The major scientific focus of this project is to determine the Ras-initiated signaling pathways and their relevant transcriptional targets that contribute to human epithelial cell transformation and metastasis. Ras is mutated in approximately one quarter of all human cancers with the highest incidence in pancreatic, lung, colon, and thyroid tumors. In addition, there is considerable experimental evidence that persistent upstream signaling in other epithelial cancers may activate Ras. Transformation functions associated with Ras effector pathways are being analyzed in experimental models of mouse melanocytes and human breast epithelial cells. Because multiple autocrine or paracrine growth factor pathways contribute to the transformation of epithelial cells and their colonization of distant tissues during the development of metastasis, Ras signaling pathways are expected to provide broadly applicable diagnostic markers and therapeutic targets. Multiple downstream effectors mediate Ras signaling, and there is a growing appreciation that the signaling outcomes of Ras activation demonstrate species and cell context differences. We have shown that ectopic Ras activation leads to the new expression of a bone metastatic phenotype in the DU145 xenograft model of human prostate cancer, and furthermore, that bone tropism can be mediated by a specific Ras effector pathway. We have investigated the role of signaling pathways initiated by oncogenic Ras including Raf/ERK, PI3-kinase, and Ral guanine nucleotide exchange factors (RalGEFs), in stimulating tissue-specific experimental metastasis of DU145. Although the RalGEF pathway appears to be required for Ras-initiated tumorigenesis in human epithelial cells, a role in metastasis has not been addressed. Oncogenic Ras promoted metastasis of hematogenously delivered DU145 to multiple organs including bone, brain, and other soft tissues. Activation of the Raf/ERK pathway stimulated metastatic colonization of the brain, while activation of the RalGEF pathway led to bone metastases, the most common organ site for prostate cancer. The generality of RalGEF pathway activation in prostate cancer bone metastasis was confirmed in two additional xenograft models. Similarly to DU145, nonmetastatic C42B cells demonstrated substantially increased bone metastasis following activation of the RalGEF pathway. In addition, shRNA-mediated knockdown of RalA in the metastatic PC3 cell line inhibited bone metastasis but did not affect subcutaneous tumor growth. Interestingly, knockdown of RalA in breast or colon cancer cells did not inhibit bone metastatic activity, suggesting some specificity for RalGEF function in the context of prostate cells. The existence of 3 model prostate cancer metastasis systems provides a strong platform to identify both general functional categories and specific gene products associated with RalGEF-mediated bone metastasis. Comparative microarray analyses are on-going. An important question is the mechanism of RalGEF-dependent bone metastasis. Genetically manipulated loss and gain of Ral function in either the DU145 or PC3 systems suggested that Ral affects expansive growth in the bone, whereas homing and initial colonization are less affected. The role of Ras-mediated signaling with respect to osteoclast induction is being investigated. Enhanced anchorage-independent growth was a cell biological correlate of bone, but not brain, metastasis. We have taken advantage of this property to develop an in vitro DU145 cell line that is being used in a tetracycline-inducible shRNAi mediated library selection for Ral-dependent gene functions. Our data begin to identify signaling pathways relevant for organ-specific metastasis. There are very few models of metastatic prostate cancer, and our DU145 model is unique in that the initiating genetic event, i.e. Ras activation, can be manipulated. The DU145(Ras) system provides an experimental system for the identification of genes mediating metastasis to different organs. It also provides an efficient and predictable model system of tissue-specific metastases for preclinical testing of therapeutic agents
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