Progesterone modulates the DSCAM-AS1/miR-130a/ESR1 axis to suppress cell invasion and migration in breast cancer

Background A preoperative-progesterone intervention increases disease-free survival in patients with breast cancer, with an unknown underlying mechanism. We elucidated the role of non-coding RNAs in response to progesterone in human breast cancer. Methods Whole transcriptome sequencing dataset of 30 breast primary tumors (10 tumors exposed to hydroxyprogesterone and 20 tumors as control) were re-analyzed to identify differentially expressed non-coding RNAs followed by real-time PCR analyses to validate the expression of candidates. Functional analyses were performed by genetic knockdown, biochemical, and cell-based assays. Results We identified a significant downregulation in the expression of a long non-coding RNA, Down syndrome cell adhesion molecule antisense DSCAM-AS1, in response to progesterone treatment in breast cancer. The progesterone-induced expression of DSCAM-AS1 could be effectively blocked by the knockdown of progesterone receptor (PR) or treatment of cells with mifepristone (PR-antagonist). We further show that knockdown of DSCAM-AS1 mimics the effect of progesterone in impeding cell migration and invasion in PR-positive breast cancer cells, while its overexpression shows an opposite effect. Additionally, DSCAM-AS1 sponges the activity of miR-130a that regulates the expression of ESR1 by binding to its 3’-UTR to mediate the effect of progesterone in breast cancer cells. Consistent with our findings, TCGA analysis suggests that high levels of miR-130a correlate with a tendency toward better overall survival in patients with breast cancer. Conclusion This study presents a mechanism involving the DSCAM-AS1/miR-130a/ESR1 genomic axis through which progesterone impedes breast cancer cell invasion and migration. The findings highlight the utility of progesterone treatment in impeding metastasis and improving survival outcomes in patients with breast cancer. Supplementary Information The online version contains supplementary material available at 10.1186/s13058-022-01597-x.


Introduction
Progesterone and estrogen, naturally occurring hormones, are known to modulate the progression and disease outcome of breast cancer [1][2][3]. Approximately 70% of breast cancer patients-positive for estrogen receptor (ER) and progesterone receptor (PR)-receive hormone therapy, such as blocking ER to inhibit estrogen signaling, as the first-line treatment for patients with luminal breast cancer [4,5]. Previous studies have highlighted the beneficial effects of the progesterone-high luteal phase on surgical outcomes in patients with breast cancer [6][7][8]. However, how progesterone modulates the downstream signaling remains sparsely understood.
The role of ER has been extensively studied in breast cancer due to its prognostic significance [9,10], along with its role in increasing the invasion and migration of breast cancer cells [11]. The PR, on the other hand, is a known ER target. The presence of PR is described as an indication of ER activity [12]. In vitro studies suggest that progesterone inhibits the invasion and migration of breast cancer cells [13,14]. Progesterone also induces cell cycle arrest and mild apoptosis in the cells mediated by PR that can function as a transcription factor to induce gene expression [15][16][17]. Additionally, PR alters ER binding sites in the genome in response to progesterone, and thus, could modify the expression pattern of ER-responsive genes in breast cancer cells [18].
Here, we identify DSCAM-AS1 as a progesteroneresponsive lncRNAs in breast cancer using an integrated functional genomics approach. DSCAM-AS1 acts as a sponge for miR-130a to regulate the expression of ESR1 in hormonal receptor-positive breast cancer cells. The study also suggests that targeting these ncRNAs may help improve survival outcomes in patients with breast cancer.

Transcriptome analysis of breast cancer patient samples
Whole transcriptome sequencing data from 30 breast tumors samples were re-analyzed. Ten tumors were derived from patients who were administered a single dose of 500 mg of hydroxyprogesterone within 15 days prior to surgery, with varying duration for individual patients, while 20 tumors were obtained from patients who were not exposed to hydroxyprogesterone [37]. Gene expression was quantified using Salmon [38].
Genes with expression > 5 reads in at least 20% of the cancer samples were retained. Design matrices were created based on progesterone treatment, and differential gene expression analyses were performed with progesterone-treated (n = 10) and control (n = 20) tumor samples, using DESeq2 [39]. Data were assessed in the R environment. ENSEMBL IDs were converted using bioconductor packages (org.Hs.eg.db), and gene names not matching the ENSEMBL IDs were obtained from LNCipedia.
Tissue culture and cancer cell line maintenance T47-D, BT-474, MCF7 and MDA-MB-231 breast cancer cells were procured, confirmed, cultured, and maintained as explained previously [13,40]. The human embryonic kidney 293FT cell line was purchased from Invitrogen (Cat No. R70007), cultured in DMEM with 10% FBS, and maintained at 37℃ with 5% CO 2 .

Progesterone and mifepristone treatment, RNA isolation, cDNA synthesis, and qPCR
Cells were serum-starved and treated with 10 nM progesterone (6 h), 100 nM mifepristone (2 h), or an equal volume of ethanol (vehicle control) as explained previously [13]. RNA isolation, DNaseI treatment, and cDNA synthesis for genes/lncRNAs and microRNAs were performed as explained previously [29,40]. Further, the cDNAs were used to study gene/miRNA expression patterns by quantitative real-time PCR (qPCR) method using the KAPA SYBR real-time PCR master mix (Sigma, Cat No. KK4601) and QuantStudio 5 real-time PCR system (Applied Biosystems, Cat no. A34322). GAPDH or ACTB and U6 were used as internal controls to normalize the expression of genes and miRNAs, respectively. Differential gene expression changes were calculated as fold change values using the 2 −∆∆CT method. The sequences for qPCR primers were manually designed using Snap-Gene sequence viewer. Designed primers were tested and optimized using OligoCalc (Sigma), UCSC In Silico PCR, and NCBI blast. The primer sequences for the genes and miRNAs are listed in Additional file 2: Table S1.

RNA-sequencing of progesterone-treated breast cancer cells
Total RNA was isolated from progesterone treated and untreated T47-D and MDA-MB-231 cells. Good quality RNA samples (RNA integration number > 9) were used to prepare the sequencing library using TruSeq library prep kit v2 (Illumina) with ribosomal RNA depletion. Libraries were sequenced on HiSeq4000 with 100 bp pair-end chemistry. A minimum of 60 million paired-end reads were obtained for each RNA sample with good Phred scores (score > 30). Differential gene expression analysis was performed using the salmon-DeSeq2 pipeline.
Briefly, all the raw reads were corrected using the trimmomatic version V0.32 [41], followed by alignment to the human reference pseudo-genome (GRCh38) using Salmon (version: 0.8.2) [38] and differential expression analysis using DeSeq2 [39]. Genes/lncRNAs with fold change > 2 and < 0.5 with p-value < 0.05 were considered to be significantly deregulated in response to progesterone.

ChIP-sequencing data analysis
ChIP-sequencing data with PR, ER, and p300 pulldown for progesterone-treated T47-D cell line were downloaded from the SRA database [18]. The raw data for these experiments were analyzed as described earlier [40]. Briefly, reads were aligned to the gencode (v30) human reference genome (GRCh38) using a BWA aligner (version 0.7.17). Peak calling was performed using the MACS tool (version 2.0) [42]. Aligned reads were used for differential protein binding in the genome using Diff-Bind (version 3.0) [43]. The 5 kb upstream and downstream regions for annotated genes/lncRNAs were analyzed for PR, ER, and p300 binding, and annotation of the peaks was performed using Uropa [44].

siRNA-mediated knockdown
Sense and antisense DNA oligonucleotides with T7 RNA promoter sequences were designed and synthesized by Sigma-Aldrich to prepare siRNAs targeting DSCAM-AS1, PGR, and ESR1. The complete method for synthesis of small RNA transcripts using T7 RNA polymerase has been described previously [51]. Briefly, sense and antisense strands of DNA oligonucleotides with T7 RNA promoter complementary sequences were annealed in a Thermocycler for synthesizing dsDNA. The dsRNAs were subjected to in vitro transcription reaction (37 ℃ for 2 h) using T7 RNA polymerase (Promega, Cat no. P2075) in 1 × T7 Transcription Buffer (Promega, Cat no. P118B). The single-stranded sense and antisense siR-NAs were further annealed to prepare double-stranded siRNAs. The complete list of DNA oligonucleotides for siRNA synthesis is provided in Additional file 2: Table S1. Synthesized siRNAs were transfected in breast cancer cells using Lipofectamine 3000 kit (Invitrogen, Cat No. L3000015) in serum-free media. Progesterone treatment was given to transfected cells 48 h post-transfection and collected for downstream analysis.

Overexpression of genomic elements
For transient overexpression of miR-130a in breast cancer cells, the precursor miRNA sequence with 200 bp flanking gene sequence was amplified from T47-D genomic DNA and cloned in pJET1.2/blunt vector (Thermo Scientific, Cat no. K1232) followed by sub-cloning in pcDNA3.1(-) mammalian expression vector under CMV promoter. XbaI (NEB, Cat no. R0145) and HindIII (NEB, Cat no. R0104) recognition sequences in multiple cloning sites of pcDNA3.1(-) were used for cloning. For transient overexpression of DSCAM-AS1, the complete cDNA sequence was cloned in the pcDNA3.1( +) expression vector using XbaI and BamHI (NEB, Cat no. R0136). Cloned constructs were confirmed by restriction digestion and Sanger sequencing. Further, the overexpression plasmids were transfected into breast cancer cells using Lipofectamine 3000. Empty pcDNA3.1(-) vector was transfected as vector control. Cells were collected 48 h post-transfection and RNA was extracted. Overexpression was confirmed by qPCR analysis.
For stable overexpression of DSCAM-AS1 in breast cancer cells, a complete cDNA sequence was cloned in the pBABE-puro expression vector using BamHI and SalI (NEB, Cat no. R0138). Cloning was confirmed using restriction digestion and Sanger sequencing. The primer sequences used for cloning and Sanger sequencing are provided in Additional file 2: Table S1. The 293FT cells were used for transfection and retrovirus production. Transductions were performed for 16 h in T47-D cells, followed by a selection of positive clones using 1 µg/mL puromycin (HiMedia, Cat no.TC198-10MG). Puromycinresistant clones were further confirmed for DSCAM-AS1 overexpression by qPCR analysis.

Transwell cell invasion and migration assay
Transwell cell migration and invasion assays were performed as described previously [13]. Briefly, cell invasion assay was performed with Matrigel loaded onto the inserts in Boyden chambers; while, cell migration assay was performed without Matrigel. The number of cells that migrated or invaded through the membrane was counted and the total fraction of cells was plotted as percent cell migration or invasion, respectively.

Luciferase reporter assay
Full-length DSCAM-AS1 cDNA sequence was amplified from T47-D cells and cloned in pJET1.2/blunt vector, followed by sub-cloning in pGL3-promoter vector (Promega, Luciferase expressing vector) downstream to firefly luciferase using XbaI. DSCAM-AS1 mutant construct was generated with mutated overlapping primers by sitedirected mutagenesis using DpnI (NEB, Cat no. R0176) and PrimeSTAR GXL DNA polymerase (TaKaRa, Cat no. R050B). DSCAM-AS1 mutant construct contained mutations in miR-130a MRE in DSCAM-AS1 cDNA to prevent miR-130a binding. Wild-type and mutated DSCAM-AS1 constructs were confirmed by Sanger sequencing. A 620 bp fragment of 3'-UTR of ESR1 with miR-130a binding site was amplified from T47-D cDNA and cloned in pJET1.2/blunt vector. The cDNA was subcloned in the pGL3-promoter vector using XbaI. The primer sequences for cloning and generating mutant construct are provided in Additional file 2: Table S1.

Gene-miRNA correlation analysis
The total RNA and miRNA sequencing data for patients with breast cancer were downloaded from The Cancer Genome Atlas (TCGA). Data from 751 breast cancer samples sequenced for total RNA and miRNAs were considered for further analysis. The samples with normally distributed DSCAM-AS1 or ESR1 expression values were segregated into quartiles. The upper and lower quartile samples were compared. The miRNA levels were compared between patients with ESR1-high and -low expression (the upper and lower quartiles, respectively). A similar analysis was performed for miRNAs in patients with DSCAM-AS1-high and -low expression. The significance of differences between both the groups was calculated using the Wilcoxon-Mann-Whitney test.

Survival analysis
The TCGA breast cancer samples with high and low miRNA expression were compared for survival outcomes. The KM plotter [52] and GEPIA [53] were used for Kaplan-Meier survival analysis within specified breast cancer groups. Overall and relapse-free survival of patients was calculated based on the levels of lncRNAs and miRNAs in the samples.

Statistical analysis
GraphPad Prism version 8 (GraphPad Software, La Jolla, CA) was used to calculate statistical significance between different experimental groups in qPCR, cellbased assays, and luciferase reporter assays. The student's unpaired t-test was used to investigate statistical significance. A p-value < 0.05 was considered to be statistically significant.

Results
We previously reported that progesterone inhibits breast cancer invasion and migration via the deactivation of several kinases [13,29,40]. Here, we describe the regulatory role of non-coding RNAs in response to progesterone to mediate the cellular changes.

Identifying significantly deregulated lncRNAs in response to progesterone in breast cancer
First, we analyzed 30 whole transcriptome datasets to identify differentially expressed lncRNAs upon progesterone treatment. Of the 30 tumor samples, 10 had received a single 500 mg dose of hydroxyprogesterone and 20 were controls [37,54]. Sequencing of these samples generated 17.2-60.7 million reads per sample (median, 37.4 million), wherein > 94-96% reads aligned to the human genome. Differential gene expression analysis between the control and progesterone-treated patients aided in identifying 2,222 differentially expressed genes (FDR < 0.1; 764 up-and 1,458 down-regulated), containing 537 lncRNAs (287 up-and 250 down-regulated), while a majority of the deregulated genes were of proteincoding category (Fig. 1A, Additional file 2: Table S2).
Further, to better understand the underlying mechanisms of action of progesterone, we performed whole transcriptome sequencing of T47-D (PR + /ER + / Her2-) and MDA-MB-231 (PR-/ER-/Her2-) breast cancer cells in response to progesterone treatment. A minimum of 60 million pair-end reads were obtained for each sample with > 90% of reads with a Phred score > 30, suggesting good quality of the data. Of 382 and 206 differentially expressed genes in T47-D and MDA-MB-231 cells, respectively, 18 lncRNAs were significantly deregulated in response to progesterone (-1 < log2FC > 1; p-value < 0.05) ( Fig. 1A; Additional file 1: Figure S1; Additional file 2: Tables S3, S4). MDA-MB-231, a PR-negative cell line, also showed active transcriptional response to progesterone treatment, likely due to the PR-independent mode of action of progesterone mediated by glucocorticoid receptor (GR) [40]. Interestingly, expression of a few lncRNAs was consistently deregulated in the progesterone-treated breast tumor and cell line transcriptome data, viz.., DSCAM-AS1, PCED1B-AS1, RP11-21L23.2, RP11-363E7.4, and AC012358.8 (Fig. 1A). Of these, expression of DSCAM-AS1 was considerably downregulated in progesterone-treated breast cancer patients transcriptome data. Moreover, the normalized DSCAM-AS1 expression in progesterone untreated samples range from 3-860, compared to 3-450 in progesterone treated samples (Additional file 1: Figure S2). This suggests the variable DSCAM-AS1 expression across progesterone treated and untreated primary breast tumor samples. Further, consistent with our previous study, SGK1 was found to be significantly upregulated [13,29,40], in addition to deregulated expression of some lncR-NAs, in progesterone-treated breast cancer samples (Fig. 1A). Taken together, the transcriptome analyses of tumor and cell lines identified novel progesteroneresponsive lncRNAs in breast cancer.

Progesterone downregulates the expression of DSCAM-AS1 to suppress migration and invasion of PR-positive breast cancer cells
An orthologous validation of the differentially expressed lncRNAs by real-time PCR identified a long noncoding RNA Down syndrome cell adhesion molecule antisense, DSCAM-AS1, as downregulated in ER/ PR-positive T47-D and BT-474 cells upon progesterone treatment compared to ER/PR-negative MDA-MB-231 cells (Fig. 1B-D). In contrast to T47D and BT474 cells, we detected no significant change in the expression of DSCAM-AS1 in MCF7 cells (Additional file 1: Figure S3), consistent with its distinct transcriptional landscape, as described earlier, in response to progesterone treatment [18,55]. The downregulation of DSCAM-AS1 could be effectively blocked by mifepristone, an antagonist of progesterone receptor (PR) and glucocorticoid receptor (GR) (Fig. 2A, B). However, the siRNA-mediated knockdown of PR, but not GR, rescued the down-regulation of DSCAM-AS1 in response to progesterone treatment, suggesting that PR mediates the downregulation of DSCAM-AS1 in response to progesterone in PR-positive cells (Fig. 2C). Next, we tested whether DSCAM-AS1 affects the inhibition of migration and invasion ability of breast cancer cells in response to progesterone [13]. Interestingly, DSCAM-AS1 knockdown could mimic the effect of progesterone by inhibiting breast cancer cell migration and invasion comparable to the extent obtained following treatment of the PR-positive T47-D and BT474 cells with progesterone ( Fig. 2D-G).

DSCAM-AS1 downregulates the expression of ESR1 in response to progesterone in PR-positive breast cancer cells
Estrogen receptor (ER) has previously been shown to regulate DSCAM-AS1 expression via binding near the promoter region [27]. Consistent with the literature, we observed a significantly higher expression of DSCAM-AS1 transcript in TCGA breast cancer patient samples and ER/PR-positive T47-D and BT-474 cells than in MDA-MB-231 cells (Fig. 3A, B, Additional file 1: Figure  S4). We hypothesized that ER/PR could modulate the DSCAM-AS1 expression in response to progesterone by binding to its upstream regulatory or distal regions. We analyzed chromatin immunoprecipitation ChIPsequencing data following progesterone treatment in PR-positive T47-D cells, as described earlier [18,40]. We identified enrichment of PR, ER, and p300 binding peak upon progesterone treatment at the "region 3" regulatory sequence of DSCAM-AS1 (Additional file 1: Figure  S5). This suggests that progesterone alters the binding occupancy of PR and ER near DSCAM-AS1. Surprisingly, siRNA-mediated knockdown of DSCAM-AS1 in turn led to a significant decrease in the expression of ESR1 transcript, comparable to progesterone treatment, suggesting a possible feedback mechanism by which DSCAM-AS1 regulates the expression of ESR1 in T47-D and BT-474 cells (Fig. 3C, D). In contrast, overexpression of DSCAM-AS1 in T47-D cells, but not MDA-MB-231 cells, led to overexpression of ESR1 (Fig. 3E-H), suggesting that progesterone reduces expression of DSCAM-AS1 that further suppresses expression of ESR1 to inhibit cell migration and invasion in PR-positive breast cancer cells.

DSCAM-AS1 sponges miR-130a targeting 3'-UTR of ESR1 to suppress migration and invasion of PR-positive breast cancer cells
LncRNAs are known to sponge miRNAs, and thus, reduce the availability of miRNAs for target gene suppression [56,57]. We thus tested whether DSCAM-AS1 could sponge miRNAs targeting the 3'-UTR of ESR1. Using the DIANA-LncBase v2 database prediction module, we identified 167 miRNAs that could bind DSCAM-AS1 with miTG-score > 0.7 (Additional file 2: Table S5). Concomitantly, we identified 72 miRNAs predicted to target the 3'-UTR of ESR1 from the miRTarBase database (Additional file 2: Table S6), with 9 overlapping miRNAs, viz. miR-548x, miR-548aj, miR-335, miR-129,  miR-4422, miR-3121, miR-193b, miR-130a, and miR-301a (Fig. 4A). A real-time PCR-based validation of these 9 miRNAs in response to progesterone or genetic knockdown of DSCAM-AS1 identified miR-130a as significantly upregulated in T47-D and BT-474 cells (Fig. 4B, Additional file 1: Figure S6). Interestingly, in BT474 cells, a greater number of miRNAs were downregulated in response to silencing DSCAM-AS1 than in response to progesterone treatment. This may be related to the derepression of miRNAs upon silencing DSCAM-AS1, a miRNA sponge, as well as the activation and inhibition of various pathways in response to progesterone, such as the up-regulation of miR-129-2, which in turn regulates the expression of PR, as demonstrated before [29]. Next, to investigate the function, we ectopically expressed miR-130a in T47-D and BT-474 cells. The ectopic expression of miR-130a led a significant decrease in ESR1 transcript than in vector control (Fig. 4C, D), with a concomitant decrease in invasion and migration of T47-D and BT-474 cells. miR-130a overexpression could mimic progesterone treatment Expression of miR-130a is normalized with respect to that of U6. p-value calculated using Student's t-test. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; ns non-significant or DSCAM-AS1 knockdown in PR-positive T47-D and BT-474 cells (Fig. 4E-H). Furthermore, to test a direct interaction between miR-130a and DSCAM-AS1, DSCAM-AS1 cDNA was cloned downstream to the luciferase reporter gene. The findings revealed a decrease in normalized luciferase activity upon overexpression of wild-type miR-130a, but not with miR-130a construct with a mutated binding site. As a positive control, the 3'-UTR of ESR1 cloned downstream to the luciferase gene showed similar inhibition of luciferase activity (Fig. 4I). In contrast, overexpression of DSCAM-AS1 in T47-D, but not MDA-MB-231, cells led to a significant reduction in miR-130a levels than that in vector control (Fig. 4J, K, Additional file 1: Figure S7 A-C). Interestingly, miR-130a also showed a significant inverse correlation to DSCAM-AS1 and ESR1 expression in 752 TCGA breast cancer samples (Additional file 1: Figure S8 A-B). Taken together, these results validate the association between DSCAM-AS1 and miR-130a to maintain ESR1 levels in PR-positive breast cancer cells with a consistent inverse correlation of miR-130a with the expression of DSCAM-AS1 and ESR1 in the TCGA patient samples.

Upregulation of miR-130a correlates with better survival outcome in breast cancer patients
The prognostic value of DSCAM-AS1 and miR-130a expression in survival prediction was further tested in TCGA breast cancer datasets (n = 1062) generated by whole transcriptome sequencing to perform the Kaplan-Meier (KM) survival analysis. Patients in the datasets were divided into high-and low-expression classes by the median expression value of DSCAM-AS1 and miR-130a, and a log-rank test was performed for stratifying patients with different prognoses. The analysis showed a significantly better overall survival in patients with breast carcinoma with high miR-130a expression than those with low miR-130a expression (log-rank p = 0.02). Patients with high expression of miR-130a survived better (87 months) than those with low expression of miR-130a (69 months). Overall, we observed a survival benefit of 18 months in the miR-130a high expression cohort. Similar results were observed in patients with ER-positive subtype cancer (log-rank p = 0.05) (Fig. 5A, B). In contrast, KM analysis of patients with breast cancer did not show statistically significant change in overall survival in patients who exhibit high and low levels of DSCAM-AS1 (Fig. 5C,  D). These findings imply that a high expression of miR-130a influence survival of patients with breast cancer.

Discussion
Progesterone confers better survival outcomes in patients with breast cancer, especially in those with lymph node involvement [58]. These early clinical observations have increased interest in researchers globally to investigate the mechanisms by which progesterone affects breast cancer pathophysiology. We have previously shown that progesterone reduces breast cancer cell invasion and migration [13] by regulating a tight network of proteincoding genes that reduce the activity of kinases that are known to induce cellular stress [40]. The present study highlights the multiplicity of genomic mediators, especially ncRNAs, recruited by progesterone and PR in breast cancer to abrogate cell invasion and migration.
To begin with, this is the first study to describe progesterone-responsive lncRNAs in breast tumor samples and cell lines. Interestingly, the analyses identified DSCAM-AS1 as a novel target of progesterone in breast cancer. Progesterone downregulates the expression of DSCAM-AS1 specifically in PR-positive breast cancer cells, wherein PR modulates the genomic binding pattern of ER, the classical activator of DSCAM-AS1 [27], in response to progesterone. This also highlights the importance of PR in clinical outcome of breast cancer prognosis and confirms the previous findings that PR modulates ER binding in breast cancer cells treated with progesterone [18,59]. However, recent report suggests that progesterone treatment may have varied response on tumor growth in patient derived xenograft mouse models [60]. Consistent with this, we also observed variability in DSCAM-AS1 expression in response to progesterone.
Second, the findings suggest that DSCAM-AS1 functions as a miRNA sponge to help maintain the high expression of ER in breast cancer cells. DSCAM-AS1 has previously been shown to function as a miRNA sponge for miR-101 [61] and miR-186 [62] in osteosarcoma, and miR-136 in endometrial cancer [63]. Interestingly, we show that progesterone opposes the DSCAM-AS-1-ESR1 feedback loop, and thus essentially the ER signaling pathway, by employing two synergistic mechanisms-it decreases the expression of DSCAM-AS1 and increases the expression of miR-130a that binds to both DSCAM-AS1 and 3'UTR of ESR1 in breast cancer cells. This strengthens the role of progesterone in regulating the expression of non-coding genomic elements in breast cancer [29,64], in addition to regulating the expression of protein-coding elements. The results of the present study also emphasize the necessity of PR expression in breast cancer cells for progesterone to alter the expression of DSCAM-AS1 and miR-130a, as these effects were not observed in PR-negative MDA-MB-231. Additionally, the expression pattern of miR-130a was found to be inversely correlated with that of ESR1 and DSCAM-AS1 in cell lines and patients with breast cancer.
Third, the cellular experiments indicated that silencing of DSCAM-AS1 or overexpression of miR-130a led to a significant reduction in breast cancer cell migration and invasion than that in vehicle control cells, comparable to the effect induced by progesterone-alone. Furthermore, progesterone treatment of cells with high miR-130a levels led to a greater reduction in cell invasion and migration than progesterone treatment of vehicle-treated control cells; this result demonstrates that variation in expression of these ncRNAs modifies other genomic components that augment the effects of progesterone on breast cancer cells, as described previously [13,29,40]. Further, miR-130a has been reported to be involved in mitigating progression in breast cancer stem cells [65], and its expression has been reported to be downregulated in breast cancer [66,67]. Finally, using the TCGA datasets, we show that patients with breast cancer with high miR-130a levels correlate with a tendency toward better overall survival (that could not attain statistical significance). Therefore, the findings may help clinicians to better categorize patients with luminal A/B subtype based on the expression of DSCAM-AS1 or miR-130a to receive appropriate care and aid in prolonging their survival outcomes.
In conclusion, this study elucidates an underlying mechanism for a clinical consequence in response to progesterone treatment among patients with breast cancer. Progesterone downregulates the expression of DSCAM-AS1, a known ncRNA member of the ER signaling survival curves indicate differences in overall survival based on miR-130a high and low levels in patients with breast carcinoma (BRCA) of A all subtypes and B estrogen receptor (ER)-positive subtype. C, D KM survival plots indicate differences in overall survival based on DSCAM-AS1 high and low levels in patients with C all BRCA subtypes and D ER-positive subtypes. The probability of survival is plotted on Y-axis and survival time (in months) is represented on X-axis. The red curve represents survival probability in patients with high expression; whereas, the black curve represents that in patients with low expression. Log-rank p < 0.05 is considered the cutoff for calculating the significance value. The hazard ratio (HR) for each KM plot is also denoted pathway, and increases the expression of miR-130a that inhibits ESR1, to suppress breast cancer cell invasion and migration. Additionally, high miR-130a levels are associated with improved overall survival outcomes in patients with breast cancer, similar to that observed in the randomized controlled trial with preoperative progesterone. Thus, progesterone treatment under hormonal therapy in the adjuvant and neoadjuvant settings may help in impeding cell migration and invasion of breast cancer cells, and in improving the overall and relapse-free survival outcomes in patients with breast cancer.
Additional file 1. Fig. S1. Differentially expressed genes upon progesterone treatment in breast cancer cell lines (A, B) Volcano plot depicting differentially expressed genes upon progesterone treatment in (A) T47-D and (B) MDA-MB-231 breast cancer cell lines, identified in RNA-sequencing data. X-and Y-axes represent log2(fold change) and -log10(p-value), respectively. Each dot represents expression fold change for an individual gene. All genes above the horizontal red line and outside central blue quadrant are significantly deregulated upon progesterone treatment. The total number of significantly up-regulated and down-regulated genes are represented on the top right and top left of the plot respectively. Fig. S2. DSCAM-AS1 expression in progesterone-treated and -untreated primary breast tumor samples. Gene expression normalization was performed using median of ratios method (DESeq2). The normalized values are plot on Y-axis. X-axis indicates breast cancer patient samples (samples #1 to #30). Median DSCAM-AS1 expression in each group is indicated. Fig. S3. Real time PCR analysis of differentially expressed lncRNAs in MCF7 cells treated with progesterone. Data are normalized with expression of GAPDH and relative fold changes with respect to vehicle control are plotted on Y-axis. Changes in the normalized expression of lncRNAs upon treatment are plotted as relative fold change (2-ΔΔCT) with respect to expression in vehicle control for the same cell line. This consist of data from three biological replicates. The horizontal black line represents normalized expression of lncRNAs in vehicle-treated cells. SGK1, a progesterone-responsive gene, is used as a positive control. p-value calculated using Student's t-test. *, p<0.05; **,p<0.01; ***,p<0.001; ****,p<0.0001; ns, non-significant. Fig. S4. DSCAM-AS1 is up-regulated in breast cancer patient samples (A) Box-plot indicating DSCAM-AS1 expression in breast cancer patients and normal tissue sample data obtained from the TCGA. Red boxes denote the expression of DSCAM-AS1 in cancer samples, whereas black boxes represent expression in normal tissue samples. Fig. S5. Differential binding of PR, ER, and p300 near DSCAM-AS1 genomic region upon progesterone treatment (A) Differential binding of ER, PR, and p300 (histone acetyltransferase) near the DSCAM-AS1 regulatory regions (within 5 kb upstream and downstream) in T47-D cells upon progesterone treatment. Differential peak calling at each binding location upon progesterone treatment is calculated in three biological replicates. FDR<0.05 is considered the significance value for each peak. Relative fold change of expression of gene/lncRNA and miR130a with respect to that of ACTB and U6, respectively, is plotted. p-value calculated using Student's t-test. *, p<0.05; **,p<0.01; ***,p<0.001; ****,p<0.0001; ns, non-significant. Fig. S8. Expression of miR-130a is inversely correlated with DSCAM-AS1 and ESR1 expression in the TCGA breast cancer RNA-seq dataset (A, B) Expression plot for miR-130a in the TCGA breast cancer samples expressing high and low levels of (A) DSCAM-AS1 and (B) ESR1 high and low TCGA breast cancer samples. Upper and lower quartile patient groups in terms of DSCAM-AS1 or ESR1 expression are included in the analysis. Normalized expression of miR-130a is plotted on Y-axis.