Development and validation of a selenium metabolism regulators associated prognostic model for hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is a highly malignant cancer and was the third leading cause of death worldwide in 2020, with approximately 906,000 new cases and 830,000 deaths [1]. The incidence rate of HCC is extremely high in China, with approximately 370,000 new cases in China in 2015 [2]. Many risk factors, including hepatitis B virus (HBV) and hepatitis C virus (HCV), can influence the tumorigenesis of HCC [3]. Although current therapeutic options include surgical resection and systemic therapy, the overall survival (OS) of patients remains unsatisfactory [4].

Recently, cancer cell metabolism has been shown to play a pivotal role in tumour progression [5]. One important feature of cancer cell metabolism is the acquisition of abnormal amounts of nutrients to maintain cell viability and build new biomass [6]. In addition to the principal nutrients glucose and glutamine, various micronutrients, including trace metals and trace minerals such as selenium, contribute to tumorigenesis. Selenium is an essential trace mineral that is fundamental to human health [7]. Selenium can be not only incorporated into selenoproteins to form the rare amino acid selenocysteine [8], which is essential for the survival of cancer [9], but also methylated for the detoxification of excess selenium [10]. Thus, selenium metabolism seems to play an important role in cancer but further investigation, especially related to its role in liver cancer, is needed.

Oncogene-driven metabolic reprogramming allows cancer cells to maintain deregulated proliferation [11]. Thus, genes involved in selenium metabolism regulation also play important roles in cancer cells. Some studies have examined the expression of selenoprotein genes in colorectal adenoma, colorectal cancer [12] and breast cancer [13]. However, an integrated understanding of selenium metabolism genes in HCC, including the interactions between selenium metabolism regulators and the tumour environment, is lacking.

Our study aimed to systematically assess the correlations of selenium metabolism regulators with prognosis, immune check-point and the tumour immune microenvironment. We ultimately elucidated the role of indolethylamine n-methyltransferase (INMT) in HCC progression.

Tumorigenesis is dependent on the reprogramming of cellular metabolism [6]. Altered tumour metabolism reprogramming has profound effects on gene expression and the tumour environment [6]. Selenium is an essential trace element in humans. Inadequate amounts of selenium or selenium deficiency has been linked to an elevated risk of disease, especially cancer [21]. Thus, the regulated whole-body pool of selenium and selenium metabolism are extremely important. Selenoproteins are the most important factors involved in selenium metabolism. Almost 25 human genes encode selenoproteins [22], and nearly all selenoproteins are redox enzymes and glutathione peroxidases that protect cells against oxidative injury [23]. According to the selenocysteine biosynthesis pathway [9], three key enzymes, SEPHS2, PSTK, and SEPSECS, were analysed; moreover, four selenoproteins, GPX1, TXNRD1, GPX4, and SELH, were included in this study. In addition to selenoproteins, excess selenium can be transformed to trimethylselenonium ions (TMSe) through methylation regulation [24, 25]. Therefore, two methyltransferases, INMT and TPMT [10], were analysed in our study.

The liver is the body’s largest metabolism and detoxification organ and is known as the main target organ of selenium deficiency [26]. The liver is also the most important organ regulating the metabolism of carbohydrates, proteins, and other lipids to maintain nutrient balance [27]. Moreover, the liver is sensitive to dietary selenium [28] and is the central organ for selenium regulation and maintaining selenium balance. Liver cancer is a highly malignant form of cancer characterized by altered metabolism [29]. Higher selenium concentrations have been reported to be associated with a significantly lower HCC risk, and the liver appears to be particularly sensitive to selenium supply [30]. Selenium status and selenium intake are negatively associated with hepatitis, cirrhosis, and liver cancer [31]. Yu et al. also found that selenium supplementation protects hepatitis B antigen carriers against the development of HCC [32]. Recently, selenium nanoparticles combined with sorafenib were shown to inhibit HCC progression [33]. The selenium compound CU27 inhibits cancer stem cell generation and reduces primary tumour size in liver cancer [34]. These studies indicate a protective role of selenium in liver cancer. Higher concentrations of selenium may protect against HCC progression. However, at present, the exact role of selenium metabolism regulators in HCC has not been fully reported. Our study focused on the role of the 9 selenium regulators in the TCGA LIHC dataset. Based on the expression profile, we further constructed a selenium metabolism model for the first time. Only two regulators, INMT and SEPSECS, were identified in this model. This model performed well both in the training cohort and in the validation cohort. This model could also predict prognosis of liver cancer patients in both cohorts.

INMT and TPMT are two critical human methyltransferases in selenium methylation. TPMT catalyses the conversion of nonmethylated selenium to the monomethylated form [25]. TPMT expression is reported to be different between normal and pancreatic cancer cells, and knockdown of TPMT sensitizes Panc1 cells to 6-thioguanine [35]. Low TPMT activity is associated with severe toxicity in acute lymphoblastic leukaemia patients [36]. INMT, also named aromatic N-methyltransferase, can transfer one or more methyl groups from S-adenosylmethionine (SAM) to substrates and subsequently create S-adenosylhomocysteine (SAH) [37]. SAM is abundant in the liver, but injury and HCC occur if the level of SAM is chronically excessive [38]. Increasing SAH clearance through the activation of flux via methionine metabolism can extend the lifespan by impacting SAM/SAH levels [39]. Therefore, cells must maintain low concentrations of SAH. Deregulation of INMT expression in primary lung cancer and prostate cancer has been reported [40‐42]. Moreover, the expression of INMT has been found to be associated with castration-resistant prostate cancer [43]. In our study, we demonstrate that the expression of INMT is lower in liver cancer, which is consistent with the finding of Carlos and his colleagues [44]. Knockdown of INMT significantly promoted liver cancer cell proliferation and colony formation. The expression of the proliferation-related markers PCNA and E2F1 was significantly elevated after INMT knockdown. All these results indicate that INMT may function as a tumour suppressor in HCC and that INMT affects liver cancer cell proliferation. Previous studies [39] revealed that INMT affects tumour cell apoptosis. The training cohort and validation cohort analysis also revealed that high expression of INMT was correlated with low-risk patients. Low-risk patients may have longer survival times than high-risk patients. The TME in the low-risk group was also more conducive to a positive antitumour response than that in the high-risk group. All these studies revealed that the lower expression of INMT may promote tumour progression. Selenium concentration and SAM/SAH levels may also be abnormal in the TME after INMT knockdown. However, whether the role of INMT in HCC is dependent on its function in selenium metabolism should be further investigated and explained.

SEPSECS catalyses the final step of selenocysteine formation [45]. SEPSECS influences human acute myeloid leukaemia progression [46]. Moreover, Jia Yi et al. reported that SEPSECS expression is decreased in HCC, which is consistent with our findings [47]. Downregulation of SEPSECS may result in the accumulation of selenium and a decrease in selenoproteins. Selenoproteins, such as the GPx family, play an important role in maintaining cellular redox homeostasis and protecting cancer cells against ferroptosis or chemotherapeutic drugs [48, 49].

The prognosis of HCC patients is poor, and the median OS of patients receiving immunotherapy is less than two years [50]. Therefore, we focused on the 1- and 3-year AUC values of this model. In the training cohort, the 1- and 3-year AUCs of the selenium metabolism gene model were 0.691 and 0.641, respectively. In the validation cohort, the 1- and 3-year AUCs of the selenium metabolism model reached 0.704 and 0.685 respectively. All these results suggested that the selenium metabolism model performed well for predicting the prognosis of HCC patients.

Furthermore, the effect of selenium metabolism regulators on immune cell infiltration in the tumour environment is still unclear. Previous studies have shown that tumour-infiltrating lymphocytes serve as prognostic biomarkers and targets for immunotherapy in HCC [51]. In our research, the difference in immune cells and the corresponding immune function between the two risk groups was significant. Most immune checkpoints, chemokines, MHC molecules and some costimulators were significantly higher in the high-risk group than in the low-risk group. Huang et al. [51] also reported the effects of selenium levels on immune-related diseases, including antiviral immunity, autoimmunity, and chronic inflammatory disorders, which further verified our results.

Recently, the role of selenium in cancer immunology has attracted increasing attention. For T cells, supranutritional selenium suppresses RANKL-expressing osteoclastogenic CD4 T cells [52]. Selenium supplementation increases follicular helper T-cell numbers [53], activated Treg cells [54] and Treg subset CD4⁺CD25⁻FOXP3⁺ Tregs [55]. Selenium nanoparticles combined with aerobic exercise training increases Th1 cytokines in splenocytes [56]. Selenium supplementation may also suppress Th1 cell differentiation [57]. Selenium deficiency reduces the number of CD3⁺ and CD8⁺ T-cell populations [58]. For macrophages, selenium nanoparticles induce M1 macrophage polarization [59] and the expression of the fusion receptors CD47 and CD172α and the adhesion molecules CD54 and ICAM-1. Selenium deficiency enhances the migration of immature dendritic cells (DCs), while high selenium levels inhibit immature DC migration [60]. For mature DCs, low selenium levels impair free migration, and high selenium levels inhibit chemotactic migration [60]. Moreover, selenium deficiency may inhibit DC differentiation by downregulating the expression of CD11c, CD40, CD86, and MHCII [61]. Selenium also regulates the immune function of NK cells. Selenium-containing nanoemulsions effectively potentiate the recognition of cancer cells by NK cells [62]. Selenium-containing nanosystems can deliver pemetrexed to tumour sites and increase the sensitivity of human NSCLC cells to NK cells [63].

This study had several limitations. First, the study did not analyse all selenium-related proteins, which limits the understanding of the role of these proteins in HCC. Second, we only validated the INMT gene in the cell experiments. Other genes, such as SEPSECS, may also be prognostic genes for HCC. Finally, we verified that INMT was a prognostic marker for HCC, which was consistent with the results in The Human Protein Atlas. However, together with these findings, the prognostic significance of INMT in HCC is strongly confirmed.

In summary, this study systematically evaluated the expression of selenium metabolism regulators in HCC. Then, we developed a novel model based on these regulators to predict the prognosis of LIHC patients. The difference in immune cell infiltration in the tumour environment was also evaluated. More importantly, experiments validated that INMT might be a suppressor of HCC progression. In the future, more investigations should be conducted to further reveal the mechanism of selenium regulation in HCC.