Kevin Adrian

Concurrent PEDF deficiency and Kras mutation induce invasive pancreatic cancer and adipose-rich stroma in mice

Background and aims Pigment epithelium-derived factor (PEDF), a non-inhibitory SERPIN with potent antiangiogenic activity, has been recently implicated in metabolism and adipogenesis, both of which are known to influence pancreatic cancer progression. Increased pancreatic fat in human pancreatic tumour correlates with greater tumour dissemination while PEDF deficiency in mice promotes pancreatic hyperplasia and visceral obesity. Oncogenic Ras, the most common mutation in pancreatic ductal adenocarcinoma (PDAC), has similarly been shown to promote adipogenesis and premalignant lesions. Methods In order to determine whether concurrent loss of PEDF is sufficient to promote adipogenesis and tumorigenesis in the pancreas, the authors ablated PEDF in an EL-KrasG12D mouse model of non-invasive cystic papillary neoplasms. Results EL-KrasG12D/PEDF deficient mice developed invasive PDAC associated with enhanced matrix metalloproteinase (MMP)-2 and MMP-9 expression and increased peripancreatic fat with adipocyte hypertrophy and intrapancreatic adipocyte infiltration (pancreatic steatosis). In support of increased adipogenesis, the stroma of the pancreas of EL-KrasG12D/PEDF deficient mice demonstrated higher tissue levels of two lipid droplet associated proteins, tail-interacting protein 47 (TIP47, perilipin 3) and adipose differentiation-related protein (ADRP, Pperilipin 2), while adipose triglyceride lipase, a key factor in lipolysis, was decreased. In patients with PDAC, both tissue and serum levels of PEDF were decreased, stromal TIP47 expression was higher and the tissue VEGF to PEDF ratio was increased (p<0.05). Conclusions These data highlight the importance of lipid metabolism in the tumour microenvironment and identify PEDF as a critical negative regulator of both adiposity and tumour invasion in the pancreas.

A high omega-3 fatty acid diet mitigates murine pancreatic precancer development.

BACKGROUND Diets containing omega-3 (ω-3) fat have been associated with decreased tumor development in the colon, breast, and prostate. We assessed the effects of a diet rich in ω-3 fat on the development of pancreatic precancer in elastase (EL)-Kras transgenic mice and examined the effect of an ω-3 fatty acid on pancreatic cancer cells in vitro. MATERIALS AND METHODS Two cohorts of EL-Kras mice were fed a high ω-3 fat diet (23% menhaden oil) for 8 and 11 mo and compared with age-matched EL-Kras mice fed standard chow (5% fat). Pancreata from all mice were scored for incidence and frequency of precancerous lesions. Immunohistochemistry was performed for proliferating cell nuclear antigen (PCNA) to assess proliferative index in lesions of mice fed either a high ω-3 or standard diet. In vitro, the effect of the ω-3 fatty acid, docosahexaenoic acid (DHA), on two pancreatic cancer cell lines was assessed. Cancer cell proliferation was assessed with an MTT assay; cell cycle analysis was performed by flow cytometry; and apoptosis was assessed with annexin/PI staining. RESULTS The incidence, frequency, and proliferative index of pancreatic precancer in EL-Kras mice was reduced in mice fed a high ω-3 fat diet compared with mice fed a standard chow. In vitro, DHA treatment resulted in a concentration-dependent decrease in proliferation through both G1/G0 cell cycle arrest and induction of apoptosis. CONCLUSIONS A high ω-3 fat diet mitigates pancreatic precancer by inhibition of cellular proliferation through induction of cell cycle arrest and apoptosis.

Modeling pancreatic cancer in vivo: from xenograft and carcinogen-induced systems to genetically engineered mice.

In the last 10 years, there has been a relative explosion of new rodent systems that recapitulate both genetic and cellular lesions that lead to the development of pancreatic cancer. These models now need to be considered when selecting an appropriate in vivo system to study disease etiology, cell signaling, and drug development. The majority of these evaluations have used transplantation of cancer cells and the use of carcinogens, which still maintain their value when investigating human cancer and epigenetic contributors. Xenograft models utilize cultured or primary pancreatic cancer cells that are placed under the skin or implanted within the pancreas of immunocompromised mice. Carcinogen-induced systems rely on administration of certain chemicals to generate cellular changes that rapidly lead to pancreatic cancer. Genetically modified mice are more advanced in their design in that relevant genetic mutations can be inserted into mouse genomic DNA in both a conditional and inducible manner. Generation of mice that develop spontaneous pancreatic cancer from a targeted genetic mutation is a valuable research tool, considering the broad spectrum of genes and cell targets that can be used, producing a variety of neoplastic lesions and cancer that can reflect many aspects of human pancreatic ductal adenocarcinoma.

Tgfbr1 Haploinsufficiency Inhibits the Development of Murine Mutant Kras-Induced Pancreatic Precancer

To dissect the role of constitutively altered Tgfbr1 signaling in pancreatic cancer development, we crossed Elastase-Kras(G12D) (EL-Kras) mice with Tgfbr1 haploinsufficient mice to generate EL-Kras/Tgfbr1(+/-) mice. Mice were euthanized at 6 to 9 months to compare the incidence, frequency, and size of precancerous lesions in the pancreas. Only 50% of all EL-Kras/Tgfbr1(+/-) mice developed preinvasive lesions compared with 100% of EL-Kras (wild-type Tgfbr1) mice. The frequency of precancerous lesions was 4-fold lower in haploinsufficient than in control mice. Paradoxically, the precancerous lesions of EL-Kras/Tgfbr1(+/-) mice were considerably larger than those in EL-Kras mice. Yet, the mitotic index of precancerous cells and the observable levels of fibrosis, lipoatrophy, and lymphocytic infiltration were reduced in EL-Kras/Tgfbr1(+/-) mice. We conclude that Tgfbr1 signaling promotes the development of precancerous lesions in mice. These findings suggest that individuals with constitutively decreased TGFBR1 expression may have a decreased risk of pancreatic cancer.

Alteration of strain background and a high omega-6 fat diet induces earlier onset of pancreatic neoplasia in EL-Kras transgenic mice†

Diets containing omega‐6 (ω‐6) fat have been associated with increased tumor development in carcinogen‐induced pancreatic cancer models. However, the effects of ω‐6 fatty acids and background strain on the development of genetically‐induced pancreatic neoplasia is unknown. We assessed the effects of a diet rich in ω‐6 fat on the development of pancreatic neoplasia in elastase (EL)‐KrasG12D (EL‐Kras) mice in two different backgrounds. EL‐Kras FVB mice were crossed to C57BL/6 (B6) mice to produce EL‐Kras FVB6 F1 (or EL‐Kras F1) and EL‐Kras B6 congenic mice. Age‐matched EL‐Kras mice from each strain were compared to one another on a standard chow. Two cohorts of EL‐Kras FVB and EL‐Kras F1 mice were fed a 23% corn oil diet and compared to age‐matched mice fed a standard chow. Pancreata were scored for incidence, frequency, and size of neoplastic lesions, and stained for the presence of mast cells to evaluate changes in the inflammatory milieu secondary to a high fat diet. EL‐Kras F1 mice had increased incidence, frequency, and size of pancreatic neoplasia compared to EL‐Kras FVB mice. The frequency and size of neoplastic lesions and the weight and pancreatic mast cell densities in EL‐Kras F1 mice were increased in mice fed a high ω‐6 fatty acid diet compared to mice fed a standard chow. We herein introduce the EL‐Kras B6 mouse model which presents with increased frequency of pancreatic neoplasia compared to EL‐Kras F1 mice. The phenotype in EL‐Kras F1 and FVB mice is promoted by a diet rich in ω‐6 fatty acid.

Visualization of mouse pancreas architecture using MR microscopy.

Pancreatic diseases, which include diabetes, pancreatitis, and pancreatic cancer, are often difficult to detect and/or stage, contributing to a reduced quality of life and lifespan for patients. Thus, there is need for a technology that can visualize tissue changes in the pancreas, improve understanding of disease progression, and facilitate earlier detection in the human population. Because of low spatial resolution, current clinical magnetic resonance imaging (MRI) at low field strength has yet to fully visualize the exocrine, endocrine, vascular, and stromal components of the pancreas. We used high field strength magnetic resonance microscopy (μMRI) to image mouse pancreas ex vivo without contrast agents at high spatial resolution. We analyzed the resulting high-resolution images using volume rendering to resolve components in the pancreas, including acini, islets, blood vessels, and extracellular matrix. Locations and dimensions of pancreatic components as seen in three-dimensional μMRI were compared with histological images, and good correspondence was found. Future longitudinal studies could expand on the use of in vivo μMRI in mouse models of pancreatic diseases. Capturing three-dimensional structural changes through μMRI could help to identify early cellular and tissue changes associated with pancreatic disease, serving as a mode of improved detection in the clinic for endocrine and exocrine pathologies.

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 A84: ω3 rich diet protects against Kras-induced metaplasia via repression of NF-κB

Expression of mutant Kras is observed and often implicated in both pancreatitis and pancreatic cancer. Yet, the means by which inflammation occurs is relatively unexplored, although a previous report supports that induction of inflammation in the presence of oncogenic Kras initiates an NF-κB–mediated positive feedback loop that incorporates Cox-2 and production of PGE2 which further amplify mutant Kras. The involvement of PGE2 is interesting, as recent data suggests diets rich in ω6 fatty acids promote Kras induced pancreatic inflammation and neoplastic lesions. However, the precise mechanism and role of epigenetic events derived from diets rich in fatty acids have yet to be completely resolved. In this work, we demonstrate that a key step in this process is the induction and accumulation of NF-κB in pancreatic epithelia, a factor sufficient to promote chronic inflammation and progression to pancreatic adenocarcinoma. In HPDE cells, inhibition of NF-κB shows promise, as treatment with the IKK inhibitor Bay11-7085 noticeably reduced levels of the pro-proliferative factor pAkt. In EL-Kras mice fed a diet rich in ω6 fatty acids, NF-kB is upregulated in the pancreas, predominantly in metaplastic lesions. Conversely, these transgenic mice fed an ω3 fatty acid diet show little, if any, NF-kB expression, which is similar to the response in mice fed an intermediate, isocaloric high fat diet. pAkt is known to act upstream of NF-κB, implicating that NF-κB may be involved in a positive feedback loop with a variety of Kras effectors culminating in pancreatitis and eventually pancreatic neoplasia/adenocarcinoma. It has also been demonstrated that the ω6 Cox2-metabolite PGE2 is mediating this response, though in cultured human pancreatic epithelia, Cox-2 inhibition showed no significant change on levels of NF-κB. Yet, Cox2 inhibition in the stroma may be synergistic with epithelial inhibition of NF-κB, offering protection against the progression from inflammation to cancer. This data provides support to multiple targets of chemoprevention and therapy, including Ras and IKK/NF-κB, for pancreatic neoplasia and cancer. Citation Format: Daniel R. Principe, Windel Emman T. Mascarinas, Chintan Chheda, Kevin Adrian, Riley Mangan, Lindsay C. Boven, Paul J. Grippo. ω3 rich diet protects against Kras-induced metaplasia via repression of NF-κB. [abstract]. In: Proceedings of the Eleventh Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2012 Oct 16-19; Anaheim, CA. Philadelphia (PA): AACR; Cancer Prev Res 2012;5(11 Suppl):Abstract nr A84.

Abstract 3144: Tgfbr1 haploinsufficiency inhibits the development of murine mutant Kras-induced pancreatic precancer

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Abstract Transforming Growth Factor Beta (TGFβ) signaling can play dual roles in pancreatic tumor development. In human pancreatic cancer, loss of DPC4/smad4 has been observed in more than half of all pancreatic cancers. In vivo loss of either Smad4 or Tgfbr2 promotes pancreatic adenocarcinoma in mice expressing mutant Kras. Conversely, pharmacologically-induced reduction in Tgfbr1 kinase activity attenuates growth and metastasis of Panc1 cells orthotopically injected into mouse pancreas. To dissect the role of constitutively altered Tgfbr1 signaling in pancreatic cancer development, we crossed Elastase-KrasG12D (EL-Kras) mice with Tgfbr1 haploinsufficient mice to generate EL-Kras/Tgfbr1+/− mice. Mice were euthanized at 6-9 months to compare the incidence, frequency, and size of precancerous lesions in the pancreas. Only 50% of all EL-Kras/Tgfbr1+/− mice developed preinvasive lesions compared to 100% of EL-Kras (wild type Tgfbr1) mice. The frequency of precancerous lesions was 4-fold lower in haploinsufficient than in control mice. The mitotic index of precancerous cells and the observable levels of fibrosis, lipoatrophy, and lymphocytic infiltration were reduced in EL-Kras/Tgfbr1+/− mice. Tgfbr1 haploinsufficiency was demonstrated by decreased levels of psmad2, psmad3, and nuclear localization of psmad4 and was culminated by a reduction in the Tgfbr1: Tgfbr2 ratio in whole mouse pancreas, which was predominantly caused by increased Tgfbr2. Paradoxically, the precancerous lesions of EL-Kras/Tgfbr1+/− mice were considerably larger than those in EL-Kras mice, which was due, in part, to a reduced level of apoptosis. We conclude that Tgfbr1 signaling promotes the development of precancerous lesions in mice. These findings correlate with TGF-β signals that increase ECM deposition and augment immune responses and strongly suggest that Tgfbr1 haploinsufficiency may have a protective role with respect to early pancreatic cancer development. It is also possible that individuals with constitutively decreased TGFBR1 expression may have a decreased risk of pancreatic cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3144.