Lysine butyrylation of HSP90 regulated by KAT8 and HDAC11 confers chemoresistance

Abstract

Posttranslational modification dramatically enhances protein complexity, but the function and precise mechanism of novel lysine acylation modifications remain unknown. Chemoresistance remains a daunting challenge to successful treatment. We found that lysine butyrylation (Kbu) is specifically upregulated in chemoresistant tumor cells and tissues. By integrating butyrylome profiling and gain/loss-of-function experiments, lysine 754 in HSP90 (HSP90 K754) was identified as a substrate for Kbu. Kbu modification leads to overexpression of HSP90 in esophageal squamous cell carcinoma (ESCC) and its further increase in relapse samples. Upregulation of HSP90 contributes to 5-FU resistance and can predict poor prognosis in cancer patients. Mechanistically, HSP90 K754 is regulated by the cooperation of KAT8 and HDAC11 as the writer and eraser, respectively; SDCBP increases the Kbu level and stability of HSP90 by binding competitively to HDAC11. Furthermore, SDCBP blockade with the lead compound V020-9974 can target HSP90 K754 to overcome 5-FU resistance, constituting a potential therapeutic strategy.

Fig. 1. Lysine butyrylome profile in cancer chemoresistance and the important role of Kbu modification of HSP90.

a Scheme of the lysine butyrylome in 5-FU-resistant cell line KYSE150-FR and the parental cells KYSE150. b DEKPs and DEKSs identified by butyrylome analysis of resistant and parental cells. c Subcellular distribution of DEKPs in 5-FU-resistant and parental cells. d Heatmap indicating the enrichment (red) and depletion (green) of amino acids at each position flanking the Kbu sites. e The DEKSs were classified into four clusters by the Q classification method for further enrichment analysis. f GO enrichment analysis showing the enriched protein domains of DEKPs. g PPI network showing the relationships among the DEKPs. h Top 10 hub proteins in the PPI network. i Enrichment analysis of DEKPs identified by butyrylome analysis revealed the important role of the HSP90AA1-PI3K/AKT pathway in chemoresistance in cancer. j HSP90 expression levels in paired ESCC biopsy and post-5-FU-treatment relapse surgical specimens. k Kbu modification of HSP90 was increased in 5-FU-resistant cells.

Fig. 2. Butyrylation of HSP90 at K754 is essential for cancer chemoresistance.

a The MS/MS spectrum of the modified HSP90 peptide. b KYSE150 and KYSE410 were transiently transfected with the pcDNA3.1-Flag plasmid expressing wild-type HSP-90 (HSP90-WT) or the HSP90-K754R mutant and were then subjected to immunoprecipitation/immunoblotting (IB) with the indicated antibodies. c The sequences surrounding K754 in HSP90 among seven species were aligned. Lysine 754 of HSP90 was colored in red. d KYSE150 and KYSE410 were transiently transfected with the pcDNA3.1-Flag plasmid expressing HSP90-WT or the HSP90-K754R mutant. Colony formation assay showing that the HSP90 K754R mutation abolished the promoting effect of HSP90-WT on 5-FU resistance in ESCC cells. e Representative images and quantitative analyses of tumor xenografts established with ESCC cells as indicated in the presence or absence of 5-FU. HSP90-WT or the HSP90-K754R mutant was re-overexpressed in HSP90-deficient ESCC cells. f, g The effect of HSP90 K754R mutation on its interaction with client proteins and cochaperones. h A CHX (100 μg/mL) chase assay was used to compare the stability of the wild-type HSP90 (HSP90-WT) and HSP90-K754R mutant proteins in ESCC cells. i Representative images and expression pattern of HSP90 in 170 ESCC and 145 paired adjacent normal tissues. j Kaplan–Meier analysis of overall survival for 170 ESCC patients stratified by the tumor HSP90 level. Bars, SDs; *P < 0.05; **P < 0.01; ***P < 0.001.

Fig. 3. HDAC11 and KAT8 are the eraser and writer enzymes for Kbu modification of HSP90.

a Diagram of enzymatic reaction for acetyl-lysine and the hypothesized mechanism for butyryllysine. b HDAC11 decreased Kbu modification of HSP90. c The interaction between endogenous HDAC11 and HSP90 was investigated by immunoprecipitation. d Purified GST (lane 1) or HSP90-GST recombinant protein (lane 2) was immobilized on Glutathione-Sepharose beads and incubated with HDAC11-His recombinant protein, followed by immunoblotting. e Knockdown of HDAC11 increased Kbu modification of HSP90. f Effect of the HDAC11-H143A mutant on the HSP90 Kbu level. g In vitro deacylase assay was used to determine the Kbu levels of HSP90 in the presence of wild-type HDAC11 or the catalytically inactive mutant of HDAC11 (HDAC11-H143A). h Quantification of protein stability after CHX treatment in ESCC cells transfected with wild-type HDAC11 or the HDAC11-H143A mutant. i Effect of HDAC11 and HDAC11-H143A on the ubiquitination of HSP90. j An immunoprecipitation assay was performed to confirm the endogenous interaction between HSP90 and KAT8. k Purified GST (lane 1) or HSP90-GST recombinant protein (lane 2) was immobilized on Glutathione-Sepharose beads and incubated with KAT8-His recombinant protein, followed by immunoblotting. l KAT8 increased Kbu modification of HSP90. Effect of KAT8 knockdown (m) and KAT8-K274R mutant (n) on HSP90 Kbu level. o In vitro butyrylation of HSP90. HSP90 was incubated with the purified KAT8 or KAT8-K274R in the presence of buty-CoA as indicated. p Quantification of protein stability after CHX treatment in ESCC cells transfected with wild-type KAT8 or HAT-dead KAT8-K274R mutant. q Effect of KAT8 and KAT8-K274R on the ubiquitination of HSP90. Bars, SDs; *P < 0.05; **P < 0.01; ***P < 0.001.

Fig. 4. SDCBP is a key regulator of HSP90 Kbu modification.

a Diagram showing the strategy used to screen for the candidate proteins that are not only upregulated in 5-FU-resistant cells but also interact with HSP90. IP-MS was performed in KYSE150 cells with HSP90 overexpression. Systematic proteomic analysis was conducted in the 5-FU-resistant cell line KYSE150-FR and the parental cells KYSE150. b The interaction between endogenous SDCBP and HSP90 was determined by immunoprecipitation. c HSP90 Kbu levels were determined in SDCBP-overexpressing ESCC cells. d SDCBP knockdown decreased the HSP90 Kbu level. Detection of HSP90 expression in SDCBP-overexpressing (e) and SDCBP-knockdown ESCC cells (f). g Analysis of HSP90 stability in SDCBP-overexpressing and SDCBP-knockdown ESCC cells by a CHX chase assay. h Upper panel, schematic diagram of different SDCBP truncation mutant constructs; lower panel, ESCC cells cotransfected with Flag-tagged SDCBP mutants as indicated and Myc-tagged HSP90 were collected for immunoprecipitation. i Upper panel, in silico docking simulation showing the interaction between SDCBP and HSP90; middle panel, schematic of the HSP90 mutant constructs; lower panel, cells cotransfected with Flag-tagged SDCBP and Myc-tagged HSP90 mutants were subjected to immunoprecipitation. j Co-IP results showing the binding of HDAC11 to wild-type or mutant HSP90. k The interactions among HSP90, SDCBP and HDAC11 were determined by co-IP. l Comparison of the HSP90 Kbu levels in ESCC cells overexpressing SDCBP-WT, SDCBP-PDZ2, SDCBP-△PDZ2 or vector control. m HSP90 Kbu levels were determined in ESCC cells transfected with indicated plasmids. Bars, SDs; *P < 0.05; **P < 0.01; ***P < 0.001.

Fig. 5. HSP90 mediates the role of SDCBP in promoting cancer chemoresistance.

a, b Cell viability assay and colony formation assay showing the effect of SDCBP on the 5-FU sensitivity of ESCC cells. c, d Subcutaneous xenografts were established with SDCBP-overexpressing and SDCBP-knockdown cells, and the mice were treated with 5-FU or vehicle (n = 6). Representative images of tumors are shown, and tumor growth were quantified. Bars, SDs; *P < 0.05; **P < 0.01; ***P < 0.001. e Western blot analysis of SDCBP, HSP90, p-AKT, AKT and TS in the indicated ESCC cell lines. f Cell viability assay comparing 5-FU sensitivity in ESCC cells with manipulated SDCBP and/or HSP90 expression. g Expression pattern of SDCBP in a tumor tissue microarray consisting of 170 ESCC tissues and 145 normal tissues. h Kaplan–Meier analysis of overall survival for 170 ESCC patients stratified according to tumor SDCBP expression. i Correlation analysis between the expression of HSP90 and SDCBP. j Expression pattern of SDCBP in paired pre- and post-treatment samples from ESCC patients who received 5-FU-based neoadjuvant chemotherapy and subsequent surgery. Bars, SDs; *P < 0.05; **P < 0.01; ***P < 0.001.

Fig. 6. Identification of a lead compound targeting HSP90 Kbu to suppress chemoresistance.

a Flow chart showing the strategy used to screen SDCBP inhibitors. b 3D structure of the SDCBP protein. c Comparison of the survival inhibitory activity of 30 candidate compounds. d Biacore analysis showing the binding between V020-9974 and the SDCBP protein. e Western blot analysis of p-AKT, AKT, HSP90 and TS in ESCC cells treated with different concentrations of V020-9974. f Predicted model of V020-9974 binding to the PDZ2 domain of SDCBP. g The mutant SDCBP protein was purified and subjected to Biacore analysis to evaluate its binding to V020-9974. h, i IP assay showing that V020-9974 disrupted the SDCBP-HSP90 interaction but enhanced the HDAC11-HSP90 interaction. j V020-9974 decreased Kbu modification of HSP90. k The establishment of the PDX models and the treatment strategy. Representative images are shown, and tumor growth was monitored. l Representative images showing the expression of SDCBP in PDX#9, #14, #55 and #57 (right panel). m Summary diagram. Bars, SDs; *P < 0.05; **P < 0.01; ***P < 0.001.