Supriya Shah

Bisphenol A facilitates bypass of androgen ablation therapy in prostate cancer

Prostatic adenocarcinomas depend on androgen for growth and survival. First line treatment of disseminated disease exploits this dependence by specifically targeting androgen receptor function. Clinical evidence has shown that androgen receptor is reactivated in recurrent tumors despite the continuance of androgen deprivation therapy. Several factors have been shown to restore androgen receptor activity under these conditions, including somatic mutation of the androgen receptor ligand-binding domain. We have shown previously that select tumor-derived mutants of the androgen receptor are receptive to activation by bisphenol A (BPA), an endocrine-disrupting compound that is leached from polycarbonate plastics and epoxy resins into the human food supply. Moreover, we have shown that BPA can promote cell cycle progression in cultured prostate cancer cells under conditions of androgen deprivation. Here, we challenged the effect of BPA on the therapeutic response in a xenograft model system of prostate cancer containing the endogenous BPA-responsive AR-T877A mutant protein. We show that after androgen deprivation, BPA enhanced both cellular proliferation rates and tumor growth. These effects were mediated, at least in part, through androgen receptor activity, as prostate-specific antigen levels rose with accelerated kinetics in BPA-exposed animals. Thus, at levels relevant to human exposure, BPA can modulate tumor cell growth and advance biochemical recurrence in tumors expressing the AR-T877A mutation. [Mol Cancer Ther 2006;5(12):3181–90]

Malic enzyme tracers reveal hypoxia-induced switch in adipocyte NADPH pathway usage

The critical cellular hydride donor NADPH is produced through various means, including the oxidative pentose phosphate pathway (oxPPP), folate metabolism and malic enzyme. In growing cells, it is efficient to produce NADPH via the oxPPP and folate metabolism, which also make nucleotide precursors. In nonproliferating adipocytes, a metabolic cycle involving malic enzyme holds the potential to make both NADPH and two-carbon units for fat synthesis. Recently developed deuterium ((2)H) tracer methods have enabled direct measurement of NADPH production by the oxPPP and folate metabolism. Here we enable tracking of NADPH production by malic enzyme with [2,2,3,3-(2)H]dimethyl-succinate and [4-(2)H]glucose. Using these tracers, we show that most NADPH in differentiating 3T3-L1 mouse adipocytes is made by malic enzyme. The associated metabolic cycle is disrupted by hypoxia, which switches the main adipocyte NADPH source to the oxPPP. Thus, (2)H-labeled tracers enable dissection of NADPH production routes across cell types and environmental conditions.

Targeting Pioneering Factor and Hormone Receptor Cooperative Pathways to Suppress Tumor Progression

Nuclear receptors and pioneer factors drive the development and progression of prostate cancer. In this disease, aggressive disease phenotypes and hormone therapy failures result from resurgent activity of androgen receptor (AR) and the upregulation of coactivator protein p300 and pioneer factors (e.g., GATA2 and FOXA1). Thus, a major emphasis in the field is to identify mechanisms by which castrate-resistant AR activity and pioneer factor function can be combinatorially suppressed. Here we show that the turmeric spice isoflavone curcumin suppresses p300 and CBP occupancy at sites of AR function. Curcumin reduced the association of histone acetylation and pioneer factors, thereby suppressing AR residence and downstream target gene expression. Histone deacetylase inhibitors reversed the effects of curcumin on AR activity, further underscoring the impact of curcumin on altering the chromatin landscape. These functions precluded pioneer factor occupancy, leading ultimately to a suppression of ligand-dependent and ligand-independent AR residence on chromatin. Moreover, these functions were conserved even in cells with heightened pioneer factor activity, thus identifying a potential strategy to manage this subclass of tumors. Biological relevance was further identified using in vivo xenograft models mimicking disease progression. Curcumin cooperated in vivo with androgen deprivation as indicated by a reduction in tumor growth and delay to the onset of castrate-resistant disease. Together, our results show the combinatorial impact of targeting AR and histone modification in prostate cancer, thus setting the stage for further development of curcumin as a novel agent to target AR signaling.

2,2-Bis(4-Chlorophenyl)-1,1-Dichloroethylene Stimulates Androgen Independence in Prostate Cancer Cells through Combinatorial Activation of Mutant Androgen Receptor and Mitogen-Activated Protein Kinase Pathways

Therapy resistance represents a major clinical challenge in disseminated prostate cancer for which only palliative treatment is available. One phenotype of therapy-resistant tumors is the expression of somatic, gain-of-function mutations of the androgen receptor (AR). Such mutant receptors can use noncanonical endogenous ligands (e.g., estrogen) as agonists, thereby promoting recurrent tumor formation. Additionally, selected AR mutants are sensitized to the estrogenic endocrine-disrupting compound (EDC) bisphenol A, present in the environment. Herein, screening of additional EDCs revealed that multiple tumor-derived AR mutants (including T877A, H874Y, L701H, and V715M) are sensitized to activation by the pesticide 2,2-bis(4-chlorophenyl)-1,1-dichloroethylene (DDE), thus indicating that this agent may impinge on AR signaling in cancer cells. Further investigation showed that DDE induced mutant AR recruitment to the prostate-specific antigen regulatory region, concomitant with an enhancement of target gene expression, and androgen-independent proliferation. By contrast, neither AR activation nor altered cellular proliferation was observed in cells expressing wild-type AR. Activation of signal transduction pathways was also observed based on rapid phosphorylation of mitogen-activated protein kinase (MAPK) and vasodilator-stimulated phosphoprotein, although only MAPK activation was associated with DDE-induced cellular proliferation. Functional analyses showed that both mutant AR and MAPK pathways contribute to the proliferative action of DDE, as evidenced through selective abrogation of each pathway. Together, these data show that exposure to environmentally relevant doses of EDCs can promote androgen-independent cellular proliferation in tumor cells expressing mutant AR and that DDE uses both mutant AR and MAPK pathways to exert its mitogenic activity. (Mol Cancer Res 2008;6(9):1507–20)

Targeting ACLY sensitizes castration-resistant prostate cancer cells to AR antagonism by impinging on an ACLY-AMPK-AR feedback mechanism

The androgen receptor (AR) plays a central role in prostate tumor growth. Inappropriate reactivation of the AR after androgen deprivation therapy promotes development of incurable castration-resistant prostate cancer (CRPC). In this study, we provide evidence that metabolic features of prostate cancer cells can be exploited to sensitize CRPC cells to AR antagonism. We identify a feedback loop between ATP-citrate lyase (ACLY)-dependent fatty acid synthesis, AMPK, and the AR in prostate cancer cells that could contribute to therapeutic resistance by maintaining AR levels. When combined with an AR antagonist, ACLY inhibition in CRPC cells promotes energetic stress and AMPK activation, resulting in further suppression of AR levels and target gene expression, inhibition of proliferation, and apoptosis. Supplying exogenous fatty acids can restore energetic homeostasis; however, this rescue does not occur through increased β-oxidation to support mitochondrial ATP production. Instead, concurrent inhibition of ACLY and AR may drive excess ATP consumption as cells attempt to cope with endoplasmic reticulum (ER) stress, which is prevented by fatty acid supplementation. Thus, fatty acid metabolism plays a key role in coordinating ER and energetic homeostasis in CRPC cells, thereby sustaining AR action and promoting proliferation. Consistent with a role for fatty acid metabolism in sustaining AR levels in prostate cancer in vivo, AR mRNA levels in human prostate tumors correlate positively with expression of ACLY and other fatty acid synthesis genes. The ACLY-AMPK-AR network can be exploited to sensitize CRPC cells to AR antagonism, suggesting novel therapeutic opportunities for prostate cancer.

A cancerous web: signaling, metabolism, and the epigenome

Histone acetylation is sensitive to the availability of acetyl-CoA. However, the extent to which metabolic alterations in cancer cells impact tumor histone acetylation has been unclear. Here, we discuss our recent findings that oncogenic AKT1 activation regulates histone acetylation levels in tumors through regulation of acetyl-CoA metabolism.

Abstract B40: Oncogenic Kras induces histone acetylation in pancreatic ductal adenocarcinoma

Deregulation of cellular epigenetics is essential for malignant transformation. However, the mechanisms that cause epigenetic alterations in cancer cells are incompletely understood. Recent evidence has shown that cellular metabolism has a direct impact on the epigenome, since many chromatin-modifying enzymes rely on intracellular metabolites as cofactors or donor substrates. We have previously shown that the metabolic enzyme ATP-citrate lyase (ACLY), which generates nuclear-cytoplasmic acetyl-CoA from glucose, is required for maintaining histone acetylation levels in multiple mammalian cell types, suggesting that alterations in acetyl-CoA metabolism in cancer cells might also impact histone acetylation levels. Cellular metabolism is massively rewired in multiple cancer types, including pancreatic cancer, although the impact of metabolic alterations on the tumor epigenome is poorly understood. We postulated that tumor cell histone acetylation levels are determined in part by changes in acetyl-CoA availability mediated by oncogenic metabolic reprogramming. In this study, we demonstrate that acetyl-CoA abundance in cancer cells is dynamically regulated by glucose availability and that histone acetylation levels are responsive to the ratio of acetyl-CoA:coenzyme A within the nucleus. To test whether oncogene activation could mediate changes in histone acetylation in vivo, we performed immunohistochemical analysis comparing pancreata from mice expressing Kras G12D with those from mice with WT Kras. Whereas the acinar cells of WT mice exhibited very low levels of histone H4 (K5/8/12/16) acetylation, in KPC (LSL-KrasG12D; p53L/+; Pdx1-Cre) mice, acinar H4 acetylation was markedly increased, prior to the appearance of histological abnormalities or aberrant cell proliferation. High H4 acetylation persisted in pancreatic intraepithelial neoplasia (PanIN) and PDA. Histone acetylation in PanIN-derived primary cells was selectively impaired by PI3K and Akt inhibitors, correlating with suppression of glucose consumption and cellular acetyl-CoA levels. Moreover, addition of supraphysiological doses of acetate, a source of acetyl-CoA alternative to glucose, restored histone acetylation levels. These data suggest that oncogenic Kras promotes elevated histone acetylation preceding tumor development through Akt-dependent regulation of cellular acetyl-CoA levels. Further mechanistic analysis suggests that Akt promotes elevated histone acetylation through combined effects on promoting glucose uptake and phosphorylation and activation of ATP-citrate lyase, a metabolic enzyme that produces nuclear-cytoplasmic acetyl-CoA. The aberrant activation of the PI3K/Akt pathway occurs in broad variety of human malignancies. pAkt(Ser473) levels correlate significantly with histone acetylation marks in human gliomas and prostate tumors, suggesting that PI3K-Akt-dependent promotion of acetyl-CoA metabolism may contribute to histone acetylation levels in multiple cancer types. Our data implicate acetyl-CoA metabolism as a key determinant of histone acetylation levels in tumors and offer novel insights on Kras-induced pancreatic carcinogenesis. Citation Format: Alessandro Carrer, Joyce V. Lee, Supriya Shah, Nathaniel W. Snyder, Ellen Jackson, Nicole M. Aiello, Benjamin A. Garcia, Lewis A. Chodosh, Ben Z. Stanger, Ian A. Blair, Kathryn E. Wellen. Oncogenic Kras induces histone acetylation in pancreatic ductal adenocarcinoma. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr B40.

Abstract 1013: Quantification of NADPH balance in cancer

NADPH is a key cofactor involved in antioxidant defense and reductive biosynthesis. The quantitative contribution of different NADPH pathways, either in normal tissues or in tumors, remains unclear. Here we enable tracing of NADPH production using deuterium-labeled nutrients and mass spectrometry, providing methods that for the first time can track each of the major cytosolic pathways: malic enzyme, isocitrate dehydrogenase, folate metabolism, and the pentose phosphate pathway. This is achieved via a battery of different 2H-tracers. A linear algebra method for deducing the fractional contribution of each pathway, even when a specific tracer for that pathway is not available, will be described. Utility of these methods will be illustrated through examples drawn from both normal cellular physiology and cancer. Specifically, we find that most NADPH in differentiating adipocytes is made by malic enzyme. Examining cancer cells, we find that the pentose phosphate pathway is typically the largest cytosolic NADPH source, but that the collective contribution of alternative pathways is often larger than the pentose phosphate pathway. The specific other pathways involved differ strongly depending on the cancer cell type. We also provide examples where NADPH production routes in both normal and cancer cells vary with nutrient availability, including hypoxia. Given the heightened redox stress of cancer cells, understanding NADPH production routes is likely to illuminate new avenues for therapeutic intervention. Citation Format: Ling Liu, Li Chen, Greg Ducker, Junyoung Park, Supriya Shah, Kathryn E. Wellen, Joshua D. Rabinowitz. Quantification of NADPH balance in cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1013.

Abstract PR03: Exploring the link between Kras and histone acetylation

KRAS mutations in pancreatic ductal adenocarcinoma (PDAC) have been shown to extensively rewire cellular metabolism to promote macromolecular biosynthesis and maintain redox homeostasis. Recently, compelling evidence has emerged demonstrating that many epigenetic modifications are sensitive to the availability of cellular metabolites. For example, acetyl-CoA is the donor substrate for lysine acetylation, and histone acetylation is responsive to levels of acetyl-CoA, which is produced largely from glucose metabolism. We thus hypothesized that oncogenic metabolic reprogramming alters metabolite levels in a way that impacts the epigenome and could thus contribute to tumorigenesis. Indeed, we find that oncogenic Akt activation promotes elevated histone acetylation, and that this effect is mediated by ATP-citrate-lyase (ACLY), a nucleocytoplasmic enzyme that converts glucose-derived citrate into acetyl-CoA. In order to understand what cellular processes are affected by such AKT-ACLY-mediated increase in histone acetylation, we employed a mouse model of pancreatic ductal adenocarcinoma, driven by oncogenic Kras (Pdx1-Cre; LSL-KrasG12D; TP53L/+). In this model, the PI3K/Akt pathway is activated downstream of Kras. By immunohistochemistry we found that, in mice with WT Kras, pancreatic acinar cells exhibit very low levels of histone H4 (K5/8/12/16) acetylation (AcH4), although AcH4 is clearly detectable in ductal epithelial cells and islets. By contrast, in mice expressing oncogenic Kras in the pancreas (LSL-KrasG12D; p53L/+; Pdx1-Cre), acinar H4 acetylation is dramatically increased. Remarkably, this overt increase in histone acetylation precedes the appearance of histological abnormalities and persists during several steps in tumor progression. Moreover, Kras and Akt-dependent reuglation of histone acetylation levels can be recapitulated in ex vivo acinar cell culture, suggesting that this is a primary effect of oncogene activation in these cells. Akt inhibition also significantly reduced glucose consumption and phosphorylation of ACLY and ultimately decreased acetyl-CoA levels. Akt inhibition in vitro reduces Kras-induced acinar cell hyperacetylation and block acinar-to-ductal metaplasia, an initiating event of pancreatic carcinoma. Our data indicate that Akt orchestrates a metabolic rewiring in pancreatic tumorigenesis, which promotes histone hyperacetylation, a phenotype evident before the manifestation of the disease and that might conceivably contribute to cell plasticity and tumor progression. Citation Format: Alessandro Carrer, Joyce V. Lee, Supriya Shah, Nicole M. Aiello, Nathaniel W. Snyder, Andrew J. Worth, Ian A. Blair, Ben Z. Stanger, Kathryn E. Wellen. Exploring the link between Kras and histone acetylation. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Sep 24-27, 2015; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2016;76(2 Suppl):Abstract nr PR03.

Abstract A31: Akt-dependent metabolic reprogramming regulates tumor cell histone acetylation

Histone acetylation plays important roles in gene regulation, DNA replication, and the response to DNA damage, and it is frequently deregulated in tumors. We postulated that tumor cell histone acetylation levels are determined in part by changes in acetyl-CoA availability mediated by oncogenic metabolic reprogramming. Here, we demonstrate that acetyl-CoA is dynamically regulated by glucose availability in cancer cells and that the ratio of acetyl-CoA: coenzyme A within the nucleus modulates global histone acetylation levels. In vivo, expression of oncogenic Kras or Akt stimulates histone acetylation changes that precede tumor development. Furthermore, we show that Akt9s effects on histone acetylation are mediated through the metabolic enzyme ATP-citrate lyase (ACLY), and that pAkt(Ser473) levels correlate significantly with histone acetylation marks in human gliomas and prostate tumors. The data implicate acetyl-CoA metabolism as a key determinant of histone acetylation levels in cancer cells. Citation Format: Supriya Shah, Joyce V. Lee, Alessandro Carrer, Nathaniel W. Snyder, Kathryn E. Wellen. Akt-dependent metabolic reprogramming regulates tumor cell histone acetylation. [abstract]. In: Proceedings of the AACR Special Conference: Targeting the PI3K-mTOR Network in Cancer; Sep 14-17, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(7 Suppl):Abstract nr A31.