Abstract
Abstract Microtubules (MTs) are essential architectural components of cells whose dynamic structures are critical for multiple functions including cell division, intracellular signaling, and trafficking. Many proteins are associated with MTs or their motors, including molecular chaperones such as heat shock protein 90 (HSP90), a key component of the cellular proteostatic machinery. The coordinated interplay of these processes is crucial for maintaining normal or malignant cell phenotypes. Alterations in protein levels, their localization and/or post-translational modifications (PTMs), such as phosphorylation or acetylation, are recognized as targets for disruption and may impact cell viability. To that end, we investigated the levels of HSP90 and pHSP90αT5/7 (a unique PTM previously associated with DNA damage) in both prostate (22RV1, PC3), and colorectal (HCT116wt, HCT116 DNA-PK -/-) carcinoma cell lines after treatment with microtubule-targeting agents (MTAs) or DNA-damaging agents (DDAs). We observed a dose dependent increase in the level of pHSP90αT5/7 protein in each of the cell lines treated with an MTA (paclitaxel (PTX), vincristine) or a DDA (gemcitabine, oxaliplatin) compared to the untreated control, whereas the levels of HSP90 remained monotonous throughout. The increases in pHSP90αT5/7 were independent of DNA-PK status for the HCT116 cells. These observations were accompanied by increasing levels of γ-H2AX and acetylated tubulin, the latter prominent following treatment with PTX. When cell lysates were separated into cytoplasmic (C) and nuclear (N) fractions, HSP90 and pHSP90αT5/7 were located predominantly in the C fractions while acetylated tubulin was surprisingly distributed between the C and N fractions as was γ-H2AX. Markers of cytoplasmic localization, including glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and the voltage-dependent anion channel (VDAC), and of nuclear localization, including phospho-Histone H3 and acetylated-Histone H3, were each segregated to their appropriate cellular compartments. HSP90α was previously shown to be a substrate and chaperone of the nuclear kinase DNA-PK and its DNA-PK dependent phosphorylation in the nucleus at T5 and T7 was necessary for the apoptotic response to TRAIL (Solier S, et al PNAS, 105:32, 2012). In contrast, our data demonstrate that HSP90αT5/7 phosphorylation also occurs in response to MTA, but is localized in cytosol and is independent of DNA-PK. Meanwhile, others have reported that tubulin acetylation favors HSP90 recruitment to MTs, stimulating the signaling function of HSP90 clients (Giustiniani, J, et al Cell Sig, 21, 2009). The unique effects of MTAs and DDAs on pHSP90αT5/7 and tubulin acetylation have implications for malignant cell survival and drug resistance mechanisms. Experiments are underway to determine the significance of cytosolic pHsp90αT5/7 and nuclear acetylated tubulin in these cellular tumor models. Citation Format: Marianne S. Poruchynsky, Michael Moses, Len Neckers. Comparison of the effects of tubulin-targeting and DNA-damaging agents on the modulation and distribution of HSP90, pHSP90αT5/7, and tubulin in cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-110. doi:10.1158/1538-7445.AM2017-LB-110
Published Version
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