Untangling the ATR-CHEK1 network for prognostication, prediction and therapeutic target validation in breast cancer

Abstract

ATR-CHEK1 signalling is critical for genomic stability. ATR-CHEK1 signalling may be deregulated in breast cancer and have prognostic, predictive and therapeutic significance. We investigated ATR, CHEK1 and phosphorylated CHEK1 (Ser345) protein (pCHEK1) levels in 1712 breast cancers. ATR and CHEK1 mRNA expression was evaluated in 1950 breast cancers. Pre-clinically, biological consequences of ATR gene knock down or ATR inhibition by the small molecule inhibitor (VE-821) were investigated in MCF7 and MDA-MB-231 breast cancer cell lines and in non-tumorigenic breast epithelial cells (MCF10A). High ATR and high cytoplasmic pCHEK1 levels were significantly associated with higher tumour stage, higher mitotic index, pleomorphism and lymphovascular invasion. In univariate analyses, high ATR and high cytoplasmic pCHEK1 levels were associated with poor breast cancer specific survival (BCSS). In multivariate analysis, high ATR level remains an independent predictor of adverse outcome. At the mRNA level, high CHEK1 remains associated with aggressive phenotypes including lymph node positivity, high grade, Her-2 overexpression, triple negative, aggressive molecular phenotypes and adverse BCSS. Pre-clinically, CHEK1 phosphorylation at serine(345) following replication stress was impaired in ATR knock down and in VE-821 treated breast cancer cells. Doxycycline inducible knockdown of ATR suppressed growth, which was restored when ATR was re-expressed. Similarly, VE-821 treatment resulted in a dose dependent suppression of cancer cell growth and survival (MCF7 and MDA-MB-231) but was less toxic in non-tumorigenic breast epithelial cells (MCF10A). We provide evidence that ATR and CHEK1 are promising biomarkers and rational drug targets for personalized therapy in breast cancer.

Keywords: ATR; Biomarker; Breast cancer; CHEK1.

Figure 1

ATR and pCHEK1 protein level in breast cancer. A. Microphotograph of ATR and pCHEK1 negative and positive breast cancer tissue. B. Kaplan Meier curves showing breast cancer specific survival (BCSS) and ATR level (B1). Kaplan Meier curves showing breast cancer specific survival (BCSS) and cytoplasmic pCHEK1 level (B2). Kaplan Meier curves showing breast cancer specific survival (BCSS) and combined nuclear/cytoplasmic pCHEK1 level (B3). Kaplan Meier curves showing breast cancer specific survival (BCSS) and combined ATR/pCHEK1 level (B4).

Figure 2

A. Kaplan Meier curves showing breast cancer specific survival (BCSS) and combined p53/ATR level. B. Kaplan Meier curves showing breast cancer specific survival (BCSS) and combined p53/cytoplasmic pCHEK1 level. C. Kaplan Meier curves showing breast cancer specific survival (BCSS) and ATR mRNA expression. D. Kaplan Meier curves showing breast cancer specific survival (BCSS) and CHEK1 mRNA expression.

Figure 3

A. ATR is responsible for CHEK1 phosphorylation at serine345 following replication stress. A1. MCF7 cells were subjected to 10 nM siRNA for 48 h before being treated with 1 μM gemcitabine (Gem) or 10 mM hydroxyurea (HU) for 1 h. Cells were harvested, lysed and the proteins separated using gel electrophoresis. ATR, pCHEK1Ser345 and β‐actin were detected using western blotting. Bands were quantified using densitometry (A2 and A3). Figure 3A3 shows data from a single representative experiment where 10 mM hydroxyurea had been used as this is the most frequently used inducer of ATR activity. The data for gemcitabine was a pooled analysis of three independent experiments. * = significant. See text for details. B. VE‐821 inhibits gemcitabine‐induced ATR activity as measured by pCHEK1Ser345. MCF7, MDA‐MB‐231 or MCF10A cells were treated with 1 μM gemcitabine ± VE‐821 for 1 h before being harvested and lysed. Proteins were separated and detected using western blotting. Blot shown is in MDA‐MB‐231 cells and is representative of all experiments (B1). Concentration‐response curve (B2) data shown is the mean ± standard deviation of three individual experiments in each cell line. IC50 values from the 3 independent experiments are shown in B3.

Figure 4

ATR is required for cell growth in MCF7 cells. Cells with doxycycline (Dox) – inducible shATR were incubated with or without Dox for 3 days. Then Dox was removed and cells were cultured for further indicated days. Cell growth was analysed using DAPI fluorescence (A1). ATR level was monitored by western blotting (A2). Knockdown of ATR following Dox induction suppressed growth, which was restored when ATR was re‐expressed. B. ATR inhibitor VE‐821 reduces breast cancer cell growth. MCF7 or MDA‐MB‐231 were treated for 24 h with a dose range of VE‐821. Cells were then allowed to grow for 5 days in fresh media. Cell growth was measured by DAPI fluorescence. C. There was a direct correlation between growth inhibition and ATR inhibition in MCF7 and MDA‐MB‐231 cells. D. VE‐821 is selective against breast cancer cells compared to non‐cancer cells. MCF7, MDA‐MB‐231 and MCF10A cells were seeded into 6‐well tissue culture plates and allowed to adhere for 24 h. Cells were treated with VE‐821 for 24 h before being counted and re‐seeded for colony formation. Cells were then allowed to grow for 14 days. Colonies were then fixed, stained and counted.