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Archive for the ‘Anaerobic Glycolysis’ Category


Agios Pharmaceuticals target the metabolism of cancer cells for making drugs that essentially try to repair cancer cells

Reporter: Aviva Lev-Ari, PhD, RN

A small biotech behind a groundbreaking approach to tackling cancer just got its first drug approved

http://www.businessinsider.com/fda-approves-agios-pharmaceuticals-drug-targeting-cancer-cell-metabolism-2017-8

See

Cancer Metabolism

http://www.agios.com/research/cancer-metabolism/

Metabolic Immuno-Oncology

http://www.agios.com/research/metabolic-immuno-oncology/

 

 

The VOICE of Larry H. Bernstein, MD, FCAP

Cancer cells didn’t need as much oxygen to metabolize sugar as normal cells. 

Not correct. Cancer cells metabolize glucose by aerobic glycolysis (4 ATP) with an impaired mitochondrial oxygen utilization (36 ATP). 

There is a reverse Warburg effect in which the underlying stromal cell carries out crosstalk with the epithelial cell. 

There is also a 3rd dimension. Cells undergo a series of adaptive changes tied to proteostasis. This involves the sulfur amino acid cysteine and disulfide bonds, which is involved with protein oligomerization in the ER, and also signaling in the mitochondria with mDNA and the nucleus. 

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Inactivation of an enzyme needed to metabolize glucose by Vitamic C deprives tumor cells of energy

Reporter: Aviva Lev-Ari, PhD, RN

 

 

Vitamin C did kill cultured colon cancer cells with BRAF or KRAS mutations by raising free radical levels, which in turn inactivate an enzyme needed to metabolize glucose, depriving the cells of energy.

 

Vitamin C selectively kills KRAS and BRAF mutant colorectal cancer cells by targeting GAPDH

Glucose Deprivation Contributes to the Development of KRAS Pathway Mutations in Tumor Cells

A few years ago, Jihye Yun, then a graduate student at Johns Hopkins University in Baltimore, Maryland, found that colon cancer cells whose growth is driven by mutations in the gene KRAS or a less commonly mutated gene,BRAF, make unusually large amounts of a protein that transports glucose across the cell membrane. The transporter, GLUT1, supplies the cells with the high levels of glucose they need to survive. GLUT1 also transports the oxidized form of vitamin C, dehydroascorbic acid (DHA), into the cell, bad news for cancer cells, because Yun found that DHA can deplete a cell’s supply of a chemical that sops up free radicals. Because free radicals can harm a cell in various ways, the finding suggested “a vulnerability” if the cells were flooded with DHA, says Lewis Cantley at Weill Cornell Medicine in New York City, where Yun is now a postdoc.

Cantley’s lab and collaborators found that large doses of vitamin C did indeed kill cultured colon cancer cells with BRAF or KRAS mutations by raising free radical levels, which in turn inactivate an enzyme needed to metabolize glucose, depriving the cells of energy. Then they gave daily high dose injections—equivalent to a person eating 300 oranges—to mice engineered to develop KRAS-driven colon tumors. The mice developed fewer and smaller colon tumors compared with control mice.

SOURCE
http://www.sciencemag.org/news/2015/11/vitamin-c-kills-tumor-cells-hard-treat-mutation

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Targeted Therapy for Triple Negative Breast Cancer

Curator: Larry H. Bernstein, MD, FCAP

LPBI

 

Triple-Negative Breast Cancer Target Is Found

May 17, 2016   Researchers at UC Berkeley discover a target that drives cancer metabolism in triple-negative breast cancer.
http://www.technologynetworks.com/Genotyping/news.aspx?ID=191502

UC Berkeley researchers have found a long-elusive Achilles’ heel within “triple-negative” breast tumors, a common type of breast cancer that is difficult to treat. The scientists then used a drug-like molecule to successfully target this vulnerability, killing cancer cells in the lab and shrinking tumors in mice.

“We were looking for targets that drive cancer metabolism in triple-negative breast cancer, and we found one that was very specific to this type of cancer,” said Daniel K. Nomura, an associate professor of chemistry and of nutritional sciences and toxicology at UC Berkeley and senior author for the study, which is published online ahead of print in Cell Chemical Biology.

Triple-negative breast cancers account for about one in five breast cancers, and they are deadlier than other forms of breast cancer, in part because no drugs have been developed to specifically target these tumors.

Triple-negative breast cancers do not rely on the hormones estrogen and progesterone for growth, nor on human epidermal growth factor receptor 2 (HER2). Because they do not depend on these three targets, they are not vulnerable to modern hormonal therapies or to the HER2-targeted drug Herceptin (trastuzumab).

Instead, oncologists treat triple-negative breast cancer with older chemotherapies that target all dividing cells. If triple-negative breast cancer spreads beyond the breast to distant sites within the body, an event called metastasis, there are few treatment options.

Tumor cells develop abnormal metabolism, which they rely on to get the energy boost they need to fuel their rapid growth. In their new study, the research team used an innovative approach to search for active enzymes that triple-negative breast cancers use differently for metabolism in comparison to other cells and even other tumors.

Inhibiting cancer metabolism

They discovered that cells from triple-negative breast cancer cells rely on vigorous activity by an enzyme called glutathione-S-transferase Pi1 (GSTP1). They showed that in cancer cells, GSTP1 regulates a type of metabolism called glycolysis, and that inhibition of GSTP1 impairs glycolytic metabolism in triple-negative cancer cells, starving them of energy, nutrients and signaling capability. Normal cells do not rely as much on this particular metabolic pathway to obtain usable chemical energy, but cells within many tumors heavily favor glycolysis.

Co-author Eranthie Weerapana, an associate professor of chemistry at Boston College, developed a molecule named LAS17 that tightly and irreversibly attaches to the target site on the GSTP1 molecule. By binding tightly to GSTP1, LAS17 inhibits activity of the enzyme. The researchers found that LAS17 was highly specific for GSTP1, and did not attach to other proteins in cells.

According to Nomura, LAS17 did not appear to have toxic side effects in mice, where it shrank tumors grown to an invasive stage from surgically transplanted, human, triple-negative breast cancer cells that had long been maintained in lab cultures.

The research team intends to continue studying LAS17, Nomura said, with the next step being to study tumor tissue resected from human triple-negative breast cancers and transplanted directly into mice.

“Inhibiting GSTP1 impairs glycolytic metabolism,” Nomura said. “More broadly, this inhibition starves triple-negative breast cancer cells, preventing them from making the macromolecules they need, including the lipids they need to make membranes and the nucleic acids they need to make DNA. It also prevents these cells from making enough ATP, the molecule that is the basic energy fuel for cells.”

Beyond the metabolic role they first sought to track down, GSTP1 also appears to aid signaling within triple-negative breast cancer cells, helping to spur tumor growth, the researchers found.

Technique identifies Achilles’ heels

Nomura said it was surprising that a single, unique target emerged from the research team’s search.

The method used by the researchers, called “reactivity-based chemoproteomics,” can quickly lead to specific targetable sites — the Achilles’ heels — on proteins of interest, and eventually to drug development strategies, Nomura said.

The approach is to search for protein targets that are actively functioning within cells, instead of first using the well-trod path of surveying all genes to identify the specific genes that have taken the first step toward protein production. With that more conventional strategy, the switching on, or “expression,” of genes is evidenced by the easily quantified molecule called messenger RNA, made by the cell from a gene’s DNA template.

Nomura’s team instead first used chemical probes that can react with certain configurations of two of the amino acid building blocks of protein — cysteine and lysine — known to be involved in several kinds of important structural and functional transitions that active proteins can undergo.

“A lot can happen after the first step in protein production, and we believe our method for identifying fully formed, active proteins is more useful for tracking down relevant differences in cellular physiology,” Nomura said.

The researchers analyzed and compared cells from five distinct triple-negative breast cancers that had been grown in cell cultures for generations, along with cells from four distinct breast cancers that were not triple negative.

The scientists used a chemical identification technique known as mass spectrometry to narrow down the set of proteins that had active lysines and cysteines to just those that were metabolic enzymes. Only then did they use the more conventional approach of measuring gene expression in the different cancer cell types.

GSTP1 was the only metabolically active enzyme that was specifically expressed only in triple-negative breast cancer cells compared to other breast cancer cell types, the researchers found. Separate analysis of databases of human breast cancer by UC San Francisco co-authors confirmed that GSTP1 is overexpressed in patients with triple-negative breast cancers in comparison to patients with other breast cancers.

In addition to Nomura and Weerapana, study authors included Sharon Louie, Elizabeth Grossman, Lucky Ding, Tucker Huffman and David Miyamoto, from UC Berkeley; Roman Camarda and Andrei Goga, from UC San Francisco, and Lisa Crawford, from Boston College. Study funders included the National Institutes of Health, the American Cancer Society, the U.S. Department of Defense, and the Searle Scholar Foundation.

 

Triple-negative breast cancer target is found

UC Berkeley researchers have found a long-elusive Achilles’ heel within “triple-negative” breast tumors, a common type of breast cancer that is difficult to treat. The scientists then used a drug-like molecule to successfully target this vulnerability, killing cancer cells in the lab and shrinking tumors in mice.

“We were looking for targets that drive cancer metabolism in triple-negative breast cancer, and we found one that was very specific to this type of cancer,” said Daniel K. Nomura, an associate professor of chemistry and of nutritional sciences and toxicology at UC Berkeley and senior author for the study, which is published online ahead of print on May 12 in Cell Chemical Biology.

Triple-negative breast cancers account for about one in five breast cancers, and they are deadlier than other forms of breast cancer, in part because no drugs have been developed to specifically target these tumors.

Triple-negative breast cancers do not rely on the hormones estrogen and progesterone for growth, nor on human epidermal growth factor receptor 2 (HER2). Because they do not depend on these three targets, they are not vulnerable to modern hormonal therapies or to the HER2-targeted drug Herceptin (trastuzumab).

Instead, oncologists treat triple-negative breast cancer with older chemotherapies that target all dividing cells. If triple-negative breast cancer spreads beyond the breast to distant sites within the body, an event called metastasis, there are few treatment options.

Tumor cells develop abnormal metabolism, which they rely on to get the energy boost they need to fuel their rapid growth. In their new study, the research team used an innovative approach to search for active enzymes that triple-negative breast cancers use differently for metabolism in comparison to other cells and even other tumors.

Inhibiting cancer metabolism

They discovered that cells from triple-negative breast cancer cells rely on vigorous activity by an enzyme called glutathione-S-transferase Pi1 (GSTP1). They showed that in cancer cells, GSTP1 regulates a type of metabolism called glycolysis, and that inhibition of GSTP1 impairs glycolytic metabolism in triple-negative cancer cells, starving them of energy, nutrients and signaling capability. Normal cells do not rely as much on this particular metabolic pathway to obtain usable chemical energy, but cells within many tumors heavily favor glycolysis.

for mor see.. http://news.berkeley.edu/2016/05/12/triple-negative-breast-cancer-target-is-found/

 

GSTP1 Is a Driver of Triple-Negative Breast Cancer Cell Metabolism and Pathogenicity

Sharon M. Louie, Elizabeth A. Grossman, Lisa A. Crawford….., Eranthie Weerapana, Daniel K. Nomura
Figure thumbnail fx1
  • We used chemoproteomics to profile metabolic drivers of breast cancer
  • GSTP1 is a novel triple-negative breast cancer-specific target
  • GSTP1 inhibition impairs triple-negative breast cancer pathogenicity
  • GSTP1 inhibition impairs GAPDH activity to affect metabolism and signaling

Breast cancers possess fundamentally altered metabolism that fuels their pathogenicity. While many metabolic drivers of breast cancers have been identified, the metabolic pathways that mediate breast cancer malignancy and poor prognosis are less well understood. Here, we used a reactivity-based chemoproteomic platform to profile metabolic enzymes that are enriched in breast cancer cell types linked to poor prognosis, including triple-negative breast cancer (TNBC) cells and breast cancer cells that have undergone an epithelial-mesenchymal transition-like state of heightened malignancy. We identified glutathione S-transferase Pi 1 (GSTP1) as a novel TNBC target that controls cancer pathogenicity by regulating glycolytic and lipid metabolism, energetics, and oncogenic signaling pathways through a protein interaction that activates glyceraldehyde-3-phosphate dehydrogenase activity. We show that genetic or pharmacological inactivation of GSTP1 impairs cell survival and tumorigenesis in TNBC cells. We put forth GSTP1 inhibitors as a novel therapeutic strategy for combatting TNBCs through impairing key cancer metabolism and signaling pathways.

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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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Cyclic Dinucleotides and Histone deacetylase inhibitors

Curators: Larry H. Bernsten, MD, FCAP and Aviva Lev-Ari, PhD, RN

LPBI

 

New Class of Immune System Stimulants: Cyclic Di-Nucleotides (CDN): Shrink Tumors and bolster Vaccines, re-arm the Immune System’s Natural Killer Cells, which attack Cancer Cells and Virus-infected Cells

Reporter: Aviva Lev-Ari, PhD, RN

The Immunotherapeutics and Vaccine Research Initiative (IVRI), a UC Berkeley effort funded by Aduro Biotech, Inc.

https://pharmaceuticalintelligence.com/2016/04/24/new-class-of-immune-system-stimulants-cyclic-di-nucleotides-cdn-shrink-tumors-and-bolster-vaccines-re-arm-the-immune-systems-natural-killer-cells-which-attack-cancer-cells-and-virus-inf/

A new class of immune system stimulants called cyclic di-nucleotides have shown promise in shrinking tumors and bolstering vaccines against tuberculosis, and research that could help re-arm the immune system’s natural killer cells, which normally attack cancer cells and virus-infected cells, to better fight tumors.

Much of the excitement around combining these two areas — the immunology of cancer and the immunology of infectious disease — comes from the amazing success of immunotherapy against cancer, which started with the work of James Allison when he was a professor of immunology at UC Berkeley and director of the Cancer Research Laboratory from 1985 to 2004. Allison, now at the University of Texas MD Anderson Cancer Center, discovered a way to release a brake on the body’s immune response to cancer that has proved highly successful at unleashing the immune system to attack melanoma and is being tried against other types of cancer. Allison’s technique uses an antibody that blocks an immune suppressor called CTLA4, antibodies that block another immune suppressor, PD1. This has been successful in treating melanoma, renal cancer and a type of lung cancer. Both CTLA4 and PD1 antibodies are now FDA-approved as cancer therapies.

Another promising avenue involves a protein in cells that responds to foreign DNA to launch an innate immune response — the first response of the body’s immune system. The protein, dubbed STING, is triggered by small molecules called cyclic di-nucleotides (CDN), and makes immune cells release interferon and other cytokines that activate disease-fighting T cells and stimulate the production of antibodies that together kill invading pathogens and destroy cancer cells. Listeria bacteria, for example, secrete a CDN directly into infected cells that activates STING.

Russell Vance, a UC Berkeley professor of molecular and cell biology and current head of the Cancer Research Laboratory, discovered several years ago that the chemical structure of these di-nucleotides is critical to their ability to work in humans. Aduro has since developed a CDN designed to work in humans and found that injecting it directly into a tumor in mice caused the tumor to shrink.

Sarah Stanley, an assistant professor of public health, has found evidence that CDNs can help improve the imperfect vaccines we have today against tuberculosis.

 

Researchers at UC Berkeley will have access to Aduro’s novel technology platforms for research use, including its STING pathway activators, proprietary monoclonal antibodies and the engineered listeria bacteria, referred to as LADD (listeria attenuated double-deleted). David Raulet, professor of molecular and cell biology and director of IVRI has contributed to making these cells a new focus of cancer research. As tumors advance, NK cells inside the tumors appear to become desensitized, he said. Recent research shows that some cytokines and other immune mediators Raulet discovered are able to “wake them up” and get them to resume their elimination of cancer cells.

 

Histone deacetylase inhibitors: molecular mechanisms of action

W S Xu1,2, R B Parmigiani1,2 and P A Marks1

Oncogene (2007) 26, 5541–5552; http://dx.doi.org:/10.1038/sj.onc.1210620

This review focuses on the mechanisms of action of histone deacetylase (HDAC) inhibitors (HDACi), a group of recently discovered ‘targeted’ anticancer agents. There are 18 HDACs, which are generally divided into four classes, based on sequence homology to yeast counterparts. Classical HDACi such as the hydroxamic acid-based vorinostat (also known as SAHA and Zolinza) inhibits classes I, II and IV, but not the NAD+-dependent class III enzymes. In clinical trials, vorinostat has activity against hematologic and solid cancers at doses well tolerated by patients. In addition to histones, HDACs have many other protein substrates involved in regulation of gene expression, cell proliferation and cell death. Inhibition of HDACs causes accumulation of acetylated forms of these proteins, altering their function. Thus, HDACs are more properly called ‘lysine deacetylases.’ HDACi induces different phenotypes in various transformed cells, including growth arrest, activation of the extrinsic and/or intrinsic apoptotic pathways, autophagic cell death, reactive oxygen species (ROS)-induced cell death, mitotic cell death and senescence. In comparison, normal cells are relatively more resistant to HDACi-induced cell death. The plurality of mechanisms of HDACi-induced cell death reflects both the multiple substrates of HDACs and the heterogeneous patterns of molecular alterations present in different cancer cells.

histone deacetylase, histone deacetylase inhibitor, apoptosis, mitotic cell death, senescence, angiogenesis

Acetylation and deacetylation of histones play an important role in transcription regulation of eukaryotic cells (Lehrmann et al., 2002;Mai et al., 2005). The acetylation status of histones and non-histone proteins is determined by histone deacetylases (HDACs) and histone acetyl-transferases (HATs). HATs add acetyl groups to lysine residues, while HDACs remove the acetyl groups. In general, acetylation of histone promotes a more relaxed chromatin structure, allowing transcriptional activation. HDACs can act as transcription repressors, due to histone deacetylation, and consequently promote chromatin condensation. HDAC inhibitors (HDACi) selectively alter gene transcription, in part, by chromatin remodeling and by changes in the structure of proteins in transcription factor complexes (Gui et al., 2004). Further, the HDACs have many non-histone proteins substrates such as hormone receptors, chaperone proteins and cytoskeleton proteins, which regulate cell proliferation and cell death (Table 1). Thus, HDACi-induced transformed cell death involves transcription-dependent and transcription-independent mechanisms (Marks and Dokmanovic, 2005Rosato and Grant, 2005Bolden et al., 2006;Minucci and Pelicci, 2006).

Table 1 – Nonhistone protein substrates of HDACs (partial list).   Full table

http://www.nature.com/common/images/table_thumb.gif

In humans, 18 HDAC enzymes have been identified and classified, based on homology to yeast HDACs (Blander and Guarente, 2004;Bhalla, 2005Marks and Dokmanovic, 2005). Class I HDACs include HDAC1, 2, 3 and 8, which are related to yeast RPD3 deacetylase and have high homology in their catalytic sites. Recent phylogenetic analyses suggest that this class can be divided into classes Ia (HDAC1 and -2), Ib (HDAC3) and Ic (HDAC8) (Gregoretti et al., 2004). Class II HDACs are related to yeast Hda1 (histone deacetylase 1) and include HDAC4, -5, -6, -7, -9 and -10 (Bhalla, 2005Marks and Dokmanovic, 2005). This class is divided into class IIa, consisting of HDAC4, -5, -7 and -9, and class IIb, consisting of HDAC6 and -10, which contain two catalytic sites. All class I and II HDACs are zinc-dependent enzymes. Members of a third class, sirtuins, require NAD+ for their enzymatic activity (Blander and Guarente, 2004) (see review by E Verdin, in this issue). Among them, SIRT1 is orthologous to yeast silent information regulator 2. The enzymatic activity of class III HDACs is not inhibited by compounds such as vorinostat or trichostatin A (TSA), that inhibit class I and II HDACs. Class IV HDAC is represented by HDAC11, which, like yeast Hda 1 similar 3, has conserved residues in the catalytic core region shared by both class I and II enzymes (Gao et al., 2002).

HDACs are not redundant in function (Marks and Dokmanovic, 2005Rosato and Grant, 2005Bolden et al., 2006). Class I HDACs are primarily nuclear in localization and ubiquitously expressed, while class II HDACs can be primarily cytoplasmic and/or migrate between the cytoplasm and nucleus and are tissue-restricted in expression.

The structural details of the HDAC–HDACi interaction has been elucidated in studies of a histone deacetylase-like protein from an anerobic bacterium with TSA and vorinostat (Finnin et al., 1999). More recently, the crystal structure of HDAC8–hydroxamate interaction has been solved (Somoza et al., 2004Vannini et al., 2004). These studies provide an insight into the mechanism of deacetylation of acetylated substrates. The hydroxamic acid moiety of the inhibitor directly interacts with the zinc ion at the base of the catalytic pocket.

This review focuses on the molecular mechanisms triggered by inhibitors of zinc-dependent HDACs in tumor cells that explain in part: (I) the effects of these compounds in inducing transformed cell death and (II) the relative resistance of normal and certain cancer cells to HDACi induced cell death. HDACi, for example, the hydroxamic acid-based vorinostat (SAHA, Zolinza), are promising drugs for cancer treatment (Richon et al., 1998Marks and Breslow, 2007). Several HDACi are in phase I and II clinical trials, being tested in different tumor types, such as cutaneous T-cell lymphoma, acute myeloid leukemia, cervical cancer, etc (Bug et al., 2005Chavez-Blanco et al., 2005Kelly and Marks, 2005;Duvic and Zhang, 2006) (Table 2). Although considerable progress has been made in elucidating the role of HDACs and the effects of HDACi, these areas are still in early stages of discovery.

Table 2 – HDACi in clinical trials.  Full table

http://www.nature.com/common/images/table_thumb.gif

Recent phylogenetic analyses of bacterial HDACs suggest that all four HDAC classes preceded the evolution of histone proteins (Gregoretti et al., 2004). This suggests that the primary activity of HDACs may be directed against non-histone substrates. At least 50 non-histone proteins of known biological function have been identified, which may be acetylated and substrates of HDACs (Table 1) (Glozak et al., 2005Marks and Dokmanovic, 2005;Rosato and Grant, 2005Bolden et al., 2006Minucci and Pelicci, 2006Zhao et al., 2006). In addition, two recent proteomic studies identified many lysine-acetylated substrates (Iwabata et al., 2005Kim et al., 2006). In view of all these findings, HDACs may be better called ‘N-epsilon-lysine deacetylase’. This designation would also distinguish them from the enzymes that catalyse other types of deacetylation in biological reactions, such as acylases that catalyse the deacetylation of a range of N-acetyl amino acids (Anders and Dekant, 1994).

Non-histone protein targets of HDACs include transcription factors, transcription regulators, signal transduction mediators, DNA repair enzymes, nuclear import regulators, chaperone proteins, structural proteins, inflammation mediators and viral proteins (Table 1). Acetylation can either increase or decrease the function or stability of the proteins, or protein–protein interaction (Glozak et al., 2005). These HDAC substrates are directly or indirectly involved in many biological processes, such as gene expression and regulation of pathways of proliferation, differentiation and cell death. These data suggest that HDACi could have multiple mechanisms of inducing cell growth arrest and cell death (Figure 1).

Figure 1.  Full figure and legend (90K)

Multiple HDACi-activated antitumor pathways. See text for detailed explanation of each pathway. HDAC6, histone deacetylase 6; HIF-1, hypoxia-induced factor-1; HSP90, heat-shock protein 90; PP1, protein phosphatase 1; ROS, reactive oxygen species; TBP2, thioredoxin binding protein 2; Trx, thioredoxin; VEGF, vascular endothelial growth factor.

http://www.nature.com/onc/journal/v26/n37/images/1210620f1.jpg

HDACi have been discovered with different structural characteristics, including hydroximates, cyclic peptides, aliphatic acids and benzamides (Table 2) (Miller et al., 2003Yoshida et al., 2003Marks and Breslow, 2007). Certain HDACi may selectively inhibit different HDACs. For example, MS-275 preferentially inhibits HDAC1 with IC50, at 0.3 m, compared to HDAC3 with an IC50 of about 8 m, and has little or no inhibitory effect against HDAC6 and HDAC8 (Hu et al., 2003). Two novel synthetic compounds, SK7041 and SK7068, preferentially target HDAC1 and 2 and exhibit growth inhibitory effects in human cancer cell lines and tumor xenograft models (Kim et al., 2003a). A small molecule, tubacin, selectively inhibits HDAC6 activity and causes an accumulation of acetylated -tubulin, but does not affect acetylation of histones, and does not inhibit cell cycle progression (Haggarty et al., 2003). No other HDACi for a specific HDAC has been reported.

HDACi selectively alters gene expression

HDACi-induced antitumor pathways

  • HDACi induces cell cycle arrest
  • HDACi activates the extrinsic apoptotic pathways
  • HDACi activates the intrinsic apoptotic pathways
  • HDACi induces mitotic cell death
  • HDACi induces autophagic cell death and senescence
  • ROS, thioredoxin and Trx binding protein 2 in HDACi-induced cell death
  • Antitumor effects of HDAC6 inhibition
  • Activation of protein phosphatase 1
  • Disruption of the function of chaperonin HSP90
  • Disruption of the aggresome pathway
  • HDACi inhibits angiogenesis

HDACi can block tumor angiogenesis by inhibition of hypoxia inducible factors (HIF) (Liang et al., 2006). HIF-1 and HIF-2 are transcription factors for angiogenic genes (Brown and Wilson, 2004). The oxygen level can control HIF activity through two mechanisms. First, under normoxic conditions, HIF-1 binds to von Hippel–Lindau protein (pVHL) and is degraded by the ubiquitination–proteasome system. Second, HIF activity depends on its transactivation potential (TAP), which is affected by the interaction with the coactivator p300/CBP among others. This complex can be disrupted by Factor Inhibiting HIF (FIH). Hypoxic conditions activate HIF through repression of the hydroxylases responsible for HIF degradation and loss of function.

 

Combination of HDACi with other antitumor agents

The HDACi have shown synergistic or additive antitumor effects with a wide range of antitumor reagents, including chemotherapeutic drugs, new targeted therapeutic reagents and radiation, by various mechanisms, some unique for particular combinations (Rosato and Grant, 2004Bhalla, 2005Marks and Dokmanovic, 2005Bolden et al., 2006).

Clinical development of HDACi

At least 14 different HDACi are in some phase of clinical trials as monotherapy or in combination with retinoids, taxols, gemcitabine, radiation, etc, in patients with hematologic and solid tumors, including cancer of lung, breast, pancreas, renal and bladder, melanoma, glioblastoma, leukemias, lymphomas, multiple myeloma (see National Cancer Institute website for CTEP clinical trials, ctep.cancer.gov or clinicaltrials.gov, and website of companies developing HDACi; Table 2).

The resistance to HDACi

Conclusions and perspectives

HDACs have multiple substrates involved in many biological processes, including proliferation, differentiation, apoptosis and other forms of cell death. Indeed, the fact that HDACs have histone and multiple nonhistone protein substrates suggests these enzymes should be referred to as ‘lysine deacetylases’. HDACi can cause transformed cells to undergo growth arrest, differentiation and/or cell death. Normal cells are relatively resistant to HDACi. HDACi are selective in altering gene expression, which may reflect, in part, the proteins composing the transcription factor complex to which HDACs are recruited. Both altered gene expression and changes in non-histone proteins caused by HDACi-induced acetylation play a role in the antitumor activity of HDACi. This is reflected in the different inducer-activated antitumor pathways in transformed cells (Figure 1). The functions of HDACs are not redundant. Thus, a pan-HDAC inhibitor such as vorinostat may activate more antitumor pathways and have therapeutic advantages compared to HDAC isotype-specific inhibitors.

Almost all cancers have multiple defects in the expression and/or structure of proteins that regulate cell proliferation and death. Compared to other antitumor reagents, the plurality of action of HDACi potentially confers efficacy in a wide spectrum of cancers, which have heterogeneity and multiple defects, both among different types of cancer and within different individual tumors of the same type. The multiple defects in a cancer cell may be the reason for transformed cells being more sensitive than normal cells to HDACi. Thus, given the relatively rapid reversibility of vorinostat inhibition of HDACs, normal cells may be able to compensate for HDACi-induced changes more effectively than cancer cells.

HDACi have synergistic or additive antitumor effects with many other antitumor reagents – suggesting that combination of HDACi and other anticancer agents may be very attractive therapeutic strategies for using these agents. Complete understanding of the mechanisms underlying the resistance and sensitivity to HDACi has obvious therapeutic importance. Targeting resistant factors will enhance the antitumor efficacy of HDACi. Identifying markers that can predict response to HDACi is a high priority for expanding the efficacy of these novel anticancer agents.

References  ….

NEWS AND VIEWS   Blocking HDACs boosts regulatory T cells

Nature Medicine News and Views (01 Nov 2007)

RESEARCH   

Dimethyl sulfoxide to vorinostat: development of this histone deacetylase inhibitor as an anticancer drug

Nature Biotechnology Research (01 Jan 2007)

Comments of reviewer:

 

The complexity of cancer has been known for almost a century, in large part from the seminal work of Otto Warburg in the 1920s using manometry, and following the work of Louis Pasteur 60 years earlier with fungi.

 

The volume of work and our unlocking of mitotic activity, apoptosis, mitochondria, and the cytoskeleton has taken us further into the cell interior, cell function, metabolic regulation, and pathophysiology.  Despite the enormous contributions to our knowledge of genomics, there is a large body of work in pathways of cell function that resides in no small part in activity of protein catalysts and enzymes.

 

The work that has been described covers only cyclic dinucleotides and HDACi’s.  Some of the activities described have relevance to microorganisms as well as cancer.  As we have seen, blocking HDACs boosts the activity of regulatory T-cells. There are many specific functional alterations elucidated above.

 

The first presentation is of an antibody that blocks an immune suppressor called CTLA4, antibodies that block another immune suppressor, PD1. This also involves a protein in cells that responds to foreign DNA to launch an innate immune response — the first response of the body’s immune system. The protein, dubbed STING, is triggered by small molecules called cyclic di-nucleotides (CDN), and makes immune cells release interferon and other cytokines that activate disease-fighting T cells and stimulate the production of antibodies that together kill invading pathogens and destroy cancer cells. Listeria bacteria, for example, secrete a CDN directly into infected cells that activates STING.

 

The second is resident in acetylation status of histones and non-histone proteins is determined by histone deacetylases (HDACs) and histone acetyl-transferases (HATs). HATs add acetyl groups to lysine residues, while HDACs remove the acetyl groups. In general, acetylation of histone promotes a more relaxed chromatin structure, allowing transcriptional activation. HDACs can act as transcription repressors, due to histone deacetylation, and consequently promote chromatin condensation. HDAC inhibitors (HDACi) selectively alter gene transcription, in part, by chromatin remodeling and by changes in the structure of proteins in transcription factor complexes (Gui et al., 2004).  The description focuses on the molecular mechanisms triggered by inhibitors of zinc-dependent HDACs in tumor cells that explain in part: (I) the effects of these compounds in inducing transformed cell death and (II) the relative resistance of normal and certain cancer cells to HDACi induced cell death.

 

HDACs have multiple substrates involved in many biological processes, including proliferation, differentiation, apoptosis and other forms of cell death. Indeed, the fact that HDACs have histone and multiple nonhistone protein substrates suggests these enzymes should be referred to as ‘lysine deacetylases’. HDACi can cause transformed cells to undergo growth arrest, differentiation and/or cell death. Normal cells are relatively resistant to HDACi. HDACi are selective in altering gene expression, which may reflect, in part, the proteins composing the transcription factor complex to which HDACs are recruited. Both altered gene expression and changes in non-histone proteins caused by HDACi-induced acetylation play a role in the antitumor activity of HDACi.

 

 

 

 

 

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Cholesterol metabolism in pancreatic cancer

Larry H. Bernstein, MD, FCAP, Curator

LPBI

 

 

New Pancreatic Treatment Shows Promise

http://www.genengnews.com/gen-news-highlights/new-pancreatic-treatment-shows-promise/81252686/

Study demonstrates how controlling cholesterol metabolism in pancreatic cancer cells reduces metastasis. [NIH].   http://www.genengnews.com/Media/images/GENHighlight/thumb_May4_2016_NIH_PancreaticCancerCells8616346835.jpg

Scientists say they have shown how controlling cholesterol metabolism in pancreatic cancer cells reduces metastasis, pointing to a potential new treatment using drugs previously developed for atherosclerosis.

“We show for the first time that if you control the cholesterol metabolism you could reduce pancreatic cancer spread to other organs,” said Ji-Xin Cheng, Ph.D., a professor in Purdue University’s Weldon School of Biomedical Engineering and Department of Chemistry. “We chose pancreatic cancer to test this approach because it is the most aggressive disease of all the cancers.”

Dr. Cheng had previously led a team of researchers discovering a link between prostate cancer’s aggressiveness and the accumulation of a compound produced when cholesterol is metabolized in cells, findings that could bring new diagnostic and treatment methods. The new study involved researchers at the Purdue Center for Cancer Research and School of Biomedical Engineering, the Indiana University Simon Cancer Center and School of Medicine, and Purdue’s Department of Biological Sciences, Department of Comparative Pathobiology, and Department of Biochemistry.

The findings, detailed in a paper (“Abrogating Cholesterol Esterification Suppresses Growth and Metastasis of Pancreatic Cancer”) just published in Oncogene, suggest that a class of drugs previously developed to treat atherosclerosis could be repurposed for treatment of pancreatic cancer and other forms of cancer. Atherosclerosis is the buildup of fats, cholesterol, and other substances in arteries, restricting blood flow.

The researchers found accumulations of the compound cholesteryl ester in human pancreatic cancer specimens and cell lines, demonstrating a link between cholesterol esterification and metastasis. Excess quantities of cholesterol result in cholesteryl ester being stored in lipid droplets within cancer cells.

“The results of this study demonstrate a new strategy for treating metastatic pancreatic cancer by inhibiting cholesterol esterification,” said Jingwu Xie, Ph.D., the Jonathan and Jennifer Simmons Professor at the Indiana University School of Medicine and a researcher at the Indiana University Melvin and Bren Simon Cancer Center.

The paper’s lead author is Purdue postdoctoral fellow Junjie Li, Ph.D. Purdue researchers have developed an analytical tool, Raman spectromicroscopy, that allows compositional analysis of single lipid droplets in living cells.

“We identified an aberrant accumulation of cholesteryl ester in human pancreatic cancer specimens and cell lines,” Dr. Li said. “Depletion of cholesterol esterification significantly reduced pancreatic tumor growth and metastasis in mice.”

Findings show that drugs like avasimibe, previously developed for treatment of atherosclerosis, reduced the accumulation of cholesteryl ester. Pancreatic cancer usually kills within a few months of diagnosis. It is hoped the potential new treatment might extend life of these patients for a year, Cheng said.

The accumulation of cholesteryl ester is controlled by an enzyme called acyl-coenzyme A acyltransferase-1 (ACAT-1), and findings have correlated a higher expression of the enzyme with a poor survival rate for patients. The researchers analyzed tissue samples from pancreatic cancer patients and then tested the drug treatment in a type of laboratory mice referred to as an orthotopic mouse model, developed at the IU School of Medicine. Specimens of human pancreatic tissues were obtained from the Simon Cancer Center Solid Tissue Bank.

Imaging showed a decrease of the number of lipid droplets, and Raman spectral analysis verified a significant reduction of cholesteryl ester in the lipid droplets, suggesting that avasimibe acted by blocking cholesterol esterification. The drug did not induce weight loss, and there was no apparent organ toxicity in the liver, kidney, lung and spleen, Dr. Cheng said.

Findings also showed that blocking storage of cholesteryl ester causes cancer cells to die, specifically due to damage to the endoplasmic reticulum, a workhorse of protein and lipid synthesis.

“By using avasimibe, a potent inhibitor of ACAT-1, we found that pancreatic cancer cells were much more sensitive to ACAT-1 inhibition than normal cells,” added Dr. Cheng.

Additional research confirmed that the anticancer effect of avasimibe is specific to ACAT-1 inhibition. The researchers performed various biochemical assays and “genetic ablation” to confirm the drug’s anticancer effect.

“The results showed that avasimibe treatment for four weeks remarkably suppressed tumor size and largely reduced tumor growth rate,” said paper co-author Timothy Ratliff, the Robert Wallace Miller Director of Purdue’s Center for Cancer Research. “Metastatic lesions in lymph nodes and distant organs also were assessed at the end of the study. A much higher number of metastatic lesions in lymph nodes were detected in the control group than the avasimibe-treated group.”

Each mouse in the control group showed at least one metastatic lesion in the liver. In contrast, only three mice in the avasimibe-treated group showed single lesion in liver.

 

Abrogating cholesterol esterification suppresses growth and metastasis of pancreatic cancer

J Li1, D Gu2, S S-Y Lee1, B Song1, S Bandyopadhyay3, S Chen4, S F Konieczny3,5, T L Ratliff5,6, X Liu5,7, J Xie2 and J-X Cheng1,5
O
ncogene 2 May 2016;                                         http://dx.doi.org:/10.1038/onc.2016.168

Cancer cells are known to execute reprogramed metabolism of glucose, amino acids and lipids. Here, we report a significant role of cholesterol metabolism in cancer metastasis. By using label-free Raman spectromicroscopy, we found an aberrant accumulation of cholesteryl ester in human pancreatic cancer specimens and cell lines, mediated by acyl-CoA cholesterol acyltransferase-1 (ACAT-1) enzyme. Expression of ACAT-1 showed a correlation with poor patient survival. Abrogation of cholesterol esterification, either by an ACAT-1 inhibitor or by shRNA knockdown, significantly suppressed tumor growth and metastasis in an orthotopic mouse model of pancreatic cancer. Mechanically, ACAT-1 inhibition increased intracellular free cholesterol level, which was associated with elevated endoplasmic reticulum stress and caused apoptosis. Collectively, our results demonstrate a new strategy for treating metastatic pancreatic cancer by inhibiting cholesterol esterification.

Metastasis is the major cause of cancer-related mortality. Though localized tumors can often be treated by surgery or other therapies, treatment options for metastatic diseases are limited. Cancer metastasis has been revealed to be a multiple step process, including cancer cell migration, local invasion, intravasation, circulation through blood and lymph vessels, extravasation, survival and colonization in distant organs.1, 2, 3Mediators identified in these processes have provided the basis for the development of therapies to target metastasis. Current therapeutic strategies for treating metastatic tumors mainly focus on targeting the adhesive molecules and extracellular proteases.4However, these therapeutics have not been proven to be effective in clinical trials, partially owing to the various escape mechanisms used by the metastatic cancer cells.2, 5, 6 Thus, an unmet need exists to develop new therapeutic strategies for treating metastatic cancers.

Recent advances in cancer metabolism have unveiled many potential therapeutic targets for cancer treatment. Metabolic reprogramming, a strategy used by cancer cells to adapt to the rapid proliferation, is being recognized as a new hallmark of cancer.7 Substantial studies have found increased glycolysis, glutaminolysis, nucleotide and lipid synthesis in cancer cells.7, 8, 9,10 Considering that altered metabolic pathways only happen in cancer cells but not in normal cells, targeting these pathways may provide cancer-specific treatments. A number of inhibitors of metabolic enzymes, such as glycolysis inhibitors, are under clinical trials as targeted cancer therapeutics.11

Of various metabolic pathways, lipid metabolism has been suggested to have an important role in cancer cell migration, invasion and metastasis.12 A recent study reported that surrounding adipocytes provide energy source for ovarian cancer cells to promote its rapid growth and metastasis.13 Blocking lipidde novo synthesis pathway has been shown to suppress tumor regrowth and metastasis after anti-angiogenesis treatment withdrawal.14 In parallel, lipolysis by the enzyme monoacylglycerol lipase was shown to regulate the fatty acid network, which promotes cancer cell migration, invasion and growth.15

Cholesterol, a critical component of the plasma membrane, is also implied to be correlated to cancer metastasis.16 It has been shown that prostate cancer bone metastases contain a high level of cholesterol.17 Modulation of cholesterol level in plasma membrane was shown to regulate the capability of cell migration.18, 19Moreover, cholesterol-enriched lipid rafts were shown to have an essential role in cancer cell adhesion and migration.20 Mammalian cells obtain cholesterol either from de novo synthesis or from the uptake of low-density lipoprotein (LDL).21 Inside cells, excess free cholesterol is esterified and stored as cholesteryl ester (CE) in lipid droplets (LDs), which is mediated by acyl-CoA cholesterol acyltransferase (ACAT).22 Increased CE level has been reported in breast cancer,23 leukemia,24 glioma25 and prostate cancer.26Despite these advances, the role of cholesterol esterification in cancer progression, especially in cancer metastasis, is not well understood.

In this article, we report a link between cholesterol esterification and metastasis in pancreatic cancer. Using stimulated Raman scattering (SRS) microscopy and Raman spectroscopy to map LDs stored inside single cells and analyze the composition of individual LDs, we identified an aberrant accumulation of CE in human pancreatic cancer specimens and cell lines. Abrogation of cholesterol esterification, either by inhibiting ACAT-1 enzyme activity or by shRNA knockdown of ACAT-1 expression, significantly reduced pancreatic tumor growth and metastasis in an orthotopic mouse model. Mechanistically, inhibition of cholesterol esterification disturbed cholesterol homeostasis by increasing intracellular free cholesterol level, which was associated with elevated endoplasmic reticulum (ER) stress and eventually led to apoptosis.

In this study, we revealed a link between CE accumulation and pancreatic cancer metastasis. Accumulation of CE via ACAT-1 provides a mechanism to keep high metabolic activity and avoid toxicity from excess free cholesterol. Previously, CE has been reported in breast cancer,23 leukemia,24 glioma25 and prostate cancer.26 Inhibition of cholesterol esterification was shown to suppress tumor growth or cancer cell proliferation.24, 25, 26 Here, we demonstrate that inhibition of cholesterol esterification can be used to treat metastatic pancreatic cancer.

Cholesterol is an essential lipid having important roles in membrane construction, hormone production and signaling.21Aberrant cholesterol metabolism is known to be associated with cardiovascular diseases and cancers.35, 36 Statins, inhibitors of HMG-CoA reductase, have been explored as potential therapies for pancreatic cancer.37 However, statins were not associated with a reduced risk of pancreatic cancer in clinical trials.38 One possible reason is that HMG-CoA reductase is also required for downstream protein prenylation, a critical process for protein activation.39Thus, the effect of statin is not just inhibiting cholesterol synthesis, but also other pathways which may render toxicity to normal cells. This non-specific toxicity is a possible reason for the limited anti-cancer outcome of statin in clinical trials.

Our study identified cholesterol esterification as a novel target for suppression of pancreatic cancer proliferation and metastasis. Inhibitors of ACAT-1 are expected to have great value as cancer-targeting therapeutics, as CE accumulation only occurs in cancer tissues or cell lines. Our animal studies with avasimibe treatment showed no adverse effect to the animals at a dosage of 15mg/kg. More importantly, modulation of cholesterol esterification suppressed not only tumor growth but also tumor metastasis. These results are expected to stimulate further biological studies to fully appreciate the role of cholesterol metabolism in cancer initiation and progression. As CE accumulation happens in several types of aggressive cancer, blocking cholesterol esterification could be pursued as a therapeutic strategy for other types of cancers. By combining with existing chemotherapies, such as gemcitabine, we believe this metabolic treatment possesses high possibilities to extend patients’ survival time by retarding cancer progression and metastasis.

The molecular mechanism that links CE accumulation to cancer aggressiveness needs further studies. One possible mechanism is that cholesterol esterification keeps signaling pathways active by maintaining a low free cholesterol environment. One of the possible targets is the caveolin-1 signaling pathway. Caveolin-1, a regulator of cellular cholesterol homeostasis, is considered as a marker for pancreatic cancer progression.11 Particularly, a promoting role of caveolin-1 in pancreatic cancer metastasis has been reported.40 Our preliminary studies showed ACAT-1 inhibition reduced the expression level of SREBP1, caveolin-1 and phosphorylated ERK1/2 (unpublished data). The effect on caveolin-1 is probably mediated by SREBP1, which senses the intracellular cholesterol homeostasis.41 Meanwhile, caveolin-1 may have an important role in mediating the action of SREBP1 on MAPK pathways,42, 43 which are known to have essential roles in cancer cell metastasis.44 Therefore, it is possible that increased free cholesterol level induced by ACAT-1 inhibition inactivates SREBP1, leading to downregulation of caveolin-1/MAPK pathway, which contributes to the reduced cancer aggressiveness.

Besides the caveolin-1/MAPK signaling, other possibilities include the potential alteration of the membrane composition, such as lipid rafts, by ACAT-1 inhibition. Lipid rafts are known to provide platforms for multiple cellular signaling pathways.20 Thus, modulation of cholesterol metabolism is likely to have more profound effects via other signaling pathways. Future studies are needed to fully elucidate the molecular mechanism.

 

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Metabolic insight into cancer cell survival

Larry H. Bernstein, MD, FCAP, Curator

LPBI

 

Revised 4/20/2016

AACR 2016: Novel Epigenetic Drug Therapeutics Revealed

http://www.genengnews.com/gen-news-highlights/aacr-2016-epigenetic-drug-therapeutics/81252634/

As the 2016 American Association for Cancer Research meeting begins to downshift toward a close, the presentation sessions certainly did not suffer from a lack of enthusiasm from attendees or high-quality research from presenters. Of particular note was a major symposium that discussed next-generation epigenetic therapeutics.

In the past several years, there have been a variety of epigenetic targets exploited by newly developed drug compounds, many of which have already progressed into clinical trials. Often these compounds will target specific classes of epigenetic regulators like acetylases and histone demethylases, for instance, the small-molecule inhibitors of protein interacting bromodomains—implicated in a diverse range of cancers—and methyltransferase inhibitors, such as lysyl demethylases (KDM).

However, for all of their recently achieved success, researchers are continually searching for increasingly rapid methods to validate epigenetic drug targets. Session Chairperson Udo Oppermann, Ph.D., principal investigator at the University of Oxford, stressed that open access research and continued investigator cooperation were key factors for driving rapid development of novel therapeutics in the field. Anecdotally, Dr. Oppermann noted that if biologists were a bit more like the international cooperative teams of physicists that discovered the Higgs boson or gravitational waves, many biological endeavors would advance rather quickly.

After providing the audience with a brief introduction to the symposium’s topic, Dr. Oppermann described his current research on histone demethylase inhibitors in multiple myeloma and the connection to metabolic pathways. He surmised that tricarboxylic acid (TCA) cycle-derived metabolites can link cellular metabolism to cancer—impacting epigenetic landscapes. Specifically, the TCA intermediates are inhibitors of KDMs, ultimately controlling epigenetic and metabolic regulation.

Furthermore, Dr. Oppermann’s group was able to show that treatment of myeloma cell lines with the potent and specific histone demethylase inhibitor GSK-J4 was able to reverse the Myc-driven metabolic dependency, forcing a selected amino acid depletion. This deficiency led to the integrated stress response and the activation of proapoptotic genes. This work helps to solidify further the potent nature of GSK-J4 in cancer while simultaneously uncovering the metabolic mechanisms that cancer cells employ to keep their overproliferative phenotypes progressing forward.

Next, Tomasz Cierpicki, Ph.D., assistant professor at the University of Michigan, described his work on targeting leukemic stem cells with small-molecule inhibitors of the protein regulator of cytokinesis 1 (PRC1). Dr. Cierpicki took the audience through his research design, which was to target BMI1, an oncogene that determines the proliferative capacity and self-renewal potential of normal and leukemic stem cells. BMI1 has been implicated in a variety of tumors and is essential for the Polycomb Repressive Complex 1 (PRC1). Moreover, BMI1 interacts with the RING1B protein to form an active E3 ubiquitin ligase that targets histone H2A, modifying epigenetic regulation mechanisms.

Dr. Cierpicki’s laboratory looked at inhibitors of the RING1B–BMI1 E3 ligase complex as potential therapeutic agents targeting cancer stem cells. Using an array of techniques from fragment screening to medicinal chemistry, the researchers were able to identify potent compounds that bound to RING1B–BMI1 and inhibit its E3 ubiquitin ligase activity with low micromolar affinities. When testing in vitro, the inhibitors revealed robust downregulation of H2A ubiquitination. Dr. Cierpicki and his colleagues found that the RING1–BMI1 inhibitor blocked the self-renewal capacity of the stem cells and induced cellular differentiation—validating RING ligases as a novel epigenetic drug target.

Finishing up the session was William Sellers, M.D., vice president and global head of oncology for the Novartis Institutes for BioMedical Research. Dr. Sellers’ research is focused on what genes are necessary for epigenetic regulation of cancer and how they are linked to essential metabolic processes. He and his colleagues accomplished their studies through the use of large-scale shRNA screening across a diverse set of 390 cancer cell lines.

Utilizing deep small hairpin RNA (shRNA) screening libraries, at 20 shRNAs per genome, provided the investigators with highly robust gene-level data, which resulted in the emergence of several distinct classes of cancer-dependent genes. For example, Dr. Sellers’ group found that several known oncogenes fell into the genetic dependence class, whereas other genes were sorted into lineage, paralog, and collateral synthetic lethality dependent classes.

An interesting example from Dr. Sellers’ work was the link his laboratory discovered between polyamine metabolism and salvage and the protein arginine methyltransferase 5 (PRMT5). In particular, the loss of methylthioadenosine phosphorylase (MTAP)—which has been observed in many solid tumors and hematologic malignancies—resulted in the accumulation of S-methyl-5′-thioadenosine (MTA), which specifically inhibited the epigenetic mechanisms of PRMT5. The culmination Dr. Sellers’ analysis led to the finding that PRMT5 is a novel target for therapeutic development in MTAP deleted cancers.

These three presentations represented some of the excellent, cutting-edge research that is not only looking to develop novel drug therapeutics but also trying to uncover the underlying molecular mechanisms of epigenetic regulation and cancer.

 

 

 

A Metabolic Twist that Drives Cancer Survival

http://www.technologynetworks.com/Metabolomics/news.aspx?ID=190262

A novel metabolic pathway that helps cancer cells thrive in conditions that are lethal to normal cells has been identified.

 

“It’s long been thought that if we could target tumor-specific metabolic pathways, it could lead to effective ways to treat cancer,” said senior author Dr. Ralph DeBerardinis, Associate Professor of CRI and Pediatrics, Director of CRI’s Genetic and Metabolic Disease Program, and Chief of the Division of Pediatric Genetics and Metabolism at UT Southwestern. “This study finds that two very different metabolic processes are linked in a way that is specifically required for cells to adapt to the stress associated with cancer progression.”

The study, available online today in the journal Nature, reveals that cancer cells use an alternate version of two well-known metabolic pathways called the pentose phosphate pathway (PPP) and the Krebs cycle to defend against toxins. The toxins are reactive oxygen species (ROS) that kill cells via oxidative stress.

This work builds on earlier studies by Dr. DeBerardinis’ laboratory that found the Krebs cycle, a series of chemical reactions that cells use to generate energy, could reverse itself under certain conditions to nourish cancer cells.

Dr. DeBerardinis said most normal cells and tumor cells grow by attaching to nutrient-rich tissue called a matrix. “They are dependent on matrix attachment to receive growth-promoting signals and to regulate their metabolism in a way that supports cell growth, proliferation, and survival,” he said.

Detachment from the matrix results in a sudden increase in ROS that is lethal to normal cells, he added. Cancer cells seem to have a workaround.

The destruction of healthy cells when detached from the matrix was reported in a landmark 2009 Nature study by Harvard Medical School cell biologist Dr. Joan Brugge. Intriguingly, that same study found that inserting an oncogene – a gene with the potential to cause cancer – into a normal cell caused it to behave like a cancer cell and survive detachment, said Dr. DeBerardinis, who also is affiliated with the Eugene McDermott Center for Human Growth & Development, holds the Joel B. Steinberg, M.D. Chair in Pediatrics, and is a Sowell Family Scholar in Medical Research at UT Southwestern.

“Another Nature study, this one from CRI Director Dr. Sean Morrison’s laboratory in November 2015, found that the rare skin cancer cells that were able to detach from the primary tumor and successfully metastasize to other parts of the body had the ability to keep ROS levels from getting dangerously high,” Dr. DeBerardinis said. Dr. Morrison, also a CPRIT Scholar in Cancer Research and a Howard Hughes Medical Institute Investigator, holds the Mary McDermott Cook Chair in Pediatrics Genetics at UT Southwestern.

Working under the premise that the two findings were pieces of the same puzzle, a crucial part of the picture seemed to be missing, he said.

It was known for decades that the PPP was a major source of NADPH, which provides a source of reducing equivalents (that is, electrons) to scavenge ROS; however, the PPP produces NADPH in a part of the cell called the cytosol, whereas the reactive oxygen species are generated primarily in another subcellular structure called the mitochondria.

“If you think of ROS as fire, then NADPH is like the water used by cancer cells to douse the flames,” Dr. DeBerardinis said. But how could NADPH from the PPP help deal with the stress of ROS produced in a completely different part of the cell? “What we did was to discover how this happens,” Dr. DeBerardinis said.

The current study in Nature demonstrates that cancer cells use a “piggybacking” system to carry reducing equivalents from the PPP into the mitochondria. This movement involves an unusual reaction in the cytosol that transfers reducing equivalents from NADPH to a molecule called citrate, similar to a reversed reaction of the Krebs cycle, he said. The citrate then enters the mitochondria and stimulates another pathway that results in the release of reducing equivalents to produce NADPH right at the location of ROS creation, allowing the cancer cells to survive and grow without the benefit of matrix attachment.

“We knew that both the PPP and Krebs cycle provide metabolic benefits to cancer cells.  But we had no idea that they were linked in this unusual fashion,” he said. “Strikingly, normal cells were unable to transport NADPH by this mechanism, and died as a result of the high ROS levels.”

Dr. DeBerardinis stressed that the findings were based on cultured cell models, and more research will be necessary to test the role of the pathway in living organisms. “We are particularly excited to test whether this pathway is required for metastasis, because cancer cells need to survive in a matrix-detached state in the circulation in order to metastasize,” he said.

CRI scientists find novel metabolic twist that drives cancer survival
http://www.utsouthwestern.edu/newsroom/news-releases/year-2016/april/cancer-metabolism.html

https://pharmaceuticalintelligence.com/2016/04/09/programmed-cell-death-and-cancer-therapy/5 days ago Cancer Cell Survival Driven by Novel Metabolic Pathway … This new study describes an alternate version of two wellknown metabolic pathways, the pentose phosphate pathway (PPP) and the Krebs cycle,…

http://www.nature.com/nature/journal/v481/n7381/abs/nature10642.html Jan 19, 2012 Nature | Letter …. DeBerardinis, R. J. et al. Beyond aerobic …. Andrew R. Mullen,; Pei-Hsuan Chen,; Tzuling Cheng &; Ralph J. DeBerardinis ..   

Haematopoietic stem cells require a highly regulated protein – Nature
http://www.nature.com/nature/journal/v509/n7498/abs/nature13035.html May 1, 2014 Nature | Article. Print; Share/ ….. synthesis. Nature Methods 6, 275–277 (2009) …. Robert A. J. Signer,; Jeffrey A. Magee &; Sean J. Morrison …       
http://www.nature.com/nature/journal/v527/n7577/abs/nature15726.html Nov 12, 2015 Nature | Article ….. Multistep nature of metastatic inefficiency: dormancy of solitary cells after successful extravasation and ….. Sean J. Morrison …     
Deep imaging of bone marrow shows non-dividing stem Nature
http://www.nature.com/nature/journal/v526/n7571/abs/nature15250.html   Oct 1, 2015 Nature | Letter. Print; Share/ …… Morrison, S. J. & Scadden, D. T. The bone marrow niche for ….. Kiranmai S. Kocherlakota &; Sean J. Morrison …
https://pharmaceuticalintelligence.com/category/cancer-biology-innovations-in-cancer-therapy/genomic-expression/Glutamine and cancer: cell biology, physiology, and clinical opportunities …. Metabolism of glutamine-derived α-ketoglutarate in the TCA cycle serves … known as hexosamines, that are used to glycosylate growth factor receptors and …… of two wellknown metabolic pathways, the pentose phosphate pathway (PPP )
The Mitochondrial Warburg Effect: A Cancer Enigma – IBC Journal
http://www.ibc7.org/article/file_down.php?pid=48&mode=article This feature of cancer cells is known as the Warburg effect, named … new paradigm of collaboration and a well-designed systemic approach will supply … Krebs cycle. … The pentose phosphate pathway uses glucose to produce ribose, which is used … glucose is taken up into cells, it is used in two main metabolic pathways

 

New paper offers intriguing insights into tumor metabolism     William G. Gilroy    August 19, 2009

Posted In: Research

A paper appearing in this week’s edition of the journal Nature by a team of researchers that includes University of Notre Dame biologist Zachary T. Schafer has important new implications for understanding the metabolism of tumors.

Schafer, an assistant professor of biological sciences and Coleman Junior Chair of Cancer Biology, points out that in the early stages of tumor formation some cells become detached from their normal cellular matrix. These “homeless” cells tend to develop certain defects that stop them from becoming cancerous. In a process known as apoptosis, these precancerous cells essentially kill themselves, allowing them to be destroyed by immune system cells.

The prevailing wisdom among researchers has been that apoptosis was the only way that cells could die.

In studies conducted prior to the research described in the Nature paper, it was found that even when apoptosis was inhibited in detached, precancerous cells, they still eventually died. Intrigued by these results, a team of researchers led by Joan S. Brugge, Louise Foote Pfieffer Professor of Cell Biology at Harvard Medical School, and Schafer decided to take a closer look.

They report in this week’s Nature paper that they found that even when apoptosis was inhibited in detached cells endowed with a cancer-causing gene, they still were incapable of absorbing glucose, their primary energy source. Additionally, the cells displayed signs of oxidative stress, which is a harmful accumulation of oxygen-derived molecules called reactive oxygen species (ROS). The research also revealed decreased ATP production, a key factor in energy transport in the cells.

Schafer notes that this combination of loss of glucose transport, decreased ATP production and heightened oxidative stress reveal a manner of cell death that hadn’t been previously demonstrated to play a role in this context.

In the next phase of the study, Schafer engineered the cells to express a high level of HER2, a gene known to be hyperactive in many breast cancer tumors. He also treated the cells with antioxidants to relieve oxidative stress.

Both approaches helped the cells survive. The HER2-treated cells regained glucose transport, avoided oxidative stress and recovered ATP levels.
Most surprisingly, the antioxidants restored metabolic activity in the cells by allowing fatty acids to be effectively used instead of glucose as an energy source, providing them with a chance to survive.

“Our results raise the possibility that antioxidant activity might allow early stage tumor cells to survive where they would otherwise die from these metabolic defects,” Schafer said.

He also cautions that while the antioxidant findings were surprising, their research was done solely in cell cultures and more research needs to be done before there are clear implications for individuals and their diets.

The paper does, however, offer important new clues about the metabolism of tumor cells and important information that may lead to drugs that can developed to target them.

 

Antioxidant and Oncogene Rescue of Metabolic Defects Caused by
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2931797/Aug 19, 2009
Nature. Author manuscript; available in PMC 2010 Sep 2. Published in … Y. Irie,1 Sizhen Gao,1 Pere Puigserver,1,2 and Joan S. Brugge1,*.

http://www.nature.com/cdd/journal/v10/n8/full/4401251a.html
Proteasome inhibitors have been shown to be effective in cancer treatment, an ability … a specific inhibitor of 26S proteasome, also reduced cell viability ( 80% with 10 mu …
be a consequence of the increased generation of ROS caused by MG132. …. vectors endowed with the wild type forms of RB or p53 genes (Figure 1f).

 

The Metastasis-Promoting Roles of Tumor-Associated Immune Cells

Tumor metastasis is driven not only by the accumulation of intrinsic alterations in malignant cells, but also by the interactions of cancer cells with various stromal cell components of the tumor microenvironment. In particular, inflammation and infiltration of the tumor tissue by host immune cells, such as tumor-associated macrophages, myeloid-derived suppressor cells, and regulatory T cells have been shown to support tumor growth in addition to invasion and metastasis. Each step of tumor development, from initiation through metastatic spread, is promoted by communication between tumor and immune cells via the secretion of cytokines, growth factors and proteases that remodel the tumor microenvironment. Invasion and metastasis requires neovascularization, breakdown of the basement membrane, and remodeling of the extracellular matrix for tumor cell invasion and extravasation into the blood and lymphatic vessels. The subsequent dissemination of tumor cells to distant organ sites necessitates a treacherous journey through the vasculature, which is fostered by close association with platelets and macrophages. Additionally, the establishment of the pre-metastatic niche and specific metastasis organ tropism is fostered by neutrophils and bone marrow-derived hematopoietic immune progenitor cells and other inflammatory cytokines derived from tumor and immune cells, which alter the local environment of the tissue to promote adhesion of circulating tumor cells. This review focuses on the interactions between tumor cells and immune cells recruited to the tumor microenvironment, and examines the factors allowing these cells to promote each stage of metastasis.

 

Once established, tumors are quite adept at preventing anti-tumor immune responses, and several defense mechanisms to circumvent immune detection have been described including antigen loss, down-regulation of major histocompatibility molecules (MHC), deregulation or loss of components of the endogenous antigen presentation pathway, and tumor-induced immune suppression mediated through cytokine secretion or direct interactions between tumor ligands and immune cell receptors [2]. These mechanisms contribute to the process of immunoediting in which tumor cell subpopulations susceptible to immune recognition are lysed and eliminated, while resistant tumor cells proliferate and increase their frequency in the developing neoplasia [3]. However, tumors not only effectively escape immune recognition, they also actively subvert the normal anti-tumor activity of immune cells to promote further tumor growth and metastasis.

During early stages of cancer development, infiltrating immune cell populations are primarily tumor suppressive, but depending on the presence of accessory stromal cells, the local cytokine milieu, and tumor-specific interactions, these immune cells can undergo phenotypic changes to enhance tumor cell dissemination and metastasis. For instance, CD4+ T cells, macrophages, and neutrophils have all been shown to possess opposing properties depending on the inflammatory state of the tumor environment, the tissue context, and other cellular stimuli intrinsic to the altered tumor cells [4, 5]. These features are dependent upon the inherent plasticity of immune cells in response to stimulatory or suppressive cytokines [6]. Notably, the switch from a Th1 tumor-suppressive phenotype such as CD4+ “helper” T cells, which aid cytotoxic CD8+ T cells in tumor rejection, to a Th2 tumor-promoting “regulatory” phenotype, which blocks CD8+ T-cell activity, is a characteristic outcome in the inflammatory, immune-suppressive tumor microenvironment [5, 7]. Likewise, M1 macrophages and N1 neutrophils are known to have pronounced anti-tumor activity; however, these immune cells are often subverted to a tumor-promoting M2 and N2 phenotype, respectively, in response to immune-suppressive cytokines secreted by tumor tissue [8].

 

The crosstalk that occurs between tumor and immune cells within the tumor microenvironment, the circulation, or at distant metastatic sites has been clearly shown to foster metastatic dissemination. Immune cells as well as the suppressive factors that they secrete represent potential targets for therapeutic intervention. Regardless of their source, cytokines, chemokines, proteases, and growth factors are some of the main factors contributing to immunosuppression and immune-mediated tumor progression. These molecules can be produced by immune, stromal, or malignant cells and can act in paracrine and autocrine fashion to promote each stage of tumor cell invasion and metastasis by enhancing inflammation, angiogenesis, tumor proliferation, and recruitment of additional immunosuppressive and tumor-promoting immune cells. These secreted factors provide the malignant cells with an abundant source of growth and survival signals that perpetuate a supportive microenvironment for tumor metastasis and represent some of the most attractive targets for directed anti-tumor therapy. Immune pathways provide numerous soluble targets for cancer treatment, and indeed, many drugs to target immune-suppressive molecules are moving forward in clinical trials. For instance, the anti-RANKL (Denosumab) antibody has been shown to effectively inhibit bone metastasis in prostate cancer patients [201], while a variety of neutralizing antibodies to IL-1β and IL-1 receptor have been shown to have efficacy in treating metastasis in pre-clinical animal models [202]. Several agents that target IL-1 or other immune-suppressive cytokines are already approved for the treatment of some inflammatory diseases and are prime candidates for human trails [202]. Additionally, other proteins involved in tumor progression that are induced directly or indirectly by immune cell populations, such as EMT-associated transcription factors, adhesion molecules, and tumor receptors and ligands which mediate immune suppression, could also be targeted with small molecules or blocking antibodies. Antibodies against two surface molecules expressed by suppressive lymphoid cells, anti-CTLA-4 (ipiliumimab) [203, 204] and anti-PD-1 have been recently gaining increasing support from clinical trials for their effective treatment for many forms of cancer including advanced melanoma and prostate cancer [205, 206]. Specifically, anti-CTLA-4 has been shown to be particularly efficacious in metastatic melanoma, while anti-PD-1 has only just begun a comprehensive evaluation in clinical trials [204, 207]. Likewise, non-steroidal anti-inflammatory drugs (NSAIDS) to prevent or treat chronic inflammation and lymphangiogenesis [208210], and anti-coagulants to prevent platelet aggregation on circulating tumor cells [211] are just two examples of a multitude of therapeutic agents that could be utilized to prevent immune-mediated tumor progression at unique stages of metastasis. Of course, new methods or biomarkers for the detection of patients at risk of tumor progression or metastasis are also desperately needed to tailor personalized therapy for patients to obtain the best possible clinical outcome.

 

  1. https://pharmaceuticalintelligence.com/category/cancer-and-therapeutics/Mar 26, 2016 This turns your immune systems ability to attack and kill cancer cells back on” …. the rare skin cancer cells that were able to detach from theprimary tumor and successfully metastasize to other parts of the body had the ability to keep ROS levels from getting dangerously high,” Dr. DeBerardinis remarked.

  2. https://pharmaceuticalintelligence.com/tag/histone-deacetylase-inhibitors-hdac/The HDAC-inhibiting agent romidepsin significantly increased T-cell tumor … skin cancer cells that were able to detach from the primary tumor and successfully … of the body had the ability to keep ROS levels from getting dangerously high,” Dr. …. Sensitivity for EGFR or KRAS was higher in patients with multiplemetastatic …

  3. https://pharmaceuticalintelligence.com/category/cancer-biology-innovations-in-cancer-therapy/genomic-expression/In a study involving 320 patients, the researchers were able to infer cell death in …. Glutamine and cancer: cell biology, physiology, and clinical opportunities … On the other hand, GLS2 expression is enhanced in some neuroblastomas, …… of the body had the ability to keep ROS levels from getting dangerously high,” Dr.

 

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