TOM40 as a prognostic oncogene for oral squamous cell carcinoma prognosis

Abstract

Background: To investigate the role of the translocase of the outer mitochondrial membrane 40 (TOM40) in oral squamous cell carcinoma (OSCC) with the aim of identifying new biomarkers or potential therapeutic targets.

Methods: TOM40 expression level in OSCC was evaluated using datasets downloaded from The Cancer Genome Atlas (TCGA), as well as clinical data. The correlation between TOM40 expression level and the clinicopathological parameters and survival were analyzed in TCGA. The signaling pathways associated with TOM40 were identified through gene set enrichment analysis. A network of genes co-expressed with TOM40 was constructed and functionally annotated by gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses. The immune infiltration pattern in OSCC was analyzed in the TCGA-OSCC cohort using the CIBERSORT algorithm. Clinically significant factors of OSCC were screened through the expression levels of TOM40 and a clinically relevant nomogram was constructed. The TCGA-OSCC cohort was divided into the TOM40^high and TOM40^low groups and the correlation between TOM40 expression level and the sensitivity to frequently used chemotherapeutic drugs was evaluated. CCK-8 and colony formation assays were applied to determine the cell growth.

Results: TOM40 was highly expressed in OSCC tissues and correlated negatively with the overall survival (P < 0.05). Patients with high TOM40 expression level showed worse prognosis. Furthermore, GO and KEGG enrichment analyses of the differentially expressed genes related to TOM40 showed that these genes are mainly associated with immunity and tumorigenesis. Immunological infiltration analysis has found that the expression levels of TOM40 are correlated with the proportions of several immune cells. Moreover, we found that TOM40 knockdown inhibited cell growth in OSCC cell lines.

Conclusions: Our results uncovered that TOM40 is a reliable prognostic marker and therapeutic target in OSCC.

Keywords: Cell growth; OSCC; Prognostic marker; TOM40; Therapeutic target.

Fig. 1

TOM40 is upregulated in OSCC and correlates with poor prognosis. (A) Pan-cancer TOM40 mRNA expression level in TIMER 2.0 database. (*: P value < 0.05; **: P value < 0.01; ***: P value < 0.001). TCGA = the cancer genome atlas. (B) Expression of TOM40 gene in tumor and normal tissues in TCGA database. (C) TOM40 expression level in OSCC and paired non-tumor tissues. (D) OS curves of OSCC patients demarcated by TOM40 expression level in the TCGA database. (E) ROC curve for the prognostic value of TOM40

Fig. 2

Construction of Cox regression model. (A) The heatmap of risk score level and clinical characteristics. (B and C) Univariate cox regression and multivariate cox regression according to risk score and clinical characteristics. (D) The calibration curve of the nomogram. € The development of a nomogram based on the clinical characteristics in the TCGA training cohort. N stands for N classification in TNM system, and T stands for T classification in TNM system

Fig. 3

Network ofTOM40 and KEGG and GO biological function enrichment analyses ofTOM40-related genes. (A) The TOM40 gene co-expression network. Red and green represent genes with positive and negative correlation to TOM40 respectively. (B) PPI network of TOM40-related DEGs. Red and green represent the positively and negatively correlated genes respectively. The size of the graph is proportional to the correlation of TOM40. (C) The heatmap shows the DEGs associated with TOM40 in the TCGA-OSCC cohort. (D) KEGG signaling pathway enrichment analysis. (E) GO biological function enrichment analysis (when P-value < 0.05, q-value < 0.05, the results were statistically significant)

Fig. 4

TOM40 is associated with the immune landscape of OSCC. (A) The differences in immune cell expression between TOM40^high and TOM40^low expression (B) The association between TOM40 and immune cells in the TCGA-OSCC cohort. P < 0.05, **P < 0.01, ***P < 0.001. (C) Heatmap showing the correlation between 16 immune checkpoint molecules and TOM40. The numbers in each box indicate the correlation coefficient between two cells. Red represents positive correlation and blue represents negative correlation. The darker the color, the more significant the correlation. (D) Violin plot revealing the distinction between low and high express TOM40 in TMB. (E) Estimate the score of the expression profile in the low and high express TOM40. *P < 0.05

Fig. 5

TOM40 expression can predict the chemotherapy response in OSCC. The sensitivity of TOM40 ^high and TOM40^low samples to (A) 5-fluorouracil, (B) etoposide, (C) methotrexate, (D) cisplatin, (E) doxorubicin, (F) mitomycin, (G) zibotentan, (H) epothilone, and (I) gemcitabine are indicated

Fig. 6

TOM40 knockdown inhibited the proliferation of OSCC cells in vitro. (A) and (B) TOM40 protein and mRNA expression in HN4 and SCC9 cells transfected with siControl, siTOM40#1 or siTOM40#2. (C) and (D) Viability of HN4 and SCC9 cells transfected with siControl, siTOM40#1 or siTOM40#2. (E) and (F) Representative images and number of colonies formed by HN4 and SCC9 cells transfected with siControl, siTOM40#1 or siTOM40#2. (G) Signaling connections involved in the TOM40 regulated OSCC development. The data represent the mean ± SEM of at least three independent experiments. * p < 0.05, versus siControl