Novel role of BRCA1 interacting C‐terminal helicase 1 (BRIP1) in breast tumour cell invasion

Abstract Breast cancer (BC) is the most common malignancy and the leading cause of death in women worldwide. Only 5%‐10% of mutations in BRCA genes are associated with familial breast tumours in Eastern countries, suggesting the contribution of other genes. Using a microarray gene expression profiling study of BC, we have recently identified BRIP1 (fivefold up‐regulation) as a potential gene associated with BC progression in the Omani population. Although BRIP1 regulates DNA repair and cell proliferation, the precise role of BRIP1 in BC cell invasion/metastasis has not been explored yet; this prompted us to test the hypothesis that BRIP1 promotes BC cell proliferation and invasion. Using a combination of cellular and molecular approaches, our results revealed differential overexpression of BRIP1 in different BC cell lines. Functional assays validated further the physiological relevance of BRIP1 in tumour malignancy, and siRNA‐mediated BRIP1 knockdown significantly reduced BC cell motility by targeting key motility‐associated genes. Moreover, down‐regulation of BRIP1 expression significantly attenuated cell proliferation via cell cycle arrest. Our study is the first to show the novel function of BRIP1 in promoting BC cell invasion by regulating expression of various downstream target genes. Furthermore, these findings provide us with a unique opportunity to identify BRIP1‐induced pro‐invasive genes that could serve as biomarkers and/or targets to guide the design of appropriate BC targeted therapies.

Qatar encompasses numerous risk factors such as late menopause, prolonged hormone replacement therapy, older age at first live childbirth, family history of BC at a young age and the mutations of the breast cancer associated gene 1 and 2 (BRCA1/2) genes. 4,5 New prognostic biomarkers need to be developed better-targeted therapies against invasive stages of BC to enhance the chance for long-term survival and patient's quality of life. To achieve this goal, it is imperative to understand the exact signalling pathways associated with the multistage process of metastasis. 6 Invasion, the hallmark of malignancy, is the recurring and defining event in the metastatic process, and elucidation of its mechanisms is critical for developing effective anti-metastatic therapies.
In GCC countries, where the rate of consanguinity is quite high (~50%), a significant number of BC patients are younger and show advanced tumours. In addition, a previous study conducted among Omani females with BC, showed no significant pathogenic BRCA1 gene missense mutations, suggesting the involvement of other genes in BC development. Based on the observations described above, we have previously used microarray analysis to compare RNA samples isolated from 40 malignant breast tumour tissues and 40 normal/ benign breast tissues, and identified BRCA1-interacting C-terminal helicase 1 (BRIP1), showing fivefold induction, as a potential gene that might promote BC progression.. 7 BRIP1, also known as Fanconi Anaemia Group J Protein (FANCJ) or BRCA1-associated C-terminal helicase (BACH1), was first identified using tandem mass spectrometry by its physical interaction with BRCA1 and also belongs to the Fanconi anaemia (FA) genes family. 8 BRIP1 is located on chromosome 17q22, spanning a region greater than 180 kb, starting from 61 679 185 to 61 863 558 base pair with 20 exons and 19 introns. 8 Interestingly, BRCA1 is also located on chromosome 17q21 region, hence in close proximity with BRIP1. BRIP1 plays major role in DNA repair, type J Fanconi anemia, development of various cancers, including BC. 8 BRIP1, a DNAdependent ATPase and a 5'-3' helicase that belongs to the DNAdependent RecQ DEAH helicase family, interacts with BRCA1 and is involved in double-stranded DNA breaks (DSB) repair during the G2-M phase of the cell cycle as well as in tumour suppression. 9,10 BRIP1 is expressed in both normal and malignant cells, and controls genome integrity via regulation of replication and homologous recombination, 11 DNA damage responses and checkpoints, which are crucial for genomic stability. 9 While BRCA1 and BRIP1 work as tumour suppressor genes, 10 when BRIP1 fails to bind BRCA1 in certain conditions, cells become sensitive to different genotoxic stresses with aberrant homologous DNA repair function. 12 Identification of BRIP1 germline mutations in BC patient with wild-type BRCA1 and BRCA2 suggests a major link between moderate penetrance of BC and BRIP1 mutation. [13][14][15][16] Based on these observations, we hypothesized that beyond its function as a DNA repair gene, BRIP1 plays a novel role in tumour cell invasion/metastasis. In addition, to providing a better understanding of the mechanisms that underpin BRIP1-mediated tumour cell invasion, this study has validated BRIP1 as a target gene that can be used to design efficient therapeutic strategies against BC. Human BC cell lines HCC-2218, T47D, BT474, MCF-7, CAMA-1 and   the immortalized human breast cell line MCF 10A were obtained   from the American Type Culture Collection (ATCC)

| Western blotting
RIPA buffer supplemented with protease inhibitors (Pierce) was used to prepare whole cell lysates. Protein quantification was determined using Pierce™ BCA Protein Assay Kit (Thermo Scientific™) according to the manufacturer's detailed protocol. Approximately 20 µg of each denatured cell lysates was loaded on a 7.5% SDS-PAGE.

| Real-time qRT-PCR
According to the manufacturer's protocol, 1 μg of total RNA (ex-

| siRNA transfection
Breast cancer cells were transfected with smart pool BRIP1 siRNA

| In vitro cell invasion and migration assays
Cell migration and invasion assays were performed as we have previously described. 17,18 Briefly, for migration assay, 3 × inverted microscope). Fields were quantified and analysed using ImageJ software (NIH Image Software).

| Wound healing assay
Briefly, MCF-7, CAMA-1 and MDA-MB-231 cells were transfected with BRIP1 siRNA or non-targeted siRNA (si-Ctrl). Then, the cells were counted and seeded in a 24-well plate with serum-free media.
A straight scratch was made in each well using a sterile 10 μL white tip. Cells were washed gently with sterile PBS to remove debris and then cultured in their corresponding complete growth media. The plates were then incubated and photographs were taken at 0, 24 and 48 hours, depending on the cell line used in reference to a marker line for accurate imaging. Results were analysed using the ImageJ software (NIH Image Software).

| Cell cycle
Cell cycle analysis was carried out using fluorescence-activated cell sorting (FACS) method. In Both siRNA-BRIP1 and si-Ctrl-transfected, cells were trypsinized using TrypLE reagent (Gibco), fixed and incubated overnight at 4°C. Next day, the cells were centrifuged, washed and then incubated with 40 μg/mL propidium iodide (PI) and 100 μg/ mL RNase (Sigma) in the dark at room temperature for 30 minutes.

| Metastasis-associated genes profiling
Predesigned tumour Metastasis Fast 96-well plates (4414098, Life Technologies) containing lyophilized TaqMan ® Gene Expression assay was used to determine the expression levels of pro-metastatic genes in BRIP1-siRNA transfected cells compared with si-Ctrl. Briefly, total RNA was converted into cDNA as described above. The metastasis-associated genes profiling reaction was performed using QuantStudio™ 6 Flex Real-Time PCR System (Applied Biosystems, Inc), according to the manufacturer instructions. The results were first normalized to GAPDH and further analysed to obtain the Relative mRNA expression levels, using the formula 2 − ΔΔCt . All the reactions were carried out in triplicates and repeated twice.

| Statistical analysis
In proteomics analysis, Student's two-tailed test was used to assess the statistical significance of the protein quantification. Statistical significance between groups was assessed using ANOVA tests and Student's t test. For other experiments, a non-parametric Student's t test was applied using Microsoft Excel 2013 and GraphPad Prism 8. Data of three independent experiments were presented as means ± SD unless otherwise indicated in the legends. P values ≤.05 were considered statistically significant.

| BRIP1 is highly expressed in different breast cancer cell lines
Both BRIP1 protein and mRNA levels were differentially overexpressed in various BC cell lines compared with control HuMEC and MCF 10A cells (Figure 1). At the protein level, Western blotting analysis followed by densitometric quantification showed relative overexpression of BRIP1 compared with control ( Figure 1B). Similarly, BRIP1 mRNA levels showed 1.8 to 14-fold increase in the tested BC cell lines compared with normal/control cells ( Figure 1C).

| BRIP1 facilitates proliferation in breast cancer cell lines
To examine the effect of BRIP1 inhibition on BC cell proliferation, cells were treated with either BRIP1-siRNA or si-Ctrl, and cellular proliferation was assessed at 24, 48, and 72 hours post-treatment using Alamar blue assay. As shown in Figure 3

| BRIP1 promotes migration and invasion ability of breast cancer cell lines
Wound healing assay results revealed that BRIP1 suppression significantly attenuated the ability of cells to close the gap. Compared with control cells, a relative wound closure of 60% in MCF-7 and MDA-MB-231, and 50% in CAMA-1 was observed ( Figure 5).

| BRIP1 promotes cell invasion by modulating expression of an array of related genes
In order to identify potential molecular signalling pathway(s) underpinning the novel role of BRIP1 in BC progression, TaqMan RT-qPCR of 92 different metastasis-associated genes was performed to determine differentially expressed genes in BRIP1-suppressed MCF-7 and MDA-MB-231 cells compared with controls. Analysis of our results showed a significant up-regulation/down-regulation of a group of genes known to regulate both cell growth and cell motility. Among several differentially expressed genes, the present study focused on genes associated with extracellular matrix, adhesion molecules, cell proliferation and motility (C-X-C motif chemokine 12 [CXCL12],

| BRIP1 overexpression in breast cancer
The mutational spectrum of BRIP1 was recently investigated in various BC cell lines using the Estimate algorithm of genome-wide copy number analysis and hybrid capture sequencing of 1651 genes 20 showed no BRIP1 mutations in our selected model for functional characterization. Up-regulated BRIP1 levels in malignant breast tumours contradict its role as a tumour suppressor. As an example, the tumour protein 53 (TP53) tumour suppressor gene is overexpressed in colorectal cancer, which is not predictive with its mutational status as an early event, 21 suggesting that TP53 has an oncogenic role independent of the tumour suppression activity. This dual behaviour was also reported for other genes, such as Wilms' tumour 1 (WT1); mutated WT1 led to the onset of kidney tumours, and its overexpression was detected in a subset of human cancers. 22 Although gene amplification plays a major role in tumorigenesis, especially for solid tumours (reviewed in, 23 oncogenes, 27 BC poor prognosis and tumour progression. 27,28 Gain of function resulting from amplification of this region has been reported in other cancers, including liver, 29 pancreas, 30 bladder, 31 testis, 32 ovary, 33 lung 34 and brain tumours. 35 Interestingly, the 17q23 amplicon where BRIP1 gene lies, encompasses known oncogenes RPS6KB1, TBX2 and PPM1D. 35 In particular, TCGA showed a 3.2-fold up-regulation of BRIP1 in breast tumours compared with normal breast tissues. 36 Similarly, our microarray data revealed that BRIP1 had an average of fivefold overexpression as compared to normal breast tissue samples. 7 Consistent with these observations, our results showed clearly that both the protein and mRNA levels of BRIP1 were overexpressed in various BC cell lines compared with control normal/immortalized and normal/control breast cells, suggesting that BRIP1 might act as an oncogenic driver in BC. Interestingly, previous study revealed the presence of BRIP1 germline mutations in BC patients without any BRCA mutation, suggesting a link between BRIP1 mutations and BC susceptibility. 19 Collectively, although BRIP1 is considered as a tumour suppressor gene, it is amplified in sporadic cancers, 36 thus supporting our previous 7 and current findings, suggesting that BRIP1 amplification in sporadic cancers could explain the oncogenic role of BRIP1.

| BRIP1 promotes breast cancer cell proliferation
In order to understand the mechanisms that underpin BRIP1promoted BC cell proliferation, our results revealed that siRNA In G1/S, Ras-activation drives Myc accumulation and regulates proliferation-related genes through E2F transcriptional activity. 37,38 Normally, c-Myc is expressed during active cell division phase only, regulates the transition of cells from G1 to S phase and is associated with poor prognosis 39 (Reviewed in 40 ). RNAi-inhibited c-Myc reduced MCF-7 BC cells by 30% as well as tumour development in nude mice. 41 Although our results showed that inhibition of BRIP1 reduced RB expression levels, ongoing experiments aim to evaluate the expression levels of phosphorylated Rb, expected to decrease with increased BRIP1 expression to arrest cell cycle. However, during BC metastasis, Rb is expected to increase to promote cell growth and invasion. Ras GTPase pathway appears to be required in all decisions during both G1 and G2 phases. 42 Ongoing pharmacological and functional approaches aim to further validate whether these genes underpin BRIP1-promoted cell growth.

| BRIP1 promotes breast tumour cell invasion
On the other hand, and to our knowledge, no study has linked BRIP1 to cancer metastasis yet. Our siRNA experiments suggest that BRIP1 up-regulation may promote breast tumour metastasis. In fact, previous studies revealed that overexpression of RhoA GTPase was associated with carcinogenesis. 43 In the present study, RNAi-inhibited TA B L E 1 BRIP1 promotes cell growth and metastasis by modulating the expression of an array of related genes In conclusion, BRCAness status and family history support the idea of the association of other moderate/low penetrance genes in the onset of BC, such as BRIP1, which binds to BRCA1 to maintain genome integrity. 3 The present study revealed a novel role of BRIP1 in promoting BC cell growth and invasion. In addition, a number of BRIP1-transcriptional target genes associated with cell growth and cell invasion were identified. While these findings support our hypothesis that BRIP1 promotes BC progression, ongoing structural and functional validation experiments in our laboratory aim to shed light on the exact mechanisms by which BRIP1-downstream signalling promotes BC cell growth and cell invasion.

ACK N OWLED G M ENTS
We

DATA AVA I L A B I L I T Y S TAT E M E N T
All the data needed to evaluate the conclusion section are provided in the paper and/or the Supplementary Materials. Additional data related to this paper could be made available upon request from the corresponding author.