Ashley M. Mohr

Sonic hedgehog paracrine signaling regulates metastasis and lymphangiogenesis in pancreatic cancer.

Sonic hedgehog (SHH) expression is tightly regulated throughout development. In the adult, aberrant expression of SHH is associated with the onset and progression of pancreatic cancer, as evidenced by increased levels of expression in premalignant and malignant lesions of the pancreas. We investigated the hypothesis that SHH, secreted from pancreatic tumors, functions in a paracrine manner to influence the biological condition of mesenchymal and endothelial cells. Orthotopic implantation of a pancreatic tumor cell line expressing SHH (Capan-2) and a transformed primary cell line that overexpresses SHH (T-HPNE.SHH) were used to show that overexpression of SHH increased primary tumor size and metastasis. Treatment with a neutralizing antibody, 5E1, decreased primary tumor volume and inhibited metastasis. Lyve-1+ vessels and stromal fibroblasts in tumors expressed primary cilium and showed localization of the receptor Smoothened to the primary cilium, providing evidence of active SHH signaling through this pathway. Although primary cilia are present on normal ductal cells of the pancreas, we did not observe primary cilium on the ductal tumor cells, suggesting decreased autocrine signaling through pathways mediated by the primary cilium in pancreatic cancer. These data support the hypothesis that SHH, secreted from pancreatic epithelia, is critical in establishing and regulating the tumor microenvironment and thereby contributes to progression of pancreatic cancer.

Molecular pathology of early pancreatic cancer.

We describe the pathology of early pancreatic cancer and present an overview of known molecular alterations that occur in these lesions. There are three defined precursor lesions in current models of pancreatic cancer: pancreatic intraepithelial neoplasia (PanIN), mucinous cystic neoplasms (MCN), and intraductal papillary mucinous neoplasms (IPMN). Molecular alterations detected in these lesions include: telomeres, K-ras and downstream targets, p16/CDKN2A, p53, SMAD4/DPC4, microRNAs, mucins and their post-translational processing, inflammatory cytokines, CEACAM, and epigenetic alterations. We summarize previous analyses of these markers as diagnostic markers of disease, and suggest areas of future study.

MicroRNA-200c Modulates the Expression of MUC4 and MUC16 by Directly Targeting Their Coding Sequences in Human Pancreatic Cancer

Transmembrane mucins, MUC4 and MUC16 are associated with tumor progression and metastatic potential in human pancreatic adenocarcinoma. We discovered that miR-200c interacts with specific sequences within the coding sequence of MUC4 and MUC16 mRNAs, and evaluated the regulatory nature of this association. Pancreatic cancer cell lines S2.028 and T3M-4 transfected with miR-200c showed a 4.18 and 8.50 fold down regulation of MUC4 mRNA, and 4.68 and 4.82 fold down regulation of MUC16 mRNA compared to mock-transfected cells, respectively. A significant reduction of glycoprotein expression was also observed. These results indicate that miR-200c overexpression regulates MUC4 and MUC16 mucins in pancreatic cancer cells by directly targeting the mRNA coding sequence of each, resulting in reduced levels of MUC4 and MUC16 mRNA and protein. These data suggest that, in addition to regulating proteins that modulate EMT, miR-200c influences expression of cell surface mucins in pancreatic cancer.

MUC1 Regulates Expression of Multiple microRNAs Involved in Pancreatic Tumor Progression, Including the miR-200c/141 Cluster

MUC1 is a transmembrane glycoprotein that modulates transcription via its cytoplasmic domain. We evaluated the capacity of MUC1 to regulate the global transcription of microRNAs in pancreatic cancer cells expressing MUC1. Results indicated that MUC1 regulated expression of at least 103 microRNAs. We evaluated further regulation of the microRNA transcript cluster miR-200c/141, which was among the most highly regulated microRNAs. We found that MUC1 directly interacted with ZEB1, a known transcriptional repressor of the miR-200c/141 cluster, at the promoter of miR-200c/141, and further reduced transcript production. These data indicate that signaling through MUC1 influences cancer progression by regulating transcription of microRNAs that are associated with the process of metastasis.

Expression of core 3 synthase in human pancreatic cancer cells suppresses tumor growth and metastasis

Core 3‐derived glycans, a major type of O‐glycan expressed by normal epithelial cells of the gastrointestinal tract, are downregulated during malignancy because of loss of expression of functional β3‐N‐acetylglucosaminyltransferase‐6 (core 3 synthase). We investigated the expression of core 3 synthase in normal pancreas and pancreatic cancer and evaluated the biological effects of re‐expressing core 3 synthase in pancreatic cancer cells that had lost expression. We determined that pancreatic tumors and tumor cell lines have lost expression of core 3 synthase. Therefore, we re‐expressed core 3 synthase in human pancreatic cancer cells (Capan‐2 and FG) to investigate the contribution of core 3 glycans to malignant progression. Pancreatic cancer cells expressing core 3 synthase showed reduced in vitro cell proliferation, migration and invasion compared to vector control cells. Expression of core 3 O‐glycans induced altered expression of β1 integrin, decreased activation of focal adhesion kinase, led to the downregulation of expression of several genes including REG1α and FGFR3 and altered lamellipodia formation. The addition of a GlcNAc residue by core 3 synthase leads to the extension of the tumor‐associated Tn structure on MUC1. Orthotopic injection of FG cells expressing core 3 synthase into the pancreas of nude mice produced significantly smaller tumors and decreased metastasis to the surrounding tissues compared to vector control FG cells. These findings indicate that expression of core 3‐derived O‐glycans in pancreatic cancer cells suppresses tumor growth and metastasis through modulation of glycosylation of mucins and other cell surface and extracellular matrix proteins.

Saturated Free Fatty Acids Induce Cholangiocyte Lipoapoptosis

Recent studies have identified a cholestatic variant of nonalcoholic fatty liver disease (NAFLD) with portal inflammation and ductular reaction. Based on reports of biliary damage, as well as increased circulating free fatty acids (FFAs) in NAFLD, we hypothesized the involvement of cholangiocyte lipoapoptosis as a mechanism of cellular injury. Here, we demonstrate that the saturated FFAs palmitate and stearate induced robust and rapid cell death in cholangiocytes. Palmitate and stearate induced cholangiocyte lipoapoptosis in a concentration‐dependent manner in multiple cholangiocyte‐derived cell lines. The mechanism of lipoapoptosis relied on the activation of caspase 3/7 activity. There was also a significant up‐regulation of the proapoptotic BH3‐containing protein, PUMA. In addition, palmitate‐induced cholangiocyte lipoapoptosis involved a time‐dependent increase in the nuclear localization of forkhead family of transcription factor 3 (FoxO3). We show evidence for posttranslational modification of FoxO3, including early (6 hours) deacetylation and dephosphorylation that coincide with localization of FoxO3 in the nuclear compartment. By 16 hours, nuclear FoxO3 is both phosphorylated and acetylated. Knockdown studies confirmed that FoxO3 and its downstream target, PUMA, were critical for palmitate‐ and stearate‐induced cholangiocyte lipoapoptosis. Interestingly, cultured cholangiocyte‐derived cells did not accumulate appreciable amounts of neutral lipid upon FFA treatment. Conclusion: Our data show that the saturated FFAs palmitate and stearate induced cholangiocyte lipoapoptosis by way of caspase activation, nuclear translocation of FoxO3, and increased proapoptotic PUMA expression. These results suggest that cholangiocyte injury may occur through lipoapoptosis in NAFLD and nonalcoholic steatohepatitis patients. (Hepatology 2014;60:1941–1955)

Targeting the NF-κB and mTOR Pathways with a Quinoxaline Urea Analog That Inhibits IKKβ for Pancreas Cancer Therapy

Purpose: The presence of TNF-α in approximately 50% of surgically resected tumors suggests that the canonical NF-κB and the mTOR pathways are activated. Inhibitor of IκB kinase β (IKKβ) acts as the signaling node that regulates transcription via the p-IκBα/NF-κB axis and regulates translation via the mTOR/p-S6K/p-eIF4EBP axis. A kinome screen identified a quinoxaline urea analog 13-197 as an IKKβ inhibitor. We hypothesized that targeting the NF-κB and mTOR pathways with 13-197 will be effective in malignancies driven by these pathways. Experimental Design: Retrospective clinical and preclinical studies in pancreas cancers have implicated NF-κB. We examined the effects of 13-197 on the downstream targets of the NF-κB and mTOR pathways in pancreatic cancer cells, pharmacokinetics, toxicity and tumor growth, and metastases in vivo. Results: 13-197 inhibited the kinase activity of IKKβ in vitro and TNF-α–mediated NF-κB transcription in cells with low-μmol/L potency. 13-197 inhibited the phosphorylation of IκBα, S6K, and eIF4EBP, induced G1 arrest, and downregulated the expression of antiapoptotic proteins in pancreatic cancer cells. Prolonged administration of 13-197 did not induce granulocytosis and protected mice from lipopolysaccharide (LPS)-induced death. Results also show that 13-197 is orally available with extensive distribution to peripheral tissues and inhibited tumor growth and metastasis in an orthotopic pancreatic cancer model without any detectable toxicity. Conclusion: These results suggest that 13-197 targets IKKβ and thereby inhibits mTOR and NF-κB pathways. Oral availability along with in vivo efficacy without obvious toxicities makes this quinoxaline urea chemotype a viable cancer therapeutic. Clin Cancer Res; 19(8); 2025–35. ©2013 AACR.

FoxO3 increases miR-34a to cause palmitate-induced cholangiocyte lipoapoptosis

Nonalcoholic steatohepatitis (NASH) patients have elevated plasma saturated free fatty acid levels. These toxic fatty acids can induce liver cell death and our recent results demonstrated that the biliary epithelium may be susceptible to lipotoxicity. Here, we explored the molecular mechanisms of cholangiocyte lipoapoptosis in cell culture and in an animal model of NASH. Treatment of cholangiocytes with palmitate (PA) showed increased caspase 3/7 activity and increased levels of cleaved poly (ADP-ribose) polymerase and cleaved caspase 3, demonstrating cholangiocyte lipoapoptosis. Interestingly, treatment with PA significantly increased the levels of microRNA miR-34a, a pro-apoptotic microRNA known to be elevated in NASH. PA induction of miR-34a was abolished in cholangiocytes transduced with forkhead family of transcription factor class O (FoxO)3 shRNA, demonstrating that FoxO3 activation is upstream of miR-34a and suggesting that FoxO3 is a novel transcriptional regulator of miR-34a. Further, anti-miR-34a protected cholangiocytes from PA-induced lipoapoptosis. Direct and indirect targets of miR-34a, such as SIRT1, receptor tyrosine kinase (MET), Kruppel-like factor 4, fibroblast growth factor receptor (FGFR)1, and FGFR4, were all decreased in PA-treated cholangiocytes. SIRT1 and MET were partially rescued by a miR-34a antagonist. Cholangiocyte apoptosis and miR-34a were dramatically increased in the liver of mice with early histologic features of NASH. Our study provides evidence for the pro-apoptotic role of miR-34a in PA-induced cholangiocyte lipoapoptosis in culture and in the liver.

MicroRNA Function in Human Diseases

MicroRNAs are emerging as a hot topic in research, and rightfully so. They show great promise as targets of treatment and as markers for common human diseases, such as cancer and metabolic diseases. In this review, we address some of the basic questions regarding microRNA function in human disease and the clinical significance of microRNAs. Specifically, microRNAs in epigenetics, cancer, and metabolic diseases are discussed, with examples taken from cholangiocarcinoma and nonalcoholic fatty liver disease.

Enhanced colorectal cancer metastases in the alcohol-injured liver

Metastatic liver disease is a major cause of mortality in colorectal cancer (CRC) patients. Alcohol consumption is a noted risk factor for secondary cancers yet the role of alcoholic liver disease (ALD) in colorectal liver metastases (CRLM) is not defined. This work evaluated tumor cell colonization in the alcoholic host liver using a novel preclinical model of human CRC liver metastases. Immunocompromised Rag1-deficient mice were fed either ethanol (E) or isocaloric control (C) diets for 4 weeks prior to intrasplenic injection of LS174T human CRC cells. ALD and CRLM were evaluated 3 or 5 weeks post-LS174T cell injection with continued C/E diet administration. ALD was confirmed by increased serum transaminases, hepatic steatosis and expression of cytochrome P4502E1, a major ethanol-metabolizing enzyme. Alcohol-mediated liver dysfunction was validated by impaired endocytosis of asialoorosomucoid and carcinoembryonic antigen (CEA), indicators of hepatocellular injury and progressive CRC disease, respectively. Strikingly, the rate and burden of CRLM was distinctly enhanced in alcoholic livers with metastases observed earlier and more severely in E-fed mice. Further, alcohol-related increases (1.5–3.0 fold) were observed in the expression of hepatic cytokines (TNF-α, IL-1 beta, IL-6, IL-10) and other factors noted to be involved in the colonization of CRC cells including ICAM-1, CCL-2, CCL-7, MMP-2, and MMP-9. Also, alcoholic liver injury was associated with altered hepatic localization as well as increased circulating levels of CEA released from CRC cells. Altogether, these findings indicate that the alcoholic liver provides a permissive environment for the establishment of CRLM, possibly through CEA-related inflammatory mechanisms.