Prostate Cancer (PCa) is the second leading cause of cancer death in American men. PCa patients' mortality is mainly attributed to complications caused by metastasis of the disease to organs critical for survival, such as bone. As such, it is important to understand cancer-bone microenvironment interactions in order to develop therapeutics that will slow or halt the process of cancer metastasis. Snail1 is a zinc-finger transcription factor that induces epithelial-mesenchymal transition (EMT) which is associated with cell migration and metastasis in cancer. We hypothesized that cancer cell-bone interactions would promote higher calcium release from bone, more specifically by cancer cells overexpressing Snail, which would lead to increased paracrine cell signaling and migration. For this study, we utilized various prostate cancer cell lines: LNCaP (Snail-low), C4-2 (Snail-high), E006AA (Snail-high), E006AA HT (Snail-high) and C4-2 with stable Snail knockdown. Cancer cells were co-cultured with Hydroxyapatite (HA; inorganic component of bone) of different densities to represent the African American vs Caucasian bone ratio (since African Americans have higher bone density than any other race). The conditioned media was then used to assay calcium levels, perform paracrine migration assays using LNCaP cells, and examine paracrine signaling effects of the various conditioned media on LNCaP cells by western blotting. We observed that calcium levels were elevated in conditioned media from cancer cell-bone co-cultures, compared to media or cancer cells alone, and this could be antagonized by EGTA, a calcium chelator. C4-2 cancer-bone co-culture conditioned media increased paracrine cell migration which was decreased by Snail knockdown as well as lower bone density. We also observed increased STAT3 phosphorylation and paracrine cell migration in LNCaP cells incubated with conditioned media from C4-2, E006AA or E006AA HT cells co-cultured with HA; this phosphorylation and cell migration could be antagonized by Snail knockdown or STAT3 inhibitor (WP1066). An in vivo study was also done using nude mice that were surgically implanted with either 40mg HA or 100mg HA and subcutaneously injected with either C4-2 NS or C4-2 Snail shRNA cells into a position close to the shoulder from the surgical HA implant site in the mouse. At week 2 and week 4, Snail shRNA cells injected mice showed larger tumors than C4-2 NS injected mice, however, mice implanted with cancer cells plus higher bone density resulted in larger tumors than those with lower bone density. In conclusion, our study shows that Snail can mediate cancer-bone microenvironment interactions via STAT3 signaling, that can possibly promote increased paracrine cell migration towards bone of high mineral density. Therefore, targeting cancer-bone micronenvironmental interactions is an important avenue to consider for therapeutic targeting of prostate cancer.

Citation Format: Veronica M. Henderson, Ohuod Hawsawi, Liza J. Burton, Kennedi Trice, Jodi Dougan, Simone M. Howard, Valerie A. Odero-Marah. STAT3 pathway regulates the cancer-bone microenvironment interactions mediated by Snail [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1096.