Indiana University-Purdue University Indianapolis (IUPUI) / Ovarian cancer (OC) is a lethal disease due to metastasis and chemoresistance. Our laboratory previously reported that tissue transglutaminase (TG2) is overexpressed in OC and enhances OC peritoneal metastasis. TG2 is a multifunctional protein which catalyzes Ca2+-dependent cross-linking of proteins. The purpose of this study was to explore the mechanism by which TG2 is upregulated in OC and its role in OC progression. We demonstrated that transforming growth factor (TGF)-β1 is secreted in the OC milieu and regulates the expression and function of TG2 primarily through the canonical Smad signaling pathway. Increased TG2 expression level correlates with a mesenchymal phenotype of OC cells, suggesting that TGF-β1 induced TG2 promotes epithelial-to-mesenchymal transition (EMT). TG2 induces EMT by negatively regulating E-cadherin expression. TG2 modulates E-cadherin transcriptional suppressor Zeb1 expression by activating NF-κB complex, which leads to increased cell invasiveness in vitro and tumor metastasis in vivo. The N-terminal fibronectin (FN) binding domain of TG2 (tTG 1-140), lacking both enzymatic and GTPase function, induced EMT in OC cells, suggesting the interaction with FN involved in EMT induction. A TGF-β receptor kinase inhibitor, SD-208, blocked TGF-β1 induced TG2 upregulation and EMT in vitro and tumor dissemination in vivo, which confirms the link between TGF-β1 and TG2 in EMT and tumor metastasis. TG2 expression was correlated with the number and size of self-renewing spheroids, the percentage of CD44+CD117+ ovarian cancer stem cells (CSCs) and with the expression level of stem cell specific transcriptional factors Nanog, Oct3/4, and Sox2. These data suggest that TG2 is an important player in the homeostasis of ovarian CSCs, which are critical for OC peritoneal metastasis and chemoresistance. TG2 expression was also increased in CSCs isolated from human ovarian tumors, confirming the implication of TG2 in CSCs homeostasis. Further, we demonstrated that TG2 protects OC cells from cisplatin-induced apoptosis by regulating NF-κB activity. We proposed a model whereby TGF-β-inducible TG2 modulates EMT, metastasis, CSC homeostasis and chemoresistance in OC. These findings contribute to a better understanding of the mechanisms of OC metastasis modulated by TG2.

<p>Metastasis of primary mammary tumors to vital secondary organs is the primary cause of breast cancer-associated death, with no effective treatment. Metastasis is a highly selective process that requires cancer cells to overcome multiple barriers to escape the primary tumor, survive in circulation, and eventually colonize distant secondary organs. One of the important aspects of metastatic cancers is the ability to undergo epithelial-mesenchymal transition (EMT) and the reverse process mesenchymal-epithelial transition (MET) process. Constant interconversion of tumor cells between these phenotypes creates epithelial-mesenchymal heterogeneity (EMH) and interaction between these tumor cell types and the stromal cell compartment is clearly important to metastasis. In healthy tissues, stromal cells maintain the composition and structure of the tissue through the production of extracellular matrix (ECM) proteins and paracrine signaling with epithelial cells. However, little is known about how EMH promotes changes in the ECM to promote breast cancer progression and metastasis. Cancer cells also secret exosomes, nano-size extracellular vesicles, to establish intercellular communication with distant organs in order to induce metastasis. These exosomes contain a plethora of different proteins including extracellular matrix proteins and matrix crosslinking enzymes. Fibronectin, an important ECM protein, plays an active role in tumor progression and is often crosslinked by tissue transglutaminase 2 (TGM2) to promote fibrosis in cancer. Both FN and TGM2 exist in exosomes and are expressed by heterogenous breast tumors. Although FN and TGM2 have been reported to play essential roles in cancer, their involvement in metastasis remains unclear. This work utilizes a variety of approaches to investigate the role of tumor heterogeneity and ECM proteins in promoting breast cancer metastasis. In this dissertation, we establish that mesenchymal cells expressing intracellular FN are held in a stable non-metastatic mesenchymal phenotype and produce cellular fibrils containing functionalized FN capable of supporting the growth of metastatic competent epithelial cells. We introduce a novel 3D culture system consisting of a tessellated scaffold which is capable of recapitulating cellular and matrix phenotypes <i>in vivo. </i>Further, we also demonstrate breast tumor cells secrete exosomes containing TGM2 crosslinked FN fibrils to promote premetastatic niche formation and induction of metastasis.<i> </i>Using genetic approaches, we establish TGM2 is essential and sufficient to drive metastasis. Finally, we demonstrate pharmacological inhibition of TGM2 offers a potential therapeutic strategy to treat metastatic breast cancer. Altogether, our research provides insights into the mechanism through which TGM2 promotes metastatic breast cancer. This work will help in developing new drugs to target TGM2 aimed at reducing breast cancer metastasis.<br></p>

La transglutaminase tissulaire (TG2) est une enzyme de la famille des transglutaminases (EC 2.3.2.13) qui est exprimée de manière ubiquitaire chez les mammifères. Cette enzyme catalyse la formation d'une liaison amide intra- ou intermoléculaire entre un résidu glutamine et un résidu lysine. Ce processus biologique conduit à la modification post-traductionnelle des protéines. Un nombre croissant de publications associe la surexpression de cette enzyme et la déréglementation de son activité, avec un certain nombre de pathologiques humaines telles que les maladies neurodégénératives (maladie d’Alzheimer, maladie de Huntington, maladie de Parkinson), la fibrose tissulaire, certains cancers et la maladie cœliaque. Le développement d'inhibiteurs puissants et sélectifs de la TG2 est primordial pour identifier soit des outils pharmacologiques pour comprendre les processus biologiques dépendant de cette enzyme ou soit des candidats médicaments pour traiter les pathologies liées à la surexpression de la TG2. La majorité des composés inhibiteurs synthétisés jusqu'à présent agissent en bloquant de manière irréversible la réaction de transamidification de la TG2 en ciblant spécifiquement la cystéine 277 présente dans le site actif de la TG2.L’objectif de ce travail a été d’identifier et de sélectionner des molécules de faible poids moléculaire inhibant de façon sélective et puissante l’activité de transamidification de la TG2. Nous présenterons l’optimisation de deux séries originales de composés (synthèse, études de relation de structure-activité) comportant un noyau aromatique central de type naphtalénique ou indolique et une fonction acrylamide comme accepteur de Michael pour piéger la fonction thiol de la cystéine 277. Un certain nombre de composés synthétisés montre une inhibition nanomolaire de la TG2 (IC50 = 1.7-6 nM) avec un excellent profil de sélectivité vis-à-vis de TG1, TG6 et FXIIIa (IC50 > 10 µM). Ces inhibiteurs inhibent efficacement la TG2 dans des extraits de tissus et de cellules. Aucune toxicité apparente n’a été observée pour des concentrations inférieures à 10 µM d’inhibiteur sur les lignées vSMCs et SH-SY5Y. Les valeurs de KI, kinact et kinact/KI ont été également déterminés sur deux inhibiteurs sélectionnés (23b et 78f) pour leurs activités biologiques. La formation d’une liaison covalente entre la cystéine 277 de la TG2 et ces deux inhibiteurs a été prouvée par digestion trypsique suivie d’une analyse LC-MS/MS / Tissue transglutaminase (TG2) is a ubiquitously expressed enzyme of the mammalian transglutaminase (TG) family which catalyzes the formation of an intra- or inter-molecular isopeptide bond between a glutamine and a lysine, leading to the post-translational modification of proteins. An increasing number of literature has associated the over-expression of this enzyme, and the deregulation of its activity, with a number of human physio-pathological states like neurodegenerative disorders (Alzheimer’s disease, Huntington’s disease, Parkinson’s disease), tissue fibrosis, certain cancers, and celiac disease. The development of potent and selective TG2 inhibitors has become primordial to reach either a pharmacological probe, to understand the biological processes that depend on this enzyme, or a drug candidate, to treat the pathologies related with its overexpression. The majority of the inhibitory compounds synthesized so far act by irreversibly blocking the transamidation reaction of TG2. These TG2 inhibitors specifically target the cysteine 277 present in the TG2 active site. The aim of this work was the identification and selection of new potent and selective small molecules to inhibit the TG2 transamidation activity. We present the optimization of two new series of compounds (synthesis, structure-activity relationship studies) bearing naphthalene or indole aromatic rings as the central backbone structure. Both series present an acrylamide group as the Michael acceptor in order to react with the thiol group of cysteine 277. Several of the synthesized compounds showed a nanomolar inhibition over TG2 (1.7-6 nM) with an excellent selectivity profile over TG1, TG6 and FXIIIa (IC50 > 10 µM). These inhibitors showed high specificity on inhibiting TG2 in tissue and cell extracts. No apparent toxicity up to 10 µM was observed in vSMCs and SH-SY5Y cell lines. Their KI, kinact et kinact/KI were also determined on two selected inhibitors (23b and 78f) for their biological activities. The formation of a covalent bond between the cysteine 277 of TG2 and these two inhibitors was proven by tryptic digestion followed by LC-MS/MS analysis