PCGF4 is an important regulator of cancer cell proliferation and CSC self-renewal, and it is considered an important therapeutic target . Previous studies have found that PCGF4 is regulated by another PcG protein, PCGF2 . Our study suggested that the two homologous proteins PCGF2 and PCGF4 reversely regulate the stem cell population through p38 pathway (Fig. 5C). Expression of PCGF2 and PCGF4 show an opposite trend in gastric cancer and breast cancer [39, 40]. However, regulation of the activity of LCSCs by PCGF2 or PCGF4 has not been verified. In this study, we observed that PCGF2 and PCGF4 negatively regulate the activity of LCSCs. It was also found that PCGF4 protein expression can be used as an independent prognostic indicator of overall survival but that PCGF2 expression cannot. However, inconsistent with other reports, we did not find that low expression of PCGF2 is associated with poor prognosis. This discrepancy may be due to the limited number of samples in our study or different types of cancer studied.
In addition, our data show that PCGF2 or PCGF4 not only play a role in the prognosis of HCC but are also related to the stemness and drug resistance of LCSCs. This reliable result indicates that low expression of PCGF4 or high expression of PCGF2 can reduce the population of LCSCs and sphere formation. Similar to HCC cells, LCSCs have heterogeneity, we used different LCSCs markers to compare effects on expression of PCGF2 and PCGF4 based on changes in LCSCs populations. Remarkably, low expression of PCGF4 reduced the abundance of CD13+, EpCAM+, and CD133+ cells, but high expression of PCGF2 only affected that of CD13+ and EpCAM+ cells. In line with our findings, currently reported markers for LCSCs are not specific to LCSCs. To facilitate separation of LCSCs, various types of markers should be examined . Our results indicate that PCGF2 or PCGF4 can alter the population and self-renewal ability of LCSCs. To determine whether low expression of PCGF2 leads to poor prognosis, we conducted IHC, and the results showed that PCGF2 is expressed at lower levels in HCC tissues than in paracancerous tissues but that PCGF2 cannot be used as an independent prognostic marker for patients with HCC.PCGF2, which is a component of the polycomb protein complex PRC1, regulates cell proliferation and senescence through transcriptional inhibition of PCGF4 and c-Myc oncoproteins . However, no significant difference in the half-life of PCGF4 in control and PCGF2-over-expressing cells was observed, further indicating that PCGF4 is not regulated at the protein level by PCGF2 . In our tumor samples, we did not detect a tendency for PCGF4 to be over-expressed in the same patient, but PCGF2 to be lower-expressed. It should be noted that we assessed the protein level and not the transcription level.
Regarding the HCC cell line, PCGF4 affects the proliferation of HCC cells, which is the same as the results of Wu et al [9, 10, 16, 22, 43, 44]. Over-expression of PCGF2 had no significant effect on the proliferation of HCC cells but enhanced sensitivity to sorafenib. Similarly, PCGF2 has been proposed as a tumor suppressor in breast cancer . PCGF2 over-expression restored ER-α expression in triple-negative breast cancer (TNBC). Furthermore, mice bearing PCGF2-overexpressing MDA MB-468 TNBC cell tumors acquired sensitivity to tamoxifen treatment, although no difference in tumor growth was observed between the control and PCGF2-overexpressing cell xenografts .
We also found that PCGF4 and PCGF2 negatively regulate tumor sphere formation. It is suggested that PCGF2, as a tumor-suppressor gene, also affects the maintenance of LCSCs stemness.
We demonstrated that PCGF4 KD or PCGF2 OE regulate the stem phenotype of LCSCs through the p38 pathway. Some study explored the role of p38 MAPK in maintenance of CSC phenotype, therapy resistance and DNA damage repair and response in HNSCC . The role of p38 in cancer has been extensively studied. Some reports indicate that p38 acts as an anti-tumor factor, especially in promoting cell cycle arrest and differentiation, as a tumor suppressor. However, some articles discuss that p38 promotes cancer by enhancing tumor cell survival, migration, or resistance to stress and chemotherapy drugs . There is a dual function of p38 MAPKs in colon cancer: inhibiting initiation of colitis-related tumors but promoting cancer cell survival . Some studies demonstrate that p16Ink4a inhibits lung adenocarcinoma through PCGF4 when p38 is activated .
Our data indicated that the PCGF4 KD or PCGF2 OE could inhibit stem-like properties in vitro via downregulating the expression of p38 pathway. More importantly, PCGF4 KD or PCGF2 OEcould increase the sensitivity of HCC cells to sorafini. Therefore, further studies are required to develop a combination therapy as a potential therapeutic strategy.