Melanoma progresses as a multistep process where the thickness of the lesion and depth of tumor invasion are the best prognostic indicators of clinical outcome. Degradation of the interstitial collagens in the extracellular matrix is an integral component of tumor invasion and metastasis, and much of this degradation is mediated by collagenase-1 (MMP-1), a member of the matrix metalloproteinase (MMP) family. MMP-1 levels increase during melanoma progression where they are associated with shorter disease-free survival. The Ras/Raf/ MEK/ERK mitogen-activated protein kinase (MAPK) pathway is a major regulator of melanoma cell proliferation. Recently, BRAF has been identified as a common site of activating mutations, and, although many reports focus on its growth-promoting effects, this pathway has also been implicated in progression toward metastatic disease. In this study, we describe four melanoma cell lines that produce high levels of MMP-1 constitutively. In each cell line the Ras/Raf/MEK/ERK pathway is constitutively active and is the dominant pathway driving the production of MMP-1. Activation of this pathway arises due to either an activating mutation in BRAF (three cell lines) or autocrine fibroblast growth factor signaling (one cell line). Furthermore, blocking MEK/ ERK activity inhibits melanoma cell proliferation and abrogates collagen degradation, thus decreasing their metastatic potential. Importantly, this inhibition of invasive behavior can occur in the absence of any detectable changes in cell proliferation and survival. Thus, constitutive activation of this MAPK pathway not only promotes the increased proliferation of melanoma cells but is also important for the acquisition of an invasive phenotype.

Increased matrix metalloproteinase-1 (MMP-1) expression is associated with advanced stages of breast cancer and may be a predictive marker for the development of invasive disease. In this report, we used short hairpin RNA (shRNA) molecules to investigate whether MMP-1 production in MDA-231 breast cancer cells contributed to the degradation of a collagen matrix or tumor formation in nude mice. We created two groups of MDA-231 cell lines. MDA-231 cells containing a vector producing shRNA specific for MMP-1 had a >90% decrease in MMP-1 mRNA and protein compared with cells containing an empty vector, and blocking MMP-1 expression inhibited the in vitro collagenolytic activity of MDA-231 cells. When the cells were injected into the mammary fat pad, there was no difference in the frequency of tumor formation in mice. However, the average tumor size was larger in mice injected with cells containing the empty vector (1,216 F 334 mm 3 ) than in mice injected with cells expressing the MMP-1 shRNA (272 F 117 mm 3 ; P = 0.027). We conclude that MMP-1 expression is essential for the ability of MDA-231 cells to invade and destroy a collagen matrix and in vivo experiments suggest an important role for MMP-1 in breast tumor growth. (Cancer Res 2005; 65(23): 11101-8)

Most cases of hepatitis C virus (HCV) infection result inHepatitis C virus (HCV) is the most important causative agent of blood-borne non-A non-B hepatitis, infecting a million people a year worldwide.

Objectives:Pancreatic insufficiency (PI) and malabsorption of fats lead to reduced caloric intake, inability to maintain weight, and increased gastrointestinal symptoms. Thus, enteral nutrition (EN) is used in patients with cystic fibrosis (CF) and poor nutritional status. The current study evaluated safety, tolerability, and improvement of fatty acid (FA) status in red blood cell (RBC) membranes, a marker of long-term FA absorption, with an in-line digestive cartridge (RELiZORB) that hydrolyzes fat in enteral formula.Methods:Patients with CF receiving EN participated in a multicenter, 90-day open-label study during which RELiZORB was used with overnight EN. The primary endpoint was change over time in RBC uptake of docosahexaenoic acid (DHA)+ eicosapentaenoic acid (EPA). Gastrointestinal symptoms were collected to evaluate safety and tolerability. Several clinical and anthropometric parameters were also assessed throughout the study.Results:A total of 36 subjects completed the study with a mean age of 13.8 years, body mass index of 17.7 and 6.2 years mean use of overnight EN. Fat absorption significantly improved as shown by increased RBC levels of DHA+EPA, improved ω-6/ω-3 ratio, and increased plasma levels of DHA+EPA. RELiZORB use was not associated with any unanticipated adverse events.Conclusions:RELiZORB use was found to be safe, well tolerated, and resulted in increased levels of FAs in RBCs and plasma. This is the first prospective study to show EN can improve FA abnormalities in CF. Because improvement in omega-3 levels has been shown to help pulmonary and inflammatory status as well as anthropometric parameters in CF, RELiZORB may have important long-term therapeutic benefits in patients with CF.

Matrix metalloproteinase-1 (MMP-1) is one of only a few enzymes with the ability to degrade the stromal collagens (types I and III) at neutral pH, and high expression of MMP-1 has been associated with aggressive and invasive cancers. We recently reported a single nucleotide insertion/deletion polymorphism (SNP) in the collagenase-1 (MMP-1) promoter (Rutter et al. [1998] Can. Res. 58:5321-5325), where the insertion of an extra guanine (G) at -1607 bp creates the sequence, 5'-GGAA-3 (2G allele), compared to the sequence 5'-GAA-3' (1G allele). The presence of 2G constitutes a binding site for the ETS family of transcription factors, and increases MMP-1 transcription in fibroblasts and A2058 melanoma cells cultured in vitro. In addition, the presence of the 2G allele has been linked to several aggressive malignancies as well as to enhanced expression of MMP-1. In this study, we describe a melanoma cell line, VMM5, that is 1G homozygous, but that is invasive and expresses high levels of MMP-1 constitutively. The high level of MMP-1 expression in VMM5 cells is due to the utilization of both the p38 and ERK1/2 transduction pathways. In contrast, in the A2058 cell line, which also expresses MMP-1 constitutively and which is 2G homozygous, only the ERK pathway is activated. Thus, our data suggest that in the absence of 2G allele and in the presence of the appropriate transcription factors, tumor cells may use alternative signal/transduction pathways and cis-acting sequences to achieve high levels of MMP-1 expression, which contribute to the ability of tumor cells to invade, regardless of their genotype.

We have previously reported that chimpanzees chronically infected with hepatitis C virus (HCV) could be reinfected, even with the original infecting strain. In this study we tested the hypothesis that this might reflect the presence of minor quasispecies to which there was little or no immunity. To evaluate this hypothesis, we sequenced multiple clones taken at intervals after primary infection and rechallenge from four chronically infected chimpanzees. The inoculum used in these studies (HCV-H, genotype 1a) revealed 17 separate variants among 46 clones sequenced. Following challenge, each of the four challenged animals showed marked alterations of their quasispecies distribution. The new variants, which appeared 1 to 6 weeks after challenge, were either identical to or closely resembled variants present in the challenge inoculum. These results, paralleled by an increase in viremia in some of the challenged animals, suggest that quasispecies in the challenge inoculum were responsible for signs of reinfection and that there was little immunity. However, the newly emerged quasispecies completely took over infection in only one animal. In the remaining three chimpanzees the prechallenge quasispecies were able to persist. The natural evolution of infection within chimpanzees resulted in variants able to compete with the inoculum variants. Whether through reexposure or the natural progression of infection, newly emerged quasispecies are likely to play a role in the pathogenesis of chronic HCV infection.

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.

SUPPLEMENTARY NOTES 12a. DISTRIBUTION / AVAILABILITY STATEMENTApproved for Public Release; Distribution Unlimited 12b. DISTRIBUTION CODE Abstract (Maximum 200 Words) (abstract should contain no proprietary or confidential information)The lethality of breast cancer is derived from its ability to metastasize, and matrix metalloproteinases (MMPs) facilitate cellular invasion by degrading the extracellular matrix. One component of MMP regulation is transcription. We have identified a single nucleotide polymorphism (SNP) that enhances the transcriptional activity of MMP-1 promoters in transient transfections.The SNP consists of an extra guanine nucleotide that creates an ETS family transcription factor binding site (2G). We utilized cotransfection experiments, and determined that MMP-1 promoters containing the 2G polymorphism can interfere with transcriptional activity of promoters with the IG polymorphism, but only at high concentrations of DNA. Analysis of five breast cancer cell lines with transient transfections reveals that the transcriptional effect of the 2G SNP is only observed in one cell line, and this cell line was the only one examined that expressed endogenous MMP-1. To better address the role of the polymorphism in endogenous gene expression, more samples are needed, and a source of those samples is human foreskin fibroblasts (HFFs References 9 Appendices 10 Introduction:The lethality of breast cancer is derived from its ability to metastasize, and a critical component of the metastatic mechanism is the degradation of extracellular matrix (ECM). Degradation of ECM is largely mediated by a family of proteins known as matrix metalloproteinases (MMPs). One subfamily of MMPs, the collagenases, are able to degrade stromal collagens type I, II, and III, and the most widely expressed collagenase is collagenase 1 (MMP-1). We have identified a single nucleotide polymorphism (SNP) that enhances the transcriptional activity of the MMP-1 promoter in tumor cell lines and normal stromal cells. The SNP consists of an additional guanine nucleotide creating an Ets transcription factor-binding site (5'-AGGA-3') at position -1607 in the MMP-1 promoter. When this 2G polymorphism is present, transcriptional activity from the MMP-1 promoter, as measured by transient transfections, is increased an average of 6 fold over the IG polymorphism. A possible role for the SNP in cancer was suggested by observations of increased frequency of the 2G polymorphism in breast cancer cell lines and Japanese cancer patients as compared to normal DNA samples and non-patients(l, 2, 3). The MMP-1 gene is located on chromosome 11 (1 lq22.2-22.3) in a region associated with loss of heterozygosity in breast cancer patients. The studies outlined in this proposal are designed to characterize the mechanism of enhanced transcription from promoters with the 2G polymorphism, and to determine the effect of the polymorphism on transcription of the endogenous MMP-1 gene. We also hope to elucidate the role of the polymorphism and increased MMP-1 production...