Proteomic identification of in vivo substrates for matrix metalloproteinases 2 and 9 reveals a mechanism for resolution of inflammation.

Clearance of allergic inflammatory cells from the lung through matrix metalloproteinases (MMPs) is necessary to prevent lethal asphyxiation, but mechanistic insight into this essential homeostatic process is lacking. In this study, we have used a proteomics approach to determine how MMPs promote egression of lung inflammatory cells through the airway. MMP2- and MMP9-dependent cleavage of individual Th2 chemokines modulated their chemotactic activity; however, the net effect of complementing bronchoalveolar lavage fluid of allergen-challenged MMP2(-/-)/MMP9(-/-) mice with active MMP2 and MMP9 was to markedly enhance its overall chemotactic activity. In the bronchoalveolar fluid of MMP2(-/-)/MMP9(-/-) allergic mice, we identified several chemotactic molecules that possessed putative MMP2 and MMP9 cleavage sites and were present as higher molecular mass species. In vitro cleavage assays and mass spectroscopy confirmed that three of the identified proteins, Ym1, S100A8, and S100A9, were substrates of MMP2, MMP9, or both. Function-blocking Abs to S100 proteins significantly altered allergic inflammatory cell migration into the alveolar space. Thus, an important effect of MMPs is to differentially modify chemotactic bioactivity through proteolytic processing of proteins present in the airway. These findings provide a molecular mechanism to explain the enhanced clearance of lung inflammatory cells through the airway and reveal a novel approach to target new therapies for asthma.

Activation of matrix metalloproteinase -9 (MMP-9) by heregulin in human breast cancer cells involves multiple signaling pathways

Matrix metalloproteinase-9 (MMP-9) plays an important role in tumor invasion and angiogenesis. Secretion of MMP-9 has been reported in various cancer types including lung cancer, brain cancer, colon cancer, and breast cancer. Heregulin is a growth factor that regulates growth and differentiation of normal breast cells as well as mammary tumor cells. To study the role of heregulin in breast cancer metastasis, we tested whether heregulin may regulate MMP-9 secretion. By screening a panel of breast cancer cell line for their ability to respond to heregulin and produce MMP-9, we have found that MMP-9 secretion can be induced by heregulin-β1 in two breast cancer cell lines, SKBr3 and MCF-7. In both cell lines, increase of MMP-9 activity as shown by zymography was accompanied by increased protein level as well as mRNA level of MMP-9. Using a reporter luciferase assay, we have identified that proximal −670bp promoter of MMP-9 had similar activity to a 2.2kb MMP-9 promoter in response to heregulin stimulation. Heregulin treatment of SKBr3 and MCF-7 activated multiple signaling pathways inside cells. These include the Erk pathway, the p38 kinase pathway, PKC pathway, and PI-3K pathway. To examine which pathways are involved in MMP-9 activation by heregulin, we have used a panel of chemical inhibitors to specifically inhibit each one of these pathways. Ro-31-8220 (PKC inhibitor) and SB203580 (p38 kinase inhibitor) completely blocked heregulin activation of MMP-9. On the other hand, PD098059 (MEK-1 inhibitor) partially blocked MMP-9 activation, whereas PI-3K inhibitor, wortmannin, had no effect. Therefore, at least three signaling pathways are involved in activation of MMP-9 by heregulin. Since MMP-9 is tightly associated with metastatic potential, our study also suggests that heregulin may enhance breast tumor metastasis through induction of MMP-9 expression. ^

Functional studies of insulin-like growth factor binding protein 2 pathway in glioma progression

Comparison of gene expressing profiles between gliomas with different grades revealed frequent overexpression of insulin-like growth factor binding protein 2 (IGFBP2) in glioblastomas (GBM), in which uncontrolled cell proliferation, angiogenesis, invasion and anti-apoptosis are hallmarks. Using the glia-specific gene transfer transgenic mouse and the stable LN229(BP2) GBM cell lines, we found that IGFBP2 by itself cannot transform cells in vitro and in vivo. IGFBP2 had growth inhibitory effects on mouse primary neural progenitors, but overexpression of IGFBP2 had no effect on GBM cells. ^ Although IGFBP2 does not initiate gliomagenesis, using tissue array technology, we observed strong correlation between IGFBP2 overexpression and VEGF up-regulation in human diffuse gliomas. Furthermore, overexpression of IGFBP2 in GBM cells not only enhanced VEGF expression but also increased the malignant potential of U87 MG cells in our angiogenesis xenograft animal model. ^ In parallel to these studies, using established stable SNB19 GBM cells that overexpress IGFBP2, we found that IGFBP2 significantly increased invasion by induction of matrix metalloproteinase-2 (MMP-2) as well as other invasion related genes, providing evidence that IGFBP2 contributes to glioma progression in part by enhancing MMP-2 gene transcription and in turn tumor cell invasion. ^ Finally, we found that primary filial cells infected with an anti-sense IGFBP2 construct have markedly increased sensitivity to γ irradiation and reduced Akt activation. On the other hand, SNB19(BP2) stable lines have consistently increased levels of Akt and NFkB activation, suggesting that one possible mechanism for anti-apoptosic function of IGFBP2 is through the activation of Akt and NFkB. Beside this, what is especially interesting is the finding that Akt protein was cleaved and inactivated during apoptosis by caspases, and IGFBP2 can prevent Akt cleavage, revealing another possible mechanism through it IGFBP2 exhibit strong antiapoptotic effects. Our data showed that IGFBP2 is a specific substrate for caspase-3, raising the possibility that IGFBP2 may inhibit apoptosis by a suicide mechanism. ^ In summary, using cellular, genomics, and molecular approaches, this thesis documented the potential roles of IGFBP2 in glioma progression. Our findings shed light on an important biological aspect of glioma progression and may provide new insights useful for the design of novel mechanism-based therapies for GBM. ^

Matrix metalloproteinase-1 as a prognosticator in malignant glioma

The overall purpose of this study was to assess the relationship between the promoter region polymorphism (-2607 1G/2G) of matrix metalloproteinase-1 (MMP-1) polymorphism and outcome in brain tumor patients diagnosed with a primary brain tumor between 1994 and 2000 at The University of Texas M. D. Anderson Cancer Center. The MMP-1 polymorphism was genotyped for all brain tumor patients who participated in the Family Brain Tumor Study and for whom blood samples were available. Relevant covariates were abstracted from medical records for all cases from the original protocol, including information on demographics, tumor histology, therapy and outcome was obtained. The hypothesis was that brain tumor patients with the 2G allele have a poorer prognosis and shorter survival than brain tumor patients with the 1G allele. ^ Experimental Design: Genetic variants for the MMP-1 enzyme were determined by a polymerase chain reaction-restriction fragment length polymorphism assay. Comparison was made between the overall survival for cases with the 2G polymorphism and overall survival for cases with the 1G polymorphism using multivariable Cox Proportional-Hazard analysis, controlling for age, sex, Karnofsky Performance Scale (KPS), extent of surgery, tumor histology and treatment received. Kaplan-Meier and Cox Proportional-Hazard analyses were utilized to assess if the MMP-1 polymorphisms were related to overall survival. Results: Overall survival was not statistically significantly different between the 2G allele brain tumor patients and the 1G allele patients and there was no statistically significant difference between tumor types. ^ Conclusions: No association was found between MMP-1 polymorphisms and survival in patients with malignant gliomas. ^

p75 receptor-mediated neurotrophism: A new mechanism of melanoma cell survival and invasion

Trophism as a "clonal dominance" support mechanism for tumor cells is an unexplored area of tumor progression. This report presents evidence that the human melanoma low-affinity neurotrophin receptor (p75) can signal independently of its high-affinity tyrosine kinase counterparts, the TRK family of kinases. Signaling may be accomplished by a p75-associated purine-analog-sensitive kinase and results in enhanced invasion into a reconstituted basement membrane with a corresponding stimulation of matrix metalloproteinase-2 expression. Additionally, a "stress culture" survival assay was developed to mimic the growth limiting conditions encountered by melanoma cells in a rapidly growing primary tumor or metastatic deposit prior to neoangiogenesis. Under these conditions, p75, promotes the survival of high p75 expressing brain-colonizing melanoma cells. Extensive 70W melanoma cell-cell contact, which downregulates p75, immediately precedes the induction of cell death associated with diminished production of two key cell survival factors, bcl-2 and the p85 subunit of phosphoinositol-3-kinase, and an elevation in apoptosis promoting intracellular reactive oxygen species (ROSs). Since one function of bcl-2 may be to control the generation of ROSs via the antioxidant pathway, these cells may receive a apoptosis-prompting "double hit". 70W melanoma cell death occurred by an apoptotic mechanism displaying classical morphological changes including plasma membrane blebbing, loss of microvilli and redistribution of ribosomes. 70W apoptosis could be pharmacologically triggered following anti-p75 monoclonal antibody-mediated clustering of p75 receptors. 70W cells fluorescently sorted for high-p75 expression (p75$\sp{\rm H}$ cells) exhibited an augmented survival potential and a predilection to sort with the S + G2/M growth phase, relative to their low p75 expressing, p75$\sp{\rm L}$ counterparts. Apoptosis is significantly delayed by p75$\sp{\rm H}$ cells, whereas p75$\sp{\rm L}$ cells are exquisitely prone to initiate apoptosis. Importantly, the p75$\sp{\rm L}$ cells that survive apoptosis, highly re-expressed p75 and were remarkably responsive to exogenous NGF.^ These are the first data to implicate p75-mediated neurotrophism as an invasion and survival support mechanism employed by brain-metastatic cells. In particular, these results may have implications in little understood phenomena of tumor progression, such as the emergence of "clonal dominance" and tumor dormancy. ^

The role and mechanism of supressor of cytokine signaling 1 in brain metastasis

Brain metastasis, which occurs in 40%-60% of patients with advanced melanoma, has led directly to death in the majority of cases. Unfortunately, little is known about the biological and molecular basis of melanoma brain metastases. In our previous study, we developed a model to study human melanoma brain metastasis and found that Stat3 activity was increased in human brain metastatic melanoma cells when compared with that in cutaneous melanoma cells. The increased activation of Stat3 is also responsible for affecting melanoma angiogenesis in vivo and melanoma cell invasion in vitro and significantly affecting the expression of bFGF, VEGF, and MMP-2 in vivo and in vitro. Interestingly, a member of a new family of cytokine-inducible inhibitors of signal transduction, termed suppressors of cytokine signaling 1 (SOCS1) was found to negatively regulate the Janus kinase signal transducer and activator of transcription (Jak/STAT) signaling cascade. Here we report that restoration of SOCS1 expression by transfecting of SOCS1-expressing vector effectively inhibited melanoma brain metastasis through inhibiting Stat3 activation and further affecting melanoma angiogenesis and melanoma cell invasion in vitro, and significantly affected the expression of vascular endothelial growth factor (VEGF) and matrix metalloproteinase-2 (MMP-2) in vitro and in vivo. In addition, we used cDNA array to compare mRNA expression in the SOCS1-transfected and vector-transfected cell lines and found some genes are tightly correlated to the restoration of SOCS1. One of them is Caveolin-1 (Cav-1). Cav-1 was reported to function as a tumor suppressor gene by several groups. Finally, the Cav-1 expression is up-regulated in SOCS1-overexpressing cell line. Further study found the regulation of Cav-1 by SOCS1 occurs through inhibiting Stat3 activation. Activated Stat3 binds directly to Cav-1 promoter and the Cav-1 promoter within -575bp is essential for active Stat3 binding. My studies reveal that Stat3 activation and SOCS1 expression play important roles in melanoma metastases. Moreover, the expression between SOCS1, Stat3 and Cav-1 forms a feedback regulation loop. ^

MMP-9/TIMP-1 imbalance induced in human dendritic cells by Porphyromonas gingivalis.

Matrix metalloproteinase-9 (MMP-9) cleaves collagen, allowing leukocytes to traffic toward the vasculature and the lymphatics. When MMP-9 is unregulated by tissue inhibitor of metalloproteinase-1 (TIMP-1), this can lead to tissue destruction. Dendritic cells (DCs) infiltrate the oral mucosa increasingly in chronic periodontitis, characterized by infection with several pathogens including Porphyromonas gingivalis. In this study, human monocyte-derived DCs were pulsed with different doses of lipopolysaccharide of P. gingivalis 381 and of Escherichia coli type strain 25922, as well as whole live isogenic fimbriae-deficient mutant strains of P. gingivalis 381. Levels of induction of MMP-9 and TIMP-1, as well as interleukin-10 (IL-10), which reportedly inhibits MMP-9 induction, were measured by several approaches. Our results reveal that lipopolysaccharide of P. gingivalis, compared with lipopolysaccharide from E. coli type strain 25922, is a relatively potent inducer of MMP-9, but a weak inducer of TIMP-1, contributing to a high MMP-9/TIMP-1 ratio.Whole live P. gingivalis strain 381, major fimbriae mutant DPG-3 and double mutant MFB were potent inducers of MMP-9, but minor fimbriae mutant MFI was not. MMP-9 induction was inversely proportional to IL-10 induction. These results suggest that lipopolysaccharide and the minor and the major fimbriae of P. gingivalis may play distinct roles in induction by DCs of MMP-9, a potent mediator of local tissue destruction and leukocyte trafficking.

Effects of galectin-1 and galectin-3 expression on prostate cancer cell adhesion, differentiation, proliferation, and tumorigenicity

The vertebrate $\beta$-galactoside-binding lectins galectin-1 and galectin-3 have been proposed to function in diverse cellular processes such as adhesion, proliferation, differentiation, and tumorigenesis. Experiments were initiated to further study the functional properties of these molecules. A prostate cancer cell line, LNCaP, was identified which expressed neither galectin. This line was stably transfected with cDNA for either galectin-1 or galectin-3. The resultant clones were used to study effects on critical cell processes. LNCaP cells expressing galectin-1 on the surface were found to bind more rapidly than control lines to the human extracellular matrix proteins laminin and fibronectin, although overall binding was not increased. To analyze effects on differentiation, LNCaP cells were studied which had either been transfected with galectin-1 or which had been induced to express endogenous galectin-1 by treatment with the differentiation agent sodium butyrate. In both cases, cells displayed a slower rate of growth and increased rate of apoptosis. A transient decrease in expression of prostate specific antigen was seen in the butyrate treated cells but not in the transfected cells. To investigate the role of galectins in the process of malignant transformation and progression, immunohistochemical analysis was performed on formalin-fixed, paraffin-embedded sections of human prostate tissue, the premalignant lesion prostatic intraepithelial neoplasia, primary adenocarcinoma of the prostate, and foci of metastatic prostate cancer. Galectin-1 expression was relatively constant throughout in contrast to galectin-3 which demonstrated significantly less expression in primary and metastatic tumors. LNCaP cells transfected with galectin-3 cDNA displayed lower proliferation rates, increased spontaneous apoptosis, and G1 growth phase arrest compared to controls. Four of six galectin-3 lines tested were less tumorigenic in nude mice than controls. The following conclusions are drawn regarding the role of galectin-1 and galectin-3 expression in the context of prostate cancer: (1) galectin-1 may participate in the early stages of cancer cell adhesion to extracellular matrix proteins; (2) galectin-1 expression results in a differentiated phenotype and may contribute to differentiation induction by butyrate; (3) galectin-3 expression correlates inversely with prostate cell tumorigenesis and prostate cancer metastasis. ^

Non-sexual household transmission of HCV infection: A cohort study

Objective. This study was designed to determine the prevalence and incidence of HCV infection among non-sexual household contacts of HCV-infected women and to describe the association between HCV infection and potential household risk factors in order to examine whether non-sexual household contact is a route of transmission for HCV infection. ^ Methods. A baseline prevalence survey included 409 non-sexual household contacts of 241 HCV-infected index women in the Houston area from 1994 to 1997. A total of 470 non-sexual household contacts with no evidence of HCV infection at baseline investigation were re-assessed approximately three years after baseline enrollment. Information on potential risk factors was collected through face to face interviews and blood samples were tested for anti-HCV with ELISA-2 and Matrix/RIBA-2. The relationships between HCV infection and potential risk factors were examined by using univariate and multivariate logistic regression analyses. ^ Results. The overall prevalence of anti-HCV positivity among 409 non-sexual household contacts was 4.4%. The highest prevalence of anti-HCV was found in parents (19.5%), followed by siblings (8.1%) and other relatives (5.6%); the children had the lowest prevalence of anti-HCV (1.2%). The univariate analysis showed that IDU, blood transfusion, tattoos, sexual contact with injecting drug users, more than 3 sexual partners in a lifetime, history of a STD, incarceration, previous hepatitis, and contact with hepatitis patients were significantly associated with HCV infection, however, sharing razors, nail clippers, toothbrushes, gum, food or beds with HCV-infected women, and history of dialysis, health care job, body piercing, and homosexual activities were not. Multivariate analysis found that IDU (OR = 221.7 with 95% CI of 22.8 to 2155.7) and history of a STD (OR = 11.7 with 95% CI of 1.2 to 113.1) were the only variables significantly associated with HCV infection. No such associations remained for other risk factors. The three-year cumulative incidence of anti-HCV among 352 non-sexual household contacts of HCV-infected women was zero. ^ Conclusion. This study has provided no evidence that non-sexual household contact is a likely route of transmission for HCV infection. The risk of sharing razors, nail clippers, toothbrushes, gum, food and/or beds with HCV-infected women is not evident and has not been shown to be the likely mode for HCV spread among family members. This study does suggest that IDU is the likely route of transmission for most HCV infection. Association also has been shown independently with a history of STD. The prevalence of anti-HCV among non-sexual household contacts was low. Exposure to common parenteral risk factors and sexual transmission between sexual partners may account for HCV spread among household members of HCV-infected persons. ^

HRGbeta1 inhibits metastasis of breast cancer cell lines, one of which exhibits lineage infidelity

Recent publications have questioned the origin of the MDA-MB-435 breast cancer cell line and have suggested that it is of melanocyte origin rather than breast epithelial origin. The data presented herein show unequivocally that MDA-MB-435 does express breast epithelial markers and produces milk-specific lipids. The data also indicated that MDA-MB-435 does express some melanocyte proteins but this expression occurs in the same MDA-MB-435 cells that express breast epithelial proteins. Although MDA-MB-435 does not strictly adhere to a breast lineage, it does retain breast specific markers and is thus valid as an experimental cell line in breast cancer studies. ^ Heregulinβ1 (HRGβ1) has been shown to both stimulate and inhibit breast tumorigenic and metasastasic phenotypes. Some studies used only the EGF-like domain of the extracellular domain of HRGβ1 while others used bacterially-expressed HRGβ1. Our in vitro data demonstrated that the full-length extracellular domain of human HRGβ1 reduced clonal growth of MDA-MB-435 breast cancer cells but stimulated apoptosis in MDA-MB-435 and MCF-7 breast cancer cells. In addition, mammalian-expressed HRGβ1 did not dramatically affect matrix metalloproteinase-9 activity but did inhibit cell motility of MDA-MB-435 and MCF-7 cells. Taken together, the in vitro data indicated that HRGβ1 inhibits metastasis-associated properties. ^ The in vivo data demonstrated that inducible expression of the full-length extracellular domain of human HRGβ1 in MDA-MB-435 cells reduced tumor volume and cell proliferation but increased apoptosis of cells injected at the mammary fat pad in nude mice. More importantly, HRGβ1 reduced the number of metastases observed by a spontaneous metastasis assay. Taken together, these data indicate that the full-length extracellular domain of human HRGβ1 has the net effect of inhibiting breast cancer metastasis. ^

Fibulin-2 stabilizes tumor extracellular matrix and drives malignant progression of lung adenocarcinoma

The ECM of epithelial carcinomas undergoes structural remodeling during periods of uncontrolled growth, creating regional heterogeneity and torsional stress. How tumors maintain ECM integrity in the face of dynamic biophysical forces is still largely unclear. This study addresses these deficiencies using mouse models of human lung adenocarcinoma. Spontaneous lung tumors were marked by disorganized basement membranes, dense collagen networks, and increased tissue stiffness. Metastasis-prone lung adenocarcinoma cells secreted fibulin-2 (Fbln2), a matrix glycoprotein involved in ECM supra-molecular assembly. Fibulin-2 depletion in tumor cells decreased the intra-tumoral abundance of matrix metalloproteinases and reduced collagen cross-linking and tumor compressive properties resulting in inhibited tumor growth and metastasis. Fbln2 deposition within intra-tumoral fibrotic bands was a predictor of poor clinical outcome in patients. Collectively, these findings support a feed-forward model in which tumor cells secrete matrix-stabilizing factors required for the assembly of ECM that preferentially favors malignant progression. To our knowledge, this is the first evidence that tumor cells directly regulate the integrity of their surrounding matrix through the secretion of matrix-stabilizing factors such as fibulin-2. These findings open a new avenue of research into matrix assembly molecules as potential therapeutic targets in cancer patients.