Fig 2: Downregulation of MORC2 by HSP90 N‐terminal inhibitors is independent on HSP90. (A) Both HSP90α and HSP90β genes were knocked out in MCF‐7 and T47D cells using CRISPR/Cas9 technology. Immunoblotting analysis was performed with the indicated antibodies. (B–D) WT and HSP90 KO MCF‐7 and T47D cells were treated with DMSO or 1 μM of 17‐AAG, AUY922 or STA‐9090 for 24 h. Total cellular lysates were harvested for immunoblotting analysis. (E,F) Lysates from MCF‐7 and T47D cells were subjected to Co‐IP assays and immunoblotting analysis with the indicated antibodies. (G) MCF‐7 cells were treated with or without 5 μM of trichostatin A for the indicated times. Total cellular lysates were harvested for immunoblotting analysis with the indicated antibodies. (H) MCF‐7 cells were treated with or without 10 mM of sodium butyrate (NaBu) for the indicated times. Total cellular lysates were harvested for immunoblotting analysis with the indicated antibodies. (I) MCF‐7 cells were treated with or without 5 mM of NAM for the indicated times. Total cellular lysates were harvested for immunoblotting analysis with the indicated antibodies

Fig 3: The proposed working model. HSP90 N‐terminal inhibitors target MORC2 for lysosomal degradation through disrupting its N‐terminal homodimerization, and consequently, block MORC2‐mediated breast cancer progression (left). In contrast, in the absence of HSP90 N‐terminal inhibitors, dimer formation leads to an increase of protein stability of MORC2, thus manifesting its oncogenic functions (right)

Fig 4: HSP90 N‐terminal inhibitors induce lysosomal degradation of MORC2 through the CMA pathway. (A) MCF‐7 and T47D cells were treated with DMSO or 1 μM of 17‐AAG, AUY922, STA‐9090 alone or in combination with 100 nM Baf A1 for 24 h. Total cellular lysates were harvested for immunoblotting analysis with the indicated antibodies. (B) MCF‐7 and T47D cells were treated with DMSO or 1 μM of 17‐AAG, AUY922, STA‐9090 alone or in combination with 10 μM MG‐132 for 24 h. Total cellular lysates were harvested for immunoblotting analysis with the indicated antibodies. (C,D) MCF‐7 and T47D cells were transfected with control siRNA (siNC) or two independent siRNAs targeting HSPA8 (siHSPA8) or LAMP2A (siLAMP2A). After 24 h of transfection, cells were treated with DMSO or 1 μM of 17‐AAG, AUY922 or STA‐9090 for 24 h. Total cellular lysates were harvested for immunoblotting analysis. (E) MCF‐7 and T47D cells were transfected with Flag‐MORC2. After 24 h of transfection, cells were treated with DMSO or 1 μM of 17‐AAG, AUY922 or STA‐9090 for 24 h, and then subjected to immunofluorescent staining with the anti‐Flag (red), anti‐HSPA8 (green) and anti‐LAMP2A (green) antibodies. DNA was counterstained with DAPI. Scale bar, 10 μm. The dark blue arrows in the merged images indicate the co‐localization of MORC2 with HSPA8 or LAMP2A in the cytoplasm (yellow colour)

Fig 5: HSP90 N‐terminal inhibitors disrupt MORC2 dimerization in breast cancer cells. (A) Graphic representation of MORC2 domains and site mutagenesis. (B) Summary of the effects of Y18A, N39A and S87L mutations on the dimer formation and ATPase activities of MORC2. (C,D) HEK293T cells were transfected with various HA‐MORC2 plasmids (WT, Y18A, N39A or S87L). After 36 h of transfection, cells were treated with 100 μg/ml of CHX for the indicated times and then subjected to immunoblotting analysis with the indicated antibodies (C). Relative HA‐MORC2 expression levels (HA‐MORC2/Vinculin) are shown in (D). (E) HEK293T cells were transfected with Flag‐MORC2 (WT, Y18A, N39A and S87L). After 24 h of transfection, cells were treated with DMSO or 1 μM of 17‐AAG, AUY922 or STA‐9090 for 24 h, and then subjected to immunofluorescent staining with an anti‐Flag antibody. The nuclei were counterstained with DAPI. Scale bar, 10 μm. (F) HEK293T cells we transfected with Flag‐MORC2. After 48 h of transfection, cells were subjected to cytosol‐nucleus cell fractionation assays, followed by chemical cross‐linking experiment. The quantitative results (dimer/monomer ratio) are shown below. (G,H) MCF‐7 and T47D cells were treated with DMSO or 1 μM of 17‐AAG, AUY922, STA‐9090 or 10 μM of NB for 12 h. Total cellular lysates were harvested for cross‐linking reactions with glutaraldehyde (G). Dimer/monomer ratio is shown (H). (I) Immunoprecipitated Flag‐MORC2 was incubated with 1 μM 17‐AAG, AUY922, STA‐9090 and NB for 1 h at room temperature in the presence of 4 mM ATP. The absorbance at 620 nm was read and analysed