STEM-05GPR133 IS ENRICHED IN GLIOBLASTOMA STEM CELLS AND REGULATES THE RESPONSE TO HYPOXIA

Abstract

Intratumoral heterogeneity in glioblastoma (GBM) is exemplified by the diversity of tumor microenvironments, which include normoxic hypervascular areas and necrotic regions, which are considered hypoxic. GBM stem cells (GSCs) play a central role in tumor growth and therapy resistance. How GSCs adapt to diverse GBM microenvironments remains an important and unanswered question. We recently discovered that CD133-expressing GSCs are metabolically adept at expanding in hypoxic conditions and do not require Notch signaling for their self-renewal. Transcriptional analysis indicated that CD133+ GSCs have 17.8 ± 8.8-fold enriched expression of GPR133 (n = 3 biospecimens), a member of the adhesion family of G protein-coupled receptors. Immunostaining with GPR133 antibody revealed that GPR133 expression is restricted to hypoxic regions within GBM tumors (12/12 GBM biospecimens) and not present in normal brain. We observed that GPR133 mRNA expression correlates with hypoxia-induced transcripts, such as CA9 and VEFGA (n = 3 primary cultures). To test the hypothesis that GPR133 expression is regulated by oxygen tension, we subjected GBM cultures to 1% O2 in vitro and found that GPR133 transcript was consistently upregulated (n = 5 cultures). To examine whether GPR133 is important for GSC self-renewal, we tested the effect of shRNA-mediated knockdown on in vitro tumorsphere formation ability. GPR133 knockdown depleted CD133+ GSCs and inhibited tumorsphere formation under both normoxic and hypoxic conditions (p < 0.05). GPR133 knockdown also reduced in vivo tumorigenicity and increased survival of implanted mice (n = 3). Using colorimetric assays, we found that CD133+ GSCs have 26.2 ± 12.53% higher cAMP levels compared to CD133- GBM cells (n = 3) and that GPR133 knockdown downregulated cAMP levels to 47.25 ± 27.27% of scramble control (n = 3), suggesting that GPR133 signals through activation of adenylate cyclase and cAMP elevation.