- Open Access
RETRACTED ARTICLE: LncRNA BCAR4 promotes migration, invasion, and chemo-resistance by inhibiting miR-644a in breast cancer
Journal of Experimental & Clinical Cancer Research volume 42, Article number: 14 (2023)
Metastasis and drug resistance of breast cancer have become a barrier to treating patients successfully. Long noncoding RNAs (lncRNAs) are known as vital players in cancer development and progression.
The RT-qPCR were used to detect the gene expression. Colony formation assay, would healing assay, and transwell assay were performed to investigate oncogenic functions of cells. CCK8 assay was used to detect the cell viability. Western blot was applied to detect the protein level. Dual-luciferase reporter assay was used to determine the relationship between molecules. Mouse orthotopic xenograft tumor models were established to evaluate the effects of BCAR4 on tumor growth and metastasis in vivo.
LncRNA BCAR4 was significantly increased in breast cancer patients’ tissues and plasma and upregulated in breast cancer cell lines. BCAR4 upregulation was correlated with the TNM stages and decreased after surgical removal of breast tumors. Silencing of BCAR4 suppressed breast cancer cell colony formation, migration, invasion, and xenograft tumor growth and promoted chemo-sensitivity. Mechanistically, BCAR4 facilitates breast cancer migration and invasion via the miR-644a-CCR7 axis of the MAPK pathway. BCAR4 promotes ABCB1 expression indirectly by binding to and down-regulating miR-644a to induce chemo-resistance in breast cancer.
Our findings provide insights into the oncogenic role of BCAR4 and implicate BCAR4 as a potential diagnostic biomarker and a promising therapeutic agent to suppress metastasis and inhibit chemo-resistance of breast cancer.
In 2020, female breast cancer surpassed lung cancer as the most commonly diagnosed cancer, with an estimated 2.3 million new cases, representing 11.7% of all cancer cases. Breast cancer is also the leading cause of cancer-associated death in women . Although surgery, chemotherapy, and radiotherapy have greatly improved the overall survival rate of breast cancer patients, some patients still suffer metastasis, resistance to chemoradiotherapy, and recurrence [2, 3]. Doxorubicin (DOX) is a widely-used therapeutic agent to treat metastatic or advanced breast cancer. However, the clinical efficiency is often limited by acquired resistance . Therefore, research on key signaling pathways and therapeutic targets is necessary to improve the outcomes of breast cancer patients.
Long noncoding RNAs (lncRNAs) are noncoding RNAs with a length of more than 200 nucleotides. They are involved in various biological processes, such as apoptosis, proliferation, migration, drug resistance, and differentiation [5,6,7]. Long noncoding RNA breast cancer anti-estrogen resistance 4 (BCAR4), located in chromosome 16p13.13, was first identified by screening endocrine resistance-related genes in breast cancer cells, and its expression has a positive correlation with anti-estrogen resistance . BCAR4 is overexpressed in multiple cancers, and it inhibits tumor cell apoptosis and promotes cell proliferation and migration in bladder cancer , colorectal cancer , and lung cancer . BCAR4 activates the signaling pathways of ErbB2/ErbB3 and Wnt/β-catenin to promote breast cancer cell resistance to tamoxifen  and gastric cancer cell resistance to cisplatin . Moreover, emerging evidence support that elevated BCAR4 is associated with a worse prognosis for cancer patients; thus, it is a potential prognostic biomarker [14,15,16]. BCAR4 is implicated in tumor cell proliferation, metastasis, and endocrine resistance in breast cancer [17,18,19], yet the underlying mechanisms remain elusive.
In this study, we explored BCAR4 expression levels in the tissues and plasma of breast cancer patients and in breast cancer cell lines. BCAR4 was involved in breast cancer cell viability, migration, invasion, drug resistance, and xenograft tumor metastasis. Furthermore, we found that BCAR4 sponged to miR-644a, thereby upregulating CCR7 and ABCB1 to promote breast cancer progression. Our study sheds light on the oncogenic BCAR4 as a promising therapeutic target to suppress metastasis and drug resistance of breast cancer.
Materials and methods
Cell culture and clinical specimens
The human breast cancer cell lines (BT-549, T-47D, MDA-MB-468, MDA-MB-231, MCF-7), the noncancerous breast epithelial cell line HBL-100, and the human embryonic kidney (HEK) 293T cells were purchased from the American Type Culture Collection (ATCC). All cell lines were cultured following the instructions of ATCC. The DOX resistant MCF-7 was purchased from shanghai Meixuan Biotechnology Co., LTD. The DOX resistant MDA-MB-231 was constructed by our laboratory using conventional continuous exposure to DOX with dose-stepwise increment. DOX resistant MCF-7 (MCF-7/DOX) were cultured in RPMI-1640 with 10% FBS containing 430 nM DOX. DOX resistant MDA-MB-231 (MDA-MB-231/DOX) were cultured in DMEM with 10% FBS containing 350 nM DOX.
Blood samples were obtained from the Central Hospital of Wuhan. Eighty breast cancer patients and 80 healthy controls were included in the study. Twenty paired blood samples were collected, including preoperative and postoperative blood from breast cancer patients. The newly diagnosed breast cancer patients had received no chemotherapy or radiotherapy before sample collection. All healthy subjects did not have any diseases or injuries. Peripheral blood samples from participants were collected in tubes containing EDTA and were processed within 4 h by centrifugation at 1000 g for 15 min at 4 °C. The supernatant was then gently transferred to a fresh RNase-free 1.5-ml tube and cryopreserved at − 80 °C immediately. The study was approved by the Ethical and Scientific Committees of the Central Hospital of Wuhan, and all participants provided informed consent during enrollment.
RNA isolation and qRT-PCR
RNA was extracted by TRIzol reagent (Invitrogen, Carlsbad, CA, USA). BCAR4 was measured by SYBR Green (Applied Biosystems, Carlsbad, CA, USA) in StepOne™ Real-Time PCR System (Applied Biosystems). MiR-644a was measured with a bulge-loop miRNA real-time PCR kit (RiboBio, Guangzhou, China). GAPDH was used as the endogenous control for BCAR4 and U6 for miR-644a. The relative expression of BCAR4 and miR-644a was calculated by 2–△△Ct in breast cancer cells and by 2–△Ct for that in plasma. The primers for BCAR4 were as follows: sense, ATA CAA TGG CGT AAT CAT AGC, and antisense, AGA CAT TCA GAG CAA GAC A. ABCB1: sense, CAG GCT TGC TGT AAT TAC CCA, and antisense, TCA AAG AAA CAA CGG TTC GG. GAPDH: sense, GGG AGC CAA AAG GGT CAT, and antisense, GAG TCC TTC CAC GAT ACC AA.
MiR-644a mimic, or miR-644a inhibitor, small interfering RNAs (siRNAs) targeting BCAR4, and their respective negative controls were all obtained from RiboBio (Guangzhou, China). The sequence of siRNAs targeting BCAR4 were as follows: si-1#: CCT GTG ATG TGT TGA TAA A, si-2#: CAC AAT TGA TGT TCT CTA A, and si-3#: GAG TTC TGC AAT CCA CAA T. The full-length of BCAR4 was cloned into BamH1 and Xho1 sites in a pcDNA-3.1 vector ((Invitrogen), which was confirmed by sequencing. Transfection was performed using ExFect® Transfection reagent (Vazyme, Nanjing, China) according to the manufacturer’s guidelines.
Colony formation assay
MCF-7 or MDA-MB-231 cells were seeded into 12-well plates with 500 cells/well and incubated for ten days. Plates were then gently washed with PBS and stained with 0.1% crystal violet (Beyotime, Nantong, China). A colony containing more than 50 cells was counted as one colony.
Wound healing assay
For cell mobility assay, MCF-7 or MDA-MB-231 cells were seeded into 6-well plates (4 × 105 cells/well). An artificial wound was made by scratching the confluent cell monolayer with a sterile 200 μl pipette tip 24 h after transfection. Then, cells were incubated in a medium containing 1% FBS. After scratching for 0 h and 48 h, wound healing images were photographed at magnification 100 × using an IX81 microscope (Olympus, Tokyo, Japan). The migration rate was determined by ImageJ software.
To further investigate breast cancer cell migration and invasion, transwell chambers (Corning, NY, USA) were used. MCF-7 or MDA-MB-231 cells were detached and suspended in 200 μL serum-free medium 24 h after transfection. A total of 2 × 104 cells were added into the upper chamber for migration assay or a total of 3 × 104 cells were added into the upper chamber coated with Matrigel (BD Biosciences, CA, USA) for invasion assay. The bottom chambers were filled with the 600 μL complete culture medium. After being cultured for 48 h, cells that migrated or invaded the lower filter surfaces were fixed and then stained with 0.1% crystal violet (Beyotime). Images (magnification, × 100) were taken from each membrane, and the numbers of migrated or invaded cells were counted using an IX81 microscope (Olympus).
Drug sensitivity assay and CCK8 assay
The DOX (Selleck Chemicals) sensitivity of cells was detected using the CCK8 kit (Beyotime) by measuring the IC50 value (the concentration of DOX resulted in 50% decline of the absorbance compared with control). MCF-7 or MDA-MB-231 cells were seeded into 96-well plates with 5000 cells/well and incubated with different concentrations of DOX (5 nM, 10 nM, 30 nM, 100 nM, 200 nM, 500 nM, 1000 nM and 2000 nM). After treatment for 48 h, CCK8 solution was then added to wells and incubated for another 4 h. The absorbance was recorded at 450 nm by the EnSpire multimode reader (PerkinElmer, Waltham, MA).
Western blot assay
Western blot analysis was performed according to the description previously . The following primary antibodies were used: rabbit polyclonal anti-ABCB1 (1: 500, Proteintech, Rosemont, USA), rabbit monoclonal anti-CCR7 (1:10,000, Abcam, Cambridge, MA), mouse monoclonal anti-ERK1/2 (1:2000, Proteintech), mouse monoclonal anti-P38 MAPK (1: 2000, Proteintech). rabbit monoclonal anti-phospho-p44/42 MAPK(Erk1/2)(Thr202/Tyr204, 1: 1000, Cell Signaling Technology, Danvers, MA, USA), rabbit monoclonal anti-phospho-p38 MAPK (Thr180/Tyr182, 1: 1000, Cell Signaling Technology), mouse monoclonal anti-E-cadherin (1:2000, Proteintech), mouse monoclonal anti-Vimentin(1:5000, Proteintech), rabbit polyclonal anti-VEGF(1: 1000, Proteintech). Rabbit polyclonal anti-GAPDH (1:5000, Proteintech) was used as an internal control.
Luciferase reporter assay
For the BCAR4 luciferase reporter assay, pRL-TK-BCAR4 or pRL-TK-BCAR4 mutant vectors and pGL3 control (Promega, Madison, WI) were transfected into HEK-293T cells along with miR-644a mimic or miR-644a inhibitor using ExFect® Transfection reagent (Vazyme) according to the instructions of the manufacturer. For miR-644a target gene CCR7 luciferase reporter assay, pRL-TK-CCR7-3΄UTR or pRL-TK- CCR7-3΄UTR mutant vectors and pGL3 control (Promega) were transfected into HEK-293T cells along with miR-644a mimic or miR-644a inhibitor using ExFect® Transfection reagent (Vazyme). The luciferase activity was measured 48 h after transfection using the Dual-Glo luciferase reporter assay system (Promega) according to the guidelines of the manufacturer. Luminescence values were recorded by a Victor X2 multilabel reader (PerkinElmer).
In vivo assay
Female athymic four-week-old BALB/c nude mice were purchased from HFK Bio-Technology Co. Ltd. (Beijing, China). A total of 5 × 106 MDA-MB-231 cells, resuspended in Opti-MEM, were injected into the mammary fat pad. Tumor volume was analyzed as V = D × d2 × 0.5 (D, the longer diameter; d, the shorter diameter). When the tumor reached about 60 mm3, the mice were divided into a si-control group (n = 5) and a si-BCAR4 group (n = 5) randomly. The in vivo siRNA for BCAR4 knockdown was synthesized by RiboBio (Guangzhou, China). The sequence of the in vivo BCAR4 knockdown was as follows: si-BCAR4: CAC AAT TGA TGT TCT CTA A. In vivo si-BCAR4 or the control (2.5 nmol / 20 g body weight) was directly administered into the implanted tumor two times per week for three weeks. One month after the first injection of si-BCAR4 or the control, all mice were anesthetized with 1% pentobarbital sodium. The tumors, lungs, and livers were obtained and then fixed in formalin for hematoxylin and eosin (HE) staining or immunohistochemistry. The rabbit polyclonal anti-CCR7 (1:200, Proteintech) was used to determine CCR7 expression. The mouse monoclonal anti-E-cadherin (1:1000, Proteintech), mouse monoclonal anti-Vimentin (1: 1000, Proteintech), rabbit polyclonal anti-VEGF (1:200, Proteintech) was used to detect E-cadherin, Vimentin and VEGF expression respectively.
All statistical analyses were performed using SPSS 22.0 software. The one-way analysis of variance (ANOVA), χ2 test or the student’s t-test was carried out for comparison between groups. All experiments were independently repeated at least three times. P value < 0.05 represents statistically significance. *P < 0.05, ** P < 0.01, ***P < 0.001.
BCAR4 is significantly upregulated in breast cancer tissues and plasma samples
We examined the expression of BCAR4 in breast cancer tissues from the TCGA database and the StarBase V3.0. In the TCGA database, BCAR4 expression was higher in 1,097 breast cancer tissues than in 114 noncancerous breast tissues (Fig. 1A). So did that in the StarBase V3.0 with 1,104 breast cancer tissues and 113 noncancerous breast tissues (Fig. 1B). Next, we tested BCAR4 expression levels in plasma samples from 80 breast cancer patients and 80 healthy controls and found that BCAR4 level was significantly increased in breast cancer plasma samples (Fig. 1C). The plasma levels of BCAR4 were significantly higher in stages III and IV than those in stages I and II (Fig. 1D). The clinical parameters of the breast cancer patients are shown in Table 1. We found that BCAR4 level is significantly associated with tumor size, TNM stage, lymphnode status, but it has no diference among the age, ER status, PR status, HER status, and molecular subtypes (Table 1, Supplementary Fig. 1). Furthermore, we analyzed 20 paired preoperative and postoperative plasma samples from breast cancer patients. The levels of BCAR4 were remarkably reduced in the postoperative plasma samples (Fig. 1E). Finally, we measured the expression of BCAR4 in different breast cancer cells. BCAR4 was highly expressed in MDA-MB-231 and MCF-7 (Fig. 1F), so we chose these two lines for the subsequent experiments. These results demonstrate that BCAR4 is significantly upregulated in breast cancer tumors and plasma samples.
Knockdown of BCAR4 inhibits migration, invasion and DOX resistance in breast cancer
To explore the functions of BCAR4 in breast cancer, BCAR4 siRNA was transfected into MCF-7 and MDA-MB-231 cells. A RT-qPCR assay showed that BCAR4 siRNAs (si-1#, si-2#, and si-3#) markedly inhibited BCAR4 expression, with si-2# exhibiting the maximal inhibition (Fig. 2A and E). Transwell assay revealed that si-2# decreased the migration and invasion of MCF-7 and MDA-MB-231 cells (Fig. 2B, C, D, F, G, and H). In addition, a wound healing assay demonstrated that knockdown of BCAR4 decreased the migration of breast cancer cells (Fig. 2I and J). The number of colonies was significantly reduced in breast cancer cells by si-2# (Fig. 2K and L). Besides, the 50% inhibitory concentration (IC50) of DOX were decreased in MCF-7 and MDA-MB-231 cells by BCAR4 inhibition (Fig. 2M and N). ABCB1 (also known as the P-glycoprotein 1 [P-gp]), a member of the ATP-binding cassette (ABC) superfamily of transporters and encoded by the multidrug resistance 1 (MDR1) gene, is closely associated with drug resistance in a plethora of cancers because it pumps many foreign substances such as cytotoxic drugs out of cells [21, 22]. Compared with MCF-7 and MDA-MB-231, the expression of ABCB1 and BCAR4 were signigicantly increased in MCF-7/DOX and MDA-MB-231/DOX (Supplementary Fig. 2A and B). A western blot assay showed that BCAR4 inhibition downregulated the expression of ABCB1 and two mesenchymal markers (Vimentin and VEGF) and upregulated the epithelial marker E-cadherin (Fig. 2O). Collectively, these results support that knockdown of BCAR4 inhibits breast cancer cell migration, invasion, chemo-resistance, and potentially epithelial-mesenchymal transition.
BCAR4 downregulates miR-644a expression
To investigate the underlying mechanisms by which BCAR4 promotes breast cancer progression, we searched for miRNAs that bind to BCAR4. MiR-644a was predicted to interact with BCAR4 in the StarBase V3.0 (Fig. 3A). A luciferase reporter assay in HEK-293T, MDA-MB-231, and MCF-7 showed that overexpression of miR-644a repressed the luciferase activity in a BCAR4 wildtype (WT) construct but not in a mutant (MUT, Fig. 3B, D, and F). Downregulation of miR-644a markedly increased the luciferase activity with WT BCAR4 but not MUT (Fig. 3C, E, and G). BCAR4 knockdown resulted in a significant upregulation of miR-644a in MCF-7 and MDA-MB-231 cells (Fig. 3H). In addition, miR-644a levels in plasma samples from 80 breast cancer patients were lower than those from healthy controls (Fig. 3I). Moreover, there was a negative correlation between the expression of BCAR4 and miR-644a in the plasma of 80 breast cancer patients (Fig. 3J). MiR-644a was also downregulated in different breast cancer cells compared with the noncancerous breast epithelial cell line HBL-100 (Fig. 3K). These results demonstrate that BCAR4 directly binds to and negatively regulates miR-644a in breast cancer.
BCAR4 promotes migration, invasion, and chemo-resistance via miR-644a downregulation in breast cancer cells
We transfected MCF-7 and MDA-MB-231 cells with BCAR4 siRNA plus miR-644a inhibitor. The reduced colony formation led by BCAR4 silencing was reversed by miR-644a inhibition in breast cancer cells (Fig. 4A, B). Wound healing and Transwell assays showed that BCAR4 siRNA-mediated decreased migration and invasion of both MCF-7 and MDA-MB-231 cells were overturned by cotransfection with miR-644a inhibitor (Fig. 4C-4F). Additionally, BCAR4 silencing reduced the cell viability and IC50 of DOX, which were reversed by miR-644a inhibitor in MDA-MB-231 cells (Fig. 4G). BCAR4 overexpression inceased the cell viability and IC50 of DOX, which were also reversed by miR-644a mimic in MDA-MB-231 cells (Supplementary Fig. 3). Moreover, downregulation of ABCB1, Vimentin and VEGF and E-cadherin upregulation by BCAR4 inhibition were inverted in MCF-7 and MDA-MB-231 cells by miR-644a inhibitor (Fig. 4H). These results demonstrate that BCAR4 promotes breast cancer cell migration, invasion, and chemo-resistance, at least partially, by miR-644a downregulation.
MiR-644a directly targets CCR7 to regulate migration and invasion via MAPK signaling
We introduced miR-644a mimic and inhibitor into MDA-MB-231 cells for gain- and loss‐of‐function studies. The colony formation assay showed that there were fewer colonies formed in MDA-MB-231 transfected with miR-644a mimic than the control cells. Adversely, the number of colonies was clearly higher in MDA-MB-231 cells transfected with miR-644a inhibitor than in the control cells (Fig. 5A). Overexpression of miR-644a significantly inhibited cell migration and invasion, whereas inhibition of miR-644a promoted them (Fig. 5B, C). Cell viability and IC50 to DOX were decreased in MDA-MB-231 transfected with miR-644a mimic, while they were increased by miR-644a inhibitor (Fig. 5D). These results suggested that miR-644a inhibited cell migration, invasion, and chemo-resistance in breast cancer.
The 3′UTRs of 13 candidate genes had a miR-644a binding site, as predicted by TargetScan, miRTarBase, and miRDB (Fig. 5E). We focused on the chemokine receptor 7 (CCR7) gene (Fig. 5E). CCR7 and its ligand CCL19 control the EMT progression in breast cancer cells and facilitate the invasion and migration process of tumor cells by triggering several signaling pathways, such as PI3K/AKT, MAPK, and JAK/STAT3 . In the TCGA dataset, the expression of CCR7 was significantly elevated in breast cancer tissues (n = 1,097), compared to the noncancerous breast tissues (n = 114; Fig. 5F). Notablely, the expression of CCR7 was higher in tumors of stage I (n = 183), stage II (n = 615), and stage III (n = 247), but not in stage IV (n = 20) (Fig. 5G). In particular, When breast tumors were stratified into molecular subtypes, CCR7 expression in luminal (n = 566), HER2-positive (n = 37), triple-negative (n = 116) tumors was evidently higher than in noncancerous breast tissues (n = 114; Fig. 5H). The luciferase reporter assay in HEK-293T cells showed that miR-644a negatively regulated the luciferase activity from CCR7 WT 3′ UTR, but not the mutant (Fig. 5I). Conversely, when miR-644a was inhibited, there was an obvious increase in luciferase activities in HEK-293T cells with the wildtype 3′UTR but not in those with the mutant 3′UTR (Fig. 5J). In MDA-MB-231 cells, overexpression of miR-644a significantly downregulated ABCB1, Vimentin, VEGF, CCR7, p-ERK, and p-p38 and upregulated E-cadherin. Inhibition of miR-644a exhibited opposite effects on these protein levels (Fig. 5K). Moreover, downregulation of CCR7, p-ERK, and p-p38 by BCAR4 inhibition were reversed in MDA-MB-231cells cotransfected with miR-644a inhibitor (Fig. 5L).
Downregulation of BCAR4 reduces tumor growth and metastasis in vivo
To evaluate whether the downregulation of BCAR4 reduces tumor growth and metastasis in vivo, we injected MDA-MB-231 cells into the mammary fat pad of BALB/c nude mice. Xenografted tumors were treated with the in vivo BCAR4 siRNA or a control for three weeks. We found that tumor development in mice treated with si-BCAR4 was slower than the control (Fig. 6A and B). Lung and liver biopsies revealed fewer lung and liver metastases in the si-BCAR4 mice (Fig. 6C and D). Furthermore, the immunohistochemistry analysis indicated that the expression of E-cadherin was markedly increased and the expression of CCR7, VEGF and Vimentin was deceased by si-BCAR4 (Fig. 6E). These results suggest that downregulation of BCAR4 signifcantly reduces tumor growth and metastasis of breast cancer by elevating CCR7 expression in vivo.
Breast cancer is the leading cause of cancer death in women worldwide . Metastasis and drug resistance are major drivers for the high mortality, underlying the urgency to explore new therapeutic targets for metastasis and drug resistance. LncRNAs take part in gene expression at epigenetic, transcriptional, and post-transcriptional levels. Their different functions on gene regulation act as a signaling mediator, decoy, guide, or scaffold . Besides, lncRNAs interact with miRNAs to regulate the target genes of the miRNAs. Accumulating evidence supports the oncogenic or tumor-suppressive roles of lncRNAs in breast cancer. The expression of lncRNA MALAT1, PVT1, and HUMT was elevated in breast cancer, and their downregulation inhibited cell growth, migration, and invasion [25,26,27]. LncRNAs ANCR, LINC00675, and PHACTR2-AS1 were downregulated in breast cancer, and their upregulation led to reduced cell proliferation and metastasis [28,29,30].
In the present study, we found that BCAR4 was significantly increased in the tissues and plasma of breast cancer patients. BCAR4 upregulation was correlated with the TNM stages of breast cancer. Plasma BCAR4 levels in breast cancer patients decreased after surgical removal of tumors. Inhibition of BCAR4 in MCF7 and MDA-MB-231 cells restrained cell viability, migration, and invasion. BCAR4 was increased in MCF-7/DOX and MDA-MB-231/DOX which overexpressed ABCB1 compared with MCF-7 and MDA-MB-231. BCAR4 downregulation increased breast cancer cells’ sensitivity to DOX by downregulating the drug resistance related protein ABCB1. These findings imply that BCAR4 is a promising therapeutic target for breast cancer.
LncRNAs act as molecular sponges to absorb miRNAs to relieve the inhibition of target mRNAs at the post-transcriptional level . Hence, we used the StarBase V3.0 database to find potential target miRNAs of BCAR4. Among the predicted target miRNAs, miR-644a is a tumor suppressor that represses multiple oncogenes [32,33,34]. In bladder cancer, BCAR4 sponges miR-644a to modulate the expression of TLX1 and promote cancer development . The miR-644a-CTBP1-p53 axis suppresses drug resistance by inhibiting tumor cell survival and epithelial-mesenchymal transition in breast cancer . In our work, bioinformatics analysis and luciferase reporter assays indicated that miR-644a directly interacts with BCAR4. MiR-644a expression was increased in breast cancer cells upon BCAR4 knockdown. In addition, miR-644a was significantly decreased in breast tumors, and there was a negative correlation between BCAR4 and miR-644a levels. Knockdown of BCAR4 suppressed the migration, invasion, and DOX resistance of breast cancer cells, which was reversed following miR-644a inhibition. BCAR4 overexpression induced resistance of breast cancer to DOX, which was reversed by miR-644a upregulation in MDA-MB-231 cells. These results provide evidence that BCAR4 promotes breast cancer cell metastasis and chemo-resistance, at least partially through miR-644a blocking.
Further study of miR-644a in breast cancer showed that the overexpression of miR-644a enhanced MDA-MB-231 cells viability, migration, invasion, and DOX resistance, while inhibition of miR-644a had the opposite effects. To investigate the underlying mechanisms of miR-644a in promoting breast cancer cell progression, we identified a new miR-644a target gene, CCR7. CCR7 belongs to the G-protein-coupled receptors (GPCRs) family and is implicated in inflammatory diseases, tumor growth, and metastasis . Upon its ligand CCL19 stimulation, CCR7 is activated, resulting in phosphorylation of MAPK members (ERK1/2 and JNK) to promote cell migration and invasion in head and neck carcinoma . Ectopic expression of CCR7 promoted prostate cancer cell metastasis via phosphorylation of ERK1/2, P38, JNK, and P65 . We found that CCR7 expression was significantly elevated in breast cancer tissues, especially in the more aggressive triple-negative tumors. Overexpression of miR-644a significantly downregulated ABCB1, CCR7, CCR7-related proteins in the MAPK pathway (p-ERK and p-p38), Vimentin, and VEGF and upregulated E-cadherin expression. Additionally, BCAR4 downregulation decreased CCR7 and CCR7 downstream proteins p-ERK and p-p38. In vivo mouse xenograft experiments verified that BCAR4 inhibition led to decreased tumor volume, lung metastases, and liver metastases, as well as CCR7 expression. These data support that BCAR4 enhances metastasis in breast cancer cells via the miR-644a-CCR7 axis and promote chemo-resistance by binding to miR-644a. This is in line with a previous report revealing that BCAR4 contributes to breast cancer metastasis mediated by directing cooperative epigenetic regulation downstream of chemokine signals .
Our study demonstrates the oncogenic role of BCAR4 in breast cancer. We reveal that BCAR4 accelerates breast cancer migration and invasion by the miR-644a-CCR7 axis and the MAPK pathway (Fig. 7). BCAR4 also upregulates ABCB1 to induce chemo-resistance in breast cancer (Fig. 7). These findings implicate BCAR4 as a promising therapeutic target for breast cancer metastasis and drug resistance.
Availability of data and materials
All data generated or analyzed during this study are included in this published article.
Long noncoding RNAs
Real-time quantitative polymerase chain reaction
Cell counting kit 8
Breast cancer anti-estrogen resistance 4
Tumor Node Metastasis
Chemokine receptor 7
Mitogen-activated protein kinase
Erb-b2 receptor tyrosine kinase 2/ Erb-b2 receptor tyrosine kinase3
ATP binding cassette subfamily B member 1
Human embryonic kidney 293T
American Type Culture Collection
Ethylene diamine tetraacetic acid
Small interfering RNAs
Hematoxylin and eosin
Vascular endothelial cell growth factor
One-way analysis of variance
Human epidermal growth factor receptor
Phosphorylated- extracellular regulated MAP kinase
50% Inhibitory concentration
Multidrug resistance 1
Metastasis associated lung adenocarcinoma transcript 1
Plasmacytoma variant translocation 1
Differentiation antagonizing non-protein coding RNA
T cell leukemia homeobox 1
C-Terminal Binding Protein 1
Sung H, Ferlay J, Siegel RL, Laversanne M, Soerjomataram I, Jemal A, Bray F. Global Cancer Statistics 2020: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries. CA Cancer J Clin. 2021;71(3):209–49.
Maishman T, Cutress RI, Hernandez A, Gerty S, Copson ER, Durcan L, Eccles DM. Local Recurrence and Breast Oncological Surgery in Young Women With Breast Cancer: The POSH Observational Cohort Study. Ann Surg. 2017;266(1):165–72.
Tufail M, Cui J, Wu C. Breast cancer: molecular mechanisms of underlying resistance and therapeutic approaches. Am J Cancer Res. 2022;12(7):2920–49.
Li Y, Gao X, Yu Z, Liu B, Pan W, Li N, Tang B. Reversing Multidrug Resistance by Multiplexed Gene Silencing for Enhanced Breast Cancer Chemotherapy. ACS Appl Mater Interfaces. 2018;10(18):15461–6.
Ye P, Feng L, Shi S, Dong C. The Mechanisms of lncRNA-Mediated Multidrug Resistance and the Clinical Application Prospects of lncRNAs in Breast Cancer. Cancers (Basel). 2022;14(9):2101.
Xue ST, Zheng B, Cao SQ, Ding JC, Hu GS, Liu W, Chen C. Long non-coding RNA LINC00680 functions as a ceRNA to promote esophageal squamous cell carcinoma progression through the miR-423-5p/PAK6 axis. Mol Cancer. 2022;21(1):69.
Ashrafizaveh S, Ashrafizadeh M, Zarrabi A, Husmandi K, Zabolian A, Shahinozzaman M, Aref AR, Hamblin MR, Nabavi N, Crea F, et al. Long non-coding RNAs in the doxorubicin resistance of cancer cells. Cancer Lett. 2021;508:104–14.
Meijer D, van Agthoven T, Bosma PT, Nooter K, Dorssers LC. Functional screen for genes responsible for tamoxifen resistance in human breast cancer cells. Mol Cancer Res. 2006;4(6):379–86.
Wang X, He H, Rui W, Xie X, Wang D, Zhu Y. Long Non-Coding RNA BCAR4 Binds to miR-644a and Targets TLX1 to Promote the Progression of Bladder Cancer. Onco Targets Ther. 2020;13:2483–90.
Ouyang S, Zhou X, Chen Z, Wang M, Zheng X, Xie M. LncRNA BCAR4, targeting to miR-665/STAT3 signaling, maintains cancer stem cells stemness and promotes tumorigenicity in colorectal cancer. Cancer Cell Int. 2019;19:72.
Yang H, Yan L, Sun K, Sun X, Zhang X, Cai K, Song T. lncRNA BCAR4 Increases Viability, Invasion, and Migration of Non-Small Cell Lung Cancer Cells by Targeting Glioma-Associated Oncogene 2 (GLI2). Oncol Res. 2019;27(3):359–69.
Godinho MF, Sieuwerts AM, Look MP, Meijer D, Foekens JA, Dorssers LC, van Agthoven T. Relevance of BCAR4 in tamoxifen resistance and tumour aggressiveness of human breast cancer. Br J Cancer. 2010;103(8):1284–91.
Wang L, Chunyan Q, Zhou Y, He Q, Ma Y, Ga Y, Wang X. BCAR4 increase cisplatin resistance and predicted poor survival in gastric cancer patients. Eur Rev Med Pharmacol Sci. 2021;25(7):2822.
Ju L, Zhou YM, Yang GS. Up-regulation of long non-coding RNA BCAR4 predicts a poor prognosis in patients with osteosarcoma, and promotes cell invasion and metastasis. Eur Rev Med Pharmacol Sci. 2016;20(21):4445–51.
Gan FJ, Li Y, Xu MX, Zhou T, Wu S, Hu K, Li Y, Sun SH, Luo Q. LncRNA BCAR4 expression predicts the clinical response to neoadjuvant chemotherapy in patients with locally advanced breast cancer. Cancer Biomark. 2021;32(3):339–51.
Zhao W, Wang Z, Fang X, Li N, Fang J. Long noncoding RNA Breast cancer antiestrogen resistance 4 is associated with cancer progression and its significant prognostic value. J Cell Physiol. 2019;234(8):12956–63.
van Agthoven T, Dorssers LC, Lehmann U, Kreipe H, Looijenga LH, Christgen M. Breast Cancer Anti-Estrogen Resistance 4 (BCAR4) Drives Proliferation of IPH-926 lobular Carcinoma Cells. PLoS ONE. 2015;10(8):e0136845.
Godinho M, Meijer D, Setyono-Han B, Dorssers LC, van Agthoven T. Characterization of BCAR4, a novel oncogene causing endocrine resistance in human breast cancer cells. J Cell Physiol. 2011;226(7):1741–9.
Xing Z, Lin A, Li C, Liang K, Wang S, Liu Y, Park PK, Qin L, Wei Y, Hawke DH, et al. lncRNA directs cooperative epigenetic regulation downstream of chemokine signals. Cell. 2014;159(5):1110–25.
Wu T, Chen W, Liu S, Lu H, Wang H, Kong D, Huang X, Kong Q, Ning Y, Lu Z. Huaier suppresses proliferation and induces apoptosis in human pulmonary cancer cells via upregulation of miR-26b-5p. FEBS Lett. 2014;588(12):2107–14.
Tian F, Dahmani FZ, Qiao J, Ni J, Xiong H, Liu T, Zhou J, Yao J. A targeted nanoplatform co-delivering chemotherapeutic and antiangiogenic drugs as a tool to reverse multidrug resistance in breast cancer. Acta Biomater. 2018;75:398–412.
Liang BJ, Lusvarghi S, Ambudkar SV, Huang HC. Use of photoimmunoconjugates to characterize ABCB1 in cancer cells. Nanophotonics. 2021;10(12):3049–61.
Rizeq B, Malki MI. The Role of CCL21/CCR7 Chemokine Axis in Breast Cancer Progression. Cancers (Basel). 2020;12(4):1036.
Yao RW, Wang Y, Chen LL. Cellular functions of long noncoding RNAs. Nat Cell Biol. 2019;21(5):542–51.
Zhao C, Ling X, Xia Y, Yan B, Guan Q. The m6A methyltransferase METTL3 controls epithelial-mesenchymal transition, migration and invasion of breast cancer through the MALAT1/miR-26b/HMGA2 axis. Cancer Cell Int. 2021;21(1):441.
Liu S, Chen W, Hu H, Zhang T, Wu T, Li X, Li Y, Kong Q, Lu H, Lu Z. Long noncoding RNA PVT1 promotes breast cancer proliferation and metastasis by binding miR-128-3p and UPF1. Breast Cancer Res. 2021;23(1):115.
Zheng S, Yang L, Zou Y, Liang JY, Liu P, Gao G, Yang A, Tang H, Xie X. Long non-coding RNA HUMT hypomethylation promotes lymphangiogenesis and metastasis via activating FOXK1 transcription in triple-negative breast cancer. J Hematol Oncol. 2020;13(1):17.
Fan S, Wang L. N(6)-Methyladenosine-regulated LINC00675 suppress the proliferation, migration and invasion of breast cancer cells via inhibiting miR-513b-5p. Bioengineered. 2021;12(2):10690–702.
Li Z, Hou P, Fan D, Dong M, Ma M, Li H, Yao R, Li Y, Wang G, Geng P, et al. The degradation of EZH2 mediated by lncRNA ANCR attenuated the invasion and metastasis of breast cancer. Cell Death Differ. 2017;24(1):59–71.
Chu W, Zhang X, Qi L, Fu Y, Wang P, Zhao W, Du J, Zhang J, Zhan J, Wang Y, et al. The EZH2-PHACTR2-AS1-Ribosome Axis induces Genomic Instability and Promotes Growth and Metastasis in Breast Cancer. Cancer Res. 2020;80(13):2737–50.
Tay Y, Rinn J, Pandolfi PP. The multilayered complexity of ceRNA crosstalk and competition. Nature. 2014;505(7483):344–52.
Li Y, Yan X, Ren L, Li Y. miR-644a Inhibits Cellular Proliferation and Invasion via Suppression of CtBP1 in Gastric Cancer Cells. Oncol Res. 2018;26(1):1–8.
Zhang JX, Chen ZH, Xu Y, Chen JW, Weng HW, Yun M, Zheng ZS, Chen C, Wu BL, Li EM, et al. Downregulation of MicroRNA-644a Promotes Esophageal Squamous Cell Carcinoma Aggressiveness and Stem Cell-like Phenotype via Dysregulation of PITX2. Clin Cancer Res. 2017;23(1):298–310.
Ebron JS, Shankar E, Singh J, Sikand K, Weyman CM, Gupta S, Lindner DJ, Liu X, Campbell MJ, Shukla GC. MiR-644a Disrupts Oncogenic Transformation and Warburg Effect by Direct Modulation of Multiple Genes of Tumor-Promoting Pathways. Cancer Res. 2019;79(8):1844–56.
Raza U, Saatci O, Uhlmann S, Ansari SA, Eyupoglu E, Yurdusev E, Mutlu M, Ersan PG, Altundag MK, Zhang JD, et al. The miR-644a/CTBP1/p53 axis suppresses drug resistance by simultaneous inhibition of cell survival and epithelial-mesenchymal transition in breast cancer. Oncotarget. 2016;7(31):49859–77.
Liu FY, Safdar J, Li ZN, Fang QG, Zhang X, Xu ZF, Sun CF. CCR7 regulates cell migration and invasion through MAPKs in metastatic squamous cell carcinoma of head and neck. Int J Oncol. 2014;45(6):2502–10.
Du R, Tang G, Tang Z, Kuang Y. Ectopic expression of CC chemokine receptor 7 promotes prostate cancer cells metastasis via Notch1 signaling. J Cell Biochem. 2019;120(6):9639–47.
We would like to thank all laboratory members for their critical discussion of this manuscript.
This project was supported by grants from the Natural Sciences Foundation of Hubei Province (2021CFB230), the Yellow Crane Talent Plan Foundation, Research Fund of Wuhan Public Health Bureau (WZ22Q44 and WX21Q49).
Ethics approval and consent to participate
The present study was approved by the Ethics and Scientific Committees of the Central Hospital of Wuhan and complied with the Declaration of Helsinki. All procedures involving animal care and use were approved by the Institutional Animal Care and Usage Committee of Huazhong University of Science and Technology, and were in accordance with the National Policy on Use of Laboratory Animals.
Consent for publication
The authors declare that they have no competing interests.
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
This article has been retracted. Please see the retraction notice for more detail:https://doi.org/10.1186/s13046-023-02658-5
Additional file 1:
Supplementary Figure 1. The expression of LncRNA BCAR4 on breast cancer subclasses from TCGA database. Supplementary Figure 2. The expression of ABCB1 and LncRNA BCAR4 in MCF-7, MCF-7/DOX, MDA-MB-231 and MDA-MB-231/DOX cell lines. Supplementary Figure 3. MDA-MB-231 transfected with pcDNA-BCAR4 and/or miR-644a mimic was treated with gradient doses of DOX(5 nM, 10 nM, 30 nM, 100 nM, 200 nM, 500 nM, 1000 nM and 2000 nM). Cell viability and IC50 were detected by CCK8 assay (n=5). The concentration of DOX (X axis) was presented on a logarithmic scale.
Rights and permissions
Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.
About this article
Cite this article
Wu, T., Li, X., Yan, G. et al. RETRACTED ARTICLE: LncRNA BCAR4 promotes migration, invasion, and chemo-resistance by inhibiting miR-644a in breast cancer. J Exp Clin Cancer Res 42, 14 (2023). https://doi.org/10.1186/s13046-022-02588-8
- Breast cancer