Pancreatic Ductal Adenocarcinoma Maintenance Research Articles

Regulation of GLI Underlies a Role for BET Bromodomains in Pancreatic Cancer Growth and the Tumor Microenvironment.

The initiation, progression, and maintenance of pancreatic ductal adenocarcinoma (PDAC) results from the interplay of genetic and epigenetic events. While the genetic alterations of PDAC have been well characterized, epigenetic pathways regulating PDAC remain, for the most part, elusive. The goal of this study was to identify novel epigenetic regulators contributing to the biology of PDAC. In vivo pooled shRNA screens targeting 118 epigenetic proteins were performed in two orthotopic PDAC xenograft models. Candidate genes were characterized in 19 human PDAC cell lines, heterotopic xenograft tumor models, and a genetically engineered mouse (GEM) model of PDAC. Gene expression, IHC, and immunoprecipitation experiments were performed to analyze the pathways by which candidate genes contribute to PDAC. In vivo shRNA screens identified BRD2 and BRD3, members of the BET family of chromatin adaptors, as key regulators of PDAC tumor growth. Pharmacologic inhibition of BET bromodomains enhanced survival in a PDAC GEM model and inhibited growth of human-derived xenograft tumors. BET proteins contribute to PDAC cell growth through direct interaction with members of the GLI family of transcription factors and modulating their activity. Within cancer cells, BET bromodomain inhibition results in downregulation of SHH, a key mediator of the tumor microenvironment and canonical activator of GLI. Consistent with this, inhibition of BET bromodomains decreases cancer-associated fibroblast content of tumors in both GEM and xenograft tumor models. Therapeutic inhibition of BET proteins offers a novel mechanism to target both the neoplastic and stromal components of PDAC. Clin Cancer Res; 22(16); 4259-70. ©2016 AACR.

InVivo Functional Platform Targeting Patient-Derived Xenografts Identifies WDR5-Myc Association as a Critical Determinant of Pancreatic Cancer.

Current treatment regimens for pancreatic ductal adenocarcinoma (PDAC) yield poor 5-year survival, emphasizing the critical need to identify druggable targets essential for PDAC maintenance. We developed an unbiased and invivo target discovery approach to identify molecular vulnerabilities in low-passage and patient-derived PDAC xenografts or genetically engineered mouse model-derived allografts. Focusing on epigenetic regulators, we identified WDR5, a core member of the COMPASS histone H3 Lys4 (H3K4) MLL (1-4) methyltransferase complex, as a top tumor maintenance hit required across multiple human and mouse tumors. Mechanistically, WDR5 functions to sustain proper execution of DNA replication in PDAC cells, as previously suggested by replication stress studies involving MLL1, and c-Myc, also found to interact with WDR5. We indeed demonstrate that interaction with c-Myc is critical for this function. By showing that ATR inhibition mimicked the effects of WDR5 suppression, these data provide rationale to test ATR and WDR5 inhibitors for activity in this disease.

Abstract IA26: Identifying metabolic liabilities in obesity-associated pancreatic cancer

Abstract Obesity is a rising pandemic that is increasingly being recognized for its striking correlation with the incidence of/mortality from cancers of different tissues, particularly that of the pancreas. Ranking as the fourth leading cause of cancer death in the United States, pancreatic ductal adenocarcinoma (PDAC) is a highly lethal, insidious malignancy whose poor prognosis is reflected in a 5-year survival rate of only 5%. In an obese state characterized by elevated circulating levels of insulin and insulin-like growth factor-1 (IGF-1), metabolic signaling pathways, including PI3K/Akt might be aberrantly regulated, altering tumor cell metabolism and driving the maintenance of PDAC. Recognized as a hallmark of cancer formation, altered cellular metabolism is evident in Kras-driven PDAC tumors. However, the specific metabolic impact of obesity and increased insulin and IGF-1 levels on PDAC tumors has not been thoroughly investigated. We performed a large orthotopic xenograft experiment involving the injection of human pancreatic cancer cells modified to either express constitutively active, myristoylated Akt, or a control fluorescent protein (CFP) into the pancreata of lean or diet-induced obese immunodeficient mice. We find that control CFP-tumors grown in obese mice and all Akt-tumors, independent of obesity, are significantly larger than CFP-tumors grown in lean mice. Gene expression and metabolomics analyses demonstrate a striking representation of distinct metabolic pathways, including the nitrogen/arginine or urea cycle pathway in pancreatic tumors grown in obese versus lean mice. This pathway enrichment partially overlaps with that observed in the Akt-driven tumors, independent of obesity. Here we describe the validation of these results by altering urea cycle gene expression in the human PDAC cells and assessing their effect on PDAC cell proliferation, survival, metabolism, and response to arginine deprivation both in tissue culture in vitro and as orthotopic xenografts in the pancreata of lean or diet-induced obese mice in vivo. These studies will help identify metabolic dependencies in Akt-driven or non-Akt-driven pancreatic tumors grown under either obese/hyperinsulinemic or lean states, that could be targeted therapeutically in PDAC patients. Citation Format: Tamara Zaytouni, Cory Dubois, Daniel Hitchcock, Nicholas Stylopoulos, Clary Clish, Nada Kalaany. Identifying metabolic liabilities in obesity-associated pancreatic cancer. [abstract]. In: Proceedings of the AACR Special Conference: Metabolism and Cancer; Jun 7-10, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(1_Suppl):Abstract nr IA26.

Oncogene ablation-resistant pancreatic cancer cells depend on mitochondrial function.

Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest cancers in western countries, with a median survival of 6 months and an extremely low percentage of long-term surviving patients. KRAS mutations are known to be a driver event of PDAC, but targeting mutant KRAS has proved challenging. Targeting oncogene-driven signalling pathways is a clinically validated approach for several devastating diseases. Still, despite marked tumour shrinkage, the frequency of relapse indicates that a fraction of tumour cells survives shut down of oncogenic signalling. Here we explore the role of mutant KRAS in PDAC maintenance using a recently developed inducible mouse model of mutated Kras (Kras(G12D), herein KRas) in a p53(LoxP/WT) background. We demonstrate that a subpopulation of dormant tumour cells surviving oncogene ablation (surviving cells) and responsible for tumour relapse has features of cancer stem cells and relies on oxidative phosphorylation for survival. Transcriptomic and metabolic analyses of surviving cells reveal prominent expression of genes governing mitochondrial function, autophagy and lysosome activity, as well as a strong reliance on mitochondrial respiration and a decreased dependence on glycolysis for cellular energetics. Accordingly, surviving cells show high sensitivity to oxidative phosphorylation inhibitors, which can inhibit tumour recurrence. Our integrated analyses illuminate a therapeutic strategy of combined targeting of the KRAS pathway and mitochondrial respiration to manage pancreatic cancer.

Stromal reengineering to treat pancreas cancer.

Pancreatic ductal adenocarcinoma co-opts multiple cellular and extracellular mechanisms to create a complex cancer organ with an unusual proclivity for metastasis and resistance to therapy. Cell-autonomous events are essential for the initiation and maintenance of pancreatic ductal adenocarcinoma, but recent studies have implicated critical non-cell autonomous processes within the robust desmoplastic stroma that promote disease pathogenesis and resistance. Thus, non-malignant cells and associated factors are culprits in tumor growth, immunosuppression and invasion. However, even this increasing awareness of non-cell autonomous contributions to disease progression is tempered by the conflicting roles stromal elements can play. A greater understanding of stromal complexity and complicity has been aided in part by studies in highly faithful genetically engineered mouse models of pancreatic ductal adenocarcinoma. Insights gleaned from such studies are spurring the development of therapies designed to reengineer the pancreas cancer stroma and render it permissive to agents targeting cell-autonomous events or to reinstate immunosurveillance. Integrating conventional and immunological treatments in the context of stromal targeting may provide the key to a durable clinical impact on this formidable disease.

Dormant Cancer Cells Contribute to Residual Disease in a Model of Reversible Pancreatic Cancer

The initiation and progression of pancreatic ductal adenocarcinoma (PDAC) is governed by a series of genetic and epigenetic changes, but it is still unknown whether these alterations are required for the maintenance of primary and metastatic PDAC. We show here that the c-Myc oncogene is upregulated throughout the entire process of neoplastic progression in human PDAC and in genetically engineered mice that express mutant Kras. To experimentally address whether c-Myc is essential for the growth and survival of cancer cells, we developed a novel mouse model that allows a temporally and spatially controlled expression of this oncogene in pancreatic progenitors and derived lineages of the exocrine pancreas. Unlike previous reports, upregulation of c-Myc was sufficient to induce the formation of adenocarcinomas after a short latency without additional genetic manipulation of cell survival pathways. Deficiency in Cdkn2a increased the rate of metastasis but had no effect on tumor latency or c-Myc-mediated cancer maintenance. Despite a macroscopically complete regression of primary, metastatic, and transplantable tumors following the ablation of c-Myc, some cancer cells remained dormant. A significant number of these residual neoplastic cells expressed cancer stem cell markers, and re-expression of exogenous c-Myc in these cells led to rapid cancer recurrence. Collectively, the results of this study suggest that c-Myc plays a significant role in the progression and maintenance of PDAC, but besides targeting this oncogene or its downstream effectors, additional therapeutic strategies are necessary to eradicate residual cancer cells to prevent disease recurrence.

Highlights of This Issue

The protease urokinase-type plasminogen activator (uPA) is an established prognostic marker and a potential therapeutic target in cancer. To circumvent the specificity problems inherent in inhibitors of the catalytic activity of proteases, we have generated a highly specific uPA-binding 35 nucleotides long 2′-fluoro-pyrimidine RNA molecule. This aptamer inhibits the conversion of the inactive zymogen form to active uPA, the binding of uPA to its cellular receptor uPAR, and uPA-dependent tumor cell dissemination in an in vivo model system. Our results are of general interest for the design of new modalities for inhibition of cancer dissemination.Rummel and colleagues address the controversy as to whether the breast microenvironment is genetically stable. Using a panel of microsatellite markers, they evaluated the genetic changes in high-quality, research-grade stromal DNA, compared them to data generated from archival specimens, and found that high-quality specimens harbored significantly fewer alterations than did archived tissues. These data, which demonstrate a lack of genetic changes, support the idea that development and use of therapeutics targeting the breast stroma will provide a new avenue of effective treatment for patients with breast cancer.Glioblastoma multiforme (GBM) is an aggressive primary brain tumor that is highly resistant to chemotherapy. Svilar and colleagues conducted a synthetic lethal siRNA screen in a chemotherapy-resistant GBM cell line with the alkylator temozolomide (TMZ). A comparison with alkylation screens done in Escherichia coli and Saccharomyces cerevisiae suggested that alkylation resistance mechanisms were evolutionarily conserved. Functional analysis showed that high-dose TMZ treatment initiated the formation of reactive oxygen species and revealed an epistatic relationship between UNG and UBE3B in response to TMZ. These studies provide potential new targets to overcome alkylating agent resistance.The RAS-activated ERK–MAP kinase pathway is critical for the initiation and maintenance of pancreatic ductal adenocarcinoma (PDA), but the mechanism(s) by which this pathway contributes to the aberrant pathobiology of PDA cells remains obscure. By expression profiling of a panel of PDA cell lines in the absence or presence of pharmacologic pathway blockade, Gysin and colleagues detail the full complexity of mRNAs regulated by this pathway. As expected, mRNAs with protein products that promote the cell division cycle feature prominently. However, surprisingly, many of the antiproliferative effects of RAF–MEK–ERK pathway inhibition can be ascribed to regulatory effects on the c-MYC oncoprotein, a noted master regulator of general mRNA expression within the cell.

A Central Role for RAF→MEK→ERK Signaling in the Genesis of Pancreatic Ductal Adenocarcinoma

KRAS mutation is a hallmark of pancreatic ductal adenocarcinoma (PDA) but remains an intractable pharmacologic target. Consequently, defining RAS effector pathway(s) required for PDA initiation and maintenance is critical to improve treatment of this disease. Here, we show that expression of BRAF(V600E), but not PIK3CA(H1047R), in the mouse pancreas leads to pancreatic intraepithelial neoplasia (PanIN) lesions. Moreover, concomitant expression of BRAF(V600E) and TP53(R270H) result in lethal PDA. We tested pharmacologic inhibitors of RAS effectors against multiple human PDA cell lines. Mitogen-activated protein (MAP)/extracellular signal-regulated (ERK) kinase (MEK) inhibition was highly effective both in vivo and in vitro and was synergistic with AKT inhibition in most cell lines tested. We show that RAF→MEK→ERK signaling is central to the initiation and maintenance of PDA and to rational combination strategies in this disease. These results emphasize the value of leveraging multiple complementary experimental systems to prioritize pathways for effective intervention strategies in PDA. PDA is diffi cult to treat, in large part, due to recurrent mutations in the KRAS gene. Here, we defi ne rational treatment approaches for the disease achievable today with existing drug combinations by thorough genetic and pharmacologic dissection of the major KRAS effector pathways, RAF→MEK→ERK and phosphoinositide 3′-kinase (PI3'K)→AKT.

Abstract 3262: Notch signaling pathway-targeted therapy suppresses tumor progression and prevents metastatic spread in pancreatic cancer

Abstract Background: Pancreatic ductal adenocarcinoma (PDA) has the highest mortality rate of any cancer type. The 5-year survival rate of 5% remained largely unchanged in the last three decades. The paucity of breakthroughs in treatment regimens and continued poor survival demonstrates an acute need for improvement in therapy for this lethal malignancy. Mounting evidence suggest that sustained re-activation of Notch signaling pathway contributes to the initiation, progression and maintenance of PDA. Recently, Notch signaling has been linked to the viability of putative tumor initiating cancer stem cell populations in pancreatic cancer. PF-03084014 is a selective γ-secretase inhibitor developed by Pfizer. The present study was conducted to determine the effect of PF-03084014 monotherapy and in combination with gemcitabine (GEM) to suppress tumor progression and prevent metastatic spread in preclinical models of pancreatic cancer. Methods: NOTCH-1 gene expression was determined in a panel of 30 human pancreatic cancer xenografts by Affymetrix gene array. We investigated the efficacy of PF-03084014 and in combination with GEM in subcutaneously (s.c) implanted as well as orthotopic models of pancreatic cancer. We also determined the treatment effect on putative pancreatic cancer stem cell (CSC) populations. TUNEL and Ki-67 staining were performed to determine whether the therapy induce apoptosis and suppresses tumor cell proliferation. Results: A combination of PF-03084014 and GEM is highly effective in producing durable tumor regression compared to GEM in subcutaneously implanted pancreatic cancer xenografts. Tumor re-growth curves plotted after 4 weeks of drug treatment demonstrate that the effect of the combination therapy is longer lasting than that of GEM. Notably, in a highly aggressive Panc265 orthotopic model, PF-03084014 and GEM combination was effective in controlling the primary tumor growth as well as preventing metastatic dissemination to organs as compared to GEM treatment. Flow cytometry of sorted tumor cells from Panc215 (after 4 weeks of treatment) showed that GEM treatment was not capable of diminishing the CSC pool as compared to the vehicle treated mice. However, PF-03084014 alone, and the combination of GEM with PF-03084014 resulted in a 3.72 and 3.04-fold decrease in CD24+CD44+ tumor cells compared with GEM treatment, respectively. TUNEL and Ki-67 staining demonstrated that combination therapy induced apoptosis and suppresses cell proliferation. Conclusions: The findings presented here provide strong rationale for the clinical investigation on notch inhibition as a chemotherapeutic strategy to combat pancreatic cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3262. doi:1538-7445.AM2012-3262

Oncogenic KRAS Activates Hedgehog Signaling Pathway in Pancreatic Cancer Cells

Hedgehog (Hh) signaling is deregulated in multiple human cancers including pancreatic ductal adenocarcinoma (PDA). Because KRAS mutation represents one of the earliest genetic alterations and occurs almost universally in PDA, we hypothesized that oncogenic KRAS promotes pancreatic tumorigenesis in part through activation of the Hh pathway. Here, we report that oncogenic KRAS activates hedgehog signaling in PDA cells, utilizing a downstream effector pathway mediated by RAF/MEK/MAPK but not phosphatidylinositol 3-kinase (PI3K)/AKT. Oncogenic KRAS transformation of human pancreatic ductal epithelial cells increases GLI transcriptional activity, an effect that is inhibited by the MEK-specific inhibitors U0126 and PD98059, but not by the PI3K-specific inhibitor wortmannin. Inactivation of KRAS activity by a small interfering RNA specific for oncogenic KRAS inhibits GLI activity and GLI1 expression in PDA cell lines with activating KRAS mutation; the MEK inhibitors U0126 and PD98059 elicit a similar response. In addition, expression of the constitutively active form of BRAF(E600), but not myr-AKT, blocks the inhibitory effects of KRAS knockdown on Hh signaling. Finally, suppressing GLI activity leads to a selective attenuation of the oncogenic transformation activity of mutant KRAS-expressing PDA cells. These results demonstrate that oncogenic KRAS, through RAF/MEK/MAPK signaling, is directly involved in the activation of the hedgehog pathway in PDA cells and that collaboration between these two signaling pathways may play an important role in PDA progression.