PDAC Development Research Articles

Dynamic Mast Cell–Stromal Cell Interactions Promote Growth of Pancreatic Cancer

Pancreatic ductal adenocarcinoma (PDAC) exists in a complex desmoplastic microenvironment, which includes cancer-associated fibroblasts [also known as pancreatic stellate cells (PSC)] and immune cells that provide a fibrotic niche that impedes successful cancer therapy. We have found that mast cells are essential for PDAC tumorigenesis. Whether mast cells contribute to the growth of PDAC and/or PSCs is unknown. Here, we tested the hypothesis that mast cells contribute to the growth of PSCs and tumor cells, thus contributing to PDAC development. Tumor cells promoted mast cell migration. Both tumor cells and PSCs stimulated mast cell activation. Conversely, mast cell-derived interleukin (IL)-13 and tryptase stimulated PSC proliferation. Treating tumor-bearing mice with agents that block mast cell migration and function depressed PDAC growth. Our findings suggest that mast cells exacerbate the cellular and extracellular dynamics of the tumor microenvironment found in PDAC. Therefore, targeting mast cells may inhibit stromal formation and improve therapy.

Abstract 2741: NF-κB activation by IL-1α and p62 feedforward loops is required for Kras-induced pancreatic ductal adenocarcinoma.

Abstract Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer death in the United States with a less than six month mean survival period and less than five percent of an overall five-year survival rate. Despite developments in the management of pancreatic cancer, the survival rate has remained unchanged in the past 50 years. Thus, a better understanding of the mechanism of pancreatic cancer inception and development is the key to identifying prevention strategies and therapeutic targets for increasing patient survival rates, which is one of the greatest challenges in pancreatic cancer research. The mutational activation of Kras is an early event in PDAC development and has been detected in 90% of PDAC and 50% of pancreatic intraepithelial neoplasia (PanIN). The genetic mutation of oncogenic Kras in the pancreas induces chronic pancreatitis and pancreatic cancer. However, the key signaling pathway of the downstream Kras remains unidentified. Here, we report that pancreas-specific targeted IKK2/β inactivation inhibited NF-κB activation and PDAC development in KrasG12D and KrasG12D; Ink4a/ArfF/F mice, demonstrating a mechanistic link between IKK2/β/NF-kB signaling pathway and KrasG12D signaling pathways in PDAC inception. Our findings reveal that KrasG12D-activated AP-1 induces IL-1α, which, in turn, activates NF-κB and its target genes IL-1α and p62, to initiate IL-1α/p62 feedforward loops for inducing and sustaining NF-κB activity. Furthermore, IL-1α overexpression correlates with Kras mutation, NF-κB activity, and poor survival in PDAC patients. Knock-down of IL-1α in mouse PDAC cell lines and lung adenocarcinoma cell lines significantly reduces tumorigenesis in orthotopic xenograft mouse and subcutaneous xenograft mouse models. Therefore, our findings demonstrate the new mechanism by which IKK2/β/NF-κB is activated by KrasG12D through dual feedforward loops of IL-1α/p62 to promote development of PDAC. This suggests that the interruption of the dual feedforward loops of IL-1α/p62 could be the therapeutic targets for inhibiting oncogenic Kras signaling in PDAC. Citation Format: Jianhua Ling, Ya'an Kang, Paul Chiao. NF-κB activation by IL-1α and p62 feedforward loops is required for Kras-induced pancreatic ductal adenocarcinoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2741. doi:10.1158/1538-7445.AM2013-2741

Abstract 2742: Activation of Wnt/β-catenin signaling accelerates progression of Kras-induced pancreatic cancer.

Abstract Pancreatic ductal adenocarcinoma (PDAC), a poor prognostic cancer, commonly develops following activating mutations in the KRAS oncogene. Activation of the Wnt signaling pathway is also commonly observed in PDAC. To ascertain the impact of postnatal activation of the Wnt signaling pathways in PDAC development, we combined the elastase-tva-based RCAS-TVA pancreatic cancer model with the established LSL-KrasG12D, Ptf1a-cre model. Delivery of RCAS viruses encoding β-cateninS37A and Wnt1 stimulated the progression of premalignant PanIN and PDAC development. Moreover, mice injected with RCAS-β-cateninS37A and Wnt1 had reduced survival relative to RCAS-GFP controls (log-rank test; p < 0.05). Meanwhile, active β-catenin or its DNA-binding partner TCF4 enhanced PDAC cell proliferation, soft-agar colony formation, as well as migration and invasion activity. In contrast, these phenotypes were significantly blocked by the introduction of Icat, an inhibitor of the β-catenin/TCF4 interaction. Interestingly, Id2 (inhibitor of differentiation/DNA binding2) was significantly up-regulated by induction of β-catenin and TCF4, whereas Id2 expression was inhibited by Icat. Furthermore, nuclear β-catenin and Id2 were mainly observed in poorly differentiated PDACs and sarcomatoid tumors. Together, these data suggest that the Wnt/β-catenin signaling pathway stimulates pancreatic tumor development and progression through the activation of Id2. Citation Format: Makoto Sano, David R. Driscoll, Wilfredo E. DeJesus-Monge, David S. Klimstra, Brian C. Lewis. Activation of Wnt/β-catenin signaling accelerates progression of Kras-induced pancreatic cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2742. doi:10.1158/1538-7445.AM2013-2742

Abstract 2729: A genetically engineered mouse model of de novo pancreatic carcinogenesis .

Abstract Multistep models of PDAC development converge on both abnormalities in KRAS and E2F activation, which are part of the Rb/E2F pathway, and dysfunction of p53, which is associated with the MDM2/p53 pathway. Recent progress in the understanding of the protective roles of the Rb/E2F and MDM/p53 pathways against carcinogenesis showed that cross talk and complementary actions of these pathways are present in many types of cancers. In this study, we addressed directly questions regarding the collaborative roles of the Rb/E2F and MDM2/p53 pathways in suppressing the progression of multistep PDAC carcinogenesis by generating TKC (loxP-βgeo-STOP-loxPtemperature-sensitive SV40 T antigen (tsTAg)), loxP-STOP-loxP-KrasG12D, and Pdx1-Cre) mice which exhibit impairment of both pathways and K-rasG12D expression in pancreatic epithelial cells. The PDAC induced in TKC mice exhibited rapid development and absence of inflammation or PanIN formation, which occurred by de novo carcinogenesis. The tumors resembled human PDAC at the histological and molecular level. In in vitro study, we established PDAC cell lines from TKC mice and immortalized pancreatic ductal cell lines from TC mice, Cytokeratin 19 expression with which was applied to select cells as pancreatic ductal cell differentiation. In collagen gel 3D culture, PDAC cells formed atypical gland structures the same as in subcutaneous transplanted tumor in nude mice, while the Cytokeratin 19 positive immortalized ductal cell lines from TC mice formed ductal structures similar to either intralobular or interloblar pancreatic ducts. The results of the analysis of the highlighted molecular mechanisms suggest that the synthetic interaction among mutated Kras, uncontrolled E2f activation, and dysfunction of p53 results in de novo pancreatic carcinogenesis. This model represents an important tool to explore molecular targets for diagnostics and cancer therapy for PDAC via de novo pancreatic carcinogenesis.This work was supported by grants from the Program for Promotion of Basic Research Activities for Innovative Biosciences (PROBRAIN) and New Energy and Industrial Technology Development Organization (NEDO) of Japan, and supported in part by Grants-in-Aid for Scientific Research on Priority Area (21590454, 24590498, and 24108006 to Y. I.) from the Ministry of Education, Culture, Sports, Science and Technology of Japan. Citation Format: Takashi Yamaguchi, Sanae K. Ikehara, Yuzuru Ikehara. A genetically engineered mouse model of de novo pancreatic carcinogenesis . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2729. doi:10.1158/1538-7445.AM2013-2729

Abstract C14: Optical imaging of integrin αVβ3 detects pancreatic cancer in endogenous mouse models.

Abstract Pancreatic ductal adenocarcinoma remains one of the most lethal malignacies in the Western World with a 5-year survival rate lower then 4%. Late diagnosis and poor response to all available chemotherapies are main causes for the devastating clinical course. Development of methods for early detection and evaluation of new therapies is of great importance. Here, we used optical imaging for detection of αVβ3 integrin in pancreatic cancer in endogenous mouse models. To model stages of PDAC development in humans, following genetically engineered animal models (GEMM) were used: mice with pancreas specific activation of oncogenic Kras (p48+/Cre Kras+/LSL-G12D, CK) that develop preneoplastic lesions preceding the cancer and mice with concomitant deletion of p53 (p48+/Cre Kras+/LSL-G12D p53Lox/Lox, CKP) that develop PDAC already at 6–8 weeks of age. Pancreatic αVβ3 integrin expression was analysed by immunohistochemistry and Western blot. For optical imaging, animals were injected with Integrisense680, Near Infrared fluorochrome labelled agent that specifically binds to αVβ3. After 24h, animals were subjected to intraoperative epi-illumination optical imaging procedure. Sacrificed animals were whole-mount frozen for analysis of Integrisense intra-tissue distribution via multispectral cryoslicing optical imaging. For comparison, T2 weighted (T2w) images of the abdomen were generated with 1,5T clinical MRI scanner and a dedicated surface coil. β3 protein expression was dramatically increased in CKP pancreatic tumors compared to CK pancreata. β3 positive cells were found among malignant cells and in the reactive stroma. In CK animals, β3 expression was specifically limited to ductal and preneoplastic lesions and surrounding stroma with no expression in normal tissue. Intraoperative optical imaging of Integrisense correlated well with the high β3 expression in the tumor and demonstrated very strong and specific fluorescence signals at the tumor surface with excellent demarcation of tumor borders and good contrast to surrounding tissues. CK pancreas was well visualized and spotty fluorescence pattern of Integrisense was observed. Cryoslicing imaging of the animals confirmed that Integrisense680 penetrated well into all parts of the tumor and accumulated very specifically in cancer tissue in CKP animals. In CK pancreata, spots of fluorescence were also observed throughout the tissue confirming intraoperative imaging. Spots of fluorescence correlated well with the foci of ductal and preneoplastic lesions embedded in healthy tissue as confirmed by histopathological analysis. In contrast, preneoplastic lesions were difficult to differentiate on T2w-MRI images due to low spatial resolution. Tumors appeared relative homogenously hyperintense on T2w images and tumor borders were in certain cases difficult to identify, when adjacent structures, i.e. gut, were of similar signal intensity. We demonstrated increased expression of αVβ3 integrin in pancreatic tumors in GEM models and ability to detect this integrin via optical imaging procedures. We were able to specifically identify the cancer, demarcate its borders and differentiate between pancreatic tissue with preneoplastic lesions and cancer. We suggest that optical imaging of αVβ3 integrin is a promising future approach for clinical practice. αVβ3 fluorescence guided surgery can assist the surgeon in precise demarcation of surgical margins and minimisation of residual tissue. Additionally, targeted Integrisense optical imaging would allow early evaluation αVβ3 targeted therapies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr C14.

SPERM ASSOCIATED ANTIGEN 1 (SPAG1) IS EXPRESSED EARLY IN PANCREATIC TUMORIGENESIS AND PROMOTES MOTILITY OF CANCER CELLS

SPAG1, Sperm associated antigen 1, was recently identified in a rare form of infertility where anti-SPAG1 antibodies in the serum of an affected woman cause sperm agglutination and subsequent infertility. Except for reported expression of SPAG1 in spermatogenesis, the function of this gene is completely unknown. The unexpected finding of high levels of SPAG1 expression in pancreatic adenocarcinoma compared to normal pancreatic tissue in our gene expression profiling studies prompted us to look in more detail at the expression and function of this gene. With in-house generated SPAG1-specific monoclonal antibody we have confirmed high levels of SPAG1 protein in both testis and in a large proportion of pancreatic ductal adenocarcinomas. In the latter, SPAG1 expression was predominantly cytoplasmic and confined to malignant cells. Furthermore, the extent and intensity of SPAG1 expression was shown to be associated with stage and tumour nodal status, while analysis of precursor lesions, PanINs (pancreatic intraepithelial neoplasia), demonstrated its increased immunoreactivity with increasing PanIN grade, suggesting that SPAG1 is a novel marker of PDAC progression. By immunocytochemistry, we have demonstrated co-localization of SPAG1 with microtubules, and confirmed their association by co-immunoprecipitation. Motility assays further substantiated a potential role of SPAG1 in cancer cell motility. Combined with the finding of its early expression in PDAC development, our data suggest that SPAG1 could contribute to the early spread and invariably poor prognosis of pancreatic adenocarcinoma.