Introduction: Different combinations of three driver mutations localized respectively to the p53, Rb and receptor tyrosine kinase networks are responsible for selective growth advantage of glioblastoma tumors (GBM) and contribute to significant intra-tumoral heterogeneity, a major cause of treatment failure. microRNA-34a (miR-34a) modulates the expression of multiple genes in all three deregulated GBM networks, suggesting it could serve as a novel therapeutic agent.
Methods: Three primary patient-derived lines (GBM 6, GBM118 and GBM 126, respectively belonging to classical, mesenchymal and proneural subtypes) and four established cell lines (T98G, U251, A172, LN229) were studied. A172, LN229, GBM6 are sensitive to temozolomide (TMZ), whereas T98G, U251, GBM118 and GBM126 show primary resistance to TMZ. In addition, TMZ-resistant lines (A172-TR, LN229-TR) were generated from parental lines by incubating cells at twice the respective EC50s for 2 weeks.
Results: Transfection with miR-34a mimics significantly reduced proliferation in all cell lines and sensitized to TMZ treatment, regardless of TMZ resistance. We identified a broadly conserved 7-mer binding site for the 5’ seed region of miR-34a in the 3’ UTR of Bcl-2 mRNA and observed significantly reduced Bcl-2 protein in cells transfected with miR-34a on western blots. Further, siRNA-mediated knockdown of Bcl-2 and inhibition with the Bcl-2 selective inhibitor ABT-199 produced sensitization approximating that of miR-34a, confirming the hypothesis that miR-34a sensitizes to TMZ in part through directly regulating Bcl-2 levels. Moreover, all cell lines examined were sensitized to radiation with dose enhancement factors ranging from 1.7-2.2. Early mechanistic studies implicate the DNA damage response protein Rad9A as an indirectly regulated miR-34a target contributing to radiation sensitization.
Conclusions: miR-34a is a promising novel therapeutic that inhibits proliferation and sensitizes to TMZ and radiation. Current work is focused on delivering miR-34a to orthotopic tumors using different types of nanoparticles.
Patient Care: Glioblastoma is a uniformly fatal disease with a mean overall survival of 14.5 months. The survival trends haven’t improved significantly over the last decade and there is a dire need for novel therapeutics. Intra-tumoral heterogeneity remains a significant challenge to develop effective therapeutics. Further, no appreciable survival enhancing drugs exist for radiation and temozolomide resistant and primary gliomas and recurrent gliomas. miR-34a can counteract heterogeneity by targeting multiple oncogenic pathways and as demonstrated by our data, can synergize with existing therapies. Thus, miR-34a loaded nanoparticles can significantly enhance progression free and overall survival in patients afflicted with GBM.
Learning Objectives: 1. miRNAs are potential therapeutic agents to counteract intra-tumor heterogeneity
2. miR-34a sensitizes to both temozolomide and radiation therapy via multiple mechanisms
3. Nanoparticles can be used as delivery vehicles for miRNA to brain tumors