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

mTOR: A Mechanistic Target of Muscle and Cancer Crosstalk

In the United States, the number of colon and breast cancer cases that are attributed to physical inactivity lead the exercise-associated cases of heart disease and type II diabetes. For the millions of people in the United States burdened with breast cancer, there is a noted substantial risk reduction with increased physical activity. Previous research has investigated skeletal muscle’s endocrine-like potential on inflammation and cancer metabolism; however there is limited investigation into exercise-facilitated suppression of cancer’s major anabolic pathway, the mechanistic target of rapamycin (mTOR) pathway. While previous cancer research has established that mTOR’s activity is dysregulated in cancer, little is known about the impact of exercise on the regulation of anabolic/proliferative features of breast cancer cells, nor has exercise been evaluated as a mediator of muscle and cancer crosstalk. This project’s objective is to determine how exercise is affecting the biological regulation of tumorgenesis (a critical component of treatment innovation) via the mTOR pathway, and how that regulation is mediated by skeletal muscle contraction. While research efforts and analyses about the mTOR pathway have led to key insights into its regulation of apoptotic and autophagic signaling in cancer, these efforts do not capture the complete profile of mTOR control on cell growth and survival, nor address preliminary data indicating that cellular proliferation rates are significantly reduced in breast cancer cells treated with excretion factors arising from contracting skeletal muscle (“exercise”). The work presented here-in utilizes a research approach consisting of cell culture and animal models to investigate key mechanistic foundations that underlie the biological regulation of breast cancer in individuals who partake in exercise. Specifically, the MCF7 epithelial breast cancer cell line, a hemicorpus hind limb perfusion (HHLP) surgery, and pharmacological interventions allow for evaluation of skeletal muscle’s endocrine ability, global protein synthesis, signal transduction and gene expression. The investigators’ consideration of musclecancer crosstalk via exercise lays the foundation for future evaluation of muscle-derived biomolecules (ie. microRNA) as a potential crosstalk mediators. This project’s successful completion proposes key mechanistic foundations that underlie the biological regulation of breast cancer, contributing greatly to science’s efforts towards novel translational investigation of the beneficial relationship between muscle and cancer crosstalk. Establishing a casual role for exercise as primary cancer prevention would have major translational impact in cancer prevention and patient survivorship, with even a small reduction in incidence of cancer resulting in multi- billion dollar health care savings

Systemic Modeling of Biomolecular Interaction Networks

For more than the entire past century, classical experimental methodologies have dominated biological research, providing a wealth of information about individual molecular species in cells and their functions. However, there is an increasing and strong level of evidence suggesting that an isolated biological function can only rarely be attributed to an individual biological molecule. Instead, more recently, it is argued that most biological characteristics are due to complex interactions between the cell’s numerous constituents, such as proteins, DNA and RNA. Therefore, a major challenge for the biological sciences in this century is to unravel the structure and the dynamics of these complex intracellular interactions at a systems level.   Many types of statistical and computational models have been built and applied to study cellular behavior and in this research work, we focus on two distinct instances, one from each of the two broad types of models used in computational systems biology: i) statistical inference models applied to gene regulatory interaction networks and ii) biochemical reaction models applied to protein-protein interaction networks.   For our first research problem, we focused on microRNA-mediated gene regulatory networks. MicroRNAs (miRNAs) are small non-coding ribonucleic acids (RNAs) that extensively regulate gene expression in metazoan animals, plants and protozoa. With the goal to gain a systemic understanding of miRNA-mediated interaction networks, we developed IntegraMiR, a novel integrative analysis method that can be used to infer certain types of regulatory loops of dysregulated miRNA/Transcription Factor (TF) interactions which appear at the transcriptional, post-transcriptional and signaling levels in a statistically over-represented manner. We demonstrate instances of the results in a number of distinct biological settings, which are known to play crucial roles in the contexts of prostate cancer and autism spectrum disorders.   To study the dynamics of biomolecular interaction networks, we focused on a protein-protein interaction network in living cells. Our collaborators at the School of Medicine planned to synthetically develop and characterize a biomaterial, which was produced by this protein-protein interaction network, and which would act as a molecular sieve to control the passage of biomolecules in living cells. And we wanted to computationally model the dynamic formation of this biomolecular sieve, termed a hydrogel, and characterize its properties that were relevant to the experimental work. The resulting model presented us and our experimental collaborators with a systemic and deeper understanding of the problem of gel synthesis, which guided the experimental design and provided further validation of the subsequent experimental findings and conclusions

Integrative approaches for systematic reconstruction of regulatory circuits in mammals

Thesis (Ph. D.)--Massachusetts Institute of Technology, Computational and Systems Biology Program, 2012.Cataloged from PDF version of thesis.Includes bibliographical references (p. 141-149).The reconstruction of regulatory networks is one of the most challenging tasks in systems biology. Although some models for inferring regulatory networks can make useful predictions about the wiring and mechanisms of molecular interactions, these approaches are still limited and there is a strong need to develop increasingly universal and accurate approaches for network reconstruction. This problem is particularly challenging in mammals, due to the higher complexity of mammalian regulatory networks and limitations in experimental manipulation. In this thesis, I present three systematic approachs to reconstruct, analyse and refine models of gene regulation. In Chapter 1, I devise a method for deriving an observational model from temporal genomic profiles. I use it to choose targets for perturbation experiments in order to determine a network controlling the responses of mouse primary dendritic cells to stimulation with pathogen components. In Chapter 2, I introduce the algorithm Exigo, for identifying essential interactions in regulatory networks reconstructed from experimental data where regulators have been silenced, using a network reduction strategy. Exigo outperforms previous approaches on simulated data, uncovers the core network structure when applied to real networks derived from perturbation studies in mammals, and improves the performance of network inference methods. Lastly, I introduce in Chapter 3 an approach to learn a module network from multiple highthroughput assays. Analysis of a diffuse large B-cell lymphoma dataset identifies candidate regulator genes, microRNAs and copy number aberrations with biological, and possibly therapeutic, importance.by Ana Paula Santos Botelho Oliveira Leite.Ph.D

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

Characterization of the cargo of circulating extracellular vesicles in patients affected with idiopathic inflammatory myopathies and evaluation of clinical correlates in a cross-sectional comparative analysis from a monocentric cohort.

openBackground Idiopathic inflammatory myopathies (IIM) are heterogeneous autoimmune disorders that comprise different clinical entities, characterized by different features. Although several myositis-specific and myositis-associated antibodies have been characterized, the molecular mechanisms underlying these conditions require further exploration. The research field on extracellular vescicles (EVs) is rapidly evolving, highlighting their role in intercellular communication. EVs convoy a cargo of proteins and nucleic acids, such as microRNA (miRNAs), that mediate immune-response regulation in autoimmune diseases. miRNAs regulate gene-expression post-transcriptionally and are involved in multiple molecular pathways of human disease. Evidence of EVs and miRNAs in IIM is still limited and undefined. Aim of the study This study aims to quantify the circulating EVs and characterize their cargo, with a specific focus on miRNAs content to propose novel biomarkers of IIM. Materials and methods A monocentric study was conducted including adult IIM patients (≥18 years old) followed at the Rheumatology Unit of Padua University Hospital, and age- and sex- matched healthy controls (HD). EVs were isolated from platelet-free plasma through size exclusion chromatography followed by ultrafiltration. EVs were quantified by nanoparticle tracking analysis (NTA) and. EV-miRNA cargo was investigated through Next-Generation Sequencing (NGS). Statistical analysis was performed with parametric Student-T test and one-way Anova (Bonferroni correction). Results Sixty-four consecutive IIM patients and sixty-five HDs were included in the study. NTA measurements of EVs concentration showed a significantly higher mean concentration of circulating EVs in IIM patients than in HD (p=0.0073). Across IIM subsets, patients affected with cancer associated myositis (CAM) displayed the highest levels of circulating EVs compared to no CAM patients (p=0.0060) and to HD (p=0.0004). Patients with circulating myositis-associated autoantibodies displayed significantly higher EV levels than HD (p=0.0363). Patients in clinical remission displayed higher levels of circulating EVs compared to those with active disease (p=0.0087). EVs levels were significantly reduced in IIM patients treated with rituximab (RTX) than in patients receiving other treatments (p<0.0001). NGS analysis detected EV-miRNAs with different expression profiles between IIM (n=47) and HDs (n=49): miR-223-3p (p=0.019), miR-15a-5p (p=0.0189), miR-451a (p=0.0074), miR-486-5p (p=0.0052), miR-32-5p (p=0.0146), and miR-222-3p (p=0.0282) were up-regulated in IIM, while miR-141-3p (p=0.0313), miR-142-3p (p=0.0244), and let-7a-5p (p=0.0003) were down-regulated in IIM patients vs. HDs. Other EV-miRNAs expression varied across IIM subsets: CAM patients displayed up-regulated expression of miR-143-3p compared to non-CAM patients (p=0.0085), while miR-148a-3p (p=0.0171) and miR-335-5p (p=0.0171) were up-regulated in dermatomyositis vs. polymyositis/ inclusion body myositis/anti-synthetase syndrome patients. Patients characterized by active disease displayed an up-regulated expression of miR-222-3p (p=0.002) and miR-151-3p (p=0.0233) and down-regulated expression of miR-363-3p (p=0.0001), miR-374a-5p (p=0.0258), miR-144-3p (p=0.0170), miR-181a-5p (p=0.0037) compared to those in clinical remission. Moreover, IIM patients receiving only glucocorticoids (GC) reported up-regulated expression of miR-4433b-5p (p=0.0439), miR-92a-3p (p=0.0111), let-7f-5p (p=0.0304), and down-regulated expression of miR-27a-3p (p=0.0486) compared to patients receiving GC in combination with immunosuppressants (IS). Conclusions Our results showed significantly increased concentration of circulating EVs in IIM patients. That is confirmed within specific disease phenotypes and pharmacological treatments. EV miRNAs exhibited a differential expression profile between IIM and HD, and significant differences were outlined among IIM subsets.Background Idiopathic inflammatory myopathies (IIM) are heterogeneous autoimmune disorders that comprise different clinical entities, characterized by different features. Although several myositis-specific and myositis-associated antibodies have been characterized, the molecular mechanisms underlying these conditions require further exploration. The research field on extracellular vescicles (EVs) is rapidly evolving, highlighting their role in intercellular communication. EVs convoy a cargo of proteins and nucleic acids, such as microRNA (miRNAs), that mediate immune-response regulation in autoimmune diseases. miRNAs regulate gene-expression post-transcriptionally and are involved in multiple molecular pathways of human disease. Evidence of EVs and miRNAs in IIM is still limited and undefined. Aim of the study This study aims to quantify the circulating EVs and characterize their cargo, with a specific focus on miRNAs content to propose novel biomarkers of IIM. Materials and methods A monocentric study was conducted including adult IIM patients (≥18 years old) followed at the Rheumatology Unit of Padua University Hospital, and age- and sex- matched healthy controls (HD). EVs were isolated from platelet-free plasma through size exclusion chromatography followed by ultrafiltration. EVs were quantified by nanoparticle tracking analysis (NTA) and. EV-miRNA cargo was investigated through Next-Generation Sequencing (NGS). Statistical analysis was performed with parametric Student-T test and one-way Anova (Bonferroni correction). Results Sixty-four consecutive IIM patients and sixty-five HDs were included in the study. NTA measurements of EVs concentration showed a significantly higher mean concentration of circulating EVs in IIM patients than in HD (p=0.0073). Across IIM subsets, patients affected with cancer associated myositis (CAM) displayed the highest levels of circulating EVs compared to no CAM patients (p=0.0060) and to HD (p=0.0004). Patients with circulating myositis-associated autoantibodies displayed significantly higher EV levels than HD (p=0.0363). Patients in clinical remission displayed higher levels of circulating EVs compared to those with active disease (p=0.0087). EVs levels were significantly reduced in IIM patients treated with rituximab (RTX) than in patients receiving other treatments (p<0.0001). NGS analysis detected EV-miRNAs with different expression profiles between IIM (n=47) and HDs (n=49): miR-223-3p (p=0.019), miR-15a-5p (p=0.0189), miR-451a (p=0.0074), miR-486-5p (p=0.0052), miR-32-5p (p=0.0146), and miR-222-3p (p=0.0282) were up-regulated in IIM, while miR-141-3p (p=0.0313), miR-142-3p (p=0.0244), and let-7a-5p (p=0.0003) were down-regulated in IIM patients vs. HDs. Other EV-miRNAs expression varied across IIM subsets: CAM patients displayed up-regulated expression of miR-143-3p compared to non-CAM patients (p=0.0085), while miR-148a-3p (p=0.0171) and miR-335-5p (p=0.0171) were up-regulated in dermatomyositis vs. polymyositis/ inclusion body myositis/anti-synthetase syndrome patients. Patients characterized by active disease displayed an up-regulated expression of miR-222-3p (p=0.002) and miR-151-3p (p=0.0233) and down-regulated expression of miR-363-3p (p=0.0001), miR-374a-5p (p=0.0258), miR-144-3p (p=0.0170), miR-181a-5p (p=0.0037) compared to those in clinical remission. Moreover, IIM patients receiving only glucocorticoids (GC) reported up-regulated expression of miR-4433b-5p (p=0.0439), miR-92a-3p (p=0.0111), let-7f-5p (p=0.0304), and down-regulated expression of miR-27a-3p (p=0.0486) compared to patients receiving GC in combination with immunosuppressants (IS). Conclusions Our results showed significantly increased concentration of circulating EVs in IIM patients. That is confirmed within specific disease phenotypes and pharmacological treatments. EV miRNAs exhibited a differential expression profile between IIM and HD, and significant differences were outlined among IIM subsets

Melanocytes, Melanocytic Nevi, and Progression to Melanoma

Benign melanocytic nevi form when melanocytes that acquire a BRAFV600E mutation undergo a period of rapid proliferation followed by subsequent growth-arrest. Constitutive activation of MAPK signaling downstream of BRAF drives the initial proliferative phenotype. However, the factors that establish and maintain growth-arrest in nevi remain elusive. Here we investigate the contributions of several potential mediators of BRAFV600E-induced growth arrest in nevus melanocytes. These mediators include the gene products of the CDKN2A-CDKN2B genetic locus, nevus-enriched microRNAs MIR211-5p and MIR328-3p, as well as transcriptional networks associated with primary cilia expression.p16INK4A, the cyclin dependent kinase inhibitor encoded by CDKN2A, has historically been associated with nevus formation. CDKN2B’s gene product, p15INK4B, remained under-studied in the context of nevus formation. Using primary melanocytes isolated directly from human nevi and naturally expressing the BRAFV600E-acitvating mutation, nevi progressing to melanoma, and normal melanocytes engineered to inducibly express BRAFV600E, we showed that p15INK4B expression is required for growth arrest in nevi and that loss promotes progression to melanoma. Investigating the role of the p16INK4A tumor suppressor in nevus formation and progression to melanoma, we found that loss of p16INK4A contributes to melanocyte hypermotility in vitro and increases invasive and metastatic behavior of melanoma cell lines in vivo. Using CRISPR-Cas9 to engineer a cellular model of melanoma initiation from primary human melanocytes, we determined that E2F1 upregulation as a consequence of CDKN2A loss drives these new phenotypes.Additional acquired genetic alterations do not distinguish proliferating BRAFV600E melanocytes from their growth-arrested nevus counterparts, suggesting a role for regulatory elements. We investigated the role of microRNAs in the initiation and maintenance of nevus growth arrest. Using primary human melanocytes, melanocytic nevi, and adjacent melanoma, we show that MIR211-5p and MIR328-3p are enriched in nevi compared to normal melanocytes, then subsequently downregulated in adjacent melanoma. Both MIR211-5p and MIR328-3p proved necessary effectors of BRAFV600E-induced growth arrest in human melanocytes. We identified microRNA target networks which, when suppressed, phenocopy BRAFV600E-induced growth arrest and converge on inhibition of AURKB to block cell cycle progression in primary human melanocytes.Similarly, expression of the primary cilium organelle has been shown to differentiate nevi from malignant melanoma. Investigations of primary cilia-associated gene expression in matched nevus and melanoma cases demonstrates that transcriptional changes in core primary cilia genes and related signaling molecules accompanies loss of the organelle during progression to melanoma.Taken together, these data suggest that the BRAFV600E mutation leads to a cascade of intracellular changes that establish and maintain of growth arrest in human melanocytic nevi

Post-transcriptional Regulation through Long Noncoding RNAs (lncRNAs)

This book is a collection of eight articles, of which seven are reviews and one is a research paper, that together form a Special Issue that describes the roles that long noncoding RNAs (lncRNA) play in gene regulation at a post-transcriptional level

Effects of Oncostatin-M Receptor overexpression on cervical squamous cell carcinoma cells and their extracellular vesicles

Cervical carcinoma remains the fourth most common cause of cancer in women worldwide. Cytological screening and introduction of prophylactic HPV vaccines have drastically decreased incidence rates in high income countries. However, it is still one of the leading causes of cancer morbidity in middle and low income countries and the prognosis for advanced or recurrent cervical carcinoma remains poor. Further research to enhance the current understanding of molecular aberrations driving cervical carcinoma is, therefore, vital in order to develop effective novel therapies. The oncostatin-M receptor (OSMR) frequently undergoes copy-number gain and overexpression in squamous cell carcinomas (SCC) at multiple sites, consistently associated with an adverse overall survival independent of tumour stage. OSM-OSMR signalling activates STAT3 and MAP-kinase pathways and induces a pro-malignant phenotype including increased cell migration, invasion and angiogenesis. Bidirectional communication between cancer cells and cells of the tumour microenvironment (TME) is essential for tumour progression. There is mounting evidence to suggest that extracellular vesicles (EVs) are key mediators of intercellular communication and can promote tumour progression through various mechanisms. The work presented in this PhD thesis aims to determine the effect of OSM-OSMR signalling on mRNA and miRNA expression in cervical SCC cells and their extracellular vesicles. Next generation sequencing (NGS) was used to investigate global changes in mRNA and miRNA expression. These experiments were performed using SW756 cells, a cervical SCC cell line with OSMR copy number gain and overexpression, and an OSMR knock down (KD) SW756 cell line generated by CRISPR-Cas9. OSM-OSMR signalling was found to be capable of modulating both cellular and EV mRNA expression. Treatment of SW756 cells with OSM resulted in significant (p≤0.01) upregulation of 225 (cell) and 88 (EV) mRNAs and downregulation of 98 (cell) and 202 (EV) mRNAs. Treatment of SW756 OSMR KD cells with OSM resulted in no significant changes in cellular or EV mRNA expression. Pathway analysis revealed that genes primarily involved in cytokine mediated signalling, hypoxia response, interferon response and negative regulation of viral lifecycle, myeloid leukocyte activation and angiogenesis were all upregulated in response to OSM, whereas, genes involved in cell cycle regulation, cellular organisation and cell differentiation were downregulated. Effects of OSM-OSMR signalling on cellular and EV miRNA expression was found to be less pronounced. The effect of OSM-OSMR signalling on tumour growth in vivo was also investigated. OSMR KD cells had a reduced growth rate following sub-cutaneous transplantation in NOD-SCID mice, compared with SW756 cells. Similarly, SW756 cells with constitutive-overexpression of OSM grew more quickly than SW756 in vivo. Together, this work indicates that OSM-OSMR signalling is an important driver of cervical SCC tumour progression both in vitro and in vivo, and demonstrates, for the first time, that OSM-OSMR signalling alters EV composition in cervical SCC cells. Subsequent work will focus on elucidating the functional importance of these EVs and their effects on cells of the TME