Title: Phase I-II study of vorinostat plus paclitaxel and bevacizumab in metastatic breast cancer: evidence for vorinostat-induced tubulin acetylation and Hsp90 inhibition in vivo
Materials and Methods for correlative studies:
Reagents and Antibodies: Mouse monoclonal anti-hsp90 and polyclonal anti-hsp70 antibodies were purchased from StressGen Biotechnologies Corp. (Victoria, British Columbia, Canada). Affinity-purified polyclonal antibody against Ac-K69-hsp90 was generated by Alpha Diagnostic (San Antonio, TX), based on the synthetic 12 amino acid peptide flanking K69 (acetylated and un-acetylated) ETLTDPSKLDSGK. [1]. Monoclonal anti-acetyl α-tubulin and anti-β-actin antibodies were purchased from Sigma-Aldrich Corporation (St. Louis, MO). Polyclonal anti-pAKT (Ser473); anti-AKT and monoclonal anti-acetyl lysine antibodies were obtained from Cell Signaling Technologies (Beverly, MA). Anti-acetylated K56 Histone H3 was obtained from Epitomics, Inc. (Burlingame, CA). All other anti-histone antibodies were obtained from Millipore (Billerica, MA). Polyclonal anti-CDK4 was obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Monoclonal anti-c-RAF, anti-p27 and anti-p21 antibodies were obtained as previously described [2]. MB231 cells were used as control.
Isolation of peripheral blood mononuclear cells: Peripheral blood samples were collected in heparinized tubes, according to the IRB approved clinical protocol. Mononuclear cells were separated using Lymphoprep (Axis-Shield, Oslo, Norway), washed once with complete RPMI-1640 media and then centrifuged to pellet the cells prior to use in the immunoblot analyses, as previously described [3].
Cell lysis and protein quantitation: Untreated or vorinostat (VS)-treated PBMCs or tumor biopsy specimens were re-suspended in 200 µL of lysis buffer (25 mM Tris [pH 7.4], 150 mM sodium chloride, 25 mmol/L sodium fluoride, 1.0 mmol/L benzamidine, 1% Triton X-100, 1 mmol/L phenylmethylsulfonyl fluoride [PMSF], 10 µg/ml leupeptin, 1 µg/ml pepstatin-A, 2 µg/ml aprotinin, 20 mmol/L p-nitrophenyl phosphate, 1.0 mmol/L sodium orthovanadate and 1.0 mmol/L 4-(2-aminoethyl) benzenesulfonylfluoride hydrochloride (AEBSF)) and incubated on ice for 30 minutes. Tumor samples were briefly sonicated (10 seconds) and the cell lysates were centrifuged. An aliquot of each cell lysate was diluted 1:10 and the protein concentrations were quantitated using a BCA protein quantitation kit (Pierce, Rockford, IL), according to the manufacturer’s protocol.
SDS-PAGE and Western Blotting: Seventy five micrograms of total cell lysate was used for the SDS-PAGE analyses. Immunoblot analyses of acetyl lysine, acetylated K69 hsp90, hsp90, hsp70, c-RAF, p-AKT, AKT, CDK4, p27, p21 and acetylated histone H3 and H4 were performed on total cell lysates, using specific anti-sera or monoclonal antibodies. Blots were washed with 1X PBST then incubated in IRDye 680 goat anti-mouse or IRDye 800 goat anti-rabbit secondary antibodies (LI-COR, Lincoln, NE) for 1 hour, washed 3X in 1X PBST and scanned with an Odyssey Infrared Imaging System (LI-COR, Lincoln, NE). The expression levels of β-actin were used as the loading control.
1. Yang Y, Rao R, Shen J, Tang Y, Fiskus W, Nechtman J, Atadja P, Bhalla K: Role of acetylation and extracellular location of heat shock protein 90alpha in tumor cell invasion. Cancer Research 2008, 68(12):4833-4842.
2. Bali P PM, Swaby R, Fiskus W, Yamaguchi H, Balasis M, Rocha K, Wang H, Richon V, Bhalla K: Activity of Suberoylanilide Hydroxamic acid against human breast cancer cells with amplificatoin of Her 2. Clinical Cancer Research 2005, 11(17):6382-6389.
3. Fiskus W, Ren Y, Mohapatra A, Bali P, Mandawat A, Rao R, Herger B, Yang Y, Atadja P, Wu J et al: Hydroxamic acid analogue histone deacetylase inhibitors attenuate estrogen receptor-alpha levels and transcriptional activity: a result of hyperacetylation and inhibition of chaperone function of heat shock protein 90. Clin Cancer Res 2007, 13(16):4882-4890.