HSP40 Interacts with Pyruvate Kinase M2 and Regulates Glycolysis and Cell Proliferation in Tumor Cells

<div><p>Pyruvate kinase M2 (PKM2) is predominantly expressed in cancers, which is considered as a key regulator of the Warburg effect. In this study, HSP40 was identified as a novel binding partner of PKM2. HSP40-PKM2 association destabilized PKM2 protein through HSC70. In the presence of HSP40, PKM2 protein level and PKM2-mediated PDK1 expression were down-regulated. Moreover, HSP40 was involved in regulating glucose metabolism on PKM2 dependent way and at the mean time had an effect on mitochondrial oxygen respiration. In line with inhibition effect of HSP40 on glycolysis, the growth of cancer cells was inhibited by HSP40.Our data provided a new regulation mechanism of PKM2, which suggested a new therapeutic target for cancer therapy.</p></div

HSP40 regulates glycolysis and mitochondrial respiration in a PKM2 dependent way.

<p>(A and B) HeLa, A549 and HepG2 cells were transfected with the siRNAs of negative control (NC), PKM2 (si-PKM2), HSP40 (si-HSP40) or both. The media were collected for analysis of glucose consumption and lactate production (mean ± S.D., n = 3). (C and D) HeLa, A549 and HepG2 cells were transfected with empty vector, HA-PKM2 or both HA-PKM2 and Flag-HSP40. The media were collected to analysis of glucose consumption and lactate production (mean ± S.D., n = 3). (E) Basal OCR of HeLa, A549 and HepG2 cells after transfected with NC or si-HSP40 were detected by seahorse XF24 extracellular flux analyzer. (F) HeLa cells were transfected with NC or si-HSP40. 24 h after transfection, the cells were replanted to detect O₂ consumption rate (OCR) by seahorse XF24 extracellular flux analyzer (mean ± S.D., n = 5). (mean ± S.D., n = 5).</p

Cellular consequences followed by HSP40 binding to PKM2.

<p>HSP40 interacts with PKM2, inhibits PKM2 protein level and pyruvate kinase activity. Consequently, glucose uptake from medium decreases and lactate consumption drops as well and mitochondrial oxygen respiration rate ascends at the same time. Interestingly, the transcriptional co-factor function of PKM2 was also impacted. As a result of all these cellular PKM2 functions change, the proliferation of tumor cells was impacted to some degree. Red arrows and dashed lines indicated a decrease and green arrow indicated an increase.</p

HSP40 impairs PKM2 stability and functions in HeLa cells.

<p>(A, B and C) HeLa, A549 and HepG2 cells were transfected with Flag-HSP40, after 48 h incubation, endogenous PKM2 protein levesl were detected by anti-PKM2 antibody. (D) HeLa cells were transfected with HSP40 siRNA, after 48 h incubation, PKM2 protein level was analyzed using anti-PKM2 antibody. (E) Cell lysates of HEK293T cells transfected with HA-HSC70 and Flag-HSP40/DNAJB1 were immunoprecipitated with anti-Flag antibody, the bound proteins were detected by anti-HA antibody. (F) HeLa cells transfected with HA-HSC70 or both HA-HSC70 and Flag-HSP40 were lysed, then western blot was analyzed using anti-PKM2, anti-HA and anti-Flag antibodies. (G) Pyruvate kinase activity of extracts prepared from HeLa cells transfected with HSP40 siRNA. The pyruvate kinase activity is detected by PK assay kit and normalized by protein measurement (mean ± S.D., n = 3). (H and I) HeLa cells were transfected with HSP40 siRNA or Flag-HSP40, after 24 h normal incubation followed with 24 hr anaerobic incubation, RT-PCR was performed to analyze PDK1 mRNA level (mean ± S.D., n = 3).</p

HSP40 impacts growth of HeLa cells via regulating PKM2 protein.

<p>(A, C and D) HeLa, A549 or HepG2 cells were transfected with HA-PKM2 or Flag-HSP40 or both. 24 h after transfection, cells were replanted and cell numbers were counted every 24 h for analysis of cell proliferation (mean ± S.D., n = 3). (B, D and E) HeLa, A549 or HepG2 cells were transfected with si-PKM2 or si-HSP40 or both. 24 h after transfection, cells were replanted and cell numbers were counted every 24 h for analysis of cell proliferation (mean ± S.D., n = 3).</p

PKM2 interacts with HSP40/DNAJB1 in vivo and in vitro.

<p>(A) Growth of yeast expressing HSP40/DNJB1-GalAD and PKM2-GalBD on nutritional deficient SD medium (-Trp-Leu or -Trp-Leu-Ade-His). Vector pGADT7-T and pGBKT7-p53 performed as positive control, pGADT7-T and pGBKT7-Lam performed as negative control. (B) Cell lysates of HEK293T cells transfected with HA-PKM2 and Flag-HSP40 were immunoprecipitated with anti-HA antibody, the bound proteins were detected by anti-Flag antibodies. (C) Cell lysates of HEK293T cells transfected with HA-PKM2 and Flag-HSP40 were immunoprecipitated with anti-Flag antibody, the bound proteins were detected by anti-HA antibodies. (D) HeLa cells were lysed and Co-IP was performed with anti-PKM2 antibody, then western blot was analyzed using anti-PKM2 and anti-HSP40 antibodies. (E) HeLa cells were lysed and Co-IP was performed with anti-HSP40 antibody, then western blot was analyzed using anti-PKM2 and anti-HSP40 antibodies. (F) Immunofluorescence analysis reveals that HSP40 and PKM2 co-localize with each other in nucleus in HeLa cells.</p

Recipients with acute rejection.

<p>Data are presented as number (percentage).</p>a<p>Glucocorticoid treatment consisted of oral methylprednisolone or oral prednisone.</p>†<p>Chi-square test;</p>‡<p>Fisher’s exact test.</p><p>MMF = mycophenolate mofetil, NA = non-available.</p

Recipients with HCC Recurrence^a.

<p>Data are presented as number (percentage).</p>a<p>The number of patients for basiliximab and steroid groups are 78 and 100, respectively unless indicated otherwise.</p>†<p>Chi-square test;</p>‡<p>Fisher’s exact test. NA: non-available.</p>b<p>Four subjects in the basiliximab group and 6 subjects in the steroid group had missing data.</p>c<p>One subject in the basiliximab group was missing data.</p><p>HCC = hepatocellular carcinoma.</p

Recipient postoperative status, complications, and immunosuppressive therapy^a.

<p>Data are presented as number (percentage), median (IRQ), or mean ± standard deviation.</p>a<p>The number of patients for the basiliximab and steroid groups are 78 and 100, respectively unless indicated otherwise.</p>b<p>The number of patients for the basiliximab and steroid groups are 44 and 58, respectively.</p>†<p>Wilcoxon rank sum test;</p>‡<p>Chi-square test;</p>¶<p>Fisher’s exact test;</p>§<p>independent t-test.</p><p>CMV = cytomegalovirus; GVHD = graft versus host disease; MMF = mycophenolate mofetil; NA = not available; PTLD = post-transplant lymphoproliferative disorder.</p

Overall survival of recipients between basiliximab and steroid groups (log-rank test, <i>P = </i>0.734).

<p>Overall survival of recipients between basiliximab and steroid groups (log-rank test, <i>P = </i>0.734).</p