USP9X inhibits metastasis in pulmonary sarcomatoid carcinoma by regulating epithelial-mesenchymal transition, angiogenesis and immune infiltration

Abstract

Background: Pulmonary sarcomatoid carcinoma (PSC) is a highly invasive pulmonary malignancy with an extremely poor prognosis. The results of previous studies suggest that ubiquitin-specific peptidase 9X (USP9X) contributes to the progression of numerous types of cancer. Nevertheless, there is little knowledge about the molecular mechanisms and functions of USP9X in the metastasis of PSC.

Methods: Immunohistochemistry and western blotting were used to detect USP9X expression levels in PSC tissues and cells. Wound healing, transwell, enzyme-linked immunosorbent assay (ELISA), tube formation, and aortic ring assays were used to examine the function and mechanism of USP9X in the metastasis of PSC.

Results: Expression of USP9X was markedly decreased and significantly correlated with metastasis and prognosis of patients with PSC. Then we revealed that USP9X protein levels were negatively associated with the levels of epithelial-mesenchymal transition (EMT) markers and the migration of PSC cells. It was confirmed that USP9X in PSC cells reduced VEGF secretion and inhibited tubule formation of human umbilical vein endothelial cells (HUVEC) in vitro. USP9X was detected to downregulate MMP9. Meanwhile, MMP9 was positively related to EMT, angiogenesis and was negatively related to immune infiltration in the public databases. USP9X was significantly negatively associated with the expression of MMP9, EMT markers, CD31, and positively associated with CD4, and CD8 in PSC tissues.

Conclusion: The present study reveals the vital role of USP9X in regulating EMT, angiogenesis and immune infiltration and inhibiting metastasis of PSC via downregulating MMP9, which provides a new effective therapeutic target for PSC.

Keywords: Angiogenesis; Epithelial-to-mesenchymal transition; Immune infiltration; Metastasis; Pulmonary sarcomatoid carcinomas; Ubiquitin-specific peptidase 9X.

Fig. 1

Low USP9X expression was positively associated with metastasis in PSC. (A) USP9X expression in 909 lung adenocarcinoma (LUAD)tissues and normal tissues, and in 894 lung squamous cell carcinoma (LUSC) tissues and normal tissues from the TCGA dataset. (B) Comparison of metastatic status of 364 LUADsamples from the TCGA dataset with different levels of USP9X expression. (C) Representative image of USP9X immunohistochemical staining in non-metastatic and metastatic PSC tissues. Black bar, 100 μm. (D) OS and PFS were also determined according to USP9X expression in NSCLC samples via Kaplan-Meier plotter. ** P < 0.01, *** P < 0.001.

Fig. 2

USP9X was low-expressed in PSC cells which were prone to EMT and migration. (A) NSCLC cells were treated with TGFβ1 for 15 days to establish in vitro PSC cell line analogs. (B) Western blot analysis of USP9X and EMT marker expression in PSC cell lines. (C) The migration of PSC cells was determined via a wound healing assay. Red bar, 100 μm. (D) Cell migration was determined via a transwell migration assay. Red bar, 200 μm. * P < 0.05, ** P < 0.01, *** P < 0.001.

Fig. 3

USP9X inhibits the EMT and migration of PSC cells. (A) PSC cells were treated with Degrasyn or DMSO, after which western blotting was used to measure the protein levels of EMT markers. (B) PSC cells were treated with the shUSP9X or shNC lentivirus, and western blotting was subsequently used to assess the protein levels of EMT markers. (C) The epithelial phenotype marker E-cadherin (green) and mesenchymal marker Vimentin (green) were detected via IF staining. Nuclei were stained with DAPI (blue). Red bar, 20 μm. (D) Cell migration was determined via wound healing assays. Red bar, 100 μm. (E) Cell migration was determined via a transwell migration assay. * P < 0.05, ** P < 0.01.

Fig. 4

USP9X inhibits angiogenesis and induces immune infiltration in PSC. (A) The tube formation ability of HMEC-1 cells cultured with CM collected from PSC cells pretreated with Degrasyn or DMSO, shUSP9X or shNC lentiviruses was tested via an endothelial cell tube formation assay. Red bar, 100 μm. (B) Rat aortic ring microvessel sprouting induced by CM, collected from PSC cells pretreated with Degrasyn or DMSO, shUSP9X or shNC was tested by a rat aortic ring assay. (C) USP9X knockdown in PSC cells increased VEGF secretion into the supernatant, as determined by ELISA. Tumor cells were cultured in serum-containing medium for 48 h, after which the CM was collected to determine the concentration of VEGF. (D) USP9X expression is positively associated with macrophage, CD4+ T cell, and CD8+ T-cell infiltration in NSCLC. * P < 0.05, ** P < 0.01.

Fig. 5

USP9X regulates EMT, angiogenesis and immune infiltration through suppressing MMP9. (A) The protein level of MMP9 was determined using a western blot assay. (B) The levels of MMP9 in the culture media were quantified via ELISA. (C) Coexpression analysis of MMP9 and EMT-related molecules, angiogenesis-related molecules and infiltrating immune cells in NSCLC. (D) The mRNA level of MMP9 was positively correlated with angiogenesis-related genes in NSCLC. (E) MMP9 expression was positively associated with Treg cells, and negatively related to CD8+ T-cell infiltration in NSCLC. * P < 0.05.

Fig. 6

Correlations between USP9X/MMP9 and EMT, angiogenesis, and immune infiltration in PSC tissues. (A) Representative IHC staining for MMP9, E-cadherin, CD4, CD8, and CD31 in PSC tissues of patients, which were divided into USP9X high-expression subgroup and USP9X low-expression subgroup. Red bar, 100 μm. (B) Kaplan-Meier analysis of OS and PFS in patients with NSCLC stratified by MMP9 expression. (C) Schematic diagram of the mechanism of USP9X in PSC metastasis. USP9X inhibits metastasis in pulmonary sarcomatoid carcinomas by regulating epithelial-mesenchymal transition, the tumor vascular system and immune infiltration.