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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

Acute pancreatitis (AP) is a disease characterized by an acute inflammatory phase followed by a convalescent phase. Diabetes mellitus (DM) was historically felt to be a transient phenomenon related to acute inflammation; however, it is increasingly recognized as an important late and chronic complication. There are several challenges that have prevented precisely determining the incidence rate of DM following AP and understanding the underlying mechanisms. The Diabetes RElated to Acute Pancreatitis and its Mechanisms (DREAM) Study is a prospective cohort study designed to address these and other knowledge gaps to provide the evidence needed to screen for, prevent, and treat DM following AP. In the following article, we summarize literature regarding the epidemiology of DM following AP, and provide the rationale and an overview of the DREAM study.

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