Anjana Bhardwaj

Identification of a MicroRNA that Activates Gene Expression by Repressing Nonsense-Mediated RNA Decay

Nonsense-mediated decay (NMD) degrades both normal and aberrant transcripts harboring stop codons in particular contexts. Mutations that perturb NMD cause neurological disorders in humans, suggesting that NMD has roles in the brain. Here, we identify a brain-specific microRNA-miR-128-that represses NMD and thereby controls batteries of transcripts in neural cells. miR-128 represses NMD by targeting the RNA helicase UPF1 and the exon-junction complex core component MLN51. The ability of miR-128 to regulate NMD is a conserved response occurring in frogs, chickens, and mammals. miR-128 levels are dramatically increased in differentiating neuronal cells and during brain development, leading to repressed NMD and upregulation of mRNAs normally targeted for decay by NMD; overrepresented are those encoding proteins controlling neuron development and function. Together, these results suggest the existence of a conserved RNA circuit linking the microRNA and NMD pathways that induces cell type-specific transcripts during development.

GATA Factors and Androgen Receptor Collaborate To Transcriptionally Activate the Rhox5 Homeobox Gene in Sertoli Cells

ABSTRACT How Sertoli-specific expression is initiated is poorly understood. Here, we address this issue using the proximal promoter (Pp) from the Rhox5 homeobox gene. Its Sertoli cell-specific expression is achieved, in part, through a negative regulatory element that inhibits Pp transcription in non-Sertoli cell lines. Complementing this negative regulation is positive regulation conferred by four androgen-response elements (AREs) that interact with the androgen receptor (AR), a nuclear hormone receptor expressed at high levels in Sertoli cells. A third control mechanism is provided by a consensus GATA-binding site that is crucial for Pp transcription both in vitro and in vivo. Several lines of evidence suggested that GATA factors and AR act cooperatively to activate Pp transcription: (i) the GATA-binding site crucial for Pp transcription is in close proximity to two of the AREs, (ii) GATA and AR form a complex with the Pp in vitro, (iii) overexpression of GATA factors rescued expression from mutant Pp constructs harboring defective AREs, and (iv) incubation of a Sertoli cell line with testosterone triggered corecruitment of AR and GATA4 to the Pp. Collectively, our results suggest that the Rhox5 gene achieves Sertoli cell-specific transcription using a combinatorial strategy involving negative and cooperative positive regulation.

The Rhox Homeobox Gene Cluster Is Imprinted and Selectively Targeted for Regulation by Histone H1 and DNA Methylation

ABSTRACT Histone H1 is an abundant and essential component of chromatin whose precise role in regulating gene expression is poorly understood. Here, we report that a major target of H1-mediated regulation in embryonic stem (ES) cells is the X-linked Rhox homeobox gene cluster. To address the underlying mechanism, we examined the founding member of the Rhox gene cluster—Rhox5—and found that its distal promoter (Pd) loses H1, undergoes demethylation, and is transcriptionally activated in response to loss of H1 genes in ES cells. Demethylation of the Pd is required for its transcriptional induction and we identified a single cytosine in the Pd that, when methylated, is sufficient to inhibit Pd transcription. Methylation of this single cytosine prevents the Pd from binding GA-binding protein (GABP), a transcription factor essential for Pd transcription. Thus, H1 silences Rhox5 transcription by promoting methylation of one of its promoters, a mechanism likely to extend to other H1-regulated Rhox genes, based on analysis of ES cells lacking DNA methyltransferases. The Rhox cluster genes targeted for H1-mediated transcriptional repression are also subject to another DNA methylation-regulated process: Xp imprinting. Remarkably, we found that only H1-regulated Rhox genes are imprinted, not those immune to H1-mediated repression. Together, our results indicate that the Rhox gene cluster is a major target of H1-mediated transcriptional repression in ES cells and that H1 is a candidate to have a role in Xp imprinting.

Regulation and Function of the Rhox5 Homeobox Gene

Abstract: Recently, a large cluster of homeobox genes was discovered on the X chromosome that is expressed in reproductive tissues after birth. It is postulated that these reproductive homeobox genes on the X chromosome (Rhox) encode transcription factors that regulate gametogenesis. In support of this, male mice lacking the founding member of this gene cluster, Rhox5, are subfertile, exhibiting increased germ‐cell apoptosis and a defect in sperm motility. To identify RHOX5 targets, microarray analyses were used to identify genes differently expressed in postnatal testes from Rhox5‐null and control littermates. Highly overrepresented were genes that encode proteins involved in cellular metabolism. Several lines of evidence indicated that one of these, insulin II, is a direct target of RHOX5. Microarray analysis was also used to identify genes differentially expressed in response to physiological levels of Rhox5 in a Sertoli‐cell line. Among the few genes identified, the netrin‐1 receptor UNC5c, a proapoptotic molecule that is inhibited by RHOX5, was also regulated in vivo, and is thus a candidate to be downstream of RHOX5 in a prosurvival germ‐cell pathway. To understand the means by which Rhox5 expression is restricted to Sertoli nurse cells in the testis, a variety of molecular approaches were used in both Sertoli‐cell lines and mice. This analysis revealed that both positive and negative cis elements collaborate to confer Sertoli cell‐specific gene expression. Acting on the positive cis elements are androgen receptor and GATA transcription factors. Collectively, the results of this study provide an initial glimpse into the regulatory networks that control spermatogenesis.

DNA Demethylation-Dependent AR Recruitment and GATA Factors Drive Rhox5 Homeobox Gene Transcription in the Epididymis

Mammalian male fertility depends on the epididymis, a highly segmented organ that promotes sperm maturation and protects sperm from oxidative damage. Remarkably little is known about how gene expression is controlled in the epididymis. A candidate to regulate genes crucial for epididymal function is reproductive homeobox gene on X chromosome (RHOX)5, a homeobox transcription factor essential for optimal sperm motility that is expressed in the caput region of the epididymis. Here, we report the identification of factors that control Rhox5 gene expression in epididymal cells in a developmentally regulated and region-specific fashion. First, we identify GATA transcription factor-binding sites in the Rhox5 proximal promoter (Pp) necessary for Rhox5 expression in epididymal cells in vitro and in vivo. Adjacent to the GATA sites are androgen-response elements, which bind to the nuclear hormone receptor androgen receptor (AR), and are responsible for the AR-dependent expression of Rhox5 in epididymal cells. We provide evidence that AR is recruited to the Pp in a region-specific and developmentally regulated manner in the epididymis that is dictated not only by differential AR availability but differential methylation of the Pp. Site-specific methylation of the Pp cytosine and guanine separated by one phosphate, most of which overlap with androgen-response elements, inhibited both AR occupancy at the Pp and Pp-dependent transcription in caput epididymal cells. Together, our data support a model in which DNA methylation, AR, and GATA factors collaborate to dictate the unique developmental and region-specific expression pattern of the RHOX5 homeobox transcription factor in the caput epididymis, which in turn controls the expression of genes critical for promoting sperm motility and function.

Annexin A1 Preferentially Predicts Poor Prognosis of Basal-Like Breast Cancer Patients by Activating mTOR-S6 Signaling

Introduction Annexin A1 (ANXA1) is an anti-inflammatory protein reported to play a role in cell proliferation and apoptosis, and to be deregulated in breast cancer. The exact role of annexin A1 in the biology of breast cancer remains unclear. We hypothesized that the annexin A1 plays an oncogenic role in basal subtype of breast cancer by modulating key growth pathway(s). Methods By mining the Cancer Genome Atlas (TCGA)-Breast Cancer dataset and manipulating annexin A1 levels in breast cancer cell lines, we studied the role of annexin A1 in breast cancer and underlying signaling pathways. Results Our in-silico analysis of TCGA-breast cancer dataset demonstrated that annexin A1 mRNA expression is higher in basal subtype compared to luminal and HER2 subtypes. Within the basal subtype, patients show significantly poorer overall survival associated with higher expression of annexin A1. In both TCGA patient samples and cell lines, annexin A1 levels were significantly higher in basal-like breast cancer than luminal and Her2/neu-positive breast cancer. Stable annexin A1 knockdown in TNBC cell lines suppressed the mTOR-S6 pathway likely through activation of AMPK but had no impact on the MAPK, c-Met, and EGFR pathways. In a cell migration assay, annexin A1-depleted TNBC cells showed delayed migration as compared to wild-type cells, which could be responsible for poor patient prognosis in basal like breast cancers that are known to express higher annexin A1. Conclusions Our data suggest that annexin A1 is prognostic only in patients with basal like breast cancer. This appears to be in part due to the role of annexin A1 in activating mTOR-pS6 pathway.

Regulation of miRNA-29c and its downstream pathways in preneoplastic progression of triple-negative breast cancer

Little is understood about the early molecular drivers of triple-negative breast cancer (TNBC), making the identification of women at risk and development of targeted therapy for prevention significant challenges. By sequencing a TNBC cell line-based breast cancer progression model we have found that miRNA-29c is progressively lost during TNBC tumorigenesis. In support of the tumor suppressive role of miRNA 29c, we found that low levels predict poor overall patient survival and, conversely, that ectopic expression of miRNA-29c in preneoplastic cell models inhibits growth. miRNA-29c exerts its growth inhibitory effects through direct binding and regulation of TGFB-induced factor homeobox 2 (TGIF2), CAMP-responsive element binding protein 5 (CREB5), and V-Akt murine thymoma viral oncogene homolog 3 (AKT3). miRNA-29c regulation of these gene targets seems to be functionally relevant, as TGIF2, CREB5, and AKT3 were able to rescue the inhibition of cell proliferation and colony formation caused by ectopic expression of miRNA-29c in preneoplastic cells. AKT3 is an oncogene of known relevance in breast cancer, and as a proof of principle we show that inhibition of phosphoinositide 3-kinase (PI3K) activity, a protein upstream of AKT3, suppressed proliferation in TNBC preneoplastic cells. We explored additional opportunities for prevention of TNBC by studying the regulation of miRNA-29c and identified DNA methylation to have a role in the inhibition of miRNA-29c during TNBC tumorigenesis. Consistent with these observations, we found 5 aza-cytadine to relieve the suppression of miRNA-29c. Together, these results demonstrate that miRNA-29c loss plays a key role in the early development of TNBC.

Suppression of Akt-mTOR Pathway-A Novel Component of Oncogene Induced DNA Damage Response Barrier in Breast Tumorigenesis

DNA damage has been thought to be directly associated with the neoplastic progression by enabling mutations in tumor suppressor genes and activating/and amplifying oncogenes ultimately resulting in genomic instability. DNA damage causes activation of the DNA damage response (DDR) that is an important cellular mechanism for maintaining genomic integrity in the face of genotoxic stress. While the cellular response to genotoxic stress has been extensively studied in cancer models, less is known about the cellular response to oncogenic stress in the premalignant context. In the present study, by using breast tissues samples from women at different risk levels for invasive breast cancer (normal, proliferative breast disease and ductal carcinoma in situ) we found that DNA damage is inversely correlated with risk of invasive breast cancer. Similarly, in MCF10A based in vitro model system where we recapitulated high DNA damage conditions as seen in patient samples by stably cloning in cyclin E, we found that high levels of oncogene induced DNA damage, by triggering inhibition of a major proliferative pathway (AKT), inhibits cell growth and causes cells to die through autophagy. These data suggest that AKT-mTOR pathway is a novel component of oncogene induced DNA damage response in immortalized ‘normal-like’ breast cells and its suppression may contribute to growth arrest and arrest of the breast tumorigenesis.

Hormone-induced and DNA Demethylation-induced Relief of a Tissue-specific and Developmentally Regulated Block in Transcriptional Elongation

Background: The regulation of transcriptional elongation in vertebrates in vivo is poorly understood. Results: The tissue-specific and developmentally regulated expression pattern of the Rhox5 homeobox gene in vivo is dictated, at least in part, by transcriptional elongation. Conclusion: Transcriptional elongation control is conferred by hormone signaling and epigenetic regulation. Significance: The Rhox5 gene provides a model system to study tissue-specific and developmentally regulated gene expression at the level of transcriptional elongation. Genome-wide studies have revealed that genes commonly have a high density of RNA polymerase II just downstream of the transcription start site. This has raised the possibility that genes are commonly regulated by transcriptional elongation, but this remains largely untested in vivo, particularly in vertebrates. Here, we show that the proximal promoter from the Rhox5 homeobox gene recruits polymerase II and begins elongating in all tissues and cell lines that we tested, but it only completes elongation in a tissue-specific and developmentally regulated manner. Relief of the elongation block is associated with recruitment of the elongation factor P-TEFb, the co-activator GRIP1, the chromatin remodeling factor BRG1, and specific histone modifications. We provide evidence that two mechanisms relieve the elongation block at the proximal promoter: demethylation and recruitment of androgen receptor. Together, our findings support a model in which promoter proximal pausing helps confer tissue-specific and developmental gene expression through a mechanism regulated by DNA demethylation-dependent nuclear hormone receptor recruitment.

Abstract P4-15-03: Regulation of miRNA-29c and its gene targets in preneoplastic progression of triple negative breast cancer

Introduction: Little is understood about the early molecular drivers of the triple negative breast cancer making identification of women at risk and development of targeted therapy for prevention a significant challenge. Methods: Here, by deep sequencing of TNBC- cell line based breast cancer progression system we have identified miRNA-29c and its functional gene targets to be potentially involved in the normal to preneoplastic transition during TNBC progression. We have used cell line based functional assays that are relevant in early tumorigenesis such cell proliferation (ki67), and colony formation assay to study the growth inhibitory potential of these miRNA and their gene targets. To identify direct gene targets of miRNA-29c, we cloned the 39untranslated region containing miRNA-29c binding sites from predicted gene targets in a luciferase reporter vector, pmiRGLO and studied the potential of miRNA-29c overexpression on the repression of luciferase reporter activity indicating their direct gene regulation. Results: Our deep sequencing results and their further validation by QPCR revealed miRNA-29c to be lost during the TNBC progression, and its forced expression to inhibit cell proliferation and colony formation of preneoplastic (MCF10AT1) and ductal carcinoma in situ (MCF10DCIS) cells. We found miRNA-29c to directly bind in 39UTR of TGIF2, CREB5, AKT3 and CDK6 and regulate their expression as shown by our luciferase assays. We also found miRNA-29c binding to 39UTR of these gene targets to be functionally relevant as TGIF2, CREB5 and AKT3 were able to rescue the inhibition in cell proliferation and colony formation assay caused by loss of miRNA-29c in preneoplastic cells. Further confirming the relevance of these miRNA-29c gene targets and pathways in TNBC tumorigenesis, inhibition of PI3K, which is upstream of AKT3, inhibits cell proliferation in MCF10AT1 and DCIS cells. We also examined the regulation of tumor suppressor miRNA-29c to study the mechanisms responsible for its loss during breast cancer development. We found c-myc and EZH2 driven epigenetic mechanism as well as DNA methylation in part to cause the loss of miRNA-29c during TNBC progression. Consistently, we found a pan HDAC inhibitor and a DNA methylation inhibitor to relieve the suppression of miRNA-29c. Conclusions: Together, these results indicate that loss of miRNA-29c plays a central role in preneoplastic development of breast cancer and efforts directed at inhibition of its target pathways or rescue of miRNA-29c itself may provide novel opportunities for prevention of TNBC.Introduction: Little is understood about the early molecular drivers of the triple negative breast cancer making identification of women at risk and development of targeted therapy for prevention a significant challenge. Methods: Here, by deep sequencing of TNBC- cell line based breast cancer progression system we have identified miRNA-29c and its functional gene targets to be potentially involved in the normal to preneoplastic transition during TNBC progression. We have used cell line based functional assays that are relevant in early tumorigenesis such cell proliferation (ki67), and colony formation assay to study the growth inhibitory potential of these miRNA and their gene targets. To identify direct gene targets of miRNA-29c, we cloned the 39untranslated region containing miRNA-29c binding sites from predicted gene targets in a luciferase reporter vector, pmiRGLO and studied the potential of miRNA-29c overexpression on the repression of luciferase reporter activity indicating their direct gene regulation. Results: Our deep sequencing results and their further validation by QPCR revealed miRNA-29c to be lost during the TNBC progression, and its forced expression to inhibit cell proliferation and colony formation of preneoplastic (MCF10AT1) and ductal carcinoma in situ (MCF10DCIS) cells. We found miRNA-29c to directly bind in 39UTR of TGIF2, CREB5, AKT3 and CDK6 and regulate their expression as shown by our luciferase assays. We also found miRNA-29c binding to 39UTR of these gene targets to be functionally relevant as TGIF2, CREB5 and AKT3 were able to rescue the inhibition in cell proliferation and colony formation assay caused by loss of miRNA-29c in preneoplastic cells. Further confirming the relevance of these miRNA-29c gene targets and pathways in TNBC tumorigenesis, inhibition of PI3K, which is upstream of AKT3, inhibits cell proliferation in MCF10AT1 and DCIS cells. We also examined the regulation of tumor suppressor miRNA-29c to study the mechanisms responsible for its loss during breast cancer development. We found c-myc and EZH2 driven epigenetic mechanism as well as DNA methylation in part to cause the loss of miRNA-29c during TNBC progression. Consistently, we found a pan HDAC inhibitor and a DNA methylation inhibitor to relieve the suppression of miRNA-29c. Conclusions: Together, these results indicate that loss of miRNA-29c plays a central role in preneoplastic development of breast cancer and efforts directed at inhibition of its target pathways or rescue of miRNA-29c itself may provide novel opportunities for prevention of TNBC. Citation Format: Bhardwaj A, Tachibana K, Ganesan N, Rajapakshe K, Singh H, Gunaratne P, Coarfa C, Bedrosian I. Regulation of miRNA-29c and its gene targets in preneoplastic progression of triple negative breast cancer [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P4-15-03.