Antifibrotic activity of an inhibitor of group IIA secretory phospholipase A(2) in young spontaneously hypertensive rats

The development of fibrosis in the chronically hypertensive heart is associated with infiltration of inflammatory cells and cardiac hypertrophy. In this study, an inhibitor of the proinflammatory enzyme, group IIA human secretory phospholipase A(2) (sPLA(2)-IIA), has been found to prevent collagen deposition as an important component of cardiovascular remodeling in a rat model of developing chronic hypertension. Daily treatment of young male spontaneously hypertensive rats (SHR) with an sPLA2-IIA inhibitor (KH064, 5-(4-benzyloxyphenyl)-4S-(phenyl-heptanoylamino)-pentanoic acid, 5 mg/kg/day p.o.) prevented increases in the content of perivascular,(SHR 20.6 +/- 0.9%, n = 5; SHR+KH064 14.0 +/- 1.2%, n = 5) and interstitial (SHR 7.9 +/- 0.3%, n = 6; SHR+KH064 5.4 +/- 0.7%, n = 6) collagen in the left ventricle of rat hearts, but did not affect numbers of infiltrating monocytes/macrophages, left ventricular hypertrophy (SHR 2.88 +/- 0.08, n = 12; SHR+KH064 3.09 +/- 0.08 mg/g body weight, n = 9), increased systolic blood pressure, or thoracic aortic responses. This selective antifibrotic activity suggests that sPLA2-IIA may have an important but specific role in cardiac fibrosis, and that its inhibitors could be useful in dissecting molecular pathways leading to fibrotic conditions.

Combined Gene Therapy and Functional Tissue Engineering for the Treatment of Osteoarthritis

The pathogenesis of osteoarthritis is mediated in part by inflammatory cytokines including interleukin-1 (IL-1), which promote degradation of articular cartilage and prevent human mesenchymal stem cell (hMSC) chondrogenesis. We combined gene therapy and functional tissue engineering to develop engineered cartilage with immunomodulatory properties that allow chondrogenesis in the presence of pathologic levels of IL-1 by inducing overexpression of IL-1 receptor antagonist (IL-1Ra) in hMSCs via scaffold-mediated lentiviral gene delivery. A doxycycline-inducible vector was used to transduce hMSCs in monolayer or within 3D woven PCL scaffolds to enable tunable IL-1Ra production. In the presence of IL-1, IL-1Ra-expressing engineered cartilage produced cartilage-specific extracellular matrix, while resisting IL-1-induced upregulation of matrix metalloproteinases and maintaining mechanical properties similar to native articular cartilage. The ability of functional engineered cartilage to deliver tunable anti-inflammatory cytokines to the joint may enhance the long-term success of therapies for cartilage injuries or osteoarthritis.

Following this, we modified this anti-inflammatory engineered cartilage to incorporate rabbit MSCs and evaluated this therapeutic strategy in a pilot study in vivo in rabbit osteochondral defects. Rabbits were fed a custom doxycycline diet to induce gene expression in engineered cartilage implanted in the joint. Serum and synovial fluid were collected and the levels of doxycycline and inflammatory mediators were measured. Rabbits were euthanized 3 weeks following surgery and tissues were harvested for analysis. We found that doxycycline levels in serum and synovial fluid were too low to induce strong overexpression of hIL-1Ra in the joint and hIL-1Ra was undetectable in synovial fluid via ELISA. Although hIL-1Ra expression in the first few days local to the site of injury may have had a beneficial effect, overall a higher doxycycline dose and more readily transduced cell population would improve application of this therapy.

In addition to the 3D woven PCL scaffold, cartilage-derived matrix scaffolds have recently emerged as a promising option for cartilage tissue engineering. Spatially-defined, biomaterial-mediated lentiviral gene delivery of tunable and inducible morphogenetic transgenes may enable guided differentiation of hMSCs into both cartilage and bone within CDM scaffolds, enhancing the ability of the CDM scaffold to provide chondrogenic cues to hMSCs. In addition to controlled production of anti-inflammatory proteins within the joint, in situ production of chondro- and osteo-inductive factors within tissue-engineered cartilage, bone, or osteochondral tissue may be highly advantageous as it could eliminate the need for extensive in vitro differentiation involving supplementation of culture media with exogenous growth factors. To this end, we have utilized controlled overexpression of transforming growth factor-beta 3 (TGF-β3), bone morphogenetic protein-2 (BMP-2) or a combination of both factors, to induce chondrogenesis, osteogenesis, or both, within CDM hemispheres. We found that TGF-β3 overexpression led to robust chondrogenesis in vitro and BMP-2 overexpression led to mineralization but not accumulation of type I collagen. We also showed the development of a single osteochondral construct by combining tissues overexpressing BMP-2 (hemisphere insert) and TGF-β3 (hollow hemisphere shell) and culturing them together in the same media. Chondrogenic ECM was localized in the TGF-β3-expressing portion and osteogenic ECM was localized in the BMP-2-expressing region. Tissue also formed in the interface between the two pieces, integrating them into a single construct.

Since CDM scaffolds can be enzymatically degraded just like native cartilage, we hypothesized that IL-1 may have an even larger influence on CDM than PCL tissue-engineered constructs. Additionally, anti-inflammatory engineered cartilage implanted in vivo will likely affect cartilage and the underlying bone. There is some evidence that osteogenesis may be enhanced by IL-1 treatment rather than inhibited. To investigate the effects of an inflammatory environment on osteogenesis and chondrogenesis within CDM hemispheres, we evaluated the ability of IL-1Ra-expressing or control constructs to undergo chondrogenesis and osteogenesis in the prescence of IL-1. We found that IL-1 prevented chondrogenesis in CDM hemispheres but did not did not produce discernable effects on osteogenesis in CDM hemispheres. IL-1Ra-expressing CDM hemispheres produced robust cartilage-like ECM and did not upregulate inflammatory mediators during chondrogenic culture in the presence of IL-1.

An Ex Vivo Model for Studying Hepatic Schistosomiasis and the Effect of Released Protein from Dying Eggs

BACKGROUND: We report the use of an ex vivo precision cut liver slice (PCLS) mouse model for studying hepatic schistosomiasis. In this system, liver tissue is unfixed, unfrozen, and alive for maintenance in culture and subsequent molecular analysis.

METHODS AND FINDINGS: Using thick naive mouse liver tissue and sterile culture conditions, the addition of soluble egg antigen (SEA) derived from Schistosoma japonicum eggs, followed 4, 24 and 48 hrs time points. Tissue was collected for transcriptional analysis and supernatants collected to quantitate liver enzymes, cytokines and chemokines. No significant hepatotoxicity was demonstrated by supernatant liver enzymes due to the presence of SEA. A proinflammatory response was observed both at the transcriptional level and at the protein level by cytokine and chemokine bead assay. Key genes observed elevated transcription in response to the addition of SEA included: IL1-α and IL1-β, IL6, all associated with inflammation. The recruitment of antigen presenting cells was reflected in increases in transcription of CD40, CCL4 and CSF1. Indications of tissue remodeling were seen in elevated gene expression of various Matrix MetalloProteinases (MMP3, 9, 10, 13) and delayed increases in TIMP1. Collagen deposition was significantly reduced in the presence of SEA as shown in COL1A1 expression by qPCR after 24 hrs culture. Cytokine and chemokine analysis of the culture supernatants confirmed the elevation of proteins including IL6, CCL3, CCL4 and CXCL5.

CONCLUSIONS: This ex vivo model system for the synchronised delivery of parasite antigen to liver tissue provides an insight into the early phase of hepatic schistosomiasis, corresponding with the release of soluble proteins from dying schistosome eggs.

Effect of dental tissue conditioners and matrix metalloproteinase inhibitors on type I collagen microstructure analyzed by Fourier transform infrared spectroscopy

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Negative regulation of the hepatic fibrogenic response by suppressor of cytokine signaling 1 (SOCS1)

Abstract: Suppressor of cytokine signaling 1 (SOCS1) is an indispensable regulator of IFN-γ signaling and has been implicated in the regulation of liver fibrosis. However, it is not known whether SOCS1 mediates its anti-fibrotic functions in the liver directly, or via modulating IFN-γ, which has been implicated in attenuating hepatic fibrosis. Additionally, it is possible that SOCS1 controls liver fibrosis by regulating hepatic stellate cells (HSC), a key player in fibrogenic response. While the activation pathways of HSCs have been well characterized, the regulatory mechanisms are not yet clear. The goals of this study were to dissociate IFN-γ-dependent and SOCS1-mediated regulation of hepatic fibrogenic response, and to elucidate the regulatory functions of SOCS1 in H SC activation. Liver fibrosis was induced in Socs1[superscript -/-]Ifng[superscript -/-] mice with dimethylnitrosamine or carbon tetrachloride. Ifng[superscript -/-] and C57BL/6 mice served as controls. Following fibrogenic treatments, Socs1[superscript -/-]Ifng[superscript -/-] mice showed elevated serum ALT levels and increased liver fibrosis com-pared to mice Ifng[superscript -/-]. The latter group showed higher alanine aminotransferase (ALT) levels and fibrosis than C57BL/6 controls. The livers of Socs1-deficient mice showed bridging fibrosis, which was associated with increased accumulation of myofibroblasts and abundant collagen deposition. Socs1-deficient livers showed increased expression of genes coding for smooth muscle actin, collagen, and enzymes involved in remodeling the extracellular matrix, namely matrix metalloproteinases and tissue inhibitor of metalloproteinases. Primary HSCs from Socs1-deficient mice showed increased proliferation in response to growth factors such as HGF, EGF and PDGF, and the fibrotic livers of Socs1-deficient mice showed increased expression of the Pdgfb gene. Taken together, these data indicate that SOCS1 controls liver fibrosis independently of IFN-γ and that part of this regulation may occur via regulating HSC proliferation and limiting growth factor availability.

The role of cancer related inflammation, Src family kinases and matrix metalloproteinase 9 in colorectal cancer

Colorectal cancer (CRC) is the third most common cancer in the UK with 41,000 new cases diagnosed in 2011. Despite undergoing potentially curative resection, a significant amount of patients develop recurrence. Biomarkers that aid prognostication or identify patients who are suitable for adjuvant treatments are needed. The TNM staging system does a reasonably good job at offering prognostic information to the treating clinician, but it could be better and identifying methods of improving its accuracy are needed. Tumour progression is based on a complex relationship between tumour behaviour and the hosts’ inflammatory responses. Sustained tumour cell proliferation, evading growth suppressors, resisting apoptosis, replicative immortality, sustained angiogenesis, invasion & metastasis, avoiding immune destruction, deregulated cellular energetics, tumour promoting inflammation and genomic instability & mutation have been identified as hallmarks. These hallmarks are malignant behaviors are what makes the cell cancerous and the more extreme the behaviour the more aggressive the cancer the more likely the risk of a poor outcome. There are two primary genomic instability pathways: Microsatellite Instability (MSI) and Chromosomal Instability (CI) also referred to as Microsatellite Stability (MSS). Tumours arising by these pathways have a predilection for specific anatomical, histological and molecular biological features. It is possible that aberrant molecular expression of genes/proteins that promote malignant behaviors may also act as prognostic and predictive biomarkers, which may offer superior prognostic information to classical prognostic features. Cancer related inflammation has been described as a 7th hallmark of cancer. Despite the systemic inflammatory response (SIR) being associated with more aggressive malignant disease, infiltration by immune cells, particularly CD8+ lymphocytes, at the advancing edge of the tumour have been associated with improved outcome and tumour MSI. It remains unknown if the SIR is associated with tumour MSI and this requires further study. The mechanisms by which colorectal cancer cells locally invade through the bowel remain uncertain, but connective tissue degradation by matrix metalloproteinases (MMPs) such as MMP-9 have been implicated. MMP-9 has been found in the cancer cells, stromal cells and patient circulation. Although tumoural MMP-9 has been associated with poor survival, reports are conflicting and contain relatively small sample sizes. Furthermore, the influence of high serum MMP-9 on survival remains unknown. Src family kinases (SFKs) have been implicated in many adverse cancer cell behaviors. SFKs comprise 9 family members BLK, C-SRC, FGR, FYN, HCK, LCK, LYN, YES, YRK. C-SRC has been the most investigated of all SFKs, but the role of other SFKs in cellular behaviors and their prognostic value remains largely unknown. The development of Src inhibitors, such as Dasatinib, has identified SFKs as a potential therapeutic target for patients at higher risk of poor survival. Unfortunately, clinical trials so far have not been promising but this may reflect inadequate patient selection and SFKs may act as useful prognostic and predictive biomarkers. In chapter 3, the association between cancer related inflammation, tumour MSI, clinicopathological factors and survival was tested in two independent cohorts. A training cohort consisting of n=182 patients and a validation cohort of n=677 patients. MSI tumours were associated with a raised CRP (p=0.003). Hypoalbuminaemia was independently associated with poor overall survival in TNM stage II cancer (HR 3.04 (95% CI 1.44 – 6.43);p=0.004), poor recurrence free survival in TNM stage III cancer (HR 1.86 (95% 1.03 – 3.36);p=0.040) and poor overall survival in CI colorectal cancer (HR 1.49 (95% CI 1.06 – 2.10);p=0.022). Interestingly, MSI tumours were associated with poor overall survival in TNM stage III cancer (HR 2.20 (95% CI 1.10 – 4.37);p=0.025). In chapter 4, the role of MMP-9 in colorectal cancer progression and survival was examined. MMP-9 in the tissue was assessed using IHC and serum expression quantified using ELISA. Serum MMP-9 was associated with cancer cell expression (Spearman’s Correlation Coefficient (SCC) 0.393, p<0.001)) and stromal expression (SCC 0.319, p=0.002). Serum MMP-9 was associated with poor recurrence-free (HR 3.37 (95% CI 1.20 – 9.48);p=0.021) and overall survival (HR 3.16 (95% CI 1.22 – 8.15);p=0.018), but tumour MMP-9 was not survival or MSI status. In chapter 5, the role of SFK expression and activation in colorectal cancer progression and survival was studied. On PCR analysis, although LYN, C-SRC and YES were the most highly expressed, FGR and HCK had higher expression profiles as tumours progressed. Using IHC, raised cytoplasmic FAK (tyr 861) was independently associated with poor recurrence free survival in all cancers (HR 1.48 (95% CI 1.02 – 2.16);p=0.040) and CI cancers (HR 1.50 (95% CI 1.02 – 2.21);p=0.040). However, raised cytoplasmic HCK (HR 2.04 (95% CI 1.11 – 3.76);p=0.022) was independently associated with poor recurrence-free survival in TNM stage II cancers. T84 and HT29 cell lines were used to examine the cellular effects of Dasatinib. Cell viability was assessed using WST-1 assay and apoptosis assessed using an ELISA cell death detection assay. Dasatinib increased T84 tumour cell apoptosis in a dose dependent manner and resulted in reduced expression of nuclear (p=0.008) and cytoplasmic (p=0.016) FAK (tyr 861) expression and increased nuclear FGR expression (p=0.004). The results of this thesis confirm that colorectal cancer is a complex disease that represents several subtypes of cancer based on molecular biological behaviors. This thesis concentrated on features of the disease related to inflammation in terms of genetic and molecular characterisation. MSI cancers are closely associated with systemic inflammation but despite this observation, they retain their relatively improved survival. MMP-9 is a feature of tissue remodeling during inflammation and is also associated with degradation of connective tissue, advanced T-stage and poor outcome when measured in the serum. The lack of stromal quantification due to TMA use rather than full sections makes the value of tumoural MMP-9 immunoreactivity in the prognostication and its association with MSI unknown and requires further study. Finally, SFK activation was also associated with SIR, however, only cytoplasmic HCK was independently associated with poor survival in patients with TNM stage II disease, the group of patients where identifying a novel biomarker is most needed. There is still some way to go before these biomarkers are translated into clinical practice and future work needs to focus on obtaining a reliable and robust scientific technique with validation in an adequately powered independent cohort.

Tissue engineering of articular cartilage with biomimetic zones

Articular cartilage damage is a persistent and increasing problem with the aging population, and treatments to achieve biological repair or restoration remain a challenge. Cartilage tissue engineering approaches have been investigated for over 20 years, but have yet to achieve the consistency and effectiveness for widespread clinical use. One of the potential reasons for this is that the engineered tissues do not have or establish the normal zonal organization of cells and extracellular matrix that appears critical for normal tissue function. A number of approaches are being taken currently to engineer tissue that more closely mimics the organization of native articular cartilage. This review focuses on the zonal organization of native articular cartilage, strategies being used to develop such organization, the reorganization that occurs after culture or implantation, and future prospects for the tissue engineering of articular cartilage with biomimetic zones.

Tissue engineering of stratified articular cartilage from chondrocyte subpopulations

Objective: To test if subpopulations of chondrocytes from different cartilage zones could be used to engineer cartilage constructs with features of normal stratification. Design: Chondrocytes from the superficial and middle zones of immature bovine cartilage were cultured in alginate, released, and seeded either separately or sequentially to form cartilage constructs. Constructs were cultured for 1 or 2 weeks and were assessed for growth, compressive properties, and deposition, and localization of matrix molecules and superficial zone protein (SZP). Results: The cartilaginous constructs formed from superficial zone chondrocytes exhibited less matrix growth and lower compressive properties than constructs from middle zone chondrocytes, with the stratified superficial-middle constructs exhibiting intermediate properties. Expression of SZP was highest at the construct surfaces, with the localization of SZP in superficial-middle constructs being concentrated at the superficial surface. Conclusions: Manipulation of subpopulations of chondrocytes can be useful in engineering cartilage tissue with a biomimetic approach, and in fabricating constructs that exhibit stratified features of normal articular cartilage. (C) 2003 OsteoArthritis Research Society International. Published by Elsevier Ltd. All rights reserved.

Vertical Inhibition of the PI3K/Akt/mTOR Pathway for the Treatment of Osteoarthritis

Osteoarthritis is characterized by degenerative alterations of articular cartilage including both the degradation of extracellular matrix and the death of chondrocytes. The PI3K/Akt pathway has been demonstrated to involve in both processes. Inhibition of its downstream target NF-kB reduces the degradation of extracellular matrix via decreased production of matrix metalloproteinases while inhibition of mTOR increased autophagy to reduce chondrocyte death. However, mTOR feedback inhibits the activity of the PI3K/Akt pathway and inhibition of mTOR could result in increased activity of the PI3K/Akt/NF-kB pathway. We proposed that the use of dual inhibitors of PI3K and mTOR could be a promising approach to more efficiently inhibit the PI3K/Akt pathway than rapamycin or PI3K inhibitor alone and produce better treatment outcome.

The association of single nucleotide polymorphisms with endometrial cancer risk and prognosis

Endometrial cancer is one of the most common female diseases in developed nations and is the most commonly diagnosed gynaecological cancer in Australia. The disease is commonly classified by histology: endometrioid or non-endometrioid endometrial cancer. While non-endometrioid endometrial cancers are accepted to be high-grade, aggressive cancers, endometrioid cancers (comprising 80% of all endometrial cancers diagnosed) generally carry a favourable patient prognosis. However, endometrioid endometrial cancer patients endure significant morbidity due to surgery and radiotherapy used for disease treatment, and patients with recurrent disease have a 5-year survival rate of less than 50%. Genetic analysis of women with endometrial cancer could uncover novel markers associated with disease risk and/or prognosis, which could then be used to identify women at high risk and for the use of specialised treatments. Proteases are widely accepted to play an important role in the development and progression of cancer. This PhD project hypothesised that SNPs from two protease gene families, the matrix metalloproteases (MMPs, including their tissue inhibitors, TIMPs) and the tissue kallikrein-related peptidases (KLKs) would be associated with endometrial cancer susceptibility and/or prognosis. In the first part of this study, optimisation of the genotyping techniques was performed. Results from previously published endometrial cancer genetic association studies were attempted to be validated in a large, multicentre replication set (maximum cases n = 2,888, controls n = 4,483, 3 studies). The rs11224561 progesterone receptor SNP (PGR, A/G) was observed to be associated with increased endometrial cancer risk (per A allele OR 1.31, 95% CI 1.12-1.53; p-trend = 0.001), a result which was initially reported among a Chinese sample set. Previously reported associations for the remaining 8 SNPs investigated for this section of the PhD study were not confirmed, thereby reinforcing the importance of validation of genetic association studies. To examine the effect of SNPs from the MMP and KLK families on endometrial cancer risk, we selected the most significantly associated MMP and KLK SNPs from genome-wide association study analysis (GWAS) to be genotyped in the GWAS replication set (cases n = 4,725, controls n = 9,803, 13 studies). The significance of the MMP24 rs932562 SNP was unchanged after incorporation of the stage 2 samples (Stage 1 per allele OR 1.18, p = 0.002; Combined Stage 1 and 2 OR 1.09, p = 0.002). The rs10426 SNP, located 3' to KLK10 was predicted by bioinformatic analysis to effect miRNA binding. This SNP was observed in the GWAS stage 1 result to exhibit a recessive effect on endometrial cancer risk, a result which was not validated in the stage 2 sample set (Stage 1 OR 1.44, p = 0.007; Combined Stage 1 and 2 OR 1.14, p = 0.08). Investigation of the regions imputed surrounding the MMP, TIMP and KLK genes did not reveal any significant targets for further analysis. Analysis of the case data from the endometrial cancer GWAS to identify genetic variation associated with cancer grade did not reveal SNPs from the MMP, TIMP or KLK genes to be statistically significant. However, the representation of SNPs from the MMP, TIMP and KLK families by the GWAS genotyping platform used in this PhD project was examined and observed to be very low, with the genetic variation of four genes (MMP23A, MMP23B, MMP28 and TIMP1) not captured at all by this technique. This suggests that comprehensive candidate gene association studies will be required to assess the role of SNPs from these genes with endometrial cancer risk and prognosis. Meta-analysis of gene expression microarray datasets curated as part of this PhD study identified a number of MMP, TIMP and KLK genes to display differential expression by endometrial cancer status (MMP2, MMP10, MMP11, MMP13, MMP19, MMP25 and KLK1) and histology (MMP2, MMP11, MMP12, MMP26, MMP28, TIMP2, TIMP3, KLK6, KLK7, KLK11 and KLK12). In light of these findings these genes should be prioritised for future targeted genetic association studies. Two SNPs located 43.5 Mb apart on chromosome 15 were observed from the GWAS analysis to be associated with increased endometrial cancer grade, results that were validated in silico in two independent datasets. One of these SNPs, rs8035725 is located in the 5' untranslated region of a MYC promoter binding protein DENND4A (Stage 1 OR 1.15, p = 9.85 x 10P -5 P, combined Stage 1 and in silico validation OR 1.13, p = 5.24 x 10P -6 P). This SNP has previously been reported to alter the expression of PTPLAD1, a gene involved in the synthesis of very long fatty acid chains and in the Rac1 signaling pathway. Meta-analysis of gene expression microarray data found PTPLAD1 to display increased expression in the aggressive non-endometrioid histology compared with endometrioid endometrial cancer, suggesting that the causal SNP underlying the observed genetic association may influence expression of this gene. Neither rs8035725 nor significant SNPs identified by imputation were predicted bioinformatically to affect transcription factor binding sites, indicating that further studies are required to assess their potential effect on other regulatory elements. The other grade- associated SNP, rs6606792, is located upstream of an inferred pseudogene, ELMO2P1 (Stage 1 OR 1.12, p = 5 x 10P -5 P; combined Stage 1 and in silico validation OR 1.09, p = 3.56 x 10P -5 P). Imputation of the ±1 Mb region surrounding this SNP revealed a cluster of significantly associated variants which are predicted to abolish various transcription factor binding sites, and would be expected to decrease gene expression. ELMO2P1 was not included on the microarray platforms collected for this PhD, and so its expression could not be investigated. However, the high sequence homology of ELMO2P1 with ELMO2, a gene important to cell motility, indicates that ELMO2 could be the parent gene for ELMO2P1 and as such, ELMO2P1 could function to regulate the expression of ELMO2. Increased expression of ELMO2 was seen to be associated with increasing endometrial cancer grade, as well as with aggressive endometrial cancer histological subtypes by microarray meta-analysis. Thus, it is hypothesised that SNPs in linkage disequilibrium with rs6606792 decrease the transcription of ELMO2P1, reducing the regulatory effect of ELMO2P1 on ELMO2 expression. Consequently, ELMO2 expression is increased, cell motility is enhanced leading to an aggressive endometrial cancer phenotype. In summary, these findings have identified several areas of research for further study. The results presented in this thesis provide evidence that a SNP in PGR is associated with risk of developing endometrial cancer. This PhD study also reports two independent loci on chromosome 15 to be associated with increased endometrial cancer grade, and furthermore, genes associated with these SNPs to be differentially expressed according in aggressive subtypes and/or by grade. The studies reported in this thesis support the need for comprehensive SNP association studies on prioritised MMP, TIMP and KLK genes in large sample sets. Until these studies are performed, the role of MMP, TIMP and KLK genetic variation remains unclear. Overall, this PhD study has contributed to the understanding of genetic variation involvement in endometrial cancer susceptibility and prognosis. Importantly, the genetic regions highlighted in this study could lead to the identification of novel gene targets to better understand the biology of endometrial cancer and also aid in the development of therapeutics directed at treating this disease.

SPARC/osteonectin induces matrix metalloproteinase 2 activation in human breast cancer cell lines

Activation of the matrix metalloproteinase 2 (MMP-2) has been shown to play a major role in the proteolysis of extracellular matrix (ECM) associated with tumor invasion. Although the precise mechanism of this activation remains elusive, levels of the membrane type 1-MMP (MT1-MMP) at the cell surface and of the tissue inhibitor of MMP-2 (TIMP-2) appear to be two important determinants. Induction of MMP-2 activation in cells cultivated on collagen type I gels indicated that the ECM is important in the regulation of this process. In this study, we show that SPARC/osteonectin, a small ECM- associated matricellular glycoprotein, can induce MMP-2 activation in two invasive breast cancer cell lines (MDA-MB-231 and BT549) but not in a noninvasive counterpart (MCF7), which lacks MT1-MMP. Using a set of peptides from different regions of SPARC, we found that peptide 1.1 (corresponding to the NH2-terminal region of the protein) contained the activity that induced NIMP-2 activation. Despite the requirement for MT1-MMP, seen in MCF-7 cells transfected with MT1-MMP, the activation of MMP-2 by SPARC peptide 1.1 was not associated with increased steady-state levels of MT1-MMP mRNA or protein in either MT1-MMP-transfected MCF-7 cells or constitutively expressing MDA- MB-231 and BT549 cells. We did, however, detect decreased levels of TIMP-2 protein in the media of cells incubated with peptide 1.1 or recombinant SPARC; thus, the induction of MMP-2 activation by SPARC might be due in part to a diminution of TIMP-2 protein. We conclude that SPARC, and specifically its NH2-terminal domain, regulates the activation of MMP-2 at the cell surface and is therefore likely to contribute to the proteolytic pathways associated with tumor invasion.

Upregulated MT1-MMP/TIMP-2 axis in the TSU-Pr1-B1/B2 model of metastatic progression in transitional cell carcinoma of the bladder

Muscle invasive transitional cell carcinoma (TCC) of the bladder is associated with a high frequency of metastasis, resulting in poor prognosis for patients presenting with this disease. Models that capture and demonstrate step-wise enhancement of elements of the human metastatic cascade on a similar genetic background are useful research tools. We have utilized the transitional cell carcinoma cell line TSU-Pr1 to develop an in vivo experimental model of bladder TCC metastasis. TSU-Pr1 cells were inoculated into the left cardiac ventricle of SCID mice and the development of bone metastases was monitored using high resolution X-ray. Tumor tissue from a single bone lesion was excised and cultured in vitro to generate the TSU-Pr1-B1 subline. This cycle was repeated with the TSU-Pr1-B1 cells to generate the successive subline TSU-Pr1-B2. DNA profiling and karyotype analysis confirmed the genetic relationship of these three cell lines. In vitro, the growth rate of these cell lines was not significantly different. However, following intracardiac inoculation TSU-Pr1, TSU-Pr1-B1 and TSU-Pr1-B2 exhibited increasing metastatic potential with a concomitant decrease in time to the onset of radiologically detectable metastatic bone lesions. Significant elevations in the levels of mRNA expression of the matrix metalloproteases (MMPs) membrane type 1-MMP (MT1-MMP), MT2-MMP and MMP-9, and their inhibitor, tissue inhibitor of metalloprotease-2 (TIMP-2), across the progressively metastatic cell lines, were detected by quantitative PCR. Given the role of MT1-MMP and TIMP-2 in MMP-2 activation, and the upregulation of MMP-9, these data suggest an important role for matrix remodeling, particularly basement membrane, in this progression. The TSU-Pr1-B1/B2 model holds promise for further identification of important molecules.

Transmembrane/cytoplasmic domain-mediated membrane type 1-matrix metalloprotease docking to invadopodia is required for cellinvasion

The invasion of human malignant melanoma cells into the extracellular matrix (ECM) involves the accumulation of proteases at sites of ECM degradation where activation of matrix metalloproteases (MMP) occurs. Here, we show that when membrane type 1 MMP (MT-MMP) was overexpressed in RPMI7951 human melanoma cells, the cells made contact with the ECM, activated soluble and ECM-bound MMP-2, and degraded and invaded the ECM. Further experiments demonstrated the importance of localization of the MT-MMP to invadopodia. Overexpression of MT-MMP without invadopodial localization caused activation of soluble MMP-2, but did not facilitate ECM degradation or cell invasiveness. Up-regulation of endogenous MT-MMP with concanavalin A caused activation of MMP-2. However, concanavalin A treatment prevented invadopodial localization of MT-MMP and ECM degradation. Neither a truncated MT-MMP mutant lacking transmembrane (TM) and cytoplasmic domains (ΔTM(MT-MMP)), nor a chimeric MT-MMP containing the interleukin 2 receptor α chain (IL-2R) TM and cytoplasmic domains (ΔTM(MT-MMP)/TM(IL-2R)) were localized to invadopodia or exhibited ECM degradation. Furthermore, a chimera of the TM/cytoplasmic domain of MT-MMP (TM(MT-MMP)) with tissue inhibitor of MMP 1 (TIMP-1/TM(MT- MMP)) directed the TIMP-1 molecule to invadopodia. Thus, the MT-MMP TM/cytoplasmic domain mediates the spatial organization of MT-MMP into invadopodia and subsequent degradation of the ECM.