Project Summary Castration-resistant prostate cancer (CRPC) arises from a failure of standard-of-care androgen deprivation (AD) therapy to suppress emergence of castration-resistant (CR) variants from the original androgen deprivation-responsive PC (ADPC). Identifying molecular drivers of emergence is critical for developing curative therapies, which are sorely needed. We have shown AD induces cell senescence (ADIS), a permanent proliferative arrest, in ADPC (LNCaP) cells. Our work was the first to show that ADIS selects for expansion of AD-resistant quiescent subpopulations, hastening early CRPC outgrowths (termed LNCaP-SB5). These LNCaP-SB5 represent the earliest CR cells and are a unique model for evaluating acute molecular changes underlying CRPC etiology. Unbiased microarray screening comparing LNCaP-SB5 with parent LNCaP (SB0) cells shows a critical subunit of the soluble guanylate cyclase (sGC) complex, GCSa3 (gene name: GUCY1A3), is increased under AD in the ADPC cells but downregulated in their early CRPC LNCaP SB5 counterparts. Riociguat is an orally-bioavailable, FDA-approved sGC stimulator used to treat pulmonary hypertension. Using BAY41-8543, a chemical analog of riociguat, we enhanced sGC signaling in our in vitro model of prostate cancer progression and in an in vivo xenograft CRPC model. BAY41- 8543 enhanced expression of p53 and p16INK4a in ADPC LNCaP cells, suggesting enforcement of AD-induced tumor suppression and reduced xenograft CRPC tumor formation by LNCaP-SB5 and LNAI (an established CRPC variant of LNCaP) proportional to the extent by which sGC signaling was enhanced, however, treatment did not affect viability in culture, suggesting a paracrine component to tumor suppression in CRPC. Based on these findings, we hypothesize stimulation of the sGC pathway will enhance AD-associated tumor suppression and limit CRPC emergence and progression. Therefore, we propose: 1) to determine whether riociguat treatment can enhance AD durability in ADPC to prevent CRPC emergence and 2) whether and how sGC stimulation creates a hostile tumor microenvironment to limit established CRPC. Our study will potentially facilitate rapid and safe clinical repurposing of riociguat in combination with standard-of-care AD for treatment of incurable prostate cancer.