Aberrant expression of CMTM family in ovarian cancer patients
The first thing worth clarifying is the expression level of the CMTM gene family. Analysis of a total of 33 different types of tumors from the GEPIA database showed that CMTMs were expressed at different levels, suggesting that different members of the CMTM family play different roles in different cancer types, both as oncogenes and antioncogenes (Fig. 1A). Among them, especially for ovarian cancer, CMTM3/4/6/7/8 had a relatively high expression in ovarian cancer compared to control normal tissues, while CMTM1/2/5 had a relatively low expression. Subsequently, analysis of the Oncomine database showed significant changes in the expression levels of CMTM3/7/8 mRNA in ovarian cancer tissues across multiple datasets (Fig. 1B). In addition, the abnormal expression of CMTM gene family was explored at the nucleic acid level and protein expression level respectively. On the one hand, the differences in the expression of CMTMs were explored through 426 ovarian cancer tumor samples and 88 normal control samples from the GEPIA database. Figure 2A showed that the mRNA expression level of CMTM1/4/6/7/8 in tumors was relatively higher compared to normal control tissues, and the higher expression of CMTM6/7/8 was statistically significant. While the mRNA expression level of CMTM2/3/5 showed lower expression compared to normal controls. After exploring the nucleic acid levels, the differences in the expression of CMTMs at the protein level were explored with the help of immunohistochemistry data from HPA database. As seen in Fig. 2B, the protein expression of CMTM1/3/4/6 showed strong staining in ovarian cancer tissues. Finally, our RT-qPCR experiment confirmed that CMTM1/6/8 were higher expressed in ovarian cancer cell lines than normal ovarian cells, but CMTM2/3/7 were lower expressed in ovarian cancer cell lines, and these differences are statistically significant (Fig. 3). In conclusion, this study revealed that CMTMs were highly expressed in ovarian cancer tumor tissues at the mRNA level and the protein level, respectively, which indicated that they may be potential oncogenes. Therefore, further studies of CMTMs are needed to explore their impact or value in OV.
Prognostic value of CMTM gene family in ovarian cancer patients
In order to further explore the prognostic value of CMTMs for patients, the Kaplan-Meier Ploter database was used for survival analysis to evaluate the relationship between the expression level of CMTMs and OS or PFS. The results in Fig. 5A show that the high expression of CMTM1/3/5/8 expression is related to the shorter overall survival of OV patients (p = 0.02, p = 0.00047, p = 0.015, p = 0.049, and HR > 1). As can be seen in Fig. 5B, the high expression of CMTM2/3/5 is related to the shorter progression-free survival of OV patients (p = 0.017, p = 0.000007, p = 0.0002, and HR > 1). In general, it was not difficult to see that the abnormally high expression of CMTMs was a risk factor for the prognosis of OV patients.
CMTM family gene alterations and its relationship with OS and DFS in patients with ovarian cancer.
Genetic alterations play a crucial role in early malignancies. Through the analysis of the cBioPortal database, this study explored the genetic alterations of the CMTM gene family in patients with ovarian cancer, such as missense mutations, structural changes, amplifications and deep deletions. Figure 6A showed that CMTM5/4/2/6 were the top four genes with genetic alterations, and their mutation rates were 3%, 2.4%, 2.4% and 2.3% respectively. Figure 6B showed the details of the genetic alterations of the CMTM gene family in three cohorts (TCGA, Pan cancer Atlas; TCGA, Nature 2011; and TCGA, Firehose Legacy). It can be seen from Figs. 6A and 6B that the genetic alterations of the CMTM gene family for ovarian cancer were mainly amplification and deep deletion. Among them, CMTM1/2/3/4 were mainly deep deletions, while CMTM5/6/7/8 were mainly amplified, which corresponds to the abnormal overexpression of CMTMs found in the previous part of this study to some extent. In addition, the impact of genetic alterations on the survival of ovarian cancer patients was analyzed. Figure 6 (C-D) showed that the disease-free survival of the altered group was significantly shortened (p = 0.004). In short, the occurrence and development of ovarian cancer may be related to genetic alterations of the CMTM family, and these alterations may lead to a poor prognosis of patients.
5. Analysis of GO and KEGG enrichment of CMTM family and co-expressed genes in patients with ovarian cancer.
To further explore the potential molecular mechanisms of CMTMs in tumorigenesis of ovarian cancer, we sought to screen a series of pathways and biological functions through GO and KEGG. The results of Go annotation analysis were shown in Fig. 7A. Among them, the enriched biological processes included cell-cell adhesion, positive regulation of cell growth, regulation of autophagy and regulation of telomere maintenance via telomerase. And the enriched molecular function included cadherin binding involved in cell-cell adhesion, protein binding and nucleic acid binding. These biological processes and molecular function may be closely related to malignant biological processes such as tumor cell proliferation, invasion, apoptosis and autophagy. KEGG pathway enrichment analysis showed that the main enriched signaling pathways included mTOR signaling pathway, AMPK signaling pathway and TGF-beta signaling pathway (Fig. 7B). Finally, Finally, Fig. 7C showed the top 20 genes that were co-expressed with members of the CMTM gene family. Among these twenty genes, there are genes closely related to immunity, such as MAL, MALL and MAL2. For example, MAL2 inhibited tumor antigens to promote tumor immune escape and functions as an oncogene. This suggested that CMTMs, which are positively correlated with the expression of MAL2, may also function as oncogenes through altered immune environment or immune process. Taken together, based on the analysis of biological processes, signal pathways and co-expressed genes, we found that the CMTM gene family may be inseparably related to cell growth, cell adhesion and immune regulation. Therefore, we ventured to speculate that the CMTM gene family may be able to promote the occurrence and development of ovarian cancer and lead to its poor prognosis by regulating cell growth, cell adhesion and immune activity.
The correlation of CMTM gene family with Immune infiltration and immune checkpoint and its prognosis.
In this study, the TIMER database was selected to detect the relationship between CMTMs expression and immune cell infiltration in ovarian cancer tissues. It could be seen that there was a certain correlation between the expression level of CMTMs and the degree of immune cell infiltration (Fig. 8A). For example, the expression levels of CMTM1 and CMTM5 were positively correlated with the degree of infiltration of B cells, macrophages and neutrophils (partial.cor > 0, p < 0.05). The expression level of CMTM2 was positively correlated with the degree of infiltration of B cells, macrophages and dendritic cells (partial.cor > 0, p < 0.05). The expression level of CMTM3 was positively correlated with the degree of infiltration of B cells, macrophages and CD4 + cells (partial.cor > 0, p < 0.05). The expression level of CMTM6 was negatively correlated with the degree of infiltration of B cells, neutrophils and dendritic cells (partial.cor < 0, p < 0.05).
At the same time, numerous studies have confirmed that immune checkpoints promote the occurrence and development of cancer to some extent by changing the immune process, and treatment methods that suppress immune checkpoints and their receptors or ligands are promising therapeutic options for the induction of effective anticancer immunity. Therefore, we analyzed the correlation between the expression level of CMTM and the genes PDCD1, CD274 and PDCD1LG2 that encode the immune checkpoints of PD1, PD-L1 and PD-L2 (Fig. 8B). The results showed that the expression level of CMTM1/2/3 was positively correlated with the gene expression levels of PDCD1, CD274 and PDCDLG2 (partial.cor > 0, p < 0.05). And the expression level of CMTM6 was positively correlated with the gene expression levels of CD274 and PDCDLG2 (partial.cor > 0, p < 0.05). As mentioned previously, In general, the expression level of the CMTM family was closely related to the level of immune infiltration and immune checkpoints that play an important role in the occurrence and development of ovarian cancer.
The previous part of this study showed that the expression level of CMTMs has an impact on the poor prognosis of OV patients (Fig. 5) and was related to immune cell infiltration (Fig. 8A). Therefore, the impact of immune cell infiltration on survival was further explored (Fig. 9A). The results showed that the different levels of infiltration of dendritic cells, CD4 + cells and macrophages could lead to shorter survival times for patients with ovarian cancer (P < 0.05). In addition, this study also deeply explored the impact of the CMTM family gene expression level represented by CMTM3 on the survival of ovarian cancer patients under different immune cell infiltration (B cells, CD4 + T cells and macrophages) (Fig. 9B). The results showed that under these three immune cell infiltration conditions, the OS of the high expression group of CMTM3 was significantly shorter than that of the low expression group (P < 0.05).
In conclusion, the immune correlation analysis results based on the TIMER database and the Kaplan-Meier plotter database showed that the CMTM family was closely related to immune cell infiltration and immune checkpoints and may lead to poor survival of ovarian cancer patients by regulating these two important parts.