Virtually all signaling pathways that play key roles in development such as the transfroming growth factor (TGF)-beta, notch, and wnt pathways also influence tumor formation, implying that cancer is in a sense development gone awry. Therefore, identification and elucidation of developmental pathways has great potential for generating new diagnostic tools and molecular therapy targets. Singleminded-2s (SIM2s), a splice variant of the basic helilx-loop-helix / PER-ARNT-SIM (bHLH/PAS) transcriptional repressor Singleminded-2, is lost or repressed in approximately 70% of human breast tumors and has a profound influence on normal mammary development. In order to gain a better understanding of the mechanisms by which SIM2s restricts malignant transformation and progression in breast cancer, we depleted SIM2 RNA in MCF-7 cells using a retroviral shRNA system and examined gene expression and functional abilities of the SIM2-depleted MCF-7 cells (SIM2i) relative to a control MCF line expressing a non-specific “scrambled” shRNA (SCR). Depletion of SIM2 resulted in an epithelial-mesenchymal transition (EMT)-like effect characterized by increased migration and invasion, altered morphology, and loss of epithelial markers concomitant with gain of mesenchymal markers. The root of this effect may be loss of SIM2- mediated repression of the E-cadherin repressor slug, as SIM2 is able to bind and repress transcription from the slug promoter, and slug expression is dramatically elevated in SIM2i MCF-7 cells. Consistent with the previously established role of slug in resistance to various cancer therapies, SIM2i cells are resistant to the radiomimetic doxorubicin and appear to have elevated self-renewal capacity under certain conditions. Intriguingly, SIM2 protein levels are elevated by treatment with DNA damaging agents, and SIM2 interacts with the p53 complex via co-regulation of specific p53- target gene such as p21/WAF1/CIP1. These results provide a plausible mechanism for the tumor suppressor activity of SIM2, and provide insight into a novel tumor suppressive transcriptional circuit that may have utility as a therapeutic target.

A Caderina-E està intimamente relacionada com a transiÃÃo epitelial-mesenquimal e com a progressÃo tumoral em muitos tipos de cÃncer, inclusive no cÃncer colorretal. O objetivo deste trabalho foi avaliar a imunoexpressÃo de Caderina-E no cÃncer colorretal primÃrio e nas respectivas metÃstases linfonodais, na mucosa colÃnica normal, e investigar possÃveis correlaÃÃes desta expressÃo com parÃmetros clÃnicopatolÃgicos. Setenta e sete casos de colectomias por carcinoma colorretal e dez casos de linfonodos metastÃticos, dos arquivos do Departamento de Patologia e Medicina Legal/Universidade Federal do CearÃ, foram utilizados. Realizou-se o Tissue Microarray e imunohistoquÃmica, com anticorpo monoclonal anti-Caderina-E. Foram avaliados os seguintes escores: 0 = ausÃncia de expressÃo; 1 = expressÃo citoplasmÃtica; 2 = expressÃo mista (citoplasmÃtica e membranar); 3 = expressÃo membranar pura. Foi utilizada tanto a classificaÃÃo proposta por Jawhari et al., agrupando os casos em expressÃo anormal (escores 0, 1 e 2) e expressÃo normal (escore 3), como os critÃrios propostos por Almeida et al., agrupando os casos como expressÃo nÃo-membranar (escores 0 e 1) e expressÃo membranar (escores 2 e 3). Os tumores primÃrios tiveram mais casos de expressÃo de Caderina-E anormal em comparaÃÃo com a mucosa normal (p < 0.0001). NÃo houve diferenÃa significante entre expressÃo de Caderina-E no tumor intestinal e em metÃstases linfonodais, embora nestas a expressÃo membranar tenha sido mais freqÃente do que no sÃtio primÃrio. Tumores de cÃlulas agrupadas apresentaram maior expressÃo de Caderina-E membranar do que os de cÃlulas isoladas, tanto utilizando a classificaÃÃo de Jawhari et al. (p = 0.0230), como os critÃrios propostos por Almeida et al. (p = 0.0043). Em conclusÃo, a expressÃo anormal de Caderina-E no tumor primÃrio, com persistÃncia freqÃente da imunomarcaÃÃo membranar associada à marcaÃÃo citoplasmÃtica (marcaÃÃo anormal heterogÃnea ou mista), reforÃa as evidÃncias de que esta alteraÃÃo no cÃncer à mais qualitativa do que propriamente quantitativa. O predomÃnio da expressÃo membranar no sÃtio primÃrio da neoplasia e na metÃstase, com ou sem expressÃo citoplasmÃtica associada, principalmente em tumores de cÃlulas agrupadas, sugere que a presenÃa da Caderina-E à essencial para a invasÃo local e progressÃo tumoral, em oposiÃÃo ao clÃssico paradigma de que a progressÃo tumoral se exacerba com a perda desta molÃcula de adesÃo. / E-cadherin is closely related to epitelial-mesenchymal transition and tumor progression in many cancers, including colorectal cancer. The aim of this study is to evaluate the expression of E-cadherin in primary colorectal cancer as well as in lymph node metastasis, establishing also a comparison with the expression of E-cadherin in normal colonic mucosa. We utilized 77 cases of colectomies for colorectal carcinoma and 10 cases of metastatic lymph nodes from the files of the Department of Pathology and Forensic Medicine/Federal University of Ceara. Tissue microarray and immunohistochemistry were performed with monoclonal anti-E-cadherin, evaluated using the following scores: 0 = no staining; 1 = cytoplasmic staining; 2 = mixed staining (cytoplasmic and membranous); 3 = membranous staining. It was used the classification proposed by Jawahri et al. which includes cases of abnormal expression (0, 1 and 2 scores) and cases of normal expression (3 score), and was also used the classification proposed by Almeida et al. which includes cases of non-membranous expression (0 and 1 scores) and membranous expression (2 and 3 scores). Primary tumors presented more cases of abnormal E-cadherin expression in comparison to normal colonic mucosa (p < 0.0001). There were no differences between E-cadherin expression in the primary tumor in comparison to lymph node metastasis. The grouped cell tumors showed increased expression of E-cadherin in comparison to isolated cell tumors, either using the classification proposed by Jawhari et al. (p = 0.0230) and the classification proposed by Almeida et al. (p = 0.0043). In conclusion, abnormal expression of E-cadherin in the primary tumor, with frequent membranar immunostaining associated with the cytoplasmic marking (abnormal heterogeneous or mixed staining), reinforces the evidence that E-cadherin expression change in cancer is more qualitative than quantitative. The predominance of membranar expression in primary tumor and lymph node metastasis, with or without associated cytoplasmatic expression, particularly in cell-grouped tumors, suggests that E-cadherin presence is essential for local invasion and tumor progression, as opposed to the classical paradigm that tumor progression is exacerbated by the loss of this adhesion molecule.

Epithelial-mesenchymal transition (EMT) is an evolutionarily conserved process during which cells lose epithelial characteristics and gain a migratory phenotype. Although downregulation of epithelial cadherins by Snail and other transcriptional repressors is generally considered a prerequisite for EMT, recent studies have challenged this view. Here we investigate the relationship between E-cadherin and P-cadherin expression and localization, Snail function and EMT during gastrulation in chicken embryos. Expression analyses show that while E-cadherin transcripts are detected in the epiblast but not in the primitive streak or mesoderm, P-cadherin mRNA and protein are present in the epiblast, primitive and mesoderm. Antibodies that specifically recognize E-cadherin are not presently available. During EMT, P-cadherin relocalizes from the lateral surfaces of epithelial epiblast cells to a circumferential distribution in emerging mesodermal cells. Cells electroporated with an E-cadherin expression construct undergo EMT and migrate into the mesoderm. An examination of Snail function showed that reduction of Slug (SNAI2) protein levels using a morpholino fails to inhibit EMT, and expression of human or chicken Snail in epiblast cells fails to induce EMT. In contrast, cells expressing the Rho inhibitor peptide C3 rapidly exit the epiblast without activating Slug or the mesoderm marker N-cadherin. Together, these experiments show that epiblast cells undergo EMT while retaining P-cadherin, and raise questions about the mechanisms of EMT regulation during avian gastrulation.

Tubulointerstitial fibrosis is the key histological predictor of the progression of declining renal function and the final common pathway of progressive kidney disease, regardless of aetiology. Despite its significance, there are currently no treatments available to abrogate this process and those that suffer with this burden eventually succumb to renal failure. Tubulointerstitial fibrosis is largely mediated by fibroblasts and myofibroblasts present in the interstitium. In response to injury, activated fibroblasts differentiate into myofibroblasts which serves as a histological hallmark of fibrosis. Myofibroblasts are characterised as the key contributors to interstitial volume and their presence ultimately leads to loss of renal function. The pathological entities leading to fibrosis inextricably depend on complex signalling pathways. Whilst many of the well-known growth factors that exert effects on renal fibroblasts (such as FGF, EGF and PDGF) involve the activation of receptor tyrosine kinases, the intracellular signalling events dictating the response of fibroblasts remain undefined. The kinase mTOR, responsible for integrating stress and amino acids and controlling cell growth, is increasingly recognised for its ability to integrate growth factor signals mediated through the upstream serine/threonine kinase PI3K. A number of recent studies have highlighted the role of PI3K and mTOR in the regulation of key events relevant to fibrosis, serving as a basis for Chapter 3: The role of PI3K and mTOR in the regulation of fibroblast proliferation and collagen synthesis, and the first part of Chapter 5: The role of PI3K and mTOR in the regulation of myofibroblast differentiation. These studies have identified a key role for PI3K and mTOR in the regulation of fibroblast proliferation, differentiation and collagen synthesis. The work described within has also attempted to examine the derivation of myofibroblasts via EMT. EMT is a process that is integral to embryogenesis and may act as an important source of myofibroblasts during fibrosis. This process is examined in Chapter 4: Development and validation of an ex vivo model of EMT. This model aims to better represent the in vivo environment and has also been used to identify novel regulators involved in EMT being utilised in the second part of Chapter 5: The role of PI3K and mTOR in EMT. Although cytokines and growth factors are thought to be chiefly responsible for tubulointerstitial fibrosis, we now know that serine proteases of the coagulation cascade may also play roles in renal disease. However, unlike their role in glomerular diseases, the role of coagulation in tubulointerstitial fibrosis is less well-known. The work described in Chapter 6: Constituents of the coagulation cascade are spatially and functionally related to experimental tubulointerstitial fibrosis has examined temporal and spatial in vivo relationships of coagulation factors and markers of fibrosis that aid our understanding of mechanisms of fibrosis. The aim of this thesis was to examine those facets of renal fibroblast function that are most devastating to renal function and culminate in an expansion of the renal interstitium during fibrosis. This work hopes to provide useful information to aid the understanding of the multifaceted mechanisms involved in renal tubulointerstitial fibrosis.

ROGDI, a novel gene, locates on human¡¦s chromosome 16p13.3. According to Gene Ontology Annotation database, ROGDI is related to hemopoiesis and positive regulation of cell proliferation. In order to investigate the function of this novel gene in pulmonary fibrosis, fibrotic models in vivo and in vitro were created. Mice which received single intra-tracheal bleomycin injection were sacrificed on various intervals. Rogdi and other pro-fibrotic mediators, including CCL2 and TGF-£]1, were up-regulated in the early phase(< 10 days). On contrary, the anti-fibrotic mediators IL-10, IFN-£^ and heme oxygenase(HO)-1 were up-regulated in the late phase(> 10 days). The precursor microRNA 21 (miR-21) was up-regulated as the fibrotic severity increased. The human embryonic fibroblasts(WI-38 cells) showed fibrogenic phenotype and up-regulation of precursor miR-21 and ROGDI after bleomycin treatment. Human embryonic fibroblasts transfected by coding sequence of ROGDI showed up-regulated precursor miR-21 and £\-SMA compared to those transfected by empty vectors after bleomycin treatment. Two signaling molecules related to positive regulation of cell proliferation, Akt and Erk, showed over-expressed after ROGDI transfection and bleomycin treatment compared to those with empty vector transfection. Our results imply that ROGDI is up-regulated in pulmonary fibrosis and turns fibroblasts into fibrogenic phenotype through positive regulation of miR-21. The increase of precursor, but not primary miR-21, after ROGDI transfection and bleomycin treatment indicates that ROGDI may regulate the TGF-£] signaling pathway in human embryonic fibroblasts. Our results support that ROGDI is a novel gene for pulmonary fibrosis and warrants for further investigation. £[

Despite the development of novel target therapy drugs in recent years, metastatic cancer remains refractory to current cancer therapies and accounts for the majority of cancer mortalities worldwide. Metastasis consists of multiple steps including angiogenesis, extravasion, escape from immune surveillance, adhesion, and clonal expansion in different organs that a systemic therapy is required for effective control of metastasis. The pro-inflammatory nuclear factor kappa B (NF£eB) pathway plays an important role during each of these metastatic events and constitutes an excellent target for metastasis control. Stress hormone pro-opiomelanocortin (POMC) and its derived neuropeptides including corticotrophin (ACTH), £\-, £]-, and £^-melanocyte¡Vstimulating hormone (£\-, £]-, and £^-MSH), £]-endorphin are potent inhibitors of NF£eB pathway. Other than the central regulation of stress response and energy homeostasis, POMC also regulates the skin pigmentation, inflammatory processes, and immune reactions in the peripheral system. Since adenovirus¡Vmediated POMC gene delivery leads to hepatic POMC expression, it seems plausible that POMC gene therapy may elicit systemic production of anti-inflammatory POMC-derived peptides and hold promises for control of primary and metastatic cancers. In B16-F10 melanoma models, POMC gene delivery elevated the circulating ACTH levels for more than 8 weeks and suppressed the growth of established melanoma, thereby prolonging the life span of tumor-bearing mice. Moreover, combination of POMC therapy with cisplatin further enhances the survival outcome. Subsequent analysis reveals that POMC gene therapy inhibits the growth and metastasis of melanoma through apoptosis, angiogenesis inhibition, and modulation of epithelial-mesenchymal transition. Besides, £\-MSH/melanortin-1 receptor (MC-1R) pathway is involved in the POMC-mediated melanoma suppression. To investigate whether POMC therapy could be applied to other types of tumor, we evaluated the therapeutic efficacy of POMC gene therapy in Lewis lung carcinoma (LLC) cells which lack MC-1R. Interestingly, POMC gene delivery effectively inhibited the proliferation and colony formation of LLC cells in vitro and the growth of established LLC in mice. Histological analysis indicated that POMC gene delivery attenuated LLC through proliferation inhibition, apoptosis induction, and angiogenesis blockade. Moreover, POMC gene delivery perturbed £]-catenin signaling by reducing protein levels of £]-catenin and its downstream proto-oncogenes, including cyclin D1 and c-myc. These results support the existence of an MC-1R-independent pathway for POMC gene therapy and expand the therapeutic spectrum of POMC therapy for multiple types of cancer. To elucidate the role of host immunity in anti-neoplastic mechanism underlying POMC therapy, we compared the treatment efficacy of POMC gene therapy for B16-F10 melanoma between severe combined immune-deficient (SCID) and immune-competent C57BL/6 mice, and found similar extent of tumor suppression in both strains of mice. In addition, POMC gene therapy reduced the spleen weight and the number of circulating lymphocytes in B6 mice. These findings suggest that POMC therapy was not dependent on host immunity, yet instead induced immune suppression of animals through ACTH/cortisol production. To minimize such side effect of POMC therapy, we generated a series of adenovirus vectors encoding POMC with mutations in ACTH domain (ACTH-K15A/R17A), which fails to stimulate cortisol synthesis in vitro and in vivo. Gene delivery of ACTH (K15A/R17A) remained capable of suppressing the primary and metastatic melanoma, but had no effect on immune functions in mice. In conclusion, we have characterized the anti-neoplastic function and mechanism of POMC therapy for cancer. Furthermore, we have developed improved POMC gene vectors to minimize its adverse effect for future cancer therapy.

No description available.

Attrition rates for anticancer drugs are much higher than any other therapeutic area. Only 5%#37; of the agents that demonstrate anticancer activity in the preclinical stages of development demonstrate clinical efficacy in phase III trials. This high attrition rate becomes alarming when we consider that the cost of research and development can amount to 1 billion dollars. To exacerbate this problem, many new cancer drugs are being discontinued, withdrawn or suspended. The reasons for this high attrition rate are complex and may be partly attributed to suboptimal preclinical strategies such as the use of two-dimensional (2D) cell culture systems to evaluate new agents during the development and testing stages. Cancer cells cultured in 2D do not mimic the complexity of the three-dimensional (3D) milieu of tumors in vivo. There is overwhelming evidence that in vitro 3D culture systems more accurately reflect the tumor microenvironment and present better predictive value for assessing the efficacy of new chemotherapeutic agents. The development of 3D culture systems for anticancer drug development remains an unmet need. Despite progress, a simple, rapid, scalable and inexpensive 3D-tumor model that recapitulates in vivo tumorigenesis is lacking. Herein, we report on the development and characterization of a 3D nanofibrous scaffold produced by electrospinning a mixture of poly(lactic-co-glycolic acid) (PLGA) and a block copolymer of polylactic acid (PLA) and mono-methoxy polyethylene glycol (mPEG) designated as 3P. Cancer cells cultured on the 3P scaffold formed tight aggregates similar to in vivo tumors, referred to as tumoroids that depended on the topography and net charge of the scaffold. 3P scaffolds induced tumor cells to undergo the epithelial-to-mesenchymal transition (EMT) as demonstrated by up-regulation of vimentin and loss of E-cadherin expression. 3P tumoroids showed higher resistance to anticancer drugs than the same tumor cells grown as monolayers. Inhibition of ERK and PI3K signal pathways prevented EMT conversion and reduced tumoroid formation, diameter and number. Fine needle aspirates, collected from tumor cells implanted in mice when cultured on 3P scaffolds formed tumoroids, but showed decreased sensitivity to anticancer drugs, compared to tumoroids formed by direct seeding. These results show that 3P scaffolds provide an excellent platform for producing tumoroids from tumor cell lines and from biopsies and that the platform can be used to culture patient biopsies, test for anticancer compounds and tailor a personalized cancer treatment.

In order for solid tumors to metastasize, tumor cells must acquire the ability to invade the surrounding tissue and intravasate into blood- or lymph vessels, survive in the circulation and then extravasate at a distant site to form a new tumor. Overexpression of the glycosaminoglycan hyaluronan, and its adhesion receptor CD44, correlate with breast cancer progression. This thesis focuses on the role of hyaluronan in tumor invasion and metastasis. In paper I, we demonstrated that upregulation of the hyaluronan synthesizing enzyme hyaluronan synthase 2 (HAS2) was crucial for transforming growth factor β (TGFβ)-induced epithelial-mesenchymal transition (EMT) in mammary epithelial cells. In paper II, we further demonstrated that silencing of HAS2 decreased the invasive behavior of bone-metastasizing breast cancer cells, via upregulation of tissue inhibitor for metalloproteinase 1 (TIMP1), and dephosphorylation of focal adhesion kinase (FAK). During tumorigenesis, stromal cells, such as fibroblasts, play important roles and several growth factors are synthesized, promoting crosstalk between different cell surface receptors. In paper III, we investigated the crosstalk between the hyaluronan receptor CD44 and the receptors for TGFβ and platelet-derived growth factor BB (PDGF-BB) in dermal fibroblasts. We found that the receptors for the three molecules form a ternary complex, and that PDGF-BB can activate the Smad pathway downstream of TGFβRI. Importantly, CD44 negatively modulated the signaling of both PDGF-BB and TGFβ. In paper IV, we studied the process by which breast cancer cells invade blood-vessels and the role of hyaluronan and CD44 in angiogenesis. Importantly, CD44, or the hyaluronan degrading enzyme hyaluronidase 2 (HYAL2), decreased the capacity of endothelial cells to form tubes in a 3D in vivo-like assay.  Collectively, our studies add to the understanding of the role of hyaluronan in tumor progression.

Ovarian cancer is the most lethal gynecological cancer, with over 200,000 women diagnosed each year and over half of those cases leading to death. These poor statistics are related to a lack of early symptoms and inadequate screening techniques. This results in the cancer going undetected until later stages when the tumor has metastasized through a process that requires the epithelial to mesenchymal transition (EMT). In lieu of traditional monolayer cell culture, EMT and cancer progression in general is best characterized through the use of 3D spheroid models. In this study, we examine gene expression changes through microarray analysis in spheroid versus monolayer ovarian cancer cells treated with TGFβ to induce EMT. Transcripts that included Coiled-Coil Domain Containing 80 (CCDC80), Solute Carrier Family 6 (Neutral Amino Acid Transporter), Member 15 (SLC6A15), Semaphorin 3E (SEMA3E) and PIF1 5'-To-3' DNA Helicase (PIF1) were downregulated more than 10-fold in the 3D cells while Inhibitor Of DNA Binding 2, HLH Protein (ID2), Regulator Of Cell Cycle (RGCC), Protease, Serine 35 (PRSS35), and Aldo-Keto Reductase Family 1, Member C1 (AKR1C1) were increased more than 50-fold. Interestingly, stress responses and epigenetic processes were significantly affected by 3D growth. The heat shock response and the oxidative stress response were also identified as transcriptome responses that showed significant changes upon 3D growth. Subnetwork enrichment analysis revealed that DNA integrity (e.g. DNA damage, genetic instability, nucleotide excision repair, and the DNA damage checkpoint pathway) were altered in the 3D spheroid model. In addition, two epigenetic processes, DNA methylation and histone acetylation, were increased with 3D growth. These findings support the hypothesis that three dimensional ovarian cell culturing is physiologically different from its monolayer counterpart. The proteotoxic stress-responsive transcription factor HSF1 is frequently overexpressed in a variety of cancers and is vital to cellular proliferation and invasion in some cancers. Upon analysis of various patient data sets, we find that HSF1 is frequently overexpressed in ovarian tumor samples. In order to determine the role of HSF1 in ovarian cancer, inducible HSF1 knockdown cell lines were created. Knockdown of HSF1 in SKOV3 and HEY ovarian cancer cell lines attenuates the epithelial-tomesenchymal transition (EMT) in cells treated with TGFβ, as determined by western blot and quantitative RT-PCR analysis of multiple EMT markers. To further explore the role of HSF1 in ovarian cancer EMT, we cultured multicellular spheroids in a non-adherent environment to simulate early avascular tumors. In the spheroid model, cells more readily undergo EMT; however, EMT inhibition by HSF1 knockdown becomes more pronounced in the spheroid model. These findings suggest that HSF1 is important in the ovarian cancer TGFβ response and in EMT.