Defining the role of stress granules in cancer initiation

Project Summary. Stress granules (SGs) – cytoplasmic aggregates formed from stalled translation initiation complexes upon cell stress – have emerged as critical regulators of oncogenesis. SGs can confer cancer cells the ability to withstand harsh biological conditions and can impart tumorigenic translational states through mRNA sequestration. However, a mechanistic understanding of how SGs endogenously regulate tumorigenesis in vivo remains elusive. My research proposal leverages a humanized zebrafish melanoma model where tumor development can be visualized and manipulated from the precursor stage to its single cell of origin and beyond. Using this system, I have found that SGs are formed in melanoma precursor cells and persist during tumor initiation and progression. Furthermore, my preliminary data shows that CRISPR/Cas9-mediated ablation of a key SG component, g3bp1, in melanoma initiating cells delays the establishment of the cancer precursor zone, an essential initiating cell phase in the emergence of melanoma. Taken together, the overarching hypothesis of my proposal is that SGs are formed in melanoma precursor cells to enhance viability upon tumorigenic stress and once formed, subsequently accelerate melanomagenesis by enabling an oncogenic translational profile. To test my hypothesis, I will first determine the impact of stress granule formation on melanomagenesis by perturbating key components using genetically engineered zebrafish and chemical biology. I will subsequently test whether modification of stress granule formation alters melanoma cell oncogenicity using flow cytometry and subcutaneous transplant assays. In addition, I will elucidate the contribution of RNA sequestration by stress granules toward melanoma development using proximity labeling and translatome profiling. I will then functionally resolve the contribution of SG-sequestered mRNA using zebrafish genetics. Lastly, I will perform immunostaining of human melanoma samples for proteins encoded by mRNAs with preferential translation in melanoma initiating cells, assessing the conservation of these molecules and their potential to serve as melanoma biomarkers. Ultimately, the results obtained herein will provide mechanistic insight toward the functional role of stress granules during cancer initiation. As stress granules are detected across many cancer types, this work will elucidate nodes of tumor biology that can be exploited to halt a broad spectrum of cancers before they arise. In this proposal, I establish training goals that will expand my conceptual and technical expertise in cancer biology and RNA processing. In addition, I present aims that will enable the acquisition of the professional skillsets necessary to achieve my long-term research goal, which is to run my own lab at an academic research institution studying the basic biology of RNA processing during cancer initiation.