PC4 Mediated Enhancement of NHEJ Repair Promotes Radioresistance in Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) is the leading cause of cancer-related deaths globally, primarily due to the late diagnosis and limited therapeutic treatments, including radiotherapy. The onset of radioresistance complicates treatment and significantly worsens patient prognosis, therefore, understanding the molecular basis of radioresistance in liver cancer is important for developing more effective treatments.

Human Positive Cofactor 4 (PC4) is a multifunctional protein that plays a significant role in transcription regulation, DNA repair, and chromatin organization. Its involvement in cancer is tied to these fundamental cellular functions. There is a need to understand better the precise mechanisms by which PC4 influences radiosensitivity, with the aim of developing targeted therapies that could enhance the effectiveness of radiotherapy in cancer treatment. To this end, a new study published in the Journal of Biological Chemistry by a research team composed of Dr. Qimei Pan, Dr. Peng Luo, and Dr. Chunmeng Shi from the Third Military Medical University (Army Medical University), the researchers investigated the role of PC4 in mediating radioresistance in HCC through enhancing DNA repair mechanisms.  First, the team used HCC cell lines (Huh7 and HepG2) and modified them to either knock down PC4 using shRNA or overexpress PC4. To assess the impact of PC4 on cell survival post-irradiation, they performed colony formation assays which showed that PC4 knockdown increased radiosensitivity, and decreased the surviving fraction of cells post-irradiation. In contrast, PC4 overexpression made the cells more resistant to radiation. Afterward, they injected Huh7 cells with either PC4 knockdown or overexpression into BALB/c nude mice to form tumors and then the authors subjected the mice to radiation. The authors observed that tumors with PC4 knockdown grew significantly slower compared to control, while PC4 overexpression led to enhanced tumor growth post-irradiation.  Tumors were extracted and weighed, and the tumor growth curves were plotted to visually demonstrate the impact of PC4 manipulation on radioresistance in vivo.

To directly visualize DNA damage repair, the researchers used laser micro-irradiation to induce DNA double-strand breaks (DSBs) and employed live cell imaging to track the recruitment of PC4 and other DNA repair proteins to damage sites. PC4 was rapidly recruited to DSB sites, indicating its active role in the early DNA damage response. Moreover, the authors studied the molecular mechanism by which PC4 influences DNA repair, and performed coimmunoprecipitation followed by mass spectrometry. These experiments showed that PC4 interacts with key DNA repair proteins, including poly (ADP-ribose) polymerase 1 (PARP1) and the Ku complex, which are central for nonhomologous end joining (NHEJ), a major DSB repair pathway. Furthermore, through the use of immunofluorescence staining for γH2AX (a marker of DSBs) and neutral comet assays (to measure DSB repair), they showed that PC4 knockdown impaired the efficiency of DSB repair, leading to increased DNA damage and reduced cell survival post-irradiation. Indeed, PC4 enhances the repair of radiation-induced DSBs through the NHEJ pathway, thereby contributing to radioresistance in HCC cells.

Additionally, the research team investigated the clinical relevance of PC4 and examined PC4 expression in human HCC samples and adjacent non-tumor liver tissues using immunohistochemistry and western blot. The authors found that PC4 is highly expressed in HCC tissues and correlated with poor prognosis which suggest that PC4 could serve as a biomarker for radioresistance and a potential therapeutic target to overcome resistance to radiotherapy.  Moreover, the study presents PC4 as a novel regulator of the DNA damage response (DDR), specifically within the context of NHEJ, a primary pathway for repairing DSBs induced by radiation. PC4’s interaction with the key DDR proteins, PARP1 and the Ku complex, highlights the mechanism wherein PC4 mediates the PARylation of the Ku complex, and how it facilitates efficient recruitment to damaged chromatin sites. This process enhances both the repair of DSBs and fosters a radioresistant phenotype in HCC cells.

The implications of study by Dr. Chunmeng Shi and colleagues are vast, and offers hope for improving the efficacy of radiotherapy in HCC treatment. Clinically, the significance of PC4 extends beyond its molecular functions to its potential as a prognostic marker for HCC. High expression levels of PC4 in tumor tissues correlate with poor patient outcomes, presenting a novel biomarker for identifying individuals at risk of radioresistant disease. Moreover, targeting PC4 offers a promising therapeutic strategy to sensitize HCC cells to radiotherapy, potentially overcoming one of the significant barriers to effective HCC management. Furthermore, the identification of PC4 as a marker of radioresistance also offers a stratification tool for clinical trials, enabling the selection of patient cohorts more likely to benefit from novel therapies targeting DNA repair pathways.