MicroRNA and transcription factor co-regulatory networks and subtype classification of seminoma and non-seminoma in testicular germ cell tumors

Recent studies have revealed that feed-forward loops (FFLs) as regulatory motifs have synergistic roles in cellular systems and their disruption may cause diseases including cancer. FFLs may include two regulators such as transcription factors (TFs) and microRNAs (miRNAs). In this study, we extensively investigated TF and miRNA regulation pairs, their FFLs, and TF-miRNA mediated regulatory networks in two major types of testicular germ cell tumors (TGCT): seminoma (SE) and non-seminoma (NSE). Specifically, we identified differentially expressed mRNA genes and miRNAs in 103 tumors using the transcriptomic data from The Cancer Genome Atlas. Next, we determined significantly correlated TF-gene/miRNA and miRNA-gene/TF pairs with regulation direction. Subsequently, we determined 288 and 664 dysregulated TF-miRNA-gene FFLs in SE and NSE, respectively. By constructing dysregulated FFL networks, we found that many hub nodes (12 out of 30 for SE and 8 out of 32 for NSE) in the top ranked FFLs could predict subtype-classification (Random Forest classifier, average accuracy ≥90%). These hub molecules were validated by an independent dataset. Our network analysis pinpointed several SE-specific dysregulated miRNAs (miR-200c-3p, miR-25-3p, and miR-302a-3p) and genes (EPHA2, JUN, KLF4, PLXDC2, RND3, SPI1, and TIMP3) and NSE-specific dysregulated miRNAs (miR-367-3p, miR-519d-3p, and miR-96-5p) and genes (NR2F1 and NR2F2). This study is the first systematic investigation of TF and miRNA regulation and their co-regulation in two major TGCT subtypes

Integrative analysis of miRNA and gene expression reveals regulatory networks in tamoxifen-resistant breast cancer

Tamoxifen is an effective anti-estrogen treatment for patients with estrogen receptor-positive (ER+) breast cancer, however, tamoxifen resistance is frequently observed. To elucidate the underlying molecular mechanisms of tamoxifen resistance, we performed a systematic analysis of miRNA-mediated gene regulation in three clinically-relevant tamoxifen-resistant breast cancer cell lines (TamRs) compared to their parental tamoxifen-sensitive cell line. Alterations in the expression of 131 miRNAs in tamoxifen-resistant vs. parental cell lines were identified, 22 of which were common to all TamRs using both sequencing and LNA-based quantitative PCR technologies. Although the target genes affected by the altered miRNA in the three TamRs differed, good agreement in terms of affected molecular pathways was observed. Moreover, we found evidence of miRNA-mediated regulation of ESR1, PGR1, FOXM1 and 14-3-3 family genes. Integrating the inferred miRNA-target relationships, we investigated the functional importance of 2 central genes, SNAI2 and FYN, which showed increased expression in TamR cells, while their corresponding regulatory miRNA were downregulated. Using specific chemical inhibitors and siRNA-mediated gene knockdown, we showed that both SNAI2 and FYN significantly affect the growth of TamR cell lines. Finally, we show that a combination of 2 miRNAs (miR-190b and miR-516a-5p) exhibiting altered expression in TamR cell lines were predictive of treatment outcome in a cohort of ER+ breast cancer patients receiving adjuvant tamoxifen mono-therapy. Our results provide new insight into the molecular mechanisms of tamoxifen resistance and may form the basis for future medical intervention for the large number of women with tamoxifen-resistant ER+ breast cancer

Cancer cells exploit an orphan RNA to drive metastatic progression.

Here we performed a systematic search to identify breast-cancer-specific small noncoding RNAs, which we have collectively termed orphan noncoding RNAs (oncRNAs). We subsequently discovered that one of these oncRNAs, which originates from the 3' end of TERC, acts as a regulator of gene expression and is a robust promoter of breast cancer metastasis. This oncRNA, which we have named T3p, exerts its prometastatic effects by acting as an inhibitor of RISC complex activity and increasing the expression of the prometastatic genes NUPR1 and PANX2. Furthermore, we have shown that oncRNAs are present in cancer-cell-derived extracellular vesicles, raising the possibility that these circulating oncRNAs may also have a role in non-cell autonomous disease pathogenesis. Additionally, these circulating oncRNAs present a novel avenue for cancer fingerprinting using liquid biopsies

Post-transcriptional regulation of Estrogen Receptor-α by miR-17-92 interaction and LMTK3 phosphorylation in Breast Cancer

Estrogen receptor-α (ERα) is expressed in two-thirds of BCs and is a well-known prognostic and predictive marker. For this reason it is one of the most studied proteins in BC. To understand how ERα positive BC develops, it is crucial to investigate both how this protein is regulated and which genes are modulated by it. MicroRNAs (miRNAs) control gene expression post-transcriptionally by interacting through sequence complementarity to their target transcripts. Through a microarray approach, we identified the subset of miRNAs modulated by ERα, that include up-regulation of miRNAs derived from the processing of two paralogous primary (pri-) transcripts, pri-miR-17-92 and pri-miR-106a-363. Characterisation of the miR-17-92 locus confirmed that the ERα target protein c-MYC binds its promoter in an estrogen-dependent manner. These findings indicated that miRNAs derived from these pri-miRNAs (miR-18a, miR-19b and miR-20b) target and down-regulate ERα, whilst a subset of pri-miRNA-derived mature miRNAs inhibit protein translation of the ERα transcriptional p160 co-activator, AIB1. Therefore, different subsets of the miRNAs identified act as part of a negative autoregulatory feedback loop. We observed that levels of pri-miR-17-92 increase earlier than the mature miRNAs derived from it, implicating precursor cleavage modulation after transcription. Pri-mir-17-92 is immediately cleaved by Drosha to pre-miR-18a, indicating that its regulation occurs during the formation of the mature molecule from the precursors. Furthermore, we wanted to explore the new kinases that regulate the ERα activity. Thereby, we performed kinome screening (by RNAi technologies) to determine kinases that regulate ERα in MCF-7 BC cells and identified a novel kinase, LMTK3, which acts as positive regulator of ERα's transcriptional activity. This could be a new therapeutic target and/or a novel biomarker for BC, although further studies are required to validate this. Together, these studies identify new transcriptional and translational factors that regulate ERα expression in BC.Open Acces

A Densely Interconnected Genome-Wide Network of MicroRNAs and Oncogenic Pathways Revealed Using Gene Expression Signatures

MicroRNAs (miRNAs) are important components of cellular signaling pathways, acting either as pathway regulators or pathway targets. Currently, only a limited number of miRNAs have been functionally linked to specific signaling pathways. Here, we explored if gene expression signatures could be used to represent miRNA activities and integrated with genomic signatures of oncogenic pathway activity to identify connections between miRNAs and oncogenic pathways on a high-throughput, genome-wide scale. Mapping >300 gene expression signatures to >700 primary tumor profiles, we constructed a genome-wide miRNA–pathway network predicting the associations of 276 human miRNAs to 26 oncogenic pathways. The miRNA–pathway network confirmed a host of previously reported miRNA/pathway associations and uncovered several novel associations that were subsequently experimentally validated. Globally, the miRNA–pathway network demonstrates a small-world, but not scale-free, organization characterized by multiple distinct, tightly knit modules each exhibiting a high density of connections. However, unlike genetic or metabolic networks typified by only a few highly connected nodes (“hubs”), most nodes in the miRNA–pathway network are highly connected. Sequence-based computational analysis confirmed that highly-interconnected miRNAs are likely to be regulated by common pathways to target similar sets of downstream genes, suggesting a pervasive and high level of functional redundancy among coexpressed miRNAs. We conclude that gene expression signatures can be used as surrogates of miRNA activity. Our strategy facilitates the task of discovering novel miRNA–pathway connections, since gene expression data for multiple normal and disease conditions are abundantly available

Global MicroRNA Expression Profiling Identifies MiR-210 Associated with Tumor Proliferation, Invasion and Poor Clinical Outcome in Breast Cancer

Aberrant microRNA (miRNA) expression is associated with cancer and has potential diagnostic and prognostic value in various malignancies. In this study, we investigated miRNA profiling as a complementary tool to improve our understanding of breast cancer (BC) biology and to assess whether miRNA expression could predict clinical outcome of BC patients.In VitroJournal ArticleResearch Support, Non-U.S. Gov'tSCOPUS: ar.jinfo:eu-repo/semantics/publishe

Insights into KRAS Biology through its Novel Interactions.

A third of all human cancers harbor mutations in the RAS family of genes, which encode members of small GTPases. RAS proteins transduce extracellular growth signals by cycling between a GTP-bound activated state and a GDP-bound basal state, regulating effector pathways through RAF/PI3K binding. Mutations lock RAS in the activated GTP bound state, leading to constitutive signaling through various pathways, leading to neoplastic transformation. In this thesis, KRAS biology was interrogated at three levels. At the chromosomal level, using an integrative genomics approach we identified UBE2L3-KRAS gene fusion in prostatic DU145 cells and functional studies of the fusion suggests proximity of KRAS to the ubiquitin machinery, a recurrent theme in various synthetic lethal screens of KRAS. At the level of the transcript, we identified frequent ‘outlier kinases’ like polo like kinase in KRAS-dependent pancreatic cancer cell lines which show increased sensitivity to PLK inhibition in combination with KRAS knockdown. PLK is part of the Anaphase promoting complex, and is known to associate with the protein degradation machinery. At the level of the protein, an unbiased mass spectrometric analysis identified Argonaute 2 (AGO2) as an unexpected RAS interacting protein. RAS co-sediments with AGO2 in membrane fractions, and co-localizes in intracellular membrane organelles. The N-terminal domain of AGO2 directly binds the Switch II domain of RAS, in a GDP/GTP independent manner. Functionally, knock-down of AGO2 attenuates KRAS-mediated cell proliferation in mutant KRAS-dependent cancer cell lines and KRAS-mediated transformation of normal cells is enhanced upon AGO2 overexpression. The intracellular KRAS-AGO2 interaction increases mutant KRAS levels and PI3K signaling. Additionally, expression of mutant KRAS attenuates the assembly of regulatory messenger ribonucleoprotein particles (mRNPs) in NIH3T3 cells. Employing NIH3T3 AGO2-/- cells, we observed that interaction with AGO2 is required for maximal KRAS-mediated transformation. This most surprising intersection of the signaling networks of KRAS with the RNA silencing machinery through its interaction with its core component protein, AGO2, expands its range of oncogenic activities. Together these studies describe the intersection of KRAS with both the ubiquitin/proteasomal degradation and RNA silencing pathways beyond its well characterized role in signal transduction thus providing new insights into RAS function.PhDMolecular and Cellular PathologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/113415/1/sunitas_1.pd

Exploring the genome-wide impact of estrogen receptor alpha and estrogen receptor beta in breast and colon cancer cells

Estrogen signaling is involved in the development and progression of breast cancer and is implicated to be protective in colon cancer. Estrogenic actions are conveyed through transcriptional regulation by ligand stimulated estrogen receptors (ERα and ERβ). ERα is upregulated in most breast cancers and is responsible for the proliferative effect of estrogen. ERβ on the other hand is usually downregulated, and studies indicate an antiproliferative function. Therapies targeting ERα are available and commonly used in the treatment of breast cancer. In the normal colonic epithelia, however, ERβ is the most abundant estrogen receptor and the suggested mediator of the protective effects of estrogen in colon cancer. The role of ERβ in breast cancer and colon cancer is not well understood. Thus, exploring the genome-wide impact and contribution of both receptors in estrogen responsive cancers would substantially help to identify novel therapeutic and preventive strategies for these cancers. In Paper 1, we examined differences in transcriptional regulation between ERα and ERβ in the breast cancer cell line T47D. We could show that ERβ often exhibited an opposing effect on ERα-regulated genes within proliferation and regulation of cell cycle. We also demonstrated a set of genes only regulated by ERβ, indicating that, despite the high homology between the two receptors, there are differences in their transcriptional targets. The fact that ERβ opposed ERα indicates that ERβ activation may be of value in the treatment of breast cancer. To further explore the transcriptional role of ERα in breast cancer, we performed large-scale analyses of microRNA in 24 hours estrogen treated ERα-expressing T47D cells, Paper II. However, we found no evidence of direct and rapid regulation of mature miRNAs by ERα. In Paper III, we studied ERβ gene regulation in colon cancer cells. We could show that ERβ-expressing xenografts grew significantly slower than those lacking ERβ. Further we demonstrated that ERβ induced a transcriptional response independently of ERα and induced inhibition of the proto-oncogene MYC and other G1-phase cell cycle genes. In Paper IV, we dissected the regulatory networks of ERβ-induced transcriptional changes in human colon cancer cells. The set of genes changed by ERβ varied in different colon cancer cell lines, however, corresponded to the same biological processes such as cell cycle regulation and kinase activity. In addition, we identified the ERβ-driven downregulation of the transcription factor PROX1 as a key mechanism behind a large proportion of the transcriptional changes. In Paper V, we studied the effect of long term expression of ERβ on the miRNA pool in SW480 colon cancer cells. While we could not show a direct and rapid effect of ERα on the miRNome, we showed that long term expression of ERβ did induce large changes in the miRNA pool in colon cancer cells. In particular, we found the oncogenic miR-17-92 cluster to be downregulated and proposed this to be a consequence of the ERβ-induced downregulation of MYC. In conclusion, we have shown that ERβ is antiproliferative in breast and colon cancer cells, both when co-expressed with ERα and alone, as well as identified key signaling pathways. We suggest that activation of ERβ will have a beneficial effect for treatment or prevention of estrogen dependent cancers

Unraveling the MYCN-driven transcriptional landscape of neuroblastoma : a cross-species integrated transcriptome approach

ince its identification in neuroblastoma, increasing evidence points towards a central role for MYCN in the biology of neuroblastoma tumors. As a consequence, targeting MYCN and its downstream functions would be a promising strategy to treat neuroblastoma patients. However, the development of inhibitors targeting MYC proteins has been challenging, as MYC/MYCN proteins are composed of two extended alpha-helices with no obvious surfaces for small molecule binding. Therefore, a thorough understanding of MYCN's functions and the upstream mechanisms regulating its expression would contribute to the development of compounds specifically targeting the impact of MYCN in neuroblastoma cells. To address this issue, I initiated this thesis with the construction of an in vivo time-resolved miRNA and gene expression dataset of MYCN-driven neuroblastoma development, using the well-established TH-MYCN mouse model. After extensive validation of the dataset, I applied an exiting transcriptome-wide cross-species approach to search for transcriptional regulators that can be regarded as master regulators of neuroblastoma development. The corresponding miRNA expression dataset was exploited during the characterization of the complete MYCN- miRNA interactome in neuroblastoma using the powerful combination of this dataset and an unbiased 3'UTR reporter screen. I next characterized how MYCN affects the expression of LIN28B, a highly conserved RNA-binding protein, which is shown to induce MYCN expression in neuroblastoma via the suppression of the MYCN-targeting let-7 family of miRNAs. Throughout this project, studying the genetic landscape of the well-established TH-MYCN mouse model has provided great insights in the perturbed miRNA-mRNA networks in neuroblastoma. Although representing an excellent and broadly used tool, this mouse model holds some limitations related to the transgene integration site, the anatomical location of the observed tumors, implementation of in vivo imaging and tumor incidence in different genetic backgrounds. I therefore present a novel mouse model with targeted Cre-conditional MYCN expression in the neural crest. Molecular characterization showed that the tumors arising in this model, strongly resemble the existing TH-MYCN mouse model and human neuroblastoma tumors. We expect that this new model, which addresses some of the major limitations of the TH-MYCN model, will play a major role in the investigation of neuroblastoma