Breaking to Understand: DNA Repair in Response to Cancer Therapy

Human DNA constantly faces endogenous and exogenous damage, with DNA double-strand breaks (DSBs) posing the greatest threat to genome integrity. However, DSBs can be leveraged to kill cancer cells, as many treatments act as DSB inducers. The dominant repair pathway, non-homologous end-joining (NHEJ), resolves the majority of DSBs. This thesis explores strategies to sensitize resistant cancer cells through combination therapy and investigates NHEJ’s response to varying DSB complexities. Paper I addresses cisplatin resistance in ovarian cancer. We found that combining cisplatin with the HSP90 inhibitor onalespib enhances sensitivity by increasing DSB levels, inducing apoptosis, and causing G2/M arrest, making it a promising strategy. Paper II focuses on glioblastoma (GBM), an aggressive brain tumor with limited treatment options. We demonstrated that onalespib enhances radiosensitivity in 2D and 3D GBM models by increasing DSB levels, promoting apoptosis, and altering protein expression, suggesting that HSP90 inhibition could improve radiotherapy outcomes. Paper III investigates the alpha emitter Ra-223, used in bone-metastatic prostate cancer. Our findings revealed that Ra-223 generates clustered DSBs, triggering NHEJ activation, growth inhibition, and apoptosis in prostate cancer cells, with no detectable cellular uptake. Paper IV explores pharmacological ascorbate (Asc) effect on NHEJ pathway. We found that Asc induces delayed DSBs, extensive pan-nuclear γH2AX formation, necrosis, and G2/M arrest in colorectal cancer cells, with stronger effects in XRCC4 KO cells. We concluded that Asc does not generate prompt DSBs, and the delayed DSBs are linked to necrotic nuclear degradation, with sensitivity influenced by cell cycle regulation rather than NHEJ deficiency. Paper V examines NHEJ’s role in repairing DSBs of varying complexity in colorectal cancer cells. Wild-type cells exhibited both fast and slow repair kinetics, while NHEJ-deficient cells showed only a fast repair phase, followed by repair failure. Non-DSB clusters increased as the DSB:SSB ratio decreased (from calicheamicin to X-rays, bleomycin, etoposide, and temozolomide). These clusters were rapidly removed, independent of NHEJ, highlighting the impact of DSB type/complexity on repair efficiency. In conclusion, this thesis presents strategies to overcome cisplatin resistance, enhance radiosensitivity in GBM, and elucidate Ra-223 toxicity mechanisms in prostate cancer. It also examines Asc’s effects on DSB induction and repair and reveals NHEJ’s role in processing complex DSBs. Our findings provide new insights into optimizing DSB repair and therapeutic strategies in cancer treatment.