Upregulated CANT1 is correlated with poor prognosis in hepatocellular carcinoma

Because of its complicated molecular mechanism, liver cancer has been one of the most dangerous malignant tumors globally. Liver cancer is characterized by rapid growth, vascular invasion, and high tolerance to chemotherapy and targeted therapies [21, 22]. Although the upgraded liver cancer detection and treatment increases the possibility of cure, the prognosis of liver cancer is still poor due to postoperative recurrence or metastasis [23]. In recent years, the development and maturity of whole genome sequencing technology have provided great help for the exploration of new biomarkers and new therapeutic targets. However, further exploration is required in the early diagnosis of HCC and prognostic molecular targets.

CANT1 is considered to be a glycosylated protein, and its encoded protein belongs to the phosphatase family. It was overexpressed in lung cancer [24], prostate cancer [8], and clear cell renal carcinoma (ccRCC) [11]. In lung cancer, CANT1 promoted the progression of lung cancer by driving the nuclear factor-k gene binding (NF-κB) signaling pathway. Moreover, a significant difference existed between the expression level of CANT1 and N stage, and the highly expressed CANT1 had a poor prognosis. In squamous cell lung carcinoma [9], mir-607/CANT1 played an important role in its progression by mediating the EMT process, and was associated with poor prognosis. In clear cell renal carcinoma, CANT1 silencing inhibited the proliferation, migration, and invasion of cells. Chen Yang et al. screened out three candidate genes in HCC with TP53 mutation, including CANT1. Many studies have demonstrated that HCC patients with mutated TP53 have adverse prognosis, which provided new insights into personalized prognostication approaches [25].The present study analyzed CANT1 expression in 33 tumors by downloading RNAseq from TCGA and GTEx databases, and revealed high expression of CANT1 in some tumors, including HCC. The high expression of CANT1 in HCC tissues was confirmed in different databases (i.e. ICGC). IHC staining showed that CANT1 expression was higher in HCC samples than in normal tissues.

We further studied the relationship between CANT1 expression level and clinicopathological features of HCC patients. CANT1 expression was significantly correlated with T stage, pathologic stage, and histologic grade in the TCGA database. Our Ditan cohort showed that CANT1 expression was only correlated with histologic grade, not pathological stage and T stage, probably due to the small number of cases we collected and the inability to collect stage IV cases. Kaplan-Meier curves indicated that patients with high CANT1 expression presented poor OS in training and validation cohorts. Univariate and multivariate Cox regression analysis also identified that CANT1 expression level was an independent prognostic biomarker of OS in HCC patients. Furthermore, we predicted time-dependent survival ROC curve of 1-, 2-, and 3-year, with AUCs greater than 0.6 in TCGA and ICGC sets. These results suggested CANT1 as a potential prognostic marker for HCC.

Increasing evidence shows that epigenetic changes, especially abnormal DNA methylation, could lead to transcriptional silencing of tumor suppressor genes or high expression of oncogenes, thus deteriorating malignant tumors [26]. Aberrant DNA methylation resulted in malignant tumors mainly by DNA hyper- or hypo-methylation [27]. We investigated the relationship between the methylation level of CANT1 and prognosis, and three CpG sites were associated with prognosis, and the three CpG sites with hypomethylation had better OS. Immune cell infiltration and tumor microenvironment(TME) have been proven to play a key role in cancer progression [28]. Tumor immunity has been a hot topic in recent years [29]. Immunotherapy may be a promising anti-liver cancer strategy. TME is the environment in which tumor cells grow and plays an important role in the occurrence and development of tumors, which is involved in the process of tumor malignant transformation, tumor growth, metastasis and drug resistance [30]. Immune cell infiltration accelerated the progression of chronic hepatitis to liver cancer, and was associated with a poor prognosis of liver cancer [19, 31, 32]. In our study, CANT1 was positively associated with the infiltrations of six immune cells (B cells, CD4 + T cells, CD8 + T cells, neutrophils, macrophages and dendritic cells). In addition, ssGSEA was used to study the relationship between CANT1 and 24 types of immune cell infiltration. The results demonstrated a significant weak-to-moderate positive correlation of CANT1 expression with Th2, T helper cell, TFH, NK CD56 bright cells, Macrophages and aDC. Moreover, the infiltration level of Th2 was highest in high expression CANT1. This was possibly attributed to Th1/Th2 imbalance induced by high expression of CANT1 as well as the inhibitory effects of Th2-generated cytokines on Th1 proliferation and differentiation and the function of cytotoxic T lymphocytes, resulting in immunosuppression and the occurrence and development of tumors [33]. Furthermore, CANT1 expression was negatively correlated with Cytotoxic cell, DC, pDC, Treg, Neutrophils,Tgd and CD8 T cells. Cytotoxic cells have the function of killing cancer cells and have anti-tumor activity. CANT1 overexpression leads to downregulation of cytotoxic cells, which leads to the occurrence of liver cancer. Dendritic cells(DC) are the strongest specialized antigen presenting cells and play a unique role in anti-tumor immunity. Overexpression of CANT1 leads to downregulation of DC, thus limiting the activity of effector T cells and promoting tumor growth. All these suggested that CANT1 might alter the immune microenvironment of HCC and impact immune regulation. In addition, Cox proportional risk models showed that B cells, CD8 + T cells, macrophages, and dendritic cells were significantly associated with adverse clinical outcomes in HCC patients. In recent years, the discovery of immune checkpoints such as PD-1, PD-L1 and CTLA4 and the development and application of corresponding immune checkpoint inhibitors have provided new options for the treatment of advanced liver cancer [34]. In HCC, high PD-L1 expression is usually associated with high PD-1 expression, and associated with cancer recurrence, metastasis, and a high risk of cancer-related death [35, 36]. CTLA4 is one of the T cell receptors and can competitively prevent CD28 from binding to B7, thereby inhibiting T cell activation [37, 38]. Our study found that the expression of CANT1 is associated with the expression of most immune checkpoints, such as PD-1, PD-L1 and CTLA4. These findings suggested that CANT1 may be involved in immunity, which may provide new insights into the immunotherapy of HCC. Finally, we studied the relationship between CANT1 expression and drug sensitivity, and the results showed that eight drugs with notable expression differences were extracted, namely Sorafenib, Cisplatin, Crizontinib, Cyclopamine, Erlotinib, Gemcitabine, Lapatinib, and Nilotinib, which provided the theoretical basis for clinical medication.

To further understand the biological function of CANT1 in HCC, we identified DEGs between HCC patients with high- and low- expression of CANT1. GO and KEGG enrichment analysis was performed for 100 up-regulated genes, showing that DEGs were mainly enriched in cell cycle, nuclear division and microtubule binding. In addition, GSEA analysis demonstrated that CANT1 chiefly participated in the cell cycle, DNA replication, riboflavin metabolism, and gap junction. These enriched pathways were related to the mechanisms of the tumor.

We first explored the relationship between CANT1 and HCC, certain limitations were unavoidable: all data analyzed by bioinformatic methods were directly downloaded from public databases, and the protein expression level of CANT1 was only verified by IHC. Research is also needed at RNA levels and oncogenic mechanism in HCC through in vitro and in vivo experiments.