PROJECT SUMMARY/ABSTRACT Colorectal cancer (CRC) remains deadly due to metastatic disease and there is a fundamental gap in understanding how CRC metastases form. Transforming growth factor-beta (TGFβ) promotes metastatic CRC at later stages and TGFβ inhibitors are in early phase clinical trials, but their suboptimal performance may be due to the lack of appreciation for its family member activin, which itself has distinct prometastatic actions. Our preliminary studies indicate that activin and TGFβ function should be interpreted as a complexly intertwined network and further, that tumor stroma potentiates prometastatic activin/TGFβ signaling. As activin has been understudied in this context, the overall goal of this application is to elucidate combined prometastatic mechanisms of activin and TGFβ signaling in CRC with a future goal of allowing metastatic risk assessment and future niche-specific effective activin/TGFβ inhibitor-based treatments. The central hypothesis is that activin/TGFβ trigger distinct signaling pathways that potentiate each other to promote metastatic CRC via stromal amplification with the rationale that understanding the underlying mechanisms in individual CRC tumors will allow risk stratification and more effective targeted treatment which minimize adverse events. This hypothesis will be investigated with these three aims: 1) to elucidate the complementary pro-invasive actions and cross-regulation of activin/TGFβ in CRC cells and surrounding stroma, 2) to examine the in vivo role of activin/TGFβ signaling in CRC metastasis and 3) to fully delineate activin/TGFβ pathway status for prediction of CRC metastasis and outcome. For this, we will use colon cancer cell models with a spectrum of activin/TGFβ signaling status from wild type to fully abrogated with and without activin/TGFβ wild type GI fibroblasts to assess the impact of disrupting TGFβ-induced pro-metastatic activin signaling. Our endpoints include examination of ligand production; canonical and non-canonical effector signaling; and growth suppression versus pro-metastatic function. To address the impact of pathway disruption in vivo, both surgical splenic implantation and transgenic APC/KRAS murine models of CRC metastasis will be used to determine effects of loss or augmentation of activin and TGFβ signaling or a combination thereof on liver metastasis and outcome. Lastly, clinical implications of activin/TGFβ cross-play on metastasis and outcome in human CRC will be examined via three complementary and comprehensive CRC cohorts. These studies are innovative as they depart from the current dogma of single pathway assessment in TGFβ and related signaling, employ novel techniques and models allowing integration of tumoral stromal effects and are significant, as they will pave the way to tailored activin/TGFβ pathway and niche-specific inhibition based on individual signaling status to target metastatic disease in CRC.