Abstract A15: The selective role of PIK3CB/p110β in temozolomide resistance in glioblastoma

Abstract

Abstract Background: The divergent roles of phosphatidylinositide 3-kinase (PI3K) family genes in cancer pathogenesis and progression have been highly studied. PI3K genes are divided into three classes (I, II, and III). Class IA PI3K includes three highly homologous catalytic subunits p110α, β, or δ, encoded by the genes PIK3CA, B, and D, respectively. Studies in glioblastoma (GBM), the most lethal and most common brain cancer, have shown PI3K to promote resistance to the front-line chemotherapeutic drug, temozolomide (TMZ) (1). We recently investigated these isoforms in GBM and found that levels of PIK3CB/p110β, but not other PI3K isoforms, present prognostic significance for GBM recurrence and that this isoform selectively activates GBM cell survival pathways (2). Our previous work also shows that a gap-junction protein, Connexin 43 (Cx43), promotes TMZ resistance in GBM by activating PI3K signaling (3). Given that PIK3CB/p110β is the most important isoform in GBM PI3K signaling, it is therefore critical to investigate whether PI3K isoforms have divergent assignments in Cx43-mediated TMZ resistance. Understanding the role of PI3K isoforms in drug resistance will foster the rational design of more effective treatments for GBM. Hypothesis: Based upon the results described above, we hypothesize that PIK3CB/p110β is selectively important for Cx43-mediated TMZ resistance in GBM. Approaches: The expression and activity of PI3K genes and Cx43 are manipulated by overexpression of active PI3K mutants, short hairpin RNA (shRNA)-mediated knockdown, a peptide compound called αCT1, or specific chemical inhibitors. Activity of signaling is assessed by immunoblotting. Cx43 channel activity is monitored by ATP release. Protein-protein interactions are measured using fluorescence microscopy and immunoprecipitation. Cell viability is determined using the MTS viability assay. Results: We find that (1) shRNA knockdown of Cx43 inactivates PI3K signaling in GBM cells, while constitutive activation of PI3K diminishes TMZ sensitization by the Cx43 peptide inhibitor, αCT1; (2) GBM patients with high levels of Cx43 also express more p110β, active AKT, and display TMZ resistance; (3) Cx43 activates PI3K independent of Cx43-formed channels; (4) p110β, but not p110α or p110δ, interacts with Cx43; and (5) selective inhibition of p110β circumvents TMZ resistance together with αCT1. Conclusion/Impact: We conclude that PIK3CB/p110β is selectively important for Cx43-mediated temozolomide resistance, presenting the possibility of selectively targeting PIK3CB/p110β as a new and effective approach to overcoming TMZ resistance in GBM.