Peptide Inhibitor Targeting the Extraterminal Domain in BRD4 Potently Suppresses Breast Cancer Both In Vitro and In Vivo.

BRD4 is associated with a variety of human diseases, including breast cancer. The crucial roles of amino-terminal bromodomains (BDs) of BRD4 in binding with acetylated histones to regulate oncogene expression make them promising drug targets. However, adverse events impede the development of the BD inhibitors. BRD4 adopts an extraterminal (ET) domain, which recruits proteins to drive oncogene expression. We discovered a peptide inhibitor PiET targeting the ET domain to disrupt BRD4/JMJD6 interaction, a protein complex critical in oncogene expression and breast cancer. The cell-permeable form of PiET, TAT-PiET, and PROTAC-modified TAT-PiET, TAT-PiET-PROTAC, potently inhibits the expression of BRD4/JMJD6 target genes and breast cancer cell growth. Combination therapy with TAT-PiET/TAT-PiET-PROTAC and JQ1, iJMJD6, or Fulvestrant exhibits synergistic effects. TAT-PiET or TAT-PiET-PROTAC treatment overcomes endocrine therapy resistance in ERα-positive breast cancer cells. Taken together, we demonstrated that targeting the ET domain is effective in suppressing breast cancer, providing a therapeutic avenue in the clinic.

LncRNA LUESCC promotes esophageal squamous cell carcinoma by targeting the miR-6785-5p/NRSN2 axis.

Esophageal squamous cell carcinoma (ESCC) is one of the most prevalent gastrointestinal malignancies with high mortality worldwide. Emerging evidence indicates that long noncoding RNAs (lncRNAs) are involved in human cancers, including ESCC. However, the detailed mechanisms of lncRNAs in the regulation of ESCC progression remain incompletely understood. LUESCC was upregulated in ESCC tissues compared with adjacent normal tissues, which was associated with gender, deep invasion, lymph node metastasis, and poor prognosis of ESCC patients. LUESCC was mainly localized in the cytoplasm of ESCC cells. Knockdown of LUESCC inhibited cell proliferation, colony formation, migration, and invasion in vitro and suppressed tumor growth in vivo. Mechanistic investigation indicated that LUESCC functions as a ceRNA by sponging miR-6785-5p to enhance NRSN2 expression, which is critical for the malignant behaviors of ESCC. Furthermore, ASO targeting LUESCC substantially suppressed ESCC both in vitro and in vivo. Collectively, these data demonstrate that LUESCC may exerts its oncogenic role by sponging miR-6785-5p to promote NRSN2 expression in ESCC, providing a potential diagnostic marker and therapeutic target for ESCC patients.

CircHIPK3 regulates fatty acid metabolism through miR-637/FASN axis to promote esophageal squamous cell carcinoma.

The oncogenic role of circRNA in cancers including esophageal cancer (EC) has been well studied. However, whether and how circRNAs are involved in cancer cell metabolic processes remains largely unknown. Here, we reported that circRNA, circHIPK3, is highly expressed in ESCC cell lines and tissues. Knockdown of circHIPK3 significantly restrained cell proliferation, colony formation, migration, and invasion in vitro and inhibited tumor growth in vivo. Mechanistically, circHIPK3 was found to act as a ceRNA by sponging miR-637 to regulate FASN expression and fatty acid metabolism in ESCC cells. Anti-sense oligonucleotide (ASO) targeting circHIPK3 substantially inhibited ESCC both in vitro and in vivo. Therefore, these results uncover a modulatory axis constituting of circHIPK3/miR-637/FASN may be a potential biomarker and therapeutic target for ESCC in the clinic.

Targeting PRMT1-mediated SRSF1 methylation to suppress oncogenic exon inclusion events and breast tumorigenesis.

PRMT1 plays a vital role in breast tumorigenesis; however, the underlying molecular mechanisms remain incompletely understood. Herein, we show that PRMT1 plays a critical role in RNA alternative splicing, with a preference for exon inclusion. PRMT1 methylome profiling identifies that PRMT1 methylates the splicing factor SRSF1, which is critical for SRSF1 phosphorylation, SRSF1 binding with RNA, and exon inclusion. In breast tumors, PRMT1 overexpression is associated with increased SRSF1 arginine methylation and aberrant exon inclusion, which are critical for breast cancer cell growth. In addition, we identify a selective PRMT1 inhibitor, iPRMT1, which potently inhibits PRMT1-mediated SRSF1 methylation, exon inclusion, and breast cancer cell growth. Combination treatment with iPRMT1 and inhibitors targeting SRSF1 phosphorylation exhibits an additive effect of suppressing breast cancer cell growth. In conclusion, our study dissects a mechanism underlying PRMT1-mediated RNA alternative splicing. Thus, PRMT1 has great potential as a therapeutic target in breast cancer treatment.

Estrogen-Induced LncRNA, LINC02568, Promotes Estrogen Receptor-Positive Breast Cancer Development and Drug Resistance Through Both In Trans and In Cis Mechanisms.

Endocrine therapy is the frontline treatment for estrogen receptor (ER) positive breast cancer patients. However, the primary and acquired resistance to endocrine therapy drugs remain as a major challenge in the clinic. Here, this work identifies an estrogen-induced lncRNA, LINC02568, which is highly expressed in ER-positive breast cancer and functional important in cell growth in vitro and tumorigenesis in vivo as well as endocrine therapy drug resistance. Mechanically, this work demonstrates that LINC02568 regulates estrogen/ERα-induced gene transcriptional activation in trans by stabilizing ESR1 mRNA through sponging miR-1233-5p in the cytoplasm. Meanwhile, LINC02568 contributes to tumor-specific pH homeostasis by regulating carbonic anhydrase CA12 in cis in the nucleus. The dual functions of LINC02568 together contribute to breast cancer cell growth and tumorigenesis as well as endocrine therapy drug resistance. Antisense oligonucleotides (ASO) targeting LINC02568 significantly inhibits ER-positive breast cancer cell growth in vitro and tumorigenesis in vivo. Furthermore, combination treatment with ASO targeting LINC02568 and endocrine therapy drugs or CA12 inhibitor U-104 exhibits synergistic effects on tumor growth. Taken together, the findings reveal the dual mechanisms of LINC02568 in regulating ERα signaling and pH homeostasis in ER-positive breast cancer, and indicated that targeting LINC02568 might represent a potential therapeutic avenue in the clinic.

CircPVT1 promotes ER-positive breast tumorigenesis and drug resistance by targeting ESR1 and MAVS.

The molecular mechanisms underlying estrogen receptor (ER)-positive breast carcinogenesis and endocrine therapy resistance remain incompletely understood. Here, we report that circPVT1, a circular RNA generated from the lncRNA PVT1, is highly expressed in ERα-positive breast cancer cell lines and tumor samples and is functionally important in promoting ERα-positive breast tumorigenesis and endocrine therapy resistance. CircPVT1 acts as a competing endogenous RNA (ceRNA) to sponge miR-181a-2-3p, promoting the expression of ESR1 and downstream ERα-target genes and breast cancer cell growth. Furthermore, circPVT1 directly interacts with MAVS protein to disrupt the RIGI-MAVS complex formation, inhibiting type I interferon (IFN) signaling pathway and anti-tumor immunity. Anti-sense oligonucleotide (ASO)-targeting circPVT1 inhibits ERα-positive breast cancer cell and tumor growth, re-sensitizing tamoxifen-resistant ERα-positive breast cancer cells to tamoxifen treatment. Taken together, our data demonstrated that circPVT1 can work through both ceRNA and protein scaffolding mechanisms to promote cancer. Thus, circPVT1 may serve as a diagnostic biomarker and therapeutic target for ERα-positive breast cancer in the clinic.

A SARS-CoV-2-specific CAR-T-cell model identifies felodipine, fasudil, imatinib, and caspofungin as potential treatments for lethal COVID-19.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-induced cytokine storm is closely associated with coronavirus disease 2019 (COVID-19) severity and lethality. However, drugs that are effective against inflammation to treat lethal COVID-19 are still urgently needed. Here, we constructed a SARS-CoV-2 spike protein-specific CAR, and human T cells infected with this CAR (SARS-CoV-2-S CAR-T) and stimulated with spike protein mimicked the T-cell responses seen in COVID-19 patients, causing cytokine storm and displaying a distinct memory, exhausted, and regulatory T-cell phenotype. THP1 remarkably augmented cytokine release in SARS-CoV-2-S CAR-T cells when they were in coculture. Based on this "two-cell" (CAR-T and THP1 cells) model, we screened an FDA-approved drug library and found that felodipine, fasudil, imatinib, and caspofungin were effective in suppressing the release of cytokines, which was likely due to their ability to suppress the NF-κB pathway in vitro. Felodipine, fasudil, imatinib, and caspofungin were further demonstrated, although to different extents, to attenuate lethal inflammation, ameliorate severe pneumonia, and prevent mortality in a SARS-CoV-2-infected Syrian hamster model, which were also linked to their suppressive role in inflammation. In summary, we established a SARS-CoV-2-specific CAR-T-cell model that can be utilized as a tool for anti-inflammatory drug screening in a fast and high-throughput manner. The drugs identified herein have great potential for early treatment to prevent COVID-19 patients from cytokine storm-induced lethality in the clinic because they are safe, inexpensive, and easily accessible for immediate use in most countries.

A Tumor-suppressive Molecular Axis EP300/circRERE/miR-6837-3p/MAVS Activates Type I IFN Pathway and Antitumor Immunity to Suppress Colorectal Cancer.

PURPOSE: The oncogenic role of circular RNAs (circRNA) has been well studied in cancers including colorectal cancer. However, tumor-suppressive circRNAs and the mechanism through which they exert their antitumor effects remain largely unknown. We aim to find out the critical tumor-suppressive circRNAs and their possibility to serve as gene therapy targets. EXPERIMENTAL DESIGN: circRNA sequencing, gain-of-function and loss-of-function experiments, and transcriptomic analysis were performed to find tumor-suppressive and antitumor immunity effects of circRERE. Molecular biology experiments were conducted for mechanism exploration. Finally, we conducted adeno-associated virus (AAV) to deliver circRERE (circRERE-AAV) and evaluated circRERE-AAV alone and in combination with anti-PD-1 antibody in C57BL/6J mice bearing subcutaneous MC38 tumors. RESULTS: circRERE is lowly expressed in colorectal cancer. Overexpression of circRERE inhibits the malignant behaviors of colorectal cancer in vitro and in vivo, while knockdown exhibits the opposite effects. The expression of circRERE is regulated by EP300, a histone acetyltransferase downregulated in colorectal cancer as well. Mechanistically, circRERE acts as a competitive endogenous RNA to sponge miR-6837-3p to upregulate MAVS expression, thereby activating type I IFN signaling and promoting antitumor immunity. Delivery of circRERE-AAV elicits significant antitumor effects, and combination treatment with circRERE-AAV and anti-PD-1 antibody exhibits synergistic effects on tumor growth in preclinical models of colorectal cancer. CONCLUSIONS: These results uncover modulatory axis constituting of EP300/circRERE/miR-6837-3p/MAVS and its essential roles in antitumor immunity, and demonstrate that circRERE-AAV might represent a new therapeutic avenue to prime immune responses and boost the effects of immunotherapy in clinic.

CPPA: A Web Tool for Exploring Proteomic and Phosphoproteomic Data in Cancer.

A tremendous amount of proteomic and phosphoproteomic data has been produced over the years with the development of mass spectrometry techniques, providing us with new opportunities to explore and understand the proteome and phosphoproteome as well as the function of proteins and protein phosphorylation sites. However, a lack of powerful tools that we can utilize to explore these valuable data limits our understanding of the proteome and phosphoproteome, particularly in diseases such as cancer. To address these unmet needs, we established CPPA (Cancer Proteome and Phosphoproteome Atlas), a web tool to mine abnormalities of the proteome and phosphoproteome in cancer based on published data sets. All analysis results are presented in CPPA with a flexible web interface to provide key customization utilities, including general analysis, differential expression profiling, statistical analysis of protein phosphorylation sites, correlation analysis, similarity analysis, survival analysis, pathological stage analysis, etc. CPPA greatly facilitates the process of data mining and therapeutic target discovery by providing a comprehensive analysis of proteomic and phosphoproteomic data in normal and tumor tissues with a simple click, which helps to unlock the precious value of mass spectrometry data by bridging the gap between raw data and experimental biologists. CPPA is currently available at https://cppa.site/cppa.

Super-enhancer-controlled positive feedback loop BRD4/ERα-RET-ERα promotes ERα-positive breast cancer.

Estrogen and estrogen receptor alpha (ERα)-induced gene transcription is tightly associated with ERα-positive breast carcinogenesis. ERα-occupied enhancers, particularly super-enhancers, have been suggested to play a vital role in regulating such transcriptional events. However, the landscape of ERα-occupied super-enhancers (ERSEs) as well as key ERα-induced target genes associated with ERSEs remain to be fully characterized. Here, we defined the landscape of ERSEs in ERα-positive breast cancer cell lines, and demonstrated that bromodomain protein BRD4 is a master regulator of the transcriptional activation of ERSEs and cognate ERα target genes. RET, a member of the tyrosine protein kinase family of proteins, was identified to be a key ERα target gene of BRD4-regulated ERSEs, which, in turn, is vital for ERα-induced gene transcriptional activation and malignant phenotypes through activating the RAS/RAF/MEK2/ERK/p90RSK/ERα phosphorylation cascade. Combination therapy with BRD4 and RET inhibitors exhibited additive effects on suppressing ERα-positive breast cancer both in vitro and in vivo, comparable with that of standard endocrine therapy tamoxifen. Furthermore, combination therapy re-sensitized a tamoxifen-resistant ERα-positive breast cancer cell line to tamoxifen treatment. Taken together, our data uncovered the critical role of a super-enhancer-associated positive feedback loop constituting BRD4/ERα-RET-ERα in ERα-positive breast cancer, and suggested that targeting components in this loop would provide a new therapeutic avenue for treating ERα-positive breast cancer in the clinic.

A specific JMJD6 inhibitor potently suppresses multiple types of cancers both in vitro and in vivo.

Jumonji C-domain-containing protein 6 (JMJD6), an iron (Fe2+) and α-ketoglutarate (α-KG)-dependent oxygenase, is expressed at high levels, correlated with poor prognosis, and considered as a therapeutic target in multiple cancer types. However, specific JMJD6 inhibitors that are potent in suppressing tumorigenesis have not been reported so far. We herein report that iJMJD6, a specific small-molecule inhibitor of JMJD6 with favorable physiochemical properties, inhibits the enzymatic activity of JMJD6 protein both in vitro and in cultured cells. iJMJD6 is effective in suppressing cell proliferation, migration, and invasion in multiple types of cancer cells in a JMJD6-dependent manner, while it exhibits minimal toxicity in normal cells. Mechanistically, iJMJD6 represses the expression of oncogenes, including Myc and CCND1, in accordance with JMJD6 function in promoting the transcription of these genes. iJMJD6 exhibits suitable pharmacokinetic properties and suppresses tumor growth in multiple cancer cell line- and patient-derived xenograft models safely. Furthermore, combination therapy with iJMJD6 and BET protein inhibitor (BETi) JQ1 or estrogen receptor antagonist fulvestrant exhibits synergistic effects in suppressing tumor growth. Taken together, we demonstrate that inhibition of JMJD6 enzymatic activity by using iJMJD6 is effective in suppressing oncogene expression and cancer development, providing a therapeutic avenue for treating cancers that are dependent on JMJD6 in the clinic.

Long non-coding RNA LINC00680 functions as a ceRNA to promote esophageal squamous cell carcinoma progression through the miR-423-5p/PAK6 axis.

BACKGROUND: Esophageal squamous cell carcinoma (ESCC) is a common invasive malignancy worldwide with poor clinical outcomes. Increasing amount of long non-coding RNAs (lncRNAs) have been reported to be involved in cancer development. However, lncRNAs that are functional in ESCC and the underlying molecular mechanisms remain largely unknown. METHODS: Transcriptomic analysis was performed to identify dysregulated lncRNAs in ESCC tissue samples. The high expression of LINC00680 in ESCC was validated by RT-qPCR, and the oncogenic functions of LINC00680 was investigated by cell proliferation, colony formation, migration and invasion assays in ESCC cells in vitro and xenografts derived from ESCC cells in mice. RNA-seq, competitive endogenous RNA (ceRNA) network analysis, and luciferase reporter assays were carried out to identify LINC00680 target genes and the microRNAs (miRNAs) bound to LINC00680. Antisense oligonucleotides (ASOs) were used for in vivo treatment. RESULTS: Transcriptome profiling revealed that a large number of lncRNAs was dysregulated in ESCC tissues. Notably, LINC00680 was highly expressed, and upregulation of LINC00680 was associated with large tumor size, advanced tumor stage, and poor prognosis. Functionally, knockdown of LINC00680 restrained ESCC cell proliferation, colony formation, migration, and invasion in vitro and inhibited tumor growth in vivo. Mechanistically, LINC00680 was found to act as a ceRNA by sponging miR-423-5p to regulate PAK6 (p21-activated kinase 6) expression in ESCC cells. The cell viability and motility inhibition induced by LINC00680 knockdown was significantly reversed upon PAK6 restoration and miR-423-5p inhibition. Furthermore, ASO targeting LINC00680 substantially suppressed ESCC both in vitro and in vivo. CONCLUSIONS: An oncogenic lncRNA, LINC00680, was identified in ESCC, which functions as a ceRNA by sponging miR-423-5p to promote PAK6 expression and ESCC. LINC00680/miR-423-5p/PAK6 axis may serve as promising diagnostic and prognostic biomarkers and therapeutic targets for ESCC.

The Mediator subunit MED20 organizes the early adipogenic complex to promote development of adipose tissues and diet-induced obesity.

MED20 is a non-essential subunit of the transcriptional coactivator Mediator complex, but its physiological function remains largely unknown. Here, we identify MED20 as a substrate of the anti-obesity CRL4-WDTC1 E3 ubiquitin ligase complex through affinity purification and candidate screening. Overexpression of WDTC1 leads to degradation of MED20, whereas depletion of WDTC1 or CUL4A/B causes accumulation of MED20. Depleting MED20 inhibits adipogenesis, and a non-degradable MED20 mutant restores adipogenesis in WDTC1-overexpressing cells. Furthermore, knockout of Med20 in preadipocytes abolishes development of brown adipose tissues. Removing one allele of Med20 in preadipocytes protects mice from diet-induced obesity and reverses weight gain in Cul4a- or Cul4b-depleted mice. Chromatin immunoprecipitation sequencing (ChIP-seq) analysis reveals that MED20 organizes the early adipogenic complex by bridging C/EBPß and RNA polymerase II to promote transcription of the central adipogenic factor, PPARγ. Our findings have thus uncovered a critical role of MED20 in promoting adipogenesis, development of adipose tissue and diet-induced obesity.

DDB1 binds histone reader BRWD3 to activate the transcriptional cascade in adipogenesis and promote onset of obesity.

Obesity has become a global pandemic. Identification of key factors in adipogenesis helps to tackle obesity and related metabolic diseases. Here, we show that DDB1 binds the histone reader BRWD3 to promote adipogenesis and diet-induced obesity. Although typically recognized as a component of the CUL4-RING E3 ubiquitin ligase complex, DDB1 stimulates adipogenesis independently of CUL4. A DDB1 mutant that does not bind CUL4A or CUL4B fully restores adipogenesis in DDB1-deficient cells. Ddb1+/- mice show delayed postnatal development of white adipose tissues and are protected from diet-induced obesity. Mechanistically, by interacting with BRWD3, DDB1 is recruited to acetylated histones in the proximal promoters of ELK1 downstream immediate early response genes and facilitates the release of paused RNA polymerase II, thereby activating the transcriptional cascade in adipogenesis. Our findings have uncovered a CUL4-independent function of DDB1 in promoting the transcriptional cascade of adipogenesis, development of adipose tissues, and onset of obesity.

BRD4 inhibition boosts the therapeutic effects of epidermal growth factor receptor-targeted chimeric antigen receptor T cells in glioblastoma.

Glioblastoma (GBM) is the deadliest brain malignancy without effective treatments. Here, we reported that epidermal growth factor receptor-targeted chimeric antigen receptor T cells (EGFR CAR-T) were effective in suppressing the growth of GBM cells in vitro and xenografts derived from GBM cell lines and patients in mice. However, mice soon acquired resistance to EGFR CAR-T cell treatment, limiting its potential use in the clinic. To find ways to improve the efficacy of EGFR CAR-T cells, we performed genomics and transcriptomics analysis for GBM cells incubated with EGFR CAR-T cells and found that a large cohort of genes, including immunosuppressive genes, as well as enhancers in vicinity are activated. BRD4, an epigenetic modulator functioning on both promoters and enhancers, was required for the activation of these immunosuppressive genes. Accordingly, inhibition of BRD4 by JQ1 blocked the activation of these immunosuppressive genes. Combination therapy with EGFR CAR-T cells and JQ1 suppressed the growth and metastasis of GBM cells and prolonged survival in mice. We demonstrated that transcriptional modulation by targeting epigenetic regulators could improve the efficacy of immunotherapy including CAR-T, providing a therapeutic avenue for treating GBM in the clinic.

The Dual Function of KDM5C in Both Gene Transcriptional Activation and Repression Promotes Breast Cancer Cell Growth and Tumorigenesis.

Emerging evidence suggested that epigenetic regulators can exhibit both activator and repressor activities in gene transcriptional regulation and disease development, such as cancer. However, how these dual activities are regulated and coordinated in specific cellular contexts remains elusive. Here, it is reported that KDM5C, a repressive histone demethylase, unexpectedly activates estrogen receptor alpha (ERα)-target genes, and meanwhile suppresses type I interferons (IFNs) and IFN-stimulated genes (ISGs) to promote ERα-positive breast cancer cell growth and tumorigenesis. KDM5C-interacting protein, ZMYND8, is found to be involved in both processes. Mechanistically, KDM5C binds to active enhancers and recruits the P-TEFb complex to activate ERα-target genes, while inhibits TBK1 phosphorylation in the cytosol to repress type I IFNs and ISGs. Pharmacological inhibition of both ERα and KDM5C is effective in inhibiting cell growth and tumorigenesis. Taken together, it is revealed that the dual activator and repressor nature of an epigenetic regulator together contributes to cancer development.

Targeting Triple-Negative Breast Cancer with Combination Therapy of EGFR CAR T Cells and CDK7 Inhibition.

EGFR-targeted chimeric antigen receptor (CAR) T cells are potent and specific in suppressing the growth of triple-negative breast cancer (TNBC) in vitro and in vivo. However, in this study, a subset of mice soon acquired resistance, which limits the potential use of EGFR CAR T cells. We aimed to find a way to overcome the observed resistance. Transcriptomic analysis results revealed that EGFR CAR T-cell treatment induced a set of immunosuppressive genes, presumably through IFNγ signaling, in EGFR CAR T-cell-resistant TNBC tumors. The EGFR CAR T-cell-induced immunosuppressive genes were associated with EGFR CAR T-cell-activated enhancers and were especially sensitive to THZ1, a CDK7 inhibitor we screened out of a panel of small molecules targeting epigenetic modulators. Accordingly, combination therapy with THZ1 and EGFR CAR T cells suppressed immune resistance, tumor growth, and metastasis in TNBC tumor models, including human MDA-MB-231 cell-derived and TNBC patient-derived xenografts, and mouse EMT6 cell-derived allografts. Taken together, we demonstrated that transcriptional modulation using epigenetic inhibitors could overcome CAR T-cell therapy-induced immune resistance, thus providing a therapeutic avenue for treating TNBC in the clinic.

Profiling PRMT methylome reveals roles of hnRNPA1 arginine methylation in RNA splicing and cell growth.

Numerous substrates have been identified for Type I and II arginine methyltransferases (PRMTs). However, the full substrate spectrum of the only type III PRMT, PRMT7, and its connection to type I and II PRMT substrates remains unknown. Here, we use mass spectrometry to reveal features of PRMT7-regulated methylation. We find that PRMT7 predominantly methylates a glycine and arginine motif; multiple PRMT7-regulated arginine methylation sites are close to phosphorylations sites; methylation sites and proximal sequences are vulnerable to cancer mutations; and methylation is enriched in proteins associated with spliceosome and RNA-related pathways. We show that PRMT4/5/7-mediated arginine methylation regulates hnRNPA1 binding to RNA and several alternative splicing events. In breast, colorectal and prostate cancer cells, PRMT4/5/7 are upregulated and associated with high levels of hnRNPA1 arginine methylation and aberrant alternative splicing. Pharmacological inhibition of PRMT4/5/7 suppresses cancer cell growth and their co-inhibition shows synergistic effects, suggesting them as targets for cancer therapy.

Estrogen-induced circRNA, circPGR, functions as a ceRNA to promote estrogen receptor-positive breast cancer cell growth by regulating cell cycle-related genes.

Estrogen and estrogen receptor (ER)-regulated gene transcriptional events have been well known to be involved in ER-positive breast carcinogenesis. Meanwhile, circular RNAs (circRNAs) are emerging as a new family of functional non-coding RNAs (ncRNAs) with implications in a variety of pathological processes, such as cancer. However, the estrogen-regulated circRNA program and the function of such program remain uncharacterized. Methods: CircRNA sequencing (circRNA-seq) was performed to identify circRNAs induced by estrogen, and cell proliferation, colony formation, wound healing, transwell and tumor xenograft experiments were applied to examine the function of estrogen-induced circRNA, circPGR. RNA sequencing (RNA-seq) and ceRNA network analysis wereperformed to identify circPGR's target genes and the microRNA (miRNA) bound to circPGR. Anti-sense oligonucleotide (ASO) was used to assess circPGR's effects on ER-positive breast cancer cell growth. Results: Genome-wide circRNA profiling by circRNA sequencing (circRNA-seq) revealed that a large number of circRNAs were induced by estrogen, and further functional screening for the several circRNAs originated from PGR revealed that one of them, which we named as circPGR, was required for ER-positive breast cancer cell growth and tumorigenesis. CircPGR was found to be localized in the cytosol of cells and functioned as a competing endogenous RNA (ceRNA) to sponge miR-301a-5p to regulate the expression of multiple cell cycle genes. The clinical relevance of circPGR was underscored by its high and specific expression in ER-positive breast cancer cell lines and clinical breast cancer tissue samples. Accordingly, anti-sense oligonucleotide (ASO) targeting circPGR was proven to be effective in suppressing ER-positive breast cancer cell growth. Conclusions: These findings reveled that, besides the well-known messenger RNA (mRNA), microRNA (miRNA), long non-coding RNA (lncRNA) and enhancer RNA (eRNA) programs, estrogen also induced a circRNA program, and exemplified by circPGR, these estrogen-induced circRNAs were required for ER-positive breast cancer cell growth, providing a new class of therapeutic targets for ER-positive breast cancer.