Oncostatin M and STAT3 Signaling Pathways Support Human Trophoblast Differentiation by Inhibiting Inflammatory Stress in Response to IFNγ and GM-CSF.

Interleukin-6 (IL-6) superfamily cytokines play critical roles during human pregnancy by promoting trophoblast differentiation, invasion, and endocrine function, and maintaining embryo immunotolerance and protection. In contrast, the unbalanced activity of pro-inflammatory factors such as interferon gamma (IFNγ) and granulocyte-macrophage colony-stimulating factor (GM-CSF) at the maternal-fetal interface have detrimental effects on trophoblast function and differentiation. This study demonstrates how the IL-6 cytokine family member oncostatin M (OSM) and STAT3 activation regulate trophoblast fusion and endocrine function in response to pro-inflammatory stress induced by IFNγ and GM-CSF. Using human cytotrophoblast-like BeWo (CT/BW) cells, differentiated in villous syncytiotrophoblast (VST/BW) cells, we show that beta-human chorionic gonadotrophin (ßhCG) production and cell fusion process are affected in response to IFNγ or GM-CSF. However, those effects are abrogated with OSM by modulating the activation of IFNγ-STAT1 and GM-CSF-STAT5 signaling pathways. OSM stimulation enhances the expression of STAT3, the phosphorylation of STAT3 and SMAD2, and the induction of negative regulators of inflammation (e.g., IL-10 and TGFß1) and cytokine signaling (e.g., SOCS1 and SOCS3). Using STAT3-deficient VST/BW cells, we show that STAT3 expression is required for OSM to regulate the effects of IFNγ in ßhCG and E-cadherin expression. In contrast, OSM retains its modulatory effect on GM-CSF-STAT5 pathway activation even in STAT3-deficient VST/BW cells, suggesting that OSM uses STAT3-dependent and -independent mechanisms to modulate the activation of pro-inflammatory pathways IFNγ-STAT1 and GM-CSF-STAT5. Moreover, STAT3 deficiency in VST/BW cells leads to the production of both a large amount of ßhCG and an enhanced expression of activated STAT5 induced by GM-CSF, independently of OSM, suggesting a key role for STAT3 in ßhCG production and trophoblast differentiation through STAT5 modulation. In conclusion, our study describes for the first time the critical role played by OSM and STAT3 signaling pathways to preserve and regulate trophoblast biological functions during inflammatory stress.

Leukemia inhibitory factor regulates the activation of inflammatory signals in macrophages and trophoblast cells.

The pleiotropic cytokine leukemia inhibitory factor (LIF) is a key gestational factor known to establish dynamic cellular and molecular cross talk at the feto-maternal interface. Previously, we described the regulatory role of the LIF-trophoblast-IL10 axis in the process of macrophage deactivation in response to pro-inflammatory cytokines. However, the direct regulatory effects of LIF in macrophage and trophoblast cell function remains elusive. In this study, we aimed to examine whether and how LIF regulates the behavior of macrophages and trophoblast cells in response to pro-inflammatory stress factors. We found that LIF modulated the activating effects of interferon-gamma (IFNγ) and granulocyte-macrophage colony-stimulating factor (GM-CSF) in macrophages and trophoblast cells by reducing the phosphorylation levels of signal transducer and activator of transcription-1 (Stat1) and -5 (Stat5). Cell activation with IFNγ inhibited cell invasion and migration but this immobilizing effect was abrogated when macrophages and trophoblast cells were deactivated with LIF; macrophage cell motility restitution could in part be explained by the positive effects of LIF in Stat3 activation and matrix metalloproteinase 9 (MMP-9) expression. Pharmacological inhibition of Stat1 and Stat3 indicated that IFNγ-induced Stat1 activation mediated macrophage motility inhibition, and that cell motility in IFNγ-activated macrophages is restored via LIF-induced Stat3 activation and Stat1 inhibition. Moreover, IFNγ-induced TNFα gene expression was also abrogated by LIF through Stat1 inhibition and Stat3 activation. Finally, we have found that cell invasion of trophoblast cells is inhibited when they were cocultured with GM-CSF-differentiated, IFNγ-stimulated macrophages. This effect, however, was inhibited when macrophages were exposed to LIF. Overall, this in vitro study reveals for the first time the anti-inflammatory and pro-gestational activities of LIF by acting directly on macrophages and trophoblast cells.

Molecular therapy with derivatives of amino benzoic acid inhibits tumor growth and metastasis in murine models of bladder cancer through inhibition of TNFα/NFΚB and iNOS/NO pathways.

Muscle-invasive bladder cancer (MIBC) is an aggressive form of urothelial bladder carcinoma (UBC) with poorer outcomes compared to the non-muscle invasive form (NMIBC). Higher recurrent rates and rapid progression after relapse in UBC is known to be linked with chronic inflammation. Here, the preclinical murine models of NMIBC (MB49) and MIBC (MB49-I) were used to assess the antitumor effects of DAB-1, an anti-inflammatory aminobenzoic acid derivative we have developed in order to target cancer-related inflammation. A subchronic toxicity study on cancer-free mice shown that DAB-1 treatment did not affect normal mouse development or normal function of vital organs. In mice bearing MB49-I tumors, whole body accumulation of the radioconjugate [131I]DAB-1 was higher than in control mice, the main sites of [131I]DAB-1 accumulation being the liver (34%), the intestines (21%), and the tumors (18%). In vivo molecular therapy of ectopic and orthotopic tumors indicated that treatment with DAB-1 efficiently inhibited tumor growth, metastasis formation, and mortality rate. The antitumor efficacy of DAB-1 was associated with strong decreased tumor cell proliferation and iNOS expression in tumor tissues and deactivation of macrophages from tumor-bearing mice. Mechanistic investigations revealed that DAB-1 efficiently inhibited i) TNFα/NFΚB and IL6/STAT3 signaling pathways activation; ii) TNFα-induced NO production by decreasing NFΚB transcriptional activation and functional iNOS expression; and iii) cellular proliferation with minimal or no effects on cell mortality or apoptosis. In conclusion, this study provides preclinical and biological/mechanistic data highlighting the potential of DAB-1 as a safe and efficient therapeutic agent for the treatment of patients with NMIBC and MIBC.

PAX2 is activated by estradiol in breast cancer cells of the luminal subgroup selectively, to confer a low invasive phenotype.

BACKGROUND: Metastasis is the leading cause of death among breast cancer patients. Identifying key cellular factors controlling invasion and metastasis of breast cancer cells should pave the way to new therapeutic strategies efficiently interfering with the metastatic process. PAX2 (paired box 2) transcription factor is expressed by breast cancer cells in vivo and recently, it was shown to negatively regulate the expression of ERBB2 (erythroblastic leukemia viral oncogene homolog 2, HER-2/neu), a well-documented pro-invasive and pro-metastastic gene, in luminal/ERalpha-positive (ERα+) breast cancer cells. The objective of the present study was to investigate a putative role for PAX2 in the control of luminal breast cancer cells invasion, and to begin to characterize its regulation. RESULTS: PAX2 activity was higher in cell lines from luminal compared to non-luminal subtype, and activation of PAX2 by estradiol was selectively achieved in breast cancer cell lines of the luminal subtype. This process was blocked by ICI 182780 and could be antagonized by IGF-1. Knockdown of PAX2 in luminal MCF-7 cells completely abrogated estradiol-induced downregulation of ERBB2 and decrease of cell invasion, whereas overexpression of PAX2 in these cells enhanced estradiol effects on ERBB2 levels and cell invasion. CONCLUSIONS: The study demonstrates that PAX2 activation by estradiol is selectively achieved in breast cancer cells of the luminal subtype, via ERα, and identifies IGF-1 as a negative regulator of PAX2 activity in these cells. Further, it reveals a new role for PAX2 in the maintenance of a low invasive behavior in luminal breast cancer cells upon exposure to estradiol, and shows that overexpression and activation of PAX2 in these cells is sufficient to reduce their invasive ability.

Transforming growth factor Beta regulates proliferation and invasion of rat placental cell lines.

Implantation of an embryo in the endometrium is a critical step for continuation of pregnancy, and implantation failure is a major cause of infertility. In rats, the implantation process involves invasion of the endometrial epithelial lining by the trophoblastic cells in order to reach the underlying stromal cells. Transforming growth factor beta (TGFB) is a multifunctional cytokine that regulates proliferation, differentiation, and invasiveness of multiple cell lineages. We used rat HRP-1 and RCHO-1 placental cell lines to perform this study. HRP-1 cells were derived from midgestation chorioallantoic placental explants of the outbred Holtzman rat, whereas RCHO-1 cells were established from a rat choriocarcinoma. MTT proliferation assays revealed that each TGFB isoform decreased HRP-1 cell growth in a dose-dependent manner, whereas RCHO-1 cells were resistant to the growth-suppressive effect of TGFB1 and TGFB3. Only TGFB2 reduced RCHO-1 cell proliferation. Activation of ERK, MAPK14 (p38 MAPK), or SMAD pathways is known to play a role in cell proliferation, and we found that TGFB activates these pathways in both HRP-1 and RCHO-1 cells in an isoform-specific manner. MTT proliferation assays revealed that ERK pathway is partially implicated in TGFB3-reduced HRP-1 cell proliferation. Hoechst nuclear staining and caspase-3 cleavage demonstrated that TGFB isoforms failed to induce apoptosis in both cell lines. Matrigel invasion assays showed that both HRP-1 and RCHO-1 cells exhibit intrinsic invasive ability under untreated conditions. The capacity of HRP-1 cells to invade the Matrigel was selectively increased by TGFB2 and TGFB3, whereas all TGFB isoforms could increase the invasiveness of RCHO-1 cells. These important functional studies progressively reveal a key role for TGFB in regulating proliferation and invasiveness of placental cells.

XIAP gene expression and function is regulated by autocrine and paracrine TGF-beta signaling.

BACKGROUND: X-linked inhibitor of apoptosis protein (XIAP) is often overexpressed in cancer cells, where it plays a key role in survival and also promotes invasiveness. To date however, the extracellular signals and intracellular pathways regulating its expression and activity remain incompletely understood. We have previously showed that exposure to each of the three TGF-beta (transforming growth factor beta) isoforms upregulates XIAP protein content in endometrial carcinoma cells in vitro. In the present study, we have investigated the clinical relevance of TGF-beta isoforms in endometrial tumours and the mechanisms through which TGF-beta isoforms regulate XIAP content in uterine cancer cells. METHODS: TGF-beta isoforms immunoreactivity in clinical samples from endometrial tumours was assessed using immunofluorescence. Two model cancer cell lines (KLE endometrial carcinoma cells and HeLa cervical cancer cells) and pharmacological inhibitors were used to investigate the signalling pathways regulating XIAP expression and activity in response to autocrine and paracrine TGF-beta in cancer cell. RESULTS: We have found immunoreactivity for each TGF-beta isoform in clinical samples from endometrial tumours, localizing to both stromal and epithelial/cancer cells. Blockade of autocrine TGF-beta signaling in KLE endometrial carcinoma cells and HeLa cervical cancer cells reduced endogenous XIAP mRNA and protein levels. In addition, each TGF-beta isoform upregulated XIAP gene expression when given exogenously, in a Smad/NF-kappaB dependent manner. This resulted in increased polyubiquitination of PTEN (phosphatase and tensin homolog on chromosome ten), a newly identified substrate for XIAP E3 ligase activity, and in a XIAP-dependent decrease of PTEN protein levels. Although each TGF-beta isoform decreased PTEN content in a XIAP- and a Smad-dependent manner, decrease of PTEN levels in response to only one isoform, TGF-beta3, was blocked by PI3-K inhibitor LY294002. CONCLUSIONS: XIAP gene expression and function is positively regulated by exposure to the three TGF-beta isoforms in a Smad-dependent manner, similar to constitutive XIAP gene expression which depends on autocrine TGF-beta/Smad signalling.

Akt isoforms regulate intermediate filament protein levels in epithelial carcinoma cells.

Keratin 8 and 18 are simple epithelial intermediate filament (IF) proteins, whose expression is differentiation- and tissue-specific, and is maintained during tumorigenesis. Vimentin IF is often co-expressed with keratins in cancer cells. Recently, IF have been proposed to be involved in signaling pathways regulating cell growth, death and motility. The PI3K/Akt pathway plays a pivotal role in these processes. Thus, we investigated the role of Akt (1 and 2) in regulating IF expression in different epithelial cancer cell lines. Over-expression of Akt1 increases K8/18 proteins. Akt2 up-regulates K18 and vimentin expression by an increased mRNA stability. To our knowledge, these results represent the first indication that Akt isoforms regulate IF expression and support the hypothesis that IFs are involved in PI3K/Akt pathway.

VP-128, a novel oestradiol-platinum(II) hybrid with selective anti-tumour activity towards hormone-dependent breast cancer cells in vivo.

We have previously reported the synthesis of VP-128, a new 17beta-oestradiol (E(2))-linked platinum(II) hybrid with high affinity for oestrogen receptor alpha (ERalpha). In the present study, we have investigated the anti-tumour activity of VP-128 towards breast cancer cells in vitro and in vivo. We used human ERalpha-positive (MCF-7) and -negative (MDA-MB-468) cells as a model for treatment with increasing doses of VP-128, cisplatin or E(2) in vitro and for xenograft experiments in nude mice in vivo. Compared with cisplatin, VP-128 showed markedly improved in vitro and in vivo anti-tumour activity towards ERalpha-positive MCF-7 breast cancer cells, without increased systemic toxicity. In these caspase-3-deficient cells, treatment with VP-128 overcame weak cellular sensitivity to cisplatin in vitro and in vivo. In these cells, only the hybrid induced apoptosis in an ERalpha-dependent manner, inactivated both X-linked inhibitor of apoptosis protein and Akt, and induced selective nuclear accumulation of ERalpha and the expression of ER-regulated genes c-myc and tff1, which was blocked by ERalpha-specific antagonist ICI 282 780. In the case of ERalpha-negative MDA-MB-468 cells, VP-128, but not cisplatin, induced nuclear accumulation of apoptosis-inducing factor and inhibited c-myc expression. However, VP-128 did not show enhanced in vivo anti-tumour activity compared with cisplatin. These results reveal two different modes of action for VP-128 in ERalpha-positive and -negative breast cancer cells, and highlight the promising therapeutic value of this unique E(2)-platinum hybrid for selective targeting of hormone-dependent cancers.

X-linked inhibitor of apoptosis protein (XIAP) regulates PTEN ubiquitination, content, and compartmentalization.

Apoptotic cell death plays a normal role in various physiological processes, and deregulated apoptosis is a hallmark of several diseases, including cancer. Cell fate is dictated by the balance between pro- and antiapoptotic factors. Akt is one of these antiapoptotic factors, which must be activated through phosphorylation. The phosphorylation of Akt has previously been shown to be promoted by X-linked inhibitor of apoptosis protein (XIAP), another antiapoptotic protein dictating the fate of normal and cancer cells. However, the underlying mechanisms are poorly understood. We have observed that XIAP associates with PTEN (phosphatase and tensin homolog deleted on chromosome ten), the best characterized negative regulator of Akt phosphorylation, in vitro and in vivo. XIAP knockdown reduces constitutive mono- and polyubiquitination of PTEN, increases PTEN protein levels, and prevents nuclear accumulation of PTEN. Overexpression of XIAP induces polyubiquitination of PTEN and proteasome-dependent decrease of PTEN protein levels. RNA interference experiments showed that XIAP-induced regulation of Akt phosphorylation is PTEN-dependent. Additional experiments confirmed that XIAP also regulates PTEN in vivo; primary mouse embryonic fibroblasts derived from XIAP(-/-) mice contain higher levels of PTEN protein, less mono- and polyubiquitinated PTEN, and less nuclear PTEN than primary mouse embryonic fibroblasts derived from XIAP(+/+) mice. Finally, we found that XIAP can directly ubiquitinate PTEN in vitro. We thus propose that XIAP acts as an E3 ubiquitin ligase for PTEN and promotes Akt activity by regulating PTEN content and compartmentalization.

X-linked inhibitor of apoptosis protein levels and protein kinase C activity regulate the sensitivity of human endometrial carcinoma cells to tumor necrosis factor alpha-induced apoptosis.

Endometrial carcinomas are often chemoresistant. TNFalpha shows potent antitumor activity against various cancers, and if it demonstrates good antitumor activity against endometrial cancer, the cytokine could represent a valuable alternative therapeutic approach. We have tested the ability of TNFalpha to induce apoptosis in endometrial carcinoma cells, and examined a putative role for X-linked inhibitor of apoptosis protein (XIAP) in regulating cellular sensitivity to the cytokine. Exposure to TNFalpha triggered TNF-R1-dependent activation of caspases-8, -9, and -3, down-regulated Akt and XIAP proteins and induced dose-dependent and time-dependent apoptosis in Ishikawa cells. On the opposite, TNFalpha up-regulated XIAP in Hec-1A cells; in these cells, the cytokine induced delayed TNF-R1-dependent activation of caspase-8, and failed to activate caspases -9 and -3 and to induce apoptosis. However, XIAP small interfering RNA restored TNFalpha-induced caspase signaling and apoptosis in Hec-1A cells; XIAP small interfering RNA also increased TNFalpha-induced apoptosis in Ishikawa cells. In addition, inhibition of protein kinase C activity enhanced TNFalpha-induced down-regulation of XIAP and potentiated apoptosis induction, in both Ishikawa and Hec-1A cells. Finally, we found XIAP immunoreactivity in epithelial cells from a large number of human endometrial tumor tissue samples, indicating that XIAP is produced by endometrial tumor cells in vivo. This could allow XIAP to play a putative in vivo role in counteracting TNFalpha-induced apoptosis in endometrial tumor cells; in this case, direct or indirect targeting of XIAP should potentiate the antitumor effect of TNFalpha.

Akt and XIAP regulate the sensitivity of human uterine cancer cells to cisplatin, doxorubicin and taxol.

We have investigated the interrelationship between two anti-apoptotic factors, XIAP and Akt, and their role in chemoresistance of uterine cancer cells. We used one cervical cancer cell line (HeLa) and two endometrial cancer cell lines (KLE and Ishikawa) as a model. The three drugs decreased Akt and XIAP content and induced apoptosis in P-Akt-negative HeLa cells. In P-Akt1/3-positive Ishikawa cells apoptosis induction correlated with XIAP decrease. P-Akt1/2/3-positive KLE cells showed maximum chemoresistance as XIAP and Akt levels/phosphorylation remained stable in response to the three drugs, and only cisplatin could significantly induce apoptosis. We found that XIAP and Akt were functionally linked in uterine cancer cells, as downregulation of XIAP with RNAi decreased P-Akt levels, and inhibition of PI3-K/Akt activity using LY294002 decreased XIAP content. Overexpression of constitutively active Akt isoforms in HeLa cells induced isoform-specific sensitivity to doxorubicin and taxol but not cisplatin. XIAP RNAi increased the cell-specific sensitivity to cisplatin and doxorubicin but not taxol. Finally, we found P-Akt immunoreactivity in epithelial cells from multiple human endometrial carcinoma tumors, suggesting that Akt may also regulate chemosensitivity in uterine cancers in vivo. Altogether these results highlight an intertwined role for specific Akt isoforms and XIAP in chemoresistance of uterine cancer cells.

Characterization of EN-1078D, a poorly differentiated human endometrial carcinoma cell line: a novel tool to study endometrial invasion in vitro.

BACKGROUND: To date, tools to study metastasis in endometrial cancers are insufficiently developed. The aim of this study was to characterize the cell line EN-1078D, a new endometrial carcinoma cell line derived from a metastasis to the ovary. METHODS AND RESULTS: Cells were characterized using cytology, transmission electron microscopy, karyotyping and morphological appearance in culture. Molecular features were determined by RT-PCR, Western Blot, FISH and sequencing. MTT proliferation assays were performed to investigate the sensitivity of EN-1078D to anticancer agents such as cisplatin and doxorubicin. Also, subcutaneous and intravenous injections in nude mice were done to test the tumorigenic and metastatic properties of EN-1078D cells. Our results indicate that EN-1078D cells express both oestrogen receptors isoforms (ER alpha and ER beta) and also low levels of progesterone receptor B (PR-B). In addition, this cell line expresses high levels of MMP-2 and MMP-14 mRNA, low levels of TIMP-1 and TIMP-2 transcripts and no detectable levels of MMP-9 mRNA. Moreover, all nude mice developed tumors by subcutaneous injections and cell invasion was observed in vitro in response to TGF-beta 3. Her-2/neu was not overamplified but mutations in the C-2 domain of PTEN gene as well as codon 12 of the K-Ras gene were found. Finally, EN-1078D shows sensitivity to drugs commonly used in chemotherapy such as cisplatin and doxorubicin: IC50 of 2.8 microM of cisplatin after 72 hours of exposure and 0.54 microM of doxorubicin after 48 hours. CONCLUSION: Taken together, these results suggest that EN-1078D will be an excellent tool to study the properties of metastatic endometrial cancer cells in vitro and their regulation by sex steroids.

Transforming growth factor-beta3 increases the invasiveness of endometrial carcinoma cells through phosphatidylinositol 3-kinase-dependent up-regulation of X-linked inhibitor of apoptosis and protein kinase c-dependent induction of matrix metalloproteinase-9.

Tumor cells often acquire intrinsic resistance to the growth inhibitory and pro-apoptotic effects of transforming growth factor-beta (TGF-beta); moreover, TGF-beta can confer invasive properties to established tumor cells. In the present study, we show that TGF-beta isoforms (TGF-beta1, TGF-beta2, and TGF-beta3) trigger proper Smad signaling in human endometrial carcinoma cell lines and efficiently inhibit cellular proliferation. These cells, however, exhibit a high degree of resistance to TGF-beta pro-apoptotic effects; we found that this resistant phenotype would be acquired through up-regulation of X-linked inhibitor of apoptosis protein (XIAP) levels. In addition, using RNA interference and pharmacological inhibitors, we show that TGF-beta increases cellular invasiveness via two distinct signaling pathways in endometrial carcinoma cells: phosphatidylinositol 3-kinase/AKT-dependent up-regulation of XIAP and protein kinase C-dependent induction of matrix-metalloproteinase-9 (MMP-9) expression. Additionally, these findings were correlated with clinical observations showing abundant TGF-beta immunoreactivity in human endometrial carcinoma tumors in vivo, extending from the epithelial compartment to the stroma upon acquisition of an invasive phenotype (gradually from grades I to III). Collectively our results describe for the first time a role for TGF-beta3 in tumor invasiveness.

Resveratrol interferes with AKT activity and triggers apoptosis in human uterine cancer cells.

BACKGROUND: Endometrial cancer is the fourth most prominent cancer among all feminine cancers in the Western world. Resveratrol, a natural anti-oxidant found in red wine emerging as a novel anticancer agent, exerts antiproliferative and pro-apoptotic activity in various cancer cell types, but its effect on uterine cancer cells is poorly understood. At the molecular level, resveratrol has been reported to inhibit cyclooxygenase (COX) expression and/or activity; in endometrial cancer cells, COX-2 is overexpressed and confers cellular resistance to apoptosis. The aim of the present study was to determine if resveratrol could exert anti-proliferative and pro-apoptotic activity over uterine cancer cells upon inhibition of COX-2 expression and/or activity. Six different human uterine cancer cell lines were used as a model (HeLa, Hec-1A, KLE, RL95-2, Ishikawa and EN-1078D). RESULTS AND DISCUSSION: High-dose of resveratrol triggered apoptosis in five out of six uterine cancer cell lines, as judged from Hoechst nuclear staining and effector caspase cleavage. In accordance, uterine cancer cell proliferation was decreased. Resveratrol also reduced cellular levels of the phosphorylated/active form of anti-apoptotic kinase AKT. Endogenous COX-2 protein levels were decreased, concomitant with a decrease in production of COX metabolites PGE2 and PGF2alpha, in each uterine cancer cell line expressing detectable levels of COX-1 and/or COX-2 in presence of resveratrol. Although COX expression was identified as a target of resveratrol in uterine cancer cells, inhibition of COX activity or exogenously added PGE2 did not modulate the effect of resveratrol on cellular proliferation. CONCLUSION: High-dose of resveratrol exerts tumoricidal activity over uterine cancer cells and regulates COX expression. In these cells, resveratrol would not directly target COX activity, but possibly other enzymes involved in prostaglandin synthesis that act downstream of the COXs.

Regulation of MMP-9 gene expression for the development of novel molecular targets against cancer and inflammatory diseases.

The need to pharmacologically control the proteolytic activity of matrix metalloproteinases (MMPs) has been commonly acknowledged, despite its limited efficacy in clinical trials. Among the reasons that explain this failure is our limited understanding of the signals that control the expression of MMPs in different cell types during different pathological conditions. Thus, future therapies must rely on more selective approaches. With the continually increasing body of proof implicating MMPs in a large number of diseases, it has become a priority to establish the pertinence of molecules involved in the signalling pathways leading to the expression of these enzymes. MMP-9 is a case in point: its dramatic overexpression in cancer and various inflammatory conditions clearly points to the molecular mechanisms controlling its expression as a potential target for eventual rational therapeutic intervention. In this article, recent progress in the signalling pathways that regulate MMP-9 expression is reviewed, and the latest strategies to be considered in the search for a specific inhibitor of its expression are presented.

Stromelysin-2 (matrix metalloproteinase 10) is inducible in lymphoma cells and accelerates the growth of lymphoid tumors in vivo.

Matrix metalloproteinase (MMP) 10 (stromelysin-2) is known to degrade various components of the extracellular matrix; however, the signals that regulate its expression and its role in lymphoma growth remain unknown. In the present work, we report the up-regulated expression of MMP10 in T lymphoma cells following contact with endothelial cells. The induction of MMP10 was found to be dependent on the specific interaction between LFA-1 and ICAM-1, which play a central role in regulating the expression of genes involved in the rate-limiting steps of lymphoma development. MMP10, but not MMP3 (stromelysin-1), was also up-regulated in human B lymphoma cells following exposure to IL-4, IL-6, and IL-13, but not to IL-1. To gain further insight into the role of MMP10 in lymphoma development, we generated lymphoma cell lines constitutively expressing high levels of MMP10 and studied these cells for their ability to form thymic lymphoma in vivo. Mice injected with lymphoma cells constitutively expressing MMP10 developed thymic lymphoma more rapidly than those injected with control lymphoma cells. These results provide the first in vivo evidence that overexpression of MMP10 promotes tumor development, and indicate that MMP10 induction is an important pathway activated not only upon ICAM-1/LFA-1-mediated intercellular contact, but also following activation of tumor cells with inflammatory cytokines.

Stromelysin-1 (MMP-3) is inducible in T lymphoma cells and accelerates the growth of lymphoid tumors in vivo.

Matrix metalloproteinase (MMP)-3 (stromelysin-1) degrades various components of the extracellular matrix as well as several non-matrix components; it has notably been shown to activate other MMPs relevant to cancer and metastasis, including MMP-9. MMP-3 gene expression in the tumor microenvironment could therefore contribute to cancer progression. Transcriptional regulation of MMP genes was often described to occur upon intercellular interactions, leading to overexpression of these genes by cancer and/or stromal cells. In the present work, we report that expression of MMP-3 in T lymphoma cells is transiently induced during specific intercellular contact with endothelial cells (EC). Moreover, mice injected with lymphoma cells expressing MMP-3 constitutively developed thymic lymphoma more rapidly than those injected with control lymphoma cells. We also found that overexpression of MMP-3 in lymphoma transfectants significantly improved their ability to migrate through the matrix when compared to cells transfected with the control vector. These results provide the first in vivo evidence that local expression of MMP-3 promotes lymphoma progression and indicate that MMP-3 expression is tightly regulated upon lymphoma cell/stromal cell interaction.

Emerging features in the regulation of MMP-9 gene expression for the development of novel molecular targets and therapeutic strategies.

Matrix metalloproteinase 9 (MMP-9; gelatinase B) belongs to the subfamily of MMPs that play an important role in tissue remodelling in normal and pathological inflammatory processes. MMP-9 is a major secretion product of macrophages and a component of cytoplasmic granules of neutrophils. The enzyme is also secreted by lymphocytes and stromal cells upon stimulation by inflammatory cytokines, or upon delivery of bi-directional activation signals following integrin-mediated cell-cell or cell-extracellular matrix (ECM) contacts. Since the integrity of the tissue architecture is closely dependent of the delicate balance between MMPs and their inhibitors, excessive production of MMP-9 is linked to tissue damage and degenerative inflammatory disorders. As a consequence, regulation of gene transcription and tissue-specific expression of MMP-9 in normal and diseased states are being actively investigated to pave the way for new therapeutic targets. The objective of this article is to provide an overview of recent developments in the field of mmp-9 gene expression in different cell types, from the triggering of cell-surface receptors, to the activation of cytoplasmic mediators and transcription factors responsible for the activation of MMP-9 promoter. We will then focus on emerging evidence showing that transcription of mmp-9 gene can be controlled by epigenetic mechanisms. The usefulness of targeting the signalling pathways regulating MMP-9 expression for the treatment of inflammatory disorders and other indications will be discussed in light of these findings.

Evidence for the role of promoter methylation in the regulation of MMP-9 gene expression.

Several studies have reported that elevated MMP-9 expression in lymphoma tissues correlated with tumor stage, grade, or prognosis. Because the DNA methylation pattern is critical for gene expression, detailed methylation analysis using genomic bisulfite sequencing was performed on a series of lymphoma cell lines. We found an inverse correlation between level of methylation of the MMP-9 promoter and the level of MMP-9 expression. Treating lymphoma cells with a DNA methylation inhibitor decreased MMP-9 promoter methylation and increased MMP-9 messenger RNA and protein secretion. This increased expression was potentiated by PMA, a known stimulus of MMP-9 in lymphoma cells. Finally, experiments using in vitro methylated MMP-9 promoter constructs confirmed the fact that DNA methylation exerts suppression on transcriptional activity. The results thus indicate that methylation may contribute to the transcriptional activity of the MMP-9 promoter.