Expression In Pancreatic Cancer Cells Research Articles

Variable Effects of Growth Factors on Developmental Gene Expression in Pancreatic Cancer Cells.

Stimulation of BxPC-3, Panc-1, and MIA PaCA-2 pancreatic cancer cells with EGF, HGF, FGF-1, FGF-2, FGF-7, and FGF-10 growth factors caused changes in the expression of master genes regulating pancreatic development (SOX9, HNF3b, GATA-4, GATA-6, and HES1). This, in turn, caused changes in the expression profile of important transcription factors, embryonic development regulators. It was also found that the master genes belonging to the same family may cause opposite effects (suppression or enhancement of expression of a particular transcriptional regulator) in the same cell line.

Abstract 1449: Aberrant expression of protein kinase D1 influences metabolic reconditioning in pancreatic cancer

Abstract Objective: Pancreatic cancer is one of the deadliest diseases, and the fourth most common cause of cancer-related deaths in the United States. Protein Kinase D1 (PKD1), a serine-threonine kinase, is an important modulator of several kinase signal transduction pathways. Although, PKD1 is known to be involved in pancreatic cancer pathogenesis, the underlying signaling mechanisms are largely unknown. Therefore, we investigated how PKD1 contributes to pancreatic tumor growth and progression. Our studies suggest a novel role of PKD1 in regulating glucose metabolism in pancreatic cancer, which drives pancreatic tumorigenesis and progression. Methods: Glucose and Lactate assays were performed in pancreatic cancer cells following PKD1 transfection. Cell culture media was collected after 48 hrs to measure the amount of glucose consumption and L-lactate production on PKD1 expression in pancreatic cancer cells using in vitro assay kits (Cayman Chemicals). Immunoblotting and qRT-PCR assays were performed to assess the expression of protein and mRNA levels, respectively, of key signaling molecules in glucose metabolism in pancreatic cancer cells. Cell proliferation and colony forming assays were performed to determine the effect of PKD1 on cell proliferation and survival under both normoxic and hypoxic conditions. In vitro functional assays for investigating migration and invasion were performed using boyden chamber and Matrigel assays, respectively, under normoxic and hypoxic conditions. Results: Our results show that PKD1 leads to altered glucose metabolism in pancreatic cancer cells. We observed increased amount of L-lactate production and glucose consumption on PKD1 expression in cells. This indicates the alterations in glucose metabolism associated with PKD1 expression in cells. These alterations were accompanied with enhanced expression of both protein and mRNA levels of Glut-1, HIF-1α, and KRAS, which are associated with glucose metabolism. These events were observed in both normoxic and hypoxic conditions, indicating that the PKD1 modulated events are independent of oxygen tension. Furthermore, we observed enhanced invasion and migration of pancreatic cancer cells on PKD1 expression, which was further increased by addition of lactate (an end product of aerobic glycolysis). Altogether, our studies indicate a role of PKD1 as a key regulator of the glucose metabolism and promoter of pancreatic cancer oncogenesis. Conclusion: These results suggest that PKD1 plays an important role in metabolic reprogramming of pancreatic cancer cell metabolism to induce cancer growth and enhanced cellular invasion and motility. This is a first study that suggests the involvement of PKD1 in metabolic remodeling of pancreatic cancer cells leading to enhanced tumor growth and progression. Citation Format: Sonam Kumari, Sheema Khan, Murali M. Yallapu, Subhash C. Chauhan, Meena Jaggi. Aberrant expression of protein kinase D1 influences metabolic reconditioning in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1449.

Orchestrating a biomarker panel with lncRNAs and mRNAs for predicting survival in pancreatic ductal adenocarcinoma.

Thelow survival of patients with pancreatic ductal adenocarcinoma (PDAC) makes the treatment of this diseaseone of the most challenging task in modern medicine. Here, by mining a large-scalecancer genome atlas data set of pancreatic cancer tissues, we identified 21 long noncoding RNAs (lncRNAs) that significantly associated with overall survival in patients with PDAC (P < .01). Further analysis revealed that 8 lncRNAs turned out to be independently correlated with patients' overall survival, and the risk score could be calculated based on their expression. To obtain a better predicting power, we integrated lncRNA data with a total of 410 differently expressed messenger RNAs (mRNAs) screened from PDAC and normal tissues in gene expression omnibus (GEO) database. The integration resulted in a much better panel including 8 lncRNAs (RP3.470B24.5, CTA.941F9.9, RP11.557H15.3, LINC00960, AP000479.1, LINC00635, LINC00636, and AC073133.1) and 8 mRNAs (DHRS9, ONECUT1, OR8D4, MT1M, TCN1, MMP9, DPYSL3, and TTN) to predict prognosis. A functional evaluation showed that these lncRNAs might play roles in pancreatic secretion, cell adhesion, and proteolysis. Using normal and pancreatic cancer cell lines, we confirmed that a majority of identified lncRNAs and mRNAs showed altered expressions in pancreatic cancer cells. Especially, LINC01589, LINC00960, TCN1, and MT1M showed a profoundly increased expression in pancreatic cancer cells, which suggests their potentially important role in pancreatic cancer. The results of our work indicate that lncRNAs have vital roles in PADC and provide new insights to integrate multiple kinds of markers in clinical practices.

Anticancer effect of HOTTIP regulates human pancreatic cancer via the metabotropic glutamate receptor 1 pathway.

The present study aimed to determine how the expression and function of HOTTIP modifies, and regulates the metabotropic glutamate receptor 1 (mGluR1) to affect human pancreatic cancer cell viability. HOTTIP expression was higher in human pancreatic cancer tissue compared with in para-carcinoma tissue. However, downregulation of HOTTIP expression was revealed to significantly reduce cell viability, induce apoptosis, promote caspase-3 and caspase-8 activities and increase Bax expression in pancreatic cancer cells. Additionally, downregulation of HOTTIP expression significantly suppressed mGluR1 and mitigated activation of the phosphoinositide 3-kinase (PI3K)/Akt/mechanistic target of rapamycin (mTOR) pathway in pancreatic cancer cells. To the best of our knowledge, the present study is the first to identify that the anticancer effect of HOTTIP against human pancreatic cancer functions the mGluR1 pathway.

The growth inhibitory effect of gambogic acid on pancreatic cancer cells.

Pancreatic cancer, the fourth most common cause of cancer-related deaths, is one of the most aggressive and devastating human malignancies with increasing incidence worldwide. To date, surgical resection is the only potentially curative therapy available for pancreatic cancer patients. Early diagnosis of pancreatic tumors is difficult, and hence, nearly 80% of patients cannot receive surgical resection. Natural products have always been a vital source for novel compounds for cancer treatment. The naturally occurring prenylated xanthone, gambogic acid, has been previously shown to exert potent anticancer, anti-inflammatory, apoptotic, antiangiogenic, and antioxidant activities. However, to our knowledge, there have been no specific studies showing its effect on the whole-genome expression in pancreatic cancer cells. Here, the anticancer activity of gambogic acid toward a panel of pancreatic cancer cells with different differentiation stages has been evaluated. Additionally, a whole-genome transcription profiling study was performed in order to identify possible candidate players modulating the antitumor effect of gambogic acid on pancreatic cancer cells. Expression analysis results showed that the pancreatic adenocarcinoma signaling pathway was specifically affected upon gambogic acid treatment. Moreover, the growth inhibitory effect of gambogic acid on pancreatic cancer cells was modulated through up-regulation of DDIT3, DUSP1, and DUSP5 and down-regulation of ALDOA, TOP2A, and ATG4B. The present work is a starting point for the generation of hypotheses on significantly regulated candidate key player genes and for a detailed dissection of the potential role of each individual gene for the activity of gambogic acid on pancreatic cancer.

The Role of Platelet-Derived ADP and ATP in Promoting Pancreatic Cancer Cell Survival and Gemcitabine Resistance.

Platelets have been demonstrated to be vital in cancer epithelial-mesenchymal transition (EMT), an important step in metastasis. Markers of EMT are associated with chemotherapy resistance. However, the association between the development of chemoresistance, EMT, and the contribution of platelets to the process, is still unclear. Here we report that platelets regulate the expression of (1) human equilibrative nucleoside transporter 1 (hENT1) and (2) cytidine deaminase (CDD), markers of gemcitabine resistance in pancreatic cancer. Human ENT1 (hENT1) is known to enable cellular uptake of gemcitabine while CDD deactivates gemcitabine. Knockdown experiments demonstrate that Slug, a mesenchymal transcriptional factor known to be upregulated during EMT, regulates the expression of hENT1 and CDD. Furthermore, we demonstrate that platelet-derived ADP and ATP regulate Slug and CDD expression in pancreatic cancer cells. Finally, we demonstrate that pancreatic cancer cells express the purinergic receptor P2Y12, an ADP receptor found mainly on platelets. Thus ticagrelor, a P2Y12 inhibitor, was used to examine the potential therapeutic effect of an ADP receptor antagonist on cancer cells. Our data indicate that ticagrelor negated the survival signals initiated in cancer cells by platelet-derived ADP and ATP. In conclusion, our results demonstrate a novel role of platelets in modulating chemoresistance in pancreatic cancer. Moreover, we propose ADP/ATP receptors as additional potential drug targets for treatment of pancreatic cancer.

Thalidomide inhibits proliferation and epithelial-mesenchymal transition by modulating CD133 expression in pancreatic cancer cells.

Pancreatic cancer is a solid malignancy with a high mortality rate, on account of the high incidence of metastasis at the time of detection. The aggressiveness of pancreatic cancer may be partly driven by cancer stem cells (CSCs), which are characterized by the ability to self-renew and recapitulate tumors in the ectopic setting. However, although a number of drugs targeting CSCs are currently under clinical investigation, few effective drugs have been developed. The present study demonstrated that thalidomide inhibited cell proliferation and metastasis in pancreatic cancer cell lines through the inhibition of epithelial mesenchymal transition. The effect of thalidomide was more pronounced in cluster of differentiation 133 (CD133)+ SW1990 cells than in Capan-2 cells, in which CD133 expression was almost undetectable. The results revealed that CD133 is likely to serve a role in the antitumor effect of thalidomide and indicated that thalidomide could be developed as a CSC-specific adjuvant chemotherapy in pancreatic cancer.

DNA-Methyltransferase 1 Induces Dedifferentiation of Pancreatic Cancer Cells through Silencing of Krüppel-Like Factor 4 Expression.

Purpose: The dismal prognosis of pancreatic cancer has been linked to poor tumor differentiation. However, molecular basis of pancreatic cancer differentiation and potential therapeutic value of the underlying molecules remain unknown. We investigated the mechanistic underexpression of Krüppel-like factor 4 (KLF4) in pancreatic cancer and defined a novel epigenetic pathway of its activation for pancreatic cancer differentiation and treatment.Experimental Design: Expressions of KLF4 and DNMT1 in pancreatic cancer tissues were determined by IHC and the genetic and epigenetic alterations of KLF4 in and KLF4's impact on differentiation of pancreatic cancer were examined using molecular biology techniques. The function of dietary 3,3'-diindolylmethane (DIM) on miR-152/DNMT1/KLF4 signaling in pancreatic cancer was evaluated using both cell culture and animal models.Results: Overexpression of DNMT1 and promoter hypermethylation contributed to decreased KLF4 expression in and associated with poor differentiation of pancreatic cancer. Manipulation of KLF4 expression significantly affected differentiation marker expressions in pancreatic cancer cells. DIM treatment significantly induced miR-152 expression, which blocked DNMT1 protein expression and its binding to KLF4 promoter region, and consequently reduced promoter DNA methylation and activated KLF4 expression in pancreatic cancer cells. In addition, DIM treatment caused significant inhibition of cell growth in vitro and tumorigenesis in animal models of pancreatic cancer.Conclusions: This is the first demonstration that dysregulated KLF4 expression associates with poor differentiation of pancreatic cancer. Epigenetic activation of miR-152/DNMT1/KLF4 signaling pathway by dietary DIM causes differentiation and significant growth inhibition of pancreatic cancer cells, highlighting its translational implications for pancreatic and other cancers. Clin Cancer Res; 23(18); 5585-97. ©2017 AACR.

MiR-377 reverses cancerous phenotypes of pancreatic cells via suppressing DNMT1 and demethylating tumor suppressor genes.

The aim was to investigate the effect of miR-377 on DNMT1 expression and cancer phenotype in pancreatic cancer cells. Real-time PCR, luciferase assay, MTT and Annexin-PI staining were used. Decreased miR-377 and increased DNMT1 (verified as a target for mir-377) levels in pancreatic cancer tissues and cell lines in comparison with normal tissues was confirmed to be influenced by promoter methylation. Also hypermethylation of BNIP3, SPARC, TFPI2 and PENK promoters was observed in tumor samples but not in normal tissues which negatively correlated with their expression. Restoration of miR-377 resulted in a reduction of the expression of DNMT1 and reactivation of BNIP3 and SPARC genes via promoter demethylation. Furthermore, enhanced expression of miR-377 could significantly inhibit cell proliferation and induce apoptosis. Our findings showed that miR-377 through targeting DNMT1 could reduce DNA methylation of some tumor suppressor genes and restore their expression in pancreatic cancer cells.

Abstract 4405: A new energy restriction mimetic decreases survival and FOXM1 expression in pancreatic cancer cells

Abstract Pancreatic cancer is a deadly disease with only 6% of the patients living longer than 5 years. Even if the disease is diagnosed when it is localized, the 5-year survival rate is approximately 19%. A characteristic of cancer cell metabolism is a shift from oxidative phosphorylation to aerobic glycolysis known as the Warburg effect. It is well known that calorie restriction (CR) is a powerful intervention that suppresses tumorigenesis in animal models by targeting glycolysis and oncogenic signaling pathways. Recent studies have shown that the metabolic responses characteristic of CR can be obtained with the use of energy restriction mimetics (ERM). The purpose of this study is to evaluate a new ERM, OSU-CG5 in altering growth of pancreatic cancer cells and nutrient-sensitive signaling pathways. Tumors including those of the pancreas have uncontrolled proliferation necessitating increased fuel demands by increasing glucose availability and altering metabolic signaling pathways. Since ERM have been reported to target glucose transport, we evaluated whether OSU-CG5 alters glucose transport (Glut) proteins and glucose uptake. Glut1, but not Glut2, 3, 4, and 6, were overexpressed in Panc-1 cells with a 6-fold higher expression compared to HPNE cells. Glut proteins were not altered in MiaPaca cells. OSU-CG5 did not alter Glut1 protein expression in either cell line. Silencing Glut1 also had no effect on the cytoxicity of OSU-CG5. OSU-CG5 did not alter glucose uptake when measured in different concentrations of glucose in the media. Cell survival, as measured with Pico green, was decreased after OSU-CG5 treatment with an IC 50 of 3uM when cultured in physiological concentration of 5 and 10mM of glucose. FOXM1 is unregulated in pancreatic cancer and a promoter of the Warburg effect. We found that OSU-CG5 significantly decreased expression of FOXM1 in Panc1 and MiaPaca cells. Our findings indicate that OSU-CG5 does not inhibit the growth of Panc1 and MiaPaca pancreatic cancer cells by decreasing glucose uptake but may target the FOXM1 signaling pathways. Citation Format: Laura Scolaro, Susan Lanza-Jacoby. A new energy restriction mimetic decreases survival and FOXM1 expression in pancreatic cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4405. doi:10.1158/1538-7445.AM2017-4405

Abstract 857: ELF3 modulates mesenchymal to epithelial transition through regulation of CD44 variant isoform expression in pancreatic cancer cells

Abstract BACKGROUND: Epithelial to Mesenchymal Transition (EMT) and Mesenchymal to Epithelial Transition (MET) may participate in pancreatic ductal adenocarcinoma (PDAC) metastasis. CD44 proteins are cell membrane proteins which function as co-receptors regulating many cellular functions. Some reports have shown that isoform switching from CD44 variant form (CD44v) to CD44 standard form (CD44s) takes place during the EMT. (Brown et al., J. Clin. Invest, 2011, Yae et al., Nat Commun., 2012). The AIM of this study was to explore the regulation of CD44 isoform switching in PDAC cells. Methods: The expression levels of CD44v and CD44s in PDAC cell lines were assessed by immunohistochemistry and immunoblot analysis. PDAC cell lines were implanted into immunodeficiency mice and treated with sulfasarazine for 14 days. Sulfasarazine, which inhibits glutamate-cysteine transport, reduces CD44v-dependent cell growth. CD44v or CD44s positive cells were isolated using flow cytometry and induced to undergo the EMT by TGF-β treatment (7.5ng/mL) for 72 hours. A doxycycline inducible system was employed to overexpress Slug, which promotes the EMT phenotype. To induce MET, the cells were released from TGF-β treatment and cultured for an additional 72 hours. E74-like ETS Transcription Factor 3 (ELF3) expression was knocked-down by siRNA transfection. RESULTS: Immunohistochemistry analysis revealed that CD44v protein is expressed specifically by PDAC with ductal structures within the complex tumor environment of these cancers. However, in contrast, CD44v expression was not observed in solitary infiltrating cancer cells. PANC-1, AsPC-1 and SUIT-2 PDAC cell lines expressed both CD44v and CD44s isoforms. Tumor mass in implanted PANC-1, AsPC-1 and SUIT-2 cells were reduced 69%, 50% and 36% respectively by sulfasarazine treatment following 2 weeks of therapy. However, CD44v null HPAC cells were resistant to sulfasarazine therapy following implantation. E-cadherin was expressed in CD44v positive cells but not in CD44s positive cells, and both CD44v and E-cadherin protein were reduced by TGF-β treatment. Slug over expressed cells were resistant to sulfasarazine treatment compared to non-Slug-induced cells. Removal of TGF-β restored both CD44v and E-cadherin expression levels which consistently occurs with MET. However, restoration of CD44v expression was diminished when ELF3 expression was reduced by siRNA transfection. CONCLUSION: The isoform switching between CD44v and CD44s was reversible. The expression of CD44v isoform appears to be controlled by ELF3 during MET. Citation Format: Ayano Kabashima-Niibe, Hiromasa Takaishi, Takanori Kanai, Hideyuki Saya, Gregory J. Gores, Hajime Higuchi. ELF3 modulates mesenchymal to epithelial transition through regulation of CD44 variant isoform expression in pancreatic cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 857. doi:10.1158/1538-7445.AM2017-857

Insulin Receptor and GPCR Crosstalk Stimulates YAP via PI3K and PKD in Pancreatic Cancer Cells.

We examined the impact of crosstalk between the insulin receptor and G protein-coupled receptor (GPCR) signaling pathways on the regulation of Yes-associated protein (YAP) localization, phosphorylation, and transcriptional activity in the context of human pancreatic ductal adenocarcinoma (PDAC). Stimulation of PANC-1 or MiaPaCa-2 cells with insulin and neurotensin, a potent mitogenic combination of agonists for these cells, promoted striking YAP nuclear localization and decreased YAP phosphorylation at Ser127 and Ser397 Challenging PDAC cells with either insulin or neurotensin alone modestly induced the expression of YAP/TEAD-regulated genes, including connective tissue growth factor (CTGF), cysteine-rich angiogenic inducer 61 (CYR61), and CXCL5, whereas the combination of neurotensin and insulin induced a marked increase in the level of expression of these genes. In addition, siRNA-mediated knockdown of YAP/TAZ prevented the increase in the expression of these genes. A small-molecule inhibitor (A66), selective for the p110α subunit of PI3K, abrogated the increase in phosphatidylinositol 3,4,5-trisphosphate production and the expression of CTGF, CYR61, and CXCL5 induced by neurotensin and insulin. Furthermore, treatment of PDAC cells with protein kinase D (PKD) family inhibitors (CRT0066101 or kb NB 142-70) or with siRNAs targeting the PKD family prevented the increase of CTGF, CYR61, and CXCL5 mRNA levels in response to insulin and neurotensin stimulation. Thus, PI3K and PKD mediate YAP activation in response to insulin and neurotensin in pancreatic cancer cells.Implications: Inhibitors of PI3K or PKD disrupt crosstalk between insulin receptor and GPCR signaling systems by blocking YAP/TEAD-regulated gene expression in pancreatic cancer cells. Mol Cancer Res; 15(7); 929-41. ©2017 AACR.

Methylation of BNIP3 in pancreatic cancer inhibits the induction of mitochondrial-mediated tumor cell apoptosis.

Bcl-2 interacting protein 3 (BNIP3) is involved in various cellular processes and is considered a key regulator of hypoxia-induced apoptosis. In the present study, the expression of BNIP3 in pancreatic cancer tissues, the correlation with clinicopathological characteristics and prognosis and the regulation of this protein in pancreatic cancer cell lines with regard to the induction of apoptosis were investigated. BNIP3 expression was significantly lower in pancreatic cancer tissues compared with normal epithelia and was associated with tumor size, clinical stage, and lymph node metastasis. The expression of BNIP3 correlated positively to the proapoptotic protein Bax and negatively to the antiapoptotic protein Bcl-2, whereas the induction of apoptosis by BNIP3 was independent of caspase 3 and 9 activation. The restoration of BNIP3 expression in pancreatic cancer cells in vitro, caused loss of ΔΨm, increase in ROS production, and apoptosis induction. The opposite effect was observed in pancreatic cancer cells, following BNIP3 silencing by RNAi. The absence of BNIP3 expression in pancreatic cancer cells was related to gene methylation that suppressed binding of HIF-1α to the BNIP3 promoter, whereas 5-Aza-2′-deoxycytidine (Aza-dC) treatment restored BNIP3 expression and sensitized pancreatic cancer cells to BNIP3-induced apoptosis. The findings indicated that BNIP3 was significantly downregulated in pancreatic cancer resulting in reduced apoptosis induction. Silencing of BNIP3 expression was associated with methylation of the hypoxia-responsive element (HRE) site that in turn inhibited the binding of HIF-1α to the BNIP3 promoter. The data suggest that BNIP3 reactivation is a potential target for therapeutic intervention against pancreatic cancer.

Histone deacetylase class-I inhibition promotes epithelial gene expression in pancreatic cancer cells in a BRD4- and MYC-dependent manner.

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive cancer with a particularly dismal prognosis. Histone deacetylases (HDAC) are epigenetic modulators whose activity is frequently deregulated in various cancers including PDAC. In particular, class-I HDACs (HDAC 1, 2, 3 and 8) have been shown to play an important role in PDAC. In this study, we investigated the effects of the class I-specific HDAC inhibitor (HDACi) 4SC-202 in multiple PDAC cell lines in promoting tumor cell differentiation. We show that 4SC-202 negatively affects TGFβ signaling and inhibits TGFβ-induced epithelial-to-mesenchymal transition (EMT). Moreover, 4SC-202 markedly induced p21 (CDKN1A) expression and significantly attenuated cell proliferation. Mechanistically, genome-wide studies revealed that 4SC-202-induced genes were enriched for Bromodomain-containing Protein-4 (BRD4) and MYC occupancy. BRD4, a well-characterized acetyllysine reader, has been shown to play a major role in regulating transcription of selected subsets of genes. Importantly, BRD4 and MYC are essential for the expression of a subgroup of genes induced by class-I HDACi. Taken together, our study uncovers a previously unknown role of BRD4 and MYC in eliciting the HDACi-mediated induction of a subset of genes and provides molecular insight into the mechanisms of HDACi action in PDAC.

Overexpression of G protein-coupled receptor GPR87 promotes pancreatic cancer aggressiveness and activates NF-\u03baB signaling pathway

BackgroundPancreatic cancer is a highly lethal disease and has the worst prognosis of any major malignancy. G protein-coupled receptor GPR87 is reported to be overexpressed in multiple cancers. The clinical significance and biological role of GPR87 in pancreatic cancer, however, remain to be established.MethodsGPR87 expression in pancreatic cancer cell lines, paired patient tissues were determined using western blotting and Real-time PCR. Ninety-six human pancreatic cancer tissue samples were analyzed by immunochemistry (IHC) to investigate the association between GPR87 expression and the clinicopathological characteristics of pancreatic cancer. Functional assays, such as anchorage-independent growth, chicken chorioallantoic membrane (CAM) assay, transwell matrix penetration assay, and Annexin V-FITC and PI staining and a xenograft tumor model were used to determine the oncogenic role of GPR87 in human pancreatic cancer progression. The effect of GPR87 on NF-κB signaling pathway was further investigated using the luciferase reporter assays, and by detection of the NF-κB signaling downstream genes.ResultsHerein, we reported that GPR87 was markedly overexpressed in pancreatic cancer cells and clinical tissues. Immunohistochemical analysis showed that the expression of GPR87 significantly correlated with patients’ clinicopathologic features, including clinical stage and tumor-nodule-metastasis (TNM) classification. Pancreatic cancer patients with higher levels of GPR87 expression had shorter overall survival compared to patients with lower GPR87 levels. We gained valuable insights into the mechanism of GPR87 expression in pancreatic cancer cells by demonstrating that overexpressing GPR87 significantly enhanced, whereas silencing endogenous GPR87 inhibited, the proliferation, angiogenesis and increased resistance to gemcitabine-induced apoptosis of pancreatic cancer in vitro and tumorigenicity of pancreatic cancer cells in vivo. Finally, we demonstrated that GPR87 enhanced pancreatic cancer aggressiveness by activating NF-κB signaling pathway. Conclusions: Taken together, these findings suggest that GPR87 plays a critical oncogenic role in pancreatic cancer progression and highlight its potential as a target for pancreatic cancer therapy.ConclusionsOur findings suggest that GPR87 plays a critical oncogenic role in pancreatic cancer progression and highlight its potential as a target for pancreatic cancer therapy.

Abstract A41: GPRC5A acts as a potent oncogene in pancreatic cancer

Abstract Background: GPRC5A is an orphan G-protein coupled receptor acting as an oncogene in some cancers and as a tumor suppressor in other. Its role in pancreatic cancer has not been studied systematically. ELAV-like protein 1, also known as “HuR,” is an RNA binding protein that is encoded by the ELAVL1 gene. HuR post-transcriptionally regulates multiple genes, plays key roles in human malignancies, and is known to mediate the efficacy of gemcitabine. Our preliminary data indicated that, in pancreatic cells, GPRC5A transcription responded to gemcitabine treatment and that its mRNA likely interacted with HuR. Methods: We immunohistochemically analyzed GPRC5A protein expression in primary and metastatic pancreatic cancer samples. We examined the effects of GPRC5A on pancreatic cells’ ability to proliferate, migrate, and invade, and how gemcitabine modulates this ability. We also studied the interaction between GPRC5A and HuR and examined how it is affected by gemcitabine. Lastly, we used microarray expression analyses to identify the genes and pathways linked to GPRC5A in pancreatic cancer cells. Results: We analyzed public mRNA expression data from 675 human cancer cell lines and 10,609 samples from The Cancer Genome Atlas (TCGA): GPRC5A mRNA abundance in pancreatic cancer was the highest (cell lines) or second highest (TCGA) among all tissues and cancer types. We analyzed an independent set of 252 pancreatic normal and cancer samples: we found GPRC5A mRNA to be up-regulated in primary tumor samples compared to normal pancreas, and even further up-regulated in metastatic pancreatic cancer. We immunostained 208 cores (103 primary and metastatic pancreatic cancer samples): we found GPRC5A protein to be barely expressed in normal pancreatic ductal cells and highly expressed in both primary and metastatic pancreatic ductal cancer cells. We carried out in vitro studies of multiple pancreatic cancer cell lines: we found that increasing GPRC5A protein levels promoted pancreatic cancer cell growth and migration whereas siRNA-mediated knockdown of GPRC5A impaired proliferation and invasion. We found that gemcitabine treatment enhanced GPRC5A expression in pancreatic cancer cells. Conversely, siRNA-mediated knockdown of GPRC5A sensitized the cells to gemcitabine. We showed that HuR/ELAVL1 up-regulated GPRC5A protein in the first 18 hours following gemcitabine treatment by stabilizing GPRC5A’s mRNA through binding to at least on site in GPRC5A’s 3´UTR. Microarray analysis showed that GPRC5A overexpression impacts several signaling networks. Conclusions: Our results indicate that GPRC5A acts as an oncogene in pancreatic cancer. Unexpectedly, gemcitabine was found to increase GPRC5A's mRNA and protein levels. We showed that this increase is mediated by HuR, a known enabler of gemcitabine efficacy, through a direct interaction between HuR and GPRC5A’s mRNA. It appears that the sensitivity of pancreatic cancer cells to gemcitabine can be augmented through down-regulation of GPRC5A. Citation Format: Honglei Zhou, Aristeidis Telonis, Yi Jing, Masaya Jimbo, Fernando Blanco, Eric Londin, Jonathan Brody, Isidore Rigoutsos.{Authors}. GPRC5A acts as a potent oncogene in pancreatic cancer. [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 A41.

FOXO3 is essential for CD44 expression in pancreatic cancer cells.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most fatal types of cancer and the 5-year survival rate is only 5%. Several studies have suggested that cancer stem cells (CSCs) are thought to be involved in recurrence and metastasis and so it is essential to establish an approach targeting CSCs. Here we have demonstrated that cyclic guanosine monophosphate (cGMP) suppressed CD44 expression and the properties of CSCs in PDAC. Microarray analysis suggested that cGMP inhibited Forkhead box O3 (FOXO3), which is known as a tumor suppressor. Surprisingly, our data demonstrated that FOXO3 is essential for CD44 expression and the properties of CSCs. Our data also indicated that patients with high FOXO3 activation signatures had poor prognoses. This evidence suggested that cGMP induction and FOXO3 inhibition could be ideal candidates for pancreatic CSC.

Leukotriene B4 induces EMT and vimentin expression in PANC-1 pancreatic cancer cells: Involvement of BLT2 via ERK2 activation

Leukotriene B4 (LTB4) is a leukocyte chemoattractant and plays a major role controlling inflammatory responses including pancreatitis. LTB4 is known to be correlated with cancer progression. LTB4 induces keratin phosphorylation and reorganization by activating extracellular regulated kinase (ERK) in PANC-1 pancreatic cancer cell lines. However, the role of LTB4 in epithelial mesenchymal transition (EMT) and vimentin expression in pancreatic cancer cells is unknown.We examined whether LTB4 induces EMT and vimentin expression by Western blot, si-RNA, and RT-PCR. LTB4 induced morphological change, decreased E-cadherin expression and increased N-cadherin and vimentin expression. LTB4 increased migration and invasion of PANC-1 cancer cells. LTB4 dose-dependently upregulated expression of vimentin in PANC-1 cancer cells. LTB4-induced vimentin expression was suppressed by LY255283 (BLT2 antagonist). Comp A, a BLT2 agonist, further increased vimentin expression. Gene silencing of BLT2 suppressed LTB4-or Comp A-induced vimentin expression in PANC-1 cells. The MEK inhibitor, PD98059 suppressed Comp A-induced vimentin expression. Comp A or transfection of plasmid containing BLT2 cDNA (pCBLT2) activated ERK, and BLT2 gene silencing suppressed Comp A-induced ERK activation. ERK2 siRNA abrogated Comp A-induced vimentin expression and ERK2 overexpression enhanced vimentin expression. One of well-known cause of ras mutation, cigarette smoke extracts increased BLT2 expression in PANC-1 cancer cells.Taken together, these results suggest that BLT2 is involved in LTB4-induced vimentin expression through ERK2 in PANC-1 cells.

KCa3.1 (IK) modulates pancreatic cancer cell migration, invasion and proliferation: anomalous effects on TRAM-34.

In the recent decades, ion channels became the focus of cancer biologists, as many channels are overexpressed in tumour tissue and functionally they are linked to abnormal cell behaviour with processes including apoptosis, chemo- and radioresistance, proliferation and migration. KCa3.1 is a Ca2+-activated K+ channel that plays a central role in tumour progression in many cancer types. Therefore, the aim of the present study was to investigate KCa3.1 expression in pancreatic cancer cells and assess possible implications to disease progression. Using qPCR technique, we found abundant expression of KCa3.1 in pancreatic cancer cell lines. Patch clamp measurements on MiaPaCa-2 cells revealed a Ca2+-activated K+ current that matched biophysical characteristics as described for KCa3.1. Moreover, the current was sensitive to the commonly used channel modulators TRAM-34, clotrimazole and DC-EBIO, and it was abolished following transient gene knockdown of KCa3.1. We utilized both pharmacology and RNAi to assess a possible role of the channel in tumour cell behaviour. We found that the channel supported MiaPaCa-2 cell proliferation. Using RNAi protocols, we also identified KCa3.1 as important entity in cell invasion. However, TRAM-34 had unexpected stimulatory effects on cell migration and invasion estimated in various assays. Moreover, TRAM-34 increased intracellular Ca2+. In conclusion, we found prominent functional expression of KCa3.1 in pancreatic cancer cells. We provide evidence that the channel has a key role in cell proliferation and for the first time identify KCa3.1 as important entity in PDAC cell migration. We further reveal anomalous effects of TRAM-34.

Antitumor activity of curcumin is involved in down-regulation of YAP/TAZ expression in pancreatic cancer cells.

Pancreatic cancer (PC) is one of the most aggressive human malignancies worldwide and is the fourth leading cause of cancer-related deaths. Curcumin (diferuloylmethane) is a polyphenol derived from the Curcuma longa plant. Certain studies have demonstrated that curcumin exerts its anti-tumor function in a variety of human cancers including PC, via targeting multiple therapeutically important cancer signaling pathways. However, the detailed molecular mechanisms are not fully understood. Two transcriptional co-activators, YAP (Yes-associated protein) and its close paralog TAZ (transcriptional coactivator with PDZ-binding motif) exert oncogenic activities in various cancers. Therefore, in this study we aimed to determine the molecular basis of curcumin-induced cell proliferation inhibition in PC cells. First, we detected the anti-tumor effects of curcumin on PC cell lines using CTG assay, Flow cytometry, clonogenic assay, wound healing assay and Transwell invasion assay. We found that curcumin significantly suppressed cell growth, weakened clonogenic potential, inhibited migration and invasion, and induced apoptosis and cell cycle arrest in PC cells. We further measured that overexpression of YAP enhanced cell proliferation and abrogated the cytotoxic effects of curcumin on PC cells. Moreover, we found that curcumin markedly down-regulated YAP and TAZ expression and subsequently suppressed Notch-1 expression. Collectively, these findings suggest that pharmacological inhibition of YAP and TAZ activity may be a promising anticancer strategy for the treatment of PC patients.