Erbin belongs to the LAP (leucine-rich repeat and PDZ domain) family of scaffolding proteins that play important roles in orchestrating cell signaling. Here, we show that Erbin functions as a tumor suppressor in colon cancer. Analysis of Erbin expression in patient specimens reveals that Erbin is downregulated at both mRNA and protein levels in tumor tissues. Functionally, knockdown of Erbin disrupts epithelial cell polarity and increases cell proliferation in 3D culture. In addition, silencing Erbin results in an increase in the amplitude and duration of signaling through Akt and RAS/RAF pathways. Moreover, Erbin-loss induces epithelial-mesenchymal transition (EMT), which coincides with a significant increase in cell migration and invasion. Erbin interacts with KSR1 and displaces it from the RAF/MEK/ERK complex to prevent signaling propagation. Furthermore, genetic deletion of Erbin in Apc knockout mice promotes tumorigenesis and significantly reduces survival. Tumor organoids derived from Erbin/Apc double knockout mice have increased tumor initiation potential along with increased Wnt target gene expression as seen by qPCR. Collectively, the studies within this dissertation identify Erbin as a negative regulator of EMT and tumor progression by directly suppressing Akt and RAS/RAF signaling in vivo.

acase@tulane.edu / Triple-negative breast cancer (TNBC) presents a clinical challenge due to the aggressive nature of the disease and a lack of targeted therapies. Constitutive activation of the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) pathway has been linked to chemoresistance and metastatic progression through distinct mechanisms, including activation of epithelial-to-mesenchymal transition (EMT) whereby cells adopt a motile and invasive phenotype through loss of epithelial markers, namely Cadherin 1/E-Cadherin (CDH1), and acquisition of mesenchymal markers, such as vimentin (VIM) and Cadherin 2/N-Cadherin (CDH2). While MAPK/ERK1/2 kinase inhibitors (MEKi) have shown promise as antitumor agents in the preclinical setting, application has had limited success clinically. Activation of compensatory signaling, potentially contributing to the emergence of drug resistance, has shifted the therapeutic strategy to combine MEK1/2 inhibitors with agents targeting oncoproteins (RAF) or parallel growth pathways (PI3K). Conventional MAPK family members have been well-characterized in modulation of cellular processes involved in tumor initiation and progression, yet the role of MEK5-ERK5 in cancer biology is not completely understood. Recent studies have highlighted the importance of the MEK5 pathway in metastatic progression of various cancer types, including those of the prostate, colon, bone and breast. Furthermore, elevated levels of ERK5 expression and activity observed in breast carcinomas are linked to worse prognosis in TNBC patients. The purpose of this work is to explore MEK5 regulation of the EMT axis and to evaluate a novel pan-MEK inhibitor on clinically aggressive TNBC cells. Our results show a distinction between the MEK1/2 and MEK5 cascades in maintenance of the mesenchymal phenotype, suggesting that the MEK5 pathway may be necessary and sufficient in EMT regulation while MEK1/2 signaling further sustains the mesenchymal state of TNBC cells. Furthermore, additive effects on MET induction are evident through the inhibition of both MEK1/2 and MEK5. Taken together, these data demonstrate the need for a better understanding of the individual roles of MEK1/2 and MEK5 signaling in breast cancer and provide rationale for combined targeting of these pathways to circumvent compensatory signaling and subsequent therapeutic resistance. / 1 / Van Hoang

How tumor cells migrate and metastasize from primary sites requires four major steps: invasion, intravasation, extravasation and proliferation from micrometastases to malignant tumor. The initiation of tumor cell invasion requires Epithelial-Mesenchymal Transition (EMT), by which tumor cells lose cell-cell interactions and gain the ability of migration. The gene expression profile during the EMT process has been extensively investigated to study the initiation of EMT. In our studies, we indicated that tyrosine phosphorylation of human p68 RNA helicase positively associated with the malignant status of tumor tissue or cells. Studying of this relationship revealed that p68 RNA helicase played a critical role in EMT progression by repression of E-cadherin as an epithelial marker and upregulation of Vimentin as a mesenchymal marker. Insight into the mechanism of how p68 RNA helicase represses E-cadherin expression indicated that p68 RNA helicase initiated EMT by transcriptional upregulation of Snail. Human p68 RNA helicase has been documented as an RNA-dependent ATPase. The protein is an essential factor in the pre-mRNA splicing procedure. Some examples show that p68 RNA helicase functions as a transcriptional coactivator in ATPase dependent or independent manner. Here we indicated that p68 RNA helicase unwound protein complexes to modulate protein-protein interactions by using protein-dependent ATPase activity. The phosphorylated p68 RNA helicase displaced HDAC1 from the chromatin remodeling MBD3:Mi2/NuRD complex at the Snail promoter. Thus, our data demonstrated an example of protein-dependent ATPase which modulates protein-protein interactions within the chromatin remodeling machine.

La transició epiteli-mesènquima (TEM) és un procés en què cèl lules epitelials, immòbils i amb polaritat apico-basal transiten cap un fenotip mesenquimal o fibroblàstic. L'expressió del factor de transcripció snail1 és suficient per induir TEM en cèl lules en cultiu i és necessari per la majoria de les TEM fisiològiques descrites. Snail1 és un membre de la família de proteïnes amb dits de Zinc que reprimeix gens epitelials (com l'E-cadherina) a través de la unió directa a seqüències especifiques dels promotors anomenades caixes E i posterior reclutament de corepressors. La TEM també es caracteritza per l'activació de gens mesenquimals, però el mecanisme pel qual snail1 indueix l'expressió d'aquests és poc conegut. En aquest treball demostrem que snail1 actua a nivell transcripcional per incrementar els nivells dels marcadors mesenquimals FN1 (fibronectina) i LEF1 (de l'anglès, lymphoid enhancer-binding factor 1) a través d'un mecanisme nou per aquesta proteïna de dits de Zn que no requereix ni caixes E ni unió directa a l'ADN. A més a més, mostrem que, per a dur a terme l'activació, snail1 coopera amb dos factors de transcripció ja descrits en relació a la TEM: beta-catenina i NF-kappa-B. Els nostres resultats també proven que l'expressió forçada de la E-cadherina evita aquesta cooperació i conseqüent activació gènica. A banda d'aquest mecanisme, també hem descrit que el factor de transcripció TFCP2c, que no havia estat prèviament relacionat amb TEM, és necessari per l'activació del gen FN1 induïda per snail1. / Epithelial-mesenchymal transition (EMT) is a cellular process by which no motile epithelial, apico-basal-polarized cells transit towards a motile mesenchymal front-backpolarized phenotype. Expression of the transcription factor snail1 is sufficient to induce EMT in cultured cells and it is required for most of the physiological EMTs described. Snail1 is a member of the Zn finger protein family that represses epithelial genes (such as E-cadherin) by directly binding to specific promoter sequences called E-boxes and subsequent recruitment of corepressors. EMT is also accompanied by activation of mesenchymal genes, however, little is known of how snail1 induces their expression.In this work we provide evidence that snail1 acts at the transcriptional level to increase the levels of the mesenchymal FN1 (fibronectin) and LEF1 (lymphoid enhancer-binding factor 1) genes through a novel mechanism for this Zn finger protein that does not require neither E-boxes nor direct binding to DNA. Furthermore, we describe a cooperative action in such mechanism between snail1 and two transcription factors previously related to EMT: beta-catenin and NF-kappaB. Our results also show that restoration of E-cadherin levels prevents such cooperation and subsequent activation. In addition, we also demonstrate that TFCP2c, which had not been previously linked to EMT, is also required for snail1-induced transcriptional activation of the FN1 gene.

Epithelial to mesenchymal transition (EMT), the transdifferentation of an epithelial cell into a mesenchymal fibroblast, is a cellular process necessary for embryonic development and wound healing. However, uncontrolled EMT can result in accumulation of myofibroblasts and excessive deposition of ECM, contributing to the pathological progression of fibrotic diseases such as pulmonary fibrosis. The ability to control EMT is important for development of novel therapeutics for fibrotic pathologies and for designing novel biomaterials for tissue engineering applications seeking to promote EMT for development of complex tissues. EMT is a highly orchestrated process involving the integration of biochemical signals from specific integrin-mediated interactions with extracellular matrix (ECM) proteins and soluble growth factors such as TGFβ. TGFβ, a potent inducer of EMT, is activated via cell contraction-mediated mechanical release of the growth factor from a macromolecular latency complex. Thus TGFβ activity and subsequent EMT may be influenced by the biochemical and biophysical state of the surrounding ECM. Based on these knowns, it was hypothesized that both changes in integrin engagement and increases in substrate rigidity would modulate EMT due to changes in epithelial cell contraction and TGFβ activation. Here we show that integrin-specific interactions with fibronectin (Fn) fragments displaying both the RGD and PHSRN binding sites facilitate cell binding through α5β1 and α3β1 integrins, and lead to maintenance of epithelial phenotype, while Fn fragments displaying only the RGD site facilitate cell binding through αv integrins and lead to EMT. An in depth investigation into α3β1 binding to Fn fragments indicates that binding is dependent on both the presence and orientation of the PHSRN site. Studies investigating the contribution of ECM stiffening on EMT responses show that increasingly rigid Fn substrates are sufficient to induce spontaneous EMT. Analysis of TGFβ-responsive genes implicate TGFβ-expression, activation or signaling as a mechanism for the observed EMT responses. Together these results suggest that the ECM micromechanical environment is a significant contributor to the onset of EMT responses and provide insights into the design of biomaterial-based microenvironments for the control of epithelial cell phenotype.

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.

The development of the mature cardiovascular system is one of the most captivating stories in embryonic development. The heart is the first organ to form in embryogenesis, and is functional early in development to perfuse the embryo with blood supplying oxygen and the nutrients required for organogenesis. The structural changes in heart development required for formation of the mature four chambered heart are under tight molecular regulation. Severe defects manifest as gross structural malformations of the valves, septa, or vessels that result in physiological consequences that my include hypertension, arrhythmia, or heart failure and may ultimately lead to lethality. According to the American Heart Association, cardiovascular disease is the leading cause of mortality worldwide. A more detailed understanding of the origin of congenital heart defects is necessary for improving prediction, diagnosis, and treatment of cardiovascular disease. Derived from the epicardium, coronary vessel formation relies on growth factor as well as extracellular matrix (ECM) influences on cells of the epicardium that regulate proliferation, motility, invasion and differentiation. The Transforming Growth Factor β (TGFβ) family of receptors have been well described in regulating cardiovascular development. The Type III TGFβ receptor (TGFβR3) has been shown to be required for development of the coronary vessels. Mouse embryos lacking TGFβR3 exhibit inhibited invasion of epicardially derived cells (EPDCs) into the myocardium. This delay of cell invasion of EPDCs and formation of coronary vessels is lethal at E 14.5. Relative to Tgfbr3+/+ cells, epicardial cells lacking TGFβR3 are hypo-proliferative, deficient in cell invasion, and deficient in executing TGFβ ligand and High-Molecular Weight Hyaluronan (HMWHA) stimulated cell invasion. Hyaluronan (HMWHA) is a glycosaminoglycan unmodified sugar extracellular matrix (ECM) molecule synthesized by the Hyaluronan Synthase (Has) family of enzymes. Mouse embryos lacking Hyaluronan Synthase 2 (Has2) are lethal at E 9.5 as a result of severely blocked cardiogenesis due to insufficient endocardial EMT. HA serves structural and bioactive functions in its capacity to stimulate signal transduction pathways required for EMT. Src kinase is a non-receptor tyrosine kinase well characterized to function in growth factor as well as ECM signal transduction, but its role in epicardial cell biology is unclear. Our hypothesis is that Src kinase is a critical regulator of TGFβ and Hyaluronan induced epicardial cell invasion, differentiation and migration during coronary vessel development. Our studies reveal that Src activity is required for TGFβ2-induced synthesis of HA in epicardial cells. We show Src is required for TGFβ2-induced vascular smooth muscle differentiation as well as TGFβ2-induced EMT, cell invasion, and filamentous actin polymerization. Src activity is sufficient to drive epicardial activation of EMT, but not vascular smooth muscle differentiation. These data show that Src is required in the context of TGFβ2-stimulated invasion and differentiation, and sufficient to drive activation of EMT. Next we demonstrate that TGFβR3 and Src are required for HMWHA induced cell invasion and filamentous actin polymerization in epicardial cells. HMWHA induces activation of Src kinase in Tgfbr3+/+ epicardial cells, but not Tgfbr3-/- epicardial cells. siRNA knockdown of TGFβR3 in Tgfbr3+/+ epicardial cells subsequently stimulated with HMWHA phenocopy this deficit in Src activation. Tgfbr3-/- epicardial cells fail to activate Rac1 or RhoA GTPases in the presence of HMWHA. Finally, we demonstrate stimulus independent activation of TGFβR3 is sufficient to activate Src. Taken together, these constitute novel findings establishing TGFβR3 as an HMWHA responsive receptor that is upstream of Src signal transduction. Migration of the epicardium to cover the looped and functioning heart tube is an early step required for development of the coronary vessels. We demonstrate that Tgfbr3-/- epicardial cells are delayed in cell migration relative to Tgfbr3+/+ cells in a wound healing model of cell migration. Tgfbr3-/- cells lack expression of BMP2 mRNA, we found that exogenous BMP2 is sufficient to drive Tgfbr3-/- (but not Tgfbr3+/+) cell migration to levels comparable to unstimulated Tgfbr3+/+ epicardial cells, without enhancing cell proliferation. We demonstrate that Src is required for this BMP2 induced cell migration and filamentous actin polymerization in Tgfbr3-/- cells. These studies demonstrate mechanisms required for TGFβ ligand as well as HMWHA stimulated epicardial cell behavior changes have a common mediator in Src kinase, and provide novel insights into early events in the development of the cardiovascular system. The adult epicardium has been demonstrated to participate in repair of ischemic myocardium in mouse models of myocardial infarction. Expression of molecules required for coronary vessel development are re-expressed in this regeneration (as discussed in chapter 5). Elucidating these pathways will constitute important future targets in aiding in adult cardiovascular regeneration and cardioprotection in adult heart disease.

The transforming growth factor beta (TGFβ)/activin signaling pathway has a number of documented roles during wound healing and is becoming increasingly appreciated as a vital component of multi-tissue regeneration. The leopard gecko (Eublepharis macularius) is able to spontaneously, and repeatedly, regenerate its tail following tail loss. We thus examined the expression and localization of several key components of the TGFβ/activin signaling pathway during tail regeneration of the leopard gecko. We observed a marked increase in phosphorylated-Smad2 expression among regenerating tissues corresponding to the location of the regenerate blastema. Interestingly, we observe that during early regeneration there appears to be an absence of TGFβ family member TGFβ1 and instead a strong upregulation of activin-βA. We also observe the expression of EMT transcription factors Snail1 and Snail2 in blastemal tissue. These observations combined with other data provide strong support for the importance of unique and non-overlapping expression patterns of different TGFβ ligands during multi-tissue regeneration

No description available.

Prostate cancer is the most frequently diagnosed non-skin cancer and the third leading cause of cancer mortality among men in the US. Androgens are functionally required for the normal growth of the prostate gland and play a critical role in prostate tumor development and progression. Epithelial-mesenchymal-transition (EMT) is an important process during normal development, and cancer cell metastasis. This study examined the ability of androgens to influence EMT of prostate cancer epithelial cells and evaluate the effect of taxol chemotherapy on androgen signaling in prostate cancer cells in prostate cancer. The EMT pattern was evaluated on the basis of expression of the epithelial markers as well as cytoskeleton reorganization in respond to DHT (1nM) and/or TGFβ (5ng/ml). Overexpressing and silencing approaches to regulate androgen receptor (AR) expression were conducted to determine the involvement of AR in EMT in the presence or absence of an AR antagonist. The AR transcriptional activity was determined on the basis of prostate specific antigen (PSA) mRNA expression and the androgen-response element (ARE) luciferase reporter assay. The interaction of AR and tubulin was investigated using immunoprecipitation, immunofluorescence as well as introduction of a truncated AR in human prostate cancer cells. Our results demonstrate that androgens induce the EMT pattern in prostate tumor epithelial cell with Snail activation and led to significant changes in prostate cancer cell migration and invasion potential. Expression levels of AR inversely correlated with androgen-mediated EMT in prostate tumor epithelial cells, pointing to a low AR content required for the EMT phenotype. Our study also reveals that treatment of prostate cancer cells with Paclitaxel or Nocodaxol inhibits androgen-dependent, as well as androgen-independent AR nuclear translocation and activation potentially via targeting the interaction of AR and microtubule cytoskeletal structures. Our findings on multiple aspects of AR function in prostate cancer development and progression may enhance the understanding of AR targeting therapy being a double-sided sword in the context of tumor microenvironment. These studies provide new insights into the mechanism of action of chemotherapy agents and the development of therapeutic resistance within tubulin/microtubule repertoire in prostate cancer cells.