The BET inhibitor JQ1 selectively impairs tumour response to hypoxia and downregulates CA9 and angiogenesis in triple negative breast cancer

Abstract

The availability of bromodomain and extra-terminal inhibitors (BETi) has enabled translational epigenetic studies in cancer. BET proteins regulate transcription by selectively recognizing acetylated lysine residues on chromatin. BETi compete with this process leading to both downregulation and upregulation of gene expression. Hypoxia enables progression of triple negative breast cancer (TNBC), the most aggressive form of breast cancer, partly by driving metabolic adaptation, angiogenesis and metastasis through upregulation of hypoxia-regulated genes (for example, carbonic anhydrase 9 (CA9) and vascular endothelial growth factor A (VEGF-A). Responses to hypoxia can be mediated epigenetically, thus we investigated whether BETi JQ1 could impair the TNBC response induced by hypoxia and exert anti-tumour effects. JQ1 significantly modulated 44% of hypoxia-induced genes, of which two-thirds were downregulated including CA9 and VEGF-A. JQ1 prevented HIF binding to the hypoxia response element in CA9 promoter, but did not alter HIF expression or activity, suggesting some HIF targets are BET-dependent. JQ1 reduced TNBC growth in vitro and in vivo and inhibited xenograft vascularization. These findings identify that BETi dually targets angiogenesis and the hypoxic response, an effective combination at reducing tumour growth in preclinical studies.

Figure 1

JQ1 downregulates the expression of several hypoxia-regulated genes, especially CA9. (a) List of DEG under hypoxia in MDA-MB-231 cells obtained from microarray. Columns at the left denote DEG under JQ1 treatment, either in normoxia (red blocks) or hypoxia (blue dots). CA9 is the most prominently downregulated gene in hypoxia (red arrow). (b) Hypoxia Network (HyN) created including pathways regulated by hypoxia. Grey dots are linking genes, included to the network remains stable. (c) Most components of HyN are upregulated by hypoxia and downregulated by JQ1 treatment in MDA-MB-231 cells. (d) Kaplan–Meier curves demonstrating the prognostic value of two genes consistently inhibited by JQ1 in both cell lines tested (MDA-MB-231 and MCF-7) for triple negative breast cancer patients. OXPHOS, oxidative phosphorylation; TCA, tricarboxylic acid cycle; PYR, pyruvate metabolism; PPP, pentose phosphate pathway.

Figure 2

JQ1 reduces TNBC monolayer and spheroid growth, regardless of MYC. (a) Cell cultures were grown in 96-well plates for 72 h in each condition. (+)-JQ1 dose-dependently reduces monolayer (two-dimensional) cell growth of TNBC cell lines MDA-MB-231 and HCC1806 in normoxia and hypoxia, while (−)-JQ1 does not. (b) JQ1 reduced c-Myc immunocontent only in the MYC-amplified cell line HCC1806, while the non-mutated cell line MDA-MB-231 showed no difference in c-Myc. (c) Representative spheroid growth curve and pictures of MDA-MB-231 spheroids following JQ1 treatment. (d) (+)-JQ1 reduces spheroid growth in MDA-MB-231 and other TNBC cell lines, while (−)-JQ1 does not. One-way analysis of variance, n=3, *P<0.05, **P<0.01, ***P<0.001.

Figure 3

JQ1 reduces CA9 and Ki67 stain in TNBC spheroids. CA9 expression is visible in untreated (UT) spheroids, but undetectable in JQ1-treated spheroids. The proliferative marker Ki67 was also reduced in spheroids treated with JQ1. For immunohistochemistry, spheroids were fixed with formalin 10%, embedded in agarose, processed, embedded in wax and cut with a microtome. Student t-test, n=3, **P<0.01, ***P<0.001.

Figure 4

JQ1 reduces CA9 expression in TNBC cell lines. (a) Hypoxia upregulates several genes and JQ1 downregulates a group of them, CA9 being the most prominent across the three cell lines. HIF expression is not altered by JQ1. (b) CA9 is consistently downregulated by JQ1 in hypoxia, without any effect on HIF. Cells were treated with JQ1 for 24 h prior to RNA or protein extraction, and then gene expression was assessed by RT–qPCR and protein immunocontent was assessed by western blot. Two-way analysis of variance, n=3, *P<0.05, **P<0.01, ***P<0.001.

Figure 5

JQ1 prevented HIF binding to the CA9 promoter and BET protein knockdowns phenocopy JQ1 treatment. (a) ChIP assay for CA9 promoter region with HIF-1β immunoprecipitation in two TNBC cell lines. Two-way analysis of variance, n=3. (b) Expression of CA9, VEGF-A and HIF-1α in hypoxia following siRNA knockdown of BET proteins. One-way analysis of variance, n=3. (c) ChIP assay for CA9 promoter region with BRD4, H3K27 acetylation or H4 acetylation immunoprecipitation in HCC1806. T-test, n=3. (d) ChIP assay for VEGF-A promoter region with BRD4, H3K27 acetylation or H4 acetylation immunoprecipitation in MDA-MB-231. T-test, n=3 (H3K27 acetylation) n=5 (BRD4 and H4 acetylation), *P<0.05; **P<0.01.

Figure 6

JQ1 reduces tumour growth, CA9, VEGF-A and additional angiogenesis-related gene expression and blood vessel count in xenograft model of TNBC. (a) Growth curve and rate in TNBC xenografts treated with JQ1 or untreated (UT). Linear regression followed by Student t-test, n=5, *P<0.05. (b) Gene expression in TNBC xenografts treated with JQ1, assessed by qRT–PCR analysis of RNA extracted from tumours. Student t-test compared with UT, n=3, *P<0.05, **P<0.01. (c) Representative CD31 immunostaining in TNBC xenografts. Non-parametric Mann–Whitney test, n=3 shCTL and n=5 JQ1-treated. Xenografts were grown using HCC1806 cells in 6–7-week-old female CD1 nude mice.