The Error-Prone DNA Polymerase κ Promotes Temozolomide Resistance in Glioblastoma through Rad17-Dependent Activation of ATR-Chk1 Signaling

Abstract

The acquisition of drug resistance is a persistent clinical problem limiting the successful treatment of human cancers, including glioblastoma (GBM). However, the molecular mechanisms by which initially chemoresponsive tumors develop therapeutic resistance remain poorly understood. In this study, we report that Pol k, an error-prone polymerase that participates in translesion DNA synthesis, was significantly upregulated in GBM cell lines and tumor tissues following temozolomide treatment. Overexpression of Pol k in temozolomide-sensitive GBM cells conferred resistance to temozolomide, whereas its inhibition markedly sensitized resistant cells to temozolomide in vitro and in orthotopic xenograft mouse models. Mechanistically, depletion of Pol k disrupted homologous recombination (HR)-mediated repair and restart of stalled replication forks, impaired the activation of ATR-Chk1 signaling, and delayed cell-cycle re-entry and progression.Further investigation of the relationship between Pol k and temozolomide revealed that Pol k inactivation facilitated temozolomide-induced Rad17 ubiquitination and proteasomal degradation, subsequently silencing ATR-Chk1 signaling and leading to defective HR repair and the reversal of temozolomide resistance. Moreover, overexpression of Rad17 in Pol k-depleted GBM cells restored HR efficiency, promoted the clearance of temozolomide-induced DNA breaks, and desensitized cells to the cytotoxic effects of temozolomide observed in the absence of Pol k. Finally, we found that Pol k overexpression correlated with poor prognosis in GBM patients undergoing temozolomide therapy. Collectively, our findings identify a potential mechanism by which GBM cells develop resistance to temozolomide and suggest that targeting the DNA damage tolerance pathway may be beneficial for overcoming resistance. Cancer Res; 76(8); 2340-53. Ó2016 AACR.

Cell-free Xenopus egg extracts for studying DNA damage response pathways

“…Consistent with this observation, Pol κ depletion facilitates temozolomide (TMZ)-induced ubiquitination and proteasome-mediated degradation of Rad17 and severely compromises ATR-Chk1 DDR pathway activation in human glioblastoma cell lines (Step 4, Fig. 4) (Wang et al, 2016). These findings suggest that TLS polymerases play a previously uncharacterized role in ATR-Chk1 DDR pathway via its catalytic and non-catalytic functions.…”

Depletion of DNA damage binding protein 2 sensitizes triple‐negative breast cancer cells to poly ADP‐ribose polymerase inhibition by destabilizing Rad51

“…The system was invented and provided as a gift by Dr Maria Jasin of the Memorial Sloan‐Kettering Cancer Center. Homologous recombination DNA repair assay was carried out as in our previous study . Briefly, the DR‐GFP HR repair substrate included 2 different GFP genes with specific mutations.…”

“…Homologous recombination DNA repair assay was carried out as in our previous study. 17 Briefly, the DR-GFP HR repair substrate included 2 different GFP genes with specific mutations. I-SceI endonuclease ectopic expression resulted in DSBs in one of the 2 differentially mutated GFP genes.…”

The role of double‐strand break repair, translesion synthesis, and interstrand crosslinks in colorectal cancer progression—clinicopathological data and survival

“…The oncological relevance of pol κ and pol η in cancer is most firmly established concerning response to treatment. Upregulation of pol κ confers resistance to temozolomide in glioblastoma, 34,35 and upregulation of pol η to platinum drugs in HNSCC, lung, gastric adenocarcinomas, and ovarian cancers. [36][37][38] Contrary to our results, low levels of POLK were previously observed in CRC.…”