The HSP90 inhibitor ganetespib has chemosensitizer and radiosensitizer activity in colorectal cancer

Abstract

The integration of targeted agents to standard cytotoxic regimens has improved outcomes for patients with colorectal cancer (CRC) over recent years; however this malignancy remains the second leading cause of cancer mortality in industrialized countries. Small molecule inhibitors of heat shock protein 90 (HSP90) are one of the most actively pursued classes of compounds for the development of new cancer therapies. Here we evaluated the activity of ganetespib, a second-generation HSP90 inhibitor, in models of CRC. Ganetespib reduced cell viability in a panel of CRC cell lines in vitro with low nanomolar potency. Mechanistically, drug treatment exerted concomitant effects on multiple oncogenic signaling pathways, cell cycle regulation, and DNA damage repair capacity to promote apoptosis. Combinations of ganetespib and low-dose ionizing radiation enhanced the radiosensitivity of HCT 116 cells and resulted in superior cytotoxic activity over either treatment alone. In vivo, the single-agent activity of ganetespib was relatively modest, suppressing HCT 116 xenograft tumor growth by approximately half. However, ganetespib significantly potentiated the antitumor efficacy of the 5-Fluorouracil (5-FU) prodrug capecitabine in HCT 116 xenografts, causing tumor regressions in a model that is intrinsically resistant to fluoropyrimidine therapy. This demonstration of combinatorial benefit afforded by an HSP90 inhibitor to a standard CRC adjuvant regimen provides an attractive new framework for the potential application of ganetespib as an investigational agent in this disease.

Fig. 1

Ganetespib activity in HCT 116 colon cancer cells in vitro and in vivo. a HCT 116 cells were treated with increasing concentrations of ganetespib or 5-FU and cell viability assessed after 72 h. b HCT 116 cells were exposed to graded concentrations of ganetespib or vehicle (V) for 24 h as indicated. Cell lysates were immunoblotted using antibodies against MET and phosphorylated Src (p-Src) as shown. GAPDH was included as a loading control. c HCT 116 cells were exposed to vehicle or ganetespib (25, 50 and 100 nM) for 24 h as indicated. Cell lysates were immunoblotted using antibodies against EGFR, IGF-1R, phosphorylated ERK (p-ERK), total ERK, phosphorylated AKT (p-AKT), total AKT, and phosphorylated 4E-BP1 (p-4E-BP1). d Mice bearing established HCT-116 xenografts (n = 8/group) were i.v. dosed with 150 mg/kg ganetespib once weekly over a 3 week cycle. % T/C values are indicated to the right of each growth curve and the error bars are the SEM; (*, p < 0.05)

Fig. 2

Ganetespib modulates cell cycle expression and induces growth arrest and apoptosis in HCT 116 colon cancer cells. a HCT 116 cells were treated with increasing concentrations of ganetespib as indicated. Cell cycle distribution was determined by flow cytometry 18 h post-treatment. b HCT 116 cells were treated with graded concentrations of ganetespib for 18 h. Cell lysates were immunoblotted using antibodies against CHK1, CHK2, p21 and p27. Arrow depicts higher molecular weight form of CHK2 induced following ganetespib treatment. c HCT 116 cells were exposed to increasing concentrations of ganetespib for 24 h. Cell lysates were immunoblotted using antibodies against RAD51, phosphorylated histone H2AX (p-histone H2AX), Bcl-XL and RIP, as indicated. d Quantification of apoptosis as assessed by Annexin V positivity. HCT 116 cells were exposed to increasing concentrations of ganetespib for 24 h and Annexin V/PI staining measured by flow cytometry

Fig. 3

Ganetespib sensitizes HCT 116 colon cancer cells to ionizing radiation. a HCT 116 cells were treated with increasing doses of ganetespib either alone or in combination with 2 Gy dosing of irradiation (Ganetespib + 2 Gy). At 48 h post-IR, quantification of apoptosis was assessed by Annexin V-positivity measured by flow cytometry. b HCT 116 cells were treated with increasing doses of ganetespib (0, 25, 50 and 100 nM), either alone or in combination with 2 Gy irradiation. At 24 h post-treatment, lysates were prepared and immunoblotted using antibodies against CHK1, CHK2, RAD51, phosphorylated histone H2AX (p-histone H2AX), cleaved Caspase 7, and cleaved PARP. GAPDH was included as a loading control. c Representative images of HCT 116 cells treated with vehicle, 100 nM ganetespib, irradiated with a single dose of 2 Gy or simultaneously treated with the combination for 48 h, as indicated. Immunofluorescence was performed on cells stained for actin (red) and DAPI (blue). Arrowheads depict examples of large, multinucleated cells. Original magnification, 20×

Fig. 4

Combination ganetespib plus capecitabine treatment confers superior antitumor efficacy in CRC xenografts. Over a 3 week cycle, mice bearing established HCT 116 xenografts (n = 8/group) were i.v. dosed with 150 mg/kg ganetespib 1×/week or p.o dosed with 400 mg/kg capecitabine daily for 14 consecutive days, either alone or in combination. % T/C values are indicated to the right of each growth curve and the error bars are the SEM. Combination ganetespib + capecitabine treatment resulted in significant tumor regression (*, p < 0.05)