Rat Mammary Adenocarcinoma Cell Line Research Articles

Identification and characterization of metastasis-associated gene/protein 1 (MTA1).

Metastasis is a complex series of sequential events involving several gene products and the regulated expression of several tumor cell genes. Using rat mammary adenocarcinoma cell lines of differing metastatic potentials and a differential complementary DNA (cDNA) hybridization method, our laboratory embarked in 1992 on a project to identify candidate metastasis-associated genes. Among the genes that were found to be abundantly overexpressed in highly metastatic rat cell lines compared to poorly metastatic cell lines, we identified a completely novel gene without any homologous or related genes in the database in 1994. The full-length cDNA of this gene was cloned, sequenced, and named mta1 (metastasis-associated gene 1), and eventually, its human cDNA counterpart, MTA1, was also cloned and sequenced by our group. MTA1 has now been identified as one of the members of a gene family (MTA gene family). The products of the MTA genes, the MTA proteins, are transcriptional co-regulators that function in histone deacetylation and nucleosome remodeling. In this review, we will briefly discuss the researches for the identification and characterization of the mta1 gene, its human counterpart MTA1, and their protein products.

Opposing roles of CXCR4 and CXCR7 in breast cancer metastasis

IntroductionCXCL12-CXCR4 signaling has been shown to play a role in breast cancer progression by enhancing tumor growth, angiogenesis, triggering cancer cell invasion in vitro, and guiding cancer cells to their sites of metastasis. However, CXCR7 also binds to CXCL12 and has been recently found to enhance lung and breast primary tumor growth, as well as metastasis formation. Our goal was to dissect the contributions of CXCR4 and CXCR7 to the different steps of metastasis - in vivo invasion, intravasation and metastasis formation.MethodsWe overexpressed CXCR4, CXCR7 or both in the rat mammary adenocarcinoma cell line MTLn3. Stable expressors were used to form tumors in severe combined immunodeficiency (SCID) mice, and in vivo invasiveness, intravital motility, intravasation, and metastasis were measured.ResultsWe found that CXCR4 overexpression increased the chemotactic and invasive behavior of MTLn3 cells to CXCL12, both in vitro and in vivo, as well as in vivo motility and intravasation. CXCR7 overexpression enhanced primary tumor growth and angiogenesis (as indicated by microvessel density and VEGFA expression), but decreased in vivo invasion, intravasation, and metastasis formation. In vitro, expression of CXCR7 alone had no effect in chemotaxis or invasion to CXCL12. However, in the context of increased CXCR4 expression, CXCR7 enhanced chemotaxis to CXCL12 but decreased invasion in response to CXCL12 in vitro and in vivo and impaired CXCL12 stimulated matrix degradation. The changes in matrix degradation correlated with expression of matrix metalloproteinase 12 (MMP12).ConclusionsWe find that CXCR4 and CXCR7 play different roles in metastasis, with CXCR4 mediating breast cancer invasion and CXCR7 impairing invasion but enhancing primary tumor growth through angiogenesis.

Visualization of Actin Polymerization in Invasive Structures of Macrophages and Carcinoma Cells Using Photoconvertible β-Actin – Dendra2 Fusion Proteins

Actin polymerization controls a range of cellular processes, from intracellular trafficking to cell motility and invasion. Generation and elongation of free barbed ends defines the regions of actively polymerizing actin in cells and, consequently, is of importance in the understanding of the mechanisms through which actin dynamics are regulated. Herein we present a method that does not involve cell permeabilization and provides direct visualization of growing barbed ends using photoswitchable β-actin - Dendra2 constructs expressed in murine macrophage and rat mammary adenocarcinoma cell lines. The method exploits the ability of photoconverted (red) G-actin species to become incorporated into pre-existing (green) actin filaments, visualized in two distinct wavelengths using TIRF microscopy. In growing actin filaments, photoconverted (red) monomers are added to the barbed end while only green monomers are recycled from the pointed end. We demonstrate that incorporation of actin into intact podosomes of macrophages occurs constitutively and is amenable to inhibition by cytochalasin D indicating barbed end incorporation. Additionally, actin polymerization does not occur in quiescent invadopodial precursors of carcinoma cells suggesting that the filaments are capped and following epidermal growth factor stimulation actin incorporation occurs in a single but extended peak. Finally, we show that Dendra2 fused to either the N- or the C-terminus of β-actin profoundly affects its localization and incorporation in distinct F-actin structures in carcinoma cells, thus influencing the ability of monomers to be photoconverted. These data support the use of photoswitchable actin-Dendra2 constructs as powerful tools in the visualization of free barbed ends in living cells.

Abstract 3395: Role of ErbB3 signaling in tumor cell motility and metastasis

Abstract The major cause of death for most cancers, including that of the breast, is from metastasis. One of the trends in anticancer therapy research has recently been inhibiting the epidermal growth factor receptor (EGFR, ErbB) family that is part of the subclass I of the receptor tyrosine kinase superfamily. ErbB receptors are involved in activating such key pathways as PI3-kinase and Ras/MAP kinase. Close to 30% of highly invasive human breast cancers show elevated expression/activity of the ErbB2 receptor, a marker for poor prognosis and decreased disease-free survival. The preferred heterodimerization partner of ErbB2 is ErbB3. Previous data from our laboratory has shown that increasing expression of ErbB3 caused a significant increase in intravasation and metastasis without affecting primary tumor growth/formation. The ligand for ErbB3 is Heregulin Beta 1 (HRGβ1) and its binding leads to heterodimerization with other ErbB's, followed by activation of signaling pathways that result in cell chemotaxis and movement. Knowing that ErbB3 is important for metastasis, our goal is to determine which signaling pathway downstream of ErbB3 is responsible for metastasis as well as what cellular property (e.g. motility or chemotaxis) and which step of metastasis is affected (invasion, intravasation, lung seeding). Stably transduced MTLn3 cells (rat mammary adenocarcinoma cell line) overexpressing WT and mutant versions of ErbB3 defective in PI3K, MAPK and Grb7 signaling were used for in vitro and in vivo experiments examining the role of ErbB3 signaling pathways. WT and mutant versions of ErbB3 were compared in their ability to chemotax in vitro. Orthotopic injection of the WT and mutant MTLn3 cell lines into the mammary fatpads of SCID mice has allowed us to examine their in vivo invasion potential by measuring the ability of cells to migrate out of the primary tumor towards a gradient of HRGβ1, intravasation - number of cells able to enter blood stream, and live imaging using multiphoton microscopy which will allow to see tumor cell polarization and movement towards blood vessels. In addition, tail vein injection tested the ability of cells to seed the lungs. While previous data from our laboratory has shown that ErbB3 overexpression significantly enhances metastasis, it is still not known how. Preliminary results from these experiments suggest a key role for tyrosine residues on the ErbB3 receptor that couple to the PI3-kinase pathway in tumor cell motility and metastasis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3395.

An Antimetastatic Role for Decorin in Breast Cancer

Decorin, a member of the small leucine-rich proteoglycan gene family, down-regulates members of the ErbB receptor tyrosine kinase family and attenuates their signaling, leading to growth inhibition. We investigated the effects of decorin on the growth of ErbB2-overexpressing mammary carcinoma cells in comparison with AG879, an established ErbB2 kinase inhibitor. Cell proliferation and anchorage-independent growth assays showed that decorin was a potent inhibitor of breast cancer cell growth and a pro-apoptotic agent. When decorin and AG879 were used in combination, the inhibitory effect was synergistic in proliferation assays but only additive in both colony formation and apoptosis assays. Active recombinant human decorin protein core, AG879, or a combination of both was administered systemically to mice bearing orthotopic mammary carcinoma xenografts. Primary tumor growth and metabolism were reduced by approximately 50% by both decorin and AG879. However, no synergism was observed in vivo. Decorin specifically targeted the tumor cells and caused a significant reduction of ErbB2 levels in the tumor xenografts. Most importantly, systemic delivery of decorin prevented metastatic spreading to the lungs, as detected by novel species-specific DNA detection and quantitative assays. In contrast, AG879 failed to have any effect. Our data support a role for decorin as a powerful and effective therapeutic agent against breast cancer due to its inhibition of both primary tumor growth and metastatic spreading.

FGF‐1‐induced matrix metalloproteinase‐9 expression in breast cancer cells is mediated by increased activities of NF‐κB and activating protein‐1

Matrix metalloproteinase-9 (MMP-9) plays a critical role in tumor invasion and metastasis. Here, we investigate the effect of fibroblast growth factor-1 (FGF-1) on the expression of MMP-9 in ENU1564, an ethyl-N-nitrosourea-induced rat mammary adenocarcinoma cell line. We observed that FGF-1 induces a dose-dependent increase in MMP-9 mRNA, protein, and activity in ENU1564 cells. To gain insight into the molecular mechanism of MMP-9 regulation by FGF-1, we investigated the role of components of PI3K-Akt and MEK1/2-ERK signaling pathways in our system since NF-kappaB and AP-1 transcription factor binding sites have been characterized in the upstream region of the MMP-9 gene. We demonstrated that FGF-1 increases Akt phosphorylation, triggers nuclear translocation of NF-kappaBp65, and enhances degradation of cytoplasmic IkappaBalpha. Pretreatment of cells with LY294002, a PI3K inhibitor, significantly inhibited MMP-9 protein expression in FGF-1-treated cells. Conversely, our data show that FGF-1 increases ERK phosphorylation in ENU1564 cells, increases c-jun and c-fos mRNA expression in a time-dependent manner, and triggers nuclear translocation of c-jun. Pretreatment of cells with PD98059, a MEK1/2 inhibitor significantly inhibited MMP-9 protein expression in FGF-1 treated cells. Finally, we observed increased DNA binding of NF-kappaB and AP-1 in FGF-1-treated cells and that mutation of either NF-kappaB or AP-1 response elements prevented MMP-9 promoter activation by FGF-1. Taken together, these results demonstrated that FGF-1-induced MMP-9 expression in ENU1564 cells is associated with increasing DNA binding activities of NF-kappaB and AP-1 and involve activation of a dual signaling pathway, PI3K-Akt and MEK1/2-ERK.

ZBP1 enhances cell polarity and reduces chemotaxis

The interaction of beta-actin mRNA with zipcode-binding protein 1 (ZBP1) is necessary for its localization to the lamellipod of fibroblasts and plays a crucial role in cell polarity and motility. Recently, we have shown that low ZBP1 levels correlate with tumor-cell invasion and metastasis. In order to establish a cause and effect relationship, we expressed ZBP1 in a metastatic rat mammary adenocarcinoma cell line (MTLn3) that has low endogenous ZBP1 levels and delocalized beta-actin mRNA. This leads to localization of beta-actin mRNA, and eventually reduces the chemotactic potential of the cells as well as their ability to move and orient towards vessels in tumors. To determine how ZBP1 leads to these two apparently contradictory aspects of cell behavior--increased cell motility but decreased chemotaxis--we examined cell motility in detail, both in cell culture and in vivo in tumors. We found that ZBP1 expression resulted in tumor cells with a stable polarized phenotype, and reduced their ability to move in response to a gradient in culture. To connect these results on cultured cells to the reduced metastatic ability of these cells, we used multiphoton imaging in vivo to examine tumor cell behavior in primary tumors. We found that ZBP1 expression actually reduced tumor cell motility and chemotaxis, presumably mediating their decreased metastatic potential by reducing their ability to respond to signals necessary for invasion.

In vivo imaging and characterization of hypoxia-induced neovascularization and tumor invasion

Hypoxia is a critical event in tumor progression and angiogenesis. Hypoxia can be detected noninvasively by a novel spectroscopic photoacoustic tomography technology (SPAT) and this finding is supported by our molecular biology investigation aimed to elucidate the etiopathogenesis of SPAT detected hypoxia and angiogenesis. The present study provides an integrated approach to define oxygen status (hypoxia) of intracranial tumor xenografts using spectroscopic photoacoustic tomography. Brain tumors can be identified based on their distorted vascular architecture and oxygen saturation (SO2) images. Noninvasive in vivo tumor oxygenation imaging using SPAT is based on the spectroscopic absorption differences between oxyhemoglobin (O2Hb) and deoxyhemoblobin (HHb). Sprague-Dawley rats inoculated intracranially with ENU1564, a carcinogen-induced rat mammary adenocarcinoma cell line, were imaged with SPAT three weeks post inoculation. Proteins important for tumor angiogenesis and invasion were detected in hypoxic brain foci identified by SPAT and were elevated compared with control brain. Immunohistochemistry, Western blotting, and semi-quantitative RT-PCR showed that HIF-1 alpha, VEGF-A, and VEGFR2 (Flk-1) protein and mRNA expression levels were significantly higher (P < 0.05) in brain tumor tissues compared to normal brain. Gelatin zymography and RT-PCR demonstrated the upregulation of MMP-9 in tumor foci compared with brain control. Together these results suggest the critical role of hypoxia in driving tumor angiogenesis and invasion through upregulation of target genes important for these functions. Moreover this report validates our hypothesis that a novel noninvasive technology (SPAT) developed in our laboratory is suitable for detection of tumors, hypoxia, and angiogenesis.

Identification of differentially expressed genes in isogenic highly metastatic and poorly metastatic cell lines of R3230AC rat mammary adenocarcinoma.

Tumour metastasis occurs as a result of a cascade of events including alterations in the expression of various genes. The identification of such genes is essential to understanding formation of metastasis. In a previous study, highly metastatic (LN4.D6) and poorly metastatic (CAb.D5) cell lines were obtained from the rat mammary adenocarcinoma cell line R3230AC. Subtractive hybridization was used to identify differentially expressed genes between these two cell lines. We identified eight cDNA clones in CAb.D5 and six cDNA clones in LN4.D6 that were differentially expressed. One of the cDNA clones in each cell line had no homology with known sequences. Expression patterns of these differentially expressed genes were examined in a pair of rat mammary and prostate adenocarcinoma cell lines. Compared with cell lines examined, cDNA FF-10 was only expressed in CAb.D5; however, cDNA RB-8, RE-1, RF-5 were only expressed in the highly metastatic LN4.D6. No correlation was observed between expression patterns of the differentially expressed genes and metastatic potential of these cells. However, differential expression of genes, especially cytokeratins (CK8 and CK5) and collagens (III and IV) between highly metastatic and low metastatic rat mammary adenocarcinoma cell lines might initiate further investigation of these genes in metastatic process.

Inhibition of rat mammary tumorigenesis by concord grape juice constituents.

The effects of Concord grape juice constituents on the promotion of chemically induced rat mammary tumor development and on the proliferation of a rat mammary adenocarcinoma cell line were studied. Isocaloric grape juice formulations provided in the drinking fluid of rats at concentrations of 489 and 651 mg of phenolics/dL of fluid significantly inhibited mammary adenocarcinoma multiplicity compared to controls. Final tumor mass also was significantly decreased for animals provided these two grape juice concentrations compared to controls. In addition, DNA synthesis of the rat mammary adenocarcinoma RBA cell line was significantly inhibited in a dose-dependent manner for cells treated with a grape extract, with an IC50 dose of approximately 14 micrograms of phenolics/mL. This inhibition of DNA synthesis was not accompanied by changes in 8-oxodeoxyguanosine formation or by substantial cell cycle arrest. These studies thus indicate that Concord grape juice constituents can inhibit the promotion stage of 7,12-dimethylbenz[a]anthracene (DMBA)-induced rat mammary tumorigenesis, in part by suppressing cell proliferation.

Tumor metastasis-associated human MTA1 gene and its MTA1 protein product: role in epithelial cancer cell invasion, proliferation and nuclear regulation.

Using differential cDNA library screening techniques based on metastatic and nonmetastatic rat mammary adenocarcinoma cell lines, we previously cloned and sequenced the metastasis-associated gene mta1. Using homology to the rat mta1 gene, we cloned the human MTA1 gene and found it to be over-expressed in a variety of human cell lines (breast, ovarian, lung, gastric and colorectal cancer but not melanoma or sarcoma) and cancerous tissues (breast, esophageal, colorectal, gastric and pancreatic cancer). We found a close similarity between the human MTA1 and rat mta1 genes (88% and 96% identities of the nucleotide and predicted amino acid sequences, respectively). Both genes encode novel proteins that contain a proline rich region (SH3-binding motif), a putative zinc finger motif, a leucine zipper motif and 5 copies of the SPXX motif found in gene regulatory proteins. Using Southern blot analysis the MTA1 gene was highly conserved, and using Northern blot analysis MTA1 transcripts were found in virtually all human cell lines (melanoma, breast, cervix and ovarian carcinoma cells and normal breast epithelial cells). However, the expression level of the MTA1 gene in normal breast epithelial cells was approximately 50% of that found in rapidly growing adenocarcinoma and atypical epithelial cell lines. Experimental inhibition of MTA1 protein expression using antisense phosphorothioate oligonucleotides resulted in inhibition of growth and invasion of human MDA-MB-231 breast cancer cells with relatively high MTA1 expression. Furthermore, the MTA1 protein was localized in the nuclei of cells transfected with a mammalian expression vector containing a full-length MTA1 gene. Although some MTA1 protein was found in the cytoplasm, the vast majority of MTA1 protein was localized in the nucleus. Examination of recombinate MTA1 and related MTA2 proteins suggests that MTA1 protein is a histone deacetylase. It also appears to behave like a GATA-element transcription factor, since transfection of a GATA-element reporter into MTA1-expressing cells resulted in 10-20-fold increase in reporter expression over poorly MTA1-expressing cells. Since it was reported that nucleosome remodeling histone deacetylase complex (NuRD complex) involved in chromatin remodeling contains MTA1 protein and a MTA1-related protein (MTA2), we examined NuRD complexes for the presence of MTA1 protein and found an association of this protein with histone deacetylase. The results suggest that the MTA1 protein may serve multiple functions in cellular signaling, chromosome remodeling and transcription processes that are important in the progression, invasion and growth of metastatic epithelial cells.

A novel short-chain alcohol dehydrogenase from rats with retinol dehydrogenase activity, cyclically expressed in uterine epithelium.

Retinoic acid is necessary for the maintenance of many lining epithelia of the body, such as the epithelium of the luminal surface of the uterus. Administration of estrogen to prepubertal rats induces in these epithelial cells the ability to synthesize retinoic acid from retinol, coincident with the appearance of cellular retinoic acid-binding protein, type two, which is normally present in these cells only at estrus in the mature, cycling animal. Here, we report the isolation, from a cDNA library prepared from uterine mRNA collected at the estrous stage and from a rat mammary adenocarcinoma cell line, of a cDNA that encodes a novel retinol dehydrogenase. A member of the short-chain alcohol dehydrogenase family, the encoded enzyme was capable of metabolizing retinol to retinal when expressed in cells after transfection of its cDNA. When cotransfected with the cDNA of human aldehyde 6, a known retinaldehyde dehydrogenase, the transfected cells synthesized retinoic acid from retinol. Immunohistochemical analysis revealed that the protein was present in the uterine lining epithelium of the mature animal only at estrus, coincident with the presence of cellular retinol-binding protein and cellular retinoic acid-binding protein, type two. Consequently, this novel short-chain alcohol dehydrogenase is an excellent candidate for the retinol dehydrogenase that catalyzes the first step in retinoic acid biosynthesis that occurs in uterine epithelial cells.

Proteomic dissection of dome formation in a mammary cell line: role of tropomyosin-5b and maspin.

In this work we extended the study of genes controlling the formation of specific differentiation structures called "domes" formed by the rat mammary adenocarcinoma cell line LA7 under the influence of DMSO. We have reported previously that an interferon-inducible gene, rat-8, and the beta-subunit of the epithelial sodium channel (ENaC) play a fundamental role in this process. Now, we used a proteomic approach to identify proteins differentially expressed either in DMSO-induced LA7 or in 106A10 cells. Two differentially expressed proteins were investigated. The first, tropomyosin-5b, strongly expressed in DMSO-induced LA7 cells, is needed for dome formation because its synthesis inhibition by the antisense RNA technology abolished domes. The second protein, maspin, strongly expressed in the uninduced 106A10 cell line, inhibits dome formation because 106A10 cells, transfected with rat8 cDNA (the function of which is required for the organization of these structures), acquired the ability to develop domes when cultured in presence of an antimaspin antibody. Dome formation in these cultures are accompanied by ENaC beta-subunit expression in the absence of DMSO. Therefore, dome formation requires the expression of tropomyosin-5b, in addition to the ENaC beta-subunit and the rat8 proteins, and is under the negative control of maspin.

Is delta-aminolevulinic acid dehydratase rate limiting in heme biosynthesis following exposure of cells to delta-aminolevulinic acid?

Understanding the regulation and control of heme/porphyrin biosynthesis is critical for the optimization of the delta-aminolevulinic-acid (ALA)-mediated photodynamic therapy of cancer, in which endogenously produced protoporphyrin IX (PPIX) is the photosensitizer. The human breast cancer cell line MCF-7, the rat mammary adenocarcinoma cell line R3230AC, the mouse mammary tumor cell line EMT-6 and the human mesothelioma cell line H-MESO-1 were used to study ALA-induced PPIX levels and their relationship to delta-aminolevulinic acid dehydratase (ALA-D) activity in vitro. Incubation of these cell lines with 0.5 mM ALA for 3 h resulted in a significant increase in PPIX accumulation, compared with control cells, but there was no significant change in ALA-D activity. Exposure of cells incubated with ALA to 30 mJ/cm2 of fluorescent light, a dose that would cause a 50% reduction in cell proliferation, did not significantly alter the activity of ALA-D. Increasing the activity of porphobilinogen deaminase (PBGD), the enzyme immediately subsequent to ALA-D, by four- to seven-fold via transfection of cells with PBGD complementary DNA did not alter the activity of ALA-D. However, incubation of cells with various concentrations of succinyl acetone, a potent inhibitor of ALA-D, caused a concomitant decline in both PPIX accumulation and ALA-D activity. These data imply that when cells are exposed to exogenous ALA, ALA-D is an important early-control step in heme/porphyrin biosynthesis and that regulation of PPIX synthesis by this dehydratase may impact the effectiveness of ALA-mediated photosensitization.

Tumor metastasis‐associated human MTA1 gene: Its deduced protein sequence, localization, and association with breast cancer cell proliferation using antisense phosphorothioate oligonucleotides

Using differential cDNA library screening techniques based on metastatic and nonmetastatic rat mammary adenocarcinoma cell lines we previously cloned and sequenced the metastasis-associated gene mta1. Using homology to the rat MTA1 gene we cloned the human MTA1 gene and found it to be overexpressed in a variety of human cell lines. We found a close similarity between the human MTA1 and rat MTA1 genes, as shown by 88% and 96% identities of the nucleotide and predicted amino acid sequences, respectively. Both genes encode novel proteins that contain a proline-rich region (SH3 binding motif), a putative zinc finger motif, a leucine zipper motif, and five copies of the SPXX motif often found in gene regulatory proteins. Using Southern blot analysis, the MTA1 gene was found to be highly conserved among all species examined; and using Northern blot analysis, MTA1 transcripts were found in virtually all cell lines of human origin that were analyzed, including melanoma and breast, cervix and ovarian carcinoma cells and normal breast epithelial cells. However, the expression level of the MTA1 gene in a normal breast epithelial cell was approximately 50% of that found in rapidly growing breast adenocarcinoma cell lines and an atypical mammary cell line. Experimental inhibition of MTA1 protein expression using antisense phosphorothioate oligonucleotides resulted in growth inhibition of human MDA-MB-231 breast cancer cells with relatively high expression of the MTA1 gene. Furthermore, the MTA1 protein was localized in the nuclei of cells transfected using a mammalian expression vector containing the full-length MTA1 gene. The results suggest that the MTA1 protein may function in cellular signaling processes important in the progression and growth of cancer cells, possibly as a nuclear regulatory factor. J. Cell. Biochem. 79:202–212, 2000. © 2000 Wiley-Liss, Inc.

Extracellular regulated kinase (ERK)-dependent regulation of sialomucin complex/rat Muc4 in mammary epithelial cells.

Sialomucin complex (SMC, rat Muc4) is a membrane mucin implicated in the protection of epithelia and the metastasis of some tumors. It is a heterodimeric complex, containing a mucin subunit with anti-adhesive activity and a transmembrane subunit with epidermal growth factor-like domains, one of which acts as an intramembrane ligand for ErbB2. Serum, insulin and insulin-like growth factor, but not epidermal growth factor, induce the expression of sialomucin complex in mammary epithelial cells. Induction correlates with sustained, but not transient, activation of extracellular-regulated protein kinase (ERK). MEK inhibitor U0126 blocked the induction, while activated MEK-1 transfected into a rat mammary adenocarcinoma cell line induced a sustained activation of ERK and up-regulated SMC/Muc4 expression. Northern and Western blotting indicated that up-regulation occurred concomitantly at the transcript and protein levels, both of which could be blocked by U0126. These results suggest that expression of SMC/Muc4 in mammary epithelial cells is regulated by selected growth factors through an ERK-dependent pathway at the transcript level.

Caveolin-1 Inhibits Epidermal Growth Factor-stimulated Lamellipod Extension and Cell Migration in Metastatic Mammary Adenocarcinoma Cells (MTLn3): TRANSFORMATION SUPPRESSOR EFFECTS OF ADENOVIRUS-MEDIATED GENE DELIVERY OF CAVEOLIN-1

Caveolin-1 is a principal component of caveolae membranes that may function as a transformation suppressor. For example, the human caveolin-1 gene is localized to a suspected tumor suppressor locus (D7S522; 7q31.1) that is deleted in human cancers, including mammary carcinomas. However, little is known about the role of caveolins in regulating cell movement, a critical parameter in determining metastatic potential. Here, we examine the role of caveolin-1 in cell movement. For this purpose, we employed an established cellular model, MTLn3, a metastatic rat mammary adenocarcinoma cell line. In this system, epidermal growth factor (EGF) stimulation induces rapid lamellipod extension and cell migration. Interestingly, we find that MTLn3 cells fail to express detectable levels of endogenous caveolin-1. To restore caveolin-1 expression in MTLn3 cells efficiently, we employed an inducible adenoviral gene delivery system to achieve tightly controlled expression of caveolin-1. We show here that caveolin-1 expression in MTLn3 cells inhibits EGF-stimulated lamellipod extension and cell migration and blocks their anchorage-independent growth. Under these conditions, EGF-induced activation of the p42/44 mitogen-activated protein kinase cascade is also blunted. Our results suggest that caveolin-1 expression in motile MTLn3 cells induces a non-motile phenotype.

Tumor metastasis-associated humanMTA1 gene: Its deduced protein sequence, localization, and association with breast cancer cell proliferation using antisense phosphorothioate oligonucleotides

Using differential cDNA library screening techniques based on metastatic and nonmetastatic rat mammary adenocarcinoma cell lines we previously cloned and sequenced the metastasis-associated gene mta1. Using homology to the rat MTA1 gene we cloned the human MTA1 gene and found it to be overexpressed in a variety of human cell lines. We found a close similarity between the human MTA1 and rat MTA1 genes, as shown by 88% and 96% identities of the nucleotide and predicted amino acid sequences, respectively. Both genes encode novel proteins that contain a proline-rich region (SH3 binding motif), a putative zinc finger motif, a leucine zipper motif, and five copies of the SPXX motif often found in gene regulatory proteins. Using Southern blot analysis, the MTA1 gene was found to be highly conserved among all species examined; and using Northern blot analysis, MTA1 transcripts were found in virtually all cell lines of human origin that were analyzed, including melanoma and breast, cervix and ovarian carcinoma cells and normal breast epithelial cells. However, the expression level of the MTA1 gene in a normal breast epithelial cell was approximately 50% of that found in rapidly growing breast adenocarcinoma cell lines and an atypical mammary cell line. Experimental inhibition of MTA1 protein expression using antisense phosphorothioate oligonucleotides resulted in growth inhibition of human MDA-MB-231 breast cancer cells with relatively high expression of the MTA1 gene. Furthermore, the MTA1 protein was localized in the nuclei of cells transfected using a mammalian expression vector containing the full-length MTA1 gene. The results suggest that the MTA1 protein may function in cellular signaling processes important in the progression and growth of cancer cells, possibly as a nuclear regulatory factor.

Transferrin receptor overexpression enhances transferrin responsiveness and the metastatic growth of a rat mammary adenocarcinoma cell line.

We previously found that breast cancer cell transferrin receptor expression and proliferative response to transferrin often correlated with metastatic capability. To further explore this, we transfected mammary tumor cells with a cDNA coding for the transferrin receptor and examined the effects of its overexpression on various cellular properties. A human transferrin receptor expression plasmid was made by excising the cDNA for the receptor from pcDTR1 and ligating it into the multiple cloning site of pcDNAINeo. The resulting construct was transfected into the poorly metastatic rat MTLn2 line that expresses low endogenous levels of rat transferrin receptor, and transfection-induced receptor expression was ascertained using antibodies specific for the human protein. Approximately 50% of the initial geneticin-resistant transfected MTLn2 cells overexpressed human transferrin receptor protein. High expressors were further isolated by four sequential FACS sorts. The final cell population expressed approximately 3-7 times more cell surface transferrin receptor than did vector transfected controls. Both lines proliferated at the same rate in normal (medium plus 5% FBS) culture conditions. However, in serum-free conditions, the transferrin receptor overexpressor cells displayed a pronounced proliferative response to transferrin whereas the control line did not. When injected into the mammary fat pads of female nude mice, cells from both lines formed micrometastases to the lung that were specifically visualized by immunohistochemical staining of rat cytokeratin 17. This revealed that the transferrin receptor transfected line formed larger lesions of this nature than did cells from the vector transfected controls. When injected into the tail vein of female nude mice, the transferrin receptor overexpressors likewise formed gross lung metastases of remarkably greater size than did the vector only transfectants. Overexpression of cell surface human transferrin receptor on MTLn2 cells appeared to affect their in vitro growth response to transferrin and their ability to grow at a secondary site in vivo.

An Intramembrane Modulator of the ErbB2 Receptor Tyrosine Kinase That Potentiates Neuregulin Signaling

The ErbB2 receptor tyrosine kinase plays a critical role in a variety of developmental processes, and its aberrant activation may contribute to the progression of some breast and ovarian tumors. ASGP2, a transmembrane glycoprotein found on the surface of the highly metastatic ascites 13762 rat mammary adenocarcinoma cell line, is constitutively associated with ErbB2 in these cells and in mammary tissue from pregnant rats. Expression studies indicate that ASGP2 interacts directly and specifically with ErbB2 through one of its epidermal growth factor-like domains and that the co-expression of the two proteins in the same cell dramatically facilitates their direct stable interaction. Ectopic expression of ASGP2 in human melanoma tumor cells potentiates the response of endogenous ErbB2 to the neuregulin-1 growth factor. These observations point to a novel intramembrane mechanism for the modulation of receptor tyrosine kinase activity.