- Open Access
Racial differences in RAD51 expression are regulated by miRNA-214-5P and its inhibition synergizes with olaparib in triple-negative breast cancer
Breast Cancer Research volume 25, Article number: 44 (2023)
Triple-negative breast cancer (TNBC) affects young women and is the most aggressive subtype of breast cancer (BC). TNBCs disproportionally affect women of African-American (AA) descent compared to other ethnicities. We have identified DNA repair gene RAD51 as a poor prognosis marker in TNBC and its posttranscriptional regulation through microRNAs (miRNAs). This study aims to delineate the mechanisms leading to RAD51 upregulation and develop novel therapeutic combinations to effectively treat TNBCs and reduce disparity in clinical outcomes.
Analysis of TCGA data for BC cohorts using the UALCAN portal and PrognoScan identified the overexpression of RAD51 in TNBCs. miRNA sequencing identified significant downregulation of RAD51-targeting miRNAs miR-214-5P and miR-142-3P. RT-PCR assays were used to validate the levels of miRNAs and RAD51, and immunohistochemical and immunoblotting techniques were used similarly for RAD51 protein levels in TNBC tissues and cell lines. Luciferase assays were performed under the control of RAD51 3’-UTR to confirm that miR-214-5P regulates RAD51 expression. To examine the effect of miR-214-5P-mediated downregulation of RAD51 on homologous recombination (HR) in TNBC cells, Dr-GFP reporter assays were performed. To assess the levels of olaparib-induced DNA damage responses in miR-214-5P, transfected cells, immunoblots, and immunofluorescence assays were used. Furthermore, COMET assays were used to measure DNA lesions and colony assays were performed to assess the sensitivity of BRCA-proficient TNBC cells to olaparib.
In-silico analysis identified upregulation of RAD51 as a poor prognostic marker in TNBCs. miRNA-seq data showed significant downregulation of miR-214-5P and miR-142-3P in TNBC cell lines derived from AA women compared to Caucasian-American (CA) women. miR-214-5P mimics downregulated RAD51 expression and induces HR deficiency as measured by Dr-GFP assays in these cell lines. Based on these results, we designed a combination treatment of miR-214-5P and olaparib in HR-proficient AA TNBC cell lines using clonogenic survival assays. The combination of miR-214-5P and olaparib showed synergistic lethality compared to individual treatments in these cell lines.
Our studies identified a novel epigenetic regulation of RAD51 in TNBCs by miR-214-5P suggesting a novel combination therapies involving miR-214-5P and olaparib to treat HR-proficient TNBCs and to reduce racial disparity in therapeutic outcomes.
Triple-negative breast cancer (TNBC) constitutes 15–20% of all breast cancers (BC), occurs at a young age (< 40 years), is more aggressive, and contributes to a relatively higher proportion of BC-related deaths in women. TNBCs are negative for both the estrogen receptor (ER) and progesterone receptor (PR) and do not overexpress epidermal growth factor receptor 2 (EGFR-2 or HER-2), which classify and define therapeutic modalities. Consequently, no targeted therapies are currently available for these patients [1,2,3,4]. Thus, most of these patients undergo cytotoxic chemotherapy; however, the responses are short-lived and the majority of patients develop resistance and undergo relapse [5,6,7]. Moreover, TNBC disproportionally affects women of African-American (AA) descent compared to other ethnicities and show the worst clinical outcomes in this population [8, 9]. The health statistics data indicate that the occurrence of TNBCs is over two times higher for AA women compared to women of European or Caucasian ancestry (EA) [10, 11]. Although socioeconomic  and comorbidity factors contribute to differences in clinical outcomes, the higher prevalence of TNBCs in young AA women, combined with higher death rates, suggests different molecular factors influence this disease .
RAD51 is a key downstream factor to BRCA1/2-mediated DNA repair  and facilitates the homology search to repair DNA double-strand breaks (DSBs)  in coordination with ATM/ATR-mediated DNA damage checkpoint responses . Moreover, RAD51 upregulation is evident in many cancers , including BCs , and is implicated in the development of tumor resistance to chemotherapeutics [19, 20]. Our previous studies showed that RAD51 is upregulated in TNBCs, and its expression is significantly higher in AA TNBCs compared to EA TNBC patients and correlates with a poor prognosis relative to EA TNBC patients . However, the mechanisms behind the upregulation of RAD51 in these patients have not been identified, particularly those contributing to the observed racial disparity.
The Food and Drug Administration (FDA) has approved PARP inhibitors (PARPi) olaparib  and talazoparib  for the treatment of BRCA-deficient BC [24, 25]. Although PARP inhibitors have shown promise, their use and effectiveness are limited to only 15–20% of TNBC patients, particularly those with BRCA mutations or homologous recombination (HR) DNA repair deficiency (HRD). In these patients, PARPi effectiveness is attributed to synthetic lethality , a process wherein the simultaneous perturbation of two compensatory genes or pathways causes cell death . Therefore, the majority (~ 80%) of the TNBC patients do not benefit from PARPi-targeted therapies. The increased expression of RAD51 observed in TNBCs, particularly in AA patients, may facilitate increased DNA repair and resistance, leading to therapeutic relapse and poor outcomes [9, 28]. Moreover, currently available data on biological and genetic differences do not explain the high mortality rate for AA women nor decode the genetic and epigenetic mechanisms that cause these discrepancies.
MicroRNAs (miRNAs) are short, non-coding RNAs with a length of 18–23 nucleotides that act as epigenetic regulators of gene expression . About 2000 miRNAs have been discovered in humans, and they regulate one-third of the human genome . miRNAs are associated with numerous human diseases and are being investigated as clinical diagnostics and therapeutic targets, particularly involving cancer progression and their influence on therapeutic responses . These observations prompted us to perform miRNA-seq analysis to find differentially regulated miRNAs in DNA repair-proficient TNBC cell lines representing both AA and EA ethnicities, particularly those targeting the RAD51 gene. Our present study identified the loss of miRNA-214-5P (miR-214-5P) in TNBCs, particularly in AA TNBC cell lines and tumors. Therefore, the goals of our current studies are to delineate miRNA-mediated epigenetic mechanisms leading to RAD51 upregulation and develop novel therapeutic combinations based on these biomarkers to effectively treat BRCA-proficient TNBCs and reduce disparity in outcomes.
In this study, our data show that miR-214-5P regulates RAD51 and suggests that this epigenetic regulation of RAD51 contributes to disparities in TNBC’s therapeutic outcomes. Additionally, our data demonstrate that miR-214-5P-mediated downregulation of RAD51 causes HRD, and synergistic lethality with the PARPi, olaparib, in HR-proficient TNBC cells.
Materials and methods
TNBC patient cohort and sample collection
To identify the RAD51 and miRNAs (miR-214-5P and miR-142-3P) differential expression in TNBCs of AA and EA women, we performed RT-PCR on a TNBC cohort. Formalin-fixed, paraffin-embedded (FFPE) TNBC samples were obtained from the Division of Anatomic Pathology in the University of Alabama at Birmingham (UAB). The UAB Institutional Review Board approved the collection and use of all samples in this study (IRB number: 060911009). Before RNA extraction, pathologists macro-dissected all tumors and corresponding normal regions.
Cell lines, culture method, and reagents
The human TNBC cell lines MDAMB231, MDAMB453, HCC1806, and MDAMB468 were purchased from ATCC, Manassas, VA. These cells were cultured in Dulbecco’s modified Eagle medium (Corning, Manassas, VA) supplemented with 10% fetal bovine serum (Omega Scientific Inc., Tarzana, CA) and 1% penicillin–streptomycin (50 U/mL, 50 μg/mL) (Invitrogen, Eugene, OR). Olaparib (Selleckchem, Houston, TX) was dissolved in DMSO and used at 25 µM for 24 h in western blot, COMET assay and immunofluorescence experiments. For the colony assay, 0–4 µM of Olaparib was used. Primary antibodies for the following were used for western blotting: pH2AX (Cat No: 05636, Millipore), RAD51 (Cat No: 8875, Cell Signaling) and GAPDH (Cat No: 32233, Santa Cruz).
Total RNA was isolated from AA (MDAMB468 and HCC1806) and EA (MDAMB231 and MDAMB453) TNBC cell lines and outsourced to Admera Health LLC for analysis. miRNA-seq data were trimmed with Cutadapt default parameters and then aligned with miRDeep2 and Bowtie. Quantification of miRNA expression was performed with miRDeep2. DE-Seq2 determined differentially expressed miRNAs. PCA plots and heatmaps were generated using R. miRNA sequencing was performed once, and RT-PCR further confirmed the results in three independent experiments.
Control miRNA (CN-001000-01-20), miR-214-5P (C-301153-01-0020), and miR-142-3P (C-300610-03-0005) were procured from Horizon Discovery Biosciences Limited. miRNAs were transfected using Lipofectamine RNAiMAX (Life Technologies, Eugene, OR) based on the protocol supplied by the manufacturer.
HR Dr-GFP assay
As previously described , the Dr-GFP reporter assay measures HR activity. pDR-GFP and pCBASceI were gifts from Maria Jasin (Addgene plasmids # 26477 and # 26475, respectively), Addgene (Watertown, MA). In brief, MDAMB468 cells were stably transfected with pDr-GFP and selected for puromycin resistance (2 μg/mL). Upon 60% confluence, the stably transfected cells were transfected with the plasmid I-Sce1 and a miR-control or miR-214-5P. Restriction enzyme I-Sce1 cuts the reporter plasmid and initiates GFP expression when the damage is repaired by HR. After transfection of miRNAs for 24 h, GFP-positive cells were measured by flow cytometry using a BD Accuri (BD Biosciences) flow cytometer. A total of three independent experiments were performed.
Protein expression by western blot
The cells were placed on ice and washed twice with ice-cold PBS, and cell lysates were collected using cytoskeletal (CSK) buffer (10 mM PIPES at pH 6.8, 100 mM NaCl, 300 mM sucrose, 3 mM MgCl2, 1 mM EGTA, 0.1 mM ATP, 0.1% Triton X-100 freshly supplemented with 1 mM dithiothreitol, 1 × protease, and phosphatase inhibitors with EDTA). Protein content was measured with Bradford reagent, and the proteins were equilibrated using CSK buffer with 6 × Laemmli buffer and heated at 100 °C for 15 min. The proteins were resolved on gradient polyacrylamide gels and then transferred onto nitrocellulose membranes using the BioRad Trans-Blot Turbo system. The membranes were blocked with a 2.5% blocking grade blocker (BioRad, USA) in 1 × Tris-buffered saline in 0.1% Tween 20 (TBST) and incubated with the primary antibody overnight on a rocking platform at 4 °C. Membranes were then washed three times with 1 × TBST before being incubated for an hour at room temperature with the secondary antibody. The membranes were then washed three times with 1 × TBST, exposed to Western lightning plus ECL (PerklinElmer, USA), and developed in a dark room with Konica Minolta equipment.
Cell cycle analysis
After transfection with miR-control or miR-214-5P, cells were trypsinized, collected in tubes, spun down, and washed with ice-cold PBS. Cells were then suspended in ice-cold ethanol and incubated overnight at − 20 °C. After incubation, cells were washed with PBS, stained with propidium iodide (PI) (Invitrogen, Eugene, OR) in the presence of RNAse A (Thermo Scientific), and processed for cell cycle profiles by flow cytometry using a BD Accuri (BD Biosciences) flow cytometer. ModFit LT 5.0 software was used to analyze the cell cycle profiles. A total of three independent experiments were performed.
Tissue sections were incubated with RAD51 (Cat No: 8349, Santa Cruz), followed by a specific biotinylated secondary antibody (1:250 dilution) using the kit from Vectastain (PK-6101), and then with conjugated horse radish peroxidase-streptavidin and 3′3-diaminobenzidine HCl chromogen, and tissues were counterstained with hematoxylin. Stained sections were imaged with an AxioCam camera, and RAD51 intensities were analyzed with a Zeiss Axioscope microscope as described previously . A total of three independent experiments were performed.
RNA isolation and real-time PCR
Using a ZYMO Research (Irvine, CA) Quick-RNA MiniPrep RNA isolation kit, total RNA was extracted from cells transfected with miR-control or miRNA-214-5P. RNA (1 µg) was reverse-transcribed using a high-capacity cDNA reverse transcription kit (Invitrogen, Eugene, OR), as per the manufacturer’s protocol and as described previously . The primers for amplified genes (RAD51 and GAPDH) were purchased from Bio-Rad. Amplification of PCR products was quantified using SYBR green dye (ABI), and fluorescence was monitored on a QuantStudio 12 K Flex detection system. Melting curve analysis was accomplished for each amplicon. Quantitation was done using the 2−ΔΔCt technique with GAPDH as an endogenous control. A total of two independent experiments in triplicate were performed in patients’ samples. A total of three independent experiments were performed in cell lines.
First, the cells were seeded into FluoroDishes (World Precision Instruments) and incubated overnight for adherence. After treatment, cells were fixed with 4% formaldehyde for 10 min at room temperature. The cells were permeabilized with PBS containing 0.2% Triton X-100 for 3 min. The cells were washed and blocked with 10% goat serum in PBS for 40 min. After three PBS washes, cells were incubated overnight at 4 °C with primary antibodies (RAD51: Cat No: 8349, Santa Cruz or pH2AX: Cat No: 05636, Millipore) in PBS, then incubated with a fluorescent secondary antibody (Molecular Probes, Eugene, OR) for 2 h at room temperature. The cells were mounted with Vectashield containing 4′,6-diamidino-2-phenylindole and analyzed for foci formation using a Nikon Eclipse TE confocal microscope. A total of three independent experiments were performed.
Comet chip assay
As previously reported  and following the manufacturer’s instructions, comet assays were performed under alkaline conditions using Comet Chip Assay Kits (Trevigen, Gaithersburg, MD). The cells transfected with miR-control or miR-214-5P were treated for 24 h with DMSO or 25 μM olaparib, harvested, suspended at a concentration of 1 X 105 cells/ml in PBS combined with 1% low melting point agarose at a ratio of 1:10 (v/v). The Comet chip was immersed in a lysis solution for 30 min and electrophoresed in a horizontal electrophoresis apparatus containing an alkaline solution. The chip was then neutralized with 0.4 M and 20 mM Tris–HCl, pH 7.4, and stained with SYBR gold overnight. The chip was again de-stained with 20 mM Tris–HCl, pH 7.4, to visualize cellular DNA with a Zeiss Axio fluorescence microscope. Fluorescence images were analyzed using the ImageJ comet plugin to demarcate each comet's “head” and “tail” regions. The comet tail areas were measured, and calculations were averaged from three independent experiments.
Clonogenic survival assay
For high-density and low-density colony formation assays, 5 × 103 cells per well and 5 × 102 cells per well, respectively, were seeded into 6-well culture plates and incubated overnight for adherence. Cells transfected with miR-control or miR-214-5P were then treated with DMSO or various concentrations of olaparib and cultured for colony formation over 7–10 days. After colony formation, the growth medium was removed, and cells were washed with ice-cold PBS three times and then fixed in ice-cold methanol for 5 min. Methanol was removed, and 1% w/v crystal violet (Invitrogen, Eugene, OR) was added for staining. After 10 min, the wells were washed under tap water, and the plates were allowed to dry at room temperature. Colonies were then imaged and counted using ImageJ software. A total of three independent experiments were performed.
Luciferase promoter assay
Individual GoClone (Luciferase with a RAD51 3’-untranslated region (3’UTR) (Cat No: S807477, Active Motif) constructs were purchased from Switch Gear Genomics. Luciferase assays were performed for MDAMB468 and HCC1806 cells using a LightSwitch Assay System kit (Cat No: LS010, Switch Gear Genomics) according to the manufacturer’s protocol. In brief, TNBC cells were transfected with miR-control or miR-214-5P for 48 h and, for the last 24 h, co-transfected with the vector expressing luciferase mRNA with the RAD51 3’UTR. After transfection/treatment, luciferase activity in the cells was measured using LightSwitch Assay reagents with a Tecan microplate reader. A total of three independent experiments were performed.
The prognostic landscapes of RAD51 in BCs were identified from the PrognoScan database. The expression profiles of RAD51 in BCs were identified from analysis of the TCGA database using the UALCAN portal.
Student’s t-test was performed to estimate statistical significance using GraphPad Prism 8.0 software.
RAD51 is overexpressed in TNBCs, particularly those of AA women, and its high expression correlates with a poor prognosis
We recently showed that RAD51 is upregulated in BCs, particularly in TNBCs . To determine whether RAD51 could be a prognostic marker for BCs, we utilized RAD51 expression data from PrognoScan for a meta-analysis. PrognoScan uses the minimum p-value approach for the grouping of patients. Patients are separated into two groups (high and low expression) at each cutpoint, and the risk differences between the two groups are assessed using the log-rank test. The best cutpoint with the most pronounced p-value is then chosen to determine high and low expression . Our analysis of RAD51 expression data for BC showed that patients with high RAD51 levels (n = 110) had low overall survival compared to the patients with low RAD51 (n = 88) (Fig. 1A). Similarly, patients with high RAD51 expression (n = 100) also had lower disease-free survival compared to the patients with low RAD51 expression (n = 149) (Fig. 1B). Consistently, BC patients with high RAD51 levels (n = 192) showed lower metastasis-free survival and relapse-free survival (n = 72) compared to the patients with low RAD51 (n = 94 and n = 87), respectively (Fig. 1C, D). We expanded our analysis to explore the expression of RAD51 protein levels in BC using the UALCAN portal. Interestingly, RAD51 levels were upregulated in the TNBC subtype (n = 16), compared to normal breast tissues (n = 18) and other subtypes (luminal: n = 64; HER2-positive: n = 10) of BC (Fig. 1E). Additionally, these data indicate relatively high RAD51 protein levels in TNBCs of AA (n = 18) patients compared to those of EAs (n = 80) (Fig. 1F). To validate CPTAC data, we analyzed RAD51 expression levels in TNBCs of our bio-specimen repository using RT-PCR and immunohistochemistry (IHC). Consistently, our results show that RAD51 transcript levels were significantly higher in AA TNBC (n = 26) samples compared to EA TNBC (n = 26) (Fig. 2A). Age at diagnosis and stage of TNBC for the samples used in Fig. 2A are listed in Table 1, showing no distinctive difference between the AA (n = 26) and EA (n = 26) TNBCs. Similarly, RAD51 protein levels were also significantly higher in AA TNBC (n = 5) specimens compared to EA TNBC (n = 5) as evidenced by the IHC data (Fig. 2B, C). These data indicate an important role for RAD51 in TNBC progression and prognosis. Additionally, RAD51’s distinct expression in TNBCs of AA and EA ethnicity implicates a role in racial disparities in these patients. However, the mechanisms that influence RAD51 expression in TNBCs, and its distinctive regulation in AA TNBC patients are not known.
RAD51 is epigenetically regulated in TNBCs, and loss of miR-214-5P and miR-142-3P leads to RAD51 upregulation
Recent studies from non-coding RNAs (ncRNAs), including miRNAs, indicate their differential regulation in different cancers and ethnicities, including TNBCs, and their attribution to disease progression and prognosis [35, 36]. To identify differentially regulated miRNAs, particularly those that target and epigenetically regulate RAD51, we performed miRNA-seq in two pairs of cell lines from each AA (MDAMB468 and HCC1806) and EA (MDAMB231 and MDAMB453)-derived TNBCs. A list of the top 30 miRNAs that were differentially regulated in AA vs EA TNBC cell lines are presented in Fig. 2D. The cell line-wise miRNA expression heatmap is shown in supplementary Fig. 1 (Additional file 1: Figure S1) and the list of miRNAs that are differentially expressed in AA TNBCs (MDAMB468 and HCC1806), compared to EA (MDAMB231 and MDAMB453) TNBCs, is presented in Table 2. To distinguish the upregulated and downregulated miRNAs based on the fold change and p-values, we presented a volcano plot analysis of the miRNA-seq data (Fig. 2E). Analysis of these data using the miRNA Target Base portal identified miR-214-5P and miR-142-3P as RAD51-targeting miRNAs based on the presence of their seed sequences (Fig. 2F). To validate these cell line data, we analyzed the expression of these miRNAs (miR-214-5P and miR-142-3P) in TNBC samples using RT-PCR. As shown in Fig. 2G, H, the expression of miR-142-3P and miR-214-5P was significantly downregulated in AA TNBCs (n = 16) compared to EA TNBCs (n = 16), respectively. Additionally, analysis of the TCGA database using the UALCAN portal further validated that miR-214 is significantly downregulated in AA TNBCs (n = 160) compared to EA TNBCs (n = 578) (Fig. 2I).
miR-214-5P downregulates RAD51 in BRCA wild-type/proficient TNBC cells
We first determined whether expression levels of miR-214-5P and miR-142-3P negatively correlate with RAD51 expression in racially distinctive TNBC cell lines by performing RT-PCR analysis. As shown in Fig. 3A, the RAD51 levels were significantly higher in two pairs of AA in TNBC cell lines compared to their respective EA counterparts, except the comparison between MDAMB453 and MDAMB468. On the contrary, miR-142-3P (Fig. 3B) and miR-214-5P (Fig. 3C) were downregulated in AA TNBC cell lines compared to the EA TNBC cell lines. Collectively, these data indicate a negative correlation between specific miRNAs (miR-214-5P and miR-142-3P) and RAD51 levels in two pairs of AA and EA TNBC cell lines (Fig. 3A–C). To investigate whether miR-214-5P and miR-142-3P target RAD51 and downregulate its expression, MDAMB468 cells were transfected with miR-control, miR-214-5P, or miR-142-3P and analyzed for RAD51 protein levels by western blots at different time points after the transfection. As expected, miR-214-5P-transfected cells showed 70% to 90% downregulation of RAD51 in the time points evaluated (Fig. 3D; Additional file 2: S2A). However, miR-142-3P was less efficient in RAD51 downregulation than miR-214-5P (Fig. 3E; Additional file 2: S2B). This could be due to multiple factors, including the presence of binding sites for miR-214-5P in two different regions (1522–1528 and 1702–1708) of RAD51 3’UTR (shown by miRNA Target Base analysis), which might influence efficient targeting. Therefore, further studies were focused only on using miR-214-5P. To rule out that these results were not cell line-specific, we further examined multiple TNBC cell lines. Consistent with MDAMB468 cells’ data (Fig. 3D), miR-214-5P downregulated the expression of RAD51 in MDAMB453 and HCC1806 TNBC cell lines (Fig. 3F). RAD51 is a critical factor in BRCA and associated protein complex mediated repair of DNA double-strand breaks (DSBs) and maintains the integrity of chromosomal DNA in replicating cells [37,38,39,40]. Since non-replicating cells tend to have low expression of the HR proteins, it is essential to rule out the contribution of any cell cycle discrepancies in miR-214-5P-transfected cells to RAD51 downregulation. In fact, our flow cytometry results from miR-214-5P-transfected MDAMB468 cells showed increased accumulation of cells in the S-phase compared to the miR-control-transfected cells (Fig. 3G; Additional file 2: S2C and S2D), which rules out the contribution of any cell cycle discrepancies to RAD51 downregulation.
miR-214-5P targets 3’UTR region of RAD51 and regulates its mRNA levels
To confirm that miR-214-5P downregulates RAD51 by targeting the 3’UTR region of the transcript, we performed luciferase assays (Promega). A luciferase reporter was designed to include the luciferase ORF under a CMV promoter, followed by the RAD51 3’UTR region, which has the predicted seed sequences (1522–1528 and 1702–1708) for miR-214-5P (Fig. 4A). Co-transfection of the luciferase reporter with either miR-control or miR-214-5P showed > 50% decreased luciferase activity in miR-214-5P-transfected MDAMB468 and HCC1806 TNBC cells compared to the miR-control (Fig. 4B). Consistently, RT-PCR data showed downregulation of RAD51 in miR-214-5P-transfected cells compared to control miRNAs (Fig. 4C), which confirms that miR-214-5P targets the 3’UTR region of RAD51 mRNA and regulates RAD51 posttranscriptionally. Additionally, this seed sequence is conserved in RAD51 mRNA (1522–1528) in different primates (Additional file 3: Figure S3A), indicating a conserved mechanism of epigenetic regulation.
miR-214-5P downregulates RAD51 and induces HR deficiency (HRD) in BRCA wild-type/proficient TNBC cells
RAD51 is an essential factor of a multi-protein complex of BRCA and related proteins that most accurately repair replication-associated DSBs [37,38,39,40]. Thus, deficiency or inhibition of RAD51 affects cells’ ability to repair DSBs due to HR deficiency [21, 40]. We performed Dr-GFP reporter assays to determine whether miR-214-5P-mediated downregulation of RAD51 affects TNBC cells’ efficiency to repair DSB by HR [41, 42]. We used BRCA-proficient MDAMB468 cells to transfect with the Dr-GFP plasmid and selected them in a puromycin selection medium. These Dr-GFP-expressing cells were then co-transfected with an ISCE-1 expression plasmid to induce DSBs and either miR-control or miR-214-5P and then analyzed for GFP expression using a flow cytometer. Consistent with RAD51 downregulation, miR-214-5P-treated MDAMB468 cells showed > 50% reduction in HR efficiency (p < 0.001) compared to the miR-control-transfected cells (Fig. 5A, B).
To further examine whether miR-214-5P-induced HRD in DNA repair-proficient TNBC cells causes increased DNA damage in response to PARPi olaparib, we transfected two RAD51 upregulated AA-derived TNBC cell lines (MDAMB468 and HCC1806) with miR-control or miR-214-5P and treated them with vehicle or olaparib. Consistent with the DNA repair deficiency, miR-214-5P-transfected MDAMB468 and HCC1806 cells showed downregulation of RAD51 and increased expression of pH2AX (p < 0.0001) protein levels in response to olaparib when compared to their respective miR-control-transfected cells treated with olaparib (Fig. 5C, D).
To further confirm that miR-214-5P-mediated downregulation of RAD51 also affects its repair foci formation in olaparib-induced DNA damage, we examined for the levels of repair foci of RAD51 and pH2AX in HCC1806 cells (Fig. 6A, C). In miR-control-transfected cells, RAD51 and pH2AX foci were undetectable. Consistent with the western blot data (Fig. 5C, D), olaparib treatment increased the number of RAD51 and pH2AX foci compared to DMSO-treated cells. Furthermore, for the miR-214-5P and olaparib combination, miR-214-5P attenuated olaparib-induced RAD51 foci formation compared to the miR-control and olaparib combination (Fig. 6B). Contrarily, pH2AX foci levels were elevated upon olaparib treatment in control cells and were further elevated by the miR-214-5P and olaparib combination, indicating increased DNA damage in these cells (Fig. 6D). These data confirm that the combination of miR-214-5P and olaparib downregulates RAD51 and increases DNA damage as measured by pH2AX.
miR-214-5P synergizes with PARPi olaparib in BRCA wild-type/proficient TNBC cells
To validate that increased pH2AX is due to increased DNA lesions, we performed alkaline COMET assays in these cell lines (MDAMB468 and HCC1806) (Fig. 7A; Additional file 3: S3B). In agreement with the pH2AX levels, miR-214-5P-transfected MDAMB468 and HCC1806 cells showed significantly elevated levels of COMET tails (p < 0.0001) in response to olaparib when compared to their respective miR-control-transfected cells treated with vehicle or olaparib (Fig. 7B, C). Together, these results suggest that miR-214-5P downregulates the HR protein RAD51, causes HR repair deficiency, and potentiates olaparib-induced DNA damage in BRCA-proficient TNBC cells. Furthermore, these data also propose a novel synergistic lethality mechanism involving miR-214-5P and olaparib combination therapy, similar to BRCA-mutant or HR-deficient TNBCs.
To confirm that the miR-214-5P-mediated downregulation of RAD51 causes synergistic lethality with the PARPi olaparib, we first performed a high-density colony assay using HCC1806 cells transfected with miR-control or miR-214-5P, and treated them with various concentrations of olaparib. As shown in Fig. 7D, HCC1806 cells transfected with miR-control showed a dose-dependent decrease in the colony-forming ability of these cells. Consistent with the DNA repair deficiency, miR-214-5P-transfected cells showed decreased colony formation capacity relative to miR-control-treated cells in response to olaparib at all the tested drug concentrations. To further confirm, we performed low-density colony assays using MDAMB468 (Fig. 7E) and HCC1806 cells transfected with miR-control or miR-214-5P, treated with or without various concentrations of olaparib and plotted the survival fractions (Fig. 7F, G). The miR-214-5P-transfected cells showed significantly decreased colony formation compared to miR-control-treated cells in response to olaparib at all the tested drug concentrations. Together, these data suggest a novel synergistic lethality combination of miR-214-5P and olaparib to effectively treat BRCA-proficient TNBCs that could aid in decreasing disparities in the therapeutic outcomes of TNBCs.
Epidemiological and other population-based studies show the existence of racial disparities relating to BC progression and therapy outcomes in AA ethnic population . In particular, the occurrence of TNBC, the most lethal form of BC, is 2.21 times higher for AA women compared to EA women, and it affects them at a younger age and has a worse prognosis [10, 11, 44, 45]. Several biological and non-biological factors have been identified to influence racial disparities . Biologically, obesity and waist–hip ratio (WHR) have been identified as increased risk for AA TNBC patients. The prevalence of TNBC in overweight and obese AA women is twofold compared to that in normal-weight AA women , and these studies are further supported by many studies implicating WHR as a strong risk factor for TNBC in AA women . However, specific endpoints like somatic mutations in prominent genes like TP53 (46% of AA patients, compared to 27% in EA patients) have been documented as the racial disparity in the overall cancer incidence but not in TNBC . These observations confirm the need to identify more potential mechanistic underpinnings that cause a racial disparity in TNBC.
MiRNAs have been identified as a potential regulator of racial disparity in multiple cancers [50,51,52], including TNBC [53, 54]. Our present study identifies that the loss of miR-214-5P, which epigenetically regulates RAD51, could be a marker for a poor prognosis in TNBC patients. It could contribute to our understanding of racial disparities in TNBCs. Particularly, miR-214-5P’s role has been documented in proliferation , migration, invasion , aggressive tumor growth  and resistance  in multiple cancers. Our studies identified a novel role for miR-214-5P in regulating critical DNA repair gene RAD51 and suggested that loss of it in AA TNBCs upregulates RAD51 and affects their response to therapeutics. As miR-214-5P is significantly decreased in AA TNBCs compared to EA TNBCs, it indicates its contribution to racial disparities in TNBC therapeutic outcomes and could serve as a biomarker for these patients. Notably, the mechanism identified in our study paves a way to treat BRCA wild-type TNBC patients who do not benefit from single-agent PARPi-targeted therapies.
Upon treatment with PARPi, cells with HRD display extensive DNA lesions and synthetic lethality . PARP inhibitors are a class of agents approved first for BRCA-deficient ovarian cancer and BC and later for other cancers . The biochemical effects of deleterious germline mutations in BRCA1 and BRCA2 compromise their essential role in maintaining functional, high-fidelity DNA repair via homologous recombination, indicating their potential clinical utility [61, 62]. However, later studies established that not only BRCA genes but proteins with loss-of-function mutations involved in HR (including RAD51, FANCD2, and PALB2) also synergize with PARPi [63,64,65]. However, PARP inhibitors are currently effective only in 15–20% of TNBC patients with genetically compromised HR. Several researchers have identified alternative ways to induce HR non-genetically by targeting various proteins involved in HR, including RAD51 [66,67,68]. Accordingly, our previous study identified that CHK1 inhibitor prexasertib inhibits RAD51 and synergizes with olaparib . Similarly, inhibition of Myc , glycosylase OGG1 , nicotinamide phosphoribosyltransferase , CDK  and topoisomerase 1  have been shown to combine with PARPi in TNBCs synergistically. However, none of these combinations addresses the potential racial disparity observed in AA TNBC patients. Based on our results, we propose a novel synergistic lethality mechanism involving the combination of miR-214-5P and PARPi to effectively treat TNBC, especially in AA TNBC patients, as they express significantly low miR-214-5P and increased RAD51 compared to EA TNBCs. Additionally, a recent clinical study evaluating olaparib against metastatic BCs in patients with BRCA mutations shows promising results . Since olaparib is already FDA-approved, targeting RAD51 either by miRNAs (e.g., miR-214-5P) or by drugs that promote its degradation would be an effective combination to treat HR-proficient TNBCs to overcome therapeutic resistance.
Our studies provide preclinical evidence for targeting RAD51 by miR-214-5P to induce HR deficiency in BRCA-wild-type or HR-proficient TNBCs and shows synergistic lethality with an FDA-approved PARPi olaparib. Since significantly decreased expression of miR-214-5P and compensatory upregulation of RAD51 were observed in AA TNBCs compared to EA TNBCs, these observations could serve as biomarkers for racial disparity in TNBC. Importantly, these results also suggest that a novel combination of miR-214-5P and olaparib could be an effective therapy for BRCA-wild-type TNBCs and reduce racial disparities in TNBC outcomes.
Availability of data and materials
All data generated or analyzed during this study are included in this research article and its supplementary information files.
Triple-negative breast cancer
The Cancer Genome Atlas
The University of Alabama at Birmingham Cancer Data Analysis Portal
Green fluorescent protein
Human epidermal growth factor receptor 2
Breast cancer susceptibility protein
Breast cancer gene 1/2
Ataxia telangiectasia and rad3-related
DNA double-strand break
Food and Drug Administration
Poly-ADP ribose polymerase (PARP) inhibitors
Homologous recombination deficiency
Real-time polymerase chain reaction
Ethylenediamine tetra acetic acid
Ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetra acetic acid
Tris-buffered saline in 0.1% tween 20
Cancer stem cell
Checkpoint kinase 1
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This work was supported by grants from Weitlauf Endowment for Cancer Research and partly by cancer health disparities supplements to NIH grant CA219187 (KP), and by CPRIT: Texas Regional Excellence in Cancer Award RP210154 to CM.
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Additional file 1.
Column heat map analysis of miRNA sequencing between EA (MDAMB231 and MDAMB453) and AA (MDAMB468 and HCC1806) cells.
Additional file 2.
miR-214-5P regulates the expression of RAD51 in a cell cycle independent manner. (A) Densitometry analysis for RAD51 expression in MDAMB468 cells transfected with miR-214-5P in three independent experiments are represented in the histogram with standard deviation as error bars. (B) Densitometry analysis for RAD51 expression in MDAMB468 cells transfected with miR-142-3P in three independent experiments isrepresented in the histogram with standard deviation as error bars. Cell cycle profile of MDAMB468 cells treated with miR-control (C) or miR-214-5P (D).
Additional file 3.
Bioinformatic analysis of protein sequence conservity. (A) Protein alignment analysis using UNIPORT shows a highly conserved seed sequence 1522- 1528 in RAD51 mRNA. (B) Comet assay representative images in HCC1806 cells transfected with miR-control or miR-214-5P and treated with or without 25M olaparib for 24 h.
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Mani, C., Acharya, G., Saamarthy, K. et al. Racial differences in RAD51 expression are regulated by miRNA-214-5P and its inhibition synergizes with olaparib in triple-negative breast cancer. Breast Cancer Res 25, 44 (2023). https://doi.org/10.1186/s13058-023-01615-6
- Racial disparity
- HR deficiency