HSP70 regulates Eg5 distribution within the mitotic spindle and modulates the cytotoxicity of Eg5 inhibitors

Abstract

The heat shock protein 70 (HSP70) is a conserved molecular chaperone and proteostasis regulator that protects cells from pharmacological stress and promotes drug resistance in cancer cells. In this study, we found that HSP70 may promote resistance to anticancer drugs that target the mitotic kinesin, Eg5, which is essential for assembly and maintenance of the mitotic spindle and cell proliferation. Our data show that loss of HSP70 activity enhances Eg5 inhibitor-induced cytotoxicity and spindle abnormalities. Furthermore, HSP70 colocalizes with Eg5 in the mitotic spindle, and inhibition of HSP70 disrupts this colocalization. Inhibition or depletion of HSP70 also causes Eg5 to accumulate at the spindle pole, altering microtubule dynamics and leading to chromosome misalignment. Using ground state depletion microscopy followed by individual molecule return (GSDIM), we found that HSP70 inhibition reduces the size of Eg5 ensembles and prevents their localization to the inter-polar region of the spindle. In addition, bis(maleimido)hexane-mediated protein-protein crosslinking and proximity ligation assays revealed that HSP70 inhibition deregulates the interaction between Eg5 tetramers and TPX2 at the spindle pole, leading to their accumulation in high-molecular-weight complexes. Finally, we showed that the passive substrate-binding activity of HSP70 is required for appropriate Eg5 distribution and function. Together, our results show that HSP70 substratebinding activity may regulate proper assembly of Eg5 ensembles and Eg5-TPX2 complexes to modulate mitotic distribution/function of Eg5. Thus, HSP70 inhibition may sensitize cancer cells to Eg5 inhibitor-induced cytotoxicity.

Mdivi-1 induces spindle abnormalities and augments taxol cytotoxicity in MDA-MB-231 cells

“…The numbers of cells with indicated spindle types were counted using a Zeiss Axioplan 2 Imaging MOT fluorescence microscope. Mitotic spindles at prophases, prometaphases, and metaphases, judged as described previously 64 , 70 , were counted as normal spindles. Multipolar spindle, monopolar spindle, disorganized spindle, and bipolar spindle with misaligned chromosomes were judged as mitotic spindle abnormalities.…”

“…The effect of mdivi-1 on the instability of microtubules was examined by cold treatment assay 71 . MDA-MB-231 cells were treated with mdivi-1 as indicated and subjected to 10 min cold treatment, followed by immunostaining, as described previously 64 , 70 . Mitotic cells with microtubule remnants at the spindle pole (pole-MT) were counted; a decrease in number indicates microtubule instability.…”

GRP75-driven, cell-cycle-dependent macropinocytosis of Tat/pDNA-Ca2+ nanoparticles underlies distinct gene therapy effect in ovarian cancer

“…As chaperone protein complexed with mitotic kinases can modulate their activity during the progression of cell-cycle [ 57 , 58 ], we next tested whether centrosome-targeting of MPS1 was dependent on the presence of GRP75. Confocal checking the association of GRP75 and MPS1 at centrosome showed that these two targets were unable to simultaneously localize at centrosomes in GRP75-KD and phosphorylation-inactivation (T62A/S65A) cell.…”

“…S10, S11) suggests that GRP75 is also required for centrosome duplication and mitotic checkpoint response. Additionally, centrosome is a highly unstable macromolecular complex and Hsp70–Hsp90 chaperone machinery is essential to maintain centrosome integrity for cell-cycle progression [ 57 , 58 ]. Thus, centrosome-recruiting of GRP75 from S- to M-phase (Figs.…”

Mitotic checkpoint regulator RAE1 promotes tumor growth in colorectal cancer

“…To investigate whether RAE1 could be a therapeutic target, we performed a comprehensive analysis of 276 genes encoding microtubule‐regulating proteins 19 , 23 , 24 , 25 , 26 , 27 , 28 , 29 , 30 , 31 using TCGA data and evaluated their potential efficacy as a therapeutic target. We calculated the fold change in mRNA expression of each gene in tumor tissues compared to normal tissues, the correlation between mRNA expression and DNA copy number, and whether the high expression group was associated with poor prognosis.…”