Normal Pancreatic Duct Epithelial Cells Research Articles

Abstract 4807: Pimavanserin suppresses pancreatic tumor growth and resistance to gemcitabine by inhibiting Akt/Gli1 signaling

Abstract Despite major advances in cancer treatment, pancreatic cancer is still incurable and the treatment outcomes are limited. In the current study, we evaluated the anti-cancer effects of pimavanserin tartrate (PVT), a drug used for the treatment of Parkinson’s disease psychosis. Our observations indicated that, PVT significantly suppressed the proliferation of pancreatic cancer cells by inducing apoptosis without exerting any cytotoxic effects in normal human pancreatic ductal epithelial (HPDE-6) cells. The colony forming ability of pancreatic cancer cells was significantly inhibited with PVT treatment. Anti-proliferative and apoptosis inducing effects of PVT were mediated by the inhibition of pAkt (Ser473), Akt, Gli1, Oct-4, SOX-2, NANOG and c-Myc. Akt and Oct-4 inhibition by PVT treatment was further validated by immunofluorescence analysis. Moreover, PVT inhibited the formation of tumorspheres in PANC1 pancreatic cancer cells. Pharmacologically inhibiting or genetically knocking out Akt or Gli1 enhanced the growth suppressive effects of PVT in pancreatic cancer cells. Subsequently, we evaluated the effects of PVT in gemcitabine resistant cells. Our results demonstrated that, PVT reduced the survival of MIAPaCa2 gemcitabine resistant cells in a concentration and time-dependent manner. Further mechanistic analysis indicated that, PVT suppressed the phosphorylation of Akt at Ser 473 and inhibited the expression of Gli1, Oct-4 and c-Myc in MIAPaCa2 gemcitabine resistant cells. Moreover, PVT increased the cleavage of caspase-3 and PARP as an indicator of apoptosis. Oral administration of PVT suppressed BxPC3 tumor xenografts by 50% in athymic nude mice. In another in vivo experiment, PVT treatment inhibited the growth of orthotopically implanted PANC1 tumors by 75%. Chronic administration of PVT did not exhibit any general signs of toxicity or behavioral side effects in mice. Taken together, our results indicate that pancreatic tumor growth suppression by PVT is orchestrated by inhibition of Akt/Gli-1 signaling. Since PVT is already available in the clinic with an established safety profile, our results will accelerate its clinical development for the treatment of patients with pancreatic cancer. Citation Format: Sharavan Ramachandran, Sanjay K. Srivastava. Pimavanserin suppresses pancreatic tumor growth and resistance to gemcitabine by inhibiting Akt/Gli1 signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4807.

The expression of long non-coding RNA-LINC01410 in pancreatic cancer and its effect on proliferation and migration of pancreatic cancer cells

Objective: To explore the expression of long non-coding RNA-LINC01410 in the pancreatic cancer tissues, corresponding paracancerous tissues, human pancreatic cancer cell lines and normal pancreatic ductal epithelial cell line, and analyze the effect of LINC01410 on pancreatic cancer cell proliferation and migration. Methods: Real-time fluorescent quantitative PCR (RT-qPCR) was used to detect the expression level of LINC01410 in 16 cases of pancreatic cancer tissue and its adjacent tissues. RT-qPCR was performed to analyze LINC01410 expression in the pancreatic cancer cell lines AsPC-1, CAPAN-1, SW1990, BxPC-3 and CFPAC-1, and human normal pancreatic ductal epithelial cell line HPDE6-C7. Transfection of interference plasmid (shRNA) in the pancreatic cancer cell line with the highest expression level of LINC01410 were used to knock-down the expression of LINC01410. CCK-8 assay, colony formation assay, and transwell chamber assay were performed to detect the proliferation and migration of pancreatic cancer cells. The complementary paired miRNAs and downstream genes of LINC01410 were predicted by bioinformatics. The expression of miRNA and downstream genes was detected by RT-qPCR, and the protein expression of downstream genes was determined by Western blot. Results: The expression of LINC01410 in the pancreatic cancer tissues was significantly higher than that in the adjacent tissues [(3.46±0.32) vs (0.65±0.08), P<0.01]. The expression levels of LINC01410 in the pancreatic cancer cell lines were significantly higher than that in the normal human pancreatic ductal epithelial cells (P<0.05). The expression of LINC01410 was highest in BxPC-3 cells (P<0.01). After knock-down of the LINC01410 expression in the pancreatic cancer cell line BxPC-3, the cell proliferation was significantly inhibited (P<0.05), and the cell migration ability was decreased (P<0.05). LINC01410 complementarily paired with miR-497-5p, and miR-497-5p complementarily paired with IFITM3. After inhibiting the expression of LINC01410, the expression of miR-497-5p was increased [(1.04±0.17) vs (5.79±0.43), P<0.01], the mRNA expression of IFITM3 was decreased [(0.39±0.05) vs (1.00±0.03), P<0.01], and the protein expression of IFITM3, CDK6, Cyclin D2, PCNA, Vimentin, and N-cadherin was decreased. Conclusions: The expression of LINC01410 was increased in pancreatic cancer tissues and cell lines. Down-regulation of LINC01410 expression inhibits the proliferation and migration of pancreatic cancer BxPC-3 cells, and its mechanism may be closely related to regulating the miR-497-5p and IFITM3 gene expression.

Pimavanserin tartrate: A potential drug for pancreatic cancer therapy in future

Despite major advances in cancer treatment, pancreatic cancer is still incurable and the treatment outcomes are limited. In the current study, we evaluated the anti‐cancer effects of pimavanserin tartrate (PVT), a drug used for the treatment of Parkinson's disease psychosis. Our observations indicated that, PVT significantly suppressed the proliferation of pancreatic cancer cells by inducing apoptosis without exerting any cytotoxic effects in normal human pancreatic ductal epithelial (HPDE‐6) cells. The colony forming ability of pancreatic cancer cells was significantly inhibited with PVT treatment. Anti‐proliferative and apoptosis inducing effects of PVT were mediated by the inhibition of pAkt (Ser473), Akt, Gli1, Oct‐4, SOX‐2, NANOG and c‐Myc. Akt and Oct‐4 inhibition by PVT treatment was further validated by immunofluorescence analysis. Apoptotic effects of PVT was confirmed by increase in cleavage of caspase‐3 and PARP. Moreover, PVT inhibited the formation of tumorspheres in PANC1 pancreatic cancer cells. Pharmacologically inhibiting or genetically knocking out Akt or Gli1 enhanced the growth suppressive effects of PVT in pancreatic cancer cells. Subsequently, we evaluated the effects of PVT in gemcitabine‐resistant cells. Our results demonstrated that, PVT reduced the survival of MIAPaCa2 gemcitabine‐resistant cells in a concentration and time‐dependent manner. Further mechanistic analysis indicated that, PVT suppressed the phosphorylation of Akt at Ser 473 and inhibited the expression of Gli1, Oct‐4 and c‐Myc in MIAPaCa2 gemcitabine‐resistant cells. Moreover, PVT increased the cleavage of caspase‐3 and PARP as an indicator of apoptosis. Oral administration of PVT suppressed BxPC3 tumor xenografts by 50% in athymic nude mice. In another in vivo experiment, PVT treatment inhibited the growth of orthotopically implanted PANC1 tumors by 75%. Chronic administration of PVT did not exhibit any general signs of toxicity or behavioral side effects in mice. Taken together, our results indicate that pancreatic tumor growth suppression by PVT is orchestrated by inhibition of Akt/Gli1 signaling. Since PVT is already available in the clinic with an established safety profile, our results will accelerate its clinical development for the treatment of patients with pancreatic cancer.Support or Funding InformationSupported in part by R01 grant CA129038, awarded to (S.K.S.) by the National Cancer Institute, Syngenta fellowship grant awarded by Syngenta pharmaceuticals.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Knockdown of KDM1B inhibits cell proliferation and induces apoptosis of pancreatic cancer cells

Pancreatic cancer (PC) is one of the common malignant tumors in digestive tract with a high fatality rate. The oncogenic role of lysine-specific demethylase1 (LSD1/KDM1 A) has been well recognized in PC. While, the role of its homolog LSD2 (KDM1B) in regulating PC progression is poorly understood. In this study, we attempted to evaluate the functional role of KDM1B in PC cells. The expression of KDM1B was detected by immunohistochemistry and immunoblotting in PC tissues and cells. Lentivirus-mediated shRNA was applied to silence KDM1B in PANC-1 and SW1990 cells. Cell proliferation was measured by MTT and Celigo assay. Cell apoptosis was determined by both Caspase-Glo®3/7 assay and Flow cytometry. Intracellular signaling molecules were detected using a PathScan intracellular signaling array kit. In this study, we found KDM1B was highly expressed in PC tissues compared to paracancerous tissues. Moreover, elevated expression of KDM1B was detected in PC cell lines (BxPC-3, CFPAC-1, PANC-1 and SW1990) as compared with a normal human pancreatic duct epithelial cell line (HPDE6-C7). Further investigations revealed that KDM1B knockdown significantly inhibited PC cell proliferation. Furthermore, the apoptosis of PANC-1 and SW1990 cells was significantly increased after KDM1B knockdown. Notably, the activations of p-ERK1/2, p-Smad2, p-p53, cleaved PARP, cleaved Caspase-3, cleaved Caspase-7, p-eIF2a and Survivin were promoted by KDM1B knockdown, while IkBa was suppressed. Taken together, our findings provided new insights into the critical and multifaceted roles of KDM1B in the regulation of cell proliferation and apoptosis, and offered a potentially novel target in preventing the progression of PC.

Inhibition of pancreatic cancer stem cell characteristics by α-Mangostin: Molecular mechanisms involving Sonic hedgehog and Nanog.

The current investigation was intended to elucidate the molecular mechanism of α‐Mangostin in the regulation of pancreatic cancer stem cell (CSC) characteristics. Here, we demonstrate that α‐Mangostin inhibited cell proliferation in pancreatic CSCs and cancer cell lines while it showed no effect on human pancreatic normal ductal epithelial cells. Also, α‐Mangostin inhibited colony formation and induced apoptosis in these cells. Further, α‐Mangostin inhibited the self‐renewal capacity of CSCs isolated from human primary tumours and KrasG12D mice. Furthermore, α‐Mangostin inhibited the invasive and metastatic ability of pancreatic CSCs by suppressing the epithelial‐to‐mesenchymal transition (EMT) via up‐regulation of E‐cadherin and down‐regulation of mesenchymal phenotype by inhibiting N‐cadherin, Snail and Slug expression. Interestingly, the pluripotency maintaining factors and CSC markers were inhibited by α‐Mangostin thus suggesting that α‐Mangostin can target CSCs to inhibit pancreatic cancer effectively. Gli signalling plays a crucial role in the self‐renewal and pluripotency of CSCs. α‐Mangostin inhibited the Gli transcription and the expression of Gli target genes (Nanog, Oct4, c‐Myc, Sox‐2 and KLF4) in CSCs. Using ChIP assay, we demonstrated that Nanog could directly bind to promoters of Cdk2, Cdk6, FGF4, c‐Myc and α‐Mangostin inhibited Nanog binding to these promoters. Conversely, the inhibitory effects of the α‐Mangostin on CSC proliferation and Gli or Nanog transcription and their targets were abrogated by either enforced activation of sonic hedgehog (Shh) or by the overexpression of Nanog. Taken together, our studies suggest that α‐Mangostin may act as Gli inhibitor and establishes the pre‐clinical significance of α‐Mangostin for the prevention and treatment of pancreatic cancer.

Triacetyl resveratrol upregulates miRNA‑200 and suppresses the Shh pathway in pancreatic cancer: A potential therapeutic agent.

Trans‑3,4',5‑trihydroxystilbene (resveratrol) is a naturally occurring polyphenolic phytoalexin with marked anticancer activities, and is mainly found in grapes, berries and peanuts. However, due to a low bioavailability, it has not progressed to clinical practice for cancer treatment. Therefore, the aims of the present study were to examine the anticancer activities of the resveratrol derivative, triacetyl resveratrol(TCRV), in pancreatic cancer cells. Apoptosis was measured by fluorescence‑activated cell sorting and terminal deoxynucleotidyl transferase(TdT)‑mediated dUTP nick‑end labeling assays. Gene expression was measured by reverse transcription‑quantitative polymerase chain reaction. TCRV inhibited colony formation and induced apoptosis through caspase‑3 activation in human pancreatic cancer AsPC‑1 and PANC‑1 cells, whereas it exerted no effect on human pancreatic normal ductal epithelial cells (HPNE). TCRV inhibited epithelial‑mesenchymal transition (EMT) by upregulating the expression of E‑cadherin and suppressing the expression of N‑cadherin and the transcription factors, Snail, Slug and Zeb1. TCRV inhibited Zeb1 3'UTR‑luciferase activity through the upregulation of microRNA (miR)‑200 family members. The inhibitory effects of TCRV on pancreatic cancer cell migration and invasion were counteracted by anti‑miR‑200 family members. The inhibitory effects of TCRV on EMT and the induction of apoptosis were exerted through the suppression of the sonic hedgehog (Shh) pathway, and through the modulation of cyclinD1 and Bcl‑2 expression. The hyperactivation of the Shh pathway by either Shh protein or Gli1 overexpression abrogated the biological effects of TCRV. Taken together, the results of this study demonstrate that TCRV inhibits pancreatic cancer growth and EMT by targeting the Shh pathway and its downstream signaling mediators. TCRV inhibited EMT through the upregulation of miR‑200 family members. Since TCRV effectively inhibited the growth of human pancreatic cancer cells by modulating the Shh pathway, without affecting the growth of HPNE cells, our findings suggest the possible use of TCRV as a promising candidate for the treatment and/or prevention of pancreatic cancer.

ProNGF siRNA inhibits cell proliferation and invasion of pancreatic cancer cells and promotes anoikis

BackgroundPrecursor of nerve growth factor (proNGF) was previously considered biologically inactive; however, it has recently been identified as having important roles in the pathology of cancer development. AimThis study aimed to explore the therapeutic effects of proNGF siRNA on the proliferation, invasion, and anoikis of pancreatic cancer cells and determine the functions of proNGF. MethodsPancreatic ductal adenocarcinoma (PDAC) and paired paracancerous tissue samples were collected from 60 patients for evaluation of proNGF expression by immunohistochemistry staining, qPCR, and western blotting. PDAC cell proliferation, migration, apoptosis, and anoikis following proNGF siRNA knockdown were investigated in two pancreatic cancer cell lines, Panc-1 and Bxpc-3, using BrdU incorporation assays, EdU staining, Ki-67 immunofluorescence (IF) staining, wound-healing assays, transwell invasion assays, and EthD-1 IF staining. Autophagy-related proteins were also measured by western blotting. ResultsLevels of proNGF protein were higher in pancreatic cancer tissues and cells lines than those in paracancerous tissues and normal pancreatic duct epithelial cells, respectively. In vitro, ProNGF knockdown by siRNA led to significantly reduced cell proliferation, remarkably inhibited wound-healing, and reduced the number of invaded PDAC cells in migration and transwell assays. Treatment with proNGF siRNA also downregulated ATG5 and Beclin 1 protein levels, increased those of P62, and increased EthD-1 staining in PDAC cells. ConclusionProNGF expression is elevated in PDAC tissues and cell lines, and proNGF siRNA can inhibit cell proliferation, migration, and invasion, and promote anoikis of pancreatic cancer cells, in which decreased proNGF may participate.

The synergic inhibitory effects of dark tea (Camellia sinensis) extract and p38 inhibition on the growth of pancreatic cancer cells.

Background: Dark tea is one of the most popular types of Chinese tea, which has been reported to exhibit anti-obesity, anti-oxidation and antitumor activities in according human cell lines. In terms of tumorigenesis, the systemic study of the physiological effect of specific fraction of dark tea and the relevant molecular mechanism warrant more attention.Methods: Dark tea was firstly isolated through solvent extraction method. Dissolved ethyl acetate extract was further fractioned by elution with various concentration of ethyl alcohol. The cytotoxicity effect of dark tea on cell proliferation was evaluated by CCK8 assay in HPDE human normal pancreatic duct epithelial cells, SW1990 and PANC-1 human pancreatic cancer cells, and SW1116 human colorectal cancer cells. Immunoblotting and flow cytometry analysis were utilized to examine the status of protein and reactive oxygen species respectively. Gene expression profile was analyzed by cDNA microarray and real-time PCR. The plasmid for ID1 expression was stably transfected into SW1990 cells for relevant functional analysis. The effect of dark tea extract on tumorigenesis in vivo was studied in xenograft tumor model.Results: Water eluate fraction of the ethyl acetate extract from dark tea inhibited the growth of SW1990, PANC-1 and SW1116 cells more efficiently compared with that in HPDE cells. Meanwhile, p38 activity was increased and AKT activity was dropped in cancer cells with dark tea extract treatment. Further functional analyses indicated that water eluate fraction and p38 inhibitor treatment exerted a synergic inhibitory effect on cancer cells growth, which was related to their suppressive effect on expression level of ID1 (inhibitor of differentiation protein 1), which was highly expressed in cancer cells. The analysis utilizing xenograft tumor model further indicated water eluate fraction exhibited a significantly inhibitory effect on tumorigenesis.Conclusion: Based on the sequential extraction procedure, our results reveal the inhibitory effect of water eluate fraction of the ethyl acetate extract from dark tea and its synergistic effect with p38 inhibition on the growth of pancreatic cancer cells, in which ID1 is identified as a downstream effector. This sheds insights into the physiological relevance of specific fraction of dark tea to tumorigenesis in pancreatic cancer.

MiR-210 knockdown promotes the development of pancreatic cancer via upregulating E2F3 expression.

The aim of this study was to explore the role of microRNA-210 (miR-210) and E2F3 in the development of pancreatic cancer and to investigate the possible underlying mechanism. The expression level of miR-210 in pancreatic cancer tissues, para-cancerous tissues, and normal pancreatic tissues was detected by quantitative Real Time-Polymerase Chain Reaction (qRT-PCR). The correlation between miR-210 expression and pathological indicators of pancreatic cancer was analyzed. Meanwhile, the expression of miR-210 in pancreatic cancer cells and normal pancreatic ductal epithelial cells was detected by qRT-PCR. After transfection with miR-210 mimics and inhibitor, the viability and cell cycle of pancreatic cancer cells were detected by cell counting kit-8 (CCK-8) assay and flow cytometry, respectively. The binding condition of miR-210 and E2F3 was verified by Dual-Luciferase reporter gene assay. MiR-210 was lowly expressed in pancreatic cancer tissues than that of para-cancerous tissues. The expression of miR-210 was negatively correlated with TNM stage and tumor size of pancreatic cancer. In vitro experiments showed that the miR-210 was downregulated in pancreatic cancer cells than that of normal pancreatic ductal epithelial cells. Meanwhile, overexpression of miR-210 arrested cell cycle decreased cell viability and downregulated E2F3 expression in pancreatic cancer cells. Dual-Luciferase reporter gene assay indicated that E2F3 bound to mi-210. Further experiments confirmed that E2F3 was negatively regulated by miR-210. MiR-210 knockdown promotes cell proliferation by upregulating E2F3 expression, thereby promoting the progression of pancreatic cancer.

Tumor-promoting effects of pancreatic cancer cell exosomes on THP-1-derived macrophages.

Pancreatic ductal adenocarcinoma (PDAC) tumor growth is enhanced by tumor-associated macrophages (TAMs), yet the mechanisms by which tumor cells and TAMs communicate are not fully understood. Here we show that exosomes secreted by PDAC cell lines differed in their surface proteins, lipid composition, and efficiency of fusing with THP-1-derived macrophages in vitro. Exosomes from AsPC-1, an ascites-derived human PDAC cell line, were enriched in ICAM-1, which mediated their docking to macrophages through interactions with surface-exposed CD11c on macrophages. AsPC-1 exosomes also contained much higher levels of arachidonic acid (AA), and they fused at a higher rate with THP-1-derived macrophages than did exosomes from other PDAC cell lines or from an immortalized normal pancreatic ductal epithelial cell line (HPDE) H6c7. Phospholipase A2 enzymatic cleavage of arachidonic acid from AsPC-1 exosomes reduced fusion efficiency. PGE2 secretion was elevated in macrophages treated with AsPC-1 exosomes but not in macrophages treated with exosomes from other cell lines, suggesting a functional role for the AsPC-1 exosome-delivered arachidonic acid in macrophages. Non-polarized (M0) macrophages treated with AsPC-1 exosomes had increased levels of surface markers indicative of polarization to an immunosuppressive M2-like phenotype (CD14hi CD163hi CD206hi). Furthermore, macrophages treated with AsPC-1 exosomes had significantly increased secretion of pro-tumoral, bioactive molecules including VEGF, MCP-1, IL-6, IL-1β, MMP-9, and TNFα. Together, these results demonstrate that compared to exosomes from other primary tumor-derived PDAC cell lines, AsPC-1 exosomes alter THP-1-derived macrophage phenotype and function. AsPC-1 exosomes mediate communication between tumor cells and TAMs that contributes to tumor progression.

Melittin-induced long non-coding RNA NONHSAT105177 inhibits proliferation and migration of pancreatic ductal adenocarcinoma

Long non-coding RNAs (lncRNAs) play crucial roles in the pathogenesis of pancreatic ductal adenocarcinoma (PDAC). Previously, we found that melittin treatment suppressed PDAC tumor growth. However, it is unclear whether lncRNAs have any role in the melittin-induced suppression of PDAC. In this study, we used microarray data to identify 844 lncRNAs that were significantly differentially expressed in response to melittin treatment. Of these lncRNAs, we focused on the lncRNA NONHSAT105177, which had about a 22-fold increase in expression with melittin treatment. We found that melittin treatment increased NONHSAT105177 expression in PDAC cell lines but not in normal pancreatic ductal epithelial cell line. NONHSAT105177 expression was significantly lower in PDAC cancer tissues than in adjacent noncancerous tissues. Additionally, overexpression of NONHSAT105177 inhibited PDAC cell proliferation, migration, and the epithelial–mesenchymal transition (EMT), both in vitro and in vivo. Consistent with the mechanism of action of melittin, NONHSAT105177 significantly downregulated cholesterol pathway genes, including Clusterin (CLU). Moreover, we found that NONHSAT105177 trafficking was mediated by exosomes. The combined findings of our current and previous studies suggest that NONHSAT105177 mediated the melittin-induced inhibition of PDAC cell growth and metastasis, which indicated a potential target for developing new strategies.

HMGN5 promotes proliferation and invasion via the activation of Wnt/β-catenin signaling pathway in pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive carcinoma with a poor prognosis. A recent study has demonstrated that high mobility group nucleosome binding domain 5 (HMGN5) was involved in tumorigenesis and progression of multiple types of human cancers. However, the role of HMGN5 in PDAC is unknown. The objective of the present study was to analyze the function and novel mechanism of HMGN5 involved in PDAC cell progression. It was firstly revealed that the expression of HMGN5 was significantly upregulated in PDAC cell lines and tissues, when compared with that in normal pancreatic duct epithelial cells and adjacent normal pancreatic tissues. In vitro assay revealed that HMGN5 silencing impaired PDAC cell viability, proliferation, migration and invasion. Similarly, tumor growth rate was also decreased in vivo following HMGN5 silencing. Furthermore, it was demonstrated that HMGN5 silencing significantly inhibited epithelial-mesenchymal transition in vitro. Notably, HMGN5-medicated Wnt/β-catenin signaling pathway activation was observed to be one of the critical signal transduction pathways that associates HMGN5 with EMT activation. Collectively, the results indicated the important role of HMGN5 in PDAC cell proliferation and metastasis, and provide a promising target against the transcriptional program of PDAC.

Abstract LB-047: Novel anti-psychotic drug for pancreatic cancer treatment

Abstract Pancreatic cancer is one of the most deadly malignancy with a five-year survival rate of 7%. Despite advances in cancer treatment, this disease is still incurable and the treatment outcomes are limited. Few studies have shown that neuroleptic drugs exert better anti-proliferative effects in pancreatic cancer cells when compared to conventional chemotherapeutic agents gemcitabine and oxaliplatin. In the current study, we evaluated the anti-cancer effects of pimavanserin, an anti-psychotic drug used for the treatment of Parkinson's disease psychosis. Pimavanserin significantly suppressed the proliferation of AsPC1, BxPC3, PANC1, MIAPaCa2 and PO2 pancreatic cancer cells with the IC50 between 3 - 9µM at 24, 48 and 72 hours. Pimavanserin treatment induced 2-5 fold apoptosis in pancreatic cancer cells. Notably, no significant cell death was observed in normal human pancreatic ductal epithelial (HPDE-6) cells by pimavanserin treatment. Furthermore, pimavanserin treatment inhibits the expression of key oncogenic proteins and cancer stem cell markers such as pAkt(S473), pSTAT3(Y705), β-catenin, GLI-1, GLI-2, c-Myc, Oct-4, SOX-2, NANOG and CD44. We further evaluated the efficacy of pimavanserin in two different in vivo tumor models. Oral administration of 10mg/kg pimavanserin suppressed 50% of sub-cutaneously implanted BxPC3 tumors in athymic nude mice. In another setting, 10mg/kg pimavanserin by oral gavage inhibited the growth of orthotopically implanted PANC1 pancreatic tumors by 75%. Moreover, chronic administration of pimavanserin did not show any general signs of toxicity and no apparent changes were observed in the behavioral activity of mice. Further mechanistic studies are in progress. Since pimavanserin is already available in the clinic with established safety profile, our results will accelerate its clinical development for treatment of patients with pancreatic cancer. Our study is the first to demonstrate the anti-cancer effects of pimavanserin. Citation Format: Sharavan Ramachandran, Sanjay K. Srivastava. Novel anti-psychotic drug for pancreatic cancer treatment [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 LB-047.

Chronic ethanol exposure of human pancreatic normal ductal epithelial cells induces cancer stem cell phenotype through SATB2.

The incidence of pancreatic cancer is on the rise. Risk factors for pancreatic cancer include alcohol toxicity and metabolic conditions such as obesity, hypertension, dyslipidaemia, insulin resistance and type 2 diabetes. However, the molecular mechanism by which chronic alcohol consumption contributes to pancreatic cancer is not well understood. The purpose of the study was to demonstrate the effects of long‐term chronic ethanol exposure on the transformation of human pancreatic normal ductal epithelial (HPNE) cells. Our data showed that ethanol‐transformed HPNE cells were more progressively transformed exhibiting spheroids and colonies, and anchorage‐independent growth. These transformed cells contained high levels of reactive oxygen species and induced SATB2 expression. Furthermore, during ethanol‐induced cellular transformation, cells gained the phenotypes of cancer stem cells (CSCs) by expressing pluripotency maintaining factors (Oct4, Sox2, cMyc and KLF4) and stem cell markers (CD24, CD44 and CD133). Ethanol‐induced SATB2 can bind to the promoters of KLF4, Oct4, cMyc, Sox2, Bcl‐2 and XIAP genes. Suppression of SATB2 expression in ethanol‐transformed HPNE cells inhibited cell proliferation, colony formation and markers of CSCs and pluripotency. These data suggest that chronic alcohol consumption may contribute toward the development of pancreatic cancer by converting HPNE cells to cancer stem‐like cells.

Antimicrobial Peptide Human Neutrophil Peptide 1 as a Potential Link Between Chronic Inflammation and Ductal Adenocarcinoma of the Pancreas.

Defensins are antimicrobial peptides playing a role in innate immunity, in epithelial cell regeneration, and in carcinogenesis of inflammation-triggered malignancies. We analyzed this role in pancreatic ductal adenocarcinoma (PDAC) in the context of its association with chronic pancreatitis (CP). Human tissue of healthy pancreas, CP, and PDAC was screened for defensins by immunohistochemistry. Defensin α 1 (human neutrophil peptide 1 [HNP-1]) expression was validated using mass spectrometry and microarray analysis. Human neutrophil peptide 1 expression and influences of proinflammatory cytokines (tumor necrosis factor α, interleukin 1β, and interferon γ) were studied in human pancreatic cancer cells (Colo 357, T3M4, PANC-1) and normal human pancreatic duct epithelial cells (HPDE). Accumulation of HNP-1 in malignant pancreatic ductal epithelia was seen. Spectrometry showed increased expression of HNP-1 in CP and even more in PDAC. At RNA level, no significant regulation was found. In cancer cells, HNP-1 expression was significantly higher than in HPDE. Proinflammatory cytokines significantly led to increased HNP-1 levels in culture supernatants and decreased levels in lysates of cancer cells. In HPDE cytokines significantly decreased HNP-1 levels. Inflammatory regulation of HNP-1 in PDAC tissue and cells indicates that HNP-1 may be a link between chronic inflammation and malignant transformation in the pancreas.

Expression of G protein‐coupled receptors in pancreatic cancer cells

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal forms of cancer and has an overall 5‐year survival rate ~7%. PDAC has poor response to chemotherapy and radiotherapy; thus new treatments for PDAC are an important, unmet medical need. G protein‐coupled receptor (GPCR) regulate many biological functions, including in cancer cells, but GPCR expression and the role of GPCRs in PDAC cells is unclear. We generated two primary PDAC cell lines (79E and 34E) and an immortalized normal pancreatic ductal epithelial cell line (HPDE6). Hypothesizing that GPCRs may regulate PDAC cells, we used Taqman GPCR array to assess GPCR expression in four PDAC cell lines, 79E, 34E, Miapaca, and AsPC. We identified 68 commonly expressed GPCRs in all four PDAC cell lines, among which 22 GPCRs were significantly up‐regulated (at least 2‐fold) in PDAC cells compared to normal control cell HPDE6; 13 GPCRs were significantly down‐regulated and 3 GPCRs were uniquely expressed in PDACs. We further tested the 13 most up‐regulated GPCRs using real time qPCR and confirmed that LPHN1, CHRM4, FZD4, HRH1 and CD97 have much higher expression in PDACs compared to HPED6, suggesting that these GPCRs might contribute to the malignant phenotype and perhaps could be novel treatment targets for pancreatic cancer, in particular for PDAC.Support or Funding InformationThis work is supported by NIH/NCI Research Grants R21 CA189477, R01 CA155620, and Padres Pedal the Cause PTC2017.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

HRH1: A Novel GPCR Drug Target in Pancreatic Cancer

Patients with pancreatic cancer, in particular pancreatic ductal adenocarcinoma (PDAC), have a ~6% chance of surviving 5 years after diagnosis. Approved drugs only add weeks or months to patient lifespan. PDAC is difficult to treat, in part because the tumor often has poor vascularization and dense extracellular matrix, which yield a hypoxic tumor microenvironment. Finding new drugs for PDAC is thus an unmet medical need. The G protein‐coupled receptor (GPCR) superfamily is the most frequently targeted class of proteins for FDA‐approved drugs. We hypothesized that one or more GPCR may be a novel therapeutic target for PDAC.Bioinformatic analysis of PDAC tumors in TCGA [The Cancer Genome Atlas] revealed prominent (~32‐fold) overexpression of H1 histamine receptor (HRH1) compared to normal pancreatic tissue [GTex database]. KRAS and p53 mutations occur, respectively, in ~70% and ~60% of PDAC tumors whereas increased HRH1 expression of HRH1 in 100% of tumor samples in TCGA. HRH1 is an attractive target for repurposing since many FDA approved antagonists are directed at this receptor. We thus sought to validate HRH1 expression in PDAC cells.Using qPCR, we found high HRH1 expression and low expression of HRH2‐4 in BxPC, AsPC‐1, and CAPAN‐2 (PDAC) cell lines and normal pancreatic ductal epithelial cells. Addition of histamine increased Ca++ in ASPC‐1 and CAPAN‐2 cells; this increase in Ca++ was blocked by multiple FDA‐approved HRH1 antihistamines. Histamine increased PDAC cell proliferation in cells grown in hypoxia but not normoxia. High expression of HRH1 in PDAC cells may thus confer a growth advantage for PDAC cells in hypoxic conditions. These preliminary data suggest that FDA‐approved HRH1 antihistamines are candidates for repurposing as novel therapeutics for the treatment of pancreatic cancer.Support or Funding InformationSupported by HHMI and an ASPET David Lehr AwardThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

UCA1 Regulates the Growth and Metastasis of Pancreatic Cancer by Sponging miR-135a

Pancreatic cancer (PC) is a devastating malignant disease with a poor prognosis. This study aimed to investigate the role of urothelial carcinoma associated 1 (UCA1) in the progression of PC. Our results revealed that long noncoding RNA (lncRNA) UCA1 was overexpressed in PC tissues compared with adjacent histologically normal tissues. A downregulated level of UCA1 was also detected in five human PC cell lines (SW1990, BxPC-3, MiaPaCa-2, PANC-1, and CAPAN-1) compared with normal pancreatic duct epithelial HPDE cells. The proliferation of PC cells was inhibited after UCA1 was suppressed by a lentiviral vector. The cell apoptosis rate was largely promoted by downregulating UCA1. Further research revealed that microRNA (miRNA)-135a is a direct target of UCA1. The expression of miR-135a was decreased in PC tissues and cell lines compared with control groups. In addition, the decreased level of miR-135a was elevated by adding miR-135a mimic in SW1990 cells transfected with lncRNA UCA1. Similarly, an upregulated level of miR-135a was downregulated by adding miR-135a inhibitor in SW1990 cells transfected with UCA1 siRNA. Luciferase activity assay further confirmed the targeting relationship between UCA1 and miR-135a. Moreover, miR-135a reversed the effect of UCA1 on cell apoptosis rate and cell viability in SW1990 cells. The migration and invasion capacities of PC cells were suppressed by UCA1. siRNA was then enhanced by the miR-135a inhibitor. In vivo, UCA1 siRNA effectively suppressed tumor growth and the expression of migration markers. Taken together, our research revealed that UCA1 works as an oncogene by targeting miR-135a. The UCA1–miR-135a pathway regulated the growth and metastasis of PC, providing new insight in the treatment of PC.

Exportin 1 (XPO1) inhibition leads to restoration of tumor suppressor miR-145 and consequent suppression of pancreatic cancer cell proliferation and migration.

Pancreatic ductal adenocarcinoma (PDAC) is the third leading cause of cancer related deaths in the United States with a majority of these patients dying from aggressively invasive and metastatic disease. There is growing evidence that suggests an important role for microRNAs (miRNAs) in the pathobiology of aggressive PDAC. In this study, we found that the expression of miR-145 was significantly lower in PDAC cells when compared to normal pancreatic duct epithelial cells. Here we show that inhibition of the nuclear exporter protein exportin 1 (XPO1; also known as chromosome maintenance region 1 [CRM1]) by siRNA knockdown or by the Selective Inhibitor of Nuclear Export (SINE) compound (KPT-330; selinexor) increases miR-145 expression in PDAC cells resulting in the decreased cell proliferation and migration capacities. A similar result was obtained with forced expression of miR-145 in PDAC cells. To this end, SINE compound treatment mediated the down-regulation of known miR-145 targets genes including EGFR, MMP1, MT-MMP, c-Myc, Pak4 and Sox-2. In addition, selinexor induced the expression of two important tumor suppressive miRNAs miR-34c and let-7d leading to the up-regulation of p21WAF1. These results are the first to report that targeted inhibition of the nuclear export machinery could restore tumor suppressive miRNAs in PDAC that warrants further clinical investigations.

Abstract 1139: G protein-coupled receptors (GPCRs): unrecognized potential therapeutic targets in cancer

Abstract G protein-coupled receptors (GPCRs) are the largest class of signaling receptors in humans and other species and in addition, the most widely targeted class for FDA-approved therapeutics. However, GPCRs are not commonly targeted in cancers other than endocrine tumors. We hypothesize that GPCRs may have been “missed” as targets in cancer, perhaps in part because they have not been frequently identified as driver mutations; however, GPCRs may have unappreciated utility as therapeutic targets in a variety of tumors. To test this hypothesis, we have used unbiased approaches (Taqman GPCR arrays and RNA-seq), mining of publicly available datasets (e.g., The Cancer Genome Atlas, TCGA), and validation studies (e.g., protein and functional analyses) to assess GPCRs in a variety of human tumors, cancer cells and stromal cells (cancer-associated fibroblasts, CAFs). A major focus of our efforts has been on pancreatic ductal adenocarcinoma (PDAC) cells/tumors and pancreatic CAFs (PCAFs). We find that PDAC cells and PCAFs express &amp;gt;70 different GPCRs, including many orphan GPCRs (receptors without known physiologic agonists) and that numerous GPCRs are expressed at much higher levels than in normal precursor cells (pancreatic ductal epithelial cells for PDAC cells and pancreatic stellate cells and pancreatic fibroblasts for PCAFs). We find that a cluster of GPCRs is overexpressed in PDAC tumors. Two such receptors are orphan GPCRs, Orphan 1 and Orphan A, neither of which is mutated but each has high expression, respectively, in PCAFs and PDAC cells. Orphan 1 and Orphan A have at least 2-fold higher expression in &amp;gt;90% of PDAC tumors in TCGA compared to that in normal pancreatic tissue (compiled from the GTEX database). Higher expression of orphan A is associated with decreased survival and remission, while Orphan 1 expression correlates with that of numerous fibrotic genes. Importantly, both Orphan 1 and Orphan A are functional and appear to contribute to the malignant phenotype. For example, siRNA knockdown of Orphan 1 in PCAFs blunts production of profibrotic markers and decreases ability of conditioned media from PCAFs to enhance proliferation of PDAC cells while transfection of Orphan A increases DNA synthesis of normal pancreatic ductal epithelial cells. Taken together, these and other results suggest that in addition to their role in endocrine tumors, GPCRs represent previously unrecognized contributors to cancer through actions on tumor cells and stromal cells. These data suggest the possibility that highly expressed GPCRs (such as Orphan A) may function as oncogenes. Thus, GPCRs may be druggable, novel targets for the treatment of cancer, including pancreatic cancer, a tumor for which new therapeutics are an important, unmet need. Note: This abstract was not presented at the meeting. Citation Format: Paul A. Insel, Krishna Sriram, Shu Z. Wiley, Thalia McCann, Randall P. French, Andrew M. Lowy. G protein-coupled receptors (GPCRs): unrecognized potential therapeutic targets in cancer [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 1139. doi:10.1158/1538-7445.AM2017-1139