Hypoxia-driven TNS4 fosters HNSCC tumorigenesis by stabilizing integrin α5β1 complex and triggering FAK-mediated Akt and TGFβ signaling pathways

Head and neck squamous cell carcinoma (HNSCC) remains a formidable clinical challenge due to its high recurrence rate and limited targeted therapeutic options. This study aims to elucidate the role of tensin 4 (TNS4) in the pathogenesis of HNSCC across clinical, cellular, and animal levels. We found a significant upregulation of TNS4 expression in HNSCC tissues compared to normal controls. Elevated levels of TNS4 were associated with adverse clinical outcomes, including diminished overall survival. Functional assays revealed that TNS4 knockdown attenuated, and its overexpression augmented, the oncogenic capabilities of HNSCC cells both in vitro and in vivo. Mechanistic studies revealed that TNS4 overexpression promotes the interaction between integrin α5 and integrin β1, thereby activating focal adhesion kinase (FAK). This TNS4-mediated FAK activation simultaneously enhanced the PI3K/Akt signaling pathway and facilitated the interaction between TGFβRI and TGFβRII, leading to the activation of the TGFβ signaling pathway. Both of these activated pathways contributed to HNSCC tumorigenesis. Additionally, we found that hypoxia-inducible factor 1α (HIF-1α) transcriptionally regulated TNS4 expression. In conclusion, our findings provide the basis for innovative TNS4-targeted therapeutic strategies, which could potentially improve prognosis and survival rates for patients with HNSCC.


Figure S1 .
Figure S1.TNS4 overexpression enhances malignant features of HNSCC cells in vitro and in vivo.(A-B) Validation of TNS4 overexpression in HNSCC cells via western blotting and qPCR following specific treatments.(C-D) Measurement of OD values at different time intervals for cells with enforced TNS4 expression and their control

Figure S2 .
Figure S2.Significant enrichment of integrin-related pathways in the TNS4-high subgroup from the TCGA HNSCC cohort..

Figure S3 .
Figure S3.The PTB domain of TNS4 physically interacts with the cytoplasmic domain of integrin β1 in HNSCC cells.(A) Schematic representation of TNS4 domains.(B) Interactions between TNS4 domains and integrin β1 in HNSCC cells as revealed by Co-IP assays.(C) Schematic diagram depicting the domain structure of integrin β1.(D) Interactions between integrin β1domains and TNS4 in HNSCC cells as revealed by Co-IP assays.

Figure S4 .
Figure S4.Significant enrichment of FAK pathway, focal adhesion pathway, and PI3K-AKT-mTOR pathway in the TNS4-high subgroup from the TCGA HNSCC cohort.

Figure S6 .
Figure S6.Reduction of tumorigenic potential in TNS4-overexpressing SCC-23 cells through FAK inhibition in vivo.(A-C) Comparative analysis of tumor size, weight, and volume among different treatment groups.(D) Evaluation of Ki-67 staining intensity in xenograft tumor tissues derived from SCC-23 cells subjected to specified treatments (scale bar=100 μm).

Figure S7 .
Figure S7.Akt inhibition effectively counteracts TNS4 overexpression-enhanced proliferation in HNSCC cells.(A-B) MTT assay demonstrating the proliferative capacity of HNSCC cells subjected to specified treatments.(C-D) Expression levels of p-Akt, Akt, and PCNA in SCC-1 and SCC-23 cells following respective modifications.

Figure S9 .
Figure S9.Alterations in TGFβ signaling and EMT marker expression in TNS4overexpressing SCC-23 cells with indicated treatments.(A) Western blot analysis assessing the expression levels of p-Smad2, Smad3, p-Smad3, and Smad2 in SCC-23 cells subjected to the indicated modifications.(B) Assessment of E-cadherin, Ncadherin, vimentin, and SNAI2 expression levels in TNS4-overexpressing cells following treatment with LY2109761.

Figure S10 .
Figure S10.Impact of TNS4 depletion or overexpression on key components of TGFβ signaling in HNSCC cells, with or without TGFβ supplementation.(A-B) Assessment of p-Smad2, Smad2, p-Smad3, and Smad3 expression levels in SCC-1 and SCC-23 cells following the indicated treatments.

Figure S11 .
Figure S11.Impact of TGFβ supplementation on TNS4 expression level in HNSCC cells.(A) Temporal variation in TNS4 expression following different durations of TGFβ exposure.(B) Dose-dependent effects of TGFβ on TNS4 expression levels in HNSCC cells.

Figure S12 .
Figure S12.The TNS4-FAK axis enhances TGFβ signaling by facilitating the interaction between TGFβRI and TGFβRII in SCC-23 cells.(A-B) Analysis of the FAK inhibitor's impact on the interaction between FAK and TGFβRI, and between TGFβRI and TGFβRII.(C-D) Effects of TNS4 depletion on the interaction between FAK and TGFβRI, and between TGFβRI and TGFβRII.(E-F) Effects of TNS4 overexpression on the interaction between FAK and TGFβRI, and between TGFβRI and TGFβRII.

Figure S13 .
Figure S13.Inhibition of FAK markedly reduced the levels of tyrosine phosphorylation on TGFβRI in both SCC-1 and SCC-23 cell lines.

Figure S14 .
Figure S14.Effects of TNS4 depletion or overexpression on HIF-1α expression in normoxic and hypoxic conditions.(A-D) Comparative analysis of HIF-1α expression in TNS4-depleted and TNS4-overexpressing cells relative to their respective control cells under normoxic conditions.(E-H) Comparative analysis of HIF-1α expression in TNS4-depleted and TNS4-overexpressing cells relative to their respective control cells under hypoxic conditions.