Chk1 phosphorylated at serine345 is a predictor of early local recurrence and radio-resistance in breast cancer

Abstract

Radiation-induced DNA damage activates the DNA damage response (DDR). DDR up-regulation may predict radio-resistance and increase the risk of early local recurrence despite radiotherapy in early stage breast cancers. In 1755 early stage breast cancers, DDR signalling [ATM, ATR, total Ckh1, Chk1 phosphorylated at serine(345) (pChk1), Chk2, p53], base excision repair [PARP1, POLβ, XRCC1, FEN1, SMUG1], non-homologous end joining (Ku70/Ku80, DNA-PKcs) and homologous recombination [RAD51, BRCA1, γH2AX, BLM, WRN, RECQL5, PTEN] protein expression was correlated to time to early local recurrence. Pre-clinically, radio-sensitization by inhibition of Chk1 activation by ATR inhibitor (VE-821) and inhibition of Chk1 (V158411) were investigated in MDA-MB-231 (p53 mutant) and MCF-7 (p53 wild-type) breast cancer cells. In the whole cohort, 208/1755 patients (11.9%) developed local recurrence of which 126 (61%) developed local recurrence within 5 years of initiation of primary therapy. Of the 20 markers tested, only pChk1 and p53 significantly associated with early local recurrence (p value = 0.015 and 0.010, respectively). When analysed together, high cytoplasmic pChk1-nuclear pChk1 (p = 0.039), high cytoplasmic pChk1-p53 (p = 0.004) and high nuclear pChk1-p53 (p = 0.029) co-expression remain significantly linked to early local recurrence. In multivariate analysis, cytoplasmic pChk1 level independently predicted early local recurrence (p = 0.025). In patients who received adjuvant local radiotherapy (n = 949), p53 (p = 0.014) and high cytoplasmic pChk1-p53 (p = 0.017) remain associated with early local recurrence. Pre-clinically, radio-sensitisation by VE-821 or V158411 was observed in both MCF-7 and MDA-MB-231 cells and was more pronounced in MCF-7 cells. We conclude that pChk1 is a predictive biomarker of radiotherapy resistance and early local recurrence.

Keywords: ATR inhibitor; Breast cancer; Chk1; Chk1 inhibitor; Local recurrence; Radiotherapy; Resistance; p53.

Figure 1

A. Microphotograph of pCHK1 and p53 protein expression in breast tumours [1 = negative, 2 = pCHK1 cytoplasmic staining, 3 = pCHK1 cytoplasmic & nuclear staining and 4 = p53 staining]. Kaplan Meier curves showing time taken to local recurrence based on B. pChk1 cytoplasmic expression; C. pChk1 nuclear expression D. pChk1 cytoplasmic & pChk1 nuclear co‐expression; D. p53 expression.

Figure 2

Kaplan Meier curves showing time taken to local recurrence based on A. p53 & pChk1 cytoplasmic co‐expression; B. p53 & pChk1 nuclear co‐expression.

Figure 3

Blockade of CHK1 phosphorylation by VE‐821 (ATR inhibitor) and radio‐potentiation. A. Effect of VE‐821 on breast cancer cell growth. For clonogenic assays, data are mean ± standard deviation from three separate experiments in MCF7 (B) and MDA‐MB‐231 (C). D. LD50 values (dose of IR where 50% of cells no longer survive) were calculated from each experiment and the potentiation factor at 50% cell kill (PF50) was calculated (see Supplementary Table S3 for further details). E. Exponentially growing MCF7 or MDA‐MB‐231 were seeded into 10 cm tissue culture dishes and allowed to adhere for 24 h. Where indicated, cells were pre‐treated with 1 μM VE‐821 for 1 h before IR (2 Grays) or mock treatment. After 24 h, media from cells was collected, the cells washed and harvested and fixed and frozen in ice cold methanol overnight. Cells were stained with 200 μg/ml PI and 200 μg/ml RNAase A in PBS and samples run using a FACScalibur counting a minimum of 20000 events. Single cell populations were then gated into phases of the cell cycle by DNA content. The resulting FACS profiles analysed using Cyflogic software (mean ± S.D. of three individual experiments) is show here.

Figure 4

Blockade of CHK1 activation by V158411 and radio‐potentiation. A. Representative western blot in MCF7 breast cancer cell line. Expression of Chk1 and actin in untreated controls treated for 48 h, 5 days and 7 days, cells treated with scrambled siRNA for 48 h, 5 days and 7 days, and cells treated with Chk1 siRNA for 48 h, 5 days. Note no 7 day sample with Chk1 siRNA as insufficient cells alive to extract protein for western blot analysis. B. Quantification CHK1 KD by siRNA in comparison to cell line samples treated with control scrambled siRNA. There was 80% KD after 48 h and more than 98% KD after 5 days. C. Clonogenic assays. In parallel with the samples prepared for western blotting a clonogenic assay was performed in control cells, those treated with scrambled siRNA and with CHK1 siRNA for 48 h. This shows that there was no clonogenic survival after 2 weeks in cells treated for 48 h with CHK1 siRNA. D. Dose dependent radiosensitisation of V158411in MCF‐7 cells. E. Dose dependent radiosensitisation of V158411in MDA‐MB‐231 cells. For all clonogenic assays, data are mean ± standard deviation from three separate experiments in breast cancer cells. F. FACS profiles in MDA‐MB‐231 cells treated with VE158411 either alone or in combination with IR.