LncRNA IPW inhibits growth of ductal carcinoma in situ by downregulating ID2 through miR-29c

To identify differentially expressed lncRNAs in DCIS, we isolated total RNA from eight paired normal and DCIS tissues and performed qRT-PCR for disease-related lncRNAs (Fig. 1A). Four lncRNAs were found to be downregulated with significant p value (< 0.05) and fold change higher than 5 (Fig. 1B). LncRNA IPW (Imprinted in Prader–Willi syndrome) was most significantly downregulated in DCIS as compared to normal tissues (Additional file 1: Fig. S1A). We further validated the expression of top four lncRNAs in three pairs of non-tumorigenic breast epithelial cells and DCIS cell lines: MCF10A and DCIS.com, S1 and S2, HMEC and SUM225. IPW was observed to be most significantly downregulated in DCIS cell lines when compared to its non-tumorigenic controls (Fig. 1C, Additional file 1: Fig. S1B–D). Therefore, IPW was selected for subsequent analysis. To further substantiate the clinical relevance of IPW in DCIS, we isolated RNAs from normal ducts, DCIS lesions and invasive ductal carcinoma (IDC) from patient specimens in formalin-fixed paraffin-embedded (FFPE) sections by microdissection (n = 10/group). We observed that IPW was significantly downregulated in DCIS and IDC when compared to the normal samples (Fig. 1D). IPW expression was then verified in cell lines representing non-tumorigenic (MCF10A), DCIS (DCIS.com) and IDC (MCF10CA). Similar to the clinical samples, IPW expression was significantly decreased in DCIS.com (Additional file 1: Fig. S1E), suggesting that IPW plays a role in DCIS formation. As changes in morphology of ducts are early events of carcinoma initiation [6, 11, 12], we examined whether IPW was involved in regulating duct size and morphology by performing acini formation assay in 3D culture. We silenced IPW expression in MCF10A cells using short hairpin RNAs (shRNAs) (Fig. 1E), and cultured cells in Matrigel-embedded culture plates (Fig. 1F). We observed that the average size of the ducts formed by cells silenced for IPW was significantly larger with irregular morphology and in higher number when compared to the parental MCF10A (Fig. 1G). This result suggests that IPW plays a functional role in the maintenance of duct integrity, and loss of IPW expression triggers DCIS transition. Collectively, our results strongly indicate that IPW is a clinically relevant lncRNA downregulated in DCIS and plays a critical role in its initiation.

To gain further insight into the role of IPW in DCIS growth, we examined the growth of MCF10A and MCF10A-shIPW cell lines in vitro. As shown in Fig. 2A, we found that the growth rate of IPW-silenced MCF10A cells was significantly increased when compared to the parental MCF10A. We then overexpressed IPW in DCIS.com (Fig. 2B), SUM225 (Additional file 1: Fig. S2A) and S2 (Additional file 1: Fig. S2F) cells by lentivirus-mediated transduction and studied the effect of IPW in cell growth and colony formation ability of these cells. We found that IPW expression significantly suppressed cell proliferation and colony-forming ability of these cells, demonstrating its strong tumor suppression function in vitro (Fig. 2C, D). Similar results were observed in SUM225 (Additional file 1: Fig. S2B, C) and S2 (Additional file 1: Fig. S2G, H) cell lines. Additionally, ectopic expression of IPW stalled cells in the G1 phase of cell cycle, thereby inhibiting cell cycle progression in DCIS.com, SUM225 (Fig. 2E) and S2 (Additional file 1: Fig. S2I) cell lines. IPW overexpression also upregulated p21 mRNA and protein in DCIS.com and SUM225 cell line (Fig. 2F, G). As our group and others have previously demonstrated the role of cancer stem-like cells (CSC) in DCIS growth [13‐15], we examined the effect of IPW in DCIS stemness. We found that IPW expression significantly decreased sphere formation ability in DCIS.com (Fig. 2H), SUM225 (Additional file 1: Fig. S2D, E) and S2 (Additional file 1: Fig. S2J, K) cells. However, we observed that ectopic IPW expression (Additional file 1: Fig. S2L) did not alter wound healing (Additional file 1: Fig. S2M) and migration ability of DCIS.com, MCF-7 and 10CA (Additional file 1: Fig. S2N) cells. Similarly, IPW expression did not affect caspase 3/7 expression indicating no marked change in cellular death (Fig. 2I). Furthermore, to address the effect of IPW expression in DCIS growth, we implanted luciferase-labeled DCIS.com cells transduced with either empty vector or IPW in the mammary fat pad of nude mice and examined tumor growth every week by bioluminescence (BLI). The size of lesions of the mice implanted with DCIS-IPW cells was significantly smaller as measured by BLI photon flux index (Fig. 2J). Furthermore, hematoxylin and eosin (H&E) staining of the tumor section revealed that ducts formed by DCIS-IPW tumors had lower cellularity and normal-like structures when compared to control tumors (Fig. 2K upper and middle panels). We also confirmed that these ducts were derived from DCIS.com-implanted cells, and not from the mouse mammary tissue, by positive staining of normal-like ducts using human cytokeratin antibody (Fig. 2K lower panel). Together, these results suggest that IPW expression inhibits DCIS growth by inducing cell cycle arrest and reducing stemness property. We found that IPW expression did not decrease in IDC compared to DCIS, indicating that IPW downregulation is an early event in tumorigenesis. However, when we segregated breast cancer patients based on IPW expression through KM plotter (n = 2032), (Additional file 1: Fig. S2O) dataset, we found that high IPW expression is correlated with significantly longer overall survival of patients suggesting that IPW is a clinically relevant lncRNA in cancer. In connection with these observations, we examined the p21, ID2 and miR-29c RNA expression in IDC lines 10CA and MCF7 ectopically expressed with either empty vector (control) or IPW. We observe that p21 and miR-29c were upregulated while ID2 was suppressed in 10CA (Additional file 1: Fig. S2P) and MCF7 (Additional file 1: Fig. S2Q) upon IPW overexpression. These results consolidate the tumor-suppressive role of IPW in DCIS and IDC. However, as the molecular drivers and signaling in IDC are very complex and subtype-dependent, further experiments are warranted to consolidate the mechanism of IPW in IDC.

Micro RNAs (miRNAs) are known to be involved in regulating multiple cellular processes in breast cancer [16, 17] including regulation of the downstream signaling to lncRNA, often through direct transcriptional control or sponge effect [18, 19]. In an attempt to uncover differentially expressed miRNAs in DCIS, we performed miRNA microarray in MCF10A and DCIS.com cell line. In this screening, we found that miR-29c was the most downregulated miRNA in DCIS.com as compared with MCF10A (Fig. 3A). The decrease in miR-29c expression was further verified by TaqMan qPCR in DCIS.com and SUM225 cells compared to MCF10A and HMEC, respectively (Fig. 3B, C). We then examined miR-29c expression in the same set of matching DCIS and adjacent normal tissue samples used for initial screening (Fig. 1A). miR-29c was significantly downregulated in DCIS samples compared to its the paired normal tissues (Fig. 3D). Furthermore, ectopic miR-29c expression significantly decreased CSC marker-positive population and sphere formation ability of DCIS.com cells (Fig. 3E-G). Importantly, we found a strong positive correlation between IPW and miR-29c expression in DCIS samples (Fig. 3H), indicating a potential regulatory interconnection between IPW and miR-29c. Therefore, to study whether IPW regulated miR-29c expression, we examined miR-29c expression in DCIS.com and SUM225 cells with ectopic expression of IPW. As shown in Fig. 3I, J, IPW significantly upregulated miR-29c expression in DCIS.com and SUM225 cells. On the other hand, ectopic expression of miR-29c did not affect IPW expression, suggesting that IPW is the upstream regulator of miR-29c (Additional file 1: Fig. S3). These results indicate that miR-29c plays a tumor-suppressive role in DCIS and its expression is controlled by IPW.

We then sought to decipher how IPW controlled miR-29c expression. LncRNAs are known to epigenetically regulate histones via posttranslational modifications, thereby affecting gene expression [20, 21]. Therefore, we utilized the UCSC genome browser to examine regulatory histone modifications marks in the promoter region (140 bp upstream of TSS) of miR-29c. We found that histone 3 lysine 4 trimethylation (H3K4me3), known for activating gene expression [22‐24], was highly enriched near the transcription start site (TSS) of miR-29c (Additional file 1: Fig. S4). To verify the presence of H3K4me3 in miR-29c TSS, we performed chromatin immunoprecipitation (ChIP) in DCIS and DCIS-IPW cells using H3K4me3 antibody (Fig. 4A). A significant enrichment of H3K4me3 was observed in DCIS-IPW cells (Fig. 4B left panel) in miR-29c promoter region. However, the enrichment of histone 3 lysine 27 trimethylation (H3K27Me3) was not evident in the same region (Fig. 4B right panel), suggesting that H3K4me3 enrichment in the miR-29c promoter region is specifically enhanced by IPW. As H3K4Me3 is known to be mediated by SET methyltransferase [25, 26], we immunoprecipitated SET methyltransferase in DCIS.com and DCIS-IPW cells (Fig. 4C) followed by isolating RNA and DNA bound to the protein. In the RNA fraction immunoprecipitated by SET methyltransferase, we found IPW to be significantly enriched in DCIS-IPW compared to parental DCIS.com cells, suggesting that SET methyltransferase interacts directly to IPW and this interaction is dependent on the level of IPW expressed by cells (Fig. 4D). On the other hand, we examined the miR-29c promoter region in the DNA fraction immunoprecipitated by SET methyltransferase. As shown in Fig. 4E, strong enrichment of SET methyltransferase was evident in the miR-29c promoter region of IPW-expressing cells. Similarly, the enrichment in IPW RNA and miR-29c promoter bound to SET methyltransferase was evident in MCF10A cells (Fig. 4F,G). Together, these results indicate that IPW positively regulates miR-29c expression through SET methyltransferase-mediated H3K4me3 enrichment in miR-29c promoter region (Fig. 4H).

Because IPW epigenetically regulated the expression of miR-29c, we sought to determine the downstream target of miR-29c. Serendipitously, we found a 12-bp miR-29c binding region in the 3’ UTR of ID2, a gene that we previously reported to strongly promote self-renewal of DCIS (Fig. 5A) [13]. We then mutated the 12-bp miR-29c binding site in ID2 3’UTR as shown in Fig. 5A and performed the promoter reporter assay for ID2 expression. We found that while miR-29c expression significantly decreased reporter activity, the mutation of the binding site in the 3’ ID2 UTR completely rescued the reporter activity in DCIS.com cells (Fig. 5B). In addition, ectopic expression of miR-29c strongly suppressed ID2 protein expression in DCIS.com cells (Fig. 5C). We then examined the effect of IPW on ID2 expression. Silencing of IPW in MCF10A cells significantly augmented ID2 mRNA and protein levels (Fig. 5D). Conversely, IPW expression in DCIS.com and SUM225 cells significantly reduced ID2 expression (Fig. 5E and Additional file 1: Fig. S5A). Similarly, ID2 expression was found to be decreased in tumors formed by DCIS.com cells with IPW expression (from animals in Fig. 2I) when examined by immunohistochemistry (Additional file 1: Fig. S5B, C). Additionally, the expression of transcription factors known to promote tumor stemness (SOX2, OCT4 and NANOG) [27, 28] were significantly downregulated in DCIS cells expressing IPW and rescued when ID2 was expressed (Additional file 1: Fig. S5D, Fig. 5F–H). Next, we examined whether miR-29c is crucial to mediate the downstream effect of IPW on ID2 expression and stem cell population. The miR-29c-mediated inhibition of ID2 expression was rescued when we transfected miR-29c locked nucleic acid (LNA) in DCIS.com cells expressing IPW (Fig. 5I). Similarly, ID2 expression in DCIS-IPW cells rescued the number and size of spheres (Fig. 5J) and tumor initiating stem cell population (Fig. 5K). We then examined the impact of IPW expression on self-renewal in DCIS.com cells. It was previously demonstrated by Al-Hajj et al. that CD44 + cells have the potential to self-renew and form mammary tumors in mice [29]. In corroboration with this observation, we isolated CD24^lowCD44^highESA^high—DCIS.com and DCIS-IPW cells by magnetic-assisted cell sorting (MACS) and performed mammosphere formation assay. We observed that the number of spheres formed by DCIS-IPW cells was significantly decreased in first- and second-generation mammosphere culture (Additional file 1: Fig. S5E).Together, these results indicate that IPW controls the DCIS self-renewal by targeting ID2 expression through miR-29c.

As cancer stem cells are known to drive transition of DCIS to invasive carcinoma, a potential strategy for treating DCIS and decreasing its probability of transitioning to aggressive carcinoma is to selectively target DCIS stem-like tumor-initiating cells. Our results strongly indicated that downregulated IPW in DCIS promoted self-renewal ability of DCIS stem cells. Therefore, we aimed to identify compounds that can selectively inhibit DCIS cells with a low IPW expression. We screened a natural compound library containing 390 drugs in DCIS cells and IPW-expressing DCIS cells by colony formation and MTS assay (Fig. 6A, Additional file 1: Fig. S6A). We found that toyocamycin significantly inhibited the relative cell viability and colony-forming ability of IPW-low DCIS cells at low concentration of 100 nM (Fig. 6B, C). Next, we examined the in vivo efficacy of toyocamycin on DCIS. We found that toyocamycin significantly suppressed the tumor growth in DCIS.com in animals with dosed at 2 mg/kg (Fig. 6D, E). Furthermore, no significant weight loss or change in alanine aminotransferase (AST) activity was observed in the blood of toyocamycin-treated mice (Additional file 1: Fig. S6B, C). Next, we sought to determine the mechanism of action of toyocamycin. We found that toyocamycin significantly downregulated the expression of ID2 and stem cell-associated transcription factors OCT4, SOX2 and NANOG (Fig. 6F–K) but did not affect IPW and miR-29c levels (Additional file 1: Fig. S6D, E). The effect of toyocamycin on p21, ID2, OCT4, SOX2 and NANOG expression was also verified in the tumor sections by immunohistochemistry (Additional file 1: Fig. S6F). In addition, we confirmed that the stained tumor tissues were derived from the implanted cells by staining tumor sections with human cytokeratin antibody (Additional file 1: Fig. S6F). Toyocamycin has been shown previously shown to regress the Ewing sarcoma [30] and multiple myeloma [31] by targeting the ER stress response genes. However, we observed no significant difference in expression of XBP, JNK, CHOP and ATF4 in DCIS.com cells treated with toyocamycin (Additional file 1: Fig. S6G–J). We also verified that toyocamycin does not affect the proliferation ability of non-tumorigenic cell lines MCF10A and HMEC (Additional file 1: Fig. S6K). These results suggest that toyocamycin specifically suppresses IPW-low DCIS cells by targeting ID2 and can potentially be used to treat patients with DCIS to avoid the current overtreatment practice.

DCIS.com was purchased from Asterand, Inc. S1 and S2 were purchased from Millipore Sigma, USA. Human mammary epithelial cell line (HMEC) was purchased from Lonza. SUM225 was a kind gift from Dr. Fariba Behbod, University of Kansas Medical Centre. MCF10A and HEK293T were purchased from ATCC. MCF 10A was cultured in DMEMF-12 (Gibco) supplemented with 5% horse serum (Thermo, cat no.: 16050130), 100 ng/ml cholera toxin (Sigma, cat no.: C8052), 20 ng/ml EGF (Peprotech, cat no.: AF-100-15), 100ug/ml insulin (Sigma, cat no.: 10516-5ML) and 0.5ug/ml hydrocortisone (Sigma, cat no.: H0888). DCIS.com, and MCF7 were cultured in RPMI medium, while S2 was cultured in DMEM medium supplemented with 10% FBS (Sigma), Penicillin (100 Units/ml) and Streptomycin (100ug/ml). HMEC and MCF10A were cultured in human mammary epithelial growth medium (Lonza). SUM225 was cultured in DMEM-F12 medium supplemented with 5%FBS, Penicillin (Gibco)- 100 Units/ml, Streptomycin (Gibco)- 100ug/ml, Hydrocortisone-1ug/ml, insulin-5ug/ml and HEPES-1 mM. SYBR green was purchased from Bio-Rad (Cat no:1725124). Sphere media were prepared by mixing 100 ml of DMEM/ F12 media with 2 ml of B27 supplement (Invitrogen, cat no.:17504), 100ul of 20ug/ml EGF (Peprotech, cat no. AF-100-15), 40ul of 10 mg/ml Insulin (Sigma, cat no.: 10516-5ML). For bioluminescence imaging, DCIS.com cells were transduced with lentivirus containing firefly luciferase. IPW expression was silenced by using shRNAs purchased from Abmgood (Cat no.: 2504209).

MCF10A, MCF10AshIPW and DCIS.com cells were cultured on regular 96-well plastic dishes (Fisher) in 10% Matrigel with phenol red as described before [54] In brief, the culture plates were coated with Matrigel and allowed to solidify for 20 min at 37 °C. Afterward, the cells were trypsinized and counted by hemocytometer. 1000 cells were suspended in 60 ul of medium with Matrigel (10%) and added on Matrigel-precoated wells drop wise. Cells were allowed to attach for 30 min at 37 °C. Afterward, 60ul of medium was added on the cells making final concentration of Matrigel to 5%. The medium was changed every 48 h, and cells were observed for acini between 10 and 12th day.

For wound-healing assay, cells were seeded and grown till confluence. Then, a wound is made with 200 µl tip and cells were washed with PBS. Afterward, complete media were added and cellular migration was monitored at 0 h, 12 h and 24 h. The diameter of wound was calculated by measuring the distance covered by migrated cells at 6 points around a scratch and average was derived. Each experiment was performed in triplicate, and results were represented as mean + SEM. For transwell migration assay, 5000 cells were seeded on the upper part of cell culture inserts coated with growth factor-reduced Matrigel (8.0 µM pore size, BD) and number of migrated cells were calculated after 24 h by fixing in 4% formalin followed by crystal violet staining. Cells were grown in medium without serum in the chamber, while media with 5% serum were added in the bottom as chemoattractant.

Disease-related lncRNA plate was procured from SBI (SBI, cat no. RA900A-1). The PCR array plate contained 83 lncRNAs and 11 housekeeping genes. The manufacturer’s instructions were followed. Briefly, total RNA was isolated from the DCIS and matching normal tissue with the use of Direct-zol RNA miniprep kit (Zymo, cat no: R2061) as per the manufacturer’s instructions. The tissue samples were procured from the tissue core at Wake Forest School of Medicine. All samples were pathologically confirmed and were completely anonymized. RNA purity was verified with Nano Drop spectrophotometer (Fisher Scientific). 1ug of RNA was used for cDNA synthesis with the use of Script CDNA synthesis kit (Bio-Rad, cat no 1708890) and diluted to 100 µl as per the instructions. This cDNA was further used for SYBR green-based qPCR for 40 cycles. The reaction conditions were: 50 °C-2 min, 95 °C-10 min, 95 °C-15 s, 60 °C-1 min. The results were analyzed by relative quantification. The fold change in gene expression was calculated as per the instructions relative to control samples (https://​systembio.​com/​wp-content/​uploads/​Disease_​related_​Profiler_​WEB-1.​pdf).

IPW construct was provided by Dr. Benvenisty [32]. IPW from pEGF N1 IPW was then sub-cloned in pSIN lentiviral plasmid vector. 4.5 kb of IPW was amplified with the help of primers: F: GC ACTAGT ccaaatcata gatcaagata tt, R: CTA GGATCC ttacacaggtttaagaat, Tm: 60⁰ C, 35 cycles. Amplified PCR product was purified from the gel using gel extraction kit (Qiagen, Cat no: 28506). The extracted IPW was cloned in pSIN-Pur plasmid (Addgene, cat no: 16579). Restriction enzymes were purchased from Thermo Fischer Scientific (SpeI-FD1254, BamH1- FD0054). Plasmid-expressing miR-29c was purchased from Origene. Five shRNAs spanning IPW were procured from Abmgood. A mixture of five individual shRNAs were used in to generate lentivirus in HEK293T cells and used for infecting cell lines (DCIS.com, SUM225, S2, MCF10A). For infection, cells with 60% confluence were seeded and incubated with virus particles for 8 h. Afterward, the virus solution was replaced with complete media and cells were grown for 48 h. Puromycin selection (10ug/ml) was employed to positively select the shRNAs expressing cells.

To assay cellular proliferation, 1000 cells were seeded in each well of 96-well plate and incubated for 1,3,5 and 7 days. 20ul of CellTiter 96 aqueous one solution cell proliferation assay (MTS) solution (Promega G3582) was added to cells and incubated for 90 min. The 490-nm absorbance was then quantified in the plate reader. For colony formation assay, 500 cells were seeded in each well of six-well plate. Media were replaced after every 3 days. Cells were fixed with cold methanol for 20 min at room temperature and stained with crystal violet (0.5% w/v). Colonies were counted under stereomicroscope. Alternatively, colonies were dissolved in 10% acetic acid and readouts were obtained at 595 nm. For sphere assay, 1000 cells were seeded in ultra-low attachment plates (Corning, cat no.: 3473) in sphere media. The spheres > 40 µm were quantified and imaged at day 5 by Olympus microscope.

DCIS initiating stem like cells were isolated by using magnetic-assisted cell sorting (MACS) column as described previously [55]. Briefly, DCIS.com and DCIS-IPW cells were trypsinized and stained with biotin-conjugated anti-CD24 (Miltenyi, cat no.: 130-095-951) and APC-conjugated anti-CD44 microbeads and incubated on ice for 15 min. After washing the samples with PBS, CD24-negative cells sorted by incubating with anti-biotin microbeads (Miltenyi Biotec). The flushed fraction of cells was then mixed with anti APC microbeads (Miltenyi Biotec) and biotin-conjugated anti-ESA antibody (StemCell, Cat. no.:17653) for 15 min on ice. CD44 and ESA positive fractions were collected and cultured in mammosphere media in ultra-low attachment plates. For serial passaging, mammospheres were first centrifuged and washed with PBS. Cells were then dissociated with trypsin and neutralized with FBS. The disaggregated spheres were then counted and seeded in ultra-low attachment plate with mammosphere media and number of spheres was calculated on day 5.

CellEvent Caspase3/7 green detection reagent was used to examine the cell death induced upon ectopic IPW expression in DCIS.com cells. 20,000 cells were seeded in 24-well plate and grown for 72 h. Afterwards, media was removed, cells were washed with PBS and incubated with 4 µM of Caspase3/7 green detection reagent for 1 h at 37⁰C. At the end of incubation, cells were washed twice with PBS to wash off the reagent, trypsinized and Caspase3/7 positive cells were quantified by flow cytometry.

For cell cycle analysis, IPW or empty vector-infected cells were seeded in 6-well plates. Cells were synchronized by growing in FBS-deprived media overnight. Cells were then cultured for 48 h in FBS-supplemented culture media. Cells were trypsinized, resuspended in 300ul of PBS and fixed in 700ul of cold EtOH (100%) for 2 h at -20 °C, followed by 2X PBS washing, propidium iodide (PI) staining (1 mg/ml) and RNase A treatment (1ug/ml) for 30 min at room temperature. Stained cells were analyzed by flow cytometry (Accuri-BD).

Total RNA was extracted from the cells with the use of Direct-zol RNA miniprep kit (Zymo, cat no.: R2061) as per the manufacturer’s instructions. Purity of RNA was examined by Nano Drop spectrophotometer, and 200 ng of RNA was used for cDNA synthesis by iScript CDNA synthesis kit (Bio-Rad, cat no 1708890). cDNA was amplified with the selected pair of forward and reverse primers using Bio-Rad CFX-Connect device by SYBR green-based amplification. IPW F: 5’-AGGGCAGTGCGTATTTGAAG-3’, IPW R: 5’-GGACAGTTTTGCTTCCCTAG-3’, ID2 F: 5′ -TCAGCACTTAAAAGATTCCGTG-3′, ID2 R: 5′-GACAGCAAAGCACTGTGTGG-3’, β-Actin F:5’-TGAGACCTTCAACACCCCAGCCATG-3’, β-Actin R: 5’-CGTAGATGGGCACAGTGTGGGTG-3’ XBP F: 5’- GGCCGACGGGACCCCTAAAG-3’, R: 5’- CCTCAGCGCCTTCTCCTCGG-3’, JNK F: 5’- CGGTCTGGCCAGGACTGCAG-3’, R: 5’- GCCCATGCCAAGGATGACCTCG-3’ ATF4 F: 5’- GACCAGTCGGGTTTGGGGGC-3’, R: 5’- GACTGACCAACCCATCCACAGCC-3’, CHOP F:5’- GCTGGGAGCTGGAAGCCTGG-3’, R: 5’- GCTGGTTCTGGCTCCTCCTCAG-3’. miR-29c amplification was carried out using TaqMann probes (Applied bio system-479229), and miR-361 (Applied biosystem-478056) was used as internal control. Briefly, RNA was isolated from the cells as mentioned and purity was confirmed using Nano Drop spectrophotometer (Fisher Scientific). Ten nanograms of RNA was used for cDNA synthesis. miRNA cDNA synthesis was carried out using TaqMan advanced miRNA cDNA synthesis kit (Applied Biosystems, cat no.: A28007) as per manufacturer’s instructions. Quantitative PCR was performed with TaqMan Fast Advanced Master Mix (2X) with total reaction volume of 15 µl. Reaction conditions were followed as instructed for 7500 and 7500 Fast systems for 40 cycles. The results were analyzed by relative quantification method. All qRT PCR results were normalized with respective non-tumorigenic/control experimental set. miR-29c LNA probes (miRCURY-YI04105460) were used to silence the endogenous miR-29c expression. For LNA transfection, 200,000 cells per well were seeded in 12-well plate. LNA probe (2uM) was mixed with lipofectamine RNAiMax for 5 min in opti-MEM medium and added to the cells.

Cell lysate was prepared using RIPA buffer, and debris was removed by centrifuging at 10,000 RPM for 10 min. The lysate was then quantified in NanoDrop 2000, ran on 10% SDS-polyacrylamide gel and then transferred to nitrocellulose membrane (Bio Rad) by the semi-dry method (16 V for 3 h). Membrane was blocked by 5% nonfat skim milk powder dissolved in PBST for 30 min at room temperature. Blots were incubated with ID2 (1:500, Abcam), GAPDH (1:5000, CST), OCT4 (1:1000, CST), SOX2 (1: 1000, CST), NANOG (1:1000, CST), p21 (1:1000, CST) primary and anti-rabbit (1:2000, CST) and mouse (1:2000 Sigma) HRP-conjugated secondary antibodies. Primary antibody was incubated overnight at 4⁰C, while secondary antibody was incubated for 1 h at room temperature. Blots were developed with the use of ECL western blotting detection reagent (GE healthcare, RPN2236) and Amersham Imager.

Tumor tissues were isolated from mice at the experimental endpoint and formalin-fixed paraffin-embedded, and 5-mm tissue sections were prepared. Deparaffinization was carried out by heating the slides in oven at 100⁰ C for 30 min followed by rehydration in series on xylene and ethanol. Antigen retrieval was done in sodium citrate (10 mM, pH 6) buffer for 95 °C for 30 min. Endogenous peroxidase was quenched by treating the slides with 3% H₂O₂ for 15 min at room temperature followed by PBS washes, 3 × 5 min. Afterward, slides were washed in PBS and blocked with 2% BSA for 1 h followed by incubation with ID2 antibody (1:200, Abcam), OCT4 (1:200,CST), SOX2 (1:200,CST), NANOG (1:200,CST), p21 (1:200,CST), cytokeratin (1:200, abcam) at 4⁰C overnight. This was followed by washing with PBST three times. Then, sections were incubated with secondary antibody and visualized using HRP-based Envision-plus kit (Dako Corp.). The tumor sections were ranked based on staining intensity to calculate the percentage of positive cells.

Animal experiments were performed in line with protocol approved by Wake Forest Institutional Animal Care and Use committee. Animals were housed in temperature controlled and pathogen-free environment with 12-h light/12-h dark cycle and free access to food and water. Puromycin-selected DCIS IPW and DCIS pSIN cells were transduced with pSIN luciferase virus. 1 × 10⁶ cells were resuspended in Matrigel and injected in mammary fat pad of nude mice (n = 10/group). Animals were then imaged by IVIS every week after injection. For drug treatment, animals (n = 6/group) were injected with luciferase-labelled 1 × 10⁶ DCIS.com cells in mammary fat pad. Toyocamycin treatment (2 mg/kg) was started after 2 weeks of cell implantation and administered once a week intraperitoneally. The tumor growth was quantified by IVIS imaging mice twice a week after drug injection. Animals were killed when the tumors in the control group reached 1000mm³ or morbid as instructed by the veterinarians. All animals were randomized before the experiment.