Translation Regulation Contributes to Hypoxia Adaptation in Glioblastoma

PROJECT SUMMARY Glioblastoma (GBM) is a CNS tumor derived from glial cells. It is the most common and fatal primary malignant brain tumor in adults with a median survival of 15 months. Current treatment strategies involve surgical resection followed by radiotherapy with concurrent chemotherapy. Unfortunately, GBM is highly resistant to therapy and will inevitably recur, resulting in poor patient outcomes. GBM has been described as having an oxygen-deprived niche and a perivascular, oxygen-rich, niche. Deeper characterization of these tumor environments has revealed the presence of cellular heterogeneity, angiogenesis, and invasion in both niches. The critical cellular player involved with these tumor behaviors is the glioblastoma stem-like cell (GSC). GSCs seem to reside in both hypoxic (low oxygen level) and normoxic (normal oxygen level) niches yet maintain their stemness and self- renewal capacities across both conditions. Strategies to target these GSCs have proven to be unsuccessful so far but remain a promising avenue for research. There is a need to develop a greater understanding of GSC biology, starting with the regulatory mechanisms with which they adapt to the hypoxic environment of GBM and promote tumor growth. However, most studies exploring GSCs are performed in normoxic conditions. Further, the central aspect of post-transcriptional gene regulation, translation, where genetic information is converted to functional proteins, remains relatively unexplored. The objective of the proposed research is to study the translation (mRNA-to-protein) regulatory step of protein synthesis in GSCs, as opposed to the more commonly explore transcription (DNA-to-mRNA) regulatory step, with the desired outcome of illuminating systemic programs for hypoxic adaptation. To accomplish this objective, our approach in Aim 1 will use ribosome profiling (RIBO-seq) in conjunction with RNA sequencing (RNA-seq) to offer a quantitative understanding of transcriptional and translational regulatory pressures on mRNA transcripts. I will utilize both techniques to clarify the influence on hypoxia on translational gene regulation in GSCs. I expect to find genes and gene-sets, representing biological pathways, that are modulated by hypoxia predominantly through translational regulation, revealing a novel list of candidates for future therapy. To validate this discovery framework, I will perform an array of functional studies on selected genes. In parallel, Aim 2 will focus on a particular set of translational machinery known as the Integrated Stress Response (ISR), whose members sense cellular stress and modulate translation of survival-associated transcripts. With burgeoning evidence of aberrant ISR behavior in malignant cells, interrogating the mechanism through which the ISR may mediate translational changes in hypoxic GSCs will present another avenue through which to halt such adaptive behaviors. Altogether, this proposal will promote further understanding of GSC biology, with respect to translation regulation, thereby unveiling novel targets for future GBM therapy and ultimately contribute to brain tumor patient care.