Gupta, Shiv K.

Abstract Purpose: The therapeutic benefit of temozolomide in glioblastoma multiforme (GBM) is limited by resistance. The goal of this study was to elucidate mechanisms of temozolomide resistance in GBM. Experimental Design: We developed an in vivo GBM model of temozolomide resistance and used paired parental and temozolomide-resistant tumors to define the mechanisms underlying the development of resistance and the influence of histone deacetylation (HDAC) inhibition. Results: Analysis of paired parental and resistant lines showed upregulation of O6-methylguanine-DNA methyltransferase (MGMT) expression in 3 of the 5 resistant xenografts. While no significant change was detected in MGMT promoter methylation between parental and derivative-resistant samples, chromatin immunoprecipitation showed an association between MGMT upregulation and elevated acetylation of lysine 9 of histone H3 (H3K9-ac) and decreased dimethylation (H3K9-me2) in GBM12 and GBM14. In contrast, temozolomide resistance development in GBM22 was not linked to MGMT expression, and both parental and resistant lines had low H3K9-ac and high H3K9-me2 within the MGMT promoter. In the GBM12TMZ-resistant line, MGMT reexpression was accompanied by increased recruitment of SP1, C-JUN, NF-κB, and p300 within the MGMT promoter. Interestingly, combined treatment of GBM12 flank xenografts with temozolomide and the HDAC inhibitor suberoylanilide hydroxamic acid (SAHA) favored the evolution of temozolomide resistance by MGMT overexpression as compared with treatment with temozolomide alone. Conclusion: This study shows, for the first time, a unique mechanism of temozolomide resistance development driven by chromatin-mediated MGMT upregulation and highlights the potential for epigenetically directed therapies to influence the mechanisms of resistance development in GBM. Clin Cancer Res; 18(15); 4070–9. ©2012 AACR.

Integrins are important mediators of cell adhesion and migration, which in turn are essential for diverse biological functions, including wound healing and cancer metastasis. The integrin α9β1 is expressed on numerous mammalian tissues and can mediate accelerated cell migration. As the molecular signaling mechanisms that transduce this effect are poorly defined, we investigated the pathways by which activated integrin α9β1 signals migration. We found for the first time that specific ligation of integrin α9β1 rapidly activates Src tyrosine kinase, with concomitant tyrosine phosphorylation of p130Cas and activation of Rac-1. Furthermore, activation of integrin α9β1 also enhanced NO production through activation of inducible nitric oxide synthase (iNOS). Inhibition of Src tyrosine kinase or NOS decreased integrin-α9β1-dependent cell migration. Src appeared to function most proximal in the signaling cascade, in a FAK-independent manner to facilitate iNOS activation and NO-dependent cell migration. The cytoplasmic domain of integrin α9 was crucial for integrin-α9β1-induced Src activation, subsequent signaling events and cell migration. When taken together, our results describe a novel and unique mechanism of coordinated interactions of the integrin α9 cytoplasmic domain, Src tyrosine kinase and iNOS to transduce integrin-α9β1-mediated cell migration.