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

Abstract

In preclinical models, the histone deacetylase inhibitor vorinostat sensitizes breast cancer cells to tubulin-polymerizing agents and to anti-vascular endothelial growth factor-directed therapies. We sought to determine the safety and efficacy of vorinostat plus paclitaxel and bevacizumab as first-line therapy in metastatic breast cancer (MBC), and the biological effects of vorinostat in vivo. For this purpose of this study, 54 patients with measurable disease and no prior chemotherapy for MBC received vorinostat (200 or 300 mg PO BID) on days 1-3, 8-10, and 15-17, plus paclitaxel (90 mg/m(2)) on days 2, 9, 16, and bevacizumab (10 mg/kg) on days 2 and 16 every 28 days. The primary objective of the phase I study was to determine the recommended phase II dose (RPTD) of vorinostat, and for the phase II to detect an improvement of response rate from 40 to 60% (alpha = 0.10, beta = 0.10). No dose limiting toxicities were observed, and the RPTD of vorinostat was 300 mg BID. For the primary efficacy analysis in 44 patients at the RPTD, we observed 24 objective responses (55%, 95% confidence intervals (C.I) 39%, 70%). The adverse event profile was consistent with paclitaxel-bevacizumab, with the exception of increased diarrhea with the addition of vorinostat. Analysis of serial tumor biopsies in seven patients showed increased acetylation of Hsp90 and α-tubulin following vorinostat. Vorinostat induces histone and alpha tubulin acetylation and functional inhibition of Hsp90 in breast cancer in vivo and can be safely combined with paclitaxel and bevacizumab.

Figures

Figure 1. Treatment with vorinostat (VS) induces in vivo acetylation of heat shock protein (hsp) 90, induction of hsp70 and depletion of pAKT and AKT expression levels in ER positive and ER negative breast cancer cell

A–B. Tumor biopsy specimens were collected from ER positive (#001, #010, and #53) and ER-negative (#51, 52, 54, and 55) patients prior to treatment with VS. Four hours following the third dose of VS, on Day 2, a second tumor biopsy was collected and cell lysates were prepared. Immunoblot analyses were performed for the acetylated-K69 of hsp90, total hsp90, hsp70, c-RAF, pAKT, AKT and CDK4 utilizing the tumor cell lysates. The expression levels of β-actin in the lysates served as the loading control.

Figure 2. Treatment with VS induces in vivo hyper-acetylation of histone and non-histone proteins in ER-positive and ER-negative breast cancer cells

Tumor biopsy specimens were collected from ER positive (#001, #010, and #53) and ER-negative (#51, 52, 54, and 55) patients prior to treatment with VS. Four hours following the third dose of VS, on Day 2, a second tumor biopsy was collected and cell lysates were prepared. Immunoblot analyses were performed for acetylated lysine, acetylated α-tubulin, acetylated K56 histone H3, acetylated histone H3, acetylated histone H4, p27 and p21 on the tumor cell lysates. The expression levels of β-actin in the lysates served as the loading control.

Figure 3. Inconsistent in vivo effects of VS treatment in peripheral blood mononuclear cells (PBMCs) derived from ER-positive and ER-negative patients

A. Peripheral blood was collected from patients #001 and #010 prior to the administration of VS on Day1 (Pre-Rx) and Day 2 (post-second dose) then 4 hours after dosing on Day 2 (post-third dose). PBMCs were separated by Ficoll Hypaque and cell lysates were prepared. Immunoblot analyses were performed for the acetylated-K69 of hsp90, hsp90, hsp70, and AKT on the total cell lysates. The expression levels of β-actin in the lysates served as the loading control. B. Peripheral blood was collected from patients #051, #052, #053 and #054 at the indicated time points and processed as in (A). Immunoblot analyses were performed for the acetylated-K69 of hsp90, hsp90, hsp70 and p21 on the total cell lysates. The expression levels of β-actin in the lysates served as the loading control.

Figure 4

Progression-free survival (4A) and overall survival (4B) in all eligible patients were analyzed using Kaplan-Meier method.