Pharmacological targeting of GLI1 inhibits proliferation, tumor emboli formation and in vivo tumor growth of inflammatory breast cancer cells

Abstract

Activation of the Hedgehog (Hh) pathway effector GLI1 is linked to tumorigenesis and invasiveness in a number of cancers, with targeting of GLI1 by small molecule antagonists shown to be effective. We profiled a collection of GLI antagonists possessing distinct mechanisms of action for efficacy in phenotypic models of inflammatory and non-inflammatory breast cancer (IBC and non-IBC) that we showed expressed varying levels of Hh pathway mediators. Compounds GANT61, HPI-1, and JK184 decreased cell proliferation, inhibited GLI1 mRNA expression and decreased the number of colonies formed in TN-IBC (SUM149) and TNBC (MDA-MB-231 and SUM159) cell lines. In addition, GANT61 and JK184 significantly down-regulated GLI1 targets that regulate cell cycle (cyclin D and E) and apoptosis (Bcl2). GANT61 reduced SUM149 spheroid growth and emboli formation, and in orthotopic SUM149 tumor models significantly decreased tumor growth. We successfully utilized phenotypic profiling to identify a subset of GLI1 antagonists that were prioritized for testing in in vivo models. Our results indicated that GLI1 activation in TN-IBC as in TNBC, plays a vital role in promoting cell proliferation, motility, tumor growth, and formation of tumor emboli.

Keywords: GANT61; GLI1; Hedgehog; IBC; JK184; SUM149.

Fig. 1

Effect of GLI antagonists on breast cancer cell proliferation and Hh pathway activity. (A) mRNA levels of GLI1/2/3, PTCH1, and SMO in IBC and non-IBC cell lines. Data are expressed as mean ± SD. (B) Testing of GLI antagonists for Hh pathway inhibition in C3H10T1/2 hedgehog functional assay. C3H10T12 cells were stimulated with Shh protein (2 µg/ml) in the presence of GLI antagonists (0.001 – 10 µM) and alkaline phosphatase measured at 5 d. Dose response curves were generated using non-linear regression and IC₅₀ values determined in GraphPad Prism 6. (C) SUM149, MDA-MB-231 and SUM159 cells were incubated with GLI antagonists (0.001 – 25 µM) for 72 h, stained with Hoechst-33342 nuclear stain and cell numbers determined by high content imaging. Upper: Dose response curves for relative nuclei count. For each concentration run in triplicate, percent inhibition values were calculated and data normalized to vehicle. Data is mean ± SD (n = 3). Dose response curves were generated using non-linear regression and IC₅₀ values determined in GraphPad Prism 6. Lower: Representative images. (D) SUM149, MDA-MB-231, SUM159 and SKBR3 cells were incubated with GLI antagonists (0.001 – 25 µM) for 72 h, and proliferation assessed by Presto Blue. For each concentration, percent inhibition values were calculated and data normalized to vehicle. The heat map was generated in Excel using conditional formatting with red representing 100% inhibition through to green for 0% inhibition. (E) qRT-PCR comparative expression of Hh-GLI downstream targets in SUM149, MDA-MB-231 and SUM159 cells treated with vehicle (0.1% DMSO), GANT61 (20 µM) or JK184 (30 nM) for 72 h. Mean ± SD shown, n=4, two-way ANOVA with Tukey’s post-test (*p<0.05, **p<0.005, ***p<0.0005 and ****p<0.0001; significantly different from respective controls).

Fig. 2

Effect of GLI antagonists JK184, HPI-1, HPI-4 and GANT61 on SUM149, MDA-MB-231 and SUM159 colony formation. For colony formation, cells were seeded in 6-well plates, treated with the indicated compounds for 5–14 days, fixed, colonies stained and counted. (A) Representative images for SUM149 and MDA-MB-231 colonies stained with crystal violet. A minimum of two independent experiments were carried out. (B) SUM149 and MDA-MB-231 percent colony formation relative to vehicle. (C) Representative images for SUM159 colonies and percent colony formation relative to vehicle. Data represents mean ± SD percentage of colonies relative to vehicle/untreated for a minimum of three wells. One-way ANOVA with Tukey’s post-test (*p<0.05, **p<0.005, ***p<0.0005 and ****p<0.0001; significantly different from respective controls).

Fig. 3

GANT61 disrupts formation of SUM149 tumor spheroids and tumor emboli. For the tumor spheroid assay, cells were seeded in ultralow attachment 384-well plates and treated 24 h later with either vehicle control (DMSO), staurosporine, or GANT61 at the indicated concentrations. Cells were grown for a further 72 h, spheroids stained with Hoechst/YOYO-1 and high content 3D images obtained using the CellInsight HCS platform. (A) representative images are shown for each treatment. Three independent experiments were carried out. (B) Data represent mean ± SD percent normalized to DMSO vehicle control for spheroid area (Hoechst) and spheroid YOYO-1 comprising a minimum of five replicate wells. Dose response curves were generated using non-linear regression and IC₅₀ values determined in GraphPad Prism 6. (C) Tumor emboli: Representative images (10 × – scale bar = 100 microns) of SUM149 cells grown under tumor emboli conditions. Cells were treated at the time of plating with vehicle, staurosporine or GANT61 and cells grown for 7d. Images from at least two independent experiments comprising a minimum of three replicate wells.

Fig. 4

Effect of GANT61 on formation of SUM159 and MDA-MB-231 tumor spheroids. The tumor spheroid assay was carried out as described in Fig. 3. Representative images (A) and dose response curves (B) are shown for SUM159 treated with staurosporine and GANT61. Representative images (C) and dose response curves (D) are shown for MDA-MB-231 treated with GANT61. A minimum of two independent experiments were carried out. Data represent mean ± SD percent normalized to DMSO vehicle control for spheroid area (Hoechst) and spheroid YOYO-1 comprising a minimum of five replicate wells. Dose response curves were generated using non-linear regression and IC₅₀ values determined in GraphPad Prism 6.

Fig. 5

GANT61 treatment inhibits TN-IBC SUM149-derived xenograft tumor growth. (A) Nude NU/J mic were injected orthotopically with SUM149 cells into the mammary fat pad. Once tumors reached 5 × 5 mm (6 weeks) they were treated with vehicle or GANT61 (50 mg/kg i.p. 3× per week for n = five animals). Tumor response was assessed by weekly caliper measurements. Tumor volumes (Mean ± SEM) are shown for each time point. Statistical significance relative to respective control *P<0.05, **P<0.005, ***P<0.001 (Student t-test). Comparisons in tumor growth were also made by linear regression in GraphPad Prism 6.0. Statistical significance relative to control ^#P<0.05. (B) Quantification of the study shown in (A) for GLI1 protein expression by immunoblot analysis. Mean band intensity normalized to total protein (± SD) for vehicle compared to GANT61 treated tumor lysates (left panel). *P<0.05 (Student t-test). Representative immunoblot for GLI1 protein expression from three independent analyses of tumor cell lysates (B, right panel). (C) NOD SCID mice were injected orthotopically with SUM149 cells into the mammary fat pad. Once tumors were palpable, mice were treated with vehicle or GANT61 in vehicle (50 mg/kg i.p. once per day for n = five animals). After 21 days, tumors were excised and weighed. Weights are shown as Mean ± SEM. *P<0.05, **P<0.005, ***P<0.001 (Student t-test). (D) Quantification from study shown in (C) of GLI1 mRNA in the SUM149-derived tumors (left panel). *P<0.05 (Student t-test). Representative immunoblot from three independent experiments of tumor tissue lysates for GLI1 protein expression (D, right panel). Recombinant human GLI1 protein (GLI1 protein) was included as a positive control for the Western.