Functional characterization of DNA polymerase theta in esophageal squamous cell carcinoma

Abstract

The specialized DNA polymerase theta (Pol θ), encoded by the POLQ gene, is involved in repairing DNA double-strand breaks (DSBs) through the alternative end-joining (Alt-EJ) pathway. In our previous targeted gene sequencing studies sampling familial esophageal squamous cell carcinoma (ESCC) cases from an endemic high-risk region of China, deleterious germline variants of POLQ were identified. I aim to determine the functional roles of POLQ and their underlying molecular mechanisms in the development of ESCC. By integrated analysis of one in-house and four public RNA-seq databases, I found that POLQ is predominantly upregulated in ESCC tumors. This ectopic expression of POLQ was also observed in a cohort of 25 Hong Kong ESCC patients, in whom the expression level of POLQ was negatively correlated with the patient survival before ESCC-related death (R2=0.6557, p=0.008). The CRISPR technique was implemented to knockout POLQ in three ESCC cell lines with high endogenous POLQ expression levels. The POLQ-depleted ESCC cells were significantly sensitized to stress inducers like hydroxyurea or platinum-based drugs compared with control cells. Both rH2AX foci staining and the comet assay indicate a higher level of genomic instability in POLQ knockout cells than in control cells, when exposed to ionizing radiation. Double knockout of POLQ and FANCD2, a DNA damage repair gene functioning in both Fanconi anemia and the homologous recombination (HR) DNA damage repair pathway, significantly sabotaged cell proliferation in vitro as well as in vivo, as compared with either of these single knockouts. Cells with POLQ and/or FANCD2 depletion also showed exacerbated levels of CHK2 phosphorylation. A significantly increased number of micronuclei was observed in POLQ and/or FANCD2 knockout ESCC cells. Loss of POLQ and FANCD2 also resulted in the activation of cGAS and upregulation of several interferon-stimulated genes (ISGs). Taken together, the results suggested the role of POLQ as a guardian of genome stability, as POLQ depletion sensitized ESCC cells to multiple genotoxic agents, suggesting using a POLQ inhibitor in combination with conventional chemotherapy drugs as a novel strategy in ESCC management. Meanwhile, the potential synthetic lethality relationship between POLQ and FANCD2 was described. Importantly, exciting novel findings from this study now identified new evidence linking the depletion of DNA damage repair proteins (POLQ/FANCD2) with the activation of anti-tumor immunity through the cGAS-STING-STAT1 signaling pathway.