Triple-negative breast cancer (TNBC) accounts for 15% of all diagnosed breast cancers nationally and affects African-American women 3 times more likely than any other ethnic group. Locally, African-American women in the New Orleans area see higher incidence of TNBC cases versus African-American women from the rest of the state of Louisiana, which represents an area of heightened public health interest for the metropolitan area. TNBC is a highly metastatic disease, and targeted therapies such as tamoxifen and herceptin are ineffective due to the lack of estrogen receptor (ER) and HER2/neu target expression in TNBC tumors. Chemotherapy remains the only effective drug therapy in TNBC cases. Evaluating new classes of drugs for clinical use against TNBC as well as furthering our understanding of underlying regulatory mechanisms in TNBC is a priority. Pan-deacetylase inhibitors (DACi), like panobinostat, have shown promise in clinical trials as therapies in other cancers. Pre-clinical data of panobinostat treatment in TNBC cell lines published by this lab has been positive so far, exhibiting a reduction in TNBC metastatic potential. DACi can alter multiple signaling pathways and are known to restore dysregulated microRNA (miRNA) expression patterns (miRnome) in cancers. MiRNA are a relatively new class of non-protein coding regulatory biomolecules that exhibit a variety of cancer-related properties, many still unknown in TNBC. Pan-DACi treatment combined with miRnome analysis in TNBC cell lines can be used to identify previously unknown miRNA cancer-related properties in TNBC. The specific aim of this project consists of using DACi treatment on TNBC cell lines in conjunction with miRnome analysis to identify previously undescribed anti-cancer miRNAs and elucidate their cancer-related properties in TNBC while uncovering affected cancer pathways, detecting miRNA targets, and revealing affected downstream components. Initial miRNA expression analysis of MDA-MB-231 TNBC cells treated with panobinostat or trichostatin A versus controls produced a list of potential anti-cancer miRNA candidates for further study. Among them, investigations into miR-203 and miR-335 produced unclear results as these were theorized to have anti-metastatic properties in TNBC yet enhanced cancer properties in our models and assays. Overexpression of mir-215 (a tumor suppressor in other cancers) unexpectedly enhanced tumor growth five fold in SCID mice xenografted with lentivirally-transduced MDA-MB-231 breast cancer cells stably overexpressing miR-215 (231/215+). Further qPCR analysis of 231/215+ cells uncovered upregulation of the breast cancer-associated lncRNA, HOTAIR; the breast cancer-associated miRNA, miR-196a; as well as the entire HOXC cluster in which they reside. This represents a previously unidentified regulatory mechanism of the HOXC cluster in humans. Additionally, miR-200b overexpression in MDA-MB-231 cells induced a change in cell morphology to an epithelial-like phenotype, reduced migration by 50%, and re-expressed the epithelial marker CDH1. This demonstrates a partial reversal of epithelial-mesenchymal transition (EMT), which indicates a reduction in metastatic potential by miR-200b overexpression in MDA-MB-231 cells. Additionally, these cells exhibited increased estrogen receptor alpha and related signaling pathways while also being susceptible to reduced proliferation with the anti-estrogen drug fulvestrant at high doses. Using pan-DACi treatment of TNBC cells to analyze changes in the miRnome for unknown cancer-related miRNA candidates suitable for further investigation in TNBC, we identified miR-215 overexpression in MDA-MB-231 cells as an oncogenic event that enhances tumor growth, cell proliferation, and HOXC cluster transcription while miR-200b is an anti-metastatic miRNA that partially reverses EMT and reduces fulvestrant resistance through re-expression of estrogen receptor signaling. / acase@tulane.edu

MicroRNAs (miRNAs) are small non-coding 18-22 long RNAs, which act as key mediators in many cellular processes involved in tumorigenesis including proliferation, differentiation, invasion, and apoptosis. miRNAs repress gene expression by inhibiting mRNA translation or by promoting mRNA degradation. Due to the importance of miRNAs as master regulators of gene expression many studies have emerged to distinguish both miRNA targets and regulatory mechanisms governing miRNA translation and maturation. Uncovering mechanisms that govern miRNA expression and function in cellular biology gives greater insight into pathways facilitating multiple cellular processes involved in tumor initiation and progression. miRNAs are altered in breast cancer and these alterations can lead to changes in signaling pathways involved in cancer progression. The purpose of this work is to determine the effects of miRNA expression on the cancer phenotype and to examine the roles of insulin-like growth factor 1 (IGF-1) and estrogen receptor (ER) signaling on miRNA expression and function in ER+ breast cancer cell systems. Our results demonstrate the intricate relationship between miRNAs and cell signaling networks. Through uncovering miRNA regulating networks such as IGF-1 signaling and ER induced miRNA maturation and star strand selection we have added greater insight to the complex mechanisms involved in miRNA function. miRNAs function through inhibition of mRNA targets, though the possible targets for a single miRNA can be in the thousands. Additionally, mRNA isoform variability and loss of 3’UTR greatly enhance the complexity of these networks. / acase@tulane.edu

MicroRNAs (miRNAs), an abundant class of ~22 nucleotide non-coding RNAs, are thought to play an important regulatory role in animal and plant development at the posttranscriptional level. Many miRNAs cloned from mouse bone marrow cells are differentially regulated in various hematopoietic lineages, suggesting that they might influence hematopoietic lineage differentiation. Some human miRNAs are linked to leukemias: the miR-15a/miR-16 locus is frequently deleted or down-regulated in patients with B-cell chronic lymphocytic leukemia and miR-142 is at a translocation site found in a case of aggressive B-cell leukemia. miR-181, a miRNA upregulated only in the B cell lineage of mouse bone marrow cells, promotes B cell differentiation and inhibits production of CD8⁺ T cells when expressed in hematopoietic stem/progenitor cells. In contrast miR-142s inhibits production of both CD4⁺ and CD8⁺ T cells and does not affect B cells. Collectively, these results indicate that microRNAs are components of the molecular circuitry controlling mouse hematopoiesis and suggest that other microRNAs have similar regulatory roles during other facets of vertebrate development. / Singapore-MIT Alliance (SMA)

Deciphering the rapid emergence of bilaterian animals around the time of the Cambrian Explosion and reconstructing the interrelationships of animal groups have long been two of the most elusive problems in Zoology. This thesis concerns the phylogenetic interrelationships within and among Acoela and Nemertodermatida, two groups of small worms that are believed to be basal bilaterians and which may provide important clues for understanding the early evolution of animals. In addition to trying to resolve the phylogenetic positions of these groups, major focus is put on inferring how ancestral animals might have looked, given the phylogenetic hypotheses put forward. The data used to infer phylogenies include nuclear ribosomal DNA, the mitochondrial COI gene and microRNAs. Based on phylogenetic analyses of a large number of 18S SSU ribosomal DNA sequences, it is proposed that Cnidaria is the sister taxon to Bilateria. Poor taxon sampling is suggested to be one of the reasons for why earlier assessments of the interrelationships among the most basal animal groups have yielded many conflicting results using the same gene. Analyses of new 18S SSU rDNA and 28S LSU rDNA sequences from six of the nine known species of nemertodermatids corroborate earlier indications that Acoela and Nemertodermatida are not sister taxa, as once thought. Being separate basal bilaterian animal groups, it is suggested that the last common ancestor of all bilaterians shared much of their comparatively simple morphology. Many methods are deployed to assess whether the phylogenetic results are mainly due to long-branch attraction, but no indication of this artifact is detected. The first comprehensive phylogenetic framework of Acoela is reconstructed from the 18S SSU, 28S LSU and COI genes, in combination with morphological data. The ancestral acoel worm is reconstructed using Bayesian methods and morphological observations in extant species. Two indeces, posterior similarity and reconstruction signal, are implemented to assess how similar different species are to the last common ancestor of all acoels and illustrate how clearly different characters or nodes are reconstructed. It is suggested that the ancestral acoel looked much like extant species of Diopisthoporus. The phylogenetic positions of Acoela and Nemertodermatida are assessed using new data on microRNAs in the acoel Hofstenia miamia and the nemertodermatid Meara stichopi. Acoela and Nemertodermatida are again found to be basal bilaterians, in congruence with earlier results. Using the work-flow and indeces developed earlier, it is concluded that the bilaterian ancestral microRNA repertoire can not yet be reconstructed with high confidence. All papers stress the importance of inclusive taxon sampling for making generalized inferences about ancestral features in animals.

<p>Cancer metastasis is the cause of about 90% of cancer patients' deaths. Despite significant improvements in the past three decades in understanding the molecular bases of oncogenic transformation of cancer cells, little is known about the molecular mechanisms underlying tumour cells' alteration of their microenvironment, entrance into the circulation, and colonization of distant organs. In recent years, accumulating evidence has indicated that tumour microenvironment, which consists of a variety of cell types and extracellular matrix components&#65292;plays an important role in regulating the metastatic abilities of carcinoma cells. Co-opted by cancer cells, those stromal cells promote tumour progression via multiple mechanisms, including enhancement of tumour invasiveness, elevation of angiogenesis, and suppression of immune surveillance activity. </p><p>Using a series of human breast cancer cell lines with different metastatic potentials <italic>in vivo</italic>, we performed an unbiased screen examining expression of miRNAs, and found that miR-126 and miR-126*, whose expression are regulated by methylation of the promoter of their host gene Egfl7 inside tumour cells, were significantly negatively correlated with metastatic potential. Using both mouse xenograft models and <italic>in vitro</italic> assays, we showed that this pair of miRNAs suppressed breast cancer metastasis through shaping the tumour microenvironment without changing tumour cell autonomous properties. Specifically, miR-126 and miR-126* act independently to suppress the sequential recruitment of mesenchymal stem cells (MSCs) and inflammatory monocytes into the primary tumour stroma, consequently inhibiting lung metastasis by breast tumour cells. Mechanistically, these miRNAs directly inhibit the production of stromal cell-derived factor-1 alpha (Sdf-1&alpha;, also known as Cxcl12), and indirectly suppress the expression of chemokine (C-C motif) ligand 2 (Ccl2) by the cancer cells within the tumour mass in an Sdf-1&alpha;-dependent manner. In addition, in contrast with the majority of reports which have shown incorporation of only the guiding strand of the miRNA duplex into the mRNA-targeting RNA induced silencing complex (RISC), both strands of the miR-126 RNA duplex are maintained at a similar level and suppress Sdf-1&alpha; expression independently. </p><p>Collectively, we have determined a dynamic process by which the composition of the primary tumour microenvironment could be altered via a change in the expression of two tumour-suppressive miRNAs derived from a single miRNA precursor to favor metastasis by breast cancer cells. Importantly, this work provides a prominent mechanism to explain the clinical correlation between reduced expression of miR-126/126* and poor metastasis-free survival of breast cancer patients.</p> / Dissertation

<p>MicroRNAs are key post-transcriptional regulators that have been found to play critical roles in the regulation of cellular functions. There is an emerging concept that microRNAs may be just as essential for fine-tuning physiological functions and responding to changing environments and stress conditions as for viability or development. In this dissertation, two studies are presented: The first study demonstrates a role for microRNA in the regulation of oxidative stress response in erythroid cells and the functional consequences of dysregulated microRNA expression in Sickle Cell Disease (SCD) pathobiology. The second study examines a functional role for microRNA in the cellular response to changes in cellular iron concentration. Together these studies illustrate the scope of importance of microRNAs in the coordination of cellular responses to diverse stresses. </p><p>Homozygous Sickle Cell (HbSS) erythrocytes are known to have reduced tolerance for oxidative stress, yet the basis for this phenotype has remained unknown. Here we use erythrocyte microRNA expression profiles to identify a subset of HbSS patients with higher miR-144 expression and more severe anemia. We reveal that in K562 erythroid cells and primary erythroid progenitor cells, miR-144 directly regulates NRF2, a central regulator of cellular response to oxidative stress, and modulates the oxidative stress response. We further demonstrate that increased miR-144 is associated with the reduced NRF2 levels, decreased glutathione regeneration, and attenuated antioxidant capacity found in HbSS erythroid progenitors, thereby providing a mechanism for the reduced oxidative stress tolerance and increased anemia severity seen in HbSS patients. </p><p>The post-transcriptional regulation of the IRP2 regulon in the cellular response to iron deficiency is well characterized. Here we examine the potential role for microRNA-mediated regulation in the coordinated response to cellular iron deficiency.</p> / Dissertation

El limfoma de cèl•lules del mantell (LCM) és una neoplàsia heterogènia en relació a les característiques clíniques, biològiques i moleculars que presenten els pacients. La morfologia, l’immunofenotip i el genotip no són suficients per explicar aquesta gran diversitat. Encara que es considera una neoplàsia agressiva alguns pacients segueixen un curs clínic indolent sense la necessitat de tractament en el moment del diagnòstic i presenten una supervivència llarga. Els resultats d’aquesta tesi demostren que els LCM amb elevada càrrega mutacional en els gens de les immunoglobulines (IGHV)(<97% d’identitat amb la línia germinal) utilitzen preferencialment certes famílies de gens IGHV, suggerint la presència de processos de selecció antigènica com a mecanisme de patogènesi d’aquesta neoplàsia i donant importància a la caracterització del receptor de les cèl•lules B (BCR). Els LCM amb gens IGHV mutats (M-LCM) presenten una menor complexitat genòmica, són preferencialment negatius per a l’expressió del factor de transcripció SOX11 i presenten afectació ganglionar amb més freqüència que els casos sense mutacions (U-LCM). Per altra banda, el perfil d’expressió gènica entre casos amb i sense mutacions en els gens IGHV, també mostra expressió diferencial de programes cel•lulars relacionats amb poblacions de cèl•lules B normals incidint amb la ontogènia d’ambdós grups. El perfil d’expressió de microRNAs també permet diferenciar entre dos grups de pacients amb diferent supervivència, reforçant la idea de la presència de dos subgrups que pertanyen a una mateixa entitat. La identificació i la correcte identificació d’aquests pacients de limfoma de cèl•lules del mantell són rellevants per al posterior maneig i tractament dels mateixos. / Mantle cell lymphoma is a malignant and heterogeneous disease regarding the clinical, biological and molecular characteristics presented by patients, all of them with the translocation t(11;14) and CCND1 overexpression. Although is considered an aggressive neoplasm some patients follow and indolent clinical course without treatment at the time of diagnosis and long survival. The results of this studies demonstrate that LCM with high mutational load in the immunoglobulins genes (IGHV) (<97% of identity with the germ line) use preferentially specific IGHV gene families, suggesting the presence of antigenic selection process as a mechanism of pathogenesis of this neoplasm and giving importance to the characterization of the B cell receptor. The LCM cases with IGHV mutated genes (M-LCM) present less genomic complexity are preferentially negative for the expression of the transcriptor factor SOX11 and also present lymphadenopathies more frequently compared with the IGHV unmutated cases (U-LCM). Furthermore, the gene expression profile between cases with and without IGHV mutations also shows differential expression of cellular programs related to normal B cell populations maybe related with the ontogeny for both groups. The expression profile of microRNAs also differentiates between two groups of patients with different survival and other molecular characteristics, reinforcing the idea of the presence of two subgroups that belong to the same entity. The correct identification and classification of these patients are relevant for subsequent handling and treatment of the patients.

<p>SR proteins are a family of splicing factors involved in the regulation of both constitutive and alternative splicing of pre-mRNAs. Despite years of studies, several big questions still remain: how the expression levels of SR proteins are regulated; what are the underlying mechanisms responsible for SR proteins-mediated gene regulation; what are the physiological targets of SR proteins in vivo. In my dissertation study, I am focusing on two members of the family, SF2/ASF and SRp20, to study their functional involvement in regulating microRNA/mRNA biogenesis and their own expression. </p><p>Negative feedback regulation is a common mechanism maintaining the steady-state level of SR proteins (i.e. SC35 and SRp20), and several mechanism may be involved. In order to test if miRNAs are also involved in such negative feedbacks, small RNA sequencing was used to identify differentially expressed miRNAs after SF2/ASF overexpression in an inducible stable cell line system. Among the 40 differentially expressed miRNAs, miR-7 is particularly interesting, because it is also predicted to target SF2/ASF, which forms a negative feedback regulation. This is indeed the case as shown by luciferase reporter assay and overexpression/knocking down of miR-7 in vivo. To our knowledge, this is the first identified negative feedback circuit between a SR protein and a miRNA, which may be a general mechanism in regulating SR protein homeostasis.</p><p>To characterize the mechanism underlying SF2/ASF-enhanced miRNA biogenesis, I have employed a series of molecular and biochemical approaches to pinpoint the key molecular interactions in a minigene system, which is consist of miR-7 embedded intron and the flanking exons of its host gene. By manipulating the splicing pattern of such minigene, I have uncovered a splicing-independent function of SF2/ASF in regulating miRNA biogenesis. Directly binding between SF2/ASF protein and pri-miR-7 was demonstrated by Cross-linking and immunoprecipitation assay (CLIP) and RNA affinity purification. The precise binding site was then pinpointed by combining computational prediction and mutagenesis assay. Finally, by using in vitro pri-miRNA processing assay, I showed that SF2/ASF can promote the Drosha cleavage step of pri-miR-7 through directly association with the predicted binding site. So far, this is the first SR protein discovered, which is directly involved in miRNA biogenesis. Moreover, our preliminary data also suggested that SF2/ASF may promote miRNA biogenesis in other steps after Drosha cleavage; and different SR proteins can regulate miRNA biogenesis in a substrate-specific manner. Taken together, SR family of splicing factors may be broadly involved in miRNA biogenesis through direct interactions.</p><p>In order to study the general involvement of SR proteins in RNA biogenesis, one important step stone is to have a better profile of their targets in vivo. To achieve this, I focused on SRp20, another classic SR protein. Photoactivatable-Ribonucleoside-Enhanced Cross-linking and immunoprecipitation assay combined with deep sequencing (PAR-CLIP-seq) was used to identify the binding partners of SRp20 globally, which is subsidized by candidate gene validations. Consistent with the literature, I found that SRp20 primarily targets exonic regions for splicing regulation, and such interactions are likely to be sequence dependent on the CWWCW motif. Surprisingly, I also observed extensive binding between SRp20 and the 3' UTRs of mRNA, which may affect the choice of alternative polyadenylation sites. The underlying mechanisms are being investigated by a variety of molecular methods. </p><p>In summary, I have identified a subset of miRNAs, the expression of which can be regulated by SF2/ASF in a splicing independent manner. This is the first SR protein identified in regulating miRNA biogenesis. One of the upregulated miRNAs, miRNA-7 can form a negative feedback with SF2/ASF by negatively regulating the expression of SF2/ASF on translational level. By using PAR-CLIP method, I have identified the genome-wide binding partners of SRp20 in vivo. When SRp20 binds to the exonic regions, it potentially affects the alternative splicing patterns of nearby introns. Interestingly, the 3' end choices for a subset of genes may be regulated by SRp20 through directly binding, which may be a new mechanism for the regulation of 3' end processing.</p> / Dissertation

Biotrophic plant pathogens lead to huge crop losses and they have great economical drawbacks on wheat and barley production. These pathogens rely on formation of haustoria and transfer of effector proteins into the host cells for generating disease. The main role of effector proteins is to disable plant defense mechanisms. Effector proteins contain N-terminal signal peptides and they have little sequence similarity between each other. It is vital to detect as many effector proteins as possible to understand infection and disease formation processes of biotrophic plant pathogens. To this end, sequencing of pathogen genomes are being emerged, the data will be invaluable for identifying the candidate effectors in terms of biological and biochemical roles in infection and more. There are some bioinformatics based methods available that can be utilized to classify and distinguish effectors from other pathogenic genes. It is important to understand how candidate effectors can be searched from Expressed Sequence Tags or transcriptome sequences. Hereby, our attempt is to present a pipeline in establishing a methodology. As a consequence, here we propose new candidate effectors. In plant-pathogen interactions also miRNAs are too proving to be an important factor which cannot be neglected. During disease infection, expression levels of miRNAs are varying which in turn may be a proof of miRNA regulation of pathogen genes. Therefore, cross-kingdom RNA interference may take place between plant and pathogen. We have tested plant pathogens for possible plant miRNA availability and found their most likely targets with in the pathogen genes.

Recent statistics reveal breast cancer as the most common cancer among women and accounts for approximately 41,000 mortalities per year. In diagnosis, features such as stage, grade, lymph node metastasis are important prognostic indicators that help guide physicians and oncologist towards optimal patient care. Presence of established pathological markers such as ER, PR, and Her2/neu status would indicate ideal adjuvant therapy situation. Although treatment of these types of breast cancer is well established, cancer that lack all three receptors, “triple negatives” or “basal like” do not respond to adjuvant therapy and are considered more aggressive in that patients tend to recur early and experience visceral metastasis. Although scientists have uncovered numerous molecular biology mechanisms in search of an understanding in cancer, leading to development of fields such as apoptosis or growth pathways; cell cycle; angiogenesis; metastasis; and more recently cancer stem cells, much work remains as cancer is still not eradicated. MicroRNAs (miRNAs) are post transcriptional regulators of gene expression. Their discovery and functional understanding have only been uncovered in the past ten years. Long pri-miRNAs are transcribed from the genome and processed into premiRNAs by Dicer; and then into short single stranded mature miRNAs complexed with Argonaute proteins to inhibit protein translation. The first link of miRNAs to cancer was made only relatively recently, but the field has expanded exponentially since. TGF- β induced Epithelial to Mesenchymal Transition model in Normal Mouse Mammary Gland Epithelia Cells (NMuMG) is a commonly used model to dissect the molecular processes of breast cancer metastasis. Using miRNA microarray, we demonstrated miR-155 was upregulated along with alterations of other miRNAs. This observation was validated with Northern and qRT-PCR analysis. Promoter and ChIP analysis revealed TGF- β activated the Smad4 transcriptional complex to induce the expression of miR-155. The reduction of RhoA protein levels by ubiquitination has been described to be a critical step during EMT, and we showed miR-155 down regulates RhoA proteins without degrading its mRNA levels; therefore, preventing de novo synthesis of RhoA proteins in the course of EMT. The interaction between miR-155 and RhoA’s 3’UTR was confirmed by reporter assays. In summary, we reported the importance of miR-155 during TGF β induced EMT in NMuMG cells. FOXO3a is a well studied tumor suppressor transcriptional factor and resides in the nucleus to transcribe pro-apoptotic genes such as Bim, or p27 in the active state. During conditions when cells are signaled to grow and divide, it is phosphorylated by oncogenes such as AKT or IKK β, becomes inactivated and translocates into the cytoplasm. We have shown for the first time that FOXO3a activity is also regulated by miRNAs, specifically miR-155. Western and Northern analysis revealed a correlation between FOXO3a protein and mature miR-155 RNA levels in breast cancer cell lines along with breast tumor and normal tissues. Specifically, miR-155 expression is low in BT474 and high in HS578T, and inversely correlates with endogenous FOXO3a protein levels. Overexpression of miR-155 decreased endogenous FOXO3a protein and knockdown of miR-155 HS578T rescued its expression. Reporter assay experiments validated the interaction between miR-155 and FOXO3a 3’UTR. More importantly, overexpression of miR-155 in BT474 protected the cells from apoptosis induced by drugs while knockdown of miR-155 in HS578T initiated cell death even in the absence of drugs. In summary, we have shown the importance of miR-155 in chemosensitivity by targeting FOXO3a in breast cancer. MiR-155 has been previously shown up-regulated in multiple types of malignancies, including breast cancer. In addition, miR-155 expression was reported to correlate very strongly to survival in lung and pancreatic cancer. We validated by qRTPCR and Northern analysis that miR-155 expression is detected only in breast tumors and not normal breast tissue. In situ hybridization of breast cancer tissue microarrays revealed similar results. In light of previous studies that showed a correlation between miR-155 and survival in lung and pancreatic cancers, we performed an X-tile analysis to determine an optimal cut point for miR-155 level in our breast cancer sample population that would correlate to ten years overall survival. Verification using Kaplan-Meier validated a cut point at 90.14 to significantly correlate to overall survival (P=0.007). In addition, Chi-square analysis revealed miR-155 expression to correlate with high tumor stage, grade and lymph node metastasis. However, miR-155 expression did not correspond to ER, PR, or HER2/neu status, but this is hardly surprising since computational analysis does not predict miR-155 to target these genes. In summary, we have shown deviant expression of miR-155 in breast cancer. Due to its correlation with overall survival; higher grade and stage; lymph node metastasis, and triple negative subtype, miR-155 may prove to be a valuable prognostic marker and therapeutic target for breast cancer intervention.