Caveolin-1 promotes cancer progression via inhibiting ferroptosis in head and neck squamous cell carcinoma

Abstract

Background: Head and neck squamous cell carcinoma (HNSCC) is an aggressive disease worldwide. Much progress has been made in exploring mechanisms and improving the therapy of HNSCC, but only a few studies have focused on the role of ferroptosis on HNSCC progression. The current study aimed to reveal the underlining mechanisms that caveolin-1 (CAV1)-ROS (reactive oxygen species)-ferroptosis axis affect the process of HNSCC and discover novo therapeutic targets or strategies.

Methods: The role of CAV1 in ferroptosis was analyzed by FerrDb, and its clinical significance was examined by TCGA dataset of HNSCC. The expressions of caveolin-1 (CAV1) in HNSCC tissues were measured by immunohistochemistry, western blot, and real-time PCR assay. Three siRNA sequences were designed to silence CAV1 mRNA in HNSCC cells. Cell proliferation, colony formation, wound-healing, and transwell assays were used to examine the proliferation, migration, and invasion of cancer cells. ROS evaluation and intracellular Fe²⁺ content assays were performed to examine the levels of ferroptosis.

Results: Through the analysis with published data, CAV1 was found to overexpress in HNSCC than normal tissues, and was one of the vital suppressors of ferroptosis pathway. Our study showed that CAV1 was over expressed in HNSCC tissues and the high level of CAV1 predicted poorer prognosis. Further experiments indicated that CAV1 could inhibit the ferroptosis of cancer cells and promote the proliferation, migration and invasion.

Conclusions: Overexpression of CAV1 in HNSCC inhibited the process of ferroptosis, leading to aggressive phenotypes, as well as worse prognosis. The regulatory pathway of CAV1 and ferroptosis are potential targets for designing diagnostic and combined therapeutic strategies for HNSCC patients.

Keywords: caveolin-1; ferroptosis; head and neck; squamous cell carcinoma.

FIGURE 1

CAV1 is associated with poorer prognosis and ferroptosis in TCGA HNSCC dataset. A. Heatmap showed the expression of ferroptosis driver and suppressor genes in HNSCC. B. Venn diagram showed 3 genes both expressed in selective ferroptosis suppressor group and differential expressed genes in HNSCC from TCGA. C. The GSEA analysis showed the expression of upregulated genes mediated by ROS, on the basis of highly expression of CAV1. D. Heatmap showed the different expression of genes that were related to CAV1. E. The expression of CAV1 in HNSCC cancer and normal tissues. (p < 0.0001) F. The overall survival rate was significantly different between the high and low expression of CAV1 in HNSCC patients (p = 0.0015). Data originated from TCGA database. (ns, no significant difference, · p < 0.1, ^* p < 0.05, ^** p < 0.01, ^*** p < 0.001, ^**** p < 0.0001)

FIGURE 2

CAV1 is overexpressed in HNSCC tissues and associated with poorer prognosis. A. IHC analysis of CAV1 expression levels. Images of negative, weak, moderate, and strong CAV1 staining are shown. Scale bar: 20μm. B. CAV1 expression level was significantly associated with cancer size. (p = 0.0011) C. CAV1 expression level was significantly associated with histological grade (p = 0.0452). D. The overall survival rate was different between higher and lower expression of CAV1 in HNSCC (p = 0.059). E. Western blot measured CAV1 protein expression in 5 paired HNSCC cancer tissues and adjacent normal tissues. F. Quantitative real‐time PCR evaluated the relative CAV1 mRNA levels in 30 pairs of HNSCC cancer tissues and adjacent normal tissues (p < 0.001). (ns, no significant difference, · p < 0.1, ^* p < 0.05, ^** p < 0.01, ^*** p < 0.001, ^**** p < 0.0001)

FIGURE 3

High expression of CAV1 inhibits ferroptosis of cancer cells in HNSCC. A. ROS evaluation assay was performed to detect the level of ROS in cells. DCF showed green fluorescence, and it could measure the level of ROS. Scale bar: 20 μm. B. Orange fluorescence labelled the free Fe²⁺ in cells. Scale bar: 20 μm. C. Quantitative real‐time PCR measured the expression of relative genes of ferroptosis: GPX4, FTH1, ACSL4 and NOX1. (ns, no significant difference, · p < 0.1, ^* p < 0.05, ^** p < 0.01, ^*** p < 0.001, ^**** p < 0.0001)

FIGURE 4

CAV1 promotes the proliferation and migration of HNSCC cells. A. CCK‐8 assay showed that proliferation was reduced in HN6 and CAL27 cell lines after being transfected with siCAV1. B. Transwell assays (Scale bar = 100μm). C. Colony formation assays (Scale bar = 5mm). D. Wound‐healing assays (Scale bar = 100μm). (ns, no significant difference, · p < 0.1, ^* p < 0.05, ^** p < 0.01, ^*** p < 0.001, ^**** p < 0.0001)