Background: Trop2 is a cell-surface glycoprotein overexpressed by a variety of epithelial carcinomas with reported low to restricted expression in normal tissues. Expression of Trop2 has been associated with increased tumor aggressiveness, metastasis and decreased patient survival, but the signaling mechanisms mediated by Trop2 are still unknown. Here, we studied the effects murine Trop2 (mTrop2) exerted on tumor cellular functions and some of the signaling mechanisms activated by this oncogene. Results: mTrop2 expression significantly increased tumor cell proliferation at low serum concentration, migration, foci formation and anchorage-independent growth. These in vitro characteristics translated to increased tumor growth in both subcutaneous and orthotopic pancreatic cancer murine models and also led to increased liver metastasis. mTrop2 expression also increased the levels of phosphorylated ERK1/2 mediating cell cycle progression by increasing the levels of cyclin D1 and cyclin E as well as downregulating p27. The activation of ERK was also observed in human pancreatic ductal epithelial cells and colorectal adenocarcinoma cells overexpressing human Trop2. Conclusions: These findings demonstrate some of the pathogenic effects mediated by mTrop2 expression on cancer cells and the importance of targeting this cell surface glycoprotein. This study also provides the first indication of a molecular signaling pathway activated by Trop2 which has important implications for cancer cell growth and survival.

Background: Previous studies showed that mesothelin (MSLN) plays important roles in survival of pancreatic cancer (PC) cells under anchorage dependent/independent conditions as well as resistance to chemotherapy. The recent success of intratumorally-injected adeno-encoded, chemo/radiation-inducible-promoter driven hTNF-α, (TNFerade) + gemcitabine in pre-clinical models of PC have renewed interest in use of TNF-α as a therapeutic component. To help find additional factors which might affect the therapy, we examined the resistance of MSLN-overexpressing pancreatic cancer cell lines to TNF-α-induced growth inhibition/apoptosis. Methods: Stable MSLN overexpressing MIA PaCa-2 cells (MIA-MSLN), stable MSLN-silenced AsPC-1 cells (AsPC-shMSLN) and other pancreatic cells (MIA-PaCa2, Panc 28, Capan-1, BxPC3, PL 45, Hs 766T, AsPC-1, Capan-2, Panc 48) were used. NF-κB activation was examined by western blots and luciferase reporter assay. TNF-α induced growth inhibition/apoptosis was measured by MTT, TUNEL assay and caspase activation. IL-6 was measured using luminex based assay. Results: Compared to low endogenous MSLN-expressing MIA PaCa-2 and Panc 28 cells, high endogenous MSLN-expressing Capan-1, BxPC3, PL 45, Hs 766T, AsPC-1, Capan-2, Panc 48 cells were resistant to TNF-α induced growth inhibition. Stable MSLN overexpressing MIA-PaCa2 cells (MIA-MSLN) were resistant to TNF-α-induced apoptosis while stable MSLN-silenced AsPC1 cells (AsPC-shMSLN) were sensitive. Interestingly, TNF-α-treated MIA-MSLN cells showed increased cell cycle progression and cyclin A induction, both of which were reversed by caspase inhibition. We further found that MIA-MSLN cells showed increased expression of anti-apoptotic Bcl-XL and Mcl-1; deactivated (p-Ser⁷⁵) BAD, and activated (p-Ser⁷⁰) Bcl-2. Constitutively activated NF-κB and Akt were evident in MIA-MSLN cells that could be suppressed by MSLN siRNA with a resultant increase in sensitivity of TNF-α induced apoptosis. Blocking NF-κB using IKK inhibitor wedelolactone also increased sensitivity to TNF-α-mediated cytotoxicity with concomitant decrease in Mcl-1. Blocking Akt using PI3K inhibitor also had a likewise effect presumably affecting cell cycle. MIA-MSLN cells produced increased IL-6 and were increased furthermore by TNF-α treatment. SiRNA-silencing of IL-6 increased TNF-α sensitivity of MIA-MSLN cells. Conclusions: Our study delineates a MSLN-Akt-NF-κB-IL-6-Mcl-1 survival axis that may be operative in PC cells, and might help cancer cells' survival in the highly inflammatory milieu evident in PC. Further, for the success of TNFerade + gemcitabine to be successful, we feel the simultaneous inhibition of components of this axis is also essential.

Given the high fatality rate of pancreatic cancer, an effective treatment for this devastating disease is urgently needed. We have shown that mesothelin expression was higher in human pancreatic cancer cells than in human pancreatic duct epithelial cells, and mesothelin mRNA was substantially overexpressed in 18 of 21 (86%) clinical pancreatic adenocarcinoma specimens when compared with the surrounding normal tissues. However, the biological functions of mesothelin in tumor progression are not clearly understood. Here we studied the effects of mesothelin overexpression in pancreatic cancer cell proliferation and migration in vitro and pancreatic cancer progression in vivo. We found that forced expression of mesothelin significantly increased tumor cell proliferation and migration by 90% and 300%, respectively, and increased tumor volume by 4-fold in the nude mice xenograft model when compared with the vector control cell line. Silencing of mesothelin inhibited cell proliferation and migration in pancreatic cancer cells and ablated tumor progression in vivo. Vaccination with chimeric virus-like particles that contain human mesothelin substantially inhibited tumor progression in C57BL/6J mice. The increases in mesothelin-specific antibodies and CTL activity and the decrease in regulatory T cells correlated with reduced tumor progression and prolonged survival. This study revealed novel functions of mesothelin and suggested a new therapeutic vaccine strategy whereby mesothelin is targeted to control pancreatic cancer progression. [Mol Cancer Ther 2008;7(2):286–96]

Virus-like particles (VLPs) have gained increasing interest for their use as vaccines due to their repetitive antigenic structure that is capable of efficiently activating the immune system. The efficacy of VLP immunization may lie in its ability to traffic into draining lymph nodes while activating antigen-presenting cells to initiate the orchestration of signals required for the development of a robust immune response. Currently, there is no comprehensive study showing the correlation of different VLP vaccination routes to immune outcome. In this study, we took an optical imaging approach to directly visualize the trafficking of simian-human immunodeficiency (SHIV) VLPs after immunization by commonly used routes and analyzed the corresponding humoral and cellular immune responses generated. We found that VLPs can easily enter the subcapsular sinus of draining lymph nodes with quantitative differences in the number of lymph node involvement depending on the immunization route used. Intradermal immunization led to the largest level of lymph node involvement for the longest period of time, which correlated with the strongest humoral and cellular immune responses. Flow cytometry analysis from extracted splenocytes showed that intradermal immunization led to the largest population of germinal center and activated B cells, which translated into higher antibody levels and antigen-specific cytotoxic T lymphocyte responses. Our results indicate that VLPs traffic into lymph nodes upon immunization and can be directly visualized by optical imaging techniques. Intradermal immunization showed improved responses and might be a preferable delivery route to use for viral and cancer immunotherapeutic studies involving VLPs.