SUMOylation of HSP27 regulates PKM2 to promote esophageal squamous cell carcinoma progression

Abstract

A previous proteomic screening of differentially expressed biomarkers between Kazakh patients with esophageal squamous cell carcinoma (ESCC) and normal adjacent tissues demonstrated that heat shock protein 27 (HSP27) and pyruvate kinase isoenzyme M2 (PKM2) were both highly expressed in ESCC samples compared with normal controls. However, the regulatory association between HSP27 and PKM2 in ESCC remains elusive. In the present study, immunohistochemistry and immunoblotting were adopted to examine the expression of HSP27, PKM2 and other relevant biomarkers involved in epithelial‑to‑mesenchymal transition in clinical tissue samples. The interactions between proteins were detected by co‑immunoprecipitation (Co‑IP) assay and further confirmed by immunofluorescence assay. The growth and motility of ESCC cells were examined by MTT, Transwell and wound healing assays. Overexpression of HSP27 was found to be significantly associated with T‑cell classification, lymph node metastasis and poor prognosis in ESCC. In addition, HSP27 expression was significantly correlated with PKM2 expression in ESCC specimens. Functionally, knockdown of HSP27 inhibited the growth and motility of ESCC cells. Moreover, HSP27 was found to directly interact with small ubiquitin‑related modified protein 2/3 (SUMO2/3) in ESCC cell lines, as evidenced by Co‑IP and laser confocal imaging. In addition, downregulation of HSP27 was shown to decrease PKM2 and E‑cadherin expression. Knockdown of SUMO2/3 was observed to reduce the expression of HSP27, PKM2 and EMT‑related biomarkers. The results of the present study indicated that the SUMOylation of HSP27 enhances the proliferation, invasion and migration of ESCC cells via PKM2.

Keywords: esophageal squamous cell carcinoma; heat shock protein 27; SUMOylation; pyruvate kinase isoenzyme M2.

Figure 1.

HSP27 is markedly upregulated in ESCC and is correlated with PKM2 expression and poor prognosis. (A) IHC results of HSP27 expression in ESCC tissues and NAT (magnification, ×100 and ×200). (B) Survival curve of HSP27 expression in ESCC. (C) IHC results of PKM2 in ESCC tissues and NAT (magnification, ×100 and ×200). (D) Spearman's correlation analysis between HSP27 and PKM2 expression in ESCC. Representative selected images are shown. The log-rank test was used in survival analysis (χ²=4.797, P=0.0285), and Spearman's method was employed to analyze the correlation (N=165, r=0.2526, P=0.0011). ESCC, esophageal squamous cell carcinoma; HSP, heat shock protein; PKM2, pyruvate kinase isoenzyme 2; IHC, immunohistochemistry; NAT, normal adjacent tissue.

Figure 2.

HSP27 promotes the proliferation, invasion and migration of ESCC cells. (A) Basal level of HSP27 expression in a panel of ESCC cell lines, including KYSE30, KYSE150, KYSE450, KYSE510, TE-1, Eca109 and EC9706, as detected by western blotting. (B) Detection of knockdown efficiency of HSP27 shRNA vectors in KYSE450 cells by western blotting. GAPDH was used as an internal loading control. (C and D) MTT assay of proliferation after knockdown of HSP27 in KYSE450 and KYSE150 cells. (E and F) Changes in invasion, detected by the Transwell assay, in KYSE450 and KYSE150 cells after knockdown of HSP27 (magnification, ×100). (G and H) Variations in the migratory activity of KYSE450 and KYSE150 cells analyzed using the wound healing assay after knocking down HSP27 (magnification, ×100). *P<0.05 compared with the normal control group and ^▲P<0.05 compared with the shRNA-scramble control group. ESCC, esophageal squamous cell carcinoma; HSP, heat shock protein; KD, knockdown.

Figure 3.

HSP27 modulates but does not interact directly with PKM2 in KYSE450 cells. (A) Western blotting results of downstream proteins (PKM2, vimentin and E-cadherin) following HSP27 knockdown in KYSE450 cells. GAPDH, internal loading control. (B) No interaction between HSP27 and PKM2 was observed in the co-immunoprecipitation assay. HSP, heat shock protein; PKM2, pyruvate kinase isoenzyme 2; KD, knockdown; NC, negative control; OE, overexpression.

Figure 4.

HSP27 regulates PKM2 expression through SUMO2/3 in KYSE450 and KYSE150 cells. (A) Detection of the knockdown efficiency of SUMO2/3 shRNA vectors in KYSE450 cells by western blotting. GAPDH, internal loading control. (B and C) Detection of HSP27, PKM2, vimentin and E-cadherin in KYSE450 and KYSE150 cells after SUMO2/3 silencing by western blotting. (D and E) Interaction between HSP27 and SUMO2/3, as shown by co-immunoprecipitation assay. (F) Immunofluorescence of nuclei (DAPI, blue), SUMO2/3 (green) and HSP27 (red) in KYSE450 and KYSE150 cells. HSP, heat shock protein; PKM2, pyruvate kinase isoenzyme 2; SUMO2/3, small ubiquitin-related modified protein 2/3; KD, knockdown.

Figure 5.

SUMO2/3 promotes ESCC cell proliferation, invasion and migration. (A and B) MTT assay of proliferation changes in KYSE450 and KYSE150 cells after knockdown of SUMO2/3. (C and D) Detection of invasion by Transwell assay in KYSE450 and KYSE150 cells after silencing of SUMO2/3 (magnification, ×100). (E and F) Variations in the migratory activity of KYSE450 and KYSE150 cells analyzed using the wound healing assay after SUMO2/3 knockdown (magnification, ×100). *P<0.05 compared with the normal control group and ^▲P<0.05 compared with the shRNA-scramble control group. ESCC, esophageal squamous cell carcinoma; HSP, heat shock protein; SUMO2/3, small ubiquitin-related modified protein 2/3; KD, knockdown.