Target For Pancreatic Ductal Adenocarcinoma Research Articles

LINC00346 promotes pancreatic cancer progression through the CTCF-mediated Myc transcription.

Although multiple factors are known to contribute to pancreatic ductal adenocarcinoma (PDAC) progression, the role of long non-coding RNAs (lncRNAs) in PDAC remains largely unknown. In this study, we present data that long intergenic non-coding RNA 346 (LINC00346) functions as a promoting factor for PDAC development. We first show that LINC00346 is highly expressed in pancreatic tumor specimens as compared to normal pancreatic tissue based on interrogation of The Cancer Genome Atlas (TCGA) pancreatic adenocarcinoma dataset. Of significance, this upregulation of LINC00346 is associated with overall survival (OS) and disease-free survival (DFS), respectively. We further show that knockout (KO) of LINC00346 impairs pancreatic cancer cell proliferation, tumorigenesis, migration, and invasion ability. Importantly, these phenotypes can be restored by LINC00346 re-expression in KO cells (i.e., rescue experiment). RNA precipitation assays combined with mass spectrometry analysis indicate that LINC00346 interacts with CCCTC-binding factor (CTCF), a known transcriptional repressor of c-Myc. This interaction between LINC00346 and CTCF prevents the binding of CTCF to c-Myc promoter, relieving the CTCF-mediated repression of c-Myc. Thus, LINC00346 functions as a positive transcriptional regulator of c-Myc. Together, these results suggest that LINC00346 contributes to PDAC pathogenesis by activating c-Myc, and as such, LINC00346 may serve as a potential biomarker and therapeutic target for PDAC.

Identification of novel genes associated with a poor prognosis in pancreatic ductal adenocarcinoma via a bioinformatics analysis.

Pancreatic ductal adenocarcinoma (PDAC) is a class of the commonest malignant carcinomas. The present study aimed to elucidate the potential biomarker and prognostic targets in PDAC. The array data of GSE41368, GSE43795, GSE55643, and GSE41369 were downloaded from Gene Expression Omnibus (GEO) database. The differentially expressed genes (DEGs) and differentially expressed microRNAs (DEmiRNAs) in PDAC were obtained by using GEO2R, and overlapped DEGs were acquired with Venn Diagrams. Functional enrichment analysis of overlapped DEGs and DEmiRNAs was conducted with Metascape and FunRich, respectively. The protein–protein interaction (PPI) network of overlapped DEGs was constructed by STRING and visualized with Cytoscape. Overall survival (OS) of DEmiRNAs and hub genes were investigated by Kaplan–Meier (KM) plotter (KM plotter). Transcriptional data and correlation analyses among hub genes were verified through GEPIA and Human Protein Atlas (HPA). Additionally, miRNA targets were searched using miRTarBase, then miRNA–DEG regulatory network was visualized with Cytoscape. A total of 32 DEmiRNAs and 150 overlapped DEGs were identified, and Metascape showed that DEGs were significantly enriched in cellular chemical homeostasis and pathways in cancer, while DEmiRNAs were mainly enriched in signal transduction and Glypican pathway. Moreover, seven hub genes with a high degree, namely, V-myc avian myelocytomatosis viral oncogene homolog (MYC), solute carrier family 2 member 1 (SLC2A1), PKM, plasminogen activator, urokinase (PLAU), peroxisome proliferator activated receptor γ (PPARG), MET proto-oncogene, receptor tyrosine kinase (MET), and integrin subunit α 3 (ITGA3), were identified and found to be up-regulated between PDAC and normal tissues. miR-135b, miR-221, miR-21, miR-27a, miR-199b-5p, miR-143, miR-196a, miR-655, miR-455-3p, miR-744 and hub genes predicted poor OS of PDAC. An integrative bioinformatics analysis identified several hub genes that may serve as potential biomarkers or targets for early diagnosis and precision target treatment of PDAC.

The integrin αvβ6 drives pancreatic cancer through diverse mechanisms and represents an effective target for therapy.

Pancreatic ductal adenocarcinoma (PDAC) has a 5‐year survival rate of less than 4% and desperately needs novel effective therapeutics. Integrin αvβ6 has been linked with poor prognosis in cancer but its potential as a target in PDAC remains unclear. We report that transcriptional expression analysis revealed that high levels of β6 mRNA correlated strongly with significantly poorer survival (n = 491 cases, p = 3.17 × 10−8). In two separate cohorts, we showed that over 80% of PDACs expressed αvβ6 protein and that paired metastases retained αvβ6 expression. In vitro, integrin αvβ6 promoted PDAC cell growth, survival, migration, and invasion. Treatment of both αvβ6‐positive human PDAC xenografts and transgenic mice bearing αvβ6‐positive PDAC with the αvβ6 blocking antibody 264RAD, combined with gemcitabine, significantly reduced tumour growth (p < 0.0001) and increased survival (log‐rank test, p < 0.05). Antibody therapy was associated with suppression of tumour cell activity (suppression of pErk growth signals, increased apoptosis seen as activated caspase‐3) and suppression of the pro‐tumourigenic microenvironment (suppression of TGFβ signalling, fewer αSMA‐positive myofibroblasts, decreased blood vessel density). These data show that αvβ6 promotes PDAC growth through both tumour cell and tumour microenvironment mechanisms and represents a valuable target for PDAC therapy. © 2019 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Abstract 4935: Identification of GPR115 as a key G-protein coupled receptor as a key prognostic and therapeutic target in pancreatic ductal adenocarcinoma

Abstract Purpose: Pancreatic ductal adenocarcinoma (PDAC) remains the fourth leading cause of cancer-related deaths in the United States, and the incidence of this disease continues to rise. Although recent advances in treatment modalities for this malignancy has somewhat improved patient outcomes in the recent years, the overall prognosis of patients with PDAC remains dismal. From a biological viewpoint, while the G-protein-coupled receptor (GPCRs) proteins play a multitude of important functions in tumor pathogenesis, their clinical significance if any in human cancers, especially PDAC, remains unclear. Herein we aimed to perform a comprehensive profiling of all GPCRs, in an attempt to identify the most critical protein(s) that have important biological and clinical significance in PDAC. Methods: This study included systematic analysis of 796 PDAC patients, which included data analysis from 331 patients from public datasets (TCGA, ICGC and GSE57495) and 465 patients from two, independent, clinical patient cohorts. Computational and bioinformatic analysis of genomewide expression profiling datasets led to the selection of candidate GPCR genes during the discovery and validation steps. The clinical significance of candidate genes were subsequently investigated in two in-house patient cohorts (training cohort: n = 321, validation cohort: n = 144) using qRT-PCR assays. The data were analyzed by performing appropriate statistical analyses to determine patient prognosis. Finally, using a series of functional studies, we confirmed the biological function of the candidate GPCRs in PDAC. Results: Analysis of all 33 GPCRs, led to the identification of GPR115, as the only significantly up-regulated candidate, with a prognostic significance in all three public datasets (P=0.02, 0.04 and 0.05, respectively). The patients with high-GPR115 expression exhibited significantly poorer postoperative prognosis for OS and RFS, in two, large, independent clinical cohorts (training cohort: P&amp;lt;0.01, P&amp;lt;0.01, validation cohort: P=0.01 and 0.02, respectively). More importantly, multivariate analysis indicated that high GPR115 expression was an independent prognostic factor in both cohorts (HR=1.45; P=0.01, HR=2.25; P&amp;lt;0.01). Using Cox regression, we established a risk-prediction model by incorporating GPR115 expression together with various clinicopathological factors, which was highly accurate in predicting 5-year long-term survival following surgery in PDAC patients. In addition, siRNA-mediated gene silencing of GPR115 significantly inhibited the cell proliferation and migration in human PDAC cells. Conclusion: We for the first time have identified GPR115 as a key GPCR with important prognostic significance, as well as functional role in tumor progression; providing a rationale that this may be a potential target for therapeutic targeting in patients with PDAC. Citation Format: Satoshi Nishiwada, Tadanobu Shimura, Kensuke Yamamura, Fuminori Sonohara, Takahiro Akahori, Kota Nakamura, Naoya Ikeda, Yasuhiro Kodera, Masayuki Sho, Ajay Goel. Identification of GPR115 as a key G-protein coupled receptor as a key prognostic and therapeutic target in pancreatic ductal adenocarcinoma [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 4935.

Abstract 3039: Targeting carcinoembryonic antigen cell adhesion molecule 6 (CEACAM6) reshapes the tumor-stroma in pancreatic ductal adenocarcinoma (PDA)

Abstract Background: CEACAM6, a cell adhesion receptor of the Ig-like superfamily, interacts with other CEACAMs (1, 5, 7 &amp; 8) and is over-expressed in human cancers including PDA. Over-expression of CEACAM6 causes resistance to anoikis - regulated apoptosis induced by inadequate or inappropriate cell-matrix interactions, promoting a TGFβ-mediated invasive phenotype. CEACAM6 as a predictive biomarker has not been fully investigated with regard to its potential as a candidate therapeutic target in PDA. We report a detailed analysis of CEACAM6 by expression profiling in several pancreatic tumor types, stromal cells, cell lines and mechanistic studies supporting CEACAM6 as a therapeutic target, when inhibited impacts several hallmarks key to PDA pathogenesis. Methods: RNAseq, expression arrays and clinical data was analyzed from The Cancer Genome Atlas (TCGA), International Cancer Genome Consortium (ICGC) and GEO using R (v3.4.3). Expression profiles were analyzed across PDA subtypes, stroma types and overall survival. CRISPR/Cas9 knockout (KO) of CEACAM6 in HPAF-II cells was investigated by quantitative proteomics and genomics with corroborative signaling pathway analysis. A therapeutic monoclonal antibody scFv-Fc (IgG) targeting CEACAM6 was tested in an orthotopic mouse PDA model. Results: CEACAM6 is over-expressed in ~90% of primary and metastatic PDA samples, ~10-20 fold higher vs. normal cells. Basal and classical subtypes and activated stroma over-express CEACAM6, compared to low or normal stroma. TCGA dataset shows that PDA patients with high CEACAM6 expression have a poor prognosis vs. low expression. In addition, CEACAM6 over-expression is associated with mutant KRAS and immune suppression elaborated by low cytolytic T-cell activity. Correlation studies show that CEACAM6 is associated with genes involved in cell junctions and membrane integrity. HPAF-II cells with CEACAM6 KO showed changes to cell adhesion-extracellular matrix, transmembrane activity, increase in immune signaling and changes to enzymes of mitochondrial activity with significantly decreased mitochondrial membrane potential and ATP production. In addition, CEACAM6 KO cells have a significantly decreased growth rate with reduced Src, Akt and Fak signaling. The scFv-Fc has anti-PDA activity as a single agent in an orthotopic mouse model. Conclusions: CEACAM6 over-expression is a stroma-associated poor prognostic marker and a novel therapeutic target in PDA. The scFv-Fc targeting CEACAM6 shows promising anti-PDA activity and is being developed to enhance anoikis and restore host anti-tumor immunity. Citation Format: Ritu Pandey, Muhan Zhou, Shariful Islam, Baowei Chen, Paul Langlais, Anup Srivastava, Larry S. Cooke, Eric Weterings, Daruka Mahadevan. Targeting carcinoembryonic antigen cell adhesion molecule 6 (CEACAM6) reshapes the tumor-stroma in pancreatic ductal adenocarcinoma (PDA) [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 3039.

Abstract LB-034: Myc instructs and maintains pancreatic adenocarcinoma phenotype

Abstract Pancreatic ductal adenocarcinoma (PDAC) sporadically develops from pre-cancerous pancreatic intraepithelial neoplasia (PanIN). It has a very poor prognosis and lacks effective therapies. Expression of the pleiotropic transcription factor Myc is deregulated in the majority of human cancers, including PDAC. Using a rapidly and reversibly switchable genetic mouse model to toggle Myc activity, we show that Myc drives immediate transition of indolent KRasG12D-induced PanINs into aggressive pancreatic adenocarcinomas that share all the signature features of spontaneous human PDAC. Myc induces a distinct set of instructive signals, some hitherto unknown, that together orchestrate coordinated changes in the multiple, distinct stromal and inflammatory cell types that generate the characteristic PDAC stroma. We also demonstrate that continued Myc activity is required for PDAC maintenance - Myc deactivation immediately triggers meticulous disassembly of PDAC, including its attendant stroma. Thus, both the formation and deconstruction of the complex PDAC phenotype can be mediated by a single, reversible molecular switch. We have also identified a previously unknown Myc-dependent GAS6-AXL pathway that is required for stromal changes characteristic of PDAC. Using the same pre-clinical mouse PDAC model, we show that TP-0903, a specific inhibitor of AXL kinase, profoundly blocks Myc-induced influx of neutrophils into PanINs and B220+ lymphoid cells into the PanIN periphery. This significantly retards Myc-driven stellate cell activation and proliferation and impedes PDAC growth. Hence, the GAS6-AXL-mediated signaling pathway is a promising therapeutic target for pancreatic ductal adenocarcinoma. TP-0903 is currently in a Phase I/Ib clinical study in in patients with solid tumors, including PDAC. Citation Format: Nicole M. Sodir, Roderik M. Kortlever, Valentin J.A. Barthet, Luca Pellegrinet, Tania Campos, Steven Kupczak, Laura Soucek, Mark J. Arends, Trevor D. Littlewood, Gerard I. Evan. Myc instructs and maintains pancreatic adenocarcinoma phenotype [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 LB-034.

Abstract 3614: Targeted 3DNA-siHuR nanocarrier therapy for pancreatic ductal adenocarcinoma

Abstract The poor overall survival rate of pancreatic ductal adenocarcinoma (PDAC), less than 10% over 5 years, is largely due to the majority of patients presenting with overt metastatic burden or post-surgical recurrence of micrometastases. Due to these factors, new treatment methods are needed to effectively target both primary and metastatic tumors. We have previously established that the mRNA binding protein Human antigen R (HuR), is an important molecule for PDAC progression. In a tumor microarray of matched normal and malignant patient tissues, we found active HuR staining in 79% of tumor samples, which was not seen in normal pancreatic tissue (n=70). We have shown that homozygous CRISPR knock-out of HuR results in xenograft lethality, as well as sensitization of PDAC cell lines to standard-of-care chemotherapy. Therefore, in the current experiments we have utilized a targeted 3DNA nanocarrier for the delivery of an RNA silencer against HuR in order to enhance tumor susceptibility to chemotherapy. Treatment of PDAC cell lines with this 3DNA nanocarrier was highly effective in knocking down both HuR and established HuR targets (DCK, IDH1, PIM1). To increase the specificity of the 3DNA nanocarrier, we sought to discover targeting moieties specifically upregulated in PDAC. We found increased folic acid receptor (FAR) expression in HS 766T, MIA PaCa-2, and PANC-1 PDAC cell lines with an average fold change of 2.6, in comparison to normal pancreatic cells. Additionally, patient tissue samples showed elevated FAR levels in 100% of tumors (n=80). Thus, by attaching folic acid to the 3DNA nanocarrier (FA-3DNA), we were able to target the nanocarrier specifically to tumors, both primary and metastatic. This was detected by tagging FA-3DNA with Alexa750 fluorophore and monitoring its movement in mice bearing luciferase expressing-MIA PaCa-2 tumors. FA-3DNA had a longer half-life and increased delivery to tumors, as compared to non-targeted 3DNA. We also demonstrated that treatment of mice with FA-3DNA conjugated with an RNA silencer for HuR (FA-3DNA-siHuR) led to a significant decrease in HuR mRNA and protein, in comparison to mice treated with 3DNA containing a control RNA silencer (FA-3DNA-siControl). Subsequent in vivo experiments showed prolong survival of mice treated with FA-3DNA-siHuR, as compared to mice treated with FA-3DNA-siControl (p=0.04). Our previous work has established HuR as a promising therapeutic target in PDAC. In both in vitro and in vivo models, we have shown FA-3DNA-siHuR is capable of targeting tumors directly for the inhibition of HuR. These results suggest treatment with FA-3DNA-siHuR will sensitize tumors to standard of care chemotherapy, which can be introduced systemically or incorporated into the 3DNA nanocarrier for direct delivery. The latter could potentially increase the efficiency of the chemotherapy, and also of critical importance, reduce the toxic adverse effects of systemic chemotherapy. Citation Format: Grace McCarthy, Christopher Schultz, Aditi Jain, Teena Dhir, Charles Yeo, Jessica Bowers, Lou Casta, Kelly Rhodes, Lori Getts, Robert Getts, Trevor Baybutt, Adam Snook, Jonathan Brody. Targeted 3DNA-siHuR nanocarrier therapy for pancreatic ductal adenocarcinoma [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 3614.

Abstract LB-100: HNF4G overexpression promotes PDAC progression and confers metformin sensitivity in patients with SMAD4-loss of function

Abstract BACKGROUND: Genome-wide association studies (GWAS) have identified several genetic regions associated with risk of pancreatic ductal adenocarcinoma (PDAC) while the underlying mechanisms remain largely unknown. This study investigated the functions of genes in these genetic regions and their roles as potential biomarkers or therapeutic targets for PDAC. METHODS: We reanalyzed our previous GWAS data based on referring the 108 risk loci reported in published PDAC GWAS to seek for all susceptibility genes in the Chinese population and found 44 potential risk genes. A siRNA library was used to silence these genes in PDAC cell lines and the malignant phenotypes were examined by using a high content screen method followed by a series of functional assays. RESULTS: Among the 44 genes, HNF4G was the top one who’s silencing significantly repressed PDAC cell migration and invasion. Functional assays showed that HNF4G regulates cell-cell junction pathway, which may be the underlying mechanism for its action. HNF4G was upregulated by SMAD4 loss of function and frequently overexpressed in PDACs. PDAC cell metastasis promoted by the overexpression of HNF4G could be suppressed by metformin, and this effect was seen only in PDAC cells with SMAD4 loss of function, resulting in heterogeneous vulnerability of PDAC to the drug. CONCLUSION: HNF4G overexpression might result in metformin sensitivity in PDAC with SMAD4 loss of function and thus represent as a potential therapeutic target. Citation Format: Chengcheng Wang, Chen Wu, Dongxin Lin, Yupei Zhao. HNF4G overexpression promotes PDAC progression and confers metformin sensitivity in patients with SMAD4-loss of function [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 LB-100.

Abstract 2847: Tenascin C expression in neural microenvironment enhances perineural invasion and is associated with locoregional recurrence-related poor prognosis in pancreatic ductal adenocarcinoma

Abstract Background: Perineural invasion (PNI) is commonly seen in pancreatic ductal adenocarcinoma (PDAC) and worsens the postoperative prognosis. However, the associated mechanisms in cancer progression remain unclear. Tenascin C (TNC), an extracellular matrix glycoprotein, is abundant in cancer stroma and modulates tumor progression. The aim of this study was to evaluate the functional roles of TNC in the neural microenvironment of PDAC. Methods: We immunohistochemically examined TNC expression in 78 resected PDAC specimens. TNC staining in perineural sites at the invasive front was classified as low or high intensity, by comparison with adjacent non-cancerous tissues in the same section. The relationships between TNC expression and clinicopathological features were retrospectively analyzed. Furthermore, interactions between cancer cells and nerves after supplementation with TNC were investigated when co-culturing a PDAC cell line with a mouse dorsal root ganglion (DRG) in vitro. Results: High perineural TNC expression at the invasive front, seen in 30 (38%) patients, was associated with PNI (p = 0.006), pathological T stage ≥ 3 (p = 0.01), and postoperative locoregional recurrence (p = 0.002). It was independently associated with postoperative, poor recurrence-free survival in multivariate analysis (p = 0.045). In the in vitro co-culture model, supplementation with TNC significantly enhanced both neurotropism of PDAC cells and tumor tropism of a DRG. On the other hand, when PDAC cells and a DRG were cultured separately, TNC did not affect cancer cell proliferation or neural outgrowth. Conclusions: Strong perineural TNC expression was a prognostic indicator of locoregional recurrence-related poor prognosis. The neurotropism of PDAC induced by TNC suggests that TNC could serve as a therapeutic target for PDAC. Citation Format: Satoru Furuhashi, Takanori Sakaguchi, Tomohiro Murakami, Mayu Fukushima, Yoshifumi Morita, Koji Ikegami, Hirotoshi Kikuchi, Megumi Baba, Mitsutoshi Setou, Hiyoya Takeuchi. Tenascin C expression in neural microenvironment enhances perineural invasion and is associated with locoregional recurrence-related poor prognosis in pancreatic ductal adenocarcinoma [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 2847.

Abstract 951: Setdb1 deletion protects against formation of pancreatic ductal adenocarcinoma through inducing apoptosis and upregulating p53 expression

Abstract One group of epigenetic regulators known to be altered in cancer development is histone modification. Setdb1, a histone methyltransferase that trimethylates histone H3 on lysine 9 (H3K9), has been implicated in tumorigenesis by altering the expression of oncogenes/ tumor suppressor genes. In this study, we aimed to elucidate the functional role of Setdb1 in pancreatic acinar regeneration and formation of pancreatic ductal adenocarcinoma (PDAC). Setdb1 was expressed in pancreatic ductal cells and a small subset of pancreatic acinar cells in adult wild type mice. Upon caerulein-induced pancreatitis, Setdb1 was expressed in acinar cells and acinar to ductal metaplasia (ADM). To determine the impact of pancreatic Setdb1 deletion on acinar regeneration, we generated Ptf1a-Cre; Setdb1f/f (CS) mice and induced caerulein-pancreatitis. Pancreatic deletion of Setdb1 resulted in pancreatic atrophy concomitant with increased apoptosis of acinar cells and increased expression of p53 in CS mice after caerulein-induced pancreatitis. In addition, pancreatic acinar regeneration was delayed with persistent inflammation inCS mice. To investigate the impact of pancreatic Setdb1 deletion on PDAC formation, we next generated Ptf1a-Cre; KrasG12D; Setdb1f/f (KCS) mice and compared to control Ptf1a-Cre; KrasG12D (KC) mice. Pancreatic deletion of Setdb1 dramatically accelerated spontaneous development of ADM and pancreatic intraepithelial neoplasia (PanIN), the most common precursor lesion of PDAC, in KCS mice compared to KC mice at the age of 4 weeks. Acinar cell culture experiments revealed that Setdb1 null acinar cells converted to ADM, at least in part, in a cell autonomous manner. Long term observation revealed massive pancreatic atrophy in KCS mice. Immunostaining revealed a dramatic increase in apoptotic cells and upregulated expression of p53 in KCS mice compared to KC mice at the age of 20 weeks. Consistently, microarray analysis revealed upregulated expression of p53 and apoptotic pathway genes in KCS mice compared to KC mice. We next examined the impact of pancreatic Setdb1 deletion on PDAC formation in the context of p53 deletion. Surprisingly, PDAC formation was suppressed in KCS mice in the context of heterozygous p53 deletion (KPhereroCS mice). Again, apoptosis was increased and p53 expression was upregulated in KPhereroCS mice. In contrast, in the context of homozygous p53 deletion, PDAC was formed and increased apoptosis was canceled in KPhomoCS mice. These findings suggested that Setdb1 deletion protects against PDAC formation through inducing apoptosis, which is mediated by p53. In conclusion, Setdb1 is required for normal pancreatic acinar regeneration and protects against PDAC formation though inducing apoptosis, which is mediated by p53. Thus, Setdb1 could be a therapeutic target for PDAC which retains wild p53 expression. Citation Format: Satoshi Ogawa, Akihisa Fukuda, Motoyuki Tsuda, Tomonori Masuda, Makoto Sono, Yuichi Fukunaga, Takaaki Yoshikawa, Osamu Araki, Munemasa Nagao, Norihiro Goto, Yukiko Hiramatsu, Takahisa Maruno, Yoichi Shinkai, Hiroshi Seno. Setdb1 deletion protects against formation of pancreatic ductal adenocarcinoma through inducing apoptosis and upregulating p53 expression [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 951.

Abstract 2541: Mastl regulates EGFR signaling to promote pancreatic cancer progression

Abstract Background: Anti-cancer treatments act primarily by damaging the DNA of cancer cells. Upon DNA damage, cells stop proliferation at cell cycle checkpoints, which provides them time for DNA repair. Hence, pharmacological inhibition of checkpoint kinases in combination with the DNA damaging anti-cancer therapies is now emerging as promising cancer treatment strategy. In this regard, pancreatic ductal adenocarcinoma (PDAC) remains one of the most lethal malignancies with a frightening resistance to chemotherapeutic and targeted approaches. Here, along with the KRAS-dependent signaling, receptor tyrosine kinases especially the EGF receptor (EGFR) signaling is strongly upregulated in PDAC. Importantly, Greatwall (Mastl; Microtubule-associated serine/threonine-protein kinase-like) promotes normal G2-mitosis transition and is highly upregulated in multiple cancer types, and have been demonstrated to associate with therapy resistance. However, potential role of Mastl in pancreatic cancer progression/therapy resistance remains unknown. Our central hypothesis is that MASTL expression is (a) involved in cancer progression and intrinsic drug resistance (b) constitutes a potential therapeutic target for PDAC (c) correlates with EGFR expression and regulation. Methods: We used immunoblotting, immunohistochemistry and TCGA database analysis to examine Mastl expression, its association with EGFR in pancreatic cancer progression, and patient survival. Genetic and pharmacological manipulations for Mastl expression were performed and effects on proliferation, apoptosis and cell cycle were determined. Results: A robust increase in Mastl expression characterized pancreatic cancer cells when compared with the non-transformed pancreatic cells. A similar upregulated expression of Mastl was found in samples originating from KPC mouse model of PDAC. Analysis of the human pancreatic cancer samples and the TCGA database strongly supported this outcome and suggested a positive association of the Mastl expression with cancer progression and patient mortality. Interestingly, genetic inhibition of Mastl expression in pancreatic cancer cells not only inhibited the ability of these cells to proliferate and invade through the matrix but also inhibited activation of the ErbB-family of tyrosine kinase receptors. Further studies revealed that overexpressing K-ras/mutant p53 pathway in HPNE cells (untransformed pancreatic cells) modulates Mastl expression, and thus suggested a causal correlation of Mastl with K-Ras/EGFR signaling in pancreatic cancer cells. Conclusion: Our results suggest a cross talk of Mastl with K-ras/EGFR signaling pathway in pancreatic cancer progression. We hypothesize that combinatorial therapy targeting Mastl along with Gemcitabine would overcome the drug resistance in the PDAC. Thus, this study identifies a novel approach for the treatment of tumors resistant to traditional EGFR inhibitors. Citation Format: Iram Fatima, Shailender S. Chauhan, Srijayaprakash Babu Uppada, Geoffrey A. Talmon, Amar B. Singh, Surinder K. Batra, Punita Dhawan. Mastl regulates EGFR signaling to promote pancreatic cancer progression [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 2541.

Abstract 4257: Integrated network analysis reveals potentially novel molecular pathways mechanism and therapeutic targets of pancreatic ductal adenocarcinoma

Abstract Background: Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal human cancers and shows insensitivity towards many chemotherapeutic drugs and target-based drugs. There is an urgently need to identify PDAC disease mechanism and detect novelty drug targets. With the large availability of protein interaction networks and microarray data supported, to identify the disease essential genes that have biological significance for potential drug targets is a challenge issue still. Methods: In this study, protein interaction network PathPPI, DrugBank targets and 374 transcriptome profiles from Gene Expression Omnibus (GEO) by Affymetrix HU 133 Plus 2.0 array test are collected. These microarray data includes 140 PDAC, 92 pancreatic cancer cell-line, 58 human normal pancreas tissues, and 72 patient original xenograft mouse. Based on these datasets, a novelty-integrated algorithm was developed for drug target prioritization by perturbed gene expression and network information. The integration network included data driven reconstruction network of gene regulatory (GRN) by Bayesian Network and protein interaction PathPPI network. The perturbed gene expression was refer to the differential expression genes of tumor verse adjacent normal. A novel hybrid method based on genetic algorithm (GA) is proposed to search the attractor with the maximum network perturbation for candidate drug targets in PDAC.These targets’ molecular mechanism was revealed further by network structure comparison of PDAC that is consistent with xerograph mouse model and cancer cells. Results: Highly accordance 56 genes with high perturbation score both in mRNAs and protein-protein network were identified and recommended as candidate drug targets for PDAC, including 16 novel targets, such as PSMD2, EPOR and PSMB9. Fifty-four essential genes shown strong concordance among tumor-model, patient drive xenograft mouse and pancreatic cancer cell-line by systematically comparison. We identified 1375 dysregulated genes that enriched in 25 pathways in PDAC by Gene Set Enrichment Analysis, among which 244 genes played as hubs in reconstructed PDAC regulatory network. Conclusion: In the study, we developed a global optimization-based inference of network perturbation to detect attractor for drug-target identification in PDAC tumors. The assembling Bayesian Network-based approach with protein-protein interaction provides a comprehensive information to observe energy transfer of gene perturbation in network to detect global optimum attractor for drug target selection. Citation Format: Lijun Cheng, Enze Liu, Li Lang, Xiaolin Cheng, Xiaotian Kong, Korc Murray. Integrated network analysis reveals potentially novel molecular pathways mechanism and therapeutic targets of pancreatic ductal adenocarcinoma [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 4257.

Targeting PIN1 exerts potent antitumor activity in pancreatic ductal carcinoma via inhibiting tumor metastasis.

The human prolyl isomerase PIN1, best known for its association with carcinogenesis, has recently been indicated in the disease of pancreatic ductal adenocarcinoma (PDAC). However, the functions of PIN1 and the feasibility of targeting PIN1 in PDAC remain elusive. For this purpose, we examined the expression of PIN1 in cancer, related paracarcinoma and metastatic cancer tissues by immunohistochemistry and analyzed the associations with the pathogenesis of PDAC in 173 patients. The functional roles of PIN1 in PDAC were explored in vitro and in vivo using both genetic and chemical PIN1 inhibition. We showed that PIN1 was upregulated in pancreatic cancer and metastatic tissues. High PIN1 expression is significantly association with poor clinicopathological features and shorter overall survival and disease‐free survival. Further stratified analysis showed that PIN1 phenotypes refined prognostication in PDAC. Inhibition of PIN1 expression with RNA interference or with all trans retinoic acid decreased not only the growth but also the migration and invasion of PDAC cells through regulating the key molecules of multiple cancer‐driving pathways, simultaneously resulting in cell cycle arrest and mesenchymal‐epithelial transition in vitro. Furthermore, genetic and chemical PIN1 ablation showed dramatic inhibition of the tumorigenesis and metastatic spread and then reduced the tumor burden in vivo. We provided further evidence for the use of PIN1 as a promising therapeutic target in PDAC. Genetic and chemical PIN1 ablation exerted potent antitumor effects through blocking multiple cancer‐driving pathways in PDAC. More potent and specific PIN1 targeted inhibitors could be exploited to treat this aggressive cancer.

Myoferlin Contributes to the Metastatic Phenotype of Pancreatic Cancer Cells by Enhancing Their Migratory Capacity through the Control of Oxidative Phosphorylation.

Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest malignancies with an overall survival of 5% and is the second cause of death by cancer, mainly linked to its high metastatic aggressiveness. Accordingly, understanding the mechanisms sustaining the PDAC metastatic phenotype remains a priority. In this study, we generated and used a murine in vivo model to select clones from the human Panc-1 PDAC cell line that exhibit a high propensity to seed and metastasize into the liver. We showed that myoferlin, a protein previously reported to be overexpressed in PDAC, is significantly involved in the migratory abilities of the selected cells. We first report that highly metastatic Panc-1 clones expressed a significantly higher myoferlin level than the corresponding low metastatic ones. Using scratch wound and Boyden’s chamber assays, we show that cells expressing a high myoferlin level have higher migratory potential than cells characterized by a low myoferlin abundance. Moreover, we demonstrate that myoferlin silencing leads to a migration decrease associated with a reduction of mitochondrial respiration. Since mitochondrial oxidative phosphorylation has been shown to be implicated in the tumor progression and dissemination, our data identify myoferlin as a valid potential therapeutic target in PDAC.

SF3B1 as a novel therapeutic target in pancreatic ductal adenocarcinoma: antitumoral effects of the splicing inhibitor Pladienolide B

SF3B1 as a novel therapeutic target in pancreatic ductal adenocarcinoma: antitumoral effects of the splicing inhibitor Pladienolide B

Multifunctional role of pancreatic stellate cells in pancreatic cancer

Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer related deaths with a dismal 5-year survival rate of only 8%. PDAC is characterised by extensive desmoplasia constituting about 50–80% of the tumour volume. Activated pancreatic stellate cells (PSC) are the major cellular source for stromal collagen; these cells drive pancreatic fibrosis and progression of PDAC. PSC are known to be activated by paracrine signals from several sources including injured epithelium, cancer cells, extracellular matrix, immune cells and nerve cells. Stromal-tumour interactions are now recognised as key processes in the development and progression of PDAC. Improved understanding of the mechanisms underlying stromal-tumour interactions may be the key for the discovery of new therapeutic targets in PDAC. This review summarises current knowledge regarding the role of PSCs in cancer biology and discusses the potential for development of novel therapeutic approaches targeting factors such as dysregulated signalling pathways, the stromal reaction itself and epigenetic changes in stromal and/or cancer cells.

Identification of MRP4/ABCC4 as a Target for Reducing the Proliferation of Pancreatic Ductal Adenocarcinoma Cells by Modulating the cAMP Efflux.

Pancreatic cancer is one of the most lethal types of tumors with no effective therapy available; is currently the third leading cause of cancer in developed countries; and is predicted to become the second deadliest cancer in the United States by 2030. Due to the marginal benefits of current standard chemotherapy, the identification of new therapeutic targets is greatly required. Considering that cAMP pathway is commonly activated in pancreatic ductal adenocarcinoma (PDAC) and its premalignant lesions, we aim to investigate the multidrug resistance-associated protein 4 (MRP4)-dependent cAMP extrusion process as a cause of increased cell proliferation in human PDAC cell lines. Our results from in silico analysis indicate that MRP4 expression may influence PDAC patient outcome; thus, high MRP4 levels could be indicators of poor survival. In addition, we performed in vitro experiments and identified an association between higher MRP4 expression levels and more undifferentiated and malignant models of PDAC and cAMP extrusion capacity. We studied the antiproliferative effect and the overall cAMP response of three MRP4 inhibitors, probenecid, MK571, and ceefourin-1 in PDAC in vitro models. Moreover, MRP4-specific silencing in PANC-1 cells reduced cell proliferation (P < 0.05), whereas MRP4 overexpression in BxPC-3 cells significantly incremented their growth rate in culture (P < 0.05). MRP4 pharmacological inhibition or silencing abrogated cell proliferation through the activation of the cAMP/Epac/Rap1 signaling pathway. Also, extracellular cAMP reverted the antiproliferative effect of MRP4 blockade. Our data highlight the MRP4-dependent cAMP extrusion process as a key participant in cell proliferation, indicating that MRP4 could be an exploitable therapeutic target for PDAC. SIGNIFICANCE STATEMENT: ABCC4/MRP4 is the main transporter responsible for cAMP efflux. In this work, we show that MRP4 expression may influence PDAC patient outcome and identify an association between higher MRP4 expression levels and more undifferentiated and malignant in vitro models of PDAC. Findings prove the involvement of MRP4 in PDAC cell proliferation through a novel extracellular cAMP mitogenic pathway and further support MRP4 inhibition as a promising therapeutic strategy for PDAC treatment.

Fetal bovine serum enlarges the size of human pancreatic cancer spheres accompanied by an increase in the expression of cancer stem cell markers

Pancreatic ductal adenocarcinoma (PDAC) is a major histological type of pancreatic cancer and remains one of the most lethal cancers with a high mortality rate owing to its aggressive growth, high metastatic rate, and recurrence. Recent studies on cancer stem cells (CSCs) have suggested that the aggressive growth, high metastatic rate, and recurrence might be caused by the ability of CSCs to self-renew, differentiate, and drive tumorigenesis. Thus, CSCs are expected to be a therapeutic target for PDAC. Sphere forming assay of cancer cells, including PDAC cells, is commonly performed using epidermal growth factor and fibroblast growth factor-2 containing serum-free medium to identify and isolate the enriched CSCs. Recently, we observed that PDAC spheres cultured in fetal bovine serum containing medium are morphologically similar to spheres cultured in the growth factor containing medium. In this study, we cultured two PDAC cell lines, PANC-1 and PK-1, in growth factor containing serum-free medium or serum containing medium, and compared the morphology of the spheres formed in detail by electron microscopy and examined the expression of major CSC marker genes. Both the PDAC cells formed larger spheres in the serum containing medium than in the growth factor containing medium. PK-1 cells formed more morphologically differentiated spheres, with peripheral flat lining cells, in the serum containing medium. Expression levels of most of the CSC markers were higher in the spheres of the two PDAC cells in both the culture mediums than in the cells cultured under adherent conditions. The expression levels of CSC markers in PDAC spheres cultured in the growth factor containing medium were not necessarily higher than that in the spheres cultured in the serum containing medium. These findings suggest that sphere forming assay using serum containing medium, by which large PDAC spheres with enriched CSCs are formed, may be useful for deciphering the characteristics of CSCs and for developing anti-CSC therapies for PDAC.

Protein tyrosine phosphatases: promising targets in pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is the most common type of pancreatic cancer. It is the fourth leading cause of cancer-related death and is associated with a very poor prognosis. KRAS driver mutations occur in approximately 95% of PDAC cases and cause the activation of several signaling pathways such as mitogen-activated protein kinase (MAPK) pathways. Regulation of these signaling pathways is orchestrated by feedback loops mediated by the balance between protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs), leading to activation or inhibition of its downstream targets. The human PTPome comprises 125 members, and these proteins are classified into three distinct families according to their structure. Since PTP activity description, it has become clear that they have both inhibitory and stimulatory effects on cancer-associated signaling processes and that deregulation of PTP function is closely associated with tumorigenesis. Several PTPs have displayed either tumor suppressor or oncogenic characteristics during the development and progression of PDAC. In this sense, PTPs have been presented as promising candidates for the treatment of human pancreatic cancer, and many PTP inhibitors have been developed since these proteins were first associated with cancer. Nevertheless, some challenges persist regarding the development of effective and safe methods to target these molecules and deliver these drugs. In this review, we discuss the role of PTPs in tumorigenesis as tumor suppressor and oncogenic proteins. We have focused on the differential expression of these proteins in PDAC, as well as their clinical implications and possible targeting for pharmacological inhibition in cancer therapy.

Genome-wide CRISPR screen reveals PSMA6 to be an essential gene in pancreatic cancer cells

BackgroundDespite its relatively low incidence, pancreatic ductal adenocarcinoma (PDAC) is a leading cause of cancer deaths because of the aggressive growth/metastasis of the tumor, the lack of early symptoms, and the poor treatment options. Basic research to identify potential therapeutic targets for PDAC is greatly needed.MethodsWe used a negative-selection genome-wide CRISPR screen to identify essential genes in the PANC-1 human pancreatic carcinoma cell line. We validated the top hits with follow-up siRNA screens, using the HPNE, HPAF-II, AsPC-1, and Mia PaCa-2 cell lines.ResultsThe PSMA6 gene was an identified candidate hit after the CRISPR screen, siRNA validation screen, and siRNA deconvolution screen. Spheroid formation assays and flow cytometry analysis showed that PSMA6 is critical for survival in many pancreatic ductal carcinoma cell models. Lastly, as PSMA6 protein is a proteosomal subunit of the 20S core complex, we showed that bortezomib, a proteasome inhibitor, was especially toxic in PANC-1 cells.ConclusionsFurther study of PSMA6 and the proteasome subunit that it encodes, along with other hits identified in our CRISPR screens, may provide valuable insights into potential therapeutic targets for PDAC.