The CMTM family has been found in a variety of cancers, including liver cancer. However, the mechanism in liver cancer has not been clarified. In this research, the mRNA expression, DNA methylation and prognostic value of CmTM family genes in HCC were investigated. We hope that our findings will provide novel diagnostic biomarkers and treatment designs for patients with HCC.
In the present study, all the CMTMs mRNA expressions in HCC were shown. Besides, the high level of CKLF and CMTM 1/4/7 were significantly related to worse survival outcomes in HCC patients, while CMTM2/5 was connected with better survival outcomes. Although CMTM3/6/8 wasn’t significantly concerned with OS and PFS in HCC, 47 CpGs of CMTMs demonstrated significant prognostic values. Furthermore, multivariate cox analysis indicated that CKLF and CMTM7 were independent high-risk prognostic factors for the unfavorable OS of HCC patients. Through pathway enrichment analysis, we figured out that the members of an independent prognostic value in CMTM family were involved in the immune process, tumor development via JAK-STAT and MAPKsignaling pathways. Moreover, we also studied the correlation of tumor immune infiltrating cells with CMTMs. We revealed that CKLF and CMTM1/3/6/7 were significantly associated with TIICs. However, there was no significant correlation between CMTM8 expression and TIICs.
Chemokine-like factor (CKLF), as the first discovered member of CMTM family, was cloned by suppression subtractive hybridization (SSH) technology 2001. It has been reported that CKLF was downregulated in lung cancer tissues but upregulated in ovarian cancer tissues. CKLF was overexpressed in HCC tissues and predicted poor survival and related to vascular invasion of HCC patients. Mechanistically, CKLF could trigger the IL-6/STAT3 signaling pathway to promote HCC proliferation and migration and inhibit the apoptosis induced by Doxorubicin. Previous studies demonstrated that arterial injury-induced CKLF overexpression was positive associated with the accumulation of vascular smooth muscle cells (VSMC) and neointimal hyperplasia. In vitro study indicated that reduction of CKLF1 in vascular smooth muscle cells (VSMC) could promote cell death at the G2/M phase of cell cycle progression through a PI3K/Akt/NF-κB dependent signal pathways. It was found that CKLF1 was also overexpressed in some innate immune diseases, such as lupus nephritis (LN) and arthritis[17, 18]. That the expression of CKLF could increase in activated CD4+ and CD8+ lymphocytes. In addition, CKLF modulates chemotactic activity and activates neutrophils through the MAPK signal pathway. Similarly, our study showed that CKLF mRNA expression was upregulated in HCC tissues compared with non-cancer tissues, and its expression was significantly correlated with tumor stage, lymph node metastatic status, and tumor grade of patients. In terms of prognosis, high CKLF expression was related to poor OS and PFS of HCC patients, and six CpGs of CKLF were correlated with significant OS time in HCC patients. The results indicated that CKLF was an independent prognostic marker of HCC, and its deeper mechanism and specific function in HCC needed further research.
CMTM1 has been found in a variety of cancers, such as lung, breast, liver cancers, kidney and ovarian, and high level of CMTM1 promoted cell proliferation of breast cancer and inhibited TNF-α induced apoptosis by stimulating NF-κB pathway. Additionally, NSCLC patients with higher CMTM1 expression had significantly higher pathological staging and a shorter prognosis of OS and PFS, suggesting that CMTM1 may contribute to the process of chemotherapy resistance. A previous study showed that CMTM1 was overexpressed in lymphoma cells, and the injection of CMTM1 polypeptide in vitro can specifically promote apoptosis of lymphoma cells and may serve as a potential therapeutic for lymphoma treatment. Of note, mRNA expression of CMTM1 was remarkably associated with patients’ family history and TNM stages of HCC patients. Further analysis by IHC results indicated that negative expression of CMTM1 was related to poor prognosis of HCC patients. A similar tumorigenic effect of CMTM1 in HCC was also demonstrated in our present study. Our dates found that mRNA expression of CMTM1 was up-regulated in HCC, and its expression was markedly associated with clinical features such as cancer stages, node metastasis status and tumor grades. Our data also revealed that CMTM1 prompts poor OS and PFS in HCC patients, and nine CpGs of CMTM1 were correlated with survival outcomes in patients of HCC. Given the specific studies on the role of CMTM1 in HCC remained unknown, CMTM1 is worthy of further exploration.
CMTM2 was higher expressed in diffuse-type gastric cancer and patients with shorter overall survival tended to express lower mRNA expression of CMTM2. Previous research has shown that CMTM2 was reduced in HCC tissues and correlated with vascular invasion of HCC patients. They further confirmed that CMTM2 expression was significantly associated with pathological grade and prognosis of HCC. Notably, down-regulation of CMTM2 can promote the Huh-7 and SMMC7721 cells’ proliferation and migration via inducing the EMT (Epithelial-Mesenchymal Transition) process[26, 27]. What’s more, the low expression of CMTM2 in metastatic salivary adenoid cystic carcinoma (SACC) in patients with tumor recurrence and perinerve infiltration suggests a significant role in the progression of tumor metastasis in SACC. A study shows that intracellular CMTM2 can negative regulate HIV-1 transcription in U937 (human monocyte cell line) cells and Jurkat (lymphoblastoid T-cell line) through targeting the CREB and AP-1 pathways. Likewise, our results revealed that CMTM2 was reduced in HCC tissues and the level of CMTM2 was significantly associated with patients’ cancer stages. However, CMTM2 makes no difference with tumor grades and node metastasis status. Besides, high CMTM2 expression and one CpG of CMTM2 was related to favorable survival in HCC patients. We also found that CMTM2 was positive significantly correlated with the infiltrating levels of CD8 + T cells, CD4 + T cells, neutrophils cells, macrophages cells and dendritic cells. The specific role of CMTM2 in HCC deserves further study in order to deepen the understanding of HCC and develop new therapeutic methods.
CMTM3 was significantly lower in gastric cancer tissues than in normal tissues, and the expression of CMTM3 was significantly correlated with the stage and histological grade of gastric cancer. Moreover, increased expression of CMTM3 might be associated with a favorable prognosis in gastric cancer, and CMTM3 expression was an independent positive biomarker for gastric cancer. Other studies have found that CMTM3 was obviously reduced in oral squamous cell carcinoma (OSCC), which was associated with the prognosis, recurrence and advanced TNM stage in patients with OSCC. Additionally, the frequent DNA methylation in the CMTM3 gene promoter region was a potential unfavorable prognostic factor[32–34]. For instance, CMTM3 hypermethylation was associated with poor OS in male patients with laryngeal squamous cell carcinoma. Previous research has shown that CMTM3 was found to be highly expressed in peripheral blood mononuclear cells (PBMCs), CD4+ T lymphocytes, resting B lymphocytes and monocytes. Concerning HCC research, compared with the control group (HL-7702 cell line), the expression level of CMTM3 in human liver cancer cell lines (Hep3B, HCCLM3, 97H and HepG2) was significantly down-regulated. Besides, CMTM3 prohibited proliferation and migration in HCC cells (HepG2) by inhibiting the JAK2/STAT3 signaling pathway. In our present study, conflicting findings have been observed regarding the role of CMTM3 in HCC, and the mechanism of CMTM3 involved in the regulation of HCC development remains unknown. We found that CMTM3 was up-regulated in HCC and the mRNA expression of CMTM3 was markedly accord with the presence of cancer stages, node metastasis status and tumor grades of patients. However, CMTM3 was not apparently linked with the OS and PFS of HCC patients, three CpGs of CMTM3 were related to significant OS time. Therefore, the role of CMTM3 in HCC needs in-depth studies to elucidate.
CMTM4 was reported to be down-regulated in HCC compared with the adjacent non-cancer tissues. The expression of CMTM4 was negatively correlated with clinical (TNM) stage, tumor size and tumor metastasis rate, and plays an antitumor role in HCC. Moreover, the expression of CMTM4 was significantly reduced in colorectal cancer. Further revealed that CMTM4 could significantly inhibit CRC cells growth in vivo experiments by inhibiting AKT, ERK1/2, and STAT3 signaling pathways. It was reported that CMTM4 not only as a backup molecule modulating programmed cell death ligand 1 (PD-L1) protein but also identified as one of CD8+ T cell barren tumor-associated genes[39, 40]. Recent research demonstrated that CMTM4 was highly expressed in HCC in both TCGA dataset and Gene Expression Omnibus (GEO) database. CMTM4 was described as a prognostic marker of HCC, and CMTM4 was associated with the late clinical-stage, shorter OS, and PFS. Also, the level of CMTM4 had negative correlations with influences immune cell infiltration (cytotoxic cells, CD8 + T cells, T cell and dendritic cells) in HCC tissues. A similar tumorigenic role of CMTM4 in HCC was also displayed in our present study. CMTM4 was highly expressed in HCC tissues. The study also demonstrated that CMTM4 was markedly related to the patients’ cancer stages, node metastasis status and tumor grades in patients with HCC. A correlation was identified between high CMTM4 expression and poor prognosis for PFS time, and 8 CpGs of CMTM4 have also been found to be connected with survival outcome. Besides, we also found that CMTM4 was positively significantly correlated with the infiltrating levels of CD4 + T cells, B cells, neutrophils cells, macrophages cells and dendritic cells, indicating the role of CMTM4 in immunotherapy for HCC worthy further exploitation.
CMTM5 is closely 43% homology with CMTM3 and is involved in gene silencing. CMTM5 gene showed depressing tumor activities and was usually silenced by CpG methylation in various carcinomas, such as myeloid leukemia and breast cancer[12, 42, 43]. Studies have also reported a negative correlation between CMTM5 DNA promoter hypermethylation and its mRNA expression level in breast cancer. Moreover, CMTM5 was obviously linked with differentiation, clinical staging, lymphatic metastasis and was predicted poor progression-free survival of patients in breast cancer in breast cancer[12, 44]. A case-control study observed a strong correlation between polymorphisms of rs3811178 in CMTM5 and HCC risk in the southern Chinese population. Previous studies have found CMTM5 expressed at a low level in HCC. The low expression of CMTM5 is associated with advanced TNM stage, poor tumor differentiation and poor prognosis in HCC. Similarly, another study proved that CMTM5 was lowly expressed in HCC tissues and cells. The level of CMTM5 can be inhibited after up-regulated microRNA-10b-3p and then the proliferation, migration, and invasion abilities of HCC cells were suppressed in vitro . Our study investigated that mRNA expression of CMTM5 was down-regulated in HCC tissues and CMTM5 expression was significantly associated with patients’ tumor stages. High CMTM5 expression in HCC patients predicted better OS and two CpG of CMTM5 was related to overall survival time. Besides, CMTM5 expression was positively related to CD8 + T cell and neutrophil. These findings are consistent with our studies and provide a foundation on which to perform further cell biological function assays and clinical sample validation in the future.
Recent research identified CMTM6 modulated antitumor immunity by maintaining the expression of programmed death-1 ligand 1(PD-L1). The higher expression of CMTM6 and PD-L1 in HCC, the worse the prognosis, but the better prognosis in colorectal cancer, and advanced-stage non-small cell lung cancer (NSCLC). Moreover, high CMTM6 levels also indicated that head and neck squamous cell carcinoma (HNSCC) , breast cancer, and gliomas with a bad prognosis, but predicted a favorable prognosis in lung adenocarcinoma and hepatocellular carcinoma. Besides, the high expression of CMTM6 in gliomas and malignant gliomas indicates a short survival time, which was involved in immune functions by regulation of T-lymphocyte cell-mediated anti-tumor immunity. In lung squamous carcinoma, CMTM6 expression was positively related to infiltration level of CD8 + T cell, neutrophil cell, macrophage cell and dendritic cell infiltration, and negatively correlated with infiltration of CD4 + T cell. Another study has shown that Overexpression of CMTM6 could promote cancer cell proliferation, migration invasion by triggering epithelial-mesenchymal transition (EMT) in HCC, and a better prognosis of HCC was associated significantly with lower CMTM6 expression. Furthermore, CMTM6 was found to be down-regulated in HCC tissues, and expression of CMTM6 was more likely correlated with alpha-fetoprotein (AFP), tumor staging, metastasis, and survival of HCC. Likewise, our findings revealed that mRNA expression of CMTM6 was down-regulated in HCC tissues. The CMTM6 level was associated with patients’ cancer prognosis. Although the expression of CMTM6 was not a statistical difference in OS and PFS, four CpG of CMTM6 was related to prognosis in patients with HCC. We also figured out that CMTM6 was positive significantly correlated with the infiltrating levels of CD8 + T cells, CD4 + T cells, B cells, neutrophils cells, macrophages cells and dendritic cells. Thus, CMTM6 is a potential target to improve the therapeutic of immune checkpoint inhibitors.
CMTM7 is down-regulated in gastric cancer, while over-expression of SOX10-dependent CMTM7 can impede the growing progress of cancer cells in gastric cancer. Immunohistochemical experiments on 127 lung adenocarcinoma patients showed that CMTM7 was up-regulated (43/127), down-regulated (54/127), or maintained constant (30/127) in lung adenocarcinoma patients, and knockdown CMTM7 could result in a poorer survival outcome. Moreover, Over-expression of CMTM7 could suppress the growth of cancer cells by triggering G1/S phase arrest and CMTM7 could promote EGFR internalization of repress the EGFR-mediated PI3K/AKT signaling pathway. Additionally, studies have found that CMTM7 could decrease Rab5 activation by EGFR-AKT signaling pathway to promote non-small cell lung cancer (NSCLC). A report figured out that CMTM7 was obviously lower expressed in HCC tissues and the level of CMTM7 was negatively associated with TNM staging and tumor metastasis in HCC. In addition, overexpression of CMTM7 can induce cell cycle arrest in G0/G1 phase by increasing p27 expression, decreasing cyclin D1 and cyclin dependent kinase 4/6 (CDK4/6) expression and inhibiting Akt signaling pathway. After that, the cell growth and tumorigenesis in HCC were obviously inhibited. Interestingly, CMTM7 could not only link B-cell linker protein (BLNK) and B cell receptor (BCR) but also activation of downstream BCR signal transduction to MAPKs. Previous studies have implicated that knockdown CMTM7 may promote cell death rate of B-1a (TrB-1a) cells. So, CMTM7 exerts a pivotal role in BCR expression and survival of B-1a cells. Contrary to the previous conclusion, CMTM7 mRNA was up-regulated in HCC in our research, higher mRNA expression of CMTM7 in HCC patients was associated with advanced grades or late stages. We demonstrated that high CMTM7 expression in patients with HCC means worse OS and PFS, and ten CpGs of CMTM7 were correlated with significant prognosis results in HCC patients, revealing that CMTM7 is also an independent prognostic indicator of HCC. To date, the expression of CMTM7 in HCC cell lines is still lack of fully explored. To explore the roles and specific mechanisms of CMTM7 in HCC and the development and progression of HCC needs further study.
As the database shows that CMTM8 was lower expression in bladder cancer and correlated with the progression of TNM stages. Overexpression of CMTM8 could suppress bladder cancer tissue cell growth, and possess a statistically significant predictor of better OS and PFS in bladder cancer patients[13, 67]. The functional study has shown that CMTM8 can promote cancer cells migration and invasion by reducing GSK3β to stimulate the β-catenin activation, while β-catenin knockdown suppressed these effects. Moreover, the silence of CMTM8 in HepG2 cells could induce epithelial-to-mesenchymal transition-like processes through c-MET and ERK signaling pathways, indicating CMTM8 has an essential role in regulating HepG2 cells growth and invasion. According to our research, we found that CMTM8 was up-regulated in HCC, and CMTM8 was closely correlated with patients’ tumor grades. Besides, CMTM8 has no connection with survival (OS, PFS) in patients with HCC, but four CpG of CMTM8 was related to prognosis in patients with HCC. Up to now, specific studies on the effect of CMTM8 in HCC are still unclear. Therefore, further studies are still needed to clarify the molecular mechanism and exact role of CMTM8 in liver cancer carcinogenesis and progression.