Novel role of drug-induced non homologous end joining factor 1 in the chemoresistance of hepatocellular carcinoma

Abstract

Hepatocellular carcinoma (HCC) is highly resistant to chemotherapy and one of the most common causes of cancer related death worldwide. Chemoresistance in HCC leads to restricted treatment option and poor prognosis. Acquired cellular chemoresistance mechanisms include oncogenes mutation, multi-drug resistance gene (MDR1) over expression or hypermethylation, cancer stem cells enrichment, and dysfunctional apoptosis. Most cytotoxic drugs serve as DNA damaging agents and are able to generate various DNA damages among which double strand breaks (DSBs) are the most toxic. Cellular responses such as increased DNA damage repair might imply a potential chemoresistance, which is not well defined in HCC. In this project, I aim to identify potential role of NHEJ1 mediated non homologous end joining (NHEJ) repair in HCC chemoresistance.

Chemo-drugs with diverse damaging effects (cisplatin/oxaliplatin: bulky adducts and cross link; doxorubicin: single strand breaks; 5-Fu: mismatch bases) can induce DSBs in HCC cells. In response, expressions of the key genes involved in NHEJ pathway were significantly increased. By calculating chemo-drugs IC50 concentration, we found that IC50 of HCC line 97L was significantly higher than PLC cells. Using the plasmid-based cell-free and live cell NHEJ assay, 97L cells displayed significantly enhanced NHEJ activity in both in vitro and in vivo assay, whereas PLC cell did not show drug-induced NHEJ activity. In addition, chemo drug-induced NHEJ activity was significantly increased in CD133+ HCC cell (known as HCC cancer stem cell proportion) but not in their CD133- counterparts, resulting in a more chemoresistance in CD133+ cell. These data indicate that efficient NHEJ repair might involve in chemo-resistance in HCC cells.

Non homologous end joining factor 1 (NHEJ1) is a novel component of the NHEJ pathway. NHEJ1 interacts with XRCC4 and stabilize LIG4/XRCC4/NHEJ1 complex during DSBs ends ligation—the final step of the NHEJ pathway. Based on integrative analysis of cancer genomics and clinical profiles, low genomic alteration frequency of NHEJ1 was found in many types of cancers including in HCC (0.5%). However, in my clinical study, significantly enhanced NHEJ1 expressions were found in HCC patients who received transcatheter arterial chemoembolization (TACE) but not in patients without TACE or any chemo-drug treatment, indicating a responsive over expression of NHEJ1 after chemo-drug treatment. More importantly, higher expression of NHEJ1 in TACE patients was significantly associated with larger tumor size, presence of venous infiltration, advanced stage and shorter overall survival.

Silencing NHEJ1 by siRNA or shRNA inhibited drug-induced NHEJ activity therefore restored chemo-drug sensitivity in 97L cells. The sensitive effects were shown by suppressed colony formation in vitro, and reduction of HCC tumor size in vivo using a xenograft mouse model. I also found that there was a cosuppression of LIG4 and ERCC1 in shNHEJ1 cells. Furthermore, co-inhibition of NHEJ1 and ERCC1 by siRNA synergistically enhanced oxaliplatin cytotoxicity in HCC cells.

In summary, I identified drug-induced NHEJ1 up regulation and enhanced NHEJ repair could be a novel chemo resistant mechanism in HCC. Thus, targeting NHEJ1 therefore NHEJ repair might have therapeutic potential to overcome chemoresistance in HCC.