Cytotoxic activity of tivantinib (ARQ 197) is not due solely to c-MET inhibition

Abstract

The receptor tyrosine kinase c-MET is the high-affinity receptor for the hepatocyte growth factor (HGF). The HGF/c-MET axis is often dysregulated in tumors. c-MET activation can be caused by MET gene amplification, activating mutations, and auto- or paracrine mechanisms. Thus, c-MET inhibitors are under development as anticancer drugs. Tivantinib (ARQ 197) was reported as a small-molecule c-MET inhibitor and early clinical studies suggest antitumor activity. To assess whether the antitumor activity of tivantinib was due to inhibition of c-MET, we compared the activity of tivantinib with other c-MET inhibitors in both c-MET-addicted and nonaddicted cancer cells. As expected, other c-MET inhibitors, crizotinib and PHA-665752, suppressed the growth of c-MET-addicted cancers, but not the growth of cancers that are not addicted to c-MET. In contrast, tivantinib inhibited cell viability with similar potency in both c-MET-addicted and nonaddicted cells. These results suggest that tivantinib exhibits its antitumor activity in a manner independent of c-MET status. Tivantinib treatment induced a G(2)-M cell-cycle arrest in EBC1 cells similarly to vincristine treatment, whereas PHA-665752 or crizotinib treatment markedly induced G(0)-G(1) cell-cycle arrest. To identify the additional molecular target of tivantinib, we conducted COMPARE analysis, an in silico screening of a database of drug sensitivities across 39 cancer cell lines (JFCR39), and identified microtubule as a target of tivantinib. Tivantinib-treated cells showed typical microtubule disruption similar to vincristine and inhibited microtubule assembly in vitro. These results suggest that tivantinib inhibits microtubule polymerization in addition to inhibiting c-MET.

Figure 1. Antitumor activity of tivantinib on c-MET-addicted and –independent cancer cell lines

A–C, Cells were treated with the increasing concentrations of tivantinib (A), PHA-665752 (B) or crizotinib (C) for 72 hr. Viable cells were assessed by CellTiter-Glo assay and were graphed relative to untreated cells. Experiments were carried out in sextuplet. The average values and SDs are shown. D, IC₅₀ values of each cell lines to tivantinib, PHA-665752 or crizotinib were shown. Repeated experiments gave similar results.

Figure 2. Sensitivity to tivantinib in c-MET inhibitor PHA-665752-resistant SNU638 subclones

A–C, parental SNU638 and resistant subclones SR-A1 and SR-C1 were treated with the increasing concentrations of PHA-665752 (A), crizotinib (B), or tivantinib (C), for 72 hr. Viable cells were assessed by CellTiter-Glo assay and were graphed relative to untreated cells. Experiments were carried out in sextuplet. The average values and SDs are shown. D, IC₅₀ values of each cell lines to tivantinib, PHA-665752 or crizotinib were shown. Repeated experiments gave similar results.

Figure 3. Effect of Tivantinib on c-MET phosphorylation and reactivation

A, Cells were treated with the indicated concentrations of tivantinib or crizotinib for 6 hr. Cell lysates were electrophoresed and immunoblotted with the indicated antibodies. B, MKN45 cells were pretreated with 100 nmol/L of crizotinib or 100 nmol/L of crizotinib plus 10 µmol/L of tivantinib for 6 hr. Then cells were washed three times and further incubated with RPMI growth medium containing 100 nmol/L of crizotinib, 10 µmol/L of tivantinib or no drug (Fresh medium). After the indicated time points, cells were harvested. Cell lysates were electrophoresed and immunoblotted with the indicated antibodies. For positive control, MKN45 cells were incubated with RPMI growth medium for 6 hr, washed three times with RPMI growth medium containing no drugs and then harvested. Repeated experiments gave similar results.

Figure 4. Tivantinib induces a G2/M arrest and exhibits similar activity as tubulin inhibitors across the JFCR39 cell line panel

A, EBC1 cells were treated with 1 µmol/L of tivantinib, vincristine, PHA-665752 or crizotinib for 24 hr. Cells were trypsinized, fixed and stained with propidium iodide, and the cell cycle was analyzed by flow cytometry. The histogram shows cell distribution versus DNA content. B, Growth inhibition against a panel of 39 human cancer cell lines. The mean graph was produced by computer processing of the 50% growth inhibition (GI₅₀) values as described under “Materials and Methods”. The x-axis represents the logarithm of difference between the mean of GI₅₀ values for 39 cell lines and the GI₅₀ value for each cell line. MG-MID, the mean of log GI₅₀ values for 39 cell lines; Delta, the logarithm of difference between the MG-MID and the log GI₅₀ of the most sensitive cell line; Range, the logarithm of difference between the log GI₅₀ of the most resistant cell line and the log GI₅₀ of the most sensitive cell line. Quantification of the GI₅₀ value was represented as the mean of four different experiments. Br, breast; CNS, central nervous system; Co, colon; Lu, lung; Me, melanoma; Ov, ovarian; Re, renal; St, stomach; xPg, prostate. C, The three compounds (out of 1805 compounds) which have high Pearson correlation coefficient (r) with tivantinib, and the results of two c-MET inhibitors (Crizotinib and PHA-665752) are shown in the table. Each experiment was performed as described in “Materials and Methods”.

Figure 5. Inhibition of tubulin polymerization by tivantinib

A, Tivantinib treatment disrupted microtubule in A549 cells. Control, tivantinib (10 µM, 16 hr) or vincristine (100 nM, 16 hr) treated A549 cells were fixed and stained with Alexa 488 labeled anti-alpha-tubulin antibody, Alexa 568 conjugated phalloidin (F-actin) or Hoechst33342 (nucleus). White scale bars indicate 20 µm. B, Tivantinib inhibits tubulin polymerization similar to vincristine in vitro. A tubulin polymerization assay was performed with or without indicated concentration of tivantinib, vincristine, paclitaxel, crizotinib or PHA-665752. Experiments were carried out in triplicate. The average values and SEMs are shown.