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.

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. ^

ELUCIDATING THE ROLE OF CD44 EXPRESSION ON MESENCHYMAL STEM CELLS WITHIN THE TUMOR MICROENVIRONMENT

The tumor microenvironment is comprised of a vast array of heterogeneous cells including both normal and neoplastic cells. The tumor stroma recruitment process has been exploited for an effective gene delivery technique using bone marrow derived MSC. Targeted migration of the MSC toward the tumor microenvironment, while successful, is not yet fully understood. This study was designed to assess the role of CD44 in the migration of MSC toward the tumor microenvironment and to determine the implications of CD44-deficient MSC within the tumor stroma. Inhibition of MSC migration was evaluated through a variety of methods in vitro and in vivo including CD44 receptor knockdown, CD44 antagonists, CD44 neutralizing antibodies and small molecule inhibitor of matrix metalloproteinases. Blocking CD44 signaling through MMP inhibition was characterized by lack of intracellular domain cleavage and lead to the decrease in Twist gene expression. A functional relationship between CD44 and Twist expression was confirmed by chromatin immunoprecipitation. Next, a series of murine tumor models were used to examine the role of CD44 deficient stroma within the tumor microenvironment. Labeled transgenic CD44 knockout (KO) MSC or wild type (WT) C57/B6 MSC were used to analyze the stromal incorporation within murine breast carcinomas (EO771 and 4T1). Subsequent tumors were analyzed for vessel formation (CD31), and the presence of tumor associated fibroblast (TAF) markers, α-smooth muscle actin (α-SMA), fibroblast activation protein (FAP), and fibroblast specific protein (FSP). The tumors with CD44KO MSC cells had less vessel formation than the tumors with WT MSC. The lack of fibroblastic TAF population as defined by FAP/FSP expression by the CD44KO MSC admixed tumors suggest that the bone marrow derived population of MSC were unable to contribute to the fibroblastic stromal population. Subsequently, a bone marrow transplantation experiment confirmed the endogenous migratory deficiencies of the CD44KO bone marrow derived stromal cells toward the tumor microenvironment in vivo. WT mice with CD44KO bone marrow had less CD44KOderived tumor stroma compared to mice with WT bone marrow. These results indicate that CD44 is crucial to stromal cell migration and incorporation to the tumor microenvironment as TAF.

MCAM/MUC18 REGULATES MELANOMA PROGRESSION BY MODULATING THE EXPRESSION OF ID-1

The acquisition of the metastatic melanoma phenotype is associated with increased expression of the melanoma cell adhesion molecule MCAM/MUC18 (CD146). However, the mechanism by which MUC18 contributes to melanoma metastasis remains unclear. Herein, we stably silenced MUC18 expression utilizing lentivirus-incorporated small hairpin RNA, in two metastatic melanoma cell lines, A375SM and C8161, and conducted cDNA microarray analysis. We identified and validated that the transcriptional regulator, Inhibitor of DNA Binding-1 (Id-1), previously shown to function as an oncogene in several malignancies, was downregulated by 5.6-fold following MUC18 silencing. Additionally, we found that MUC18 regulated Id-1 expression at the transcriptional level via ATF-3. Interestingly, ATF-3 was upregulated by 6.9 fold in our cDNA microarray analysis following MUC18 silencing. ChIP analysis showed increased binding of ATF-3 to the Id-1 promoter after MUC18 silencing, while mutation of the ATF-3 binding site on the Id-1 promoter increased Id-1 promoter activity in MUC18-silenced cells. These Data suggest that MUC18 silencing promotes inhibition of Id-1 expression by increasing ATF-3 expression and binding to the Id-1 promoter. Rescue of MUC18 reverted the expression of Id-1 and ATF-3, thus validating that they are not off-target effects of MUC18. To further assess the role of Id-1 in melanoma invasion and metastasis, we overexpressed Id-1 in MUC18-silenced cells. Overexpression of Id-1 in MUC18-silenced cells resulted in increased cell invasion, as well as increased expression and activity of MMP-2. Our data further reveal that Id-1 regulates MMP-2 at the transcriptional level through Sp1 and Ets-1. This is the first report to demonstrate that MUC18 does not act exclusively in cell adherence, but is also involved in cell signaling that regulates the expression of genes, such as Id-1 and ATF-3, thus contributing to the metastatic melanoma phenotype.

ROLE OF PROSTAGLANDIN E2 IN THE REGULATION OF PANCREATIC STELLATE CELLS HYPER ACTIVITY ASSOCIATED WITH PANCREATIC CANCER

Pancreatic cancer is one of the most lethal type of cancer due to its high metastasis rate and resistance to chemotherapy. Pancreatic fibrosis is a constant pathological feature of chronic pancreatitis and the hyperactive stroma associated with pancreatic cancer. Strong evidence supports an important role of cyclooxygenase-2 (COX-2) and COX-2 generated prostaglandin E2 (PGE2) during pancreatic fibrosis. Pancreatic stellate cells (PSC) are the predominant source of extracellular matrix production (ECM), thus being the key players in both diseases. Given this background, the primary objective is to delineate the role of PGE2 on human pancreatic stellate cells (PSC) hyper activation associated with pancreatic cancer. This study showed that human PSC cells express COX-2 and synthesize high levels of PGE2. PGE2 stimulated PSC migration and invasion; expression of extra cellular matrix (ECM) genes and tissue degrading matrix metallo proteinases (MMP) genes. I further identified the PGE2 EP receptor responsible for mediating these effects on PSC. Using genetic and pharmacological approaches I identified the receptor required for PGE2 mediates PSC hyper activation. Treating PSC with Specific antagonists against EP1, EP2 and EP4, demonstrated that blocking EP4 receptor only, resulted in a complete reduction of PGE2 mediated PSC activation. Furthermore, siRNA mediated silencing of EP4, but not other EP receptors, blocked the effects of PGE2 on PSC fibrogenic activity. Further examination of the downstream pathway modulators revealed that PGE2 stimulation of PSC involved CREB and not AKT pathway. The regulation of PSC by PGE2 was further investigated at the molecular level, with a focus on COL1A1. Collagen I deposition by PSC is one of the most important events in pancreatic cancer. I found that PGE2 regulates PSC through activation of COL1A1 expression and transcriptional activity. Downstream of PGE2, silencing of EP4 receptor caused a complete reduction of COL1A1 expression and activity supporting the role of EP4 mediated stimulation of PSC. Taken together, this data indicate that PGE2 regulates PSC via EP4 and suggest that EP4 can be a better therapeutic target for pancreatic cancer to reduce the extensive stromal reaction, possibly in combination with chemotherapeutic drugs can further kill pancreatic cancer cells.

Regulation of angiogenesis by interferon-beta

The purpose of these studies was to investigate the role of interferon-beta (IFN-$\beta$) in angiogenesis. IFN-$\alpha/\beta$ have been implicated in inhibiting a number of steps in the angiogenic pathway. We examined the balance of angiogenesis-regulating molecules in several systems including human infantile hemangiomas, UV-B irradiated mice, and dorsal incisional wound healing in mice. In each system, epidermal hyperplasia and cutaneous angiogenesis were directly related to the expression of positive angiogenic factors (bFGF and VEGF) and inversely related to the expression of endogenous IFN-$\beta.$ The re-expression of IFN-$\beta$ correlated with tumor regression and/or resolution of wound healing. In contrast to control mice, UV-B-induced cutaneous angiogenesis and hyperplasia persisted in IFN-$\alpha/\beta$ receptor knock-out mice. In normal mice, endogenous IFN-$\beta$ was expressed by all differentiated epithelial cells exposed to environmental stimuli. The expression of endogenous IFN-$\beta$ was necessary but insufficient for complete differentiation of epidermal keratinocytes.^ The tumor organ microenvironment can regulate angiogenesis. Human bladder carcinoma cells growing in the bladder wall of nude mice express high levels of bFGF, VEGF, and MMP-9, have higher vascular densities, and produce metastases to lymph nodes and lungs, whereas the same cells growing subcutaneously express less bFGF, VEGF, and MMP-9, have lower vascular densities, and do not metastasize. IFN-$\alpha/\beta$ was found to inhibit bFGF and MMP-9 expression both in vitro and in vivo in human bladder carcinoma cells. Systemic therapy with human IFN-$\alpha$ of human bladder cancer cells growing orthotopically in nude mice, resulted in decreased vascularity, tumorigenicity, and metastasis as compared to saline treated mice. Human bladder cancer cells resistant to the antiproliferative effects of IFN were transfected with the human IFN-$\beta$ gene. Hu-IFN-$\beta$ transfected cells expressed significantly less bFGF protein and gelatinase activity than parental or control-transfected cells and did not grow at ectopic or orthotopic sites. Collectively the data provide direct evidence that IFN-$\alpha/\beta$ can inhibit angiogenesis via down-regulation of angiogenesis-stimulating cytokines. ^

The role of the sonic hedgehog signaling pathway in epidermal homeostasis

Non-melanoma skin cancer is the most frequently diagnosed malignancy in the United States of which basal cell carcinoma (BCC) accounts for 65%. It has recently been determined that deregulation of the sonic hedgehog (shh) pathway leads to the development of BCC. Shh, gli-1, gli-2 gli-3, ptc and smo are overexpressed in BCC and overexpression of these genes in the epidermis results in formation of BCC-like tumors. Despite these observations, the mechanisms by which the pathway controls epidermal homeostasis and the development of the malignant phentotype are unknown. This study assessed the role of the shh pathway in epidermal homeostasis through regulation of apoptosis and differentiation. ^ The anti-apoptotic protein, bcl-2 is overexpressed in BCC, however transcriptional regulators of bcl-2 in the epidermis are unknown. Transient transfection of primary keratinocytes with gli-1 resulted in an increase of bcl-2 expression. Database analysis revealed seven candidate gli binding sites on the bcl-2 promoter. Cotransfection of increasing amounts of gli-1 in keratinoycytes resulted in a corresponding dose-dependent increase in bcl-2 promoter luciferase activity. An N-terminal mutant of gli-3 inhibited gli-1 transactivation of the bcl-2 promoter. The region −428 to −420 was found to be important for gli-1 regulation through gel shift, luciferase assays and site-directed mutagenesis. ^ In order to assess the ability of the shh pathway to regulate keratinocyte differentiation, HaCaT keratinocytes overexpressing sonic hedgehog, were grown in organotypic raft culture. Overexpression of shh induced a basal cell phenotype compared to vector control, as evidenced by transmural staining of cytokeratin 14 and altered Ki67 staining. Shh also induced keratinocyte invasion into the underlying collagen. This was associated with increased phosphorylation of EGFR, jnk and raf and increased expression of c-jun, mmp-9 and Ki67. Interestingly, shh overexpression in HaCaTs did not induce the typical downstream effects of shh signaling, suggesting a gli-independent mechanism. Sonic hedgehog's ability to induce an invasive phenotype was found to be dependent on activation of the EGF pathway as inhibition of EGFR activity with AG1478 and c-225 was able to reduce the invasiveness of HaCaT shh keratinocytes, whereas treatment with EGF augmented the invasiveness of the HaCaT shh clones. ^ These studies reveal the importance of the sonic hedgehog pathway in epidermal homeostasis by regulation of apoptosis through bcl-2, and control of keratinocyte differentiation and invasion through activation of the EGF pathway. They further suggest potential mechanisms by which deregulation of the shh pathway may lead to the development of the malignant phenotype. ^

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. ^

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. ^

Protease-activated receptor-1 signaling plays a major role in melanoma growth and metastasis

Overexpression of the thrombin receptor (Protease-Activated-Receptor-1), PAR-1, in cell lines and tissue specimens correlates with the metastatic potential of human melanoma. Utilizing lentiviral shRNA to stably silence PAR-1 in metastatic melanoma cell lines results in decreased tumor growth and lung metastasis in vivo. Since the use of viral technology is not ideal for clinical therapies, neutral liposomes (DOPC) were utilized as a delivery vehicle for PAR-1 siRNA. Our data suggest that PAR-1 siRNA-DOPC treatment by systemic delivery significantly decreases tumor growth and lung metastasis in nude mice. Concomitant decreases in angiogenic and invasive factors (IL-8, VEGF, MMP-2) were observed in PAR-1 siRNA-DOPC-treated mice. Utilizing a cDNA microarray platform, several novel PAR-1 downstream target genes were identified, including Connexin 43 (Cx-43) and Maspin. Cx-43, known to be involved in tumor cell diapedesis and attachment to endothelial cells, is decreased after PAR-1 silencing. Furthermore, the Cx-43 promoter activity was significantly inhibited in PAR-1-silenced cells suggesting transcriptional regulation of Cx-43 by PAR-1. ChIP analysis revealed a reduction in SP-1 and AP-1 binding to the Cx-43 promoter. Moreover, melanoma cell attachment to HUVEC was significantly decreased in PAR-1-silenced cells as well as in Cx-43 shRNA transduced cells. As both SP-1 and AP-1 transcription factors act as positive regulators of Cx-43, our data provide a novel mechanism for the regulation of Cx-43 expression by PAR-1. Maspin, a serine protease inhibitor with tumor-suppressor function, was found to be upregulated after PAR-1 silencing. Our results indicate that PAR-1 transcriptionally regulates Maspin, as the promoter activity was significantly increased after PAR-1 silencing. ChIP analysis revealed that silencing PAR-1 increased binding of Ets and c-Jun to the Maspin promoter. As Maspin was recently found to be a tumor-suppressor in melanoma by reducing the invasive capacity of melanoma cells, invasion assays revealed a decrease in invasion after PAR-1 silencing and in cells transduced with a Maspin expression vector. We propose that PAR-1 is key to the progression and metastasis of melanoma in part by regulating the expression of Cx-43 and Maspin. Taken together, we propose that PAR-1 is an attractive target for the treatment of melanoma.^

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. ^