<p>Soft tissue sarcomas (STSs) are a heterogeneous group of mesenchymal tumors comprised of >70 subtypes. An important question is how the cell of origin and the pathways to tumor development shape the broad array of STS subtypes. By forcing identical tumor-promoting mutations to different cell types in Genetically Engineered Mouse Models (GEMMs) of STS, I have a unique model system to investigate this question. In the process of performing these experiments I observed that genetic mutations are necessary, but not sufficient for rapid sarcoma formation. However, tissue injury dramatically accelerates sarcoma formation in our GEMM of STS. For my thesis, I have worked to understand how cell of origin affects sarcoma subtype and how the microenvironment in our models promotes transformation. I have observed that cell of origin plays an important, but not the only, role in defining STS subtype. Additionally, I have concluded that the microenvironment, and specifically the HGF/c-MET signaling pathway play a crucial role in promoting sarcoma development after acute tissue injury.</p> / Dissertation

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Hepatocyte Growth Factor (HGF)–induced Epithelial–Mesenchymal Transition (EMT) is a complex cellular pathway that causes epithelial cell scattering by breaking cell–cell contacts, eliminating apical–basal polarity, and replacing epithelial markers and characteristics with mesenchymal markers. Early EMT events include a brief period of cell spreading, followed by cell compaction and cell–cell contact breaks. A forward chemical genetics drug screen of 50,000 unique compounds measuring HGF–induced cell scattering identified 26 novel EMT inhibitors, including 2 proteolytic inhibitors. Here, we show that B5500–4, one of the EMT inhibitors from the screen, blocks HGF–induced EMT by a predicted blocking of the protease furin, in addition to secondarily blocking Beta–Secretase (BACE).We also show that MMP–12 and MMP–9 are required for HGF–induced EMT to progress. MMP–12 is required for cell contraction, and its inhibition produces a continuous cell spreading phenotype.We also demonstrate that both furin and BACE activity are required for HGF–induced EMT to proceed, but that they are involved in separate pathways. We show that BACE inhibition leads to a failure of cell spreading in early EMT, and that EphA2 is a member of this pathway. We also demonstrate that it is likely BACE2, and not BACE1 that is responsible for early cell spreading. Furin is also required for HGF–induced cell scattering, but does not play a role in the cell spreading process. These findings highlight the importance of proteolytic activity at the earliest stages of HGF–induced EMT.

Bone formation and remodeling require continuous generation of osteoprogenitors from bone marrow stromal cells (MSC), which are regulated by local growth factors and hormones with putative roles in mesenchymal proliferation and differentiation. Hepatocyte growth factor (HGF) and its receptor c-Met are widely expressed in MSC and are thought to play a key role in the interactions between cells. 1,25-dihydroxyvitamin D (1,25OHD) is the most active metabolite of vitamin D. 1,25OHD binds to its nuclear/membrane vitamin D receptor (VDR) and generates appropriate biological responses. The purpose of this study was to investigate the regulation of proliferation and differentiation by HGF in human bone marrow-derived stromal cells (hMSC). We examined the impact of HGF on hMSC cell-cycle regulation and the combination effects of HGF and 1,25OHD on hMSC osteogenic differentiation to enhance our knowledge of hMSC regulation. hMSC isolated from bone marrow were plated and grown in DMEM supplemented with 3% FBS incubated at 37C with 5% CO2 in air. HGF treatment of hMSCs reduced the rate of cell proliferation and this result was not due to apoptosis or cell senescence. Real-time RT-PCR and Western blot analysis showed increased gene and protein expression of the cell-cycle inhibitors p53, p21, and p27 after HGF treatment. These results appear to be specific because HGF did not significantly alter the gene expression level of other cell-cycle mediators such as RB, cyclin D1, CDK2, CDK4, or CDK6. Transfection of siRNA specific for cMet, the HGF receptor, eliminated the HGF anti-proliferation effect. cMet siRNA also eliminated the increase in p53, p21, and p27, further supporting a role for these cell-cycle inhibitors in HGF¡¯s regulation of hMSC. These results suggest that treatment of hMSC with HGF slows cell proliferation by increasing the expression of p53, p21, and p27. The reduced rate of cell proliferation did not appear to be due to cell differentiation, because treatment of hMSC with HGF alone did not induce cell differentiation. However, HGF in combination with a known osteogenic differentiation activator, 1,25OHD, significantly increased cell maturation/differentiation compared to 1,25D alone, as indicated by an increase in osteocalcin mRNA (a marker for osteogenic differentiation). Whereas HGF had no effect on 1,25OHD synthesis per se, HGF did induce 1,26OHD receptor (VDR) gene expression. HGF up-regulated the expression of the p63 gene, a member of the p53 family. Knocking down the p63 gene reduced the HGF effect on VDR expression and eliminated the HGF-induced up-regulation of the osteogenic differentiation markers osteopontin (OPN) and bone sialoprotein (BSP). Moreover, the ChIP assay shows that p63 was able to bind to the VDR promoter, possibly explaining the mechanism of p63-mediated VDR up-regulation. These results indicate that HGF can also induce hMSC osteogenic differentiation when combined with 1,25OHD by up-regulating 1,25OHD receptor VDR expression.

Hepatocyte growth factor (HGF) and its specific receptor MET play a role in many physiological functions including proliferation, migration and morphogenesis. Recently, research results in our laboratory showed that recombinant HGF variants (NK1, NK2, NK3 and NK4) became antagonists to HGF/MET pathway by suppressing proliferation, migration and invasion in human breast cancer cells (MDA-MB-435S, MDA). Similar results were achieved when HGF variants genes were introduced in MDA cells. To understand the molecular mechanism of breast cancer cells metastasis suppressed by HGF variants, MDA and five transfectants, including MDA-GFP, MDA-NK1, MDA-NK2, MDA-NK3 and MDA-NK4 cells were used for proteomic analysis using two-dimensional electrophoresis (2-DE). Differential analysis revealed that a total of 56 polypeptides were differentially expressed through five sets of comparison using wild-type MDA cells as a control. A total of 17 polypeptides were shown differential expression between MDA and MDA-GFP cells, with 11 down-regulated and 6 up-regulated. Eighteen polypeptides were differentially expressed between MDA and MDA-NK1 cells, with 15 down-regulated and 3 up-regulated. There were 22 differentially expressed polypeptides found between MDA and MDA-NK2 cells, in which 14 were down-regulated and 8 were up-regulated. Sixteen polypeptides were shown differentially expressed between MDA and MDA-NK3 cells, with 11 down-regulated and 5 up-regulated. A total of 18 polypeptides were shown differential expression between MDA and MDA-NK4 cells, with 15 down-regulated and 3 up-regulated. Proteomic analysis showed that a total of 43 polypeptides were differentially expressed through four sets of comparison (MDA-GFP and MDA-NK1, MDA-GFP and MDA-NK2, MDA-GFP and MDA-NK3, and MDA-GFP and MDA-NK4). To understand the differential expression among different HGF variants-transfected MDA cells, three sets of cross analysis were also carried out (MDA-NK1 and MDA-NK2, MDA-NK1 and MDA-NK3, and MDA-NK1 and MDA-NK4) and the results showed that a total of 37 differentially expressed polypeptides were found in the three sets of comparison. Similarly, when MDA-NK2 cells were used as a control to compare with MDA-NK3 and MDA-NK4 cells, 34 significantly differential expressed polypeptides were found. The last set of comparison between MDA-NK3 and MDA-NK4 cells, 19 polypeptides were found significantly differential expression. Therefore, our current results revealed that the differentially expressed polypeptides in MDA-MB-435S cells and HGF variants-transfected MDA cells could be related to the inhibition of proliferation and migration of human breast cancer cells by HGF variants.

Androgens are the main regulators of human hair growth stimulating larger, terminal hair development e.g. beard and causing scalp balding, androgenetic alopecia. Hair disorders cause psychological distress but are poorly controlled. Androgens probably act by altering regulatory paracrine factors produced by the mesenchyme-derived dermal papilla. This study aimed to investigate paracrine factors involved in androgen-regulated alopecia, particularly hepatocyte growth factor (HGF) family members, by investigating their in vivo status. Balding and non-balding scalp hair follicles and their component tissues were isolated and analysed by molecular biological methods (reverse transcriptase-polymerase chain reaction (RT-PCR), quantitative PCR and DNA microarray analysis), cell culture and immunohistochemistry. Scalp follicles expressed a range of paracrine messenger genes. The dermal papilla, cultured dermal papilla cells and dermal sheath expressed several HGF family genes, while matrix cells only produced the receptor RON suggesting autocrine roles for HGF and MSP, but a paracrine route only for MSP. Comparing balding and non-balding follicles from the same individuals revealed the expected reduction in several keratin and keratin-related protein genes supporting this approach's validity. There were also significant differences in paracrine factors previously implicated in androgen action by in vitro studies. Several factors believed to increase during androgen stimulation of larger, darker follicles, e.g. IGF-I and SCF, were lowered in balding follicles, while putative inhibitory factors, e.g. TGFß-1, were increased. HGF and MSP and their receptors, c-Met and RON, were significantly reduced. These results increase our understanding of androgen action in human hair follicles; this could lead to better treatments for hair disorders.

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Made available in DSpace on 2015-07-22T20:49:38Z (GMT). No. of bitstreams: 0 Previous issue date: 2006-12-31 / Objetivo: Avaliar o significado prognóstico do marcador tumoral c-met/HGF, através da técnica imuno-histoquímica, em doentes portadores de adenocarcinoma colorretal submetidos a tratamento cirúrgico. Métodos: Estudo retrospectivo de 286 doentes,portadores de adenocarcinoma colorretal, atendidos e operados no Hospital do Câncer de Barretos, de 1993 a 2002. A expressão tissular do marcador tumoral foi avaliada utilizando-se o anticorpo monoclonal antiproteína c-met/HGF, com a técnica da estreptavidina-biotina-peroxidase. A análise da positividade do marcador foi feita de maneira semiquantitativa e a leitura das lâminas, realizada por três patologistas, de forma independente e sem prévio conhecimento dos dados clínicos e histopatológicos dos doentes. Resultados: Do total de 286 doentes analisados, o marcador foi positivo em 236 (78,8%) e negativo em 50 (21,2%). Houve diferença, estatisticamente significante (p=0.004), entre os estádios l e lV, na sobrevida global (p=0.009) e no coeficiente de mortalidade por câncer (p=0.022), porém não se identificou associação do marcador com a ocorrência de recidivas (p=0.89) e o intervalo livre de doença (p=0.91). Conclusão: O marcador tumoral c-met/HGF demonstrou significância estatística, em relação à sua expressão, nos estádios l e lV da doença, na sobrevida global e no coeficiente de mortalidade por câncer, porém não se associou de forma significante com as outras variáveis prognósticas estudadas. / Objective: To evaluate the prognostic meaning of the c-met/HGF tumoral marker, through the immunohistochemical technique, in patients with colorectal adenocarcinoma who have been subjected to surgical treatment. Methods: A retrospective descriptive study of 286 patients with colorectal adenocarcinoma, who have been seen and operated at Barretos Cancer Hospital, from 1993 to 2002. The tissular expression of the tumoral marker was evaluated using the cmet/ HGF anti-protein monoclonal antibody through the estreptavidin-biotinperoxidase technique. The positivity analysis of the marker was semiquantitative, and plate reading was independently carried out by three pathologists with no previous knowledge on clinical and histopathological data of patients. Results: Out of a total of 286 patients, the marker was positive in 236 (78.8%) and negative in 50 (21.2%). It was found statistically significant difference (p=0.004) between stages l and lV, at global life span (p=0.009), and at cancer mortality rate (p=0.022); however, there was no association between the marker and recurrence (p=0.89) or the marker and disease-free period (p=0.91). Conclusion: c-met/HGF has shown significance as a tumoral marker in stages l and lV of the disease, at global life span, and at cancer mortality rate; however, there was no significant association with the remaining prognostic variables that have been studied. / TEDE / BV UNIFESP: Teses e dissertações

This paper is concerned with the use of management control systems (MCSs) within high growth firms (HGFs) to support rapid growth. Considering that a lack of MCSs has been identified as a major cause of failure among start-ups, as well as that MCSs have been claimed to enable firms to grow more during their early years, this is a highly interesting topic for scholars as well as entrepreneurs. However, despite the exceptional opportunity that HGFs provide in examining this topic, there is a lack of contemporary research on how HGFs use MCSs to support their growth. To fill this gap a multiple case study was conducted, investigating how Simons’ (1995) levers of control were used to support growth within 12 different HGFs. Through drawing upon data from interviews with 19 different key people it was then found that MCSs support growth in various ways. Different boundary systems were found to increase chances of firm survival and provide a platform for growth. Additionally, the use of interactive systems was identified as a potential characteristic of HGFs. Moreover, the research questions former assumptions on the importance of forecasting.