Peptidomimetic Src/pretubulin inhibitor KX-01 alone and in combination with paclitaxel suppresses growth, metastasis in human ER/PR/HER2-negative tumor xenografts

Abstract

Src kinase is elevated in breast tumors that are ER/PR negative and do not overexpress HER2, but clinical trials with Src inhibitors have shown little activity. The present study evaluated preclinical efficacy of a novel peptidomimetic compound, KX-01 (KX2-391), that exhibits dual action as an Src and pretubulin inhibitor. KX-01 was evaluated as a single-agent and in combination with paclitaxel in MDA-MB-231, MDA-MB-157, and MDA-MB-468 human ER/PR/HER2-negative breast cancer cells. Treatments were evaluated by growth/apoptosis, isobologram analysis, migration/invasion assays, tumor xenograft volume, metastasis, and measurement of Src, focal adhesion kinase (FAK), microtubules, Ki67, and microvessel density. KX-01 inhibited cell growth in vitro and in combination with paclitaxel resulted in synergistic growth inhibition. KX-01 resulted in a dose-dependent inhibition of MDA-MB-231 and MDA-MB-157 tumor xenografts (1 and 5 mg/kg, twice daily). KX-01 inhibited activity of Src and downstream mediator FAK in tumors that was coincident with reduced proliferation and angiogenesis and increased apoptosis. KX01 also resulted in microtubule disruption in tumors. Combination of KX-01 with paclitaxel resulted in significant regression of MDA-MB-231 tumors and reduced metastasis to mouse lung and liver. KX-01 is a potently active Src/pretubulin inhibitor that inhibits breast tumor growth and metastasis. As ER/PR/HER2-negative patients are candidates for paclitaxel therapy, combination with KX-01 may potentiate antitumor efficacy in management of this aggressive breast cancer subtype.

Figure 1

KX-01 induced growth inhibition and apoptosis in ER/PR/HER2-negative breast cancer cells and synergized with paclitaxel in vitro. A) Chemical structures of KX-01, dasatinib and paclitaxel. B,C) Growth inhibitory effects of the broad specificity Src inhibitor dasatinib (B) and KX-01 (C) at varying concentrations (10, 25, 50, 100, 250nmol/L) on ER/PR/HER2-negative MDA-MB-231, MDA-MB-157, and MDA-MB-468 cells. Cells were incubated with drugs for 48h and growth inhibition was assessed by MTT assay. Results presented as % vehicle ± SD (n=3). D) KX-01 induced synergistic growth inhibition in MDA-MB-231 cells when combined with paclitaxel (PAX). Cell growth was measured by MTT assay. Cells were incubated with KX-01 (25nmol/L), PAX (5nmol/L), or combination for 24, 48, 72h. Results presented as % vehicle ± SD (n=3). E) KX-01 in combination with PAX resulted in enhanced apoptosis in MDA-MB-231 cells. Apoptosis assay was performed following 24h incubation with vehicle KX-01 (25nmol/L), PAX (5nmol/L) or KX-01+PAX. *, P<0.05 (Student's t-test), significantly different compared to vehicle, #, P<0.05 (Student's t-test), statistical evidence of enhanced apoptosis in combination treatment compared to either drug alone. Data are representative of three independent experiments.

Figure 2

KX-01 inhibited growth of MDA-MB-231 xenografts. A) Athymic NUDE mice bearing MDA-MB-231 tumors (∼100mm³) were separated into three treatment groups randomly (n=10 tumors/group). The vehicle group received ultra-pure water and the other groups were treated with two different doses of KX-01 (1, 5mg/kg) for 28 days and tumor volumes were recorded as mm³ ± SD. *, P<0.05; (One-way ANOVA followed by Newman-keuls multiple comparison test). B,C) KX-01 inhibited phosphorylation of Src and downstream mediator FAK in MDA-MB-231 xenografts. IHC was performed as described in ‘Supplementary Methods’ for total Src, phospho-Src-Y416 (P-Y416-Src), FAK and phospho-FAK-Y861 (P-Y861-FAK). Representative photomicrographs are presented (200×). D-F) KX-01 induced apoptosis, and reduced proliferation and angiogenesis in MDA-MB-231 xenografts. Paraffin embedded tumor sections from vehicle and KX-01 (5mg/kg) groups were stained for TUNEL (D) and Ki67 (E). Fluorescent (TUNEL) and bright field (Ki67) photomicrographs were taken and representative images are presented (200×). F) Frozen tumor sections were stained for mouse CD31. Bright field microscope images (40×) were taken and representative photomicrographs are presented.

Figure 3

KX-01 combined with paclitaxel resulted in regression of MDA-MB-231 and MDA-MB-157 xenografts. Mice bearing MDA-MB-231 or MDA-MB-157 xenografts (∼100mm³) were separated into four treatment groups randomly with 5 mice/group (2 tumors/mouse). MDA-MB-231 (A) and MDA-MB-157 (B) xenograft bearing mice were treated with ultra-pure water, KX-01 alone (5mg/kg) BID, paclitaxel (PAX) (3mg/kg) once/week by IP as indicated by the arrows, and KX-01+PAX. Tumors volumes were recorded as mm³±SD. *, P<0.05; (One-Way ANOVA followed by Newman-keuls multiple comparison test).

Figure 4

Combination of high dose KX-01 with paclitaxel induced regression of established, large MDA-MB-231 tumors. A) MDA-MB-231 tumors were established in NUDE mice (tumor volume ∼300 mm³) before treatments began. Mice (n=5/group) were treated with vehicle, KX-01 (15mg/kg) by oral gavage once/daily, paclitaxel (PAX, 20mg/kg) once/week by IP (arrows), or KX-01+PAX. Tumor volumes were recorded as mm³±SD. *, P<0.05; (One-Way ANOVA followed by Newman-keuls multiple comparison test. B) Paraffin tumor sections from treatment groups were analyzed by immunofluorescence with anti–tubulin antibody conjugated with Alexafluor-488 (green) and imaged with a confocal microscope (60×). White arrows indicate fragmented microtubules in the KX-01+PAX group.

Figure 5

KX-01 inhibited invasive stellate formation, invasion, and migration of MDA-MB-231 cells in vitro. A) Invasive stellate structures of MDA-MB-231 cells in 3D culture following vehicle or KX-01 treatment were photomicrographed day4. B) MDA-MB-231 cells were incubated with vehicle (VC) or KX-01 (10, 25, 50nmol/L) for 24h and the number of cells invaded was quantified and normalized to cell number and presented as % relative invasion ±SD. *, P<0.05 statistically significant compared to VC. C) Monolayer cultures of MDA-MB-231 cells were gently scratched with a pipette tip to produce a wound. Photographs of cultures were taken immediately after the scratch (0h) and after 24h incubation with vehicle or KX-01 (5, 10, 25nmol/L). Migration is presented as % gap closure ±SD, *, p<0.05, significantly different from VC. Data are representative of three independent experiments.

Figure 6

Quantification of micrometastasis of MDA-MB-231 tumors in mouse organs. After necropsy, lung, bone marrow (femur) and liver were collected from treatment groups described in Figure 3 [vehicle control (VC), KX-01, PAX, KX-01+PAX (n=5 mice/group)] and genomic DNA isolated. Micrometastases originating from human primary MDA-MB-231 xenografts were detected by quantitative real-time RT-PCR for an α-satellite DNA sequence in the centromere region of human chromosome-17. Test samples (the number of human cells in each mouse tissue sample) were interpolated from a standard curve as described in Materials and Methods. Graphs represent the number of human cells in intact mouse lung (A), bone marrow from one femur (B) and intact mouse liver (C). *, P<0.05, significantly different compared to VC.