RECQL4 helicase has oncogenic potential in sporadic breast cancers

Abstract

RECQL4 helicase is a molecular motor that unwinds DNA, a process essential during DNA replication and DNA repair. Germ-line mutations in RECQL4 cause type II Rothmund-Thomson syndrome (RTS), characterized by a premature ageing phenotype and cancer predisposition. RECQL4 is widely considered to be a tumour suppressor, although its role in human breast cancer is largely unknown. As the RECQL4 gene is localized to chromosome 8q24, a site frequently amplified in sporadic breast cancers, we hypothesized that it may play an oncogenic role in breast tumourigenesis. To address this, we analysed large cohorts for gene copy number changes (n = 1977), mRNA expression (n = 1977) and protein level (n = 1902). Breast cancer incidence was also explored in 58 patients with type II RTS. DNA replication dynamics and chemosensitivity was evaluated in RECQL4-depleted breast cancer cells in vitro. Amplification or gain in gene copy number (30.6%), high-level mRNA expression (51%) and high levels of protein (23%) significantly associated with aggressive tumour behaviour, including lymph node positivity, larger tumour size, HER2 overexpression, ER-negativity, triple-negative phenotypes and poor survival. RECQL4 depletion impaired the DNA replication rate and increased chemosensitivity in cultured breast cancer cells. Thus, although recognized as a 'safe guardian of the genome', our data provide compelling evidence that RECQL4 is tumour promoting in established breast cancers. Copyright © 2015 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Keywords: RECQL4 helicase; breast cancer; oncogene; tumour suppressor.

Figure 1

RECQL4 copy number, mRNA and protein levels in breast cancer. A, RECQL4 gene copy number changes in PAM50. Molecular phenotypes. B, Kaplan Meier curves showing BCSS (breast cancer specific survival) in the whole cohort. C, RECQL4 mRNA levels in PAM50. Molecular phenotypes. D, Kaplan Meier curves showing BCSS (breast cancer specific survival) in the whole cohort.

Figure 2

A, Correlation between RECQL4 gene copy number and mRNA levels in the whole cohort. B, Kaplan Meier curves showing BCSS based on RECQL4 protein levels in the whole cohort. C, Effects of RECQL4 knockdown using siRNA on DNA synthesis assessed by DNA fibre assay (see Supplementary Methods for details). D, In MCF7 and MDA-MB-231 cells DNA fibre lengths were reduced by around 50% after RECQL4 knockdown. Compared with the other two lines, BT549 cells showed shorter fibre tracks to begin with, but like the other cells, RECQL4 depletion further reduced the rate of synthesis of DNA

Figure 3

RECQL4 depletion and chemosensitivity to chemotherapeutic drugs in MDA-MB453 cells. RECQL4 knockdown by adenovirus-mediated shRNA (A) and treatment with cisplatin (B), doxorubicin (C) or 5-FU (D). Cell survival was measured by the 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT) assay following the manufacturer’s instructions (Molecular Probes). Absorbance values at 540 nm were read on a Spectra Max 250 spectrophotometer (Molecular Devices). All MTT assays included 10 duplicated wells for each time-point of each cell line. The data is presented as mean ± SD from three independent experiments. *, P<0.05; **, P<0.01.