BCAS3 exhibits oncogenic properties by promoting CRL4A‐mediated ubiquitination of p53 in breast cancer

Abstract Objectives Breast cancer‐amplified sequence 3 (BCAS3) was initially found to be amplified in human breast cancer (BRCA); however, there has been little consensus on the functions of BCAS3 in breast tumours. Materials and methods We analysed BCAS3 expression in BRCA using bio‐information tools. Affinity purification and mass spectrometry were employed to identify BCAS3‐associated proteins. GST pull‐down and ubiquitination assays were performed to analyse the interaction mechanism between BCAS3/p53 and CUL4A‐RING E3 ubiquitin ligase (CRL4A) complex. BCAS3 was knocked down individually or in combination with p53 in MCF‐7 cells to further explore the biological functions of the BCAS3/p53 axis. The clinical values of BCAS3 for BRCA progression were evaluated via semiquantitative immunohistochemistry (IHC) analysis and Cox regression. Results We reported that the expression level of BCAS3 in BRCA was higher than that in adjacent normal tissues. High BCAS3 expression promoted growth, inhibited apoptosis and conferred chemoresistance in breast cancer cells. Mechanistically, BCAS3 overexpression fostered BRCA cell growth by interacting with the CRL4A complex and promoting ubiquitination and proteasomal degradation of p53. Furthermore, BCAS3 could regulate cell growth, apoptosis and chemoresistance through a p53‐mediated mechanism. Clinically, BCAS3 overexpression was significantly correlated with a malignant phenotype. Moreover, higher expression of BCAS3 correlates with shorter overall survival (OS) in BRCA. Conclusions The functional characterization of BCAS3 offers new insights into the oncogenic properties and chemotherapy resistance in breast cancer.


| INTRODUC TI ON
Breast cancer (BRCA) is the most common malignant tumour in women, mainly divided into four subtypes: ER-positive, PR-positive, HER2-positive and triple-negative breast cancer (TNBC). 1 Among breast cancers, approximately 70% are ER-positive breast cancer. 2 Clinical data have shown that tamoxifen could decrease the distant recurrence rate and contribute to improving clinical outcome. 3,4 However, breast cancer is a clinically heterogeneous cancer; it is critical to explore multiple molecular features.
BCAS3 (Breast cancer-amplified sequence 3), 98% identical to murine Rudhira, was originally identified to be amplified and overexpressed in breast cancer. 5,6 The gene is located on human chromosome 17q23, a region that carries multiple oncogenes and remains amplified in approximately 20% of primary breast carcinomas. 7 Rudhira encodes a predicted WD40 protein and is expressed in angiogenic precursors and embryonic stem (ES) cells. Similarly, previous studies implicated that BCAS3 was also distributed in human ES cells and angiogenesis. These results imply that BCAS3 is involved in the angiogenesis of tumours. In addition, evidence has shown a positive correlation between overexpression of BCAS3 and tumour progression. 8 Tumour suppressor gene p53 can effectively prevent breast cancer development. Once inactivated, oncogenic activities occur, which can exacerbate metastasis and drug resistance of cancer cells, a hallmark of cancer progression. This is consistent with several previous studies based on mouse experiments and cell-based assays, indicating that loss of p53 function increases the susceptibility of cells to tumour initiation. [9][10][11] It is widely known that p53 stability is regulated by post-translational modifications, including ubiquitination.
The ubiquitin system exerts its major function by targeting substrates for degradation and consequently maintaining cellular homeostasis. 12 The basic functions of ubiquitination are mainly dependent on the ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzyme (E2) and ubiquitin ligase (E3). MDM2 is a crucial E3 ubiquitin ligase that targets p53 for degradation; however, it can be regulated by p53. [13][14][15] In addition to MDM2, Cullin4A-RING E3 ubiquitin ligase (CRL4A) could interact with and target p53 for degradation. 16 At the N terminus of CUL4A, DCAF (DDB1-CUL4A-associated factors) are formed by binding DDB1 to recruit substrates. 17 The C terminus attaches ROC1/RBX1 to recruit E2 enzymes, which serve as catalytic centers. 16 Among additional RING-type E3 ligases, PIRH2, 18 CUL1/ SKP2, 19 COP1, 20 CARP1/2 21 and CUL5 22 all ubiquitinate p53 for proteasomal degradation. In addition, various E3 ligases, including HECT-type (ARFBP and Msl2/WWP1) and U box-type (CHIP and UBE4B), are also able to elicit p53 degradation. [23][24][25][26] In this study, we show that the WD40 repeat protein, BCAS3, is a new interaction partner of CRL4A. As a substrate-specific adaptor, BCAS3 directly interacts with DDB1 to promote p53 polyubiquitination in a CRL4A-dependent pathway. Functionally, BCAS3 regulates cell proliferation, apoptosis and chemoresistance through the degradation of p53 protein. Overall, these results reveal that BCAS3, an important substrate-specific adaptor for CRL4A to regulate the p53 stability, acts as a novel prognostic marker and guides chemotherapy regimens for patients with breast cancer. The culture medium used for these cells is composed of 89% DMEM, 10% fetal bovine serum and 1% antibiotics.

| Cell proliferation and cytotoxicity assay
Cell proliferation and cytotoxicity assay were examined using the Cell Counting Kit-8 assay (APExBio, USA). Briefly, 1 × 10 3 cells were seeded in each well of 96-well plates and incubated for 24 hours.
For cytotoxicity assay, cells were incubated for 24 hours after drug treatment, 10 μL CCK-8 solution was added to each well and incubated for 4 hours, and then, the OD 450 value was determined.

| Colony formation assays
A total of 1 × 10 3 cells were seeded in each well of 6-well plates.
The cells were placed in an incubator and incubated at 37°C for 14 days. After incubation, the medium was removed and the cells were washed twice with cold PBS. Cells were fixed for 15 minutes with methanol and then stained with 0.5% crystal violet solution for 20 minutes. The relative clone formation rate was analysed as follows: (the number of clones/the number of seeded cells) x 100%.

| Apoptosis assay
Apoptosis assays were examined using Annexin V-FITC/PI-PE apoptosis kit (Roche, Switzerland). A total of 5 × 10 5 cells were seeded in each well of 6-well plates and incubated for 24 hours at 37°C and doxorubicin (1 µmol/L) was added to each well for 24 hours. The cells were trypsinized, centrifuged and made into cell suspensions with 400 μL binding buffer. After staining and incubation for 15 minutes, the cell suspensions were analysed using a FACSCalibur flow cytometer (BD Biosciences, USA) in 1 hour.

| Immunopurification-mass spectrometry
HEK 293T cells were transfected with FLAG-BCAS3 plasmid for 48 hours and lysed for cellular extracts. The anti-FLAG M2 affinity gel (Sigma-Aldrich, USA) was added to the cellular extracts for binding. The gel was washed using cold lysis buffer, and then, FLAG peptide (Sigma-Aldrich, USA) at a concentration of 0.2 mg/mL was applied to the gel to elute the FLAG-tagged protein complex.
Samples were collected and run at 100 V on Nu-PAGE 4%-12% Bis-Tris gel (Invitrogen, USA). Gels were silver-stained using Pierce Silver Stain Kit (Thermo Scientific, USA) according to the manufacturer's instructions. The gels with target bands were cut and subjected to LC-MS/MS sequencing.

| Immunoprecipitation and Western blot
Cells were collected after washing with cold PBS and lysed with cold lysis buffer for 30 minutes at 4°C. The cell lysates were incubated with specific antibodies or normal IgG overnight at 4°C. Afterwards, protein A/G Sepharose beads were added and incubated for 2 hours at 4°C. Beads were washed three times with lysis buffer, followed by denatured in 5 x loading buffer at 95°C for 5 minutes. The immunocomplexes were subjected to SDS-PAGE and immunoblotting. Finally, enhanced chemiluminescence (ECL System, Thermo Scientific, USA) was used for immunodetection. The primary antibodies used are listed in Table S2.

| Glutathione S-transferase (GST) pull-down experiments
Plasmid expressing GST fusion proteins or GST control were transformed into E coli BL21, followed by supplemented with 1 mmol/L IPTG to induce protein expression. Bacterial bodies were ultrasonicated at 40% power to harvest the lysate. Glutathione-Sepharose 4B beads were added to the supernatant, and the mixtures were incubated for 30 minutes at 4°C. The in vitro-translated proteins were prepared using the TNT transcription/translation system (Promega, USA). The GST fusion proteins were mixed with the in vitro-transcribed/translated products and incubated in binding buffer for 2 hours at 4°C. The beads were washed five times with binding buffer and denatured in 2 × loading buffer at 95°C for 5 minutes. Protein bands were detected by Western blot using specific antibodies (Table S2).

| In vivo ubiquitination assay
MCF-7 cells were transfected with BCAS3 shRNA or Control shRNA, followed by treatment with MG132 (20 µmol/L) for 6 hours before harvesting. Briefly, cells were washed in PBS and lysed in lysis buffer supplemented with a 1 × protease inhibitor cocktail. Specific antibodies were added into cellular extracts for incubation overnight at 4°C. Afterwards, protein A/G Sepharose beads were added and incubated for 2 hours at 4°C. Beads were washed three times with lysis buffer, followed by denatured in 5 × loading buffer at 95°C for 5 minutes. The proteins were subjected to SDS-PAGE and immunoblotting with an anti-ubiquitin antibody to examine p53 ubiquitination.

| In vitro ubiquitination assay
The ubiquitin conjugation reaction buffer kit (Boston Biochem, for 60 minutes, followed by denatured in 5 × loading buffer at 95°C for 5 minutes. The lysates were subjected to SDS-PAGE and immunoblotting.

| Statistical analysis
Data were analysed using GraphPad Prism 5 (GraphPad Software Inc) and SPSS 18.0 (SPSS software Inc) and were presented as mean ± SD. A chi-square test was used to analyse the correlation between BCAS3 expression and clinicopathological parameters.
Student's unpaired t test was used to analyse differences between the two groups. Spearman's and Pearson's correlation coefficient were used to analyse correlations between groups. Kaplan-Meier analysis was used to calculate the cumulative survival time.
Univariate and multivariate analyses based on the Cox regression model were conducted. P-values <.05 were considered statistically significant.

| BCAS3 is highly expressed in BRCA tissues, and a high level of BCAS3 positively correlates with unfavourable prognosis in BRCA cases
To determine BCAS3 expression in human malignancies, we conducted a multi-cancer analysis of BCAS3 in The Cancer Genome Atlas (TCGA) multi-cancer panel downloaded from the website of cBioPortal (http://www.cbiop ortal.org). The results revealed that the BCAS3 amplification frequency was higher in breast cancer than the other four tissue-derived tumours ( Figure 1A).
We then detected BCAS3 amplification frequency in six breast cancer cohorts and found that the most frequent BCAS3 alteration was amplification ( Figure 1B). Next, we explored the protein level of BCAS3 in human breast carcinoma tissues using IHC staining in 140 cases of breast tumour tissues, and the results High BCAS3 expression is correlated with low overall survival in patients with breast carcinoma LinkedOmics (http://www.linke domics.org/admin.php) and demonstrated that BCAS3 mRNA tended to increase with disease progression ( Figure 1E). Kaplan-Meier survival curves showed that cases with elevated BCAS3 expression exhibited decreased overall survival (OS) compared with patients with low BCAS3 expression (P < .05) ( Figure 1F).

| High BCAS3 expression promotes growth, inhibits apoptosis and confers chemoresistance to MCF-7 cells
The above-mentioned observations prompted us to explore the biological properties of BCAS3 in breast tumorigenesis. First, we showed in the assays for doxorubicin ( Figure 2E). Together, these results indicate that BCAS3 overexpression makes MCF-7 cells gain more resistance to chemotherapy.

| BCAS3 interacts directly with CRL4A complex and p53
We performed affinity purification-mass spectrometric assays to identify BCAS3-associated proteins to define the mechanistic role of BCAS3 in oncogenic properties. FLAG-tagged vector and FLAG-tagged BCAS3 were stably transfected in HEK 293T cells. Extracts from cells were purified using the anti-FLAG affinity gel. Results of mass spectrometric analysis demonstrated that BCAS3 interacted with DSP, KHSRP, KIF11, HSP70, BAG5, vimentin and p53. ( Figure 3A). The functional interactions among these identified proteins were shown through a PPI network analysis using the STRING (https://strin g-db.org/cgi/input. pl; Figure 3B). As literature reported that the activation of CUL4A led to degradation of p53, 16 the substrate recruiting of CUL4A-RING E3 ubiquitin ligases imply that DDB1, which either directly interacts 27,28 or binds to the substrate protein through the association with WD40 repeat adaptor proteins. [29][30][31] BCAS3 has a WD40 domain, we explored whether there is an interaction between BCAS3 and CRL4A complex.
The interactions between the CRL4A complex and p53 were further confirmed by Western blot analysis in both MCF-7 cells and 293T cells ( Figure 3C), demonstrating that BCAS3 is associated with the CRL4A complex as well as p53 in vivo. The detailed results of the protein mass spectrometric analysis are presented in Table S1.
To explore the molecular mechanism for the interaction between BCAS3 and the p53/CRL4A complex, we conducted GST pull-down assays using GST-fused BCAS3 constructs and in vitro-transcribed/ translated p53, DDB1, CUL4A and ROC1. The results revealed that BCAS3 interacts directly with p53 and DDB1, and no interactions were observed between BCAS3 and CUL4A. Data from reciprocal GST pull-down assays verified the above results ( Figure 3D). Next, to further identify the interacting domains between BCAS3 and the CRL4A/ p53, we conducted GST pull-down assays with GSTfused BPA, BPB and BPC domains of DDB1. 32 The results revealed that the BPA domain of DDB1 interacted with BCAS3 ( Figure 3E).
Subsequently, we constructed the GST-fused BCAS3-WD40 domain  Figure 3F). Together, these data reveal the physical interaction and detailed molecular basis between BCAS3 and the p53/ CRL4A complex ( Figure 3G).

| BCAS3 promotes p53 protein ubiquitination through a CRL4A-dependent pathway
To determine the potential significance that BCAS3 physically interacted with p53, we examined the role of BCAS3 on the levels of the p53 protein. BCAS3 siRNA was transfected in MCF-7 cells, and the cellular lysates were extracted to evaluate the protein and mRNA expression levels of p53. The results indicated that p53 protein expression level was significantly increased in BCAS3 knockdown MCF-7 cells, whereas there is no difference in terms of the level of p53 mRNA ( Figure 4A). Subsequently, cycloheximide chase assays in MCF-7 cells showed that the p53 protein half-life was increased when BCAS3 was knocked down compared with control ( Figure 4B). In accordance with these results, overexpression of BCAS3 triggered a reduction in the protein levels of p53 and p21. However, the reduction in p53 protein level related to BCAS3 can be blocked after MG132 intervention, suggesting that p53 protein degradation may occur through a proteasome-mediated protein degradation mechanism ( Figure 4C).
To examine whether the CRL4A complex is involved in BCAS3mediated p53 degradation, we detected p53 levels after siRNA Together with the above-mentioned evidence that BCAS3 interacts with p53, these data suggest that BCAS3 is involved in regulating CRL4A-mediated p53 degradation, and the regulation of p53 by CRL4A complex is BCAS3-dependent.
To fully elucidate the mechanism by which BCAS3/CRL4A participates in the regulation of p53 protein degradation, we next explore whether p53 protein degradation is related to ubiquitination. MCF-7 cells were co-transfected with BCAS3 siRNA and His-tagged p53. We used anti-His antibody for immunoprecipitation and anti-ubiquitin antibody for immunoblotting, and the result demonstrated a decreased ubiquitination level of p53 after BCAS3 knockdown ( Figure 4G). In vitro ubiquitination assays showed that BCAS3 indeed enhanced the polyubiquitination of p53 when CUL4A in the reactions (lanes 5-6), but could not promote ubiquitination in the absence of CUL4A (lanes 4-5).

| BCAS3-p53 axis regulates cell growth, apoptosis and chemoresistance
To define the physiological significance of BCAS3-mediated destabilization of p53, we evaluated the effect of p53 on the malignant phenotype caused by BCAS3. BCAS3 was knocked down individually or in combination with p53 in MCF-7 cells. As the target genes of p53, p21 and BAX play an important role in cell proliferation and apoptosis. Knockdown of BCAS3 increased the expression levels of these target genes, while knockdown of BCAS3 and p53 in combination could partially restore the increased levels to original status ( Figure 5A). The proliferation rate of MCF-7 cells decreased after BCAS3 knockdown, while the rate partially increased after both BCAS3 and p53 knockdown ( Figure 5B,C). Treated with doxorubicin for 24 hours, the results revealed that the cell apoptosis rate of the BCAS3 knockdown group (37.5%) was increased compared with control (20.5%); however, this effect could be partially reversed via p53 knockdown in combination (10.8%). A similar trend can be seen in the absence of doxorubicin ( Figure 5D).
Since kinds of literature had confirmed that p53 plays an irreplaceable role in the chemoresistance of breast carcinoma, our aforementioned results demonstrated that BCAS3 promoted p53 protein ubiquitination degradation; therefore, we speculated that chemoresistance conferred by BCAS3 may be mediated by p53. To define other pathways that are involved in p53 protein homeostasis, we detected the expression of MDM2 with BCAS3 knockdown in MCF-7 cells. Our results revealed that BCAS3 knockdown could not affect the protein levels of MDM2 ( Figure 5G).

MCF-7 cells were treated with paclitaxel/doxorubicin after overex-
We propose that regulating p53 protein homeostasis by BCAS3 is

MDM2-independent.
In addition, we choose p53-mutated MDA-MB-231 cells, which had no wild-type p53 activity or non-functional p53 expression. 33  A similar trend can be seen in the absence of doxorubicin ( Figure 5J).
The results indicate that the p53-dependent function of BCAS3 in regulating proliferation and apoptosis is partially blocked in MDA-

MB-231 cells.
Overall, the above results demonstrate that the BCAS3-p53 axis is involved in biological functions, including cell proliferation, apoptosis and chemoresistance.

| High expression of BCAS3 is correlated with low levels of p53 in nuclear and is an independent parameter for predicting poor prognosis of patients with BRCA
To further explore the clinical value of BCAS3 and p53, we ex- was positively correlated with histological grades, while a negative correlation was found between nuclear p53 levels and histological grades of the breast carcinoma samples ( Figure 6A,B). Additionally, we found that BCAS3 expression was negatively correlated with p53 expression using semiquantitative IHC (r = −0.1718, P = .0432; Figure 6C). Kaplan-Meier curves showed that patients with high levels of BCAS3 were more likely to have decreased survival rate, and an opposite prognostic trend was observed in p53 expression (P < .01) ( Figure 6D).
The relationship between expression levels of BCAS3 and the clinicopathological characteristics of 140 patients with BRCA was investigated, as listed in Table 1. The results demonstrated that BCAS3 expression was associated with histological grade (P = .004), tumour size (P = .004) and TNM stage (P < .001). Multivariate analysis showed that BCAS3 expression was an independent predictor of overall survival (HR = 11.979, P < .001; Table 2). These results indicate that BCAS3 is an independent parameter for the poor prognosis of patients with breast tumours.

| D ISCUSS I ON
DNA amplification is frequently observed in several chromosomal sites allowing tumour development and progression, as well as conferring drug resistance. A previous study demonstrated that 17q23 is a common region of amplification, occurring in approximately 20% of primary breast tumours. 37 BCAS3 was found to have an amplification rate of 9.4% in primary breast tumours. Furthermore, BCAS3 was reported to be rearranged and fused to BCAS4 which was located at 20q13. The chromosomal site also tended to amplify in breast cancer. The BCAS3 and BCAS4 fusion was detected in the MCF-7 cell line and led to high levels of mRNA expression. 5 However, the function of BCAS3 in breast cancer has not yet been defined and needs to be explored experimentally. In this study, we found that BCAS3 promoted tumour cell properties linked to tumorigenesis through degrading p53 and explained the functions of BCAS3 in amplification and fusion in breast carcinoma. Compared with normal samples, we found that BCAS3 expression was remarkably elevated in breast cancer tissues. Moreover, we determined that a high level of BCAS3 positively correlated with prognosis in a cohort of 1071 patients from LinkedOmics, which was in accord with the evidence from a previous study. 38 Therefore, we speculate that high BCAS3 expression may be related to tumour malignancy.

Subsequently, we explored the biological functions of BCAS3
in MCF-7 cells, and the results demonstrated that high BCAS3 expression could promote growth, which was in accord with a previous study. 39 The proliferation rate of tumour cells is determined by the balance between proliferation and apoptosis. Further experiments revealed that BCAS3 overexpression inhibited apoptosis. It has been reported that BCAS3 excessive expression in premenopausal breast tumours seems to weaken the therapeutic effect of tamoxifen. 40 As Coping response to cellular stress and carcinogenic signals, p53 regulates the cell cycle to maintain cell's steady state, and its level is mainly regulated by ubiquitination modification. 41 The CRL4A complex is composed of CUL4A, DDB1 and ROC1. 16 The cullin subunit of CUL4A acts as a scaffold and bridges the substrate through one end, while the other end is responsible for ROC1 connection to recruit E2. In addition, several studies 16,42,43 demonstrated that inactivation of CUL4A results in an increase in p53 and downstream genes in both human and mouse cells, indicating that the CRL4A complex may be involved in p53 expression regulation. Furthermore, CUL4A remained amplified in breast carcinomas, 44 and these data may indicate that dynamic changes in the expression of the CRL4A complex contribute to breast oncogenic properties.
Mechanistically, the substrate recruiting of CRL4A complex implies that DDB1, which either directly interacts 27,28 or associates with WD40 repeat adaptor proteins, binding to the substrate protein. [29][30][31] In this study, we showed that BCAS3, a novel WD40 repeat-containing protein, regulates p53 protein stability during ubiquitin-dependent proteolysis. As CUL4A was previously found to interact with p53, bridging p53 to other E3 ligases to promote its proteasomal degradation, 31 we sought to verify whether BCAS3 acts as an adaptor and binds to p53 and CRL4A complex.
Our study further determined that BCAS3 directly interacted with both DDB1 and p53 as substrate-specific adaptors, suggesting that vivo by recruiting UbcH5c to facilitate MDM2 E3 ligase function. 46 Many factors and proteins can target the MDM2-p53 axis to regulate p53 protein levels. For example, RLIM is a downstream target of TRIM28 and functions between TRIM28 and MDM2 and thereby acts as an MDM2 inhibitor to regulate p53 protein levels. 47 TRIM45 could promote K63-linked polyubiquitination and inhibit K48-linked polyubiquitination of p53 by MDM2. In our results, no interaction between BCAS3 and MDM2 was illustrated by immunopurificationmass spectrometric analysis. 48 Knockdown of BCAS3 in MCF-7 cells also did not affect the protein levels of MDM2. Therefore, we did not pay much attention to the effect of BCAS3 on MDM2-mediated p53 degradation. In the future, we will pay more attention to whether other pathways that are involved in p53 protein homeostasis might be affected by BCAS3.
Given that BCAS3 can stabilize p53, it is not unexpected to speculate that BCAS3 could regulate cell proliferation, apoptosis and chemoresistance in a p53-dependent manner. Clinical evidence from ER-positive tumours showed that most samples with TP53 wild type (WT) were resistant to chemotherapy, while ER-negative subtypes are more sensitive to chemotherapy due to TP53 mutations. 49 Subsequently, several reports revealed that tumours with TP53 WT rarely achieved a complete response to chemotherapy. It may be due to the induction of senescence after doxorubicin treatment, while lack of growth capture in mutant tumours can lead to cell death and clinical outcome. 50,51 In recent years, there have been many studies on the chemoresistance of MCF-7, which is a breast cancer cell line expressing wild-type p53 protein. For example, a vital role of SET protein was identified in paclitaxel-induced chemoresistance in MCF-7 cells. 52 Elevated integrin α5β1 can promote doxorubicin resistance in MCF-7 cells by downregulating ERK protein kinase. 53 In this study, we showed that knockdown of BCAS3 conferred chemosensitivity to MCF-7 cells by increasing p53 protein levels. Taken together, these data provide insights into therapy for TP53 WT breast cancers through overexpression of p53 in cancer cells. Our study provided solid evidence that BCAS3 overexpression exerted vital functions in breast cancer progression via posttranslational inactivation of p53. Mechanistically, we showed that BCAS3 interacted with the CRL4A complex and promoted the degradation of p53 protein by ubiquitin-dependent proteolysis. IHC results from a cohort of patients with BRCA showed that both BCAS3 and p53 were localized in the nuclei of breast cancer cells. Clinically, patients with BRCA with elevated BCAS3 and decreased p53 levels displayed a poorer prognosis.

| CON CLUS IONS
We demonstrated that BCAS3 promoted growth and inhibited apoptosis of breast tumour cells by participating in the regulation of p53 ubiquitination mediated by the CRL4A complex. Further, BCAS3 may be a novel predictor for prognosis and guide chemotherapy regimens for patients with breast cancer.

CO N FLI C T S O F I NTE R E S T
None.

DATA AVA I L A B I L I T Y S TAT E M E N T
All data generated or analysed during this study are included in this article.