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 human prostate cancer. 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 and brain metastatic phenotype in the DU145 xenograft model of human prostate cancer. Histopathological analyses identified robust angiogenesis associated with metastasis formation. A novel receptor-ligand pathway that was observed also to be overexpressed in correlation with bone metastasis is the Tweak/Tweak Receptor pathway. Loss of the Tweak receptor inhibits bone metastasis. Since NFkappaB activation is observed downstream of Tweak receptor ligation, we have determined that the canonical NF-kappaB pathway is both necessary and sufficient for promoting bone metastasis. Relatively little is known about the regulation of FN14 expression. We have determined that androgen receptor is a transcriptional repressor of FN14 expression, which correlates with high FN14 expression in clinical samples of prostate cancer with low AR transcriptional output. We have identified the EGR transcription factor family as one positively regulated transcriptional target of FN14 signaling. This is of importance because low AR output prostate cancers are the least treatable with standard androgen deprivation therapy. This suggests that FN14 may be a useful target for castration resistant bone metastasis. A bioinformatics analysis suggested that EGR1 controls transcription of various angiogenesis factors, and further analysis of EGR1 knockdown cells confirmed that TWEAK treatment led to EGR1 dependent angiogenesis pathway component expression. In addition, we show that EGR1 regulates a variety of genes that influence osteoclastogenesis and bone metastasis development. Thus, we have determined that RAS activation of prostate cancer cells initiates angiogenesis at least in part through an FN14- EGR1 mechanism.
