Matrix metalloproteinases (MMPs) play a significant role in degrading the extracellular matrix in cancer development and metastasis. Overexpression of matrix metalloproteinases in tumour tissues relative to normal tissues has been exploited as a target for peptide-based therapeutics, to improve therapeutic index of currently used agents. The stability of MMP-activated prodrugs in normal tissue or organs is a significant challenge for their success in the clinic. In an in vitro study, the stability of twenty six prodrugs was studied in mouse liver, kidney, lung and tumour homogenates using HPLC and LC/MS. Selected agents were studied in vivo. Each prodrug has a characteristic amino acid sequence with dominant FITC N-terminal end cap. All prodrugs were conjugated to a colchicine derivative (ICT 2552) which is a vascular disrupting agent causing tumour vasculature shutdown and consequently, tumour necrosis. ICT 3146, ICT 3019, ICT 3120 and ICT 3115 prodrugs showed significant stability in normal tissues and considerable activation in certain tumour tissues compared to the lead compound ICT 2588. Also, the selectivity of promising prodrugs to the MMP family was confirmed by using leupeptin (serine, cysteine and threonine protease inhibitor), pepstatin A (aspartate protease inhibitor), phosphoramidon (nepralysin inhibitor), ilomastat (metalloproteinase inhibitor) and BML-P115 (matrix metalloproteinase inhibitor). Moreover, members of the MMP family responsible for cleaving the selected prodrugs were identified using recombinant MMP enzymes. Furthermore, a LC/MS-MS method was developed to specifically detect and quantify MMP-16 protein expression in H460 tumour. MMP- 16 was responsible for the cleavage of ICT 3146 and ICT 3115. Therefore, MMPactivated prodrugs could be a useful therapeutic approach to avoid off-site toxicities of currently used anti-tumour agents.

Matrix metalloproteinases (MMPs) play a significant role in degrading the extra- cellular matrix in cancer development and metastasis. Overexpression of matrix metalloproteinases in tumour tissues relative to normal tissues has been exploited as a target for peptide-based therapeutics, to improve therapeutic index of currently used agents. The stability of MMP-activated prodrugs in normal tissue or organs is a significant challenge for their success in the clinic. In an in vitro study, the stability of twenty six prodrugs was studied in mouse liver, kidney, lung and tumour homogenates using HPLC and LC/MS. Selected agents were studied in vivo. Each prodrug has a characteristic amino acid sequence with dominant FITC N-terminal end cap. All prodrugs were conjugated to a colchicine derivative (ICT 2552) which is a vascular disrupting agent causing tumour vasculature shutdown and consequently, tumour necrosis. ICT 3146, ICT 3019, ICT 3120 and ICT 3115 prodrugs showed significant stability in normal tissues and considerable activation in certain tumour tissues compared to the lead compound ICT 2588. Also, the selectivity of promising prodrugs to the MMP family was confirmed by using leupeptin (serine, cysteine and threonine protease inhibitor), pepstatin A (aspartate protease inhibitor), phosphoramidon (nepralysin inhibitor), ilomastat (metalloproteinase inhibitor) and BML-P115 (matrix metalloproteinase inhibitor). Moreover, members of the MMP family responsible for cleaving the selected prodrugs were identified using recombinant MMP enzymes. Furthermore, a LC/MS-MS method was developed to specifically detect and quantify MMP-16 protein expression in H460 tumour. MMP- 16 was responsible for the cleavage of ICT 3146 and ICT 3115. Therefore, MMP-activated prodrugs could be a useful therapeutic approach to avoid off-site toxicities of currently used anti-tumour agents. / The full text will be available at the end of the extended embargo: 5th March 2027

As MMPs são endopeptidases zinco dependentes que, em conjunto, podem degradar todos os componentes da MEC e gerar moléculas bioativas. São as principais responsáveis pelo remodelamento tecidual durante eventos fisiológicos normais como a embriogênese e organogênese e também em eventos patológicos como a invasão tumoral nos tecidos. As pesquisas na área de mineralização biológica têm buscado identificar os genes envolvidos nos mecanismos moleculares que regula o processo de ossificação endocondral. As MMPs e seus inibidores são responsáveis pelo controle da degradação desta matriz, como os inibidores teciduais das MMPs (TIMPs) e a proteína RECK, que, muito provavelmente, determinam o grau de remodelação da matriz extracelular. Desta forma, nosso objetivo foi delinear o perfil temporal e espacial da expressão das MMP-15/MT2-MMP, MMP-16/MT3- MMP, MMP-17/MT4-MMP, MMP-24/MT5-MMP e MMP-25/MT6-MMP durante a ossificação endocondral em embriões de camundongos e em animais recémnascidos através das técnicas de PCR em tempo real e imunohistoquímica. Por imunohistoquímica, nós não encontramos imunomarcação para a MMP-15/MT2- MMP em nenhum dos períodos analisados, apesar da padronização do anticorpo primário. Tanto a MMP-16/MT3-MMP quanto a MMP-24/MT5-MMP foram imunolocalizadas, principalmente, nos osteoblastos do fronte de ossificação da placa de crescimento. Para a MMP-17/MT4-MMP, durante a diferenciação condrocítica (E13) os condrócitos proliferativos foram imunocorados, bem como os condrócitos hipertróficos no centro da cartilagem do molde cartilaginoso (E14). Durante a invasão celular e vascular (E15), as células mesenquimais oriundas do colar ósseo, provavelmente pré-osteoblastos, foram imunocorados na cavidade medular primitiva e osteoblastos fronte de ossificação foram imunocorados, de E16 a PN1. Observamos para a MMP-25/MT6-MMP o mesmo padrão de imunomarcação das demais MT-MMPs, exceto no molde cartilaginoso, onde apenas as células do periósteo e pericôndrio foram imunocoradas, diferentemente da demais que foram localizadas apenas no centro do molde cartilaginoso. A análise da expressão dos transcritos para todas as MT-MMPs revelou o mesmo perfil de expressão, sendo alta durante a fase de diferenciação condrocítica (E13), tendo queda de expressão de E14 a E16. Em E16 há um aumento de expressão até E18 e, novamente, queda até E20 e pouca ou nenhuma expressão em PN7. Apesar deste perfil semelhante, houve uma expressão diferencial entre elas, sendo a MMP-15/MT2-MMP > MMP- 17/MT4-MMP > MMP-16/MT3-MMP > MMP-24/MT5-MMP > MMP-25/MT6/MMP. Os resultados obtidos mostram, pela primeira vez, que as MT-MMPs estão diferencialmente expressas durante a ossificação endocondral normal em camundongos, sugerindo que a atividade biológica destas enzimas esteja atuando na degradação da matriz extracelular pericelular tanto durante a fase de desenvolvimento quanto de formação óssea. / MMPs are zinc-dependent endopeptidases that, collectivelly, degrade all components of the ECM and generate bioactive molecules. They are able to remodelate the ECM during normal developmental processes such as embryogenesis and organogenesis, as well as in pathological processes such as tumoral invasion. The biological mineralization research looking for discovering the genes involved in the molecular mechanisms that control the endochondral ossification process. MMPs and their inhibitors (TIMPs and RECK) are responsable for bone matrix remodeling and, probably, determinate the level of its turnover. Thus, our goal was to evaluate the temporal-spatial expression of MMP-15/MT2-MMP, MMP-16/MT3-MMP, MMP-17/MT4-MMP, MMP-24/MT5-MMP, and MMP-25/MT6- MMP in mice embryos and newborns during endochondral ossification by Real Time PCR and immunohistochemistry. By immunohistochemistry, MMP-15/MT2-MMP signal was not detected. Both MMP-16/MT3-MMP and MMP-24/MT5-MMP were immunostained, mainly in osteoblasts at ossification front of growth plate. For MMP- 17/MT4-MMP, proliferative chondrocytes were immunopositive during chondrocyte differentiation (E13) as well as in hipertrophyc chondrocytes at the middle of cartilaginous template (E14). During cellular e vascular invasion (E15), mesenchymal cells from bone collar, probable pre-osteoblasts, were immunostained at primary bone marrow and osteoblasts at ossification front from E16 e PN1. For MMP- 25/MT6-MMP, perichondrial and periostal cellls were immunostained at cartilaginous template. All MT-MMPs evaluated showed the same transcript levels profile, being high in chondrocyte differentiation (E13), decreasing from E14 to E16. mRNA levels increased from E16 to E18 and, once more, decreasing from E18 to E20. Despite this profile, we observed difference levels: MMP-15/MT2-MMP > MMP-17/MT4-MMP > MMP-16/MT3-MMP > MMP-24/MT5-MMP > MMP-25/MT6/MMP. Our findings show, for the first time, that MT-MMPs are differentially expressed during normal endochondral ossification in mice, suggesting their biological activity act in pericellular extracellular matrix degradation in both development and bone formation.

As MMPs são endopeptidases zinco dependentes que, em conjunto, podem degradar todos os componentes da MEC e gerar moléculas bioativas. São as principais responsáveis pelo remodelamento tecidual durante eventos fisiológicos normais como a embriogênese e organogênese e também em eventos patológicos como a invasão tumoral nos tecidos. As pesquisas na área de mineralização biológica têm buscado identificar os genes envolvidos nos mecanismos moleculares que regula o processo de ossificação endocondral. As MMPs e seus inibidores são responsáveis pelo controle da degradação desta matriz, como os inibidores teciduais das MMPs (TIMPs) e a proteína RECK, que, muito provavelmente, determinam o grau de remodelação da matriz extracelular. Desta forma, nosso objetivo foi delinear o perfil temporal e espacial da expressão das MMP-15/MT2-MMP, MMP-16/MT3- MMP, MMP-17/MT4-MMP, MMP-24/MT5-MMP e MMP-25/MT6-MMP durante a ossificação endocondral em embriões de camundongos e em animais recémnascidos através das técnicas de PCR em tempo real e imunohistoquímica. Por imunohistoquímica, nós não encontramos imunomarcação para a MMP-15/MT2- MMP em nenhum dos períodos analisados, apesar da padronização do anticorpo primário. Tanto a MMP-16/MT3-MMP quanto a MMP-24/MT5-MMP foram imunolocalizadas, principalmente, nos osteoblastos do fronte de ossificação da placa de crescimento. Para a MMP-17/MT4-MMP, durante a diferenciação condrocítica (E13) os condrócitos proliferativos foram imunocorados, bem como os condrócitos hipertróficos no centro da cartilagem do molde cartilaginoso (E14). Durante a invasão celular e vascular (E15), as células mesenquimais oriundas do colar ósseo, provavelmente pré-osteoblastos, foram imunocorados na cavidade medular primitiva e osteoblastos fronte de ossificação foram imunocorados, de E16 a PN1. Observamos para a MMP-25/MT6-MMP o mesmo padrão de imunomarcação das demais MT-MMPs, exceto no molde cartilaginoso, onde apenas as células do periósteo e pericôndrio foram imunocoradas, diferentemente da demais que foram localizadas apenas no centro do molde cartilaginoso. A análise da expressão dos transcritos para todas as MT-MMPs revelou o mesmo perfil de expressão, sendo alta durante a fase de diferenciação condrocítica (E13), tendo queda de expressão de E14 a E16. Em E16 há um aumento de expressão até E18 e, novamente, queda até E20 e pouca ou nenhuma expressão em PN7. Apesar deste perfil semelhante, houve uma expressão diferencial entre elas, sendo a MMP-15/MT2-MMP > MMP- 17/MT4-MMP > MMP-16/MT3-MMP > MMP-24/MT5-MMP > MMP-25/MT6/MMP. Os resultados obtidos mostram, pela primeira vez, que as MT-MMPs estão diferencialmente expressas durante a ossificação endocondral normal em camundongos, sugerindo que a atividade biológica destas enzimas esteja atuando na degradação da matriz extracelular pericelular tanto durante a fase de desenvolvimento quanto de formação óssea. / MMPs are zinc-dependent endopeptidases that, collectivelly, degrade all components of the ECM and generate bioactive molecules. They are able to remodelate the ECM during normal developmental processes such as embryogenesis and organogenesis, as well as in pathological processes such as tumoral invasion. The biological mineralization research looking for discovering the genes involved in the molecular mechanisms that control the endochondral ossification process. MMPs and their inhibitors (TIMPs and RECK) are responsable for bone matrix remodeling and, probably, determinate the level of its turnover. Thus, our goal was to evaluate the temporal-spatial expression of MMP-15/MT2-MMP, MMP-16/MT3-MMP, MMP-17/MT4-MMP, MMP-24/MT5-MMP, and MMP-25/MT6- MMP in mice embryos and newborns during endochondral ossification by Real Time PCR and immunohistochemistry. By immunohistochemistry, MMP-15/MT2-MMP signal was not detected. Both MMP-16/MT3-MMP and MMP-24/MT5-MMP were immunostained, mainly in osteoblasts at ossification front of growth plate. For MMP- 17/MT4-MMP, proliferative chondrocytes were immunopositive during chondrocyte differentiation (E13) as well as in hipertrophyc chondrocytes at the middle of cartilaginous template (E14). During cellular e vascular invasion (E15), mesenchymal cells from bone collar, probable pre-osteoblasts, were immunostained at primary bone marrow and osteoblasts at ossification front from E16 e PN1. For MMP- 25/MT6-MMP, perichondrial and periostal cellls were immunostained at cartilaginous template. All MT-MMPs evaluated showed the same transcript levels profile, being high in chondrocyte differentiation (E13), decreasing from E14 to E16. mRNA levels increased from E16 to E18 and, once more, decreasing from E18 to E20. Despite this profile, we observed difference levels: MMP-15/MT2-MMP > MMP-17/MT4-MMP > MMP-16/MT3-MMP > MMP-24/MT5-MMP > MMP-25/MT6/MMP. Our findings show, for the first time, that MT-MMPs are differentially expressed during normal endochondral ossification in mice, suggesting their biological activity act in pericellular extracellular matrix degradation in both development and bone formation.

Prostate cancer (PCa) represents the most frequently diagnosed cancer and the second leading cause of cancer death in males. Initial development and progression of the disease is mainly regulated by androgens. However, the pathology of the disease may progress to a loss of hormone dependence, resulting in rapid growth and a metastatic phenotype. Invasion and metastasis of tumour cells results from the degradation of the basement membrane (BM) and extracellular matrix (ECM). The degradation of the BM and ECM is in part mediated by a family of proteinases called the matrix metalloproteinases (MMPs). Currently more than 20 members of the MMP family have been identified and they are further divided in to sub-classes according to their protein structure. Collectively, MMPs are capable of degrading essentially all ECM components. High expression of some MMPs correlates with a malignant phenotype of various tumours. This study focused on the expression and regulation of a sub-class of MMPs called the membrane-type MMPs (MT-MMPs) in PCa. To date 6 MT-MMPs have been identified and they are characterized by a transmembrane domain, followed by a short cytoplasmic tail (MT1-, MT2-, MT3- and MT5-MMPs) or a glycosylphosphatidylinositol (GPI) moiety (MT4- and MT6-MMPs). MT-MMPs are thought to play a key role in tumour cell invasion by virtue of their ability to activate MMP-2 (a secreted MMP, which is implicated in many metastatic tumours) and their direct degradation activity on ECM components. Elevated MT-MMP expression has been shown in breast, colon, skin, stomach, lung, pancreas and brain cancers. Until very recently there had been no studies conducted on MT-MMPs in PCa. The few studies preceding or occurring in parallel with this one, have mainly reported the mRNA expression of these enzymes in PCa. Most studies have focused on MT1-MMP. Thus, at the commencement of this project there were many unexplored aspects of the expression and regulation of the broader MT-MMP family in PCa. The aims of this study were to examine: 1 a) The expression of MT-MMPs in prostate cancer cell lines using RT-PCR and western blot analysis and b) expression of MT1-MMP and MT5-MMP in BPH (benign prostatic hyperplasia) and PCa clinical tissue sections by immunohistochemistry. 2) The regulation of MT1-MMP, MT3-MMP and MT5-MMP in PCa cell lines by Concanavalin A (Con A), phorbol-12-myristate 13-acetate (PMA), dihydrotestosterone (DHT) and insulin-like growth factors I and II (IGF I and IGF II) using western blot analysis. In this study RWPE1, a transformed but non-tumorigenic prostate cell line was used as a "normal" prostate cell model, ALVA-41 and LNCaP as androgen-dependent PCa cell models and DU-145 and PC-3 as androgen-independent PCa cell models. The mRNA expression for the 6 MT-MMPs was determined by RT-PCR. The results indicate that MT1- and MT3-MMP were detected in all cell lines. This is the first study to report MT1-MMP mRNA expression in LNCaP cells and MT3-MMP mRNA in DU-145 cells. MT2-MMP mRNA was detected in only LNCaP and DU-145 cells, whilst MT5-MMP was detected in PC-3, DU-145 and LNCaP cells. nterestingly, MT2-, MT4-, MT5- or MT6-MMP mRNA expression was not detected in the "normal" cell line RWPE1, perhaps indicating an induction in gene transcription in tumour cells. MT4-MMP mRNA was only detected in the androgen-independent cell lines, indicating a potential role in the invasion and metastasis processes of the aggressive androgen-independent PCa. In this study, very low expression of MT6-MMP was detected only in LNCaP and DU-145 cells. Previously there had been no reports on the expression of MT6-MMP in the normal or cancerous prostate. Due to the mRNA of MT1-, MT3- and MT5-MMPs being the predominant MT-MMPs expressed in the current study, and the availability of suitable antibodies against them, the protein expression of these three MT-MMPs was studied by western blot analysis. MT1-, MT3- and MT5-MMP protein expression was detected in the cell lysates and conditioned medium (CM) of RWPE1, LNCaP and PC-3 cells. For each MT-MMP, various protein species were detected including putative proforms, mature (active) forms, processed or fragmented forms as well as soluble or shed forms. The presence of soluble or shed forms of MT-MMPs in the CM of cultures of "normal" and PCa cells could imply one of the following mechanisms: ectodomain shedding by either extracellular sheddases, the secretion of intracellular processed proteins without the transmembrane domain, the release of membrane vesicles containing membrane-bound enzymes, or the presence of alternatively spliced mRNA, which gives rise to MT-MMPs without a transmembrane domain. Further characterization of these various forms, including their amino acid sequence, is required to fully elucidate their structural composition. Despite the detection of the mRNA, we did not detect the cell-associated proteins of MT1-MMP and MT5-MMP and only very low expression of MT3-MMP in DU-145 cells (CM of DU-145 cells were not screened for soluble forms of the enzymes). This is the first study to report MT5-MMP expression at the protein level in prostate derived cell lines. Immunohistochemistry was carried out on benign prostatic hyperplasia (BPH) and PCa clinical tissues using MT1- and MT5-MMP antibodies to determine their cellular localisation in benign and cancer glands. MT1- and MT5-MMPs were expressed in BPH and moderate and high grade PCa. MT1-MMP expression was highest in moderate grade cancer compared to BPH and high grade cancer. MT1-MMP expression was predominantly observed in the cytoplasm of secretory epithelial cells of both benign and cancer glands, although in cancer glands, some nuclear staining was also observed. Stromal expression of MT1-MMP was only observed in high grade cancer. This study is the first to report the immunolocalization of MT5-MMP outside the brain and in kidneys of diabetic patients. MT5-MMP was predominantly expressed in the cytoplasm of the secretory cells in benign glands. In the cancer glands, staining was heterogeneous with low to intense staining, mainly in the nuclei, plasma membrane and cytoplasm of secretory epithelial cells. Stromal expression of MT5-MMP was only observed in cancer tissues, particularly in high grade cancer. To study the regulation of MT-MMPs in PCa, we treated LNCaP and PC-3 cells, with either Con A, PMA, DHT or IGF-I and -II and studied the protein expression of MT1-, MT3- and MT5-MMPs by western blot analysis. Con A and PMA have been shown to stimulate MMP expression in other cell systems. Con A treatment showed a general increase in the protein expression of MT1-, MT3- and MT5-MMPs. By far the greatest induction by Con A observed was the nearly 4 fold increase in MT5-MMP expression caused by 40μg/mL Con A treatment of PC-3 cells. PMA treatment of LNCaP and PC-3 cells appeared to increase shedding or secretion of all three MT-MMPs in to the CM. This increase in the soluble forms corresponded to a decrease in cell-associated forms in LNCaP cells. Treatment of LNCaP with DHT alone and treatment of LNCaP and PC-3 cells with IGF-I and -II alone failed to detect any change in expression of MT1-MMP. The information gathered in this study on MT-MMPs with respect to cellular localization, expression levels and regulation by growth factors or chemicals that mimic their actions, will aid in our understanding of the role of MT-MMPs in PCa. This study provides strong preliminary data for further research, particularly with respect to functional studies of MT-MMPs in PCa. Understanding the processes which govern the actions of such proteins as these will provide potential insights into development of new management and therapeutic regimens to prevent cancer progression.