Overexpression of LIMA1 Indicates Poor Prognosis and Promotes Epithelial-Mesenchymal Transition in Head and Neck Squamous Cell Carcinoma

Abstract

Background: LIMA1 encodes LIM domain and actin binding 1, a cytoskeleton-associated protein whose loss has been linked to migration and invasion behavior of cancer cells. However, the roles of LIMA1 underlying the malignant behavior of tumors in head and neck squamous cell carcinoma (HNSC) are not fully understood.

Methods: We conducted a multi-omics study on the role of LIMA1 in HNSC based on The Cancer Genome Atlas data. Subsequent in vitro experiments were performed to validate the results of bioinformatic analysis. We first identified the correlation between LIMA1 and tumor cell functional states according to single-cell sequencing data in HNSC. The potential downstream effects of LIMA1 were explored for gene ontology and Kyoto Encyclopedia of Genes and Genomes pathways through functional enrichment analysis of the gene sets that correlated with LIMA1 in HNSC. The prognostic role of LIMA1 was assessed using the log rank test to compare difference in survival between LIMA1^High and LIMA1^Low patients. Univariate Cox regression and multivariate Cox regression were further carried out to identify the prognostic value of LIMA1 in HNSC.

Results: LIMA1 was identified as a prognostic biomarker and is associated with epithelial-mesenchymal transition (EMT) progress in HNSC. In vitro silencing of LIMA1 suppressed EMT and related pathways in HNSC.

Conclusions: LIMA1 promotes EMT and further leads to tumor invasion and metastasis. Increased expression of LIMA1 indicates poor survival, identifying it as a prognostic biomarker in HNSC.

Keywords: LIMA1; TCGA; epithelial-mesenchymal transition; head and neck squamous cell carcinoma; prognosis.

Figure 1.

The pan-cancer and proteomic analysis of LIMA1 in HNSC. LIMA1 associated tumor cell functional states in pan-cancer (A, B). LIMA1 expression and immune cell infiltration level in HNSC in pan-cancer (C). The medium expression of LIMA1 in lymph node of a 62-year-old male patient with HNSC, Patient ID: 1743 (D). The medium expression of LIMA1 in skeletal muscle of a 51-year-old male patient with HNSC, Patient ID: 2608 (E). The medium expression of LIMA1 in oral tissue of a 49-year-old male patient with HNSC, Patient ID: 4427 (F). The high expression of LIMA1 protein in tumor tissue of a 95-year-old female patient with HNSC, Patient ID: 3916 (G). HNSC indicates head and neck squamous cell carcinoma.

Figure 2.

The expression and methylation profiles of LIMA1 in HNSC. The mRNA level of LIMA1 in HNSC and paired normal samples (A). K-M (Kaplan-Meier) survival plot of LIMA1 methylation in HNSC (B). LIMA1 expression level negatively correlated with methylation level (C). Methylation data of CpG dinucleotides and its correlation with LIMA1 expression (D). The relationship between LIMA1 expression and tumor subgroups based on HPV status and TP53 mutation status in HNSC (E). The relative promoter methylation level of primary tumor and paired normal tissues (F). The relative promoter methylation level of tumor subgroups based on TP53 mutation status (G). HNSC indicates head and neck squamous cell carcinoma; HPV, human papillomavirus; TCGA, The Cancer Genome Atlas.

Figure 3.

Prognostic analysis of LIMA1 in the TCGA set. Prognostic value of LIMA1 in HNSC. Left part (from top to bottom) shows the LIMA1 expression, survival time, and survival status of TCGA HNSC dataset. Rest part (from top to bottom) shows the Kaplan-Meier survival plot of LIMA1 and time-dependent ROC (receiver operating characteristic) curve of LIMA1 (A). Univariate and multivariate Cox regression of P value, risk coefficient (HR), and confidence interval (CI) of gene expression and clinical characteristics (B and C). Nomogram to predict the 1-, 2-, and 3-year overall survival of HNSC patients (D). Calibration curve for the overall survival (OS) nomogram model in the discovery group. A dashed diagonal line represents the ideal nomogram, and the blue, red, and orange lines represent the 1-, 2-, and 3-year observed nomograms (E). HNSC indicates head and neck squamous cell carcinoma; HR, hazard ratio; TCGA, The Cancer Genome Atlas pathological TNM system, the combinations of this system are grouped in five less-detailed stages: Stage 0, Stage I, Stage II, and Stage III,Stage IV.

Figure 4.

Genomic copy number variation (CNV) events and somatic mutation associated with LIMA1 in HNSC. The related deletion or amplification gene sets associated with LIMA1 with representative histogram of top 10 genes (A and B). Gene enrichment analysis of top gene sets conducted by GeneMANIA (C). Lollipop charts display the mutation distribution and protein domains for LIMA1 in HNSC with the labeled recurrent hotspots. Somatic mutation rate and transcript names are indicated by plot title and subtitle, respectively (D). Somatic landscape of TCGA HNSC cohort. The barplot above the legend exhibits the number of mutation burden. Mutation information of each gene in each sample (ordered by LIMA1 expression) is shown by the waterfall plot, in which genes are ordered by their mutation frequencies. A –log10-transformed q values estimated by MutSigCV are shown by side (E). Cohort summary plot displays the distribution of variants with corresponding variant classification, type, and SNV class. Bottom part (from left to right) indicates mutation load for each sample, variant classification type. A stacked barplot shows top 10 mutated genes (F). CNV indicates copy number variation; HNSC, head and neck squamous cell carcinoma; SNV, single nucleotide variation; TCGA, The Cancer Genome Atlas.

Figure 5.

LIMA1 correlated gene set enrichment analysis (GSEA). The genes correlated with LIMA1 were analyzed by Pearson test in HNSC (A). The top 50 genes positively or negatively correlated with LIMA1 in HNSC (B). GO (BP, biological process; CC, cellular components; MF, molecular functions) and KEGG enrichment analysis, with P value, FDR < .05 was considered as significant (C). FDR indicates false discovery rate; GO, gene ontology; HNSC, head and neck squamous cell carcinoma; KEGG, Kyoto Encyclopedia of Genes and Genomes.

Figure 6.

Silencing LIMA1 suppresses EMT process and tumor-associated pathway in vitro. Representative images and analysis of transwell assay and wound healing assay (A and B). The expression level of proteins in the LIMA1-associated signaling pathways was determined by Western blotting assay (C). Western blot analysis of EMT-related proteins in CAL27 and HSC4 cells with LIMA1 knockdown (D). Representative images of immunofluorescent staining of N-cadherin or E-cadherin (E). (*P < .05; **P < .01; ***P < .001; ****P < .0001). EMT indicates epithelial-mesenchymal transition.