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

DOI: http://dx.doi.org/10.1016/j.cell.2016.04.033Article Info

http://www.cell.com/cms/attachment/2056388051/2061342793/fx1.jpg

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

http://www.nature.com/nrc/journal/v15/n10/carousel/nrc3985-f1.jpg

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

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

http://www.genengnews.com/Media/images/GENHighlight/thumb_115705_web4813301741.jpg

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

Petrus R. de Jong, Koji Taniguchi, Alexandra R. Harris, Samuel Bertin, Naoki Takahashi, Jen Duong, Alejandro D. Campos, Garth Powis, Maripat Corr, Michael 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-Raf^{1, 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 phosphatases^{3, 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.

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

http://www.nature.com/ncomms/2016/160517/ncomms11551/images_article/ncomms11551-f1.jpg

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ERK1/2^ΔIEC causes wasting and enterocyte dysfunction

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/

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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]

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

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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