Integrin αVβ3 silencing sensitizes malignant glioma cells to temozolomide by suppression of homologous recombination repair

// Markus Christmann 1, * , Kathrin Diesler 1 , Dragomira Majhen 2 , Christian Steigerwald 1 , Nancy Berte 1 Halima Freund 1 , Nikolina Stojanovic 2 , Bernd Kaina 1 , Maja Osmak 2 , Andreja Ambriovic-Ristov 2 , Maja T. Tomicic 1, * 1 Department of Toxicology, University Medical Center Mainz, D-55131 Mainz, Germany 2 Laboratory for Cell Biology and Signaling, Division of Molecular Biology, Ruđer Boskovic Institute, HR-10000 Zagreb, Croatia * These authors contributed equally to this work Correspondence to: Maja T. Tomicic, email: tomicic@uni-mainz.de Andreja Ambriovic-Ristov, email: Andreja.Ambriovic.Ristov@irb.hr Keywords: malignant gliomas, integrin αVβ3 silencing, temozolomide, homologous recombination repair, Rad51 Received: March 30, 2016      Accepted: July 19, 2016      Published: July 28, 2016 ABSTRACT Integrins have been suggested as possible targets in anticancer therapy. Here we show that knockdown of integrins αVβ3, αVβ5, α3β1 and α4β1 and pharmacological inhibition using a cyclo-RGD integrin αVβ3/αVβ5 antagonist sensitized multiple high-grade glioma cell lines to temozolomide (TMZ)-induced cytotoxicity. The greatest effect was observed in LN229 cells upon integrin β3 silencing, which led to inhibition of the FAK/Src/Akt/NFκB signaling pathway and increased formation of γH2AX foci. The integrin β3 knockdown led to the proteasomal degradation of Rad51, reduction of Rad51 foci and reduced repair of TMZ-induced DNA double-strand breaks by impairing homologous recombination efficiency. The down-regulation of β3 in Rad51 knockdown (LN229-Rad51kd) cells neither further sensitized them to TMZ nor increased the number of γH2AX foci, confirming causality between β3 silencing and Rad51 reduction. RIP1 was found cleaved and IκBα significantly less degraded in β3-silenced/TMZ-exposed cells, indicating inactivation of NFκB signaling. The anti-apoptotic proteins Bcl-xL, survivin and XIAP were proteasomally degraded and caspase-3/-2 cleaved. Increased H2AX phosphorylation, caspase-3 cleavage, reduced Rad51 and RIP1 expression, as well as sustained IκBα expression were also observed in mouse glioma xenografts treated with the cyclo-RGD inhibitor and TMZ, confirming the molecular mechanism in vivo . Our data indicates that β3 silencing in glioma cells represents a promising strategy to sensitize high-grade gliomas to TMZ therapy.