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Heat shock protein 90 (HSP90) functions as a well-known onco-protein to regulate protein conformation, stability and degradation. Pyruvate kinase M2 (PKM2), a critical regulator of the metabolism, growth and metastasis of cancer cells, has been confirmed to be overexpressed in various human cancer including hepatocellular carcinoma (HCC). However, the molecular mechanisms underlying the oncogenic functions of HSP90 and PKM2 overexpression in HCC remain unknown.
The expression of HSP90 and PKM2 in HCC specimens and cells were detected by immunoblotting and immunostaining. The interaction between HSP90 and PKM2 was confirmed by tandem affinity purification, co-immunoprecipitation and Glutathione S transferase (GST)-pulldown assay.
In this study, we found that HSP90 could bind to PKM2 and subsequently increased PKM2 abundance in HCC cells. Immunohistochemistry (IHC) staining showed that HSP90 level was positively correlated with PKM2 level in HCC tissues. Mechanistically, HSP90 was found to increase the phosphorylation of PKM2 at Thr-328. Protein kinase glycogen synthase kinase-3β (GSK-3β) formed a protein complex with HSP90 and PKM2, and directly mediated Thr-328 phosphorylation of PKM2 induced by HSP90. Thr-328 phosphorylation was critical for maintaining PKM2 stability and its biological functions in regulating glycolysis, mitochondria respiration, proliferation and apoptosis. Functionally, we found that HSP90 promoted the glycolysis and proliferation and inhibited apoptosis of HCC cells in a PKM2 dependent manner. In vivo experiments disclosed that PKM2 was required for the promoting effects of HSP90 on the growth of HCC cells in mice. Furthermore, we demonstrated that positive expression of HSP90 and PKM2 was correlated with poor clinicopathological features including high alpha fetoprotein (AFP) level, large tumor size, portal vein tumor thrombus (PVTT) and advanced tumor-node-metastasis (TNM) stage. Furthermore, we demonstrated that positive expression of HSP90 and PKM2, and a combination of these proteins could strongly predict the poor prognosis of HCC patients.
We suggest that HSP90 potentiates the glycolysis and proliferation, reduces the apoptosis and thus enhances the growth of HCC cells through PKM2.
Additional file 2: Figure S1. Knockdown of HSP90 reduced the protein level of PKM2. Hep3B cells were transfected with HSP90 shRNA or negative-control (NC) shRNA. 72 h after transfection, the levels of HSP90 and PKM2 protein in Hep3B cells were examined. Knockdown of HSP90 decreased PKM2 protein in Hep3B cells. (TIFF 4220 kb)
Additional file 3: Figure S2. HSP90 inhibitors reduced the protein level of PKM2. Hep3B cells were treated with 17-AAG and 17-DMAG, two kinds of HSP90 inhibitors. HSP90 inhibitors decreased PKM2 protein in Hep3B cells. (TIFF 2886 kb)
Additional file 4: Figure S3. MG132 restored the protein level of PKM2 induced by HSP90 knockdown. MG-132 was used to inhibit the proteasomal degradation in Hep3B cells. MG-132 treatment led to PKM2 accumulation in the detergent insoluble fraction of the cell lysate. (TIFF 7067 kb)
Additional file 5: Figure S4. HSP90 knockdown led to decreased Ser/Thr phosphorylation of PKM2. Hep3B cells were transfected with negative control (NC) shRNA or HSP90 shRNA. Representative IP experiments were performed to examine PKM2 Ser/Thr phosphorylation. Total cell lysates were subjected to immunoblotting analysis using specific antibodies against HSP90 and GAPDH. (TIFF 4128 kb)
Additional file 6: Figure S5. Thr-328 phosphorylation induced by HSP90 decreased the ubiquitination of PKM2 protein. Huh7 cell that were transfected with Flag-HSP90 were then transfected with HA-tagged wild type PKM2 or HA-tagged T328A PKM2. Proteins pull-down by HA antibody was subjected to western blot for ubiquitination. (TIFF 258 kb)
Additional file 7: Figure S6. Thr-328 phosphorylation-induced by HSP90 overexpression was critical for maintaining the stability of PKM2. A) PKM2 shRNA was used to knock down exogenous PKM2 in Huh7 cells. PKM2 shRNA effectively depleted the expression of endogenous PKM2 protein. B) Huh7 cells with endogenous PKM2 depleted were transfected with corresponding vectors. T328A mutant, instead of S405A, abrogated the increased phosphorylation of PKM2 induced by HSP90 overexpression. C) Huh7 cells with endogenous PKM2 depleted were co-transfected with corresponding vectors. The protein half-life of HA-tagged WT or mutated PKM2 was analyzed following treatment with cycloheximide (CHX). HSP90 overexpression increased the half-life of the wild type PKM2 while failed to increase the half time of T328A PKM2 mutant. *, P < 0.05 by t test. (TIFF 1330 kb)
Additional file 8: Figure S7. Knockdown of GSK-3β partly inhibited the elevation of PKM2 protein induced by PTEN knockdown. Huh7 cells transfected with PTEN siRNA or control siRNA along with or without GSK-3β. PTEN knockdown, which activated PT3K/AKT pathway, led to increased level of PKM2. Knockdown of GSK-3β partly inhibited the elevation of PKM2 protein induced by PTEN knockdown. (TIFF 6810 kb)
Additional file 9: Figure S8. Thr-328 phosphorylation-induced by HSP90 overexpression was critical for maintaining the function of PKM2. In Huh7 cells with endogenous PKM2 depleted, transfection of T328A PKM2 mutant significantly reduced (A) glucose consumption, (B) lactate production and (C) PK catalytic activity of Huh7 cells. Furthermore, transfection of T328A PKM2 mutant significantly reduced (D) proliferation and increased the (E) Caspase-3 activity and (F) apoptosis the of Huh7 cells. (TIFF 1030 kb)
Additional file 10: Figure S9. Thr-328 phosphorylation was required for PKM2 to regulate mitochondria respiration and co-factor function. Huh7 cells were transfected with HA-tagged PKM2-WT or T454A. Cells were re-plated into appropriate plates for analysis of O2 consumption, OCR, 6-14CO2 and ratio of 1-14CO2 to 6-14CO2. Compared with Huh7 cells transfected with wild-type PKM2, T328A PKM2 mutant significantly increased (A) O2 consumption, (B) OCR, (C) 6-14CO2 release from [6-14C] glucose while decreased (D) ratio of 1-14CO2 to 6-14CO2. E) HEK293T cells were co-transfected with wild type PKM2 or T328A PKM2 mutant, p2.1 and pSV40-Renilla. Transfected cells were exposed to 20% O2 or 1% O2 for 24 h. The ratio of firefly to renilla luciferase activity was determined. T328A PKM2 mutant significantly reduced the ability of PKM2 to promote hypoxia-induced gene transcription. Huh7 cells were transfected with wild type PKM2 or T328A PKM2 mutant. Two days after transfection, the cells were exposed to 1% O2 or 20% O2 for another 24 h. The mRNA levels of indicated genes were examined by qRT-PCR. T328A pKM2 mutant significantly reduced the ability of PKM2 in activating endogenous (F) HIF-1α target genes and (G) β-catenin target genes. *, P < 0.05 by t test. (TIFF 270 kb)
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- HSP90 promotes cell glycolysis, proliferation and inhibits apoptosis by regulating PKM2 abundance via Thr-328 phosphorylation in hepatocellular carcinoma
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