According to previous reports, CMTMs exert a crucial role in regulating tumor development in multiple cancer types [19, 20]. They are closely related to tumor growth, metastasis, and antitumor immunity [21, 22]. In this study, we systematically explored the relationship between CMTMs and HCC occurrence and prognosis. Analysis of the potential signaling pathways in which CMTMs are involved further revealed the significant biological roles of CMTMs in HCC development.
CKLF is a member of the CMTM family, and there are four alternative RNA splicing forms for CKLF: CKLF1, CKLF2, CKLF3 and CKLF4. Liu et al. concluded that CKLF1, which is highly expressed in HCC tissues, was associated with vascular invasion. Moreover, CKLF1 works as an activator of the IL-6/STAT3 signaling pathway to induce HCC development and metastasis [7]. Our findings were consistent with theirs, showing the overexpression of CKLF at the mRNA level in HCC. Furthermore, we also found that a high mRNA level of CKLF in predicted a poor prognosis in HCC patients, which confirms the prognostic value of CKLF in HCC.
Previous studies reported that CMTM1 was highly expressed in various tumors, including HCC [23]. Our results also followed the same trend. However, there was no statistically significant difference in the CMTM1 mRNA expression levels between HCC tissues and normal tissues. We considered that this might be a result of the inclusion of FC > 1.5 in the GEPIA database. We also found that patients with high mRNA levels of CMTM1 experienced short OS. This was consistent with the results of Song et al [24]. Their research suggested that the survival of HCC patients with CMTM1 positive expression was significantly shorter than those with CMTM1 negative expression. Taken together, these results suggest that CMTM1 is closely associated with HCC progression and patient prognosis. Further study on the mechanism of CMTM1 promoting the progression of HCC is one of the important research directions in the future.
CMTM2 has shown clinical significance in different tumors, such as diffuse-type gastric cancer [25]. In our study, we found that the mRNA level of CMTM2 was lower in HCC tissues than in normal tissues. CMTM2 expression levels were significantly correlated with different stages of HCC, which is consistent with Guo et al.’s findings. Similarly, they found that CMTM2 was correlated with tumor grade in HCC [26]. Furthermore, our findings revealed that patients with high mRNA expression levels of CMTM2 tended to have better DFS. Guo et al showed a similar tendency. They applied immunohistochemistry to confirm that HCC patients with negative CMTM2 expression had a significantly shorter OS time. Zhang et al. conducted an study on the mechanism by which CMTM2 down-regulation promotes the progression of HCC. They determined the epithelial-mesenchymal transition (EMT) process following down-regulation of CMTM2 expression by examining the expression of EMT-related factors. Western blot results showed that epithelial markers were decreased, while mejsenchymal markers were increased after CMTM2 was knocked-down. These results showed down-regulated CMTM2 could induce the EMT process in HCC cells. They also found a significant positive correlation between CMTM2 and E-cadherin expression in HCC tissues. These results suggested that down-regulation of CMTM2 expression in HCC tissues can promote HCC metastasis by inducing EMT process [27]. Combined with our results, CMTM2 may be a potential tumor suppressor gene, and high levels of CMTM2 expression may be related to a good prognosis. However, in-depth research is needed to determine the regulatory mechanism of CMTM2 in HCC.
CMTM3 acts as a tumor suppressor in a variety of tumors, and DNA methylation of the CMTM3 promoter can inhibit CMTM3 expression in tumor tissues [28, 29]. However, there were few studies on the role of CMTM3 in HCC. Li et al. found that CMTM3 was weakly expressed in multiple HCC cell lines included HepG2, 97H, Hep3B, and HCCLM3. Moreover, in their study, the results of Western blot analysis showed that compared with the vector group, overexpression of CMTM3 significantly down-regulated the phosphorylation level of JAK2 and the downstream JAK substrate STAT3 in HepG2 cells. Based on these results, they suggested that CMTM3 may play an important role in HCC metastasis by inhibiting the JAK2/STAT3 signaling pathway [30]. Zhao et al. found that CMTM3 might be involved in the regulation of the tumor microenvironment in HCC [31]. Our research showed that the overexpression of CMTM3 in HCC patients occurred at both the mRNA and protein levels, which is inconsistent with previous findings. Moreover, the relationship of CMTM3 with the prognosis of HCC could not be elucidated. However, CMTM3 expression was closely correlated with immune infiltration in HCC in our research. Based on the above results, we hypothesized that the role of CMTM3 in HCC may be affected by the cell or matrix components in the tumor microenvironment, and thus displayed different expression patterns from cell lines. Until now, the prognostic value of CMTM3 in HCC has not been clear. Further investigation is needed to explain the function and prognostic value of CMTM3 in HCC.
Previous studies have explored the function of CMTM4 in various types of cancer, including renal cell carcinoma [32], colorectal cancer [33], and pancreatic cancer [34]. Several studies have demonstrated the clinical importance of CMTM4 in HCC. Bei et al. found a low expression level of CMTM4 in HCC tissues and suggested that low CMTM4 expression was a risk factor for a poor prognosis [35]. In our report, we found that CMTM4 was overexpressed in HCC patients at both the mRNA and protein levels. A high expression level of CMTM4 was correlated with shorter OS and DFS, which is inconsistent with Bei’s perspectives. Furthermore, we found that CMTM4 might exert an essential role in the tumorigenesis and progression of HCC. Zhou’s findings were in line with ours. The expression of CMTM4 was significantly upregulated in HCC tissues and correlated with a poor prognosis in patients with HCC [36]. One study demonstrated that CMTM4 can participate in immune escape through the synergistic protective effect of PD-L1 and is extensively involved in the process of tumor proliferation [37]. Recently, Chui et al. reported new findings in their study on CMTM4 in HCC. They demonstrated that CMTM4 acts as a positive regulator of PD-L1 in a variety of HCC cell lines, and CMTM4 stabilizes PD-L1 expression through posttranslational mechanisms. They also found that suppression of CMTM4 can inhibit HCC growth and increased CD8 + T-cell infiltration in immunocompetent mice [38]. These findings might explain the crucial role of CMTM4 in HCC occurrence and progression, which can shorten a patient’s survival time. In our TIMER database analysis, CMTM4 was found to be positively correlated with the immune markers of exhaustion T cells included PD-1, PD-L1, CTLA4 and TIM-3. This was basically consistent with the research results of Chui et al. These results suggested that CMTM4 may promote HCC progression by inducing the immunosuppressive tumor microenvironment. Considering the existing positive research results, the role of CMTM4 in HCC is worthy of further study.
Similar to CMTM3, CMTM5 has a tumor-suppressive function, and the promoter methylation of CMTM5 can cause a tumor to evade anticancer effects [39]. Our study demonstrated that patients with high mRNA expression levels of CMTM5 experienced better OS and DFS, which was consistent with previous findings. According to the report by Guan et al., miR-10b-3p is notably upregulated in HCC cell lines based on the significant suppression of CMTM5 expression, which further promotes HCC metastasis and progression. In addition, other studies have shown that CMTM5 expression can be restored in HCC by treatment with the histone deacetylase inhibitor PXD101 [40]. Therefore, it was reasonable to assume that CMTM5 may be a valuable therapeutic target for HCC. However, the expression pattern of CMTM5 in HCC and its effect on prognosis still need to be verified by a larger sample study.
CMTM6 is regarded as a critical regulator in terms of the maintenance of PD-L1 expression in multiple cancers [41]. The researchers found that PD-L1 relied on CMTM6/4 to effectively perform its inhibitory function [37]. In this study, we found that the expression of CMTM6 and CMTM4 showed significant positive correlation. In addition, according to our research, the mRNA expression levels of CMTM6 were decreased in HCC patients, and high mRNA expression levels of CMTM6 were associated with short OS. These findings were in line with those described by Zhu et al [42]. One study showed that CMTM6 expression was positively correlated with immunosuppressive factors, such as induced T cell tolerance and regulatory T cell differentiation [43]. Yugawa et al. confirmed that CMTM6 and PD-L1 were co-expressed in HCC. They also found that high CMTM6 and PD-L1-positive expression was associated with malignant aggregates and shorter OS of HCC patients [44]. This may be due to its function in stabilizing PD-L1 expression, thereby facilitating escape from T-cell-mediated cytotoxicity. Our correlation analysis furtherly showed that there were positive correlations between CMTM6 expression and the immune markers of exhaustion T cell such as PD-L1, PD-1, CTLA4, TIM-3 and LAG3. This was consistent with Yugawa, K’s research results. Further study of the relationship between CMTM6 and PD-L1 will open up a new path for the immunotherapy of HCC.
CMTM7 is broadly expressed in normal human tissues, particularly in immune cells. Huang et al. revealed that CMTM7 was notably downregulated in HCC tissues along with cell lines and exhibited tumor-suppressor activities [45]. In addition, they concluded that CMTM7 overexpression led to cell cycle arrest at G0/G1 phase and then inhibited HCC cell growth and migration. Our results were contrary to existing findings, which showed that CMTM7 was overexpressed at the mRNA level, and low CMTM7 expression was closely correlated with a good prognosis in HCC. In addition, through immune infiltration analysis, we found that CMTM7 was significantly positively correlated with PD-1, CTLA4, LAG3, TIM-3, GZMB and PD-L1. This results suggested that CMTM7 may play an important role in immune escape and induce of immunosuppressive microenvironment. Therefore, it is necessary to focus on the function and systematic performance of CMTM7 in the HCC microenvironment.
CMTM8, which functions as a tumor suppressor, accelerates the internalization of EGFR to attenuate the EGFR-mediated signaling pathway [46]. In our research, CMTM8 expressed higher in HCC tissues, but did not differ significantly between HCC and normal tissues. In addition, the immune infiltration analysis showed that CMTM8 was almost independent of various tumor infiltrating immune cells. Prior studies revealed the possible silencing of CMTM8 in tumors, which was similar to our findings [47]. At present, the role and prognostic value of CMTM8 in HCC need to be further studied.
In this study, we carried out a systematic analysis of the expression, prognostic value and relationship of immune infiltration and CMTMs in HCC. We concluded that high expression levels of CKLF, CMTM1, CMTM4 and CMTM6-7 expression could also serve as molecular markers to identify high-risk subgroups of HCC patients. The genetic alterations in CMTMs were correlated with a poor prognosis in HCC. CKLF, CMTM1-4 and CMTM6-7 demonstrated close associations with immune infiltration in HCC.