The BRAFV600E mutation, which approaches 50% in human melanomas, constitutively activates pERK and contributes to disease progression. The BRAFV600E inhibitor, Vemurafenib (PLX4032), shows promising clinical responses, but resistance to PLX4032 usually develops within a year. Transgenic mouse models allow the study of BRafV600E melanoma in vivo, however in vitro models are necessary to better understand the molecular mechanism underlying disease progression and resistance. We established melanoma cell lines (D4M cells) from the conditional mouse model of metastatic melanoma: Tyr::CreER;BrafCA;Ptenlox/lox, which recapitulates human disease. Cultured D4M cells express high constitutive pERK. PLX4032 abrogates ERK phosphorylation, inhibits D4M proliferation, and increases expression of the melanoma associated antigen, pmel, in vitro, consistent with human BRAFV600E melanoma cell lines. D4M cells are transplantable in either immune-compromised or syngeneic B6 mice. Thus, D4M cell lines allow correlation of in vitro studies on molecular mechanisms of melanoma with in vivo investigations on pathology and immunology.
Murine BRafV600E; melanoma; pERK; Vemurafenib; PLX4032; B6 syngeneic mouse model; cell culture
We used vertical growth phase (VGP) human VMM5 melanoma cells to ask whether the tumor microenvironment could induce matrix metalloproteinase-1 (MMP-1) in vivo, and whether this induction correlated with metastasis. We isolated two clones from parental VMM5 cells: a low MMP-1 producing clone (C4) and high producing clone (C9). When these clones were injected orthotopically (intradermally) into nude mice, both were equally tumorigenic and produced equivalent and abundant amounts of MMP-1. However, the tumors from the C4 clones displayed different growth kinetics and distinct profiles of gene expression from the C9 population. The C4 tumors, which had low MMP-1 levels in vitro, appeared to rely on growth factors and cytokines in the microenvironment to increase MMP-1 expression in vivo, while MMP-1 levels remained constant in the C9 tumors. C9 cells, but not C4 cells, grew as spheres in culture and expressed higher levels of JARID 1B, a marker associated with melanoma initiating cells. We conclude that VMM5 melanoma cells exhibit striking intra-tumor heterogeneity, and that the tumorigenicity of these clones is driven by different molecular pathways. Our data suggest that there are multiple mechanisms for melanoma progression within a tumor, which may require different therapeutic strategies.
Similarities in the pathologies of autoimmune diseases and cancer have been noted for at least 30 years. Inflammatory cytokines and growth factors mediate cell proliferation, and proteinases, especially the collagenase, Matrix Metalloproteinase-1 (MMP-1), contribute to disease progression by remodeling the extracellular matrix and modulating the microenvironment. This review focuses on two cancers (melanoma and breast) and on the autoimmune disorder, rheumatoid arthritis (RA), and discusses the activated stromal cells found in these diseases. MMP-1 was originally thought to function only to degrade interstitial collagens, but recent studies have revealed novel roles for MMP-1 involving the G protein-coupled receptors: the chemokine receptor, CXCR-4, and Protease Activated Receptor-1 (PAR-1). Cooperativity between MMP-1 and CXCR4/SDF-1 signaling influences the behavior of activated fibroblasts in both RA and cancer. Further, MMP-1 is a vital part of an autocrine/paracrine MMP-1/PAR-1 signal transduction axis, a function that amplifies its potential to remodel the matrix and to modify cell behavior. Finally, new therapeutic agents directed at MMP-1 and G protein-coupled receptors are emerging. Even though these agents are more specific in their targets than past therapies, these targets are often shared between RA and cancer, underscoring fundamental similarities between autoimmune disorders and some cancers.
rheumatoid arthritis; collagenase; CXCR4; PAR-1; synovial fibroblasts; carcinoma associated fibroblasts; endothelial cells
Despite recent advances in cancer therapies, metastatic renal cell carcinoma (RCC) remains difficult to treat. Most RCCs result from inactivation of the von Hippel Lindau (VHL) tumor suppressor, leading to stable expression of Hypoxia-Inducible Factor-α (HIF-1α, -2α, -3α) and the induction of downstream target genes, including those responsible for angiogenesis and metastasis. While VHL is inactivated in the majority of RCC cases, expression of the PTEN tumor suppressor is reduced in about 30% of cases. PTEN functions to antagonize PI3K/Akt/mTOR signaling, thereby controlling cell growth and survival. Activation of PI3K/Akt/mTOR leads to increased HIF-1α expression in certain cancer cells, supporting the rationale of using mTOR inhibitors as anti-cancer agents. Notably, HIF-2α, rather than HIF-1α, has been shown to play a critical role in renal tumorigenesis. To investigate whether HIF-2α is similarly regulated by the PI3K pathway in VHL−/− RCC cells, we manipulated PI3K signaling using PTEN overexpression and siRNA knockdown studies and pharmacologic inhibition of PI3K or Akt. Our data support a novel role for wild-type PTEN in promoting HIF-2α activity in VHL null RCC cells. This mechanism is unique to the cellular environment in which HIF-2α expression is deregulated, resulting from the loss of VHL function. Our data show that PTEN induces HIF-2α transcriptional activity by inhibiting expression of Yin Yang 1 (YY1), which acts as a novel corepressor of HIF-2α. Further, PTEN suppression of YY1 is mediated through antagonism of PI3K signaling. We conclude that wild-type PTEN relieves the repressive nature of YY1 at certain HIF-2α target promoters and that this mechanism may promote early renal tumorigenesis resulting from VHL inactivation by increasing HIF-2α activity.
PTEN; YY1; VHL; HIF-2α; renal cell carcinoma; MT1-MMP
Metastatic renal cell carcinoma (RCC) resulting from the hereditary loss of the von Hippel Lindau (VHL) tumor suppressor gene is the leading cause of death in VHL patients due to the deleterious effects of the metastatic tumor(s). VHL functions in the destruction of the alpha subunits of the heterodimeric transcription factor, Hypoxia-Inducible Factor (HIF-1α and HIF-2α), in normoxic conditions. When VHL function is lost, HIF-α protein is stabilized, and target hypoxia-inducible genes are transcribed. The process of tumor invasion and metastasis involves the destruction of the extracellular matrix (ECM), which is accomplished primarily by the matrix metalloproteinase (MMP) family of enzymes. Here, we describe a connection between the loss of VHL tumor suppressor function and the upregulation of Membrane Type-1 MMP (MT1-MMP) gene expression and protein. Specifically, MT1-MMP is upregulated in VHL−/− RCC cells through an increase in gene transcription, which is mediated by the cooperative effects of the transcription factors, HIF-2 and Sp1. Further, we identify a functional HIF binding site (HBS) in the proximal promoter of MT1-MMP. To our knowledge, this is the first report to show direct regulation of MT1-MMP by HIF-2 and to provide a direct link between the loss of VHL tumor suppressor function and an increase in MMP gene and protein expression.
von Hippel Lindau; MT1-MMP; HIF-2α; renal cell carcinoma
Matrix Metalloproteinases (MMPs) comprise a family of more than 20 members, each with the ability to degrade components of the extracellular matrix. The interstitial collagenases have the unique capacity to degrade the stromal collagens, types I, II and III, the body's most abundant proteins. These collagenases include MMP-1, MMP-8, MMP-13 and MMP-14. MMP-1, with a very broad expression pattern, has major roles in mediating matrix destruction in many diseases. We have described a single nucleotide polymorphism (SNP) in the MMP-1 promoter that augments transcription. This SNP is the presence or absence of an extra guanine (G) at -1607 bp, which creates the sequence 5'-GGAA-3'(2G allele), and which is an ETS binding site. Compared to the 1G allele (5'-GAA-3'), the 2G SNP is associated with enhanced transcription of MMP-1 and increased enzymatic activity.
Although murine systems are often used to model human diseases, mice have only distant homologues of human MMP-1. Therefore, we used a technique for the targeted insertion of a single copy of a gene at the HPRT locus to compare expression of the 1G and 2G alleles. We generated transgenic mice with -4372 bp of the human MMP-1 promoter containing either the 1G or 2G SNP in front of the Lac Z (E.coli ß-galactosidase) gene. We measured relative expression of the transgenes in vitro in embryonic stem (ES) cells and in fibroblasts derived from embryonic mice. Our data show modest constitutive expression of ß-galactosidase mRNA and protein from these alleles, with the 2G allele more transcriptionally active than the 1G allele. We conclude that these mice represent a model for integration of a single copy of the human MMP-1 promoter into the murine genome, and could be used to study MMP-1 gene expression in a murine system.
mRNA; ß-galactosidase; gene expression; single nucleotide polymorphism; fibroblasts
Metastatic renal cell carcinoma (RCC) remains the leading cause of mortality in patients with clear cell RCC arising from mutations in the von Hippel Lindau (VHL) tumor suppressor. Successful RCC tumor suppression by VHL requires the negative regulation of hypoxia inducible factor alpha (HIF alpha) protein and its downstream targets. Thus, identification of HIF target genes responsible for RCC tumor progression will aid in the development of therapies for this disease. We previously identified membrane type-1 matrix metalloproteinase (MT1-MMP) as a transcriptional target of HIF-2alpha in RCC cells null for VHL and showed that MT1-MMP is overexpressed in these cells. MT1-MMP is a key regulator of tumor progression through its functions as a matrix-degrading enzyme, as well as its ability to cleave factors, such as adhesion molecules and other MMPs. The aim of this study was to investigate the contribution of MT1-MMP to the invasive potential of RCC cells using in vitro type I collagen degradation and invasion assays.
We evaluated RCC cells wild-type (WT8) and null (pRc-9) for VHL for invasive characteristics and showed that the pRc-9 cells demonstrated a greater propensity for both invasion and degradation of a type I collagen matrix. Furthermore, overexpression of either HIF-2alpha or MT1-MMP in the poorly invasive cell line, WT8, promoted collagen degradation and invasion of these cells. Finally, using RNAi, we show that inhibition of MT1-MMP suppresses tumor cell invasion of RCC cells.
Our results suggest that MT1-MMP is a major mediator of tumor cell invasiveness and type I collagen degradation by VHL RCC cells that express either MT1-MMP or HIF-2alpha. As such, MT1-MMP may represent a novel target for anti-invasion therapy for this disease.
Patients with early stage, radial growth phase (RGP) melanoma have a 97% survival rate; however, when the melanoma progresses to the invasive vertical growth phase (VGP), survival rates decrease to 15%. The targets of many clinical trials are the known genetic and molecular mechanisms involved in melanoma progression, with the most common oncogenic mutation being the BRAFV600E. However, less than half of melanomas harbor this mutation, and consequently, do not respond to the current BRAF targeted treatments. It is therefore critical to elucidate alternative mechanisms regulating melanoma progression. Increased expression of the chemokine receptor, CXCR3, on melanoma cells is correlated with increased metastasis and poor patient outcomes, suggesting a role for CXCR3 in the RGP to VGP transition. We found that endogenous CXCR3 can be induced in two RGP cell lines, BOWES (BRAFWT) and WM35 (BRAFV600E), with in vitro environmental stress and nutrient deprivation. Signaling via induced endogenous CXCR3 is linked with IL-8 expression in BOWES cells. Ectopic overexpression of CXCR3 in BOWES cells leads to increased ligand-mediated phERK, cellular migration, and IL-8 expression in vitro, and to increased tumorigenesis and lymph node metastasis in vivo. Our results demonstrate that, in BRAFWT melanomas, CXCR3 signaling mediates significant increases in IL-8 expression, suggesting that CXCR3 expression and signaling may represent a transformative event that drives the progression of BRAFWT melanomas. Implications: Expression of CXCR3 on BRAFWT melanoma cells may be a mediator of melanoma progression.
Osteoarthritic chondrocytes secrete matrix metalloproteinase-13 (MMP-13) in response to interleukin-1 (IL-1), causing digestion of type II collagen in cartilage. Using chondrocytic cells, we previously determined that IL-1 induced a strong MMP-13 transcriptional response that requires p38 MAPK, JNK and the transcription factor NF-κB. Now, we have studied the tissue-specific transcriptional regulation of MMP-13. Constitutive expression of the transcription factor Runx-2 correlated with the ability of a cell type to express MMP-13 and was required for IL-1 induction; moreover, Runx-2 enhanced IL-1 induction of MMP-13 transcription by synergizing with the p38 MAPK signaling pathway. Transiently transfected MMP-13 promoters were not IL-1 inducible. However, –405 bp of stably integrated promoter was sufficient for 5- to 6-fold IL-1 induction of reporter activity and this integrated reporter required the same p38 MAPK pathway as the endogenous gene. Finally, mutation of the proximal Runx binding site and the proximal AP-1 site blunted the transcriptional response to IL-1, and double mutation synergistically decreased reporter activity. In summary, our data suggest that the transcriptional MMP-13 response to IL-1 is controlled by the p38 pathway interacting at the MMP-13 promoter through the tissue-specific transcription factor Runx-2 and the ubiquitous AP-1 transcription factor.
Hallmarks of malignant melanoma are its propensity to metastasize and its resistance to treatment, giving patients with advanced disease a poor prognosis. The transition of melanoma from non-invasive radial growth phase (RGP) to invasive and metastatically competent vertical growth phase (VGP) is a major step in tumor progression, yet the mechanisms governing this transformation are unknown. Matrix Metalloproteinase-1 (MMP-1) is highly expressed by VGP melanomas, and is thought to contribute to melanoma progression by degrading type I collagen within the skin to facilitate melanoma invasion. Protease activated receptor-1 (PAR-1) is activated by MMP-1, and is also expressed by VGP melanomas. However, the effects MMP-1 signaling through PAR-1 have not been examined in melanoma. Here, we demonstrate that an MMP-1/PAR-1 signaling axis exists in VGP melanoma, and is necessary for melanoma invasion. Introduction of MMP-1 into RGP melanoma cells induced gene expression associated with tumor progression and promoted invasion in vitro, and enhanced tumor growth and conferred metastatic capability in vivo. This study demonstrates that both the type I collagenase and PAR-1 activating functions of MMP-1 are required for melanoma progression, and suggests that MMP-1 may be a major contributor to the transformation of melanoma from non-invasive to malignant disease.
MMP-1; PAR-1; angiogenesis; Superarray; xenograft
Tobacco-related diseases are leading causes of death worldwide, and many are associated with expression of matrix metalloproteinase-1 (MMP-1). We have reported extracellular signal–regulated kinase (ERK)1/2-dependent induction of MMP-1 by cigarette smoke in lung epithelial cells. Our objectives were to define regions of the human MMP-1 promoter required for activation by smoke, to identify differences in responses of the 1G/2G −1607 polymorphic promoters to smoke, and to identify relevant transcription factors whose activity in airway epithelial cells is increased by smoke. The responses of deletion and mutant promoter constructs were measured in transfected cells during exposure to cigarette smoke extract (CSE). DNA oligonucleotide arrays were used to identify transcription factors activated after smoke exposure. CSE activated the MMP-1 promoter, and this induction was prevented by PD98059 blockade of ERK1/2 phosphorylation. Deletion studies revealed the distal 1kb promoter region (−4438 to −3280 upstream of the transcription start site) is essential for CSE induction of MMP-1, and confers activation of a minimal promoter. Studies of 1G and 2G MMP-1 polymorphic promoter variants revealed higher 2G allele basal and CSE-responsive activities than the 1G allele. Cotransfection, mithramycin, and electrophoretic mobility shift assay studies identified activating and repressive roles for Sp1 and PEA3 transcription factors, respectively. Oligonucleotide DNA arrays confirmed activation of Sp1 and PEA3 by CSE. These data demonstrate that the MMP-1 promoter is a direct target of cigarette smoke in lung epithelial cells. This characterization of a smoke response region in the distal MMP-1 promoter has implications for smoking-related diseases such as cancer, heart disease, and emphysema.
metalloproteinase; emphysema; tobacco; chronic obstructive pulmonary disease; polymorphism
The shared characteristics of rheumatoid arthritis (RA) and cancer, particularly their unchecked growth and invasive behaviors, have been apparent for some time. However, the molecular mechanisms underlying these similarities are not clear. In a recent issue of Arthritis Research & Therapy, Abreu and colleagues link a well-studied oncogene, Ras, with expression of matrix metalloproteinase-3 (MMP-3) in RA. Their study correlates expression of the Ras guanine nucleotide exchange factor RasGRF1 with MMP-3 expression in RA synovium. They elucidate a potential mechanism of regulation of MMP-3 expression in RA, suggesting a potential target for RA treatment.
Diesel exhaust particles (DEPs) are globally relevant air pollutants that exert a detrimental human health impact. However, mechanisms of damage by DEP exposure to human respiratory health and human susceptibility factors are only partially known. Matrix metalloproteinase-1 (MMP-1) has been implied as an (etio)pathogenic factor in human lung and airway diseases such as emphysema, chronic obstructive pulmonary disease, chronic asthma, tuberculosis, and bronchial carcinoma and has been reported to be regulated by DEPs.
We elucidated the molecular mechanisms of DEPs’ up-regulation of MMP-1.
Using permanent and primary human bronchial epithelial (HBE) cells at air–liquid interface, we show that DEPs activate the human MMP-1 gene via RAS and subsequent activation of RAF-MEK-ERK1/2 mitogen-activated protein kinase signaling, which can be scaffolded by β-arrestins. Short interfering RNA mediated β-arrestin1/2 knockout eliminated formation, subsequent nuclear trafficking of phosphorylated ERK1/2, and resulting MMP-1 transcriptional activation. Transcriptional regulation of the human MMP-1 promoter was strongly influenced by the presence of the –1607GG polymorphism, present in 60–80% of humans, which led to striking up-regulation of MMP-1 transcriptional activation.
Our results confirm up-regulation of MMP-1 in response to DEPs in HBE and provide new mechanistic insight into how these epithelia, the first line of protection against environmental insults, up-regulate MMP-1 in response to DEP inhalation. These mechanisms include a role for the human –1607GG polymorphism as a susceptibility factor for an accentuated response, which critically depends on the ability of β-arrestin1/2 to generate scaffolding and nuclear trafficking of phosphorylated ERK1/2.
β-arrestin; bronchial epithelia; diesel particles; MAP kinase; MMP-1; MMP-1 promoter polymorphism; urban smog
We recently described the ability of retinoid X receptor (RXR) ligand LG100268 (LG268) to inhibit interleukin-1-beta (IL-1-β)-driven matrix metalloproteinase-1 (MMP-1) and MMP-13 gene expression in SW-1353 chondrosarcoma cells. Other investigators have demonstrated similar effects in chondrocytes treated with rosiglitazone, a ligand for peroxisome proliferator-activated receptor-gamma (PPARγ), for which RXR is an obligate dimerization partner. The goals of this study were to evaluate the inhibition of IL-1-β-induced expression of MMP-1 and MMP-13 by combinatorial treatment with RXR and PPARγ ligands and to investigate the molecular mechanisms of this inhibition.
We used real-time reverse transcription-polymerase chain reaction to measure LG268- and rosiglitazone-mediated inhibition of MMP gene transcription in IL-1-β-treated SW-1353 chondrosarcoma cells. An in vitro collagen destruction assay was a functional readout of MMP collagenolytic activity. Luciferase reporter assays tested the function of a putative regulatory element in the promoters of MMP-1 and MMP-13, and chromatin immunoprecipitation (ChIP) assays detected PPARγ and changes in histone acetylation at this site. Post-translational modification of RXR and PPARγ by small ubiquitin-like modifier (SUMO) was assayed with immunoprecipitation and Western blot.
Rosiglitazone inhibited MMP-1 and MMP-13 expression in IL-1-β-treated SW-1353 cells at the mRNA and heterogeneous nuclear RNA levels and blunted IL-1-β-induced collagen destruction in vitro. Combining LG268 and rosiglitazone had an additive inhibitory effect on MMP-1 and MMP-13 transcription and collagenolysis. IL-1-β inhibited luciferase expression in the MMP reporter assay, but rosiglitazone and LG268 had no effect. ChIP indicated that treatment with IL-1-β, but not LG268 and rosiglitazone, increased PPARγ at the proximal promoters of both MMPs. Finally, rosiglitazone or LG268 induced 'cross-SUMOylation' of both the target receptor and its binding partner, and IL-1-β-alone had no effect on SUMOylation of RXR and PPARγ but antagonized the ligand-induced SUMOylation of both receptors.
The PPARγ and RXR ligands rosiglitazone and LG268 may act through similar mechanisms, inhibiting MMP-1 and MMP-13 transcription. Combinatorial treatment activates each partner of the RXR:PPARγ heterodimer and inhibits IL-1-β-induced expression of MMP-1 and MMP-13 more effectively than either compound alone. We conclude that the efficacy of combined treatment with lower doses of each drug may minimize potential side effects of treatment with these compounds.
Matrix metalloproteinase (MMP)-1, MMP-8 and MMP-13 are interstitial collagenases that degrade type II collagen in cartilage; this is a committed step in the progression of rheumatoid arthritis and osteoarthritis. Of these enzymes, the expression of MMP-1 and MMP-13 is substantially increased in response to IL-1 and tumor necrosis factor-α, and elevated levels of these collagenases are observed in arthritic tissues. Therefore, cytokine-mediated MMP-1 and MMP-13 gene regulation is an important issue in arthritis research. In this review, we discuss current models of MMP-1 and MMP-13 transcriptional regulation, with a focus on signaling intermediates and transcription factors that may be future targets for the development of new arthritis drugs.
arthritis; matrix metalloproteinases; mitogen-activated protein kinases; nuclear factor κB; transcription
Recent work has established that IL-1β plays a central role in the inflammation and connective tissue destruction observed in both rheumatoid arthritis and osteoarthritis. These processes result from the ability of this inflammatory cytokine to activate expression of genes for neutral proteases, such as the matrix metalloproteinases. While IL-1β activates matrix metalloproteinase genes within several hours, it also activates immediate early genes, which are required for the later expression of matrix metalloproteinases and other arthritis-perpetuating genes, are also activated. To identify putative immediate early genes involved in IL-1β-mediated arthritic disease, a chondrocytic cell line (SW1353) was stimulated with this cytokine for 2 hours, total RNA was isolated, and expressed genes were identified by microarray analysis. This analysis identified alterations in the expression of multiple transcription factors, cytokines, growth factors and their receptors, adhesion molecules, proteases, and signaling intermediates that may contribute to inflammation and cartilage destruction in arthritis. Interestingly, confirmation of the expression of activating protein-1 family members by reverse transcriptase polymerase chain reaction revealed a preferential increase in junB, a known transcriptional antagonist of c-jun. The failure to observe induction of early growth response gene-1, which was detected by reverse transcriptase polymerase chain reaction to be substantially and transiently induced by 1 hour of IL-1 treatment, may be explained by the known instability of the message after early induction. However, this analysis has identified numerous IL-1β-responsive genes that warrant further investigation as mediators of disease in arthritis.
chondrocytes; interleukin-1; matrix metalloproteinases; signal transduction; transcription factors