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Cks1 proteasomal degradation is induced by inhibiting Hsp90-mediated chaperoning in cancer cells

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Abstract

Purpose

Cks1, a conformationally heterogenous 9 kDa protein, is markedly overexpressed in cancer cells and contributes to tumor development. Cks1 is an essential component of the SCF-Skp2 ubiquitin ligase complex that targets the Cdk inhibitors p27Kip1 and p21Cip1. Cks1 is known to interact with the Hsp90-Cdc37 chaperone machinery, although whether this facilitates its conformational maturation and stability is not known. To test whether abrogating the chaperone function of Hsp90 could destabilize Cks1, we examined the effects of treating different cancer cell lines with the benzoquinone ansamycin 17-allylamino geldanamycin (17-AAG), a compound that selectively binds Hsp90 and potently inhibits its ATP-dependent chaperone activity.

Methods

The effect of Hsp90 inhibition using 17-AAG on Cks1 protein and associated cell cycle proteins including Skp2, p27Kip1, p21Cip1, and Cdk1 in cancer cells was determined by Western blotting. Ubiquitination analysis was carried out by transfecting cells with an HA-ubiquitin plasmid and specifically immunoprecipitating Cks1 to examine polyubiquitinated species. Flow cytometry was utilized to examine the effects of Hsp90 inhibition on cell cycle profiles.

Results

Here, we demonstrate for the first time that inhibition of Hsp90 utilizing 17-AAG destabilizes Cks1 in cancer cells by promoting its ubiquitination and proteasomal degradation. 17-AAG-induced Cks1 depletion was accompanied by concomitant decreases in Skp2 and Cdk1. 17-AAG treatment also induced G2/M accumulation in MCF-7 breast carcinoma cells, and G1 accumulation in the colon carcinoma lines HCT116 and SW620.

Conclusions

We conclude that perturbing the Hsp90 pathway could provide a useful therapeutic strategy in tumors driven by Cks1 overexpression.

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Acknowledgments

JS is a fellow in the Howard Hughes Medical Institute Graduate Fellowship Program at UAB. Work in the authors’ laboratories was supported by grants from the Komen Breast Cancer Foundation grant (BCTR00-456, JVT), NCI Breast SPORE Developmental grant (JVT), and NIH grants ES016354 (BX) and CA133093 (BX).

Conflict of interest

None.

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Authors and Affiliations

  1. Department of Oncology, Southern Research Institute, Birmingham, AL, USA

    Vinayak Khattar, Joshua Fried, Bo Xu & Jaideep V. Thottassery

  2. Cancer Cell Biology Program UAB Comprehensive Cancer Center, Birmingham, AL, USA

    Vinayak Khattar, Joshua Fried, Bo Xu & Jaideep V. Thottassery

  3. Department of Oncology, Drug Discovery Division, Southern Research Institute, 2000 Ninth Ave South, Birmingham, AL, 35205, USA

    Jaideep V. Thottassery

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  1. Vinayak Khattar
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Correspondence to Jaideep V. Thottassery.

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Khattar, V., Fried, J., Xu, B. et al. Cks1 proteasomal degradation is induced by inhibiting Hsp90-mediated chaperoning in cancer cells. Cancer Chemother Pharmacol 75, 411–420 (2015). https://doi.org/10.1007/s00280-014-2666-7

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  • DOI: https://doi.org/10.1007/s00280-014-2666-7

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