Omega-3 Fatty Acids Prevent Early Pancreatic Carcinogenesis via Repression of the AKT Pathway.

Pancreatic cancer remains a daunting foe despite a vast number of accumulating molecular analyses regarding the mutation and expression status of a variety of genes. Indeed, most pancreatic cancer cases uniformly present with a mutation in the KRAS allele leading to enhanced RAS activation. Yet our understanding of the many epigenetic/environmental factors contributing to disease incidence and progression is waning. Epidemiologic data suggest that diet may be a key factor in pancreatic cancer development and potentially a means of chemoprevention at earlier stages. While diets high in ω3 fatty acids are typically associated with tumor suppression, diets high in ω6 fatty acids have been linked to increased tumor development. Thus, to better understand the contribution of these polyunsaturated fatty acids to pancreatic carcinogenesis, we modeled early stage disease by targeting mutant KRAS to the exocrine pancreas and administered diets rich in these fatty acids to assess tumor formation and altered cell-signaling pathways. We discovered that, consistent with previous reports, the ω3-enriched diet led to reduced lesion penetrance via repression of proliferation associated with reduced phosphorylated AKT (pAKT), whereas the ω6-enriched diet accelerated tumor formation. These data provide a plausible mechanism underlying previously observed effects of fatty acids and suggest that administration of ω3 fatty acids can reduce the pro-survival, pro-growth functions of pAKT. Indeed, counseling subjects at risk to increase their intake of foods containing higher amounts of ω3 fatty acids could aid in the prevention of pancreatic cancer.

Pigment Epithelium-Derived Factor (PEDF) as a Regulator of Wound Angiogenesis.

Although the inflammatory and proliferative phases of wound healing have been well described, much less is known about how healing resolves. During the resolution phase, pruning of the capillary bed and maturation of capillaries occurs and influences the final strength and fidelity of the wound. PEDF, an endogenous anti-angiogenic factor, is produced in wounds and may contribute to the removal of capillaries during wound resolution. This study utilized PEDF-/- mice to examine how PEDF influences wound angiogenesis, particularly capillary density and permeability. The absence of PEDF led to transient changes in dermal wound closure and collagen content, but caused substantial changes in wound angiogenesis. Compared to wild type (WT) mice, wounds from PEDF-/- mice exhibited a significant increase in capillaries during the proangiogenic phase of repair, and a delay in capillary pruning. Conversely, the addition of rPEDF caused a reduction in capillary density within skin wounds in WT mice. In vitro studies showed that PEDF inhibited migration and tube formation by dermal microvascular endothelial cells, and caused a decrease in the expression of VEGFR2, VCAM-1, and other surface receptors. The results demonstrate that loss of PEDF causes a distinctive wound healing phenotype that is characterized by increased angiogenesis and delayed resolution. The findings suggest that PEDF most likely acts through multiple mechanisms to regulate proper capillary refinement in wounds.

Loss of TGFß signaling promotes colon cancer progression and tumor-associated inflammation.

TGFß has both tumor suppressive and tumor promoting effects in colon cancer. Also, TGFß can affect the extent and composition of inflammatory cells present in tumors, contextually promoting and inhibiting inflammation. While colon tumors display intratumoral inflammation, the contributions of TGFß to this process are poorly understood. In human patients, we found that epithelial loss of TGFß signaling was associated with increased inflammatory burden; yet overexpression of TGFß was also associated with increased inflammation. These findings were recapitulated in mutant APC models of murine tumorigenesis, where epithelial truncation of TGFBR2 led to lethal inflammatory disease and invasive colon cancer, mediated by IL8 and TGFß1. Interestingly, mutant APC mice with global suppression of TGFß signals displayed an intermediate phenotype, presenting with an overall increase in IL8-mediated inflammation and accelerated tumor formation, yet with a longer latency to the onset of disease observed in mice with epithelial TGFBR-deficiency. These results suggest that the loss of TGFß signaling, particularly in colon epithelial cells, elicits a strong inflammatory response and promotes tumor progression. This implies that treating colon cancer patients with TGFß inhibitors may result in a worse outcome by enhancing inflammatory responses.

TGFß Signaling in the Pancreatic Tumor Microenvironment Promotes Fibrosis and Immune Evasion to Facilitate Tumorigenesis.

In early pancreatic carcinogenesis, TGFß acts as a tumor suppressor due to its growth-inhibitory effects in epithelial cells. However, in advanced disease, TGFß appears to promote tumor progression. Therefore, to better understand the contributions of TGFß signaling to pancreatic carcinogenesis, we generated mouse models of pancreatic cancer with either epithelial or systemic TGFBR deficiency. We found that epithelial suppression of TGFß signals facilitated pancreatic tumorigenesis, whereas global loss of TGFß signaling protected against tumor development via inhibition of tumor-associated fibrosis, stromal TGFß1 production, and the resultant restoration of antitumor immune function. Similarly, TGFBR-deficient T cells resisted TGFß-induced inactivation ex vivo, and adoptive transfer of TGFBR-deficient CD8(+) T cells led to enhanced infiltration and granzyme B-mediated destruction of developing tumors. These findings paralleled our observations in human patients, where TGFß expression correlated with increased fibrosis and associated negatively with expression of granzyme B. Collectively, our findings suggest that, despite opposing the proliferation of some epithelial cells, TGFß may promote pancreatic cancer development by affecting stromal and hematopoietic cell function. Therefore, the use of TGFBR inhibition to target components of the tumor microenvironment warrants consideration as a potential therapy for pancreatic cancer, particularly in patients who have already lost tumor-suppressive TGFß signals in the epithelium. Cancer Res; 76(9); 2525-39. ©2016 AACR.

HDAC3 mediates smoking-induced pancreatic cancer.

Smoking is a major risk factor for developing pancreatic adenocarcinoma (PDAC); however, little is known about the mechanisms involved. Here we employed a genetic animal model of early stages of PDAC that overexpresses oncogenic Kras in the pancreas to investigate the mechanisms of smoking-induced promotion of the disease in vivo. We confirmed the regulation of the interactions between the tumor microenvironment cells using in vitro cellular systems. Aerial exposure to cigarette smoke stimulated development of pancreatic intraepithelial neaoplasia (PanIN) lesions associated with a tumor microenvironment-containing features of human PDAC including fibrosis, activated stellate cells, M2-macrophages and markers of epithelial-mesenchymal transition (EMT). The pro-cancer effects of smoking were prevented by Histone Deacetylase HDAC I/II inhibitor Saha. Smoking decreased histone acetylation associated with recruitment of and phenotypic changes in macrophages; which in turn, stimulated survival and induction of EMT of the pre-cancer and cancer cells. The interaction between the cancer cells and macrophages is mediated by IL-6 produced under the regulation of HDAC3 translocation to the nucleus in the cancer cells. Pharmacological and molecular inhibitions of HDAC3 decreased IL-6 levels in cancer cells. IL-6 stimulated the macrophage phenotype change through regulation of the IL-4 receptor level of the macrophage. This study demonstrates a novel pathway of interaction between cancer cells and tumor promoting macrophages involving HDAC3 and IL-6. It further demonstrates that targeting HDAC3 prevents progression of the disease and could provide a strategy for treating the disease considering that the HDAC inhibitor we used is FDA approved for a different disease.

Characterization of Mouse Models of Early Pancreatic Lesions Induced by Alcohol and Chronic Pancreatitis.

OBJECTIVE: We describe the first mouse model of pancreatic intraepithelial neoplasia (PanIN) lesions induced by alcohol in the presence and absence of chronic pancreatitis. METHODS: Pdx1-Cre;LSL-K-ras mice were exposed to Lieber-DeCarli alcohol diet for 6 weeks with cerulein injections. The PanIN lesions and markers of fibrosis, inflammation, histone deacetylation, epithelial-to-mesenchymal transition (EMT), and cancer stemness were measured by immunohistochemistry and Western. RESULTS: Exposure of Pdx1-Cre;LSL-K-ras mice to an alcohol diet significantly stimulated fibrosis and slightly but not significantly increased the level of PanIN lesions associated with an increase in tumor-promoting M2 macrophages. Importantly, the alcohol diet did not increase activation of stellate cells. Alcohol diet and cerulein injections resulted in synergistic and additive effects on PanIN lesion and M2 macrophage phenotype induction, respectively. Cerulein pancreatitis caused stellate cell activation, EMT, and cancer stemness in the pancreas. Pancreatitis caused histone deacetylation, which was promoted by the alcohol diet. Pancreatitis increased EMT and cancer stemness markers, which were not further affected by the alcohol diet. CONCLUSIONS: The results suggest that alcohol has independent effects on promotion of PDAC associated with fibrosis formed through a stellate cell-independent mechanism and that it further promotes early PDAC and M2 macrophage induction in the context of chronic pancreatitis.

Evaluating dietary compounds in pancreatic cancer modeling systems.

With the establishment of outstanding rodent models of pancreatic neoplasia and cancer, there are now systems available for evaluating the role diet, dietary supplements, and/or therapeutic compounds (which can be delivered in the diet) play in disease suppression. Several outstanding reports, which demonstrate clear inhibition or regression of pancreatic tumors following dietary manipulations, represent a noticeable advancement in the field by allowing for the contribution of diet and natural and synthetic compounds to be identified. The real goal is to provide support for translational components that will provide true chemoprevention to individuals at higher risk for developing pancreatic cancer. In addition, administration of molecules with proven efficacy in an in vivo system will screen likely candidates for future clinical trials. Despite this growing enthusiasm, it is important to note that the mere one-to-one translation of findings in rodent models to clinical outcomes is highly unlikely. Thus, careful consideration must be made to correlate findings in rodents with those in human cells with full disclosure of the subtle but often critical differences between animal models and humans. Additional concern should also be placed on the approaches employed to establish dietary components with real potential in the clinic. This chapter is focused on procedures that provide a systematic design for evaluating dietary compounds in cell culture and animal models to highlight which ones might have the greatest potential in people. The general format for this text is a stepwise use of fairly well-known approaches covered briefly but annotated with certain considerations for dietary studies. These methods include administration of a compound or a diet, measuring the cellular and molecular effects (histology, proliferation, apoptosis, RNA and protein expression, and signaling pathways), measuring the level of certain metabolites, and assessing the stability of active compounds. Though this chapter is divided into in vitro and in vivo sections, it is not an implication as to the order of experiments but an endorsement for utilizing human cells to complement work in a rodent modeling system. The notion that cell culture can provide the basis for further in vivo work is an attractive starting point, though the lack of a response in a single cell type should not necessarily prevent diet studies in rodents. The advantage of cell culture over animal models is the human origin of these cells and the ease and directness of manipulating a single cell type (particularly when exploring mechanism of action in that cell). Of course, the full effect of a diet, diet supplement, or therapeutic can only be wholly appreciated in an intact living organism with similar anatomical and physiological relevance. Thus, both approaches are considered in this chapter as each can provide unique strengths to determining the effectiveness of various dietary compounds or supplements on pancreatic neoplasia and cancer.