Andrew M. Diaz

TGFβ signaling in the pancreatic tumor microenvironment promotes fibrosis and immune evasion to facilitate tumorigenesis

In early pancreatic carcinogenesis, TGFβ acts as a tumor suppressor due to its growth-inhibitory effects in epithelial cells. However, in advanced disease, TGFβ appears to promote tumor progression. Therefore, to better understand the contributions of TGFβ signaling to pancreatic carcinogenesis, we generated mouse models of pancreatic cancer with either epithelial or systemic TGFBR deficiency. We found that epithelial suppression of TGFβ signals facilitated pancreatic tumorigenesis, whereas global loss of TGFβ signaling protected against tumor development via inhibition of tumor-associated fibrosis, stromal TGFβ1 production, and the resultant restoration of antitumor immune function. Similarly, TGFBR-deficient T cells resisted TGFβ-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 TGFβ 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, TGFβ 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 TGFβ signals in the epithelium. Cancer Res; 76(9); 2525-39. ©2016 AACR.

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

TGFβ has both tumor suppressive and tumor promoting effects in colon cancer. Also, TGFβ 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 TGFβ to this process are poorly understood. In human patients, we found that epithelial loss of TGFβ signaling was associated with increased inflammatory burden; yet overexpression of TGFβ 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 TGFβ1. Interestingly, mutant APC mice with global suppression of TGFβ 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 TGFβ signaling, particularly in colon epithelial cells, elicits a strong inflammatory response and promotes tumor progression. This implies that treating colon cancer patients with TGFβ inhibitors may result in a worse outcome by enhancing inflammatory responses.

PEDF inhibits pancreatic tumorigenesis by attenuating the fibroinflammatory reaction

Pancreatic cancer is characterized by a pronounced fibro-inflammatory reaction that has been shown to contribute to cancer progression. Previous reports have demonstrated that pigment epithelium-derived factor (PEDF) has potent tumor suppressive effects in pancreatic cancer, though little is known about the mechanisms by which PEDF limits pancreatic tumorigenesis. We therefore employed human specimens, as well as mouse and in vitro models, to explore the effects of PEDF upon the pancreatic microenvironment. We found that PEDF expression is decreased in human pancreatic cancer samples compared to non-malignant tissue. Furthermore, PEDF-deficient patients displayed increased intratumoral inflammation/fibrosis. In mice, genetic ablation of PEDF increased cerulein-induced inflammation and fibrosis, and similarly enhanced these events in the background of oncogenic KRAS. In vitro, recombinant PEDF neutralized macrophage migration as well as inhibited macrophage-induced proliferation of tumor cells. Additionally, recombinant PEDF suppressed the synthesis of pro-inflammatory/pro-fibrotic cytokines both in vivo and in vitro, and reduced collagen I deposition and TGFβ synthesis by pancreatic stellate cells, consistent with reduced fibrosis. Combined, our results demonstrate that PEDF limits pancreatic cancer progression by attenuating the fibro-inflammatory reaction, and makes restoration of PEDF signaling a potential therapeutic approach to study in pancreatic cancer.

Thioredoxin system-mediated regulation of mutant Kras associated pancreatic neoplasia and cancer

Peroxiredoxin-1 (Prdx1), a member of the thioredoxin (Txn) system, is overexpressed and correlates with poor prognosis in pancreatic cancer patients and can suppress Kras signaling through redox-mediated inhibition of ERK and AKT in lung and breast cancer. Its redox function is maintained by Txn and sulfiredoxin (Srxn), and its tumor promoting functions are activated by post-translational modification. We studied the role of the Txn system in pancreatic neoplasia and cancer by determining how it regulates the phosphorylation of Kras effectors and by determining its association with patient survival. We found that elevated Prdx1 nuclear localization significantly correlated with better patient survival. Our data also demonstrate that the expression of the Txn system is dysregulated, with elevated Prdx1 expression and significantly decreased Txn and Srxn expression in pancreatic lesions of targeted mutant Kras mouse models. This correlated with distinct differences in the interconversion of Prdx1 oligomers that affect its ability to regulate ERK and AKT phosphorylation. Our data also suggest that Prdx1 post-translational modification and oligomerization suppress Prdx1 mediated redox regulation of ERK phosphorylation. We observed distinct differences in Txn expression and in the ability of pTyr-Prdx1 to bind to pERK in a PanIN model of pancreatic neoplasia as compared to an IPMN model, indicating a distinct difference in the function of post-translationally modified Prdx1 in cells with less Txn expression. Modified Txn system function and post-translational regulation may therefore play a significant role in pancreatic tumorigenesis by altering Kras effector phosphorylation and inhibiting the tumor suppressive redox functions of Prdx1.

The Complexity of Omega-3 Fatty acids modulation of Signaling Pathways related to Pancreatic Cancer

Cancer is a major public health problem worldwide and is the second leading cause of death in the United States. Although cancer death rate has dropped by 23% since 1991, there are certain types of cancer for which death rates are still increasing, such as pancreatic cancer. There is an urgent need to find new therapies that could help improve this dreadful outcome. In this regard, the role of nutrition in health and disease has attracted much attention. Several dietary components are involved in metabolic, physiologic and cell signaling affecting tumor growth and progression. Although lipids, and more specifically polyunsaturated fatty acids, have been traditionally studied due to their health effects in cardiovascular disease, it is now clear that they can impact an extensive array of cellular processes that influence a wide range of diseases such as type II diabetes, inflammatory disorders and cancer. These biological activities may be grouped as regulation of: (1) membrane structure and function, (2) intracellular signaling pathways, (3) transcription factor activity, (4) gene expression, and (5) production of bioactive lipid mediators. The aim of this review is to assimilate the current state of knowledge about these potential mechanism(s) of action and signaling pathways modulated by polyunsaturated fatty acids in pancreatic cancer.

Abstract 4178: Pigs as a new weapon against cancer: Modeling solid tumors in porcine

Widely-used genetically modified mice have revolutionized the field of cancer research by providing reliable in vivo systems with similar, if not identical, molecular alterations. Furthermore, Cre/Lox technology has afforded greater tissue specificity, with many of the developing cancer phenotypes recapitulating those observed in humans. However, despite these strengths, there are a number of notable limitations when comparing mice to humans including small sample sizes, dramatic differences in physiology, and often dissimilar drug responses. Pig models would alleviate these shortcomings if they were to have a repertoire of gene signatures observed in human cancer, like those engineered in mice. Therefore, to achieve this goal, we have engineered a pig model with the Cre-responsive transgene encoding KRASG12D and TP53R167H. Hence, tissue-specific targeted Cre would generate simultaneous expression of mutant KRAS and p53 alleles to induce cellular changes leading to cancer. Pig pancreases were injected with adenovirus containing a cre expression vector (adenoCre) in an attempt to generate a model of pancreatic cancer. Initial injections of adenoCre into the parenchymal body of the pancreas led to the development of acinar-ductal metaplasia (ADM), though the clear predominant histotype was leiomyosarcoma immediately adjacent to the pancreas. Pancreatic ADM was characterized by concomitant localization of amylase (acinar cell marker) and CK19 (ductal cell marker) in the same pancreatic acinus, with an occasional few cells expressing both cell markers. These lesions were further characterized and exhibited increased proliferation via PCNA staining, as well the development of surrounding desmoplastic stroma (enhanced trichrome, vimentin, and aSMA staining; a marker of pancreatic stellate cells) with a robust inflammatory component (strong CD11b and CAE staining). Based on the lack of pancreas specificity, the experiment was repeated by injecting adenoCre into the main pancreatic duct. This 10-month-old pig appears relatively healthy after, which was expected considering this was a fully adult pig prior to manipulation. We will fully access this pig in 2 months to determine if strict pancreatic duct injection of adenoCre induces ADM, neoplastic disease, and/or ductal adenocarcinoma. Based on these encouraging findings, we anticipate having a robust model of pancreatic cancer in the pig, and plan to extend this study to generate models of other solid tumors with high rates of KRAS and p53 mutations. These porcine models may significantly impact preclinical studies, as diseases induced in pigs are likely more physiologically relevant to those in humans, with a potentially similar pharmacokinetic profile following drug treatment. Indeed, due to the size of the organs and volume of blood, samples can be more readily shared among multiple groups of investigators facilitating greater collaboration and expanding future research. Citation Format: Andrew Diaz, Daniel Principe, Brian DeCant, Paul J. Grippo, Laurie Rund, Lawrence Schook. Pigs as a new weapon against cancer: Modeling solid tumors in porcine. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4178.

KRAS G12D and TP53 R167H Cooperate to Induce Pancreatic Ductal Adenocarcinoma in Sus scrofa Pigs

Although survival has improved in recent years, the prognosis of patients with advanced pancreatic ductal adenocarcinoma (PDAC) remains poor. Despite substantial differences in anatomy, physiology, genetics, and metabolism, the overwhelming majority of preclinical testing relies on transgenic mice. Hence, while mice have allowed for tremendous advances in cancer biology, they have been a poor predictor of drug performance/toxicity in the clinic. Given the greater similarity of sus scrofa pigs to humans, we engineered transgenic sus scrofa expressing a LSL-KRASG12D-TP53R167H cassette. By applying Adeno-Cre to pancreatic duct cells in vitro, cells self-immortalized and established tumors in immunocompromised mice. When Adeno-Cre was administered to the main pancreatic duct in vivo, pigs developed extensive PDAC at the injection site hallmarked by excessive proliferation and desmoplastic stroma. This serves as the first large animal model of pancreatic carcinogenesis, and may allow for insight into new avenues of translational research not before possible in rodents.

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.

Abstract 805: KRASG12Dand TP53R167Hcooperate to induce pancreatic carcinoma in conditional transgenicSus scrofta

Although survival has improved in recent years, pancreatic ductal adenocarcinoma (PDAC) remains remarkably virulent with a median survival time of only four months. As there is no effective strategy for the management of PDAC, there is a need for the development of new preclinical strategies. The majority of this work is being done in genetically modified mice, which faithfully recapitulate a variety of pancreatic cancer histotypes. While mice have allowed for unprecedented insight into pancreatic cancer etiology, due to fundamental differences in anatomy and physiology, mouse models of PDAC often fail to accurately predict responsiveness to therapy. As the pig pancreas has more similar anatomical orientation and localization to humans than that of rodents, pigs may serve as a more relevant model for PDAC and other malignancies of the pancreas. As KRAS and TP53 mutations are observed in approximately 95% and 70% of PDAC patients, respectively, we generated Cre/lox transgenic Sus scrofta expressing a LSL-KRASG12D-TP53R167H cassette and administered an Adeno-Cre particle to the pancreas gland parenchyma. This resulted in a mixed histoype of metastatic leiomysarcoma and neoplastic disease of the pancreas. To produce a similar pancreas histoype while avoiding the leiomyosarcoma, we next restricted the Adeno-Cre administration to the main pancreatic duct, which resulted in both PDAC and neuroendocrine carcinoma with no abnormality of the smooth muscle. This serves as the first large animal model of pancreatic carcinogenesis, and given the anatomical/physiological similarity of pigs and humans, may allow for insight into new avenues of research not before possible in rodents. Citation Format: Daniel Principe, Nana Overgaard, Andrew Diaz, Carolina Torres, Ronald McKinney, David Dawson, Laurie Rund, Regina Schwind, Paul J. Grippo, Lawrence Schook. KRASG12D and TP53R167H cooperate to induce pancreatic carcinoma in conditional transgenic Sus scrofta [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 805. doi:10.1158/1538-7445.AM2017-805

Abstract 2805: Protein oxidation in pancreatic neoplasia and cancer

Rationale- Peroxiredoxin-1 (Prdx1) is overexpressed in pancreatic cancer patient serum and correlates with worse prognosis. Prdx1 is an important antioxidant protein whose deletion causes the formation of malignant lesions in mice. Prdx19s redox function depends on its reduction by two other Thioredoxin system members, Thioredoxin (Txn) and Sulfiredoxin (Srxn). Prdx1 may also contribute to tumorigenesis by oligomerizing and becoming a nuclear chaperone when it9s overoxidized and its redox function is inhibited. Objective- Our objective was to investigate disruptions in the Txn system during pancreatic neoplasia and to determine how that affects phosphorylation of Kras effectors. Findings- Prdx1 expression was elevated and Srxn expression was reduced in pancreatic lesions of El-Kras, Pdx1-Cre/LSL-Kras (KC-Pdx1), and P48-Cre/LSL-Kras (KC-P48) mice. Txn was absent in lesions of KC-Pdx1 and KC-P48 mice but present and differentially located in EL-Kras lesions. In primary pancreatic acinar cells, oxidation of the Txn system with auranofin increased ERK and AKT phosphorylation in EL-Kras mice, but not in KC-P48 mice, indicating differential regulation of the Txn system in KC-P48 mice. This could be due to the presence of a higher molecular weight Prdx1 being present in KC-p48 mice and not in EL-Kras mice. This suggests that the effect of mutant Kras on the thioredoxin system in these two models may be different. Prdx1 and (p)ERK also interacted in vitro and inhibition of ERK and AKT phosphorylation modified the expression of Txn and Srxn in pancreatic cell lines. We also found that in human pancreatic cancer patient tissue, Prdx1 was overexpressed in pancreatic cancer as compared to adjacent normal tissue. Conclusions- The Txn system is disrupted during pancreatic neoplasia and may be associated with modified ERK and AKT regulation in Kras mutant pancreatic tissue. Citation Format: Michelle Schultz, Andrew Diaz, Sharon Smite, Brian DeCant, David Bentrem, Paul Grippo. Protein oxidation in pancreatic neoplasia and cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2805.