MUC5AC is a secretory mucin aberrantly expressed in various cancers. In lung cancer, MUC5AC is overexpressed in both primary and metastatic lesions; however, its functional role is not well understood. The present study was aimed at evaluating mechanistic role of MUC5AC on metastasis of lung cancer cells. Clinically, the overexpression of MUC5AC was observed in lung cancer patient tissues and was associated with poor survival. In addition, the overexpression of Muc5ac was also observed in genetically engineered mouse lung adenocarcinoma tissues (KrasG12D; Trp53R172H/+; AdCre) in comparison with normal lung tissues. Our functional studies showed that MUC5AC knockdown resulted in significantly decreased migration in two lung cancer cell lines (A549 and H1437) as compared with scramble cells. Expression of integrins (α5, β1, β3, β4 and β5) was decreased in MUC5AC knockdown cells. As both integrins and MUC5AC have a von Willebrand factor domain, we assessed for possible interaction of MUC5AC and integrins in lung cancer cells. MUC5AC strongly interacted only with integrin β4. The co-localization of MUC5AC and integrin β4 was observed both in A549 lung cancer cells as well as genetically engineered mouse adenocarcinoma tissues. Activated integrins recruit focal adhesion kinase (FAK) that mediates metastatic downstream signaling pathways. Phosphorylation of FAK (Y397) was decreased in MUC5AC knockdown cells. MUC5AC/integrin β4/FAK-mediated lung cancer cell migration was confirmed through experiments utilizing a phosphorylation (Y397)-specific FAK inhibitor. In conclusion, overexpression of MUC5AC is a poor prognostic marker in lung cancer. MUC5AC interacts with integrin β4 that mediates phosphorylation of FAK at Y397 leading to lung cancer cell migration.

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Pancreatic cancer (PC) is the fourth leading cause of cancer-related deaths in the United States. The high mortality can be accredited to late clinical presentation and early metastasis. Somatic mutations in oncogene KRAS(G12D) are the most common and earliest events during development of PC, suggesting their crucial role in tumor initiation; however, the precise functional molecular mechanism(s) of action remains unclear. CXCR2, a CXC chemokine receptor, and its ligands have also been found to be important in regulating inflammation, angiogenesis, and metastasis in various cancers. The purpose of the current study was to test the functional role of CXCR2 signaling in KRAS(G12D)-induced PC development. Immunohistochemical analysis of tumors derived from PDX-cre-LSL-kras(G12D) mice demonstrated significantly higher expression of Cxcr2 and its ligands Cxcl1 and -3. Expression of Cxcr2 and its ligands Cxcl1 and -3 was observed as early as 10 weeks and increased consistently as the lesions advanced. We demonstrated the direct link between KRAS(G12D) mutation and CXCR2 signaling using cell line models hTERT-HPNE and hTERT-HPNE -E6-E7-st with or without exogenously expressed KRAS(G12D). Real time PCR, ELISA and western blot analysis of these cells revealed that KRAS (G12D) up-regulates the expression of CXCR2 and its ligands at both the transcriptional and protein levels. In order to delineate the significance of CXCR2 in KRAS(G12D) induced autocrine growth, we treated the cell line models with various concentrations of SCH-527123 (CXCR2 antagonist) or CXCR2 neutralizing antibody. Our data shows that KRAS(G12D) bearing cells show significantly (P ≤ 0.05) greater percent inhibition in cell growth in comparison to their control counterparts. Next to evaluate the role of CXCR2 autocrine signaling in KRAS(G12D) induced cell migration, we performed a scratch assay by treating the hTERT-HPNE -E6/E7-st- KRAS(G12D) cells with SCH-527123 for 24 hours. Our data demonstrated a significant inhibition of cell migration in CXCR2 antagonist treated cultures. Together, our data demonstrate that KRAS(G12D) mutation up regulates CXCR2 and its ligands, which, in turn, play key roles in the autocrine regulation of cellular growth and migration in PC. Note: This abstract was not presented at the meeting. Citation Format: Abhilasha Purohit, Michelle Varney, Satya Rachagani, Michel Ouellette, Surinder K. Batra, Rakesh K. Singh. CXCR2 signaling axis- a helping hand to KRAS(G12D) mutation in pancreatic cancer initiation. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4874. doi:10.1158/1538-7445.AM2014-4874

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Pancreatic ductal adenocarcinoma (PDAC) has complex tumor microenvironment (TME), characterized by phenotypically and functionally heterogenous cancer-associated fibroblasts (CAFs). Different CAF subtypes have been identified that evidently contribute to desmoplasia, immunosuppression, and therapy response variably. The lack of models to study their plasticity and heterogeneity reproducibly poses a major limitation. We, thus, describe the development and characterization of a panel of immortalized patient-derived pancreatic fibroblasts (iPDPFs), studying their impact on PDAC cell lines in vitro and in vivo. Method: We developed six iPDPF lines using human telomerase reverse transcriptase (hTERT) from cancer-adjacent normal pancreas (9-26-NP), chronic pancreatitis (CPP1), and PDAC CAFs (9-17-P, 10-15-P, 10-03-P, & 10-32-P). Immunoblotting, bulk-RNAseq, and qPCR were performed for their molecular characterization. Functional characterization was done in vitro using collagen contraction assay and co-culture studies with COLO357. The impact of iPDPFs on the tumor cell behavior in vivo was studied by their co-implantation with COLO357 in athymic nude mice. Results: The iPDPFs exhibited variable expression of activation, inflammatory, and ECM markers indicative of heterogeneity across the panel. While CAFs expressed high levels of αSMA (alpha-smooth muscle actin) and fibroblast-specific protein-1 (FSP1), the expression of these markers was low in 9-26 NP & CPP-1. Also, the iPDPFs variably expressed TGFβ, PDGFRβ, VDR, COL1A, and small GTPases. High expression of inflammatory markers was observed in 9-26 NP, CPP-1, & 10-32-P suggesting their iCAF type while 9-17-P, 10-03-P, and 10-15-P were of myCAF type. These observations were recapitulated in the transcriptomic analysis. Interestingly, the marker expression changed with passage suggesting existence of plasticity in the iPDPFs. 9-17-P induced highest collagen contraction (47.9%) while 10-15-P induced the least contraction (25.89%). Co-culture of iPDPFs with COLO357 enhanced the expression of ROCK-1. In vivo, iPDPFs resulted in stroma-rich tumors; however, the extent of stroma and growth rate were variable across iPDPF lines and implantation sites. 9-17-P xenograft tumors exhibited the highest percent αSMA (14.5%), and FSP1 (30.7%) positive cells. Limited analysis showed that iPDPF co-implanted tumors exhibited variable vascularity and ECM composition suggesting that the heterogeneity observed in vitro was manifested in vivo. Conclusion: Overall, the iPDPFs recapitulate pancreatic fibroblasts heterogeneity, reflecting the overlapping nature of CAF subtypes. Also, the iPDPFs are functionally variable and plastic in vitro. Importantly, these fibroblasts exert variable effects on the tumor cells in vivo. The iPDPFs can thus serve as a useful model to reproducibly study CAF heterogeneity and plasticity in PDAC. Citation Format: Nidhi V. Dwivedi, Shailendra K. Gautam, Satya Rachagani, Ramakanth Venkata, Vipin Dalal, Maneesh Jain, Surinder K. Batra. Development and characterization of patient-derived pancreatic fibroblasts to study CAF heterogeneity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5850.