Investigation of Ras dependent signaling pathways in promoting experimental prostate cancer metastasis

Even with advances in the detection and treatment of localized prostate cancers the spread of the disease to distal sites is correlated with having a poor prognosis.[1] A subset of advanced human prostate cancers with metastatic capability have a combined loss of the tumor suppressors PTEN and P53.[2] However modeling the conditional loss of both genes in the prostates of mice only results in organ-confined disease.[3] Identifying genetic events and additional pathways that better model the phenotype and behavior of advanced cancers will create more useful preclinical models for studying the mechanisms that are important for metastasis and also for testing combinatorial therapies to help patients control their disease. The work described in this dissertation builds on and extends the current understanding of the pathways that are important for advanced prostate cancers to metastasize to the lungs, lymph nodes, bone and brain. Here we characterize the histologic phenotype and metastatic properties resulting from the overexpression of oncogenic KRASG12V in the Clone 2 cell line, which was established from the PB-Cre4(+) Pten(fl/fl)TP53(fl/fl) mouse model of prostate cancer. Adenocarcinoma that was CK8+, CK5- and P63- was the major phenotype that was observed for Clone 2-KrasG12V orthotopic tumors. Metastasis to the lung and lymph nodes occurred from Clone 2-Kras G12V orthotopic tumors. Brain metastasis occurred when Clone 2-Kras G12V cells were injected via an intracardiac route. Previous work with the Pten;Trp53 knockout mouse model, primary tumor spheres derived from the model and the Clone 2 cell line have shown that both Pten and Trp53 regulate aspects of cellular differentiation in murine prostate epithelial cells, which may also directly or indirectly affect the frequency of metastasis.[3, 4] While retaining the ability to respond to TGFbeta1 Clone 2-KrasG12V CK8+ adenocarcinoma cells did not undergo an in vivo TGFbeta1-mediated epithelial-to-mesenchymal transition (EMT) unlike Clone 2 CK8+, CK5+/-, P63- adenocarcinoma cells. When TGFbeta-SMAD dependent signaling was impaired Clone 2-KrasG12V cells became bi-phenotypic for both luminal and basal lineage markers (CK8+, CK5+, P63+) and formed lung and lymph node metastases following orthotopic implantation. The bi-potential tumor cells formed both bone and brain metastases following intracardiac injection. Additional experiments suppressing IKBalpha-p65-mediated NFKB showed a known relationship whereby Clone 2-Kras G12V cells required NFKB for both invasion and metastatic colonization. Together these data show that 1) the expression of oncogenic KrasG12V influenced the in vivo phenotype of Clone 2 cells, 2) resulted in metastasis and 3) identified the TGFbeta and NFKB pathways as being important for aspects of the metastatic phenotype of Ras dependent tumor cells as well as the ability to remain fully committed as luminal epithelial cells.