Special Section – Cancer Biomarker Update – in May Arch Pathology and Lab Med.  It is not intended to be complete, but it has quite timely content.  There are three articles I shall cover.

11.3.2.3 Cancer Biomarkers

11.3.2.3.1 Cancer Biomarkers in Structured Data Reporting

11.3.2.3.2 Cancer Biomarkers in Myeloid Malignancies

11.3.2.3.3 National Comprehensive Cancer Network Consensus on Use of Cancer Biomarkers

Cancer Biomarkers – The Role of Structured Data Reporting
Simpson,  RW; Berman, MA; Foulis PR, et al.
Arch Pathol Lab Med. 2015;139:587–593
http://dx.doi.org:/10.5858/arpa.2014-0082-RA

The College of American Pathologists has been producing cancer protocols since 1986 to aid pathologists in the diagnosis and reporting of cancer cases. Many pathologists use the included cancer case summaries as templates for dictation/data entry into the final pathology report. These summaries are now available in a computer-readable format with structured data elements for interoperability, packaged as ‘‘electronic cancer checklists.’’ Most major vendors of anatomic pathology reporting software support this model. Objectives.—To outline the development and advantages of structured electronic cancer reporting using the electronic cancer checklist model, and to describe its extension to cancer biomarkers and other aspects of cancer reporting. Data Sources.—Peer-reviewed literature and internal records of the College of American Pathologists. Conclusions.—Accurate and usable cancer biomarker data reporting will increasingly depend on initial capture of this information as structured data. This process will support the standardization of data elements and biomarker terminology, enabling the meaningful use of these datasets by pathologists, clinicians, tumor registries, and patients.

Narrative Versus Structured Data Reporting Clinical laboratory reports typically consist of discrete data elements with structured qualitative or quantitative information, often using standardized laboratory methods, data elements, and units. When discrete data elements are electronically transmitted to external clinical information systems, the transmitted information may be annotated with one or more terminologies such as Systematized Nomenclature of Medicine Clinical Terms (SNOMED CT)4 and Logical Observation Identifiers Names and Codes (LOINC),5 although the consistent application of such codes to structured laboratory data is not yet an interoperable standard. Because the structure of clinical laboratory data tends to be fixed and standardized before the point of data entry, reporting these data elements in a tabular synoptic format is a relatively simple process. The report output may not include all data collected (eg, methodologic details), but clinically relevant data can be easily extracted by computer algorithm and automatically reported in easily readable format (including custom text, result explanations, and test value trends).

Anatomic pathology reports, by contrast, have traditionally been narrative and recorded as unstructured or partially structured fields of text. Unfortunately, narrative reporting often lacks consistency in organization, content, units, terminology, and completeness.6–8 These structural inconsistencies create difficulties in finding and understanding clinically important data and increase the chance of omitting key data elements and misinterpreting information present in the narrative. This is particularly problematic when clinicians encounter reports from multiple pathology laboratories or when patients receive care at multiple institutions. Narrative reporting has equally negative effects on computer readability; the ability of computers to correctly parse and classify information contained in a narrative report is imperfect even when using advanced natural language processing software designed specifically for anatomic pathology.9,10 Natural language processing–parsed text must always undergo human review, editing, and signoff before release for patient care or research.

Ensuring consistency and readability of cancer and biomarker reports requires a reporting solution integrated into the pathologist workflow that supports entry of standardized data directly into a laboratory information system and/or electronic health record system. These systems can produce highly readable synoptic reports and can include computer-based report validation of standardized data elements to reduce or eliminate the chance of omitting required data elements. With the transition from narrative to synoptic reporting for cancer cases, many laboratories have been using modified CCPs or locally developed templates or macros, which may or may not contain all required data elements. This common mode of data entry fits well into the pathologist workflow and can result in organized, highly readable synoptic reports, but generally results in information stored as text in a single large data field. Even when results are entered as discrete data elements, subsequent storage mechanisms usually result in nonstructured text or nonstandard custom data fields in a local computer system. Unfortunately, narrative and nonstandardized data sets are very difficult to reliably aggregate and analyze for laboratory quality assurance, research, or cancer registry surveillance. Such aggregated data also remain relatively unreliable because of changes in information systems. Many of these issues can be eliminated by entering and reporting structured data with standardized electronic templates.

CAP eCC History, Development, and Adoption Efforts to bring structure to cancer pathology reporting began in the late 1980s and early 1990s1,11,12 with publication of templates that were the precursors of the current CCPs (Table). The primary goal was to improve the care of cancer patients by improving the reporting rate of clinically important data elements. The checklist approach was adopted to help standardize terminology and ensure all relevant data elements are reported. The 66 current CCPs, 3 new cancer biomarker templates,13–16 and 85 eCC templates represent the evolution of the original 1986 CCP model. The CCPs and templates are created and maintained through the ongoing work of the CAP Cancer and Cancer Biomarker Reporting Committees. The CCPs are widely adopted by laboratories and used for accreditation purposes by the American College of Surgeons–Commission on Cancer17 and CAP Laboratory Accreditation Program.18

During the past several years, the CAP has worked to create standardized pathology reporting templates that enable individual pathologists and software vendors to capture, store, retrieve, transmit, and analyze diagnostic cancer pathology information. Electronic versions of these templates, the eCCs, are based on consistent structured data representation, which enables simple yet robust computerization of cancer pathology data elements suitable for patient care, cancer registry transmission, and research. Synoptic reports are not the same as ‘‘structured data.’’ Although synoptic reports are formatted for optimum human readability and understanding, they consist of textbased questions and answers (ideally one pair per line) that present problems for computer readability and interoperability. Structured data, by contrast, refers to representation of data elements in a computer-readable data exchange format such as XML. The structured data model used by eCC XML templates assigns a unique identifier (a composite key) to every question and answer choice, template, section, and note listed in the template. Composite keys are used throughout the entire eCC life cycle to transmit the precise identity of each data element and its origin in a specific version of an eCC template.

The CAP eCC model has been implemented province wide by Cancer Care Ontario through their multiyear synoptic pathology reporting and change-management project.19–21 The Canadian Partnership Against Cancer is also currently working with several other Canadian provinces to implement population-level electronic synoptic reporting based on the CAP eCC. The Cancer Care Ontario project has shown that there is high acceptability among pathologists and clinicians22 and that data are usable for the secondary needs of tumor registries,20 but there remains room for improvement. For instance, both the Reporting Pathology Protocols project reports23–25 and the Cancer Care Ontario implementation reports22 suggest that pathologists require more time to complete the reporting task.

While this may meet quality and data reporting needs, it remains a potential barrier to acceptance. Automated human-readable report generation also could be improved, especially in terms of creating best practice guidelines for report output. Both data entry and report generation have traditionally been supported by laboratory information systems vendors, but the success of implementation has varied, and often significant effort is required to modify the resultant human readable report to satisfy local clinical needs. Because the final report remains most important for patient care, the CAP Diagnostic Intelligence in Health Information Technology and Pathology Electronic Reporting committees have initiated work on creating and promoting a standardized data structure within cancer pathology reports.

Abbreviated CAP eCC History and Milestones

2010–2012 CCO successfully implements population level electronic synoptic reporting in nearly all disease sites based on 2010 CAP eCC standards, which include AJCC 7th edition TNM staging; 97% of labs report using structured data from eCC.20

2010 CAP Laboratory Accreditation Program begins to survey institutions for inclusion of required CCP data elements in AP reports.38

2010 NAACCR Pathology Data Workgroup develops implementation guide to assist with CAP eCC-based transmissions of cancer data to central cancer registries.39

2011 CCO user acceptability data demonstrate high level of acceptance for eCC-derived synoptic reports among clinicians and pathologists.22

2012 CAP forms the multi-organizational Cancer Biomarker Reporting Workgroup, tasked to produce standardized reporting templates for breast, colorectal, and lung cancer biomarkers.40

2013 eCC-based reporting in Ontario is used to improve quality and practice performance.20

2013 The first cancer biomarker templates are produced for breast, colorectal, and lung cancer.13–16 They are available on the http://www.cap.org/cancerprotocols Web site in Word and PDF format (accessed April 28, 2014). The eCC versions are available through CAP.

2013 Launch of CAP eFRM, a software product to aid vendor integration of eCCs into AP-LIS systems or for use as a standalone product.

2014 By December 2013, CAP is maintaining current versions of 66 CCPs, 3 cancer biomarker templates, and 85 corresponding eCC templates.

References

13. Cagle PT, Sholl LM, Lindeman NI, et al. Template for reporting results of biomarker testing of specimens from patients with non–small cell carcinoma of the lung. Arch Pathol Lab Med. 2014; 138(2):171–174. http://dx.doi.org:/10.5858/arpa.2013-0232-CP

14. Cagle PT, Allen TC, Olsen RJ. Lung cancer biomarkers: present status and future developments. Arch Pathol Lab Med. 2013; 137(9):1191–1198.
http://dx.doi.org:/10.5858/arpa.2013-0319-CR

15. Bartley AN, Hamilton SR, Alsabeh R, et al. Template for reporting results of biomarker testing of specimens from patients with carcinoma of the colon and rectum. Arch Pathol Lab Med. 2014; 138(2):166–170.
http://dx.doi.org:/10.5858/arpa.2013-0231-CP

16. Fitzgibbons PL, Dillon DA, Alsabeh R, et al. Template for reporting results of biomarker testing of specimens from patients with carcinoma of the breast. Arch Pathol Lab Med. 2014; 138(5):595–601.
http://dx.doi.org:/10.5858/arpa.2013-0566-CP

20. Srigley J, Lankshear S, Brierley J, et al. Closing the quality loop: facilitating improvement in oncology practice through timely access to clinical performance indicators. J Oncol Pract. 2013; 9(5):e255–e261. http://dx.doi.org:/10.1200/JOP.2012.000818

22. Lankshear S, Srigley J, McGowan T, Yurcan M, Sawka C. Standardized synoptic cancer pathology reports—so what and who cares?: a population-based satisfaction survey of 970 pathologists, surgeons, and oncologists. Arch Pathol Lab Med. 2013; 137(11):1599–1602.
http://dx.doi.org:/10.5858/arpa.2012-0656-OA

38. College of American Pathologists. CAP cancer protocols frequently asked questions. http://www.cap.org/apps//cap.portal
39. Klein WT, Havener LA, eds. Standards for Cancer Registries Volume V: Pathology Laboratory Electronic Reporting, Version 4.0. Springfield, IL: North American Association of Central Cancer Registries; 2011:1–310.
40. Fitzgibbons PL, Lazar AJ, Spencer S. Introducing new College of American Pathologists reporting templates for cancer biomarkers. Arch Pathol Lab Med. 2014; 138(2):157–158. http://dx.doi.org:/10.5858/arpa.2013-0233-ED

41. Amin MB. The 2009 version of the cancer protocols of the College of American Pathologists. Arch Pathol Lab Med. 2010; 134(3):326–330.
http://dx.doi.org:/10. 1043/1543-2165-134.3.326.

Figure 1. (not shown) Narrative versus synoptic versus structured reporting of breast biomarker testing (excerpts). The narrative row shows a portion of a dictated biomarker report. The synoptic row satisfies the College of American Pathologists (CAP) synoptic reporting requirements, but is not computer readable.

 

 

Figure 2. (not shown) The College of American Pathologists (CAP) Cancer Protocols (CCPs) are developed by the CAP Cancer Committee. Each CCP is reformulated as question/answer structures, entered into the CAP electronic Cancer Checklist (eCC) Template Editor (not shown), and stored in the eCC template database. The eCC files in XML format are produced from this database and delivered to vendors of anatomic pathology/laboratory information system (LIS) software systems. Vendors convert the eCC files into data entry form implementations using their local technologies. In addition, most vendors create eCC-based templates for creating synoptic reports. When pathologists enter data into the eCC-based data-entry forms, the vendor software is able to run validation checks such as assessing whether all CCP-required data elements are recorded. Synoptic reports are developed from the eCC-derived data and delivered to health care providers for patient care. The eCC-structured data is stored in the vendor database, where it can be transmitted in interoperable format to other computer systems. Secondary uses of eCC-based data include cancer registry reporting, quality assurance, biospecimen annotation, research, decision support, and financial reporting. The horizontal arrows involve the exchange of eCC composite keys, preserving the fidelity of the data as part of an eCC template, providing the foundation for interoperable data transmission formats, and enabling the regeneration of eCC datasets in the exact format in which they were recorded. Activity columns that directly impact health care activities are shaded in light blue. Abbreviation: EHR, electronic health record.

Future The use of standardized, structured data elements is foundational for the development of improved reporting and clinical decision support for biomarker results. Clinicians are currently faced with synthesizing data from multiple narrative reports to decide on treatment options. Often these narrative reports are from different laboratories with very different report formats and include variable methodologic details, all of which hinders understanding of important results. For biomarkers that determine a patient’s eligibility for specific drugs, a computer-generated report that presents test results in a tabular form, similar to antibiotic susceptibility testing, may be desirable. This reporting method would allow for display of biomarker test results over time and could also link to other databases.

Structured data allows for clinical decision support such that the report displays only eligible drugs, or the report displays a note stating that a test result suggests a patient is not eligible for a specific drug. Using standardized terminology allows these rules to be the same between institutions, even if electronic health record system vendors use different means of implementation.

Figure 3. (not shown) College of American Pathologists electronic Cancer Checklist lung cancer biomarker template—anaplastic lymphoma kinase (ALK). Abbreviations: EML4, echinoderm microtubule-associated protein-like 4; KIF5B, kinesin family member 5B; KLC1, kinesin light chain 1; TFG, tropomyosin receptor kinase–fused gene.

Figure 4. (not shown) Examples of tumor biomarker dashboards. Abbreviations: ALK, anaplastic lymphoma kinase; ROS1, ROS proto-oncogene 1, receptor tyrosine kinase.

This system would allow for more efficient, more accurate, and safer methods of providing data for optimizing patient care, with all of the discrete data transmitted electronically and linked to the original tumor report. In Ontario, Canada, this vision is rapidly advancing, as demonstrated by the Cancer Care Ontario successes with eCC implementation and current plans to implement the eCC biomarker templates across the province. Future challenges include the identity and tracking of related tumor samples over time and integration of testing from different laboratories. Because testing on a given specimen can be performed at different times and in different laboratories, a future standard must address the annotation of results with tumor source, procurement dates, and other biospecimen-specific data.30 The relationship of test results from multiple specimens from the same patient needs to be recorded in a standard format so that this parent-child hierarchical relationship can be analyzed over time.

Pathologists are increasingly asked to provide biomarker information for patient care, tumor registries, epidemiologic studies, translational research, and quality improvement activities.20 The eCC model provides a pathway to meet these demands, with efficient and error-free data entry, reporting, and transmission of data elements, and with the ability to produce output that is human readable, efficient to use, and easy to interpret. As the CCPs and eCCs have matured, Ontario pathologists and cancer registries have demonstrated success with large-scale implementations. However, continued improvements are needed. As the field of pathology grows, particularly in the area of biomarkers, structured electronic reporting will become critical to helping physicians provide optimal patient care and will facilitate secondary uses of pathology data.

  1. Robb JA, Gulley ML, Fitzgibbons PL, et al. A call to standardize preanalytic data elements for biospecimens. Arch Pathol Lab Med. 2014; 138(4):526–537.

Molecular Genetic Biomarkers in Myeloid Malignancies
Matynia AP, Szankasi P, Shen W, Kelley TW.
Arch Pathol Lab Med. 2015;139:594–601
http://dx.doi.org:/10.5858/arpa.2014-0096-RA

Recent studies using massively parallel sequencing technologies, so-called next-generation sequencing, have uncovered numerous recurrent, single-gene variants or mutations across the spectrum of myeloid malignancies. Objectives.—To review the recent advances in the understanding of the molecular basis of myeloid neoplasms, including their significance for diagnostic and prognostic purposes and the possible implications for the development of novel therapeutic strategies. Data Sources.—Literature review. Conclusions.—The recurrent mutations found in myeloid malignancies fall into distinct functional categories.

These include (1) cell signaling factors, (2) transcription factors, (3) regulators of the cell cycle, (4) regulators of DNA methylation, (5) regulators of histone modification, (6) RNA-splicing factors, and (7) components of the cohesin complex. As the clinical significance of these mutations and mutation combinations is established, testing for their presence is likely to become a routine part of the diagnostic workup. This review will attempt to establish a framework for understanding these mutations in the context of myeloproliferative neoplasms, myelodysplastic syndromes, and acute myeloid leukemia.

Pathways Affected by Recurrent Mutations in Myeloid Malignancies

Cell Signaling The ability of a cell to respond to diverse physiologic stimuli, including cytokines, chemokines, growth factors, and hormones, or to the presence of bacteria and other microorganisms is mediated via the interaction of specific ligands and their corresponding cell surface receptors. Ligand binding usually results in receptor dimerization and activation of a tyrosine kinase, either intrinsically present in the cytoplasmic domain or as an associated polypeptide. Further propagation of the signal from the cell surface to the nucleus involves the formation of macromolecular complexes and the activation or inactivation of various enzymes. The final outcome of the signal transduction is modulation of the expression of certain genes and their products, which ultimately produces a cellular response. In normal cells, this process is tightly regulated owing to the involvement of negative or inhibitory signals. In tumor cells these processes may be perturbed owing to mutations that impart inappropriate activation or deactivation of enzymatic function. Genes for receptor protein tyrosine kinases, such as FLT3 and KIT, or receptor-associated kinases, such as JAK2, are the most commonly mutated cell-signaling factors in myeloid malignancies. Activating mutations in these proteins occur in narrowly defined hotspots, resulting in ligand-independent dimerization or constitutive kinase activation. An example is the protein tyrosine kinase JAK2, which transduces signals from ligand-bound cell surface receptors for thrombopoietin (TPOR/MPL) and erythropoietin (EPOR).

Activating mutations in JAK2 are commonly found in the myeloproliferative neoplasms (MPNs): polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF). These disorders appear to be driven, in large part, by the inappropriate activation of growth factor– signaling pathways, and JAK2 is central to signaling from EPOR and TPOR/MPL via STAT5, STAT3, the RAS-MAP kinase pathway, and the PI-3 kinase–AKT pathway. Many negative regulators of cytokine signaling, such as SH2B adaptor protein 3 (SH2B3, also known as LNK)1,2 and cytokine-inducible SH2-containing protein (CISH, also known as SOCS)3 keep the pathway in balance. Downstream RAS signaling is counteracted by NF1, a protein that stimulates the intrinsic RAS guanosine triphosphatase activity. An effect similar to JAK2 mutations may be achieved through mutations in other proteins involved in these pathways, including activating mutations of a surface receptor (MPL) or loss-of-function mutations in negative regulators (SH2B3, CISH), all of which promote survival, proliferation, and differentiation of committed myeloid progenitors.4 Example genes included in this group: JAK2, MPL, KIT, FLT3, CSF3R, PTPN11, KRAS, NRAS, and NF1.

Transcription Transcription is a tightly regulated process that depends on the formation and assembly of protein and protein-DNA complexes. These complexes, called transcription factors, bind to specific DNA sequences adjacent to the genes they regulate and promote (in the case of an activator) or block (in the case of a repressor) the recruitment of RNA polymerases to those genes. This regulatory activity controls the formation of messenger RNA (mRNA) transcripts. Mutations that block the activity of transcriptional activators may, in certain circumstances, lead to a block in cellular differentiation due to the lack of the necessary gene products. Many of the transcription factors that are recurrently mutated in myeloid malignancies, such as RUNX1, GATA1, GATA2, and CEBPA, are involved in fundamental aspects of myelopoiesis and it is believed that these mutations lead to a block in myeloid differentiation.  Example genes included in this group: RUNX1, CEBPA, GATA1, GATA2, ETV6, and PHF6.

Epigenetic Modifiers: Regulation of DNA Methylation DNA methylation involves the addition of a methyl group to cytosine bases in the context of cytosine-guanine sequences (so-called CpG sites), leading to the creation of 5-methylcytosine. CpG islands are usually located in or near promoter regions. Their methylation is an important epigenetic mechanism for regulating gene expression and, in the context of heritable methylation patterns, underlies the process of genomic imprinting. Additionally, it has been hypothesized that aberrant DNA methylation may contribute to the pathogenesis of cancers,5–8 including myeloid neoplasms. Although cancer genomes tend to be globally hypomethylated, in comparison with normal tissues, hypermethylation of specific CpG islands at tumor suppressor genes, resulting in their inactivation, is common in many tumors.9

ormation of compact, inactive heterochromatin.10 Several factors regulate the process of DNA methylation. Mutations in some of these factors have been found recurrently in myeloid neoplasms.11–13 DNA methyltransferases catalyze the methylation at the 50 position of cytosine. DNMT3A and DNMT3B are involved in de novo methylation, whereas DNMT1 maintains hemimethylated DNA during replication. Once created, 5-methylcytosine can be further modified by a group of methylcytosine dioxygenases (Ten Eleven Translocation dioxygenases: TET1, TET2, and TET3) to 50-hydroxymethylcytosine, a presumed short-lived intermediary that may lead to demethylation of cytosine.

Mutations in both DNMT3A and TET2 likely lead to loss of function of the respective enzyme activities. Recently, mutations in IDH1 and IDH2 have been identified in myeloid neoplasms and other cancers. Interestingly, recurring mutations of an arginine residue in the active site (R132 in IDH1 and R140 in IDH2) prevent the normal catalytic function of the enzyme (conversion of isocitrate to aketoglutarate) and appear to induce a neomorphic enzyme activity resulting in the formation of the rare oncometabolite 2-hydroxyglutarate.14 TET2 belongs to a family of dioxygenases that requires a-ketoglutarate as a cofactor.15,16 It has been shown that 2-hyroxyglutarate acts as a competitive inhibitor of a-ketoglutarate–dependent dioxygenases, which include TET2 and members of the KDM family of histone demethylases, thereby inducing epigenetic changes at both the level of DNA methylation and histone modification.14,17

Therapeutic inhibitors of mutant forms of IDH proteins and the resulting 2-hydroxyglutarate are also under investigation.18 Example genes included in this group: DNMT3A, TET2, IDH1, and IDH2.

Epigenetic Modifiers: Mutations Affecting Histone Function  Histone proteins are involved in the dynamic organization of DNA into zones of active euchromatin and inactive heterochromatin in a process that is regulated, in part, by a complex series of posttranslational modifications to histone tails, including acetylation and methylation. These modifications affect the recruitment of regulatory proteins such as transcription factors, corepressors, and coactivators, as well as histone-modifying enzymes themselves. Trimethylation of the lysine at position 27 in histone H3 (H3K27), one of the more common modifications, generally leads to reduced gene expression and it would be expected that mutations that reduce methylation at H3K27 would activate transcription. Recently, recurrent mutations in several genes encoding histone regulators, including ASXL1, EZH2, SUZ12, and KDM6A (also known as UTX), have been identified.

Perturbations in epigenetic pathways result in global, genome-wide effects and it is often difficult to identify which altered cellular function eventually leads to neoplasia. The same also holds true for perturbations in the RNA splicing machinery and the cohesion complex (see below). Example genes included in this group: ASXL1, EZH2, SUZ12, and KDM6A.

Cohesin Complex Genes  The cohesin complex is a conserved multimeric protein complex that regulates cohesion of sister chromatids during cell division,21 postreplicative DNA repair,22,23 and global gene expression.24–28
Example genes included in this group: STAG2, RAD21, SMC1A, and SMC3.

RNA-Splicing Factors  RNA splicing results in the formation of mature mRNA transcripts derived from exons, the protein coding portion of the genome. Splicing occurs in a macromolecular complex of small nuclear RNAs and proteins assembled de novo on each pre-mRNA strand in a multistep process. This complex is known as the spliceosome. Transcripts may undergo alternative splicing in a tissue, or context-specific manner and the protein products of alternatively spliced transcripts may have altered function.
Example genes included in this group: SF3B1, SRSF2, ZRSR2, and U2AF1.

Cell Cycle Regulators  Example genes included in this group: TP53 and NPM1.

Genetic Biomarkers in Myeloid Malignancies

Myeloproliferative Neoplasms  Myeloproliferative neoplasms encompass a group of clonal stem cell disorders characterized by expansion of 1 or more of the myeloid lineages resulting in bone marrow hypercellularity and increased peripheral blood myeloid cell counts. The MPN category includes chronic myelogenous leukemia, PV, ET, PMF, chronic neutrophilic leukemia, mastocytosis, and others. The underlying genetic landscape of some of these disorders is very well understood, as in the case of chronic myelogenous leukemia with t(9;22), but is much less well understood in many other entities.

The discovery of a recurrent codon 617 activating mutation (V617F) in exon 14 of the tyrosine kinase JAK237–40 and additional mutations in JAK2 exon 1241 provided the first genetic evidence of the importance of dysregulated growth factor signaling in these disorders. The prevalence of JAK2 mutations in classical MPNs varies from 95% to 99% in PV, 50% to 70% in ET, 40% to 50% in PMF,37–41,43 and molecular tests for their detection are available and widely used in clinical practice. Similarly, activating mutations in the MPL gene, encoding the thrombopoietin receptor, are present in approximately 4% of ET cases and approximately 11% of PMF cases.44–47 Recently, calreticulin (CALR), encoding an endoplasmic reticulum chaperone, has also been shown to be important. Somatic CALR mutations are found in 70% to 84% of patients with ET or PMF with wild-type JAK2 and MPL, 8% of MDS cases, and occasionally in other myeloid neoplasms.48 Clonal analyses suggest CALR mutations act as an initiating mutation in some patients.48 In ET and PMF, CALR mutations and JAK2 and MPL mutations are mutually exclusive,49 and CALR mutations appear to be absent in PV.49 CALR mutations appear to be primarily insertion or deletion mutations that result in a frameshift and the subsequent generation of a novel C-terminal peptide.48,49

Many other genes involved in intracellular signaling, such as negative regulators of the JAK2 signaling pathway, are mutated in MPNs. Among these is SH2B3, which negatively regulates JAK2 activation through its SH2 domain. Mutations in SH2B3 during the chronic phase are uncommon, fewer than 5% in ET and PMF50; however, their frequency increases during leukemic transformation, suggesting a role in disease progression.51 Another negative regulator found mutated in MPNs is the Cbl proto-oncogene, E3 ubiquitin protein ligase gene (CBL). CBL acts as a multifunctional adapter with ubiquitin ligase activity and by competitive blockade of signaling.

A shift from a simple, chronic myelogenous leukemia–like model for MPN pathophysiology to a more complex model occurred with the emergence of evidence of a ‘‘pre-JAK2’’ genetic event. This concept is based on the observation that mutations in signaling molecules are not sufficient for disease development and that several cooperating genetic hits appear to be required.4 Mutations in genes involved in epigenetic regulation, including EZH2, ASXL1, and TET2 (also found in many other myeloid neoplasms), are postulated to act as those initialing events, preceding JAK2V617F mutations.55 EZH2 mutations do not occur in ET, but are present in 3% of PVs and 13% of MFs.56 ASXL1 mutations are found in only approximately 7% of patients with ET and PV but more frequently in PMF cases (from 19%–40%).57,58 TET2 mutations occur in approximately 14% of MPNs, ranging from 11% in ETs to 19% in PMFs.59,60 Finally, there are mutations that are rarely found during the chronic phase but which may be present at transformation, and are therefore thought to play a role in disease progression. IDH1 and IDH2 mutations, for example, have a low frequency in the chronic phase (0.8% in ET, 1.9% in PV, and 4.2% in MF) but a much higher frequency in blast phase.61

Myelodysplastic Syndromes and MDS/MPN Overlap Disorders 

The myelodysplastic syndromes are a group of clonal hematopoietic stem cell disorders characterized by ineffective hematopoiesis, morphologic evidence of dysplasia in at least 1 of the myeloid lineages, peripheral cytopenias, bone marrow hypercellularity, and an increased risk of development of AML.74 Clonal cytogenetic abnormalities, including large deletions and chromosome gains, as well as balanced translocations, are observed in approximately 50% of MDS cases by routine methods,74 and their identification aids in establishing the diagnosis and may provide prognostic information.

Acute Myelogenous Leukemia  Acute myeloid leukemia is a genetically heterogeneous disease resulting in the accumulation of myeloblasts in bone marrow with a concomitant reduction in normal hematopoiesis. The diagnosis and subclassification of AML depends on detecting the presence of recurrent cytogenetic abnormalities.74 In many cases, particularly those that are cytogenetically normal (CN-AML), several single-gene mutations further aid in the stratification of disease outcomes. The significance of mutations in genes such as FLT3, NPM1, and CEBPA is well established but nextgeneration sequencing has led to the discovery of numerous additional recurrent mutations, including in TET2,12,59 ASXL1,104 IDH1/IDH2,13,105,106 DNMT3A,11,107 and PHF6.108

Among the most common mutations found in de novo AML are NMP1, FLT3, and DNMT3A mutations, present in 22% to 29%, 22% to 37%, and 15% to 26% of samples, respectively.31,110,111 Other genes less commonly targeted by mutations are IDH1/IDH2 (15%– 20%), KRAS/NRAS (12% combined), RUNX1 (5%–10%),TET2 (8%–14%), TP53 (2%–8%), CEBPA (6%–14%), WT1 (6%–8%), PTPN11 (4%), and KIT (4%–6%).31,110,111

The prognostic significance of a subset of recurrent mutations is well established. In CN-AML, biallelic CEBPA mutations127,128 and NPM1 mutations without FLT3-ITD mutations129–133 are associated with a favorable prognosis. In contrast, FLT3-ITD without NPM1 mutations112,113,129–132 and MLL–partial tandem duplication mutations134–136 portend poor outcome. KIT mutations in AML with t(8;21) or inv(16)131,137 are also associated with unfavorable outcome. The European LeukemiaNet panel138 first proposed a standardized classification according to both cytogenetic and molecular genetic data to allow a better comparison of prognosis among patients with AML. However, only mutations of NPM1, CEBPA, and FLT3 were included in their recommendations. The relevance of more recently discovered mutations, including IDH1, IDH2, WT1, TET2, ASXL1, among others, remains unclear.139–142 The presence of certain mutations may also allow for more targeted therapeutic regimens; for example, FLT kinase inhibitors may be useful in cases with mutations and IDH1 inhibitors are under investigation in patients with IDH1 mutations.143,144

An enormous amount of new information illuminating the genetic complexity of myeloid neoplasms has been generated during the last few years. Much work remains to be done but it is clear that the future role of the pathologist in collecting and interpreting this information will be an essential component of the management of these patients.

 

The Cancer Genomics Resource List 2014
Zutter MM, Bloom KJ, Cheng L, Hagemann IS, et al.
Arch Pathol Lab Med. http://dx.doi.org:/10.5858/arpa.2014-0330-C

Optimizing the Clinical Utility of Biomarkers in Oncology: The NCCN Biomarkers Compendium
Marian L. Birkeland, Joan S. McClure
Arch Pathol Lab Med. 2015;139:608–611
http://dx.doi.org:/10.5858/arpa.2014-0146-RA

The rapid development of commercial biomarker tests for oncology indications has led to confusion about which tests are clinically indicated for oncology care. By consolidating biomarker testing information recommended within National Comprehensive Cancer Network Clinical Practice Guidelines in Oncology (NCCN Guidelines), the NCCN Biomarkers Compendium aims to ensure that patients have access to appropriate biomarker testing based on the evaluations and recommendations of the expert NCCN panel members.
Objectives.—To present the recently launched NCCN Biomarkers Compendium. Data Sources.—Biomarker testing information recommended within NCCN Clinical Treatment Guidelines as well as published resources for genetic and biological information. Conclusions.—The NCCN Biomarkers Compendium is a continuously updated resource for clinicians who need access to relevant and succinct information about biomarker testing in oncology and is linked directly to the recommendations provided within the NCCN Clinical Practice Guidelines.

Most recommendations contained within the NCCN Guidelines are based upon lower-level evidence and uniform NCCN consensus (category 2A).1 This is not a deficiency of the guidelines, but is rather because high-level evidence is not available for most decisions across the continuum of care. A deeper look at what constitutes a ‘‘recommendation’’ might begin to clarify that issue. A recommendation can include all of the recommended workup, surgical options, options for chemotherapy, and tests recommended for ongoing surveillance. Although many of these options are routinely used as standard of care in clinical practice, there is often not the available body of high-level evidence that supports category 1 recommendations, thus most are category 2A levels of evidence and consensus. In other instances, recommendations for chemotherapy regimens for which there is high-level, randomized clinical trial evidence are listed as category 1.

Several derivative products arise from the NCCN Guidelines. The NCCN Drugs & Biologics Compendium (NCCN Compendium) is a resource outlining appropriate use of drugs and biologics as recommended in the NCCN Guidelines. To be included in the compendium, an agent must first be recommended in at least 1 of the NCCN Guidelines. The compendium is typically used by clinicians and payors to determine appropriate use and as a standard to determine coverage. The rapid development and commercialization of biomarker and companion diagnostic testing in cancer gave rise to the NCCN Biomarkers Compendium, to be used by both payors and clinicians to facilitate identification of biomarker tests recommended for use by NCCN guideline panels. The NCCN uses a broad definition of ‘‘biomarker’’ for the purposes of this compendium. All tests measuring genes or gene products, which are used for diagnosis, screening, monitoring, surveillance, or for providing predictive or prognostic information, are included in the biomarkers compendium. This compendium focuses on the biology of the biomarker itself and its clinical utility in supporting clinical decision making. Information is organized by the biomarker being measured, and not by listing of commercially available tests or test kits. Close to 1000 biomarker testing recommendations are included in the NCCN Biomarkers Compendium.

The NCCN Biomarkers Compendium is presented on the NCCN Web site as a series of drop-down menus, allowing users to pick from menus listing Guideline, Disease, Molecular Abnormality, or Gene Symbol.3 Users can retrieve all recommendations for a particular disease, or can select a gene-based search in order to show which diseases have a validated use for a particular gene test. Additional fields can be displayed by selecting from a series of boxes to the right of the drop-down menus (see Figure 1, which shows default fields for RAS testing in colon cancer). Once records are displayed, the resulting table can be sorted by the information in any of the displayed columns. If a searcher chooses, all records can be displayed and then searched with any text term or sorted by any of the columns for a more comprehensive picture of the contents of the database.

Figure 1. (not shown)  KRAS mutation testing recommendation from the National Comprehensive Cancer Network (NCCN) Biomarkers Compendium.3 Reproduced with permission from the NCCN Biomarkers Compendium [1] 2014 National Comprehensive Cancer Network, Inc. (NCCN.org; accessed February 21, 2014). To view the most recent and complete version of the NCCN Biomarkers Compendium, go online to NCCN.org. National Comprehensive Cancer Network, NCCN, NCCN Guidelines, and all other NCCN content are trademarks owned by the National Comprehensive Cancer Network, Inc.

Disease Description Colon cancer
Specific Indication Metastatic disease
Molecular Abnormality KRAS/NKRAS mutation
Test KRAS/NKRAS
Chromosome 1p13.2, 12p12.1
Gene Symbol KRAS/NKRAS
Test Detects Mutation
Methodology
Category of Evidence 2A
Specimen Types FFPE tumor tissue
Recommendation …Determination of RAS mutations.
Test Purpose Predictive
Guideline Page COL 4 of 5, COL 4, COL 9
Note All patients with metastatic colorectal cancer should be genotyped for RAS mutations. At the very least …

Figure 2. (Table)  Example of PDF file generated from ‘‘print’’ command of National Comprehensive Cancer Network (NCCN) Biomarkers Compendium record. Reproduced with permission from the NCCN Biomarkers Compendium [1] 2014 National Comprehensive Cancer Network, Inc (NCCN.org; accessed February 21, 2014). To view the most recent and complete version of the NCCN Biomarkers Compendium, go online to NCCN.org. National Comprehensive Cancer Network, NCCN, NCCN Guidelines, and all other NCCN content are trademarks owned by the National Comprehensive Cancer Network, Inc.

Table 2. (List) Summary of Testing Types Included in the National Comprehensive Cancer Network Biomarkers Compendiuma,b

Protein expression
Translocation
Mutation
Chromosomal defect
Gene rearrangement
Virus detection
Antigen expression
Serum proteins
Amplification
Short repeated sequences
Promoter methylation
Gene expression pattern
Helicobacter pylori

Table 3. Predictive Tests Used for Treatment Decision Making, Extracted From National Comprehensive Cancer Network Guidelines and Biomarkers Compendiuma,b

Test   Disease
21-gene RT-PCR

BCR-ABL1 translocation

Breast cancer
ABL1 mutation Ph+ acute lymphoblastic leukemia, chronic myelogenous leukemia
ALK rearrangement Non–small cell lung cancer
BRAF mutation Non–small cell lung cancer, melanoma, colon cancer, rectal cancer
EGFR mutation Non–small cell lung cancer
ERBB2 amplification/overexpression Breast cancer, esophageal and esophagogastric junction cancers, gastric cancer
ESR1 expression Breast cancer
KIT mutation Soft tissue sarcoma: GIST
KRAS mutation Colon cancer, rectal cancer, non–small cell lung cancer
MGMT promoter methylation Central nervous system cancers: anaplastic glioma/glioblastoma
MLH1, MSH2, MSH6, PMS2 expression and/or mutation, MSI testing Colon cancer, rectal cancer
PDGFRA mutation Soft tissue sarcoma: GIST
PGR expression Breast cancer
ROS1 rearrangement Non–small cell lung cancer

A large number of tests were grouped for the purposes of this simplified table into the category of gross chromosomal abnormalities. Interestingly, the guidelines so far contain only a single recommendation for the use of a gene expression profiling test, and this is the 21-gene reverse transcription–polymerase chain reaction test recommended within the breast cancer treatment guideline, where the score for this test can be used as part of a decision-making process for chemotherapy recommendations in node negative, hormone receptor–positive, HER2-negative disease.

Table 3 summarizes the biomarker tests included in the NCCN Biomarkers Compendium that are predictive for either responsiveness (eg, BRAF mutation and vemurafenib sensitivity) or nonresponsiveness (eg, KRAS mutation testing and cetuximab or panitumumab insensitivity) to a particular type of therapy. As the number of companion diagnostics and targeted therapies grows, we expect this category of test to become one of the largest categories of testing contained within the Biomarkers Compendium, and it may be surprising to note that only 15 of these types of test are currently recommended within the NCCN Guidelines.

The NCCN Biomarkers Compendium generally avoids recommendations for particular methodologies or test kits to use to assess mutations and translocations. The choice of methodology and supplier for carrying out the recommended biomarker tests remains that of the treating oncologists and pathologists.

The NCCN Biomarkers Compendium may be used by payers as a reference for coverage decisions and by clinicians as a guide to which biomarkers are appropriate to test. The Biomarkers Compendium focuses on the clinical usefulness of biomarker testing rather than specific tests or test kits that identify the presence or absence of the marker. Other groups are continuing to assess clinical and analytic validity for specific biomarker test methodologies. Even the US Food and Drug Administration approval process is limited to clinical and analytic validity, and does not specifically address clinical utility. The NCCN Biomarkers Compendium is complementary to these other efforts. By providing biomarker testing information, the NCCN Biomarkers Compendium aims to ensure that patients have coverage and access to appropriate biomarker testing, based on the evaluations and recommendations of the expert NCCN panel members.

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