Search

Summary Several approaches for overcoming immunotherapy resistance in pancreatic and colorectal cancer syngeneic models were assessed using heparin and immunotherapy. Beneficial responses were attributed to heparin-induced vascular normalization, ensuing CD8+ T-cell infiltration, and M1 macrophage polarization, suggesting the potential for heparin-anchored therapies in cold tumors such as pancreatic cancer. See related article by Wei et al., p. xxxx

PDF - 1019K, Figure S1 The CIN70 signature predicts survival in PDAC patients. Figure S2 Murine PDAC KRC cells exhibit chromosome instability and evidence of a weakened SAC. Figure S3 Clonogenic survival of PANC-1 and BxPC3 cells treated with NMS-P715. Figure S4 Growth of human adipose stem cells (ASCs) in the presence of NMS-P715. Figure S5 Murine PDAC KRC cell growth is inhibited by NMS-P715. Figure S6 Inhibition of PDAC cell growth after pre-treatment with NMS-P715. Table S1 Chromosome stability in cell lines. Table S2 PDAC CIN25 genes ranked by Cox score Supplementary methods for gene expression and survival analyses.

Aberrant activation of Cyclin D-Cdk4/6 signaling pathway is commonly found in pancreatic ductal adenocarcinoma (PDAC). Here, we show that PD-0332991, a highly specific inhibitor for Cdk4 and Cdk6, exerted growth inhibitory effects on three human PDAC cell lines. Microarray analysis revealed that PD-0332991 downregulated cell-cycle–related genes, but upregulated genes implicated in extracellular matrix (ECM) remodeling and pancreatic cancer cell invasion and metastasis. Moreover, PD-0332991 enhanced invasion in TGF-β–responsive PDAC cell lines that harbor a wild-type SMAD4 gene (COLO-357, PANC-1), but not in TGF-β–resistant AsPC-1 cells that harbor a mutated SMAD4. PD-0332991 also induced epithelial–mesenchymal transition (EMT) in COLO-357 and PANC-1, but not in AsPC-1 cells. Inhibition of CDK4/6 using shRNA mimicked the effects of PD-0332991 on EMT induction. Furthermore, PD-0332991 increased Smad transcriptional activity in luciferase readout assays and activated TGF-β signaling. SB-505124, an inhibitor of the type-I TGF-β receptor (TβRI) kinase, completely blocked EMT induction by PD-0332991. When combined with PD-0332991, SB-505124 inhibited the growth of COLO-357 and PANC-1 cells. Taken together, these data suggest that anti-Cdk4/6 therapy could induce EMT and enhance pancreatic cancer cell invasion by activating Smad-dependent TGF-β signaling, and that combining PD-0332991 and SB-505124 may represent a novel therapeutic strategy in PDAC. Mol Cancer Ther; 11(10); 2138–48. ©2012 AACR.

PDF file, 4534K, Effects of PD-0332991 and SB-505124 on growth in 3-D culture and in a clonogenic assay.

PDF file, 805K, Effects of PD-0332991 incubation on gene expression.

Most solid tumors, including pancreatic ductal adenocarcinoma (PDAC), exhibit structural and numerical chromosome instability (CIN). Although often implicated as a driver of tumor progression and drug resistance, CIN also reduces cell fitness and poses a vulnerability that can be exploited therapeutically. The spindle assembly checkpoint (SAC) ensures correct chromosome-microtubule attachment, thereby minimizing chromosome segregation errors. Many tumors exhibit upregulation of SAC components such as MPS1, which may help contain CIN within survivable limits. Prior studies showed that MPS1 inhibition with the small molecule NMS-P715 limits tumor growth in xenograft models. In cancer cell lines, NMS-P715 causes cell death associated with impaired SAC function and increased chromosome missegregation. Although normal cells appeared more resistant, effects on stem cells, which are the dose-limiting toxicity of most chemotherapeutics, were not examined. Elevated expression of 70 genes (CIN70), including MPS1, provides a surrogate measure of CIN and predicts poor patient survival in multiple tumor types. Our new findings show that the degree of CIN70 upregulation varies considerably among PDAC tumors, with higher CIN70 gene expression predictive of poor outcome. We identified a 25 gene subset (PDAC CIN25) whose overexpression was most strongly correlated with poor survival and included MPS1. In vitro, growth of human and murine PDAC cells is inhibited by NMS-P715 treatment, whereas adipose-derived human mesenchymal stem cells are relatively resistant and maintain chromosome stability upon exposure to NMS-P715. These studies suggest that NMS-P715 could have a favorable therapeutic index and warrant further investigation of MPS1 inhibition as a new PDAC treatment strategy. Mol Cancer Ther; 13(2); 307–15. ©2013 AACR.

Cell viability analysis used for normalization of pH data

PDF file, 72K, List of genes with at least two fold change upon 72 h incubation with PD.

Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer-related mortality in the United States. Aggressive treatment regimens have not changed the disease course, and the median survival has just recently reached a year. Several mechanisms are proposed to play a role in PDAC therapeutic resistance, including hypoxia, which creates a more aggressive phenotype with increased metastatic potential and impaired therapeutic efficacy. AP Endonuclease-1/Redox Effector Factor 1 (APE1/Ref-1) is a multifunctional protein possessing a DNA repair function in base excision repair and the ability to reduce oxidized transcription factors, enabling them to bind to their DNA target sequences. APE1/Ref-1 regulates several transcription factors involved in survival mechanisms, tumor growth, and hypoxia signaling. Here, we explore the mechanisms underlying PDAC cell responses to hypoxia and modulation of APE1/Ref-1 redox signaling activity, which regulates the transcriptional activation of hypoxia-inducible factor 1 alpha (HIF1α). Carbonic anhydrase IX (CA9) is regulated by HIF1α and functions as a part of the cellular response to hypoxia to regulate intracellular pH, thereby promoting cell survival. We hypothesized that modulating APE1/Ref-1 function will block activation of downstream transcription factors, STAT3 and HIF1α, interfering with the hypoxia-induced gene expression. We demonstrate APE1/Ref-1 inhibition in patient-derived and established PDAC cells results in decreased HIF1α–mediated induction of CA9. Furthermore, an ex vivo three-dimensional tumor coculture model demonstrates dramatic enhancement of APE1/Ref-1–induced cell killing upon dual targeting of APE1/Ref-1 and CA9. Both APE1/Ref-1 and CA9 are under clinical development; therefore, these studies have the potential to direct novel PDAC therapeutic treatment. Mol Cancer Ther; 15(11); 2722–32. ©2016 AACR.

APE1 expression correlates with decreased survival in PDAC and CA9 is upregulated in PDAC

Supplementary Figure. 1A. Nuclear extracts (N.E.) from various serum-starved pancreatic cancer cell lines were probed for NF-κBbinding activity. Also included were serum-starved Panc-28 cells after stimulation by IL-1. Supershift assays were performed with unstimulated Panc-28 cell extracts using anti-p65/NFκB and nonspecific (IgG) antibodies. Supplementary Figure. 1B Whole-cell extracts were prepared from untreated or TNF-treated cells and then incubated with anti- JNK1/2 antibodies. Immune complexes were washed several times with a lysis buffer and assayed for JNK kinase activity with 2 μg of GST-c-Jun (amino acids 1-79) used as substrate. Assay mixtures, which included 0.2 mM [γ-32P] ATP and 10 mM MgCl2, were incubated for 5 min at 30 {degree sign}C, at which point the reactions were terminated by the addition of a sodium dodecyl sulfate sample buffer. Radiolabeled GST- c-Jun bands were detected by autoradiography. Serum-starved Panc-28 cells were treated with or without the JNK inhibitor SP600125 for 2 h. Whole-cell extracts were probed by Western blotting for c-Jun and phospho-c-Jun (Ser63). Supplementary Figure. 1C. A collagenase/luciferase reporter gene with (-73 Col/luc) or without (-60 Col/luc) an AP-1-binding site was cotransfected with the indicated constructs into c-Jun-/- MEFs stably expressing the pMX vector, c- Jun-TA or c-Jun SAA/TA. Sixteen to twenty four hours after transfection, cells were serum-starved overnight and either harvested right away or stimulated with PDGF-BB (10 ng/ml). After PDGF treatment (24 h) cells were lysed and protein extracts assayed for luciferase activity. Each data point was obtained in triplicate and used to calculate the averages and standard deviations.

Additional methods including synthesis information for one of the inhibitors used and methods used for TCGA survival analysis found in supplemental figure 7.

PDF file, 398K, Effects of cdk4 and cdk6 knockdown on Smad transcriptional activity.

Activator protein-1 (AP-1) regulates the expression of several genes involved in human tumorigenesis. However, there is little known about this transcription factor in pancreatic ductal adenocarcinoma. We recently found high levels of AP-1-binding activities and multiple AP-1/DNA complexes containing c-Jun, JunD, Fra1, and Fra2 in pancreatic cancer cells. Transient transfection assays indicated that AP-1 was functional and capable of transactivating its gene targets. Furthermore, a c-Jun transactivation mutant inhibited anchorage-dependent and anchorage-independent proliferation, suggesting that AP-1 had an essential role in pancreatic cancer cells. Our study also uncovered a novel mechanism by which protein kinase Akt controls c-Jun activity in pancreatic cancer cells. Indeed, distinct from its known ability to induce c-fos and fra1 and to stabilize c-Jun, Akt appeared to directly regulate the transcriptional activity of c-Jun independently of the phosphorylation sites targeted by c-Jun NH2-terminal kinase (Ser63/Ser73) and glycogen synthase kinase-3 (Thr239). Our data also suggest that growth factors might use this Akt-regulated mechanism to potently induce c-Jun targets such as cyclin D1. Collectively, our findings indicate that AP-1 has an important function in pancreatic cancer cells and provide evidence for a previously unknown Akt-mediated mechanism of c-Jun activation. (Mol Cancer Res 2009;7(5):745–54)

PDF file, 1109K, Effects of cdk4 and cdk6 knockdown on EMT in PANC-1 cells.

PDF file, 2381K, Effects of PD-0332991 on colony formation.

CA9 protein levels are increased under hypoxia, but APE1 levels are not

APE1/Ref-1 protein expression affects CA9 mRNA levels in additional PDAC cell lines

Confirmation of HIF-1 deletion in MEF cells

APE1/Ref-1 interactions with STAT3 and NFκB are stimulated by IL-6 and TNFα (respectively) under normoxic conditions

PDF file, 94K.

PDF file, 735K, Effects of PD-0332991 on invasion and EMT of AsPC-1 cells.

PDF file, 83K, List of genes with at least two fold change upon 24 h incubation with PD.

APE1/Ref-1 interactions with HIF1α and STAT3 are stimulated by hypoxia in Cancer-Associated Fibroblast (CAF) cells

Figure Legends for Supplemental Figures

Purpose: High-throughput profiling experiments have linked altered expression of microRNAs (miRNA) to different types of cancer. Tumor tissues are a heterogeneous mixture of not only cancer cells, but also supportive and reactive tumor microenvironment elements. To clarify the clinical significance of altered miRNA expression in solid tumors, we developed a sensitive fluorescence-based in situ hybridization (ISH) method to visualize miRNA accumulation within individual cells in formalin-fixed, paraffin-embedded tissue specimens. This ISH method was implemented to be compatible with routine clinical immunohistochemical (IHC) assays to enable the detection of miRNAs and protein markers in the same tissue section for colocalization and functional studies.Experimental Design: We used this combined ISH/IHC assay to study a subset of cancer-associated miRNAs, including miRNAs frequently detected at low (miR-34a and miR-126) and high (miR-21 and miR-155) levels, in a panel of breast, colorectal, lung, pancreas, and prostate carcinomas.Results: Despite the distinct histopathologic alterations of each particular cancer type, general trends emerged that pinpointed distinct source cells of altered miRNA expression. Although altered expressions of miR-21 and miR-34a were manifested within cancer cells, those of miR-126 and miR-155 were predominantly confined to endothelial cells and immune cells, respectively. These results suggest a heterogeneous participation of miRNAs in carcinogenesis by intrinsically affecting cancer cell biology or by modulating stromal, vascular, and immune responses.Conclusions: We described a rapid and sensitive multicolor ISH/IHC assay and showed that it could be broadly applied as an investigational tool to better understand the etiologic relevance of altered miRNA expression in cancer. Clin Cancer Res; 16(16); 4246–55. ©2010 AACR.

Supplementary Figures S1-S7 and Supplementary Tables S1-S3.

Supplementary Data from Uncovering Growth-Suppressive MicroRNAs in Lung Cancer

CCR Translation for This Article from MicroRNA-10b Expression Correlates with Response to Neoadjuvant Therapy and Survival in Pancreatic Ductal Adenocarcinoma

Purpose: MicroRNA (miRNA) expression profiles improve classification, diagnosis, and prognostic information of malignancies, including lung cancer. This study uncovered unique growth-suppressive miRNAs in lung cancer.Experimental Design: miRNA arrays were done on normal lung tissues and adenocarcinomas from wild-type and proteasome degradation-resistant cyclin E transgenic mice to reveal repressed miRNAs in lung cancer. Real-time and semiquantitative reverse transcription-PCR as well as in situ hybridization assays validated these findings. Lung cancer cell lines were derived from each transgenic line (designated as ED-1 and ED-2 cells, respectively). Each highlighted miRNA was independently transfected into these cells. Growth-suppressive mechanisms were explored. Expression of a computationally predicted miRNA target was examined. These miRNAs were studied in a paired normal-malignant human lung tissue bank.Results: miR-34c, miR-145, and miR-142-5p were repressed in transgenic lung cancers. Findings were confirmed by real-time and semiquantitative reverse transcription-PCR as well as in situ hybridization assays. Similar miRNA profiles occurred in human normal versus malignant lung tissues. Individual overexpression of miR-34c, miR-145, and miR-142-5p in ED-1 and ED-2 cells markedly repressed cell growth. Anti-miR cotransfections antagonized this inhibition. The miR-34c target, cyclin E, was repressed by miR-34c transfection and provided a mechanism for observed growth suppression.Conclusions: miR-34c, miR-145, and miR-142-5p were repressed in murine and human lung cancers. Transfection of each miRNA significantly repressed lung cancer cell growth. Thus, these miRNAs were growth suppressive and are proposed to exert antineoplastic effects in the lung.

Supplementary Figure 3 from Pancreatic Cancer and Precursor Pancreatic Intraepithelial Neoplasia Lesions Are Devoid of Primary Cilia

XLS file - 76K, Transcriptome comparison of tumor DCs sorted from ovarian cancer-bearing mice treated with CD40 agonists plus PEI-complexed Dsi (Dmi155 vs. GFP Dsi)

Supplementary Figure 5 from Pancreatic Cancer and Precursor Pancreatic Intraepithelial Neoplasia Lesions Are Devoid of Primary Cilia

Supplementary Figure 1 from Pancreatic Cancer and Precursor Pancreatic Intraepithelial Neoplasia Lesions Are Devoid of Primary Cilia

Supplementary Figure 6 from Pancreatic Cancer and Precursor Pancreatic Intraepithelial Neoplasia Lesions Are Devoid of Primary Cilia

PDF file - 76K

Supplementary Figure 2 from Pancreatic Cancer and Precursor Pancreatic Intraepithelial Neoplasia Lesions Are Devoid of Primary Cilia

Supplementary Figure 4 from Pancreatic Cancer and Precursor Pancreatic Intraepithelial Neoplasia Lesions Are Devoid of Primary Cilia

PDF file - 76K

Primary cilia have been proposed to participate in the modulation of growth factor signaling pathways. In this study, we determined that ciliogenesis is suppressed in both pancreatic cancer cells and pancreatic intraepithelial neoplasia (PanIN) lesions in human pancreatic ductal adenocarcinoma (PDAC). Primary cilia were absent in these cells even when not actively proliferating. Cilia were also absent from mouse PanIN cells in three different mouse models of PDAC driven by an endogenous oncogenic Kras allele. Inhibition of Kras effector pathways restored ciliogenesis in a mouse pancreatic cancer cell line, raising the possibility that ciliogenesis may be actively repressed by oncogenic Kras. By contrast, normal duct, islet, and centroacinar cells retained primary cilia in both human and mouse pancreata. Thus, arrested ciliogenesis is a cardinal feature of PDAC and its precursor PanIN lesions, does not require ongoing proliferation, and could potentially be targeted pharmacologically. [Cancer Res 2009;69(2):422–30]

PDF file - 178K, Enhanced local and splenic anti-tumor responses in mice treated with anti-CD40 and Dmi155-PEI.

Supplementary Figure Legends 1-6 from Pancreatic Cancer and Precursor Pancreatic Intraepithelial Neoplasia Lesions Are Devoid of Primary Cilia

PDF file - 107K

Purpose: Fibroblast growth factor receptor 4 (FGFR4), one of the four tyrosine kinase receptors for FGFs, is involved in various cellular processes. Activation of the FGF19/FGFR4 signaling is strongly associated with cancer development and progression. BLU9931 is an irreversible and highly selective small-molecule inhibitor of FGFR4. In this study, we aimed to elucidate the role of FGF19/FGFR4 signaling using BLU9931 in human PDAC. Experimental procedures: The expression of FGFR4 in eight human PDAC cell lines were examined using real-time PCR, immunoblotting, and FACS analysis. The effects of BLU9931 on FGFR4-positive PDAC cells were examined using growth assay, cell cycle assay, immunoblotting of downstream targets of FGF19/FGFR4 signaling, real-time PCR analysis of stemness markers, and sphere forming assays. Motility was evaluated using migration and invasion assays. Matrix metalloproteinase activity was evaluated using gelatin zymography. Induction of senescence was evaluated using senescence-associated β-galactosidase activity and real-time PCR analysis of senescence-associated, phenotype-related secretory genes. The synergistic effects of senolytic drugs on BLU9931-treated cells were evaluated using ATP assay. Expression and clinicopathological features of FGFR4 in PDAC patients were examined using immunohistochemical analysis of tissue microarrays. Results: In human PDAC cases, FGFR4 expression was positively correlated with larger primary tumors and more advanced disease stage. Among the eight PDAC cell lines, FGFR4 was expressed at the highest level in PK-1 cell line, in which the single-nucleotide polymorphism G388R in FGFR4 was detected. Inhibition of signal transduction through the ERK, AKT, and STAT3 pathways by BLU9931 reduced proliferation of FGF19/FGFR4 signaling-activated PDAC cells. In contrast, BLU9931 did not alter stemness features, including stemness marker expression, anticancer drug resistance, and sphere-forming ability of PK-1 cells. However, BLU9931 inhibited cell invasion, in part, by downregulating membrane-type matrix metalloproteinase-1 (MT1-MMP) expression in FGF19/FGFR4 signaling-activated PDAC cells. Furthermore, downregulation of SIRT1 and SIRT6 by BLU9931 contributed to senescence induction, priming these cells for quercetin-induced death, a process termed senolysis. Conclusions: We demonstrated that inhibition of signal transduction pathways through the ERK, AKT, and STAT3 pathways by BLU9931 inhibited PDAC cell proliferation and invasion, in part by downregulating MT1-MMP expression in autocrine/paracrine FGF19/FGFR4 signaling-positive PDAC cells. Furthermore, downregulation of SIRT1 and SIRT6 by BLU9931 may have contributed to senolysis in these cells. Thus, we propose that BLU9931 may be a promising drug for the treatment of FGFR4-positive PDAC. Citation Format: Norihiko Sasaki, Fujiya Gomi, Hisashi Yoshimura, Masami Yamamoto, Yoko Matsuda, Masaki Michishita, Hitoshi Hatakeyama, Yoichi Kawano, Yoko Itakura, Masashi Toyoda, Murray Korc, Toshiyuki Ishiwata. FGFR4 inhibitor BLU9931 induces cellular senescence in pancreatic ductal adenocarcinoma cell lines promoting sensitivity to senolytic therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2449.

Pancreatic ductal adenocarcinoma (PDAC) is a treatment-recalcitrant malignancy that harbors several key driver mutations at a high frequency (e.g., KRAS, TP53, CDKN2A, and SMAD4 Citation Format: Samantha McElyea, Murray Korc. The glycosaminoglycan syndecan-4 facilitates pancreatic cancer progression and biologic aggressiveness [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2019 Sept 6-9; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2019;79(24 Suppl):Abstract nr C31.

Modulating the activity of miRNAs provides opportunities for novel cancer interventions. However, low bioavailability and poor cellular uptake are major challenges for delivering miRNA mimetics specifically to tumor cells. Here, we took advantage of the spontaneous enhanced endocytic activity of ovarian cancer-associated dendritic cells (DC) to selectively supplement the immunostimulatory miRNA miR-155. In vivo processing of nanoparticles carrying oligonucleotide duplexes mimicking the bulged structure of endogenous pre-miRNA (but not siRNA-like oligonucleotides) dramatically augmented miR-155 activity without saturating the RNA-induced silencing complex. Endogenous processing of synthetic miR-155 favored Ago2 and, to a lesser extent, Ago4 loading, resulting in genome-wide transcriptional changes that included silencing of multiple immunosuppressive mediators. Correspondingly, tumor-infiltrating DCs were transformed from immunosuppressive to highly immunostimulatory cells capable of triggering potent antitumor responses that abrogated the progression of established ovarian cancers. Our results show both the feasibility and therapeutic potential of supplementing/replenishing miRNAs in vivo using nonviral approaches to boost protective immunity against lethal tumors. Thus, we provide a platform, an optimized design, and a mechanistic rationale for the clinical testing of nonviral miRNA mimetics. Cancer Res; 72(7); 1683–93. ©2012 AACR.

Introduction: Pancreatic ductal adenocarcinoma (PDAC) is a lethal malignancy. Diagnosis and management of PDAC are hampered by the absence of sensitive and specific disease biomarkers. MicroRNAs (miRNA) are noncoding regulatory RNAs involved in initiation and progression of human cancers. In this study, we sought to determine whether miR-10b could serve as a biomarker for PDAC. Experimental Design: miRNA expression was characterized by fluorescence-based in situ hybridization using locked nucleic acid–modified DNA probes against miR-10b, miR-21, miR-155, miR-196a, and miR-210, followed by codetection of proteins by immunohistochemistry on the same tissue sections. miRNA expression in surgically resected PDAC tissues and in endoscopic ultrasonography (EUS)-guided fine-needle aspirate (EUS-FNA) samples was analyzed in cytokeratin 19 (CK19)–positive epithelial cells using optical intensity analysis. Results: In 10 resected PDAC samples, miR-10b was the most frequently and consistently overexpressed miRNA among characterized miRNAs, exhibiting a four-fold increase in the cancer cells (P = 0.012). Given this preferential overexpression of miR-10b, we sought to determine whether miR-10b expression was clinically relevant. Accordingly, miR-10b expression was examined in 106 EUS-FNA samples obtained from pancreatic lesions. miR-10b expression was increased in cancer cells compared with CK19-positive epithelial cells in benign lesions (P = 0.0001). In patients with PDACs, lower levels of miR-10b were associated with improved response to multimodality neoadjuvant therapy, likelihood of surgical resection, delayed time to metastasis, and increased survival. Conclusion: miR-10b is a novel diagnostic biomarker for PDACs when assessing pancreatic lesions. Expression of miR-10b is predictive of response to neoadjuvant therapy and outcome in this disease. Clin Cancer Res; 17(17); 5812–21. ©2011 AACR.

Colorectal cancer (CRC) and pancreatic ductal adenocarcinoma (PDAC) are among the most commonly diagnosed forms of cancer in the United States. Due to their widespread prevalence and high mortality rate, it is vital to develop effective therapeutic drugs to combat these deadly diseases. In both CRC and PDAC, the multifunctional factor nuclear factor kappa B (NF-kB), a central coordinator of immune responses, is activated abnormally, leading to tumorigenesis and cancer progression. Therefore, controlling NF-kB activity is critical in the treatment of these cancers. Previously, we discovered a novel mechanism by which NF-kB is activated through methylation by an epigenetic enzyme known as protein arginine methyltransferase 5 (PRMT5). We showed that overexpression of PRMT5 significantly promoted several characteristics associated with cancer, including increased cell proliferation, migration, and anchorage-independent growth in both CRC and PDAC cells, thus, putting forward PRMT5 as a novel therapeutic target in these cancers. In this study, we successfully adapted AlphaLISA technique into a high-throughput screen platform, and further employed this approach to successfully identify PR5-LL-FDA1 as a potent PRMT5 inhibitor from commercially-available screening libraries. Furthermore, we confirmed that treatment of PDAC and CRC cells with PR5-LL-FDA1 led to decreased NF-kB activation, and reduced cancer associated properties in PDAC and CRC cells. Importantly, we show that PR5-LL-FDA1 is more efficacious than the commercial PRMT5 inhibitor, EPZ015666 in both PDAC and CRC. Our work clearly highlights the significant potential of PRMT5 as a therapeutic target in PDAC and CRC and hold the promise to establish PR5-LL-FDA1 as a promising basis for new drug development in the future. Citation Format: Lakshmi Prabhu, Lan Chen, Ahmad Safa, Murray Korc, Zhong-Yin Zhang, Tao Lu. Protein arginine methyltransferase 5 as a tumor promoter and therapeutic target in gastrointestinal cancers [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 4862.

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive cancer characterized by a high frequency of key driver mutations, (e.g. KRAS, TP53, CDKN2A, and SMAD4), as well many less frequent driver mutations. In addition, there is overexpression of growth factors such as TGF-βs, growth factor receptors such as the EGF receptor (EGFR), a markedly desmoplastic stroma, and a propensity to develop metastases and chemoresistance. Consequently, the 5-year survival rate for PDAC is 8-9% with current therapeutic modalities such as gemcitabine plus nab-paclitaxel or FOLFIRINOX. There is an urgent need, therefore, to devise novel therapies in PDAC. In this regard, we previously reported that high microRNA-10b (miR-10b) levels in the cancer cells from PDAC tissues are associated with decreased patient survival and earlier appearance of metastatic disease following neoadjuvant radiochemotherapy (Preis, et al, Clin Cancer Res, 2011), and that miR-10b targets the 3'UTR of tat-interacting protein 30 (TIP30) and downregulates TIP30 mRNA levels, and that these alterations enhance EGF- and TGF-β-mediated pancreatic cancer cell (PCC) invasion and migration (Ouyang et al, Oncogene, 2013). We therefore analyzed genetic alterations in TIP30 in PDAC data from The Cancer Genome Atlas (TCGA) and found that TIP30 exhibited both copy number gains (23%, n=109) and losses (26%, n=109), with parallel changes in TIP30 mRNA levels. Reduction of TIP30 mRNA levels correlated with increased PDAC survival when compared to PDAC with increased mRNA expression (p=0.004, n=182). However, in the absence of TIP30 expression levels in the normal pancreas in TCGA, the significance of this observation is not clear. We therefore evaluated the effects of TIP30 downregulation in human PCCs (hPCCs) using siRNA targeting TIP30 or a vector expressing miR-10b and empty vector controls. Both manipulations increased EGFR and AKT protein levels. To delineate the potential role of TIP30 in PDAC, we generated compound mutant Pdx1-Cre; LSL-KrasG12D; Tip30+/- (KTip30+/-C) mice carrying mutated KrasG12D and heterozygous Tip30 deletion in the pancreas. Heterozygous Tip30 deletion alone did not alter pancreatic cyto-architecture, but accelerated Kras-associated malignant transformation and progression to murine PDAC. Derived murine PCCs exhibited accelerated proliferation and increased EGFR expression. Our results indicate that TIP30 loss enhances PDAC aggression, in part through effects on EGFR. In conclusion, partial loss of TIP30 in miR-10b overexpressing hPCCs or heterozygous loss of Tip30 in a murine model of PDAC resulted in an enhanced PCC aggressive phenotype and increased EGFR levels. To advance our understanding of the role of TIP30 in PDAC we are now evaluating a cohort of mice with homozygous Tip30 deletion. Supported, in part by National Cancer Institute grant CA-075059 to M.K. and CA075059-17S2 Diversity Supplement to IIW. Citation Format: Imade E. Imasuen-Williams, Francis Enane, Murray Korc. Perturbations in tat-interacting protein 30 (TIP30) levels contribute to pancreatic cancer aggressiveness [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 4079.

Historically, chemotherapy often attempts to activate apoptosis in tumor cells even when master regulators of apoptosis (e.g., TP53) are inactivated by genetic mutations, leading to multi-drug resistance. A potential form of non-toxic therapy is to engage p53-independent terminal differentiation. Exponential proliferation in lineage committed progenitors, coordinated by the transcription factor MYC, is succeeded by terminal differentiation, whereas MYC-coordinated aberrant proliferation in human malignancies is not succeeded by terminal differentiation. The mechanisms that suppress terminal differentiation are mostly unclear, impeding development of differentiation therapies. We performed a systematic review of published literature between January 2007 – January 2017 to identify genetic alterations that produce candidate molecular targets for differentiation induction in three treatment-recalcitrant cancers: hepatocellular carcinoma (HCC), ovarian cancer (OVC) and pancreatic ductal adenocarcinoma (PDAC). We then validated candidate molecular targets in-vitro with FANA molecules and CRISPR-Cas9, and by in-vivo validation of small molecule drugs in HCC. By systematic review, lineage tracing studies suggested master transcription factors of cell fate and their cofactors as links to suppression of cell cycle exit by differentiation. Indeed, sequence analysis of HCC, OVC and PDAC identified frequent genetic haploinsufficiency in lineage transcription factors (e.g., GATA4 67% hetloss HCC, n=442, FOXO1 63% hetloss, OVC n=302, PDX1 58% hetloss, PDAC n=109), while their coactivators (e.g. ARID1A/B, and ARID3A/C) were frequently inactivated by genetic mutation/deletion. Interestingly, corepressors (e.g., DNMT1, EED, BAZ1A/B, and SUZ12) that cooperate with these transcription factors to turn off genes were frequently amplified but not inactivated. By immunoprecipitation and mass spectroscopy, we identified in HCC that corepressors (e.g., DNMT1) were aberrantly recruited to wild-type hepatocyte transcription factors (FOXA1/2) and this interaction epigenetically suppressed hepatocyte differentiation genes (e.g., HNF4A). Pre-clinical trials evaluating DNMT1 therapeutic efficacy - by DNMT1-FANA or DNMT1-CRISPR-Cas9 - suppressed HCC proliferation but increased hepatocyte differentiation. Non-cytotoxic treatment (0.2µM) of decitabine (DEC) - DNMT1 inhibitor - terminated HCC proliferation without inducing apoptosis. This inhibition enhanced FOXA1/2 interaction with coactivators, decreased corepressors, increased hepatocyte differentiation by decreasing MYC protein, increasing p27 protein and activating HNF4A. Using 0.2mg/Kg of DEC in vivo - in obesity induced mouse model of HCC - DEC reduced HCC tumor burden, without apoptosis induction. Thus, aberrant corepressors are rational molecular targets that engage terminal differentiation in cancer cells. Citation Format: Francis O. Enane, Xiaorong Gu, Murray Korc, Yogen Saunthararajah. Differentiation therapy and the mechanisms that terminate malignant proliferation [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 2917.