Mitochondrial outer membrane protein MTUS1/ATIP1 exerts antitumor effects through ROS-induced mitochondrial pyroptosis in head and neck squamous cell carcinoma

We showed that microtubule-associated tumor suppressor gene (MTUS1/ATIP) downregulation correlated with poor survival in head and neck squamous cell carcinoma (HNSCC) patients and that MTUS1/ATIP1 was the most abundant isoform in HNSCC tissue. However, the location and function of MTUS1/ATIP1 have remain unclear. In this study, we confirmed that MTUS1/ATIP1 inhibited proliferation, growth and metastasis in HNSCC in cell- and patient-derived xenograft models in vitro and in vivo. MTUS1/ATIP1 localized in the outer mitochondrial membrane, influence the morphology, movement and metabolism of mitochondria and stimulated oxidative stress in HNSCC cells by directly interacting with MFN2. MTUS1/ATIP1 activated ROS, recruiting Bax to mitochondria, facilitating cytochrome c release to the cytosol to activate caspase-3, and inducing GSDME-dependent pyroptotic death in HNSCC cells. Our findings showed that MTUS1/ATIP1 localized in the outer mitochondrial membrane in HNSCC cells and mediated anticancer effects through ROS-induced pyroptosis, which may provide a novel therapeutic strategy for HNSCC treatment.


Figure S2 .
Figure S2.MTUS1/ATIP1 influenced mitochondrial function and metabolism in HNSCC cells.(A-B) MitoTracker Red staining or Immunofluorescence staining with COX IV were used to detect the distribution of mitochondria under a confocal microscope in MTUS1/ATIP1-overexpressed or control HNSCC cells.The collapsed mitochondria numbers detected by MitoTracker staining (A) or COX IV staining (B) were quantified respectively.(C-D) MTUS1/ATIP1-overexpressed or control HSC3 cells was determined using oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) assays.(E) Intracellular ATP levels were measured by a bioluminescence ATP determination assay in MTUS1/ATIP1-knockdwon or control HNSCC cells.(F) MTUS1/ATIP1-overexpressed or control HNSCC cells were stained with DCFH-DA and then ROS levels were detected using flow cytometry.All data are presented as the mean ± SEM of three independent experiments.*P < 0.05; **P < 0.01; ***P < 0.001.

Figure S7 .
Figure S7.MTUS1/ATIP1 exerted anticancer effects on HNSCC in vitro and in vivo.(A) MTUS1/ATIP1 overexpression inhibited migration and colony formation abilities in HNSCC cells.(B) MTUS1/ATIP1 overexpression inhibited cell proliferation, migration and colony formation abilities in HSC3 cells.(C) Quantification of fluorescence intensities at tumor sites in subcutaneous xenograft or in sito tongue tumor in MTUS1/ATIP1 overexpressed mice.FL, fluorescence intensity.All data are presented as the mean ± SEM of three independent experiments.*P < 0.05; **P < 0.01; ***P <0.001.

Figure S8 .
Figure S8.MTUS1/ATIP1 knockdown enhanced the proliferation, migration and growth in HNSCC in vitro and in vivo.

(
A) MTUS1/ATIP1 knockdown enhanced migration and colony formation abilities in SCC9 cells.(B) MTUS1/ATIP1 knockdown enhanced cell proliferation, migration and colony formation abilities in HSC3 cells.(C) Quantification of fluorescence intensities at tumor sites in subcutaneous xenograft or in sito tongue tumor in MTUS1/ATIP1 knockdowned mice.FL, fluorescence intensity.All data are presented as the mean ± SEM of three independent experiments.*P < 0.05; **P < 0.01; ***P <0.001.

Figure S10 .
Figure S10.The correlation between MTUS1/ATIP1 and GSDME expression in HNSCC.(A) GSDME expression level in HOK cells and different HNSCC cell lines.(B) Transcriptomic analysis of the expression of GSDME between normal tissue and HNSCC tumor samples.Analysis was performed using data from the TCGA with the UALCAN analysis software.(C) Transcriptomic analysis of the expression of MTUS1 between normal tissue and HNSCC tumor samples from the TCGA.(D) Transcriptomic analysis of the correlation between GSDME and MTUS1 expression level between normal tissue and HNSCC tumor samples in HNSCC from the TCGA.Analysis was performed using data from the TCGA with the UALCAN analysis software.GAPDH was used as loading proteins.All data are presented as the mean ± SEM of three independent experiments.**P < 0.01; ***P < 0.001.Human oral keratinocytes (HOK) were purchased from ScienCell (Carlsbad, USA) and cultured with RPMI-1640 medium containing 15% fetal bovine serum (FBS) and 1% penicillin/streptomycin (P/S).