USP8-Dependent Family Tyrosine Kinase Promotes the Malignant Progression of Esophageal Squamous Cell Carcinoma by Upregulating Protein Tyrosine Kinase 2 Expression

Abstract

Background: Esophageal squamous cell carcinoma (ESCC) is a lethal malignancy, and the molecular underpinnings of its aggressive behavior are not fully understood. FYN proto-oncogene, Src family tyrosine kinase (FYN) has been linked to cancer progression, yet its role in ESCC remains elusive. This study investigated the influence of FYN on ESCC malignancy.

Methods: Quantitative real-time polymerase chain reaction was used to assess the mRNA expression of FYN, while western blotting and immunohistochemistry (IHC) assays were performed to detect the protein expression of FYN, ubiquitin specific peptidase 8 (USP8) and protein tyrosine kinase 2 (PTK2). Cell viability was measured with a cell counting kit-8 assay, and cell apoptosis was evaluated using flow cytometry.

Results: FYN expression was increased in ESCC tissues and cells when compared with normal esophageal tissues and normal esophageal epithelial cells. Knockdown of FYN inhibited cell invasion, migration, stem-like traits, and glycolysis, while promoting apoptosis. USP8 was shown to stabilize FYN protein expression through its deubiquitinating activity in ESCC cells. Overexpression of FYN reversed the effects of USP8 silencing on the malignant phenotypes of ESCC cells in vitro and in vivo. FYN upregulated PTK2 expression in both TE1 and KYSE150 cell lines. Furthermore, PTK2 overexpression reversed the effects of FYN silencing on the malignant phenotypes of ESCC cells. Further, USP8 silencing-induced inhibitory effect on PTK2 protein expression was counteracted after FYN overexpression.

Conclusion: USP8-dependent FYN contributed to the malignant progression of ESCC by interacting with PTK2. Targeting this pathway may offer a novel therapeutic strategy for ESCC treatment.

Keywords: ESCC; FYN; PTK2; USP8.

FIGURE 1

FYN expression was upregulated in ESCC tissues and cells. (A) The expression property of FYN in ESCC tissues and normal esophageal tissues was analyzed by qRT‐PCR. (B) The Kaplan–Meier method was performed to analyze the prognosis of ESCC patients with high or low FYN expression. The median expression of FYN in these ESCC patients was used as the cutoff value for high or low expression of FYN. (C and D) FYN protein expression was detected by western blotting assay in ESCC tissues (N = 7), normal esophageal tissues (N = 7), ESCC cells (KYSE30, KYSE180, TE1, and KYSE150), and normal esophageal epithelial cells (HEEC). *p < 0.05, **p < 0.01, and ***p < 0.001.

FIGURE 2

FYN knockdown inhibited ESCC cell invasion, migration, stem‐like trait, and glycolysis and induced cell apoptosis. Both TE1 and KYSE150 cells were divided into two groups, including si‐NC group and si‐FYN group. (A) FYN protein expression was analyzed by western blotting assay. (B) Cell viability was analyzed by CCK‐8 assay. (C) Cell apoptosis was analyzed by flow cytometry. (D) Cell invasion was assessed by transwell assays. (E and F) Cell migration was detected by wound‐healing assay. (G) Cancer stem‐like cell property was analyzed by sphere formation assay. (H–J) Colorimetric methods were used to analyze glucose consumption, lactate production, and the value of ATP/ADP. **p < 0.01 and ***p < 0.001.

FIGURE 3

USP8 stabilized FYN protein expression through its deubiquitinating activity in ESCC cells. (A) The UbiBrowser database was used to analyze the association between FYN and USP8. (B) The efficiency of USP8 knockdown was analyzed through the western blotting assay. (C) The effect of USP8 knockdown on FYN mRNA expression was detected by qRT‐PCR. (D) Western blotting assay was used to analyze FYN protein levels in both TE1 and KYSE150 cells transfected with si‐USP8 or si‐NC, with or without MG132 treatment. (E) The efficiency of USP8 overexpression in both TE1 and KYSE150 cells was analyzed by western blotting assay. (F) CHX assay for the protein stability of FYN in both TE1 and KYSE150 cells transfected with USP8 overexpression plasmid or vector. (G and H) The association between FYN and USP8 was analyzed by the Co‐IP assays. **p < 0.01 and ***p < 0.001.

FIGURE 4

FYN overexpression attenuated USP8 silencing‐induced effects on the malignant phenotypes of ESCC cells in vitro. Both TE1 and KYSE150 cells were transfected with si‐NC, si‐USP8, si‐USP8 + vector, or si‐USP8 + FYN. (A) FYN protein expression was analyzed by western blotting assay. (B) Cell viability was analyzed by CCK‐8 assay. (C and D) Cell apoptosis was analyzed by flow cytometry. (E) Cell invasion was assessed by transwell assays. (F) Cell migration was detected by wound‐healing assay. (G) Cancer stem‐like cell property was analyzed by sphere formation assay. (H–J) Colorimetric methods were used to analyze glucose consumption, lactate production and the value of ATP/ADP. **p < 0.01 and ***p < 0.001.

FIGURE 5

FYN upregulated PTK2 expression in both TE1 and KYSE150 cells. (A) The association between FYN and PTK2 was predicted through the STRING database. (B and C) PTK2 expression was analyzed through the online databases including the GEPIA and TCGA. (D) The effect of FYN silencing on PTK2 protein expression was detected by western blotting assay. (E and F) The Co‐IP assay and immunofluorescence assay were used to analyze the association between FYN and PTK2 in both TE1 and KYSE150 cells after FYN silencing. *p < 0.05 and ***p < 0.001.

FIGURE 6

PTK2 overexpression attenuated FYN silencing‐induced effects on the malignant phenotypes of ESCC cells. Both TE1 and KYSE150 cells were transfected with si‐NC, si‐FYN, si‐FYN + vector, or si‐FYN + PTK2. (A) PTK2 protein expression was analyzed by western blotting assay. (B) Cell viability was analyzed by CCK‐8 assay. (C and D) Cell apoptosis was analyzed by flow cytometry. (E) Cell invasion was assessed by transwell assays. (F) Cell migration was detected by wound‐healing assay. (G) Cancer stem‐like cell property was analyzed by sphere formation assay. (H–J) Colorimetric methods were used to analyze glucose consumption, lactate production, and the value of ATP/ADP. *p < 0.05, **p < 0.01 and ***p < 0.001.

FIGURE 7

FYN overexpression attenuated USP8 knockdown‐induced effects on the malignant growth of ESCC cells in vivo. KYSE150 cells stably transfected with sh‐NC, sh‐USP8, or sh‐USP8 + FYN were injected into nude mice, and tumor volume was measured every 4 days after 8 days of injection (A). After 28 days, the forming tumors from KYSE150 cells were harvested for tumor weight (B) and FYN and PTK2 protein expression analysis (C and D). (E) The positive expression rates of Ki67, FYN, and PTK2 were detected by IHC assay in the forming tumors from the sh‐NC group, sh‐USP8 group, and sh‐USP8 + FYN group. *p < 0.05, **p < 0.01, and ***p < 0.001.