Hsp90 Promotes Gastric Cancer Cell Metastasis and Stemness by Regulating the Regional Distribution of Glycolysis-Related Metabolic Enzymes in the Cytoplasm

Abstract

Heat-shock protein 90 (Hsp90) plays a crucial role in tumorigenesis and tumor progression; however, its mechanism of action in gastric cancer (GC) remains unclear. Here, the role of Hsp90 in GC metabolism is the focus of this research. High expression of Hsp90 in GC tissues can interact with glycolysis, collectively affecting prognosis in clinical samples. Both in vitro and in vivo experiments demonstrate that Hsp90 is able to regulate the migration and stemness properties of GC cells. Metabolic phenotype analyses indicate that Hsp90 influences glycolytic metabolism. Mechanistically, Hsp90 interacts with glycolysis-related enzymes, forming multienzyme complexes to enhance glycolysis efficiency and yield. Additionally, Hsp90 binds to cytoskeleton-related proteins, regulating the regional distribution of glycolytic enzymes at the cell margin and lamellar pseudopods. This effect could lead to a local increase in efficient energy supply from glycolysis, further promoting epithelial-mesenchymal transition (EMT) and metastasis. In summary, Hsp90, through its interaction with metabolic enzymes related to glycolysis, forms multi-enzyme complexes and regulates regional distribution of glycolysis by dynamic cytoskeletal adjustments, thereby promoting the migration and stemness of GC cells. These conclusions also support the potential for a combined targeted approach involving Hsp90, glycolysis, and the cytoskeleton in clinical therapy.

Keywords: Hsp90; combination therapy; glycolysis; multienzyme complex; regionalized distribution.

Figure 1

Upregulation of Hsp90 in gastric cancer (GC) may potentially synergize with glycolysis to affect clinical prognosis. A) The expression of Hsp90 in patients with GC was derived from TCGA database. B) GSEA analysis was performed on HALLMARK_GLYCOLYSIS gene sets in gastric cancer. C) Venn diagram shows the intersection of glycolytic‐related genes with GC differential genes in the TCGA dataset. D,E) Lasso regression of glycolysis‐related genes (GEGs) and cross‐validation to determine the optimal penalty parameter λ. F,G) Prognostic analysis of GC patients was performed in combination with glycolysis and Hsp90. The KM plots show overall survival in F) 4 groups and G) 2 newly defined groups. H) Prognostic analysis was performed for glycolysis scores in GC patients with high and low Hsp90 expression.

Figure 2

High Hsp90 promotes proliferation, metastasis and other stem‐related features of gastric cancer in vivo and in vitro. A,B) Quantitative RT‐PCR and western blot for Hsp90 expression indicated in GC transfected cell lines with stable Hsp90 knockdown and overexpression. C) CCK‐8 show cell proliferation capacity of transfected cells. D) Analysis of the self‐renewal abilities of MGC803 and HGC27 cells stably expressing nc, oeHsp90, shcon, or shHsp90. Scale bar, 1000 µm. E) Analysis of the invasion abilities of MGC803 and HGC27 cells stably expressing nc, oeHsp90, shcon, or shHsp90. Scale bar, 100 µm. F) Expression of stemness markers and EMT‐related markers in MGC803 and HGC27 stable cell lines was detected by western blot. G) Tumorigenicity was evaluated in MGC803 and HGC27 cells stably expressing nc, oeHsp90, shcon, or shHsp90 (n = 5). H) IHC for Hsp90 and Ki67 in serial sections of tumor tissues from mice injected with MGC803 and HGC27 cells stably expressing nc, oeHsp90, shcon, or shHsp90. Scale bar, 100 µm. Error bars indicate mean ± SD. *p < 0.05, **p < 0.01 and ***p < 0.001.

Figure 3

Hsp90 can interact with metabolic enzymes associated with glycolysis and positively regulates glycolysis levels in GC cells. A) Cellular lysates from MGC803 and HGC27 cells were separated and purified by antibodies and magnetic beads. The eluates were separated by SDS‐PAGE and stained with silver. B) Preliminary analysis of mass spectrometry results and the pull‐down proteins of the two cell lines were shown by Venn diagrams. C) The differential protein bands were retrieved and analyzed by mass spectrometry. Analysis of proteomic data showed that it could be enriched to carbon metabolism. D) MGC803 and HGC27 cells were immunoprecipitated with normal IgG, anti‐Hsp90 or other antibody, and precipitates were analyzed by immunoblotting (IB) with indicated antibodies. E) The colocalization of Hsp90, ENO1 and PKM2 in MGC803 and HGC27 cells was demonstrated by immunofluorescence. Scale bar, 100 µm. F) Glucose consumption, lactate production and intracellular ATP production in MGC803 and HGC27 cells stably expressing nc, oeHsp90, shcon, or shHsp90. G) ECAR were examined in MGC803 and HGC27 cells stably expressing nc, oeHsp90, shcon, or shHsp90. Error bars indicate mean ± SD. *p < 0.05, **p < 0.01 and ***p < 0.001.

Figure 4

The regulation of Hsp90 on gastric cancer depends on glycolysis. A) Glucose consumption, lactate production and intracellular ATP production in MGC803 and HGC27 stable cell lines were detected before and after 2‐DG inhibition of glycolysis. B) ECAR were examined in MGC803 and HGC27 stable cell lines were detected before and after 2‐DG inhibition of glycolysis. C) The colony‐formation ability of stable cell lines was detected before and after 2‐DG inhibition of glycolysis. D) The self‐renewal ability of stable cell lines was detected before and after 2‐DG inhibition of glycolysis. Scale bar, 1000 µm. E) The migration ability of stable cell lines was detected before and after 2‐DG inhibition of glycolysis. F) The invasion ability of stable cell lines was detected before and after 2‐DG inhibition of glycolysis. Scale bar, 100 µm. Error bars indicate mean ± SD. *p < 0.05, **p < 0.01 and ***p < 0.001.

Figure 5

Hsp90 forms protein complexes with glycolytic‐related metabolic enzymes to improve glycolytic efficiency and regulate the regionalized distribution of glycolytic enzyme complexes. A) Expression of glycolytic enzymes in MGC803 and HGC27 stable cell lines was detected by western blot. B) Detection of PKM2 enzyme activity in MGC803 and HGC27 stable cell lines. C) Detection of protein expression of glycolytic enzyme and Hsp90 interacting glycolytic enzyme after treatment with Hsp90 inhibitor. D) The existence of glycolysis‐related enzyme complex was demonstrated by Co‐IP. E) Detection of protein expression of glycolytic enzyme and PKM2 interacting glycolytic enzyme after treatment with Hsp90 inhibitor. F) The colocalization of Hsp90 and PKM2 in MGC803 and HGC27 cells was demonstrated by immunofluorescence. Scale bar, 30 µm. G) MGC803 and HGC27 cells were immunoprecipitated with normal IgG or anti‐Hsp90, and precipitates were analyzed by immunoblotting (IB) with indicated antibodies. H) The interaction between glycolytic enzymes and cytoskeleton were demonstrated by Co‐IP. I) The changes of co‐localization positions of Hsp90 and PKM2 were detected by immunofluorescence after treatment with Hsp90 inhibitor geldanamycin (GA) for 12 h and 24 h. Scale bar, 30 µm. J) The changes of co‐localization positions of Hsp90 and PKM2 were detected by immunofluorescence after treatment with MYH9 inhibitor blebbistatin for 6 h and 12 h. Scale bar, 30 µm. K) Glucose consumption and lactate production in MGC803 and HGC27 cell were detected after GA inhibition of Hsp90. L) Glucose consumption and lactate production in MGC803 and HGC27 cell were detected after blebbistatin inhibition of MYH9. Error bars indicate mean ± SD. *p < 0.05, **p < 0.01 and ***p < 0.001.

Figure 6

Combination therapy experiment targeting Hsp90 and glycolysis using TAS‐116 and 2‐DG in vivo and in vitro. A) CCK‐8 show cell proliferation capacity of MGC803 and HGC27 after treatment with the Hsp90 inhibitor TAS and the glycolysis inhibitor 2‐DG. B) The colony‐formation ability of MGC803 and HGC27 was detected before and after treatment with the Hsp90 inhibitor TAS‐116 and the glycolysis inhibitor 2‐DG. C) The self‐renewal ability of MGC803 and HGC27 was detected before and after treatment with the Hsp90 inhibitor TAS‐116 and the glycolysis inhibitor 2‐DG. Scale bar, 1000 µm. D) The migration and invasion ability of MGC803 and HGC27 was detected before and after treatment with the Hsp90 inhibitor TAS‐116 and the glycolysis inhibitor 2‐DG. Scale bar, 100 µm. E) Expression of stemness markers and EMT‐related markers was detected before and after treatment with the Hsp90 inhibitor TAS‐116 and the glycolysis inhibitor 2‐DG by western blot. F) In vivo subcutaneous xenograft tumor model of MGC803 with combination therapy in vivo. Tumor appearance, total tumor weights and tumor volumes were assessed. G) Representative IHC staining of Ki‐67, CD44 and Vimentin in xenografted tumors. Scale bar, 100 µm. H) Expression of stemness markers and EMT‐related markers was detected in xenografted tumors. Error bars indicate mean ± SD. *p < 0.05, **p < 0.01 and ***p < 0.001.

Figure 7

Combination therapy experiment targeting Hsp90 and glycolysis regionalization using TAS‐116 and blebbistatin in vivo and in vitro. A) CCK‐8 show cell proliferation capacity of MGC803 and HGC27 after treatment with the Hsp90 inhibitor TAS‐116 and the MYH9 inhibitor blebbistatin. B) The colony‐formation ability of MGC803 and HGC27 was detected before and after treatment with the Hsp90 inhibitor TAS‐116 and the MYH9 inhibitor blebbistatin. C) The self‐renewal ability of MGC803 and HGC27 was detected before and after treatment with the Hsp90 inhibitor TAS‐116 and the MYH9 inhibitor blebbistatin. Scale bar, 1000 µm. D) The migration and invasion ability of MGC803 and HGC27 was detected before and after treatment with the Hsp90 inhibitor TAS‐116 and the MYH9 inhibitor blebbistatin. Scale bar, 100 µm. E) Expression of stemness markers and EMT‐related markers was detected before and after treatment with the Hsp90 inhibitor TAS‐116 and the MYH9 inhibitor blebbistatin by western blot. F) In vivo subcutaneous xenograft tumor model of MGC803 with combination therapy in vivo. Tumor appearance, total tumor weights and tumor volumes were assessed. G) Representative IHC staining of Ki‐67, CD44 and Vimentin in xenografted tumors. Scale bar, 100 µm. H) Expression of stemness markers and EMT‐related markers was detected in xenografted tumors. Error bars indicate mean ± SD. *p < 0.05, **p < 0.01 and ***p < 0.001.