Receptors In Pancreatic Cancer Research Articles

Abstract 2691: Mapping the landscape of innate immune pattern recognition receptors in pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma (PDAC) accounts for 90% of all pancreatic cancer cases, and is among the most aggressive and lethal malignancies worldwide. PDAC is driven by genetic alterations in the pancreatic epithelium (e.g. KRAS, TRP53) coupled with a dysregulated innate immune response, the latter leading to an inflammatory tumor microenvironment enriched in innate immune cells (e.g. macrophages). However, immune-based treatment regimens for PDAC patients have primarily focused on immunotherapy with adaptive immune checkpoint inhibitors (e.g. PD-1) which disappointingly have yielded minimal clinical benefit. Thus, there is an unmet clinical need to identity disease-associated innate immune regulators as therapeutic targets in PDAC. Innate immune responses depend on a series of cell surface, endosomal and cytosolic pattern recognition receptors (PRRs) that are expressed in immune and non-immune cells. PRRs are classified into several structurally and functionally conserved subfamilies, including Toll-like receptors (TLRs), Absent in melanoma 2 (AIM2)-like receptors (ALRs), and Nod-like receptors (NLRs). A subset of NLRs and AIM2 are also well documented for their formation of multiprotein complexes called ‘inflammasomes’ - comprising the adaptor ASC and Caspase-1 - which direct the maturation and release of bioactive pro-inflammatory cytokines, IL-18 and IL-1β. However, the role of these critical innate immune regulators in PDAC has been underexplored, and is virtually unknown. By coupling endoscopic ultrasound-guided fine needle aspiration (EUS FNA) to capture tumor biopsies from all stages of PDAC, with whole transcriptome profiling of PDAC patient primary tumors, we reveal specific enrichment of the innate immune TLR2 and ASC inflammasome molecular pathways. Augmented TLR2 and ASC expression was prognostic for low survival, and a “TLR2 activation” signature was also predictive for chemoresistance. Antibody-mediated anti-TLR2 therapy suppressed tumor growth in human PDAC tumor xenografts (patient and cell line) in both immunocompromised and ‘humanized’ mice. With respect to ASC, using the genetically engineered KPC PDAC mouse model, we show that Caspase-1 activation, a read-out for inflammasome activation, is upregulated in pancreatic tumors. Moreover, genetic ablation of ASC in KPC mice markedly suppressed the development of pancreatic tumors, which was associated with significant reductions in levels of both Caspase-1 activation and inflammation. Transcriptome profiling further identified unique downstream molecular networks associated with a range of oncogenic cellular processes. Taken together, our results support innate immune TLR2- and ASC inflammasome-based therapeutic targeting in PDAC, with further clinical utility that ASC inflammasome and TLR2 activation is prognostic, with TLR2 also predictive for chemoresponsiveness. Citation Format: Bassam Kashgari, Louise McLeod, Linden J. Gearing, Daniel Croagh, Brendan J. Jenkins. Mapping the landscape of innate immune pattern recognition receptors in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2691.

Semaphorins and their receptors in pancreatic cancer: Mechanisms and therapeutic opportunities.

Pancreatic cancer (PC) is a malignant tumor with high malignancy that is difficult to diagnose and treat. PC is a major medical problem because of its low early diagnosis rate, high surgical mortality rate, low cure rate, and expensive related testing cost. Therefore, the significance of finding new markers for PC is self-evident. Semaphorins (Semas) have been shown to affect angiogenesis and lymphangiogenesis and can also directly affect the behavior of tumor cells. The expression and related action targets of its family members on PC are summarized in this review.

Novel role of the Mu-opioid receptor in pancreatic cancer: potential link between opioid use and cancer progression.

Opioids are the most popular drugs for both acute and chronic pain management. The G protein-coupled mu-opioid receptor (MOR) is the therapeutic target for most clinically used opioids, including morphine. A mounting number of publications suggest a relationship between the MOR and possible cancer progression and recurrence extending to managing chronic cancer pain. In this study, we studied the possible link between opioid use and pancreatic cancer (PC) progression. We found increased MOR expression in murine and human PC cell lines, human PC-derived organoids, and in the undifferentiated or poorly differentiated areas of surgically resected PC tissues. Direct stimulation of MOR by morphine (MOR agonist) caused a significant dose-dependent increase in proliferation, invasion, and levels of stemness markers in PC cells. In a co-culture system, MOR stimulation of macrophages also resulted in increased proliferation of PC cells. MOR overexpression increased proliferation and cancer stemness, whereas knock-down of MOR followed opposite results in the PC cells. Morphine induced chemoresistance to conventional chemotherapeutic agents used for PC treatment. Overall, our results suggest that MOR is expressed in pancreatic cancer and may be involved in tumor progression and chemoresistance.

Targeting the Endocannabinoid System: From the Need for New Therapies to the Development of a Promising Strategy. What About Pancreatic Cancer?

Pancreatic cancer is one of the most fatal malignancies, and therefore, new strategies, which aim at the improvement of the prognosis of this lethal disease, are needed. Many clinical trials have failed to improve overall survival. Nowadays, research is focused on advances provided by novel potential targets to efficiently enhance life expectancy. Cannabinoids, the active components of Cannabis sativa L., and their derivatives, have been reported as palliative adjuvants to conventional chemotherapeutic regimens. Cannabinoid effects are known to be mediated through the activation of cannabinoid receptors. To date, two cannabinoid receptors, cannabinoid receptor 1 and 2, have been cloned and identified from mammalian tissues. Cannabinoids exert a remarkable antitumoral effect on pancreatic cancer cells, due to their ability to selectively induce apoptosis of these cells. This review strengthens the perception that cannabinoid receptors might be useful in clinical testing to prognose and treat pancreatic cancer. Many studies have tried to describe the mechanism of cell death induced by cannabinoids. The aim of this review is to discuss the effects of cannabinoid receptors in pancreatic cancer in order to provide a brief insight into cannabinoids and their receptors as pancreatic cancer biomarkers and in therapeutic strategies.

Involvement of IL-4, IL-13 and Their Receptors in Pancreatic Cancer

Interleukin (IL)-4 and IL-13 are known as pleiotropic Th2 cytokines with a wide range of biological properties and functions especially in immune responses. In addition, increasing activities have also been determined in oncogenesis and tumor progression of several malignancies. It is now generally accepted that IL-4 and IL-13 can exert effects on epithelial tumor cells through corresponding receptors. Type II IL-4 receptor (IL-4Rα/IL-13Rα1), predominantly expressed in non-hematopoietic cells, is identified to be the main target for both IL-4 and IL-13 in tumors. Moreover, IL-13 can also signal by binding to the IL-13Rα2 receptor. Structural similarity due to the use of the same receptor complex generated in response to IL-4/IL-13 results in overlapping but also distinct signaling pathways and functions. The aim of this review was to summarize knowledge about IL-4 and IL-13 and their receptors in pancreatic cancer in order understand the implication of IL-4 and IL-13 and their receptors for pancreatic tumorigenesis and progression and for developing possible new diagnostic and therapeutic targets.

Screening of Focused Compound Library Targeting Liver X Receptors in Pancreatic Cancer Identified Ligands with Inverse Agonist and Degrader Activity.

Pancreatic ductal adenocarcinoma (PDAC) is the predominant form of pancreatic cancer. PDACs harbor oncogenic mutations in the KRAS gene, and ongoing efforts to directly target its mutant protein product to inhibit tumor growth are a priority not only in pancreatic cancer but in other malignancies such as lung and colorectal cancers where KRAS is also commonly mutated. An alternative strategy to directly targeting KRAS is to identify and target druggable receptors involved in dysregulated cancer hallmarks downstream of KRAS dysregulation. Liver X receptors (LXRs) are members of the nuclear receptor family of ligand-modulated transcription factors and are involved in the regulation of genes which function in key cancer-related processes, including cholesterol transport, lipid and glucose metabolism, and inflammatory and immune responses. Modulation of LXRs via small molecule ligands has emerged as a promising approach for directly targeting tumor cells or the stromal and immune cells within the tumor microenvironment. We have previously shown that only one of the two LXR subtypes (LXRβ) is expressed in pancreatic cancer cells, and targeting LXR with available synthetic ligands blocked the proliferation of PDAC cells and tumor formation. In a screen of a focused library of drug-like small molecules predicted to dock in the ligand-binding pocket of LXRβ, we identified two novel LXR ligands with more potent antitumor activity than current LXR agonists used in our published studies. Characterization of the two lead compounds (GAC0001E5 and GAC0003A4) indicates that they function as LXR inverse agonists which inhibit their transcriptional activity. Prolonged treatments with novel ligands further revealed their function as LXR "degraders" which significantly reduced LXR protein levels in all three PDAC cell lines tested. These findings support the utility of these novel inhibitors in basic research on ligand design, allosteric mechanisms, and LXR functions and their potential application as treatments for advanced pancreatic cancer and other recalcitrant malignancies.

Gene expression profile of sigma-1 (s1R) and sigma-2 (s2R) receptors in pancreatic cancer

Gene expression profile of sigma-1 (s1R) and sigma-2 (s2R) receptors in pancreatic cancer

The role of selected chemokines and their specific receptors in pancreatic cancer

Pancreatic carcinoma is a highly malignant disease associated with an extremely poor prognosis, which is caused by late presentation, aggressive invasion and metastases, as well as the detection of pancreatic carcinoma in its advanced stages. Thus, better understanding of the tumour biology of this malignancy is sorely needed to improve the clinical outcome. A great challenge for the medical practice is finding a new biomarker of pancreatic carcinoma that will be helpful in diagnosis, in prognosis and in making clinical decisions, including the assessment of patients' response to therapy. It is suggested that selected chemokines and their specific receptors play an important role in tumour progression, such as tumour growth, angiogenesis, proliferation and development of metastasis. In the present review, general characteristics of chemokines and their specific receptors as well as the significance of these molecules in tumour development are described. The crucial issue of this review is to summarise the importance of various chemokines and their specific receptors in pancreatic carcinoma. Understanding the role of chemokines in the pathogenesis of pancreatic carcinoma is extremely important since these proteins may be used as a potential tool in the diagnosis and prognosis of pancreatic carcinoma patients.

Regulation of KRas‐Mediated Signaling by G‐Protein Coupled Receptors in Pancreatic Cancer

Pancreatic ductal adenocarcinoma is a highly lethal malignancy with few effective treatment options. We used computational approaches to identify new drug targets within this heterogeneous patient population, focusing on the G‐protein coupled receptors (GPCRs). GPCRs transduce extracellular signals from a variety of ligands through the plasma membrane, resulting in the modulation of intracellular signaling pathways and regulation of many physiological processes. This wide array of functions has resulted in the extensive utilization of GPCR targeted therapeutics, accounting for 30–50% of all currently used drugs. However, drugs targeting GPCRs are rarely utilized in cancer treatment, despite evidence that GPCRs mediate many aspects of tumorigenesis. Genomic analyses have uncovered GPCR mutations, copy number alterations and gene expression and methylation changes in a wide variety of cancers. Determining the biological implication of these genomic alterations will allow utilization of GPCR targeted therapeutics in those patients with GPCR‐driven tumors. Our preliminary evidence has uncovered the orphan GPCR GPRC5A as a critical regulator of the progression from early stage PanIN lesions to PDA.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

648 - Repurposing Penfluridol: An Old Antipsychotic Drug that Therapeutically Targets Prolactin Receptor in Pancreatic Cancer

648 - Repurposing Penfluridol: An Old Antipsychotic Drug that Therapeutically Targets Prolactin Receptor in Pancreatic Cancer

The significance of Trk receptors in pancreatic cancer.

This study investigated the Trk receptor family as a therapeutic target in pancreatic ductal adenocarcinoma and assessed their prognostic significance. Global gene expression analysis was investigated in prospectively collected pancreatic ductal adenocarcinomas that had either undergone neoadjuvant chemoradiation or were treated by surgery. PANC-1 and MIA-PaCa-2 cell lines were investigated to establish whether fractionated radiation altered expression of four neuroendocrine genes and whether this resulted in subsequent changes in radiosensitivity. A specific inhibitor of TrkA, B, and C, AstraZeneca 1332, was investigated in vitro and in vivo in combination with radiation. A tissue microarray was constructed from 77 pancreatic ductal adenocarcinoma patients who had undergone neoadjuvant chemoradiation and the Trk receptor, and neurogenic differentiation 1 expression was assessed and correlated with overall survival. A total of 99 genes were identified that were differentially expressed in the chemoradiation patients with neuroendocrine genes and pathways, in particular the neurogenic differentiation 1 and Trk receptor family, being prominent. Fractionated radiation upregulated the expression of neuroendocrine genes, and AstraZeneca 1332 treatment in vitro enhanced radiosensitivity. No added effect of AstraZeneca 1332 was observed in vivo. Trk receptor expression varied between isoforms but did not correlate significantly with clinical outcome. Radiation treatment upregulated neuroendocrine gene expression but the Trk receptor family does not appear to be a promising treatment target.

Targeting of the P2X7 receptor in pancreatic cancer and stellate cells.

The ATP‐gated receptor P2X7 (P2X7R) is involved in regulation of cell survival and has been of interest in cancer field. Pancreatic ductal adenocarcinoma (PDAC) is a deadly cancer and new markers and therapeutic targets are needed. PDAC is characterized by a complex tumour microenvironment, which includes cancer and pancreatic stellate cells (PSCs), and potentially high nucleotide/side turnover. Our aim was to determine P2X7R expression and function in human pancreatic cancer cells in vitro as well as to perform in vivo efficacy study applying P2X7R inhibitor in an orthotopic xenograft mouse model of PDAC. In the in vitro studies we show that human PDAC cells with luciferase gene (PancTu‐1 Luc cells) express high levels of P2X7R protein. Allosteric P2X7R antagonist AZ10606120 inhibited cell proliferation in basal conditions, indicating that P2X7R was tonically active. Extracellular ATP and BzATP, to which the P2X7R is more sensitive, further affected cell survival and confirmed complex functionality of P2X7R. PancTu‐1 Luc migration and invasion was reduced by AZ10606120, and it was stimulated by PSCs, but not by PSCs from P2X7‐/‐ animals. PancTu‐1 Luc cells were orthotopically transplanted into nude mice and tumour growth was followed noninvasively by bioluminescence imaging. AZ10606120‐treated mice showed reduced bioluminescence compared to saline‐treated mice. Immunohistochemical analysis confirmed P2X7R expression in cancer and PSC cells, and in metaplastic/neoplastic acinar and duct structures. PSCs number/activity and collagen deposition was reduced in AZ10606120‐treated tumours.

Germline Mutation of the CCK Receptor: A Novel Biomarker for Pancreas Cancer.

Objectives:Today, genetic biomarkers have been demonstrated to play an important role in identifying at-risk subjects for familial or inherited cancers. We have identified a single-nucleotide polymorphism (SNP) that results in missplicing of the cholecystokinin (CCK) receptor gene and expressing a larger mutated receptor in pancreatic cancer. The purpose of this study was to evaluate the significance and specificity of this SNP as a potential biomarker in patients with pancreatic cancer compared with other gastrointestinal (GI) cancers that also have CCK receptors.Methods:DNA was isolated and genotyped for the CCK receptor SNP from frozen tumor tissue from banked specimens of patients with pancreas, gastric, or colon cancer and from human cancer cell lines. Genotype and allelic frequencies were compared between the cancer cohort and two normal control databases using Fisher's exact test and odds ratio (OR). The Kaplan–Meier method was used to estimate the survival for patients with the CCK-B receptor SNP compared with those with the wild-type genotype. Immunohistochemical staining of cancer cells was done to detect the mutated receptor.Results:Colon and gastric cancer patients had similar genotype frequencies for the CCK receptor SNP as that reported in the normal population. In contrast, the prevalence of the SNP in subjects with pancreatic cancer was twice that of controls and other GI cancers. Survival was adversely affected by the presence of the SNP only in those with pancreatic cancer. Immunoreactivity for the mutated receptor was positive in pancreatic cancer tissues with the SNP but absent in other GI cancers.Conclusions:A SNP of the CCK receptor is significantly increased in patients with pancreatic cancer but not in those with other GI malignancies. Therefore, this SNP may be a potential biomarker for pancreatic cancer.

The Role of Gastrin and CCK Receptors in Pancreatic Cancer and other Malignancies.

The gastrointestinal (GI) peptide gastrin is an important regulator of the release of gastric acid from the stomach parietal cells and it also plays an important role in growth of the gastrointestinal tract. It has become apparent that gastrin and its related peptide cholecystokinin (CCK) are also significantly involved with growth of GI cancers as well as other malignancies through activation of the cholecystokinin-B (CCK-B) receptor. Of interest, gastrin is expressed in the embryologic pancreas but not in the adult pancreas; however, gastrin becomes re-expressed in pancreatic cancer where it stimulates growth of this malignancy by an autocrine mechanism. Strategies to down-regulate gastrin or interfere with its interface with the CCK receptor with selective antibodies or receptor antagonists hold promise for the treatment of pancreatic cancer and other gastrin - responsive tumors.

A novel protein isoform of the RON tyrosine kinase receptor transforms human pancreatic duct epithelial cells.

The MST1R gene is overexpressed in pancreatic cancer producing elevated levels of the RON tyrosine kinase receptor protein. While mutations in MST1R are rare, alternative splice variants have been previously reported in epithelial cancers. We report the discovery of a novel RON isoform discovered in human pancreatic cancer. Partial splicing of exons 5 and 6 (P5P6) produces a RON isoform that lacks the first extracellular immunoglobulin-plexin-transcription (IPT) domain. The splice variant is detected in 73% of pancreatic adenocarcinoma patient derived xenografts and 71% of pancreatic cancer cell lines. Peptides specific to RON P5P6 detected in human pancreatic cancer specimens by mass spectrometry confirms translation of the protein isoform. The P5P6 isoform is found to be constitutively phosphorylated, present in the cytoplasm, and it traffics to the plasma membrane. Expression of P5P6 in immortalized human pancreatic duct epithelial (HPDE) cells activates downstream AKT, and in human pancreatic epithelial nestin-expressing (HPNE) cells activates both the AKT and MAPK pathways. Inhibiting RON P5P6 in HPDE cells using a small molecule inhibitor BMS-777607 blocked constitutive activation and decreased AKT signaling. P5P6 transforms NIH3T3 cells and induces tumorigenicity in HPDE cells. Resultant HPDE-P5P6 tumors develop a dense stromal compartment similar to that seen in pancreatic cancer. In summary, we have identified a novel and constitutively active isoform of the RON tyrosine kinase receptor that has transforming activity and is expressed in human pancreatic cancer. These findings provide additional insight into the biology of the RON receptor in pancreatic cancer and are clinically relevant to the study of RON as a potential therapeutic target.

Significance of expression of glucagon-like peptide 1 receptor in pancreatic cancer

Glucagon-like peptide 1 (GLP-1) induces insulin secretion and proliferation of pancreatic β-cells, and inhibits their apoptosis through the GLP-1 receptor (GLP-1R), thus providing a foundation for using GLP-1-based therapies for the treatment of type 2 diabetes. However, doubts have emerged regarding the drug safety of these therapies. We investigated the potential role of GLP-1R in pancreatic ductal adenocarcinoma (PDAC). GLP-1R expression was semi-quantitatively evaluated by immunohistochemistry in 48 PDAC samples, and its correlations with clinicopathological features were investigated. CFPAC-1 cells were used for GLP-1R knockdown to evaluate its effects on cell proliferation, migration and invasion. GLP-1R expression was positive in 23 tumors and negative in 25 tumors. No correlations were found between GLP-1R expression status and clinicopathological characteristics. Furthermore, GLP-1R expression status did not affect the patient prognosis (P=0.74). The majority of lymph node metastases (11 of 15 samples examined; 73%) were positive for GLP-1R expression. Immunoreactivity for GLP-1R was also noted in sites of perineural and lymphovascular invasion. GLP-1R knockdown significantly reduced the proliferation, migration and invasion of CFPAC-1 cells (P<0.05). In conclusion, although GLP-1R is not an independent prognostic factor in PDAC patients, it appears to have some implications for PDAC metastatic ability.

Abstract B119: Triptolide abrogates expression of the Met and epidermal growth factor receptors in pancreatic cancer

Abstract Background: Minnelide, a pro-drug of the Chinese herb triptolide, is currently in Phase I clinical trials against pancreatic cancer. Preclinical studies with Minnelide have demonstrated its efficacy in preventing tumor formation and causing tumor regression in multiple animal models of pancreatic cancer. However, its mechanism of action remains unclear. Pancreatic cancer cells overexpress several receptor tyrosine kinases including the Met and EGF receptors which drive proliferation, migration and invasion of these cells. Here, using human pancreatic cancer cells as well as the KRasG12D; Trp53R172H; Pdx-1 Cre (KPC) animal model, we show the ability of triptolide, or its water soluble pro-drug, Minnelide, to down-regulate the Met and EGF receptors, both in vitro and in vivo. Methods:Pancreatic cancer cells were treated with triptolide for varying times (0-24h) and receptor levels evaluated using western blotting or confocal microscopy. The KPC mouse model recapitulates human disease progression and is therefore the most relevant model for chemotherapeutic drug testing. KPC animals, which spontaneously form pancreatic tumors, were treated with Minnelide for one week and tumors harvested. mRNA expression was analyzed using real time PCR and protein expression assessed by immunohistochemistry. Results: Treatment with triptolide causes an increase in tyrosine phosphorylation at earlier time points (0.5 - 6h), accompanied by an increase in levels of the Met and EGF receptor tyrosine kinases. However, longer treatments (12-24h) resulted in marked decrease in levels of both Met and EGF receptors. Further investigation using tumors from KPC animals treated with Minnelide show a decrease in gene expression of both the Met and EGF receptors compared with tumors from saline injected animals. Furthermore, immunohistochemical analysis of formalin fixed paraffin embedded sections from these tumors show a decrease in levels of the Met receptor. Conclusion: Treatment of cells or KPC pancreatic tumor bearing animals with triptolide or Minnelide leads to decreased expression of the Met and EGF receptor tyrosine kinases. These results lend further insight into the mechanism of action of Minnelide, a pro-drug currently in clinical trials against pancreatic cancer. Citation Format: Veena Sangwan, Kelsey M. Jensen, Vikas Dudeja, Rohit Chugh, Sulagna Banerjee, Selwyn M. Vickers, Morag Park, Ashok K. Saluja. Triptolide abrogates expression of the Met and epidermal growth factor receptors in pancreatic cancer. [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 B119.

Molecular Signatures of G-Protein Coupled Receptors in Pancreatic Cancer using Super-Resolution Microscopy

Emerging data indicate that G protein-coupled receptors (GPCRs) play important roles in the pathogenesis of cancers. Many GPCRs are constitutively overexpressed in various cancers, and their aberrant signaling is implicated in different stages of tumorigenesis and metastasis. For GPCRs to mediate signaling, one proposed mechanism involves formation of GPCR homo- and hetero-oligomers. However, certain details of the process remain unknown. To address this, we combine pointillistic super-resolution methods and pair-correlation analysis to quantitatively characterize GPCR oligomerization domains both with single molecule sensitivity and at nanometer scale precision. With this approach, we systematically investigated healthy/cancerous cells and multicellular tumor spheroids - a 3D tumor model. Our data suggests that overexpression of mu and kappa opioid receptors induces formation of dense oligomers with both short- and long-distance organization (also supported by fluorescence correlation spectroscopy measurements). Additionally, endogenous oligomers of various sizes are evident in pancreatic cancer cells; these include opioid receptors, somatostatin receptor 2, chemokine C-X-C motif receptor 4, and chemokine C-C motif receptor 5. Importantly, the results suggest that somatostatin receptor 2 associates with mu opioid receptor (i.e. forms hetero-oligomers) in pancreatic cancer cells and multicellular tumor spheroids. In the future, successful application of this innovative approach can be used as a tool for quantitative screening of GPCRs and drug discovery. Ultimately, this strategy may produce novel markers and therapeutic targets for pancreatic cancer.

MicroRNA-410 functions as a tumor suppressor by targeting angiotensin II type 1 receptor in pancreatic cancer.

MicroRNAs (miRNAs) act as key regulators of gene expression in diverse biological processes and are intimately involved in tumorigenesis. However, the underlying molecular mechanisms of miR-410 in pancreatic cancer remain poorly understood. In this study, we found that miR-410 overexpression suppressed pancreatic cancer cell growth in vitro and in vivo as well as cell invasion and migration. miR-410 also resulted in G1/S cell-cycle arrest. We then showed that angiotensin II type 1 receptor (AGTR1) was a direct target of miR-410, with miR-410 suppressing AGTR1 expression levels. In contrast, inhibition of miR-410 increased the expression of AGTR1. Silencing of AGTR1 inhibited cell growth and invasion, similar to miR-410 overexpression. In addition, we found that the induction of vascular endothelial growth factor and the activation of the ERK signaling pathway by angiotensin II were blocked by miR-410, similar to the angiotensin II inhibitor losartan. miR-410 overexpression inhibited angiogenesis in mice through the repression of CD31 expression. ERK pathway knockdown suppressed pancreatic cancer cell proliferation, invasion, and angiogenesis. Finally, we found that miR-410 was downregulated in pancreatic cancer tissues compared to adjacent nontumor tissues, whereas AGTR1 was upregulated in pancreatic cancer tissues. Pearson correlation analysis showed that miR-410 and AGTR1 were inversely expressed. In conclusion, our data indicate that miR-410 suppresses pancreatic cancer growth, cell invasion, migration, and angiogenesis via the downregulation of AGTR1, acting as a tumor-suppressive miRNA. In addition, our results suggest that miR-410 is a potential diagnostic biomarker and therapeutic target for patients with pancreatic cancer.

Abstract 2315: Demethylating therapy modulates expression of the RON tyrosine kinase receptor in pancreatic cancer

Abstract Introduction - The RON tyrosine kinase receptor is overexpressed in 80% of Pancreatic Ductal Adenocarcinomas (PDAC) and mediates invasiveness, resistance to apoptosis, and VEGF secretion. Though point mutations are rare a wide variety of RON protein isoforms have been identified, some of which are constitutively active and oncogenic. Previous studies of the DNA methylation patterns of pancreatic cancer specimens identified the gene for RON, MST1R, to be controlled by epigenetic modification. Methylation is one mechanism to regulate expression of RON, and we seek to investigate the effects of hypomethylating therapy on isoform expression and alteration of PDAC biology. Methods - Established pancreatic cancer cell lines (MiaPaca2, BxPC3, AsPC1, HPDE, Panc1) and 2 novel cell lines derived from patient tumor specimens (34E, 79E) were treated with 5-Aza-2′-deoxycytidine (Decitabine, DAC) for 96 hours. Genomic DNA was analyzed using the Methylflash (Epigentek) assay to determine genomic methylation percentage. Reverse Transcriptase PCR and western blot analysis was used to determine treatment effects on RON transcription and expression. Results - Genomic methylation analysis of pancreatic cancer cell lines revealed that compared to the control HPDE line (1.2% methylation) some lines were hypomethylated (ASPC1-1.13%, 34E-1.17%, Panc1-1.17%) while others were hypermethylated (BxPC3-1.6%, MiaPaca2-1.98%, 79E-2.06%). Hypomethylating therapy was effective in decreasing genomic methylation to below normal levels (0.4%-0.9%) in all cell lines and depleted DNA MethylTransferase 1 (DNMT1) protein levels. The effects of demethylating therapy on RON expression were differential. The amount of RON transcripts and protein expression in the RON expressing lines (79E and 34E) was decreased but increased in MiaPaca2 and Panc1. MiaPaca2 does not normally express RON, but demethylation induced RON expression. Transcripts specific for the Δ55 isoform of RON decreased in 34E, 79E, and HPDE cell lines but did not change in the others. Transcription and expression of other RON isoforms which are constitutively active decreased in 34E, 79E, ASPC1, and BxPC3 lines, with less exon 11 skipping and partial splicing of exon 5 and 6. Though RON protein expression was decreased overall in the expressing lines, phosphorylation was still detectable following DAC treatment. Conclusions - Genomic methylation of pancreatic cancer cell lines is variable and RON expression is modulated by the methylation status of its promoter. Demethylating therapy has variable effects on RON expression and is context specific. RON isoform expression was decreased in response to treatment, linking DNA methylation to alternative splicing events. These findings suggest that the consequences of demethylating therapy on PDAC biology are complex and additional studies are required to further elucidate definitive mechanisms. Citation Format: Jeffery Chakedis, Randall French, Dawn Jaquish, Evangeline Mose, Andrew Lowy. Demethylating therapy modulates expression of the RON tyrosine kinase receptor in pancreatic cancer. [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 2315. doi:10.1158/1538-7445.AM2014-2315