Signaling In Pancreatic Ductal Adenocarcinoma Research Articles

Stromal Annexin A2 expression is predictive of decreased survival in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDA) is renowned for high rates of metastasis and poor survival. Its notoriously dense fibrotic stroma contributes to chemoresistance. Stromal signaling in PDA is recognized for its multiple roles in regulating tumor invasion and metastasis. However, no stromal biomarker which can predict survival in PDA exists. Annexin A2 (AnxA2) was formerly identified as a metastasis-associated protein in PDA and tumoral overexpression is associated with poor survival. In this study, we examined AnxA2 expression in the tumor microenvironment in a preclinical model of PDA which suggests its role in tumor colonization.We injected wild-type (KPC) and AnxA2 knockout (KPCA) pancreatic cells into C57BL/GJ (B6) and AnxA2 knockout (KO) mice using the hemi-spleen model and observed their survival. We performed quantitative immunohistochemistry examining stromal AnxA2 expression in 56 patients who had surgically resected PDA and correlated expression with clinical outcomes.B6 mice injected with KPC cells demonstrated decreased median survival compared to those injected with KPCA cells (90 days vs. not reached, p < 0.0001) whereas there was no survival difference in the AnxA2 KO mice (p = 0.63). In patient specimens, we found that high stromal AnxA2 expression (≥80th percentile) was associated with significantly reduced disease-free survival (p = 0.002) and overall survival (p < 0.001). Using multivariate Cox models, there were no significant associations between other clinical covariates apart from high stromal AnxA2 expression.This study highlights the role that stromal AnxA2 expression plays as a prognostic marker in PDA and its potential as a predictive biomarker for survival outcomes in PDA.

RAGE maintains high levels of NFκB and oncogenic Kras activity in pancreatic cancer

Oncogenic KRas activity is central to several cancer types including pancreatic ductal adenocarcinoma (PDAC) but has been determined to be “undruggable”. Recent studies have indicated that oncogenic KRas is not constitutively active but relies on a feed-forward stimulatory mechanism involving NFκB mediated inflammation. In the current study, we investigated the role of the receptor for advanced glycation end-products (RAGE) in maintaining oncogenic signaling in PDAC. We observed that there was a shift in the levels of specific RAGE isoforms and altered cellular localization in PDAC. Furthermore, inhibition of RAGE using a pharmacological antagonist, FPS-ZM1, or a blocking antibody, decreased phosphorylation of IKBα and inhibited Erk activity down-stream of Kras in PDAC cell lines. In vivo, inhibition of RAGE using FPS-ZM1 reduced the growth of PDAC syngeneic orthotopic xenografts and prolonged survival. These data indicate that RAGE plays a central role in maintaining inflammatory signaling in PDAC that benefits tumor growth. These observations support the development of approaches to inhibit the carcinogenic actions of Kras indirectly by blocking the mechanisms which maintain its activity.

Abstract A34: Oncogenic KRAS regulates pancreatic cancer cell signaling via stromal reciprocation

Abstract Pancreatic Ductal Adenocarcinoma is characterized by a reactive stroma, which modifies tumor progression and response to therapy. Oncogenic mutations regulate signaling both within tumor cells and adjacent stromal cells. However, defining whether oncogenes can regulate tumor cell signaling and phenotypic behavior via stromal cells is of importance to understand mechanisms of disease progression and response to therapy. Here we show that oncogenic KRAS (KRASG12D) regulates pancreatic cancer cell signaling via stromal stellate cells. By combining cell-specific proteome labeling with multivariate phosphoproteomics, we analyzed heterocellular KRASG12D signaling in Pancreatic Ductal Adenocarcinoma (PDA) cells. Tumor cell KRASG12D engages heterotypic stellate cells, which subsequently instigate reciprocal signaling in the tumor cells. Reciprocal signaling employs additional kinases and doubles the number of regulated signaling nodes from cell-autonomous KRASG12D. Consequently, reciprocal KRASG12D produces a tumor cell phosphoproteome and total proteome that is distinct from cell-autonomous KRASG12D alone. Reciprocal signaling regulates tumor cell proliferation, apoptosis, and increases mitochondrial capacity via an IGF1R/AXL-AKT axis. These results demonstrate that oncogene signaling should be viewed as a heterocellular process and that our existing cell-autonomous perspective underrepresents the extent of oncogene signaling in cancer. Citation Format: Christopher Tape, Stephanie Ling, Maria Dimitriadi, Kelly McMahon, Jonathan Worboys, Hui S. Leong, Ida Norrie, Crispin Miller, George Poulogiannis, Douglas Lauffenburger, Claus Jorgensen.{Authors}. Oncogenic KRAS regulates pancreatic cancer cell signaling via stromal reciprocation. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2016 May 12-15; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(24 Suppl):Abstract nr A34.

The PRKD1 promoter is a target of the KRas-NF-κB pathway in pancreatic cancer.

Increased expression of PRKD1 and its gene product protein kinase D1 (PKD1) are linked to oncogenic signaling in pancreatic ductal adenocarcinoma, but a direct functional relationship to oncogenic KRas has not been established so far. We here describe the PRKD1 gene promoter as a target for oncogenic KRas signaling. We demonstrate that KRas-induced activation of the canonical NF-κB pathway is one mechanism of how PRKD1 expression is increased and identify the binding sites for NF-κB in the PRKD1 promoter. Altogether, these results describe a novel mechanism governing PRKD1 gene expression in PDA and provide a functional link between oncogenic KRas, NF-κB and expression of PRKD1.

Negative control of TRAIL-R1 signaling by transforming growth factor β1 in pancreatic tumor cells involves Smad-dependent down regulation of TRAIL-R1

Pancreatic ductal adenocarcinoma (PDAC) is characterized by both, overexpression of transforming growth factor (TGF)β and resistance of the tumor cells to many apoptosis-inducing stimuli. The latter negatively impacts the outcome of therapeutic efforts and represents one important mechanism which tumor cells utilize to escape the immune surveillance. Since TGFβ acts as a tumor promoter in advanced tumor stages and suppression of apoptosis is a known driver of tumor progression, it is possible that TGFβ functions as a crucial determinant of tumor cell sensitivity to apoptosis in PDAC. Here, we have studied the impact of TGFβ on TNF-related apoptosis inducing ligand (TRAIL)-induced signaling in PDAC cells.In TGFβ-responsive Panc1 and Colo357 cells, TGFβ1 reduced total and plasma membrane-associated levels of TRAIL-R1 but not those of TRAIL-R2. Consistent with the known predominant role of TRAIL-R1 in TRAIL-mediated signaling in PDAC, TGFβ1 inhibited TRAIL-induced DISC formation and apoptosis as well as phosphorylation of MAPKs and IκBα. Similarly, it also reduced signaling of TRAIL-R1 following its specific activation with an agonistic antibody. In contrast, specific TRAIL-R2 signaling remained unchanged. The TGFβ1 effect on TRAIL-R1 expression was mimicked by ectopic expression of a kinase-active version of the TGFβ type I receptor ALK5 (ALK5-T204D) but not by ALK5 double mutant lacking the ability to phosphorylate Smad proteins (RImL45-T204D). Moreover, TGFβ regulation of TRAIL-R1 was absent in two PDAC cell lines lacking the Smad4 gene DPC4 and siRNA-mediated silencing of Smad4 in Smad4-positive Panc1 cells abolished the TGFβ-mediated decrease in TRAIL-R1 expression, together showing that ALK5/Smad4 signaling is crucial for TGFβ regulation of TRAIL-R1 expression. Our results suggest a novel tumor-promoting function of TGFβ1. By downregulating TRAIL-R1, TGFβ1 may not only promote tumor escape from immune surveillance but also negatively impact on TRAIL- or TRAIL-R1-based therapy regimens for treatment of PDAC.

Resolution of Novel Pancreatic Ductal Adenocarcinoma Subtypes by Global Phosphotyrosine Profiling

Comprehensive characterization of signaling in pancreatic ductal adenocarcinoma (PDAC) promises to enhance our understanding of the molecular aberrations driving this devastating disease, and may identify novel therapeutic targets as well as biomarkers that enable stratification of patients for optimal therapy. Here, we use immunoaffinity-coupled high-resolution mass spectrometry to characterize global tyrosine phosphorylation patterns across two large panels of human PDAC cell lines: the ATCC series (19 cell lines) and TKCC series (17 cell lines). This resulted in the identification and quantification of over 1800 class 1 tyrosine phosphorylation sites and the consistent segregation of both PDAC cell line series into three subtypes with distinct tyrosine phosphorylation profiles. Subtype-selective signaling networks were characterized by identification of subtype-enriched phosphosites together with pathway and network analyses. This revealed that the three subtypes characteristic of the ATCC series were associated with perturbations in signaling networks associated with cell-cell adhesion and epithelial-mesenchyme transition, mRNA metabolism, and receptor tyrosine kinase (RTK) signaling, respectively. Specifically, the third subtype exhibited enhanced tyrosine phosphorylation of multiple RTKs including the EGFR, ERBB3 and MET. Interestingly, a similar RTK-enriched subtype was identified in the TKCC series, and 'classifier' sites for each series identified using Random Forest models were able to predict the subtypes of the alternate series with high accuracy, highlighting the conservation of the three subtypes across the two series. Finally, RTK-enriched cell lines from both series exhibited enhanced sensitivity to the small molecule EGFR inhibitor erlotinib, indicating that their phosphosignature may provide a predictive biomarker for response to this targeted therapy. These studies highlight how resolution of subtype-selective signaling networks can provide a novel taxonomy for particular cancers, and provide insights into PDAC biology that can be exploited for improved patient management.

Abstract 2490: Smoking enriches cancer stem cell population and activates Sox9 through NF-kB signaling in pancreatic ductal adenocarcinoma

Abstract Introduction: Recent studies have demonstrated a clear association between smoking and the incidence of pancreatic ductal adenocarcinoma (PDAC); however, the effect of cigarette smoke in the activation of stem cell (SC) or cancer stem cell (CSC) genes and their involvement in the initiation and progression of PDAC have not yet been studied. It is well known that CSCs are responsible for the drug resistance and aggressiveness of the disease including PDAC. In this study, we investigated the effects of smoking on enrichment of SC/CSCs in pancreatic normal and ductal adenocarcinoma cells, and we also examined whether smoking can activate NF-kB signaling, which is in part leads to enrichment of CSC and induction of CSC markers in PDAC. Methods: Cigarette smoke extract (CSE) was prepared, and HPNE (Human pancreatic nestin positive cells) and Capan-1 pancreatic cancer (PC) cells were treated with CSE for up to ∼15 weeks. Side population (SP) were analyzed by Hoechst staining using Flow-cytomer, and various CSC markers such as PD2 (a stem cell maintenance marker), CD44, ALDH-1, SOX-9 (a multipotent SC marker) and Oct-3/4 (a pluripotent marker), and NF-kB signaling molecules were analyzed by western blotting. ALDH1+ cells, CD44+CD24+ CSCs and G0/G1 phase low cycling quiescent cells were analyzed by flow cytometer. An in-vitro sphere culture was also performed to further confirm the smoke induced CSC properties. Smoke exposed pancreatic tissues excised from unfloxed littermate control (LSL-K-Ras G12D) pancreatic tissue sections were immunostained for SOX-9 using immunohistochemistry (IHC), and for SOX9 and CD44 using immunofluorescence. Results: Our results showed increased SC/CSCs and more number of spheres by CSE treated cells as compared to their untreated controls and displayed elevated protein expressions of SC/CSC markers. We also observed an elevated CD44+CD24+ CSCs, increased ALDH1+ cells and increased G0/G1 low cycling quiescent cell population in CSE treated cells as compared to untreated controls. In addition, increased immunohistochemical staining for SOX9 and increased immunofluorescent signal for SOX-9 and CD44 were observed in smoke exposed animal tissues indicating that smoking may transform SOX-9+ multipotent SCs into SOX9+CD44+ CSCs. We also analyzed the expression levels of NF-kB signaling molecules in CSE treated HPNE and Capan-1 cells. As compared to their untreated controls, CSE treated cells showed elevated protein expression levels of phospho AKT, phospho RelA (SOX-9 promoter binding subunit of NF-kB complex) and phospho IKKα (a kinase that phosphorylates IKBα, an inhibitor of RelA) suggesting that smoking activates NF-kB signaling. Conclusion: Our results illustrate that smoking enriches SC/CSCs populations in normal pancreatic cells as well as in pancreatic cancer cells, and activates SOX9 through NF-kB signaling in PDAC. Citation Format: Ramakrishna Nimmakayala, Parthasarathy Seshacharyulu, Seema Chugh, Imayavaramban Lakshmanan, Satyanarayana Rachagani, Surinder K Batra, Moorthy P Ponnusamy. Smoking enriches cancer stem cell population and activates Sox9 through NF-kB signaling in pancreatic ductal adenocarcinoma. [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 2490.

Oncogenic KRAS Regulates Tumor Cell Signaling via Stromal Reciprocation

SummaryOncogenic mutations regulate signaling within both tumor cells and adjacent stromal cells. Here, we show that oncogenic KRAS (KRASG12D) also regulates tumor cell signaling via stromal cells. By combining cell-specific proteome labeling with multivariate phosphoproteomics, we analyzed heterocellular KRASG12D signaling in pancreatic ductal adenocarcinoma (PDA) cells. Tumor cell KRASG12D engages heterotypic fibroblasts, which subsequently instigate reciprocal signaling in the tumor cells. Reciprocal signaling employs additional kinases and doubles the number of regulated signaling nodes from cell-autonomous KRASG12D. Consequently, reciprocal KRASG12D produces a tumor cell phosphoproteome and total proteome that is distinct from cell-autonomous KRASG12D alone. Reciprocal signaling regulates tumor cell proliferation and apoptosis and increases mitochondrial capacity via an IGF1R/AXL-AKT axis. These results demonstrate that oncogene signaling should be viewed as a heterocellular process and that our existing cell-autonomous perspective underrepresents the extent of oncogene signaling in cancer.Video

MicroRNA-323-3p inhibits cell invasion and metastasis in pancreatic ductal adenocarcinoma via direct suppression of SMAD2 and SMAD3.

Pancreatic ductal adenocarcinoma (PDAC), which accounts for 96% of all pancreatic cancer cases, is characterized by rapid progression, invasion and metastasis. Transforming growth factor-beta (TGF-β) signaling is an essential pathway in metastatic progression and microRNAs (miRNA) play central roles in the regulation of various biological and pathologic processes including cancer metastasis. However, the molecular mechanisms involved in regulation of miRNAs and activation of TGF-β signaling in PDAC remain to be established. The results of this study suggested that miR-323-3p expression in PDAC tissues and cell lines was significantly decreased compared to levels in normal pancreatic tissues and primary cultured pancreatic duct epithelial cells. Further investigation revealed that miR-323-3p directly targeted and suppressed SMAD2 and SMAD3, both key components in TGF-β signaling. Lower levels of miR-323-3p predicted poorer prognosis in patients with PDAC. Ectopic overexpression of miR-323-3p significantly inhibited, while silencing of miR-323-3p increased the migration and invasion abilities of PDAC cells in vitro. Moreover, using an in vivo mouse model, we demonstrated that overexpressing of miR-323-3p significantly reduced, while knockdown of miR-323-3p enhanced lung metastatic colonization of PANC-1 cells. Furthermore, miR-323-3p-induced TGF-b signaling inhibition and cell motility suppression were partially rescued by overexpressing of Smad2 and Smad3 in PDAC cells. Our findings suggest that re-expression of miR-323-3p might offer a novel therapeutic target against metastasis in patients with PDAC.

Clinicopathological Implications of Wingless/int1 (WNT) Signaling Pathway in Pancreatic Ductal Adenocarcinoma.

Pancreatic cancer is still one of the most lethal malignancies in the world, and a more thorough understanding of its detailed pathogenetic mechanisms and the development of more effective therapeutic strategies are urgently required. Pancreatic ductal adenocarcinoma (PDA), the most common type of pancreatic cancer, is characterized by consistent genetic abnormalities such as point mutations in the Kirsten rat sarcoma viral oncogene homolog (KRAS) and in the tumor suppressor protein p53 (TP53) genes. Alterations in intracellular core signal pathways have also been shown to induce the development or progression of PDA. The Wingless/int1 (WNT) signal pathway plays a pivotal role in embryonic development, cellular proliferation and differentiation, and dysregulation of WNT signaling can lead to neoplastic transformation in a variety of organ systems, including the pancreas. Recent studies have shown that altered WNT signaling is associated with a poor prognosis in patients with PDA, suggesting that the pathway is a predictor of patients' survival and a potential therapeutic target of PDA. In this review, the clinicopathological implications of WNT signaling in PDA are highlighted.

Calcipotriol Targets LRP6 to Inhibit Wnt Signaling in Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive malignancy in need of more effective treatment approaches. One potential therapeutic target is Wnt/β-catenin signaling, which plays important roles in PDAC tumor initiation and progression. Among Wnt inhibitors with suitable in vivo biologic activity is vitamin D, which is known to antagonize Wnt/β-catenin signaling in colorectal cancer and have antitumor activity in PDAC. For this study, the relationship between vitamin D signaling, Wnt/β-catenin activity, and tumor cell growth in PDAC was investigated through the use of calcipotriol, a potent non-hypercalcemic vitamin D analogue. PDAC tumor cell growth inhibition by calcipotriol was positively correlated with vitamin D receptor expression and Wnt/β-catenin activity. Furthermore, vitamin D and Wnt signaling activity were found to be reciprocally linked through feedback regulation. Calcipotriol inhibited autocrine Wnt/β-catenin signaling in PDAC cell lines in parallel with decreased protein levels of the low-density lipoprotein receptor-related protein 6 (LRP6), a requisite coreceptor for ligand-dependent canonical Wnt signaling. Decrease in LRP6 protein seen with calcipotriol was mediated through a novel mechanism involving transcriptional upregulation of low-density lipoprotein receptor adaptor protein 1 (LDLRAP1). Finally, changes in LRP6 or LDLRAP1 expression directly altered Wnt reporter activity, supporting their roles as regulators of ligand-dependent Wnt/β-catenin signaling. This study provides a novel biochemical target through which vitamin D signaling exerts inhibitory effects on Wnt/β-catenin signaling, as well as potential biomarkers for predicting and following tumor response to vitamin D-based therapy.

Abstract B77: Vitamin D suppresses pancreatic cancer growth through inhibition of autocrine Wnt/β-catenin signaling

Abstract Epidemiological studies indicate higher vitamin D levels are associated with a reduced risk for pancreatic ductal adenocarcinoma (PDAC) and other malignancies. Although vitamin D analogs inhibit the growth of some PDAC cell lines in vitro and in vivo, a clinical trial with an active metabolite of vitamin D (seocalcitol) demonstrated no objective response in patients with inoperable PDAC. In addition to initiating vitamin D receptor (VDR) mediated transcription, vitamin D is known to inhibit Wnt/β-catenin signaling, a pathway recently shown to play a critical role in pancreatic cancer initiation and progression. For this study we chose to explore a possible link between these two signaling pathways. Initially, we found VDR expression strongly correlated with endogenous Wnt/β-catenin signaling activity across a large panel of PDAC cell lines, suggesting VDR may serve as a critical mediator of Wnt/β-catenin signaling in PDAC and/or that VDR itself may be a transcriptional target of the Wnt pathway. Therefore, we next determined whether the growth inhibitory effects of vitamin D analog calcipotriol in PDAC cell lines could be mechanistically linked to its action on Wnt/β-catenin signaling. Using a β-catenin-activated reporter, we found calcipotriol inhibited Wnt/β-catenin signaling and anchorage-dependent growth in a dose-dependent manner and with greater efficacy in those PDAC cell lines with high levels of endogenous Wnt/β-catenin signaling (Wnt high). In parallel, calcipotriol also inhibited anchorage-independent growth of the same Wnt high PDAC lines in soft agar assays, a phenotype we have previously shown to be critically linked to autocrine Wnt/β-catenin signaling activity. Previously published work in colorectal cancer finds vitamin D initiates a physical interaction between VDR and β-catenin that translocates β-catenin from the nucleus to the plasma membrane to inhibit Wnt/β-catenin mediated transcription. However, in exploring this potential biochemical interaction in PDAC cell lines we have been unable to co-immunoprecipitate VDR and β-catenin, leading us to speculate that vitamin D disrupts Wnt/β-catenin signaling through a novel alternative mechanism that we are currently investigating. In conclusion, the present study identifies a subset of PDAC cell lines defined by higher levels of Wnt/β-catenin signaling that show increased sensitivity to vitamin D-mediated growth inhibition. These results may be of clinical utility in stratifying patients most likely to benefit from vitamin D-based therapy. Citation Format: Michael Dawson Arensman, Claire Ostertag-Hill, Anna Rose Lay, David Dawson. Vitamin D suppresses pancreatic cancer growth through inhibition of autocrine Wnt/β-catenin signaling. [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 B77.

Abstract A79: Post-transcriptional regulatory networks of pancreatic tumor invasion

Abstract Guidance molecules from the Slit gene family were originally described as cues for the directional guidance of axons in the developing nervous system. More recently, members of these families have been found to play critical roles in epithelial development, angiogenesis and tumorigenesis. SLIT2 has properties of a potential tumor suppressor, is mutated at a low frequency in pancreatic tumors and is epigenetically silenced in many cancers. mir-218-1 is an intronic microRNA found within intron 15 of the SLIT2 gene. We propose that cell intrinsic mir-218-1 expression and function is independent of SLIT2 signaling in pancreatic ductal adenocarcinoma. This dynamic expression of mir-218-1 can inhibit tumor dissemination following the conversion of precursor lesions to invasive carcinoma. In normal pancreatic tissue, SLIT2 is highly expressed in the ductal compartment and expressed at a reduced level in the majority of primary pancreatic ductal adenocarcinomas (PDACs). We determined that KRAS-dependent pancreatic cancer cell lines express SLIT2 while KRAS-independent cell lines show silenced SLIT2 expression. Sequenom and chromatin immunoprecipitation (ChIP) confirmed that loss of SLIT2 in KRAS-independent lines was due to DNA hypermethylation and enrichment of silencing histone marks at the SLIT2 promoter and transcriptional start site with an inverse correlation between SLIT2 and EZH2 expression. Activating histone marks are found at both the SLIT2 promoter and transcriptional start site in KRAS-dependent lines suggesting that SLIT2 is lost during pancreatic cancer progression due to epigenetic silencing. Many intronic microRNAs are transcribed from the host gene promoter; however, mir-218-1 expression is variable in pancreatic cancer cell lines and does not correlate with SLIT2 gene expression. Therefore, we examined whether mir-218-1 had an alternative promoter independent of the SLIT2 promoter. Using ChIP for H3K4me3, a marker for promoter regions, we discovered an alternative promoter of mir-218-1. ChIP for H4ac and Sequenom showed that the chromatin structure within this putative alternative promoter is acetylated, unmethylated, and, thus, permissive for transcription. The putative alternative mir-218-1 promoter was also found to have basal transcription via enhanced luciferase reporter activity and enrichment of RNA polymerase II at the H3K4me3/H4ac peak. In silico analysis of the mir-218-1 alternative promoter region indicated that DNA binding sites for transcriptional repressors MYC/MAX and ZEB1 are present. In silico analysis also showed the presence of DNA binding sites for transcriptional activators CDX1, LEF1/TCF1 and NF-κB. The transcription factor NF-κB and oncogene KRAS frequently function cooperatively to promote tumor progression; however, maintenance of ductal architecture is also important in pancreatic cancer invasion and metastasis. Site-directed mutagenesis of the NF-κB binding site decreased luciferase reporter activity suggesting that NF-κB is an activator of mir-218-1 expression at this alternative promoter. This suggests that mir-218-1 expression is uncoupled from SLIT2 expression and may contain its own tumor suppressor properties independent of SLIT2 signaling. Matrix metalloproteinases (MMPs) are associated with tumor initiation and progression by targeting extracellular matrix (ECM) components. Invadopodia are structures that have been found to facilitate the invasive phenotype of many cancer types and are dynamic actin-rich cellular projections that localize proteases such as MMP2, MMP9, and MMP14 to degrade the ECM. In addition to ROBO1, we identified additional novel mir-218-1 target genes that control PDAC cell invasion. Our results further demonstrate that mir-218-1 functions in the suppression of invadopodia maturation and metalloproteinase matrix degradation. Overall, our data establishes that mir-218-1 is a key independent mediator of pancreatic cancer invasion through the extracellular matrix. Citation Format: Brenna A. Rheinheimer, Lukas Vrba, Bernard W. Futscher, Ronald L. Heimark. Post-transcriptional regulatory networks of pancreatic tumor invasion. [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 A79.

Pancreatic cell plasticity and cancer initiation induced by oncogenic Kras is completely dependent on wild-type PI 3-kinase p110α.

Increased PI 3-kinase (PI3K) signaling in pancreatic ductal adenocarcinoma (PDAC) correlates with poor prognosis, but the role of class I PI3K isoforms during its induction remains unclear. Using genetically engineered mice and pharmacological isoform-selective inhibitors, we found that the p110α PI3K isoform is a major signaling enzyme for PDAC development induced by a combination of genetic and nongenetic factors. Inactivation of this single isoform blocked the irreversible transition of exocrine acinar cells into pancreatic preneoplastic ductal lesions by oncogenic Kras and/or pancreatic injury. Hitting the other ubiquitous isoform, p110β, did not prevent preneoplastic lesion initiation. p110α signaling through small GTPase Rho and actin cytoskeleton controls the reprogramming of acinar cells and regulates cell morphology in vivo and in vitro. Finally, p110α was necessary for pancreatic ductal cancers to arise from Kras-induced preneoplastic lesions by increasing epithelial cell proliferation in the context of mutated p53. Here we identify an in vivo context in which p110α cellular output differs depending on the epithelial transformation stage and demonstrate that the PI3K p110α is required for PDAC induced by oncogenic Kras, the key driver mutation of PDAC. These data are critical for a better understanding of the development of this lethal disease that is currently without efficient treatment.

TGFβ signaling in pancreatic ductal adenocarcinoma.

Transforming growth factor β (TGFβ) receptor signaling plays a paradoxical effect in the tumorigenesis of pancreatic ductal adenocarcinoma (PDAC), in which its tumor-inhibitory role at early stages turns into a tumor-promoting role at later stages. The underlying mechanism remains far from clear. Here we provide strong evidence that the activation of TGFβ receptor signaling in PDAC cells increased SMAD3 phosphorylation and nuclear translocation to inhibit cell growth. Meanwhile, it also activated SMAD7 to induce nuclear translocation and retention of β-catenin, which not only attenuated the inhibition of cell growth by nuclear SMAD3 but also activated vascular endothelial growth factor A (VEGF-A) to promote vascularization. Our data thus support a model involving crosstalk of the TGFβ and Wnt signaling pathways, for regulating the complicated effect of TGFβ signaling on the tumorigenesis of PDAC.

Abstract 26: MUC1 enhances neuropilin-1 signaling in pancreatic ductal adenocarcinoma

Abstract Pancreatic ductal adenocarcinoma (PDA) has the worst prognosis of all cancers and is the 4th leading cause of cancer-related deaths in the United States. Mucin1 (MUC1) is a transmembrane glycoprotein over-expressed in more than 60% of PDA and its expression correlates with high metastases and poor prognosis. In PDA, there is a correlation between blood vessel density, tumor levels of VEGF, and disease progression. We have recently discovered a novel association between MUC1 and neuropilin-1 (NRP-1) expression. Neuropilin-1 (NRP-1) is a co-receptor for VEGF165 and blockade of NRP-1-VEGF165 interaction has been shown to inhibit angiogenesis and tumor growth in several other cancer types. Thus, we hypothesize that MUC1 induces a pro-angiogenic tumor microenvironment by increasing levels of VEGF and NRP-1, thus enhancing disease progression and metastases. We have generated Muc1-expressing KC and Muc1-null KCKO mouse PDA cell lines for this study. Compared to KCKO, KC cells secrete more VEGF and express higher levels of NRP-1 as well as VEGF receptors (1/2/3). Although the cell culture supernatants from KC do not alter the NRP-1 level on the endothelial line 2H11 cells, they significantly promote the 2H11 cell tube formation. Consistent with the in vitro results, tumor lysates from KC-bearing mice have higher levels of NRP-1 and VEGFR2 and its phosphorylations at Tyr residues 951, 996, and 1175. In human PDA cell lines, inhibition of MUC1 expression by specific siRNA reduced NRP-1 level while overexpression of full-length MUC1 increases NRP-1 expression. This suggests a direct regulation of NRP-1 by MUC1. Furthermore, the over-expression of MUC1 in BxPC3 human PDA cell line induces strong angiogenesis and promotes tumor metastasis in a zebrafish tumor xenograft model. Finally, we report that inhibition of NRP-1 by its specific blocking peptide suppresses BxPC3.MUC1 tumor growth in vivo. Taking together, MUC1 in PDA cells can enhance their own NRP-1 levels and promote the VEGF-VEGFR-NRP-1 signaling, which may favor the creation of a pro-angiogenic microenvironment for tumor growth and metastases. Our findings highlight the potential for MUC1 targeted blockade of VEGF-NRP-1 interaction in PDA by conjugating the NRP-1 inhibitory agents to a MUC1 targeting antibody. Citation Format: Ru Zhou, Jennifer Curry, Priyanka Grover, Lopamudra Das Roy, TinChung Leung, Pinku Mukherjee. MUC1 enhances neuropilin-1 signaling in pancreatic ductal adenocarcinoma. [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 26. doi:10.1158/1538-7445.AM2014-26

Abstract 127: Alternative transcription of the SLIT2/mir-218-1 signaling axis mediates pancreatic cancer invasion through the regulation of invadopodia

Abstract Guidance molecules from the Netrin, Slit, Ephrin, and Semaphorin gene families were originally described as cues for the directional guidance of axons in the developing nervous system. More recently, members of these families have been found to have critical roles in tumor invasion. The SLIT2/mir-218-1 signaling axis has properties of a tumor suppressor pathway via the inhibition of directional migration and is epigenetically silenced in many cancers. We propose that mir-218-1 expression and function is independent of SLIT2 signaling in pancreatic ductal adenocarcinoma and inhibits tumor dissemination following the conversion of precursor lesions to invasive carcinoma. We determined that KRAS-dependent pancreatic cancer cell lines express SLIT2 mRNA while KRAS-independent lines show silenced SLIT2 expression. Using chromatin immunoprecipitation (ChIP) and Sequenom, we confirmed that loss of SLIT2 in KRAS-independent lines was due to enrichment of silencing histone mark H3K27me3 and DNA hypermethylation at the SLIT2 promoter, respectively. Activating marks H3K4me2 and H4ac are found at the SLIT2 promoter in KRAS-dependent lines suggesting that SLIT2 is lost during pancreatic cancer progression due to epigenetic silencing. mir-218-1 is an intronic microRNA found within intron 15 of the SLIT2 gene. Many intronic microRNAs are transcribed from the host gene promoter; however, mir-218-1 expression in pancreatic cancer cell lines does not correlate with SLIT2 gene expression. Therefore, we examined whether mir-218-1 had an alternative promoter independent of the SLIT2 promoter. Using ChIP for H3K4me3, a marker for promoter regions, and H4ac, a marker of permissive chromatin, we discovered a putative alternative promoter of mir-218-1 in all pancreatic cancer cell lines regardless of KRAS dependence. The putative alternative mir-218-1 promoter was also found to have basal transcription via enhanced luciferase reporter activity. This suggests that mir-218-1 expression is important and may contain its own tumor suppressor properties independent of SLIT2 signaling. Since pancreatic cancer is an invasive disease we examined the role of miR-218 target genes in specialized cellular structures, termed invadopodia. These structures have been found to facilitate the invasive phenotype of many cancer types and are dynamic actin-rich cellular projections that localize proteases to degrade extracellular matrix. Transfection of a miR-218 antagomir enhanced invasion in a modified Boyden chamber while addition of mir-218 reduced cell invasion. Our results further demonstrate that mir-218 functions in the suppression of invadopodia maturation and metalloproteinase matrix degradation. Overall, our data establishes that the SLIT2/mir-218-1 signaling axis is an important mediator of pancreatic cancer invasion through the extracellular matrix. Citation Format: Brenna A. Rheinheimer, Lukas Vrba, Bernard W. Futscher, Ronald L. Heimark. Alternative transcription of the SLIT2/mir-218-1 signaling axis mediates pancreatic cancer invasion through the regulation of invadopodia. [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 127. doi:10.1158/1538-7445.AM2014-127

Prognostic significance of WNT signaling in pancreatic ductal adenocarcinoma

Pancreatic ductal adenocarcinoma (PDA) is one of the most lethal human malignancies and is associated with a variety of molecular abnormalities. Although WNT signaling through its canonical/non-canonical pathways is one of the major factors involved in oncogenesis or progression of PDA, the prognostic significance of WNT signaling still remains poorly investigated. In this study, the status of the WNT signaling pathways was immunohistochemically analyzed in 101 PDAs, and its potential association with patient postoperative survival was assessed. Nuclear expression of beta-catenin, a hallmark of the activated canonical pathway, was identified in 59 cases, and was associated with reduced survival compared to the patients lacking nuclear beta-catenin expression (P = 0.002). In contrast, activation of the non-canonical pathway (25 cases), as indicated by co-expression of WNT2/5a and nuclear NFATc1, was not correlated with reduced survival (P = 0.268). Co-activation of both pathways (16 cases) was associated with worse prognosis in comparison with cases with an activated non-canonical pathway (P = 0.034). In addition, nuclear beta-catenin expression was an independent unfavorable prognostic factor (P = 0.006). Our data indicate that activated WNT signaling through its canonical pathway has a significantly negative effect on the clinical course of PDA, and the canonical WNT pathway should be considered as a future therapeutic target for PDA.

Reduced retinoids and retinoid receptors' expression in pancreatic cancer: A link to patient survival.

Pancreatic ductal adenocarcinoma (PDAC) represents one of the deadliest cancers in the world. All-trans retinoic acid (ATRA) is the major physiologically active form of vitamin A, regulating expression of many genes. Disturbances of vitamin A metabolism are prevalent in some cancer cells. The main aim of this work was to investigate deeply the components of retinoid signaling in PDAC compared to in the normal pancreas and to prove the clinical importance of retinoid receptor expression. For the study, human tumor tissues obtained from PDAC patients and murine tumors from the orthotopic Panc02 model were used for the analysis of retinoids, using high performance liquid chromatography mass spectrometry and real-time RT-PCR gene expression analysis. Survival probabilities in univariate analysis were estimated using the Kaplan-Meier method and the Cox proportional hazards model was used for the multivariate analysis. In this work, we showed for the first time that the ATRA and all-trans retinol concentration is reduced in PDAC tissue compared to their normal counterparts. The expression of RARα and β as well as RXRα and β are down-regulated in PDAC tissue. This reduced expression of retinoid receptors correlates with the expression of some markers of differentiation and epithelial-to-mesenchymal transition as well as of cancer stem cell markers. Importantly, the expression of RARα and RXRβ is associated with better overall survival of PDAC patients. Thus, reduction of retinoids and their receptors is an important feature of PDAC and is associated with worse patient survival outcomes.

Pancreatic cancer–associated retinoblastoma 1 dysfunction enables TGF-β to promote proliferation

Pancreatic ductal adenocarcinoma (PDAC) is often associated with overexpression of TGF-β. Given its tumor suppressor functions, it is unclear whether TGF-β is a valid therapeutic target for PDAC. Here, we found that proliferating pancreatic cancer cells (PCCs) from human PDAC patients and multiple murine models of PDAC (mPDAC) often exhibit abundant levels of phosphorylated retinoblastoma 1 (RB) and Smad2. TGF-β1 treatment enhanced proliferation of PCCs isolated from KrasG12D-driven mPDAC that lacked RB (KRC cells). This mitogenic effect was abrogated by pharmacological inhibition of type I TGF-β receptor kinase, combined inhibition of MEK/Src or MEK/PI3K, and restoration of RB expression. TGF-β1 promoted epithelial-to-mesenchymal transition (EMT), invasion, Smad2/3 phosphorylation, Src activation, Wnt reporter activity, and Smad-dependent upregulation of Wnt7b in KRC cells. Importantly, TGF-β1-induced mitogenesis was markedly attenuated by inhibition of Wnt secretion. In an in vivo syngeneic orthotopic model, inhibition of TGF-β signaling suppressed KRC cell proliferation, tumor growth, stroma formation, EMT, metastasis, ascites formation, and Wnt7b expression, and markedly prolonged survival. Together, these data indicate that RB dysfunction converts TGF-β to a mitogen that activates known oncogenic signaling pathways and upregulates Wnt7b, which synergize to promote PCC invasion, survival, and mitogenesis. Furthermore, this study suggests that concomitantly targeting TGF-β and Wnt7b signaling in PDAC may disrupt these aberrant pathways, which warrants further evaluation in preclinical models.