THBS1-Mediated Degradation of Collagen via the PI3K/AKT Pathway Facilitates the Metastasis and Poor Prognosis of OSCC

Abstract

Oral squamous cell carcinoma (OSCC) is a prevalent form of malignant tumor, characterized by a persistently high incidence and mortality rate. The extracellular matrix (ECM) plays a crucial role in the initiation, progression, and diverse biological behaviors of OSCC, facilitated by mechanisms such as providing structural support, promoting cell migration and invasion, regulating cell morphology, and modulating signal transduction. This study investigated the involvement of ECM-related genes, particularly THBS1, in the prognosis and cellular behavior of OSCC. The analysis of ECM-related gene data from OSCC samples identified 165 differentially expressed genes forming two clusters with distinct prognostic outcomes. Seventeen ECM-related genes showed a significant correlation with survival. Experimental methods were employed to demonstrate the impact of THBS1 on proliferation, migration, invasion, and ECM degradation in OSCC cells. A risk-prediction model utilizing four differentially prognostic genes demonstrated significant predictive value in overall survival. THBS1 exhibited enrichment of the PI3K/AKT pathway, indicating its potential role in modulating OSCC. In conclusion, this study observed and verified that ECM-related genes, particularly THBS1, have the potential to influence the prognosis, biological behavior, and immunotherapy of OSCC. These findings hold significant implications for enhancing survival outcomes and providing guidance for precise treatment of OSCC.

Keywords: collagen degradation; extracellular matrix (ECM); immunotherapy; oral squamous cell carcinoma (OSCC); prognosis.

Figure 1

(A,B) Consensus clustering analysis of ECM-related genes. (A) Consensus clustering matrix for k = 2. (B) Consensus clustering cumulative distribution function (CDF) for k = 2–9. The area under CDF reached the largest when k = 2. (C) Heat map of ECM-related gene expression in clusters C1 and C2. (D) The oncoplot of the 20 genes with the highest mutation frequencies in the ECM-high cluster and the ECM-low cluster. (E) Kaplan–Meier survival analysis of ECM-high and ECM-low clusters.

Figure 2

(A) Forest plots of 17 ECM-DEGs associated with prognosis by univariate regression analysis. (B) Forest plots of the four ECM-DEGs were used to construct the prognostic signature by multivariate Cox regression analysis and stepwise regression analysis. (C,D) The Kaplan–Meier survival curve and ROC curve were performed in the training set of TCGA. (E,F) The Kaplan–Meier survival curve and ROC curve were performed in the test set of GEO. (G) Reordering of risk scores for each sample in the training cohort and patient survival. (H) Reordering of risk scores for each sample in the test cohort and patient survival. (I,J) Heat map of four prognosis-related ECM-DEG expression profiles in the training cohort (I) and the test cohort (J) in the low- and high-risk groups.

Figure 3

(A,B) WB was used to detect the activation of the PI3K/AKT signaling pathway after overexpression or knockdown of THBS1 in two OSCC cell lines. (C,D) Quantitative analysis of p-AKT protein expression levels in OSCC cells (n = 3, one-way ANOVA followed by Tukey’s multiple comparisons; vector vs. THBS1 and NC vs. si-THBS1, * p < 0.05, *** p < 0.001, **** p < 0.0001). (E,F) Immunofluorescence was used to detect the expression and nuclear translocation of p-AKT in OSCC cells after overexpression or knockdown of THBS1 or treatment with 10 nM LY294002 inhibitor (scale bar, 50 μm). (G,H) Quantitative analysis of p-AKT nuclear expression in two OSCC cells (n = 3, one-way ANOVA followed by Tukey’s multiple comparisons; vector vs. THBS1 and NC vs. si-THBS1, * p < 0.05, **** p < 0.0001), ns: no significant.

Figure 4

(A,B) The proliferation of OSCC cells was detected by EdU assays at 48 h after transfection and treatment with 10nM LY294002 (scale bar, 100 μm). (C,D) Quantitative analysis of the number of EdU positives (unit area = 850 μm × 500 μm) (n = 3, one-way ANOVA followed by Tukey’s multiple comparisons; vector vs. THBS1, THBS1 vs. THBS1 + LY294002, NC vs. si-THBS1, and si-THBS1 vs. si-THBS1 + LY294002, *** p < 0.001, **** p < 0.0001).

Figure 5

(A,B) The migration and invasion abilities were detected by Transwell assays after OSCC cells were treated with transfection or inhibitor for 48 h (scale bar, 100 μm). (C,D) Quantitative analysis of the number of migrated and invasive cells in (C,D) (n = 3, one-way ANOVA followed by Tukey’s multiple comparisons; vector vs. THBS1, THBS1 vs. THBS1 + LY294002, NC vs. si-THBS1, and si-THBS1 vs. si-THBS1 + LY294002, ** p < 0.01, *** p < 0.001, **** p < 0.0001). (E,F) The wound healing assay was used to detect the horizontal migration ability of OSCC cells 48 h after transfection and after inhibitor treatment (scale bar, 500 μm). (G,H) The wound closure area of OSCC cells in (E,H) was quantitatively analyzed (n = 3, one-way ANOVA followed by Tukey’s multiple comparisons; vector vs. THBS1, THBS1 vs. THBS1 + LY294002, NC vs. si-THBS1, and si-THBS1 vs. si-THBS1 + LY294002, **** p< 0.0001).

Figure 6

(A,B) Immunofluorescence double staining of cortactin and F actin was performed to detect invadopodia in OSCC cells after transfection or treatment with 10nM LY294002. Red fluorescence represents F actin, green fluorescence represents cortactin, and the merged images (yellow) show colocalization of F actin (red) and cortactin (green), indicating invadopodia formation (scale bar, 50 μm). (C,D) Quantitative analysis of the area of invadopodia (yellow) per cell (n = 3, one-way ANOVA followed by Tukey’s multiple comparisons; vector vs. THBS1, THBS1 vs. THBS1 + LY294002, NC vs. si-THBS1, and si-THBS1 vs. si-THBS1 + LY294002, * p < 0.05, ** p < 0.01, **** p < 0.0001). (E,F) WB was used to detect the expressions of MMP2, MMP9, MMP14, and PCNA in OSCC cells treated with transfection or inhibitor.