PROJECT SUMMARY/ABSTRACT Over the last five years, three cyclin-dependent kinase 4/6 inhibitors (CDK4/6i) have been approved for the treatment of metastatic breast cancer. CDK4/6i prevent cancer cells from dividing by blocking their progression through the G1/G0 phase of the cell cycle. However, results from in vitro studies reveal that both exogenous and endogenous CDK4/6i increase the invasiveness of certain forms of cancer. The consequences of G1/G0 cell cycle arrest on cancer cell behavior in vivo remain unknown. Caenorhabditis elegans anchor cell (AC) invasion provides an excellent yet simple in vivo model of cell invasive behavior that allows single-cell and subcellular analysis of cell invasion through basement membrane. Our lab determined that G1/G0-specific arrest is required for AC invasion to occur and that the nuclear hormone receptor NHR-67/TLX acts as a key regulator of this process. In the absence of NHR-67, the AC becomes non-invasive but over-proliferates, indicating that proliferation can increase as invasion is blocked. The long-term goal is to understand both the molecular mechanisms controlling G1/G0 cell cycle arrest and the in vivo effects of CDK4/6i as individual agents and in combination with inhibitors of the mammalian target of rapamycin (mTORi). Together, CDK4/6i and mTORi have been shown to induce senescence in cancer cells in vitro, suggesting that combining these pharmacological agents might be useful in the clinical setting. The overall objectives of this fellowship application are to (i) elucidate how G1/G0 is maintained in the C. elegans AC and (ii) determine whether ACs in NHR-67-deficient animals can be pharmacologically induced to transition from proliferation-to-invasion-to-senescence. Based on preliminary data and prior research, the central hypothesis is that redundant mechanisms regulate the G1/G0 state of the wild-type AC and that proliferative ACs in NHR-67-deficient animals can be prompted to undergo senescence. This central hypothesis will be tested by pursuing two specifics aims. Aim 1 will identify the components regulating G1/G0 arrest in the pro-invasive AC. Aim 2 will evaluate the transition between AC proliferation, invasion and senescence upon depletion of CDK4/6, mTOR, or both. To address these aims, this project will use established gene knockdown techniques, high-resolution imaging, and cutting-edge technologies, including a novel live cell cycle state sensor and two conditional protein depletion systems. The results of these studies can help guide the development of future treatment strategies for aggressive cancers.