Pancreatic Tumor Development Research Articles

New Genetics and Genomic Data on Pancreatic Neuroendocrine Tumors: Implications for Diagnosis, Treatment, and Targeted Therapies.

The recent findings on the roles of death-associated protein 6/α-thalassemia/mental retardation X-linked (DAXX/ATRX) in the development of pancreatic neuroendocrine tumors (PanNETs) have led to major advances in the molecular understanding of these rare tumors and open up completely new therapeutic windows. This overview aims at giving a simplified view on these findings and their possible therapeutic implications. The importance of epigenetic changes in PanNET is also underlined by recent findings of a cross-species study on microRNA (miRNA) and messenger RNA (mRNA) profiles in PanNETs.

Abstract 2656: Loss of SIRT6 reactivates the oncofetal RNA-binding protein Lin28b to drive pancreatic cancer

Abstract Chromatin remodeling proteins are frequently dysregulated in human cancer, however little is known about how they control tumorigenesis. The NAD+-dependent histone deacetylase Sirtuin 6 (SIRT6) is a nutrient sensor that reprograms the epigenome in response to nutrient stress and shows reduced expression in human pancreatic ductal adenocarcinoma (PDAC) relative to normal tissue. Thus, we aimed to determine the role of SIRT6 in PDAC and to determine how the loss of this chromatin modifier could influence PDAC formation, progression and metastasis. Here, we uncover an epigenetic program mediated by SIRT6 that is critical for suppression of PDAC. We demonstrate that SIRT6 inactivation cooperates with oncogenic KRAS to greatly accelerate the development of lethal pancreatic tumors regardless of p53 status. In addition to developing PDAC and high-grade pancreatic intraepithelial neoplasia (PanIN) at an earlier age, SIRT6-deficient tumors had a greater propensity to metastasize to the lung, compared to their SIRT6 wild-type (WT) counterparts. Through genome-wide profiling of SIRT6 WT and knock-out (KO) PDAC cell lines, we identified the oncofetal RNA-binding protein Lin28b as a target of Sirt6-mediated transcriptional repression. Loss of SIRT6 in both murine and human PDAC cell lines resulted in hyperacetylation of histone 3 lysine 9 and lysine 56 at the Lin28b promoter, MYC recruitment, and pronounced induction of Lin28b expression. Knocking down Lin28b resulted in potent suppression of cell proliferation and tumor sphere formation in Sirt6null/low murine and human cell lines, while murine and human Sirt6WT/high PDAC lines were completely insensitive to the same treatment. Functionally, Lin28b binds to and induces the degradation of the let-7 family of microRNA, thus resulting in increased expression of let-7 target genes such as the Igf2bps, which have been correlated with increased agressiveness and metastasis in pancreatic tumors. Furthermore, Gene Set Enrichment Analysis (GSEA) comparing PDAC tumors and cell lines with high versus low expression of LIN28B revealed that LIN28Bhigh tumors were strongly enriched for the expression of let-7 targets. Thus, our critical findings define SIRT6 as a tumor suppressor in PDAC, working as a transcriptional repressor of the oncofetal RNA-binding protein Lin28b. We demonstrate that loss of Sirt6 creates a permissive hyperacetylated chromatin state, allowing for MYC-dependent transactivation of Lin28b. This work not only provides new insights into the epigenetic mechanisms governing the reactivation of oncofetal proteins in cancer but also demonstrates that this pathway is well-conserved in human PDAC, where reduced SIRT6 expression defines a specific subset of tumors which may benefit from novel therapeutic approaches aimed at targeting components of the LIN28B/let-7 pathway. Citation Format: Sita Kugel, Carlos Sebastián, Julien Fitamant, Kenneth N. Ross, Supriya K. Saha, Alon Goren, Sridhar Ramaswamy, Vikram Deshpande, Nabeel Bardeesy, Raul Mostoslavsky. Loss of SIRT6 reactivates the oncofetal RNA-binding protein Lin28b to drive pancreatic 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 2656.

Metformin with everolimus and octreotide in pancreatic neuroendocrine tumor patients with diabetes.

A bidirectional relationship seems to exist between diabetes mellitus and development of pancreatic tumors. Metformin, the most widely used drug in the treatment of Type 2 diabetes mellitus, has recently emerged as a potentially active agent in cancer chemoprevention and treatment. In this article, we discuss the potential correlation between glycemic status, administration of antiglycemic treatments, such as metformin or insulin, and prognosis of pancreatic neuroendocrine tumors patients treated with everolimus and octreotide, on the basis of existing evidence and our experience.

Progranulin Stimulates Proliferation of Mouse Pancreatic Islet Cells and Is Overexpressed in the Endocrine Pancreatic Tissue of an MEN1 Mouse Model.

Progranulin (PGRN) promotes cell growth and cell cycle progression in several cell types and contributes to tumorigenesis in diverse cancers. We have recently reported PGRN expression in islets and tumors developed in an MEN1 transgenic mouse. Here we sought to investigate PGRN expression and regulation after exposure to hypoxia as well as its effects on pancreatic islet cells and neuroendocrine tumors (NETs) in MEN1(+/−) mice. Gene and protein expression were analyzed by quantitative polymerase chain reaction, immunohistochemistry, and Western blot. We also investigated PGRN expression in samples from patients carrying pancreatic NETs associated or not with the multiple endocrine neoplasia 1 syndrome, using enzyme-linked immunosorbent assay and immunohistochemistry analysis. Progranulin is upregulated in tumors and islets of the MEN1 mouse as well as in the serum of patients with pancreatic NETs associated with glucagonoma syndrome. In normal mice islets and pancreatic tumors, PGRN expression was strongly potentiated by hypoxia. Progranulin promotes cell proliferation in islet cells and βTC-6 cells, a process paralleled by activation of the mitogen-activated protein kinase signaling cascade. Our findings identify PGRN as an effective inducer of pancreatic islet cell proliferation and a possible important factor for pancreatic endocrine tumor development.

Islet Cells Serve as Cells of Origin of Pancreatic Gastrin-Positive Endocrine Tumors.

The cells of origin of pancreatic gastrinomas remain an enigma, since no gastrin-expressing cells are found in the normal adult pancreas. It was proposed that the cellular origin of pancreatic gastrinomas may come from either the pancreatic cells themselves or gastrin-expressing cells which have migrated from the duodenum. In the current study, we further characterized previously described transient pancreatic gastrin-expressing cells using cell lineage tracing in a pan-pancreatic progenitor and a pancreatic endocrine progenitor model. We provide evidence showing that pancreatic gastrin-expressing cells, found from embryonic day 12.5 until postnatal day 7, are derived from pancreatic Ptf1a(+) and neurogenin 3-expressing (Ngn3(+)) progenitors. Importantly, the majority of them coexpress glucagon, with 4% coexpressing insulin, indicating that they are a temporary subpopulation of both alpha and beta cells. Interestingly, Men1 disruption in both Ngn3 progenitors and beta and alpha cells resulted in the development of pancreatic gastrin-expressing tumors, suggesting that the latter developed from islet cells. Finally, we detected gastrin expression using three human cohorts with pancreatic endocrine tumors (pNETs) that have not been diagnosed as gastrinomas (in 9/34 pNETs from 6/14 patients with multiple endocrine neoplasia type 1, in 5/35 sporadic nonfunctioning pNETs, and in 2/20 sporadic insulinomas), consistent with observations made in mouse models. Our work provides insight into the histogenesis of pancreatic gastrin-expressing tumors.

Abstract B01: Ets-2 acts as a novel oncogene in cancer associated fibroblasts and promotes pancreatic tumor initiation and development

Abstract Purpose of Study:Pancreatic cancer remains an overwhelmingly fatal disease with approximately 95% of patients dying within 5 years of diagnosis. Many recent reports have highlighted the emerging role that stromal cells’fibroblasts in particular’have on pancreatic tumor cell biology. It has become apparent that new models of pancreatic cancer that accurately portray the tumor microenvironment (TME) are necessary to move the field forward. Advances in genetic tools to modulate the TME developed in our lab have allowed us the unique opportunity to manipulate specific stromal cell compartments. Our overarching hypothesis is: Ets-2 in the pancreatic tumor-associated stroma promotes tumor initiation and development by regulating essential signaling pathways in fibroblasts and is critical for tumor-stroma co-evolution. Research Method: In this study we use a previously validated and well-studied genetically engineered mouse model of pancreatic cancer that relies on constitutive activation of the Kras oncogene tumor cell lineage. We simultaneously employ lox-cre technology to conditionally delete our gene of interest exclusively in the fibroblast compartment of the pancreas. For this study we delete the transcription factor Ets-2, which has previously been shown to effect tumor initiation and growth in mammary stroma. Novel Findings: Here we show that Ets-2 in pancreatic tumor-associated stroma promotes tumor formation by regulating essential signaling pathways in stromal fibroblasts and is critical for tumor-stroma co-evolution. We conditionally deleted Ets-2 in cancer associated fibroblasts (CAFs), thus altering stroma-tumor crosstalk and resulting in delayed tumor initiation. Specifically, we saw a decrease in pre-cancerous acinar-to-ductal metaplasic (ADM) and pancreatic intraepithelial neoplastic (PanIN) lesions in mice null for Ets-2 in the fibroblast compartment. In order to investigate the mechanism by which Ets-2 is able to effect progression of tumors from the fibroblast compartment, we developed a method for harvesting and purifying primary pancreatic CAFs. We performed microarray and gene expression analysis on Ets-2 deleted CAFs and saw significantly decreased expression of secreted factors. The altered secretome upon Ets-2 deletion in fibroblasts was crucially lacking tumor necrosis factor alpha (TNFα), a known pro-tumor ligand in pancreatic carcinogenesis. Furthermore, we found evolutionarily conserved Ets-2 transcription factor binding sites in the proximal promoter of TNFα, suggesting that Ets-2 directly regulates TNFα transcription. Thus we have shown that Ets-2 ablation in pancreatic fibroblasts delays pancreatic tumor initiation through the pro-tumor ligand TNFα. Conclusions and Implications:This report shows that deleting a gene in pancreatic fibroblasts causes a change in tumor-stroma co-evolution and that Ets-2 is able to act as a novel oncogene in cancer associated fibroblasts to promote pancreatic carcinogenesis. We have shown that Ets-2 deletion significantly changed the biological role of the CAFs and negatively effects tumor growth. This finding is relevant to the field of pancreatic cancer because it shows that the pancreatic TME can play a driver role in overall tumor initiation and development. These collective findings contribute to our lab’s overall hypothesis that the tumor microenvironment is not a mere bystander or byproduct of tumor development, but rather that it can drive tumor evolution amongst a variety of cancers. Citation Format: Jason R. Pitarresi, Jinghai Wu, Sarah Woelke, Raleigh Kladney, Maria Cuitino, Lianbo Yu, Ostrowski C. Michael. Ets-2 acts as a novel oncogene in cancer associated fibroblasts and promotes pancreatic tumor initiation and development. [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 B01.

Abstract IA6: Identification and characterization of factors required for KRAS-driven pancreatic tumorigenesis

Abstract Activating mutations in the KRAS oncogene occur in the vast majority of pancreatic ductal adenocarcinomas (PDAC). These mutations are present in early precursor pancreatic intra-epithelial neoplasia (PanIN) lesions and are considered initiating driver mutations in this disease. Moreover, expression of an activated Kras allele potently initiates PanIN formation and PDAC development in the mouse. Therefore, inhibiting mutant KRAS-mediated signaling is potentially an effective therapeutic strategy for PDAC. While efforts directly targeting mutant KRAS are underway, an alternative approach is to target key signaling molecules that are stimulated downstream by activated KRAS. We hypothesized that factors required for the development of KRAS-driven pancreatic tumors will be similarly required during late stage disease and therefore represent potential therapeutic targets. We therefore utilize in vivo and in vitro systems to identify and characterize these factors. Mutated KRAS promotes the proliferation of primary pancreatic ductal epithelial cells (PDECs) as well as their survival following challenge with apoptotic stimuli. KRAS-induced survival in PDECs is dependent on signaling through the RAF/MEK/ERK and PI3K/AKT signaling cascades. Interestingly, we found that the insulin-like growth factor receptor (IGF1R) is required for KRASG12D-induced stimulation of the PI3K/AKT signaling axis. We determined that KRASG12D induces the expression of the IGF1R ligand IGF2 downstream of the MEK/ERK cascade and IGF2 stimulates IGF1R, and subsequently PI3K/AKT signaling, in an autocrine manner. Consistent with this, ectopic IGF2 expression restored KRASG12D-induced survival in cells treated with a MEK inhibitor but not a PI3K inhibitor. Critically, IGF1R is required for KRASG12D-induced survival after exposure to apoptotic stimuli, and inhibition of IGF1R impairs pancreatic tumor formation in an orthotopic mouse model. In PDAC cell lines, shRNA-mediated knockdown of IGF1R impairs their anchorage-independent growth and migration activity. Thus, IGF1R-mediated signaling is required for KRASG12D-driven pancreatic tumorigenesis in vivo and impacts the transformation of pancreatic cancer cells in culture. The mTOR signaling complex TORC2 promotes the phosphorylation of AKT on Ser473, an event required for full activation of AKT. Given the importance of IGF1R and PI3K/AKT signaling in promoting KRASG12D-induced survival and pancreatic tumor formation, we explored the requirement for TORC2 activity in KRAS-driven PDAC development in vivo. We inactivated TORC2 through the pancreas-specific genetic ablation of the essential subunit Rictor. Rictor deficient pancreata are architecturally normal but smaller than wild-type pancreata. Interestingly, Rictor deletion significantly impaired KRASG12D-driven PanIN formation. Moreover, the absence of Rictor also impaired KRASG12D-driven PanIN formation following caerulein-induced pancreatic injury. Together, these data indicate an important role for TORC2 signaling during PDAC initiation. We additionally characterized the role of TORC2 in PDAC cell lines. ShRNA-mediated inhibition of Rictor impaired PDAC cell proliferation and transformation as assessed by anchorage-independent growth, suggesting an important role for the TORC2 signaling complex in maintenance of the transformed phenotype of PDAC cells. Analysis of TORC substrate proteins identified reduced phosphorylation and activation of AKT and SGK1, suggesting these downstream molecules as potential mediators of TORC2-dependent phenotypes in pancreatic tumorigenesis. Together, these data suggest that TORC2 activity plays a critical role in KRAS-driven PDAC initiation and progression. Collectively, our studies have identified critical factors required for KRAS-driven pancreatic tumorigenesis, suggesting additional therapeutic strategies for targeting PDAC. Citation Format: Brian C. Lewis. Identification and characterization of factors required for KRAS-driven pancreatic tumorigenesis. [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 IA6.

ROBO3 promotes growth and metastasis of pancreatic carcinoma

Pancreatic carcinoma is a highly lethal malignancy with an extremely poor prognosis. Recent genome-wide studies have implicated axon guidance pathways, including the SLIT/ROBO pathway, in pancreatic tumor development and progression. Here we showed that ROBO3 expression is up-regulated in pancreatic cancer tissue samples and cell lines. Over-expression of ROBO3 promotes pancreatic cancer cell growth, invasion and metastasis in vitro and in mouse xenograft tumor models. We identified miR-383 as a suppressor of ROBO3, and revealed its expression to be inversely correlated with ROBO3. Over-expression of ROBO3 activates Wnt pathway components, β-catenin and GSK-3, and the expression of markers indicating an EMT. By means of immunoprecipitation, we revealed an interaction between Wnt inhibitor SFRP and ROBO3 in pancreatic cancer cell lines. Our work suggests that ROBO3 may contribute to the progression of pancreatic cancer by sequestering Wnt inhibitor SFRP, which in turn leads to increased Wnt/β-catenin pathway activity. We also confirmed that ROBO3 increases with clinical grade and miR-383 expression is inversely correlated to that of ROBO3.

Abstract B05: Thymidylate synthase cooperates with oncogenic KRAS to markedly accelerate pancreatic cancer progression in a novel KrasG12D/+ mouse model

Abstract Oncogenic KRAS is a key initiator in the development of pancreatic cancer; however, other aberrations are necessary for the progression into advanced metastatic disease. Our lab was the first to show that overexpression of Thymidylate Synthase (TS), an essential DNA synthesis and repair enzyme that is elevated in many common adult cancers, plays a direct role in promoting tumorigenesis. These findings suggest that TS is not a passive marker of proliferation, but rather an important oncogenic driver in cancer. To determine whether TS enhances the neoplastic effects of oncogenic KRAS, we established a novel animal model for pancreatic ductal adenocarcinoma (PDAC) by generating conditional KrasG12D mutant mice that express high levels of human TS (hTS) in the pancreas. We discovered that overexpression of hTS in the pancreas of Pdx1-cre-KrasG12D/+ mice significantly reduced survival of the hTS/KrasG12D/+ mice. Median survival of hTS/KrasG12D/+ mice was reduced by 50% as compared to KrasG12D/+ mice (112.5 vs 222 days, p=0.0005). To test whether TS accelerates the development of preneoplastic pancreatic ductal lesions and tumors, hTS/KrasG12D/+ and KrasG12D/+ mice were euthanized at 1 month intervals from 1.0 – 6.0 months of age and the percentage of ducts displaying normal, murine pancreatic intraepithelial neoplasia (mPanIN), or invasive PDAC was scored by a masked veterinary pathologist using world health organization (WHO) criteria. We found that hTS/KrasG12D/+ mice displayed a 10 fold increase in mPanIN2 and mPanIN3 lesions at 2 months of age as compared to KrasG12D/+ mice alone. At 3 months of age the hTS/KrasG12D/+ mice showed a 10 fold increase in PDAC formation as compared to KrasG12D/+ mice alone. To determine whether TS enhances the metastatic potential of oncogenic KRAS, lung and liver tissues were harvested from these mice and analyzed for the presence of metastasis. We found that hTS/KrasG12D/+ mice developed lung metastasis as early as 2 months of age, while KrasG12D/+ mice did not develop lung metastasis until 4 months of age. In addition, 60% of hTS/KrasG12D/+ mice in the 4 month age group displayed liver metastasis compared to 20% of KrasG12D/+ mice. Taken together, our data demonstrate that hTS overexpression in the pancreas 1) enhances the initiation of preneoplastic pancreatic ductal lesions, 2) accelerates pancreatic ductal adenocarcinoma development, 3) reduces survival, and 4) increases metastasis in hTS/KrasG12D/+ mice. These data demonstrate that hTS cooperates with oncogenic KRAS to accelerate the progression of pancreatic cancer. Therefore, we predict that combined inhibition of TS and KRAS signaling will maximize tumor regression. Our lab has identified 4 new TS allosteric inhibitors (provisional patent filed in 2013), which inhibit the catalytic activity of TS through a different binding site than 5-fluorouracil (5-FU). These lead allosteric inhibitors display mid-nanomolar to low micromolar activity in KRAS-mutant pancreatic cancer cell lines. Our goal is to combine these allosteric TS inhibitors with inhibitors of KRAS effectors, such as PI3K/AKT/mTOR and RAF/MEK/ERK pathways. Given the frequency of aberrant KRAS activation in pancreatic cancer and the striking effect of TS to enhance KRAS driven tumors in our hTS/KrasG12D/+ model, this combination strategy will be an important step in identifying novel treatments for pancreatic cancer. Citation Format: Rony A. Francois, Akbar Nawab, Min Chen, Mary K. Reinhard, Frederic J. Kaye, Maria Zajac-Kaye. Thymidylate synthase cooperates with oncogenic KRAS to markedly accelerate pancreatic cancer progression in a novel KrasG12D/+ mouse model. [abstract]. In: Proceedings of the AACR Special Conference on RAS Oncogenes: From Biology to Therapy; Feb 24-27, 2014; Lake Buena Vista, FL. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(12 Suppl):Abstract nr B05. doi: 10.1158/1557-3125.RASONC14-B05

RAGE is essential for oncogenic KRAS-mediated hypoxic signaling in pancreatic cancer.

A hypoxic tumor microenvironment is characteristic of many cancer types, including one of the most lethal, pancreatic cancer. We recently demonstrated that the receptor for advanced glycation end products (RAGE) has an important role in promoting the development of pancreatic cancer and attenuating the response to chemotherapy. We now demonstrate that binding of RAGE to oncogenic KRAS facilitates hypoxia-inducible factor 1 (HIF1)α activation and promotes pancreatic tumor growth under hypoxic conditions. Hypoxia induces NF-κB-dependent and HIF1α-independent RAGE expression in pancreatic tumor cells. Moreover, the interaction between RAGE and mutant KRAS increases under hypoxia, which in turn sustains KRAS signaling pathways (RAF-MEK-ERK and PI3K-AKT), facilitating stabilization and transcriptional activity of HIF1α. Knock down of RAGE in vitro inhibits KRAS signaling, promotes HIF1α degradation, and increases hypoxia-induced pancreatic tumor cell death. RAGE-deficient mice have impaired oncogenic KRAS-driven pancreatic tumor growth with significant downregulation of the HIF1α signaling pathway. Our results provide a novel mechanistic link between NF-κB, KRAS, and HIF1α, three potent molecular pathways in the cellular response to hypoxia during pancreatic tumor development and suggest alternatives for preventive and therapeutic strategies.

Protein breakdown precedes pancreatic tumor development.

A new study shows that plasma levels of branched-chain amino acids are elevated in subjects several years before they are diagnosed with pancreatic cancer. This may reflect breakdown of peripheral protein stores in the early stages of the disease.

Abstract 4434: Role of Deubiquitinase Usp9x in pancreatic cancers: Tumor promoter or tumor suppressor

Abstract Deubiquitinase Usp9x has been shown to promote tumor cell survival and resistance to chemo- and radiotherapy through effects on the Mcl-1 pro-survival protein. It is predicted that a small molecule Usp9x inhibitor could have a major impact in cancer therapy. However, recent data has raised caution about therapeutic targeting of Usp9X in cancer. In a study to identify genes that reduce the latency of pancreatic tumor formation in a mutant Kras model, Pérez-Mancera et al. 2012 reported that the Usp9x locus was frequently inactivated in pancreatic cancer cells, resulting in protection from anoikis and enhanced transformation. In order to clarify the role of Usp9X in pancreatic cancer, we used a newly created cell line 4668 established from mouse pancreatic tumors with doxycycline (DOX) inducible expression of KrasG12D and Tp53R172H. Stable Usp9x knockdown (KD) in 4668 cells had no effect on their proliferation or phenotype in 2D cultures. However, withdrawal of DOX from DOX-treated Usp9x KD 4668 cells resulted in 4-fold increase in colony formation, unlike DOX depleted control KD 4668 cells which undergo apoptosis resulting in colony disintegration. DOX treated control KD and Usp9x KD 4668 cells did not show any difference in basal 3D colony formation. These results suggest that Usp9x loss in the absence of continuous mutant Kras/p53 expression is sufficient to sustain 3D cancer cell growth, confirming a tumor suppressor role for Usp9x in a Kras mouse model of pancreatic cancer. To determine if Usp9x also plays a tumor suppressor role in human pancreatic tumors, we did Usp9x KD in three human pancreatic cancer cell lines with varied Kras mutational status; BxPC3 (wild type), PANC1 (heterozygous) and MIA-PACA2 (homozygous) which expressed high basal levels of Usp9x. MIA-PACA2-Usp9x KD cells undergo apoptosis and caspase activation. In contrast, BxPC3-Usp9x KD cells showed no effect on growth in 2D or in 3D culture. However, PANC1-Usp9x KD cells showed more rapid (38% p=0.004) 2D growth, but 3D colony formation was consistently reduced by >50%, (p=0.03). These results suggest that the effect of Usp9x loss in established human pancreatic tumor cells is distinct from that detected in a murine pancreatic tumor model. Distinctions may be partially dependent on their Kras mutational status or Usp9x substrate utilization in human vs. murine cells. To further assess the clinical relevance of our observations, Usp9x KD was evaluated in four recently established, low passage number human pancreatic patient tumor cell lines (UM2, UM6, UM16, UM76). Usp9x KD did not affect 2D growth but fully suppressed 3D colony growth. This activity was phenocopied by our small molecule Usp9x inhibitor, EOAI3402143 (G9), with nM efficacy. Together, these results suggest that possible species distinctions exist in the tumorigenic functions of Usp9x and necessitate further assessment of its role in pancreatic tumor development and treatment. Citation Format: Anupama Pal, Marina Pasca di Magliano, Moshe Talpaz, Michelle Dziubinski, Diane Simeone, Nicholas J. Donato. Role of Deubiquitinase Usp9x in pancreatic cancers: Tumor promoter or tumor suppressor. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4434. doi:10.1158/1538-7445.AM2014-4434

Abstract 2781: Elimination of PanIn2/3 in a GEMM for pancreatic cancer with disrupted p16 and activated Kras

Abstract When treated with clinically achievable concentrations of arsenic trioxide, ascorbic acid and antabuse (disulfiram) (AAA), we found that four human pancreatic cancer cell lines underwent robust cell death associated with ATP decline and ROS accumulation. In these cells, early apoptosis shifted to aponecrosis, and was dependent on VDAC since shRNA antagonistic to VDAC-1 abrogated the ROS accumulation and blocked cell death. In this study, assessment of AAA was performed in Pdx1-Cre transgenic mice with a conditional p16 knockout (p16flox/flox), and oncogenic KrasG12D. The KrasG12D, p16flox/flox; LSL-KrasG12D; Pdx1-Cre mice develop a full spectrum of pancreatic intraepithelial neoplasia (PanIN) lesions, pancreatic ductal adenocarcinoma (PDA), and frequent metastases. Eight week old mice were treated for 8 weeks, 3 times/week with AAA, sacrificed, and assessed for the development of pancreatic cancer. Little or no toxicity was observed in the AAA-treated mice. The development of frank pancreatic tumors was decreased from 37% in controls to 0% in AAA-treated mice. The mean number of PanIN 3 lesions/mouse declined from 14.2 in controls to 1.6 in AAA-treated mice (p=0.004); the mean number of PanIN 2 lesions declined from 5.8 in controls to 1.7 in AAA-treated mice (p=0.029), and the mean number of PanIN 1 lesions in control mice was 14.2 and in AAA-treated mice was 16.0 (p=0.82), indicating no difference in PanIN 1 occurrence between control and AAA-treated mice. The data also suggests that PanIN 2 and PanIN 3 lesions are killed after exposure to AAA, since mice prior to treatment had high levels of PanIN 2 (8.1 lesions/mouse) and PanIN 3 (11.4 lesions/mouse), similar to controls at 4 months of age. Therefore, AAA may have eliminated pre-existing PanINs rather than blocking their development. Results from immunohistochemical staining of the PanIN lesions for VDAC, indicated that PanIN 1 lesions have low, and PanIN 2 and 3 lesions have high, VDAC expression. This result parallels the in vitro human cell line data showing VDAC expression is critical to AAA sensitivity, and suggests a similar mechanism of action in the murine model, as we see in vitro. This is the first treatment that has shown potential to kill existing PanIN lesions and arrest the development of tumors in this pancreatic cancer murine model. Citation Format: Richard D. Dinnen, Wanglong Qiu, Gloria H. Su, Daniel P. Petrylak, Robert L. Fine. Elimination of PanIn2/3 in a GEMM for pancreatic cancer with disrupted p16 and activated Kras. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2781. doi:10.1158/1538-7445.AM2014-2781

Abstract 2042: Deregulated TS promotes tumor progression in diverse hematopoietic and mesenchymal cell lineages

Abstract Our laboratory recently showed that elevated TS plays a direct causal role in tumorigenesis of NIH3T3 cells in vitro and in the development of endocrine pancreatic mouse tumors in vivo. This observation changed the paradigm of TS as a passive biomarker and has significance for cancer treatment as it refocuses attention on the importance of TS as a tumor-promoting signal. To address whether TS plays an early role in tumorigenesis that is restricted to specific tumor types or if TS plays a late role associated with enhanced progression of established tumors of diverse histologic origin, we have now cross-bred human TS (hTS) transgenic mice with Ink4a/Arf null mice, a common somatic mutation frequently found in adult human cancers. Median survival of hTS/Ink4a/Arf+/- (n=15) mice was reduced 24% (313 vs. 411 days, p <0.05). Both hTS/Ink4a/Arf+/- and hTS/Ink4a/Arf-/- mice (n=16) showed an increase in lymphoma, histiocytic sarcoma (HS) and soft tissue sarcoma (SS). For example, the incidence of HS in hTS/Ink4a/Arf-/- male mice was 31% versus 12% in Ink4a/Arf-/- minus (p<0.01), and tumor cells had spread to multiple tissues (as many as 9 different organs) as compared to hTS minus mice where tumors were localized in lymph nodes and spleen. Strikingly, the tumor burden was markedly increased in hTS/Ink4a/Arf-/- mice: average spleen weight 1.11 vs. 0.49 g, p<0.001, lymph nodes, 1.06 g vs. 0.42 g, p<0.001. We also detected an increase in size and extent of tumor mass of SS (average weight 5.8 g). We detected increased aneuploidy in hTS/Ink4a/Arf-/- HS cell lines as compared to Ink4a/Arf-/- HS cell lines: chromosomal loss 32.1% vs. 16.7%, p<0.05, gain 24.5% vs. 0%, p<0.001 and translocations 9.4% vs. 0%, p<0.05 by spectral karyotyping analysis. Treatment of TS positive HS tumor cells with lenti-TS shRNAs downregulated TS expression and resulted in tumor growth inhibition in vitro as well as in mouse orthotopically transplanted xenograft. In addition, combined TS shRNAs with gemcitabine or pemetrexed treatment showed synergistic inhibition of HS tumor cell growth. These data demonstrate that deregulated TS enhanced genome instability and promote tumor progression in diverse hematopoietic and mesenchymal tumor cells. Citation Format: Min Chen, Akbar Nawab, Frederic J. Kaye, Maria Zajac-Kaye. Deregulated TS promotes tumor progression in diverse hematopoietic and mesenchymal cell lineages. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2042. doi:10.1158/1538-7445.AM2014-2042

Abstract 4426: Tumor suppressive effects of wild-type Hras on oncogenic Kras-driven pancreatic tumorigenesis

Abstract The RAS proteins are a group of small GTPases that can become constitutively activated by point mutations that are found in a quarter or more of all cancer patients, particularly in pancreatic cancer, in which over 90% of patients have an activating KRAS mutation. There are three well-characterized RAS protein family members: HRAS, NRAS, and KRAS, the latter of which is alternatively spliced at the C-terminus into two proteins, KRAS4A and KRAS4B. The RAS proteins are all nearly identical at their N-termini and core effector binding domains. However, they have divergent C-terminal membrane-binding regions that impart both different subcellular localization and subtle changes in signaling. While oncogenic RAS is well established to promote cancer, recent work has suggested that wild-type RAS proteins also participate in tumorigenesis. In this regard, we previously found that wild-type HRAS is activated downstream of oncogenic KRAS, which promoted tumor growth of human pancreatic cancer cell lines. To examine the role of wild-type Hras during de novo pancreatic tumor development, we tested whether knockout of the wild-type Hras gene altered tumorigenesis in oncogenic Kras-driven mice models of pancreatic cancer. Specifically, Hras homozygous null mice (Hras-/-) were crossed into a Pdx-Cre;LSL-KRasG12D/+ background in the absence or presence of an additional mutant p53 allele (Trp53R172H/+) to induce early and late pancreatic cancer, respectively. Surprisingly, loss of Hras led to an increase in early pancreatic lesions and reduced survival in the model of late disease. Since HRAS is activated downstream of oncogenic KRAS, and high oncogenic signaling can induce a senescent growth arrest, we tested and found that suppressing senescence by mutating both alleles of Trp53 ameliorated the survival difference between wild-type and null Hras mice. We thus hypothesize that wild-type Hras amplifies oncogenic Kras signaling, leading to the growth arrest of senescence and thereby inhibit early tumorigenesis. However, once senescence is suppressed later in tumorigenesis, wild-type RAS proteins instead promote more malignant phenotypes. Citation Format: Jamie D. Weyandt, Christopher M. Counter. Tumor suppressive effects of wild-type Hras on oncogenic Kras-driven pancreatic tumorigenesis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4426. doi:10.1158/1538-7445.AM2014-4426

PI3K Regulation of RAC1 Is Required for KRAS-Induced Pancreatic Tumorigenesis in Mice

New drug targets are urgently needed for the treatment of patients with pancreatic ductal adenocarcinoma (PDA). Nearly all PDAs contain oncogenic mutations in the KRAS gene. Pharmacological inhibition of KRAS has been unsuccessful, leading to a focus on downstream effectors that are more easily targeted with small molecule inhibitors. We investigated the contributions of phosphoinositide 3-kinase (PI3K) to KRAS-initiated tumorigenesis. Tumorigenesis was measured in the Kras(G12D/+);Ptf1a(Cre/+) mouse model of PDA; these mice were crossed with mice with pancreas-specific disruption of genes encoding PI3K p110α (Pik3ca), p110β (Pik3cb), or RAC1 (Rac1). Pancreatitis was induced with 5 daily intraperitoneal injections of cerulein. Pancreata and primary acinar cells were isolated; acinar cells were incubated with an inhibitor of p110α (PIK75) followed by a broad-spectrum PI3K inhibitor (GDC0941). PDA cell lines (NB490 and MiaPaCa2) were incubated with PIK75 followed by GDC0941. Tissues and cells were analyzed by histology, immunohistochemistry, quantitative reverse-transcription polymerase chain reaction, and immunofluorescence analyses for factors involved in the PI3K signaling pathway. We also examined human pancreas tissue microarrays for levels of p110α and other PI3K pathway components. Pancreas-specific disruption of Pik3ca or Rac1, but not Pik3cb, prevented the development of pancreatic tumors in Kras(G12D/+);Ptf1a(Cre/+) mice. Loss of transformation was independent of AKT regulation. Preneoplastic ductal metaplasia developed in mice lacking pancreatic p110α but regressed. Levels of activated and total RAC1 were higher in pancreatic tissues from Kras(G12D/+);Ptf1a(Cre/+) mice compared with controls. Loss of p110α reduced RAC1 activity and expression in these tissues. p110α was required for the up-regulation and activity of RAC guanine exchange factors during tumorigenesis. Levels of p110α and RAC1 were increased in human pancreatic intraepithelial neoplasias and PDAs compared with healthy pancreata. KRAS signaling, via p110α to activate RAC1, is required for transformation in Kras(G12D/+);Ptf1a(Cre/+) mice.

Nicotine Promotes Initiation and Progression of KRAS-Induced Pancreatic Cancer via Gata6-Dependent Dedifferentiation of Acinar Cells in Mice

Although smoking is a leading risk factor for pancreatic ductal adenocarcinoma (PDAC), little is known about the mechanisms by which smoking promotes initiation or progression of PDAC. We studied the effects of nicotine administration on pancreatic cancer development in Kras(+/LSLG12Vgeo);Elas-tTA/tetO-Cre (Ela-KRAS) mice, Kras(+/LSLG12D);Trp53+/LSLR172H;Pdx-1-Cre (KPC) mice (which express constitutively active forms of KRAS), and C57/B6 mice. Mice were given nicotine for up to 86 weeks to produce blood levels comparable with those of intermediate smokers. Pancreatic tissues were collected and analyzed by immunohistochemistry and reverse transcriptase polymerase chain reaction; cells were isolated and assayed for colony and sphere formation and gene expression. The effects of nicotine were also evaluated in primary pancreatic acinar cells isolated from wild-type, nAChR7a(-/-), Trp53(-/-), and Gata6(-/-);Trp53(-/-) mice. We also analyzed primary PDAC cells that overexpressed GATA6 from lentiviral expression vectors. Administration of nicotine accelerated transformation of pancreatic cells and tumor formation in Ela-KRAS and KPC mice. Nicotine induced dedifferentiation of acinar cells by activating AKT-ERK-MYC signaling; this led to inhibition of Gata6 promoter activity, loss of GATA6 protein, and subsequent loss of acinar differentiation and hyperactivation of oncogenic KRAS. Nicotine also promoted aggressiveness of established tumors as well as the epithelial-mesenchymal transition, increasing numbers of circulating cancer cells and their dissemination to the liver, compared with mice not exposed to nicotine. Nicotine induced pancreatic cells to acquire gene expression patterns and functional characteristics of cancer stem cells. These effects were markedly attenuated in K-Ras(+/LSL-G12D);Trp53(+/LSLR172H);Pdx-1-Cre mice given metformin. Metformin prevented nicotine-induced pancreatic carcinogenesis and tumor growth by up-regulating GATA6 and promoting differentiation toward an acinar cell program. In mice, nicotine promotes pancreatic carcinogenesis and tumor development via down-regulation of Gata6 to induce acinar cell dedifferentiation.

Parp-1 genetic ablation in Ela-myc mice unveils novel roles for Parp-1 in pancreatic cancer.

Pancreatic cancer has a dismal prognosis and is currently the fourth leading cause of cancer-related death in developed countries. The inhibition of poly(ADP-ribose) polymerase-1 (Parp-1), the major protein responsible for poly(ADP-ribosy)lation in response to DNA damage, has emerged as a promising treatment for several tumour types. Here we aimed to elucidate the involvement of Parp-1 in pancreatic tumour progression. We assessed Parp-1 protein expression in normal, preneoplastic and pancreatic tumour samples from humans and from K-Ras- and c-myc-driven mouse models of pancreatic cancer. Parp-1 was highly expressed in acinar cells in normal and cancer tissues. In contrast, ductal cells expressed very low or undetectable levels of this protein, both in a normal and in a tumour context. The Parp-1 expression pattern was similar in human and mouse samples, thereby validating the use of animal models for further studies. To determine the in vivo effects of Parp-1 depletion on pancreatic cancer progression, Ela-myc-driven pancreatic tumour development was analysed in a Parp-1 knock-out background. Loss of Parp-1 resulted in increased tumour necrosis and decreased proliferation, apoptosis and angiogenesis. Interestingly, Ela-myc:Parp-1(-/-) mice displayed fewer ductal tumours than their Ela-myc:Parp-1(+/+) counterparts, suggesting that Parp-1 participates in promoting acinar-to-ductal metaplasia, a key event in pancreatic cancer initiation. Moreover, impaired macrophage recruitment can be responsible for the ADM blockade found in the Ela-myc:Parp-1(-/-) mice. Finally, molecular analysis revealed that Parp-1 modulates ADM downstream of the Stat3-MMP7 axis and is also involved in transcriptional up-regulation of the MDM2, VEGFR1 and MMP28 cancer-related genes. In conclusion, the expression pattern of Parp-1 in normal and cancer tissue and the in vivo functional effects of Parp-1 depletion point to a novel role for this protein in pancreatic carcinogenesis and shed light into the clinical use of Parp-1 inhibitors.

Stellate cells, a point of light in the dark night of pancreatic cancer

Sir, We have read with particular attention the recent article published in the BJC by Alvarez et al (2013). They, through endoscopic ultrasound elastography system and conventional immuno-histochemical assays, perform an intrinsic characterisation of tumour stroma's stiffness, fibroblast density and architecture focusing in type I collagen fibre arrangement, cancer-associated fibroblasts (CAFs) and activated CAF quantification. The analysis of post-treatment tissues from patients treated with nab-paclitaxel and gemcitabine combination showed a decrease in CAF number and changes in collagen architecture. These results reaffirm the potential ability of nab-paclitaxel to target the stroma and to change its phenotype, altering the ‘hard of the rock', as described by Garber (2010), intensifying its permeability to deliver cytotoxic agents such as gemcitabine by increasing tumour vascularisation or enzymatic inhibition (Frese et al, 2012) that would suppose a better pharmacokinetic profile (Von Hoff et al, 2011). Even though it is known the clinical benefit of nab-paclitaxel plus gemcitabine in metastatic pancreatic cancer, the cellular or molecular mechanisms that are expressly addressing this stromal involution have not been appointed yet (Alvarez et al, 2013). In this sense, authors analysed the role of secreted protein, acidic and rich in cysteine (SPARC) although they could not find the correlation between the degree of SPARC expression and clinical or pathological responses due to its high affinity to albumin protein of nab-paclitaxel. SPARC strengthens the accumulation of nab-paclitaxel mediated by albumin in the ‘tumour's Achilles' heel' (Von Hoff, Annual meeting of the American Society of Clinical Oncology, 2009); however, the mere use of this endogenous transport system would not justify the clinical impact of this combined treatment. From our point of view the key may be in what Omary et al (2007) described as ‘a star on the rise' in pancreatic disease: the pancreatic stellate cells (PaSCs; α-SMA+ Nestin++ Vimentin++) because of their role as the main manufacturers of profibrotic extracellular matrix (ECM) components of the pancreatic tumour stroma, and which should match with the activated CAFs (SMA+ Vimentin+ fibroblasts) in Alvareźs paper. In pancreatic cancer, PaSCs show increased proliferation and migration properties, and so they could be a suitable target for nab-paclitaxel because of their ability to interfere with the mitotic activity (Gradishar, 2006). Alvarez et al (2013) demonstrate that although CAFs number decreases in patients treated with nab-paclitaxel plus gemcitabine in neoadjuvant setting, the average of activated CAFs remains unchanged. We would like to explain our hypothesis related to what is happening in the stroma: it could be a transient blockade of activated CAFs metabolism, a quiescent status forced for a pharmacologically active substance. Bachem et al (2005) show that cancer cells (CCs) induce a desmoplastic reaction in pancreatic adenocarcinoma by stimulating PaSCs in a paracrine way. So it could be an indirect elimination of principal fibrogenic mediators that stimulate proliferation (platelet-derived growth factor) and ECM synthesis (fibroblast growth factor -2 and transforming growth factor -s1) of activated PaSCs through the abrogation of CCs. In this sense, the hypothetical presence of surface cellular receptors for nab-paclitaxel in CCs could be an interesting pathway biomarker for the effectiveness of the drug as authors related. On the other hand, the ablation of physiological PaSCs functioning after nab-paclitaxel inclusion, in turn, would break the two-way communication between PaSCs and CCs (Apte et al, 2013). In this context, under normal conditions CCs recruit new PaSCs to their vicinity (Vonlaufen et al, 2008), whereas nab-paclitaxel would temporarily inhibit the main mechanism that rules the desmoplastic reaction. All these molecular mechanisms would allow to maintain and stabilise the activated CAF's number despite them being dysfunctional, and also would decline its CAF progeny. In this sense, Apte et al (2013) and Bachem et al (2005) propose that activated PaSCs can transform into a myofibroblast-like phenotype sub-population with the ability of secreting excess amounts of ECM. In conclusion, due to the dynamic nature of the stromal compartment, it is critically involved in the development and progresion of pancreatic tumours (Heinemann et al, 2013). Before using neadjuvant treatment it may be important to know the stroma's cellular activation grade with regard to PaSCs plus their number. In this sense, PaSCs or activated CAFs could give us ample information about tumoral potential of the stroma itself and so could have an important contribution for the patient's prognosis. Also, the fact that activated CAFs do not decrease after nab-paclitaxel treatment could not mean that these cells are not one of the main actors of stromal disruption but the primary target of nab-paclitaxel.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature