Project description:Pancreatic ductal adenocarcinoma (PDAC) remains a major unsolved health problem. Most drugs that pass preclinical tests fail in these patients, emphasizing the need of appropriate preclinical models to test novel anticancer strategies. We developed four orthotopic mouse models employing primary human PDAC cells expressing Firefly and Gaussia luciferases, enabling bioluminescence monitoring of tumor growth and metastasis formation. Additional tumor characterization was performed using MR and high frequency ultrasound imaging. Genomic and immunohistochemical analysis revealed c-Met amplification and overexpression in one of four models. Analysis of c-Met inhibitors in vitro showed that crizotinib had the most potent effect. Moreover, we demonstrated synergistic effects between crizotinib and gemcitabine M-bM-^@M-^S the standard of care therapeutic in PDAC patients - in vitro and in vivo. Importantly, crizotinib reduced the cytidine deaminase activity in PDAC cells causing prolonged activity of gemcitabine due to diminished metabolic inactivation, as measured by LC-MS/MS. This might at least in part explain the observed prolonged survival of concomitantly treated mice with PDAC tumors and metastases. In conclusion, our orthotopic PDAC models enabled PDAC tumor imaging, and showed genetic, histopathological and metastatic features similar to their originator tumors. This allowed the identification of c-Met as a potential therapeutic target in PDAC, and revealed a cytidine deaminase-mediated synergistic mechanism between crizotinib and gemcitabine, a combination of drugs that warrants further investigation for the potential treatment of PDAC patients. 12 test samples in total [4 PDAC models (PDAC-1, PDAC-2, PDAC-3, PDAC-4; in three panel: primary human PDAC, primary tumor culture and mouse sample)] and reference sample (healthy control (mix/pool of healthy volunteers DNA) were analyzed as following (8 hybridizations); PDAC-1 primary human-Cy3 vs PDAC-4 cultured cells -Cy5 PDAC-1 cultured cells-Cy3 vs PDAC-4 mouse-Cy5 PDAC-1 mouse-Cy3 vs reference-Cy5 PDAC-2 primary human-Cy3 vs reference-Cy5 PDAC-2_cultured cells-Cy3 vs PDAC-2 mouse-Cy5 PDAC-3_primary human-Cy3 vs reference-Cy5 PDAC-3_cultured cells-Cy3 vs PDAC-3 mouse-Cy5 PDAC-4 primary human-Cy5 vs reference-Cy3 Normalized log2 ratio of (sample/references) data were calculated for 12 samples [*txt files on Series records]. Description of experimental/analysis design in r-program to generate 12 samples from 8 raw data files is provided in README.txt [Series supplementary file]

Project description:Pancreatic ductal adenocarcinoma (PDAC) remains among the most lethal of human cancers underscoring the need to identify new therapeutic vulnerabilities. Previously, we reported high expression of transcriptional co-regulators C-terminal binding proteins (CtBP) 1 and 2 in human PDAC, however their precise role in PDAC formation or progression remains unclear. Here, we have studied PDAC tumor dependency on CtBPs for growth and metastasis using an orthotopic syngeneic pancreatic tumor mouse model. CRISPR-based homozygous deletion of Ctbp2 dramatically decreased PDAC tumor growth, drastically reduced metastatic potential, and significantly prolonged survival. Interrogating differential gene expression of the orthotopic PDAC tumors from CtBP2 WT vs. CtBP2 KO cohorts, we identified significant downregulation of the EGFR-superfamily receptor ErbB3 in CtBP2 KO tumors, and further determined that CtBP regulates expression of both the ErbB2 and 3 EGFR superfamily genes in PDAC cells. We therefore hypothesized that CtBP regulation of physiologic ErbB2/3 signaling contributes, in part, to PDAC growth and metastasis. Indeed, we observed that CtBP2 KO cells exhibited severely attenuated activation of phospho-Akt after neuregulin stimulation of ErbB2/3, and we demonstrate that human PDAC cells also show CtBP dependence of ErbB2/3 expression. Our results suggest that a subset of PDAC tumors is dependent on physiologic ErbB2/3 signaling and could be targeted by pharmacologic inhibitors of ErbB2 and/or ErbB3. Providing proof of concept, the ErbB2-targeted multikinase inhibitor lapatinib, but not the ErbB1/EGFR targeted agent erlotinib, effectively killed ErbB3 expressing PDAC cells, while CtBP2 KO cells where ErbB3 expression was extinguished, were resistant to lapatinib. Taken together, our data suggests that ErBb2/3 targeted therapeutics can effectively target a critical PDAC dependency on physiologic ErbB2/3 signaling which is the result of CtBP’s oncogenic transcriptional program that drives PDAC tumor progression.

Project description:Immunotherapeutics represent highly promising agents with the potential to improve patient outcomes in a variety of cancer types. Unfortunately, single-agent immunotherapy has achieved limited clinical benefit to date in patients suffering from pancreatic ductal adenocarcinoma (PDAC). This may be due to the presence of a uniquely immunosuppressive tumor microenvironment (TME) present in PDACs, which creates a barrier to effective immune surveillance. Critical obstacles to immunotherapy in PDAC tumors include the dense desmoplastic stroma that acts as a barrier to T-cell infiltration and the high numbers of tumor-associated immunosuppressive cells. We have identified hyperactivated focal adhesion kinase (FAK) activity in neoplastic PDAC cells as a significant regulator of the fibrotic and immunosuppressive TME. We found that FAK activity was elevated in human PDAC tissues and correlates with high levels of fibrosis and poor CD8+ cytotoxic T-cell infiltration. Single-agent FAK inhibition (VS-4718) dramatically limited tumor progression, resulting in a doubling of survival in the p48-Cre/LSL-KrasG12D/p53Flox/+ (KPC) mouse model of human PDAC. This alteration in tumor progression was associated with dramatically reduced tumor fibrosis, decreased numbers of tumor-infiltrating immature myeloid cells and immunosuppressive macrophages. We postulated that these desirable effects of FAK inhibition on the TME might render PDAC tumors more sensitive to immunotherapy. Accordingly, we found that VS-4718 rendered the previously unresponsive KPC mouse model responsive to anti-PD1 and anti-CTLA4 antagonists leading to a nearly tripling of survival times. These data suggest that FAK inhibition increases immune surveillance by overcoming the fibrotic and immunosuppressive PDAC TME thus rendering tumors more responsive to immunotherapy. We treated KP orthotopic tumor-bearing mice with vehicle and FAK inhibitor (FAKi) for 14 days, then extracted total RNA from tumor tissues.

Project description:Cyclooxygenase-2 (COX-2) is upregulated in pancreatic ductal adenocarcinomas (PDAC). However, how COX-2 promotes PDAC development is unclear. While previous studies have evaluated the efficacy of COX-2 inhibition via the use of non steroidal anti-inflammatory drugs (NSAIDs) or the COX-2 inhibitor celecoxib in PDAC models, none have addressed the cell intrinsic vs. microenvironment roles of COX-2 in modulating PDAC initiation and progression. We tested the cell intrinsic role of COX-2 in PDAC progression, using both loss-of-function and gain-of-function approaches. Cox-2 deletion in Pdx1+ pancreatic progenitor cells significantly delays the development of PDAC in mice with K-ras activation and Pten haploinsufficiency. Conversely, COX-2 over-expression promotes early onset and progression of PDAC in the K-ras mouse model. Loss of PTEN function is a critical factor in determining lethal PDAC onset and overall survival. Mechanistically, COX-2 over-expression increases P-AKT levels in the precursor lesions of Pdx1+;K-rasG12D/+;Ptenlox/+ mice in the absence of Pten LOH. In contrast, Cox-2 deletion in the same setting diminishes P-AKT levels and delays cancer progression. These data suggest an important cell intrinsic role for COX-2 in tumor initiation and progression through activation of the PI3K/AKT pathway. PDAC that is independent of intrinsic COX-2 expression eventually develops with decreased FKBP5 and increased GRP78 expression, two alternate pathways leading to AKT activation. Together, these results support a cell intrinsic role for COX-2 in PDAC development and suggest that, while anti-COX-2 therapy may delay the development and progression of PDAC, mechanisms known to increase chemoresistance through AKT activation must also be overcome. Murine mutants with pancreatic specific loss of Pten (Pten +/-) and K-ras activation (K-rasG12D) and either COX-2 over-expression (Cox-2 COE) or knockout (Cox-2 KO) under regulation of the Pdx-1 promoter developed pancreatic ductal adenocarcinoma. RNA was extracted from pancreatic tumors from individual mutants with pathology thought to closely mimic the human disease. Pancreatic tissue was subject to RNA extraction and hybridization on Affymetrix cDNA microarrays.

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