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• W2045010127 abstract "Here we used the Met-1 cell line in an orthotopic transplantation model in FVB/N mice to dissect the role of the Cav-1(P132L) mutation in human breast cancer. Identical experiments were performed in parallel with wild-type Cav-1. Cav-1(P132L) up-regulated the expression of estrogen receptor-α as predicted, because only estrogen receptor-α-positive patients have been shown to harbor Cav-1(P132L) mutations. In the context of primary tumor formation, Cav-1(P132L) behaved as a loss-of-function mutation, lacking any tumor suppressor activity. In contrast, Cav-1(P132L) caused significant increases in cell migration, invasion, and experimental metastasis, consistent with a gain-of-function mutation. To identify possible molecular mechanism(s) underlying this invasive gain-of-function activity, we performed unbiased gene expression profiling. From this analysis, we show that the Cav-1(P132L) expression signature contains numerous genes that have been previously associated with cell migration, invasion, and metastasis. These include i) secreted growth factors and extracellular matrix proteins (Cyr61, Plf, Pthlh, Serpinb5, Tnc, and Wnt10a), ii) proteases that generate EGF and HGF (Adamts1 and St14), and iii) tyrosine kinase substrates and integrin signaling/adapter proteins (Akap13, Cdcp1, Ddef1, Eps15, Foxf1a, Gab2, Hs2st1, and Itgb4). Several of the P132L-specific genes are also highly expressed in stem/progenitor cells or are associated with myoepithelial cells, suggestive of an epithelial-mesenchymal transition. These results directly support clinical data showing that patients harboring Cav-1 mutations are more likely to undergo recurrence and metastasis. Here we used the Met-1 cell line in an orthotopic transplantation model in FVB/N mice to dissect the role of the Cav-1(P132L) mutation in human breast cancer. Identical experiments were performed in parallel with wild-type Cav-1. Cav-1(P132L) up-regulated the expression of estrogen receptor-α as predicted, because only estrogen receptor-α-positive patients have been shown to harbor Cav-1(P132L) mutations. In the context of primary tumor formation, Cav-1(P132L) behaved as a loss-of-function mutation, lacking any tumor suppressor activity. In contrast, Cav-1(P132L) caused significant increases in cell migration, invasion, and experimental metastasis, consistent with a gain-of-function mutation. To identify possible molecular mechanism(s) underlying this invasive gain-of-function activity, we performed unbiased gene expression profiling. From this analysis, we show that the Cav-1(P132L) expression signature contains numerous genes that have been previously associated with cell migration, invasion, and metastasis. These include i) secreted growth factors and extracellular matrix proteins (Cyr61, Plf, Pthlh, Serpinb5, Tnc, and Wnt10a), ii) proteases that generate EGF and HGF (Adamts1 and St14), and iii) tyrosine kinase substrates and integrin signaling/adapter proteins (Akap13, Cdcp1, Ddef1, Eps15, Foxf1a, Gab2, Hs2st1, and Itgb4). Several of the P132L-specific genes are also highly expressed in stem/progenitor cells or are associated with myoepithelial cells, suggestive of an epithelial-mesenchymal transition. These results directly support clinical data showing that patients harboring Cav-1 mutations are more likely to undergo recurrence and metastasis. We and others have shown that the caveolin-1 (Cav-1) gene is commonly mutated in human breast cancers.1Hayashi K Matsuda S Machida K Yamamoto T Fukuda Y Nimura Y Hayakawa T Hamaguchi M Invasion activating caveolin-1 mutation in human scirrhous breast cancers.Cancer Res. 2001; 61: 2361-2364PubMed Google Scholar, 2Li T Sotgia F Vuolo MA Li M Yang WC Pestell RG Sparano JA Lisanti MP Caveolin-1 mutations in human breast cancer: functional association with estrogen receptor alpha-positive status.Am J Pathol. 2006; 168: 1998-2013Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar Up to one-third of estrogen receptor (ER)-α-positive breast cancers harbor Cav-1 mutations,2Li T Sotgia F Vuolo MA Li M Yang WC Pestell RG Sparano JA Lisanti MP Caveolin-1 mutations in human breast cancer: functional association with estrogen receptor alpha-positive status.Am J Pathol. 2006; 168: 1998-2013Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar indicating that it may be a very common initiating or early event in the development of breast cancers in humans. In accordance with these human genetic studies, Cav-1 (−/−)-null mice show numerous mammary gland phenotypes, including progressive mammary intraductal hyperplasia,3Lee H Park DS Razani B Russell RG Pestell RG Lisanti MP Caveolin-1 mutations (P132L and null) and the pathogenesis of breast cancer: caveolin-1 (P132L) behaves in a dominant-negative manner and caveolin-1 (−/−) null mice show mammary epithelial cell hyperplasia.Am J Pathol. 2002; 161: 1357-1369Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar, 4Park DS Lee H Frank PG Razani B Nguyen AV Parlow AF Russell RG Hulit J Pestell RG Lisanti MP Caveolin-1-deficient mice show accelerated mammary gland development during pregnancy, premature lactation, and hyperactivation of the Jak-2/STAT5a signaling cascade.Mol Biol Cell. 2002; 13: 3416-3430Crossref PubMed Scopus (98) Google Scholar and are more susceptible to mammary tumorigenesis and metastasis, when crossed with established mouse models that spontaneously develop mammary tumors.5Williams TM Cheung MW Park DS Razani B Cohen AW Muller WJ Di Vizio D Chopra NG Pestell RG Lisanti MP Loss of caveolin-1 gene expression accelerates the development of dysplastic mammary lesions in tumor-prone transgenic mice.Mol Biol Cell. 2003; 14: 1027-1042Crossref PubMed Scopus (135) Google Scholar, 6Williams TM Lee H Cheung MW Cohen AW Razani B Iyengar P Scherer PE Pestell RG Lisanti MP Combined loss of INK4a and caveolin-1 synergistically enhances cell proliferation and oncogene-induced tumorigenesis.J Biol Chem. 2004; 279: 24745-24756Crossref PubMed Scopus (74) Google Scholar, 7Williams TM Medina F Badano I Hazan RB Hutchinson J Muller WJ Chopra NG Scherer PE Pestell RG Lisanti MP Caveolin-1 gene disruption promotes mammary tumorigenesis and dramatically enhances lung metastasis in vivo: role of Cav-1 in cell invasiveness and matrix metalloproteinase (MMP-2/9) secretion.J Biol Chem. 2004; 279: 51630-51646Crossref PubMed Scopus (269) Google Scholar Thus, Cav-1 is thought to function as a tumor suppressor or modifier gene in mammary epithelia.8Williams TM Lisanti MP Caveolin-1 in oncogenic transformation, cancer, and metastasis.Am J Physiol. 2005; 288: C494-C506Crossref PubMed Scopus (464) Google Scholar, 9Bouras T Lisanti MP Pestell RG Caveolin-1 in breast cancer.Cancer Biol Ther. 2004; 3: 931-941Crossref PubMed Scopus (59) Google Scholar Although eight Cav-1 breast cancer-associated mutations have been described to date, the most common mutation is a proline to leucine change at position 132 within its putative transmembrane domain.2Li T Sotgia F Vuolo MA Li M Yang WC Pestell RG Sparano JA Lisanti MP Caveolin-1 mutations in human breast cancer: functional association with estrogen receptor alpha-positive status.Am J Pathol. 2006; 168: 1998-2013Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar The P132L mutation accounts for more than half of the breast cancer cases with Cav-1 mutations, indicating that this residue is a hot-spot for sporadic mutation in the genome, akin to the Ras (G12V) mutation. Interestingly, this proline residue is critical because it is 1 of 12 invariant caveolin residues that are conserved from worms (Caenorhabditis elegans) to humans.10Tang Z Okamoto T Boontrakulpoontawee P Katada T Otsuka AJ Lisanti MP Identification, sequence, and expression of an invertebrate caveolin gene family from the nematode Caenorhabditis elegans: implications for the molecular evolution of mammalian caveolin genes.J Biol Chem. 1997; 272: 2437-2445Crossref PubMed Scopus (82) Google Scholar The analogous proline residue is also mutated in Cav-3 (P104L), a muscle-specific caveolin-related protein, and gives rise to an autosomal dominant form of muscular dystrophy, termed limb-girdle muscular dystrophy (LGMD-type 1C).11Minetti C Sotgia F Bruno C Scartezzini P Broda P Bado M Masetti E Mazzocco P Egeo A Donati MA Volonte' D Galbiati F Cordone G Bricarelli FD Lisanti MP Zara F Mutations in the caveolin-3 gene cause autosomal dominant limb-girdle muscular dystrophy.Nat Genet. 1998; 18: 365-368Crossref PubMed Scopus (498) Google Scholar However, unlike Cav-1 (P132L), which does not undergo significant degradation, Cav-3 (P104L) is rapidly degraded by a ubiquitin/proteosomal-dependent pathway.12Galbiati F Volonte D Minetti C Bregman DB Lisanti MP Limb-girdle muscular dystrophy (LGMD-1C) mutants of caveolin-3 undergo ubiquitination and proteasomal degradation. Treatment with proteasomal inhibitors blocks the dominant negative effect of LGMD-1C mutanta and rescues wild-type caveolin-3.J Biol Chem. 2000; 275: 37702-37711Crossref PubMed Scopus (88) Google Scholar Little is known about how the Cav-1 (P132L) mutation mediates its effects. Based on biochemical studies, P132L appears to represent a loss-of-function mutation, that can also act in a dominant-negative manner, by inactivating the WT Cav-1 protein product. These initial studies showed that when the P132L mutant is transiently expressed in fibroblastic cell types, it is misfolded, forms high-molecular mass aggregates, and is retained in a perinuclear ER/Golgi-like compartment.3Lee H Park DS Razani B Russell RG Pestell RG Lisanti MP Caveolin-1 mutations (P132L and null) and the pathogenesis of breast cancer: caveolin-1 (P132L) behaves in a dominant-negative manner and caveolin-1 (−/−) null mice show mammary epithelial cell hyperplasia.Am J Pathol. 2002; 161: 1357-1369Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar It is not efficiently targeted to the plasma membrane. Interestingly, when Cav-1 (P132L) is co-expressed with WT Cav-1, both are retained intracellularly, indicating that Cav-1 (P132L) behaves in a dominant-negative manner.3Lee H Park DS Razani B Russell RG Pestell RG Lisanti MP Caveolin-1 mutations (P132L and null) and the pathogenesis of breast cancer: caveolin-1 (P132L) behaves in a dominant-negative manner and caveolin-1 (−/−) null mice show mammary epithelial cell hyperplasia.Am J Pathol. 2002; 161: 1357-1369Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar Thus, because wild-type (WT) Cav-1 is normally localized to plasmalemmal caveolae, the functional effects of the Cav-1 (P132L) mutant may be explained in part by its mislocalization and intracellular retention. Studies in NIH-3T3 fibroblasts have shown that expression of the Cav-1 (P132L) mutant is sufficient to mediate cell transformation.1Hayashi K Matsuda S Machida K Yamamoto T Fukuda Y Nimura Y Hayakawa T Hamaguchi M Invasion activating caveolin-1 mutation in human scirrhous breast cancers.Cancer Res. 2001; 61: 2361-2364PubMed Google Scholar Similarly, knock-down of Cav-1 protein expression in NIH-3T3 cells, using an anti-sense cDNA approach, also drives cell transformation and tumor formation in immune-deficient mice.13Galbiati F Volonte D Engelman JA Watanabe G Burk R Pestell RG Lisanti MP Targeted downregulation of caveolin-1 is sufficient to drive cell transformation and hyperactivate the p42/44 MAP kinase cascade.EMBO J. 1998; 17: 6633-6648Crossref PubMed Scopus (433) Google Scholar Thus, these studies also provide evidence that the P132L mutation represents a loss-of-function mutation. However, this may be an oversimplification because human breast cancer patients that harbor Cav-1 mutations are more likely to undergo recurrence and metastasis, despite the good prognosis that is usually associated with ER positivity.2Li T Sotgia F Vuolo MA Li M Yang WC Pestell RG Sparano JA Lisanti MP Caveolin-1 mutations in human breast cancer: functional association with estrogen receptor alpha-positive status.Am J Pathol. 2006; 168: 1998-2013Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar Thus, we developed an orthotopic transplantation model in mice to study the behavior of the Cav-1 (P132L) mutation in vivo. For this purpose, we chose Met-1 cells, which are a mouse luminal mammary epithelial cell line derived from an MMTV-PyMT mammary tumor.14Borowsky AD Namba R Young LJ Hunter KW Hodgson JG Tepper CG McGoldrick ET Muller WJ Cardiff RD Gregg JP Syngeneic mouse mammary carcinoma cell lines: two closely related cell lines with divergent metastatic behavior.Clin Exp Metastasis. 2005; 22: 47-59Crossref PubMed Scopus (153) Google Scholar, 15Namba R Young LJ Abbey CK Kim L Damonte P Borowsky AD Qi J Tepper CG MacLeod CL Cardiff RD Gregg JP Rapamycin inhibits growth of premalignant and malignant mammary lesions in a mouse model of ductal carcinoma in situ.Clin Cancer Res. 2006; 12: 2613-2621Crossref PubMed Scopus (68) Google Scholar Although Met-1 cells have been selected for their capacity to undergo metastasis, they also form primary mammary tumors when orthotopically implanted in FVB/N mice. We have previously shown that Cav-1 (WT) behaves as a tumor suppressor in this system,7Williams TM Medina F Badano I Hazan RB Hutchinson J Muller WJ Chopra NG Scherer PE Pestell RG Lisanti MP Caveolin-1 gene disruption promotes mammary tumorigenesis and dramatically enhances lung metastasis in vivo: role of Cav-1 in cell invasiveness and matrix metalloproteinase (MMP-2/9) secretion.J Biol Chem. 2004; 279: 51630-51646Crossref PubMed Scopus (269) Google Scholar indicating that Met-1 cells may also be a useful model for studying the properties of Cav-1 mutants associated with human breast cancer. Here, using the Met-1 cell system, we show that the Cav-1 (P132L) mutation behaves as a loss-of-function mutation in the context of primary tumor formation. However, we also show that the Cav-1 (P132L) mutation acts as a gain-of-function mutation in the context of cell migration, invasion, and experimental metastasis. Global genome-wide expression profiling studies reveal a Cav-1 (P132L)-specific expression signature that contains numerous genes previously shown to be associated with breast cancer metastasis, cell motility, or invasiveness. As such, these studies may provide a mechanistic basis for understanding why breast cancer patients with Cav-1 mutations are more prone to disease recurrence. In further support of the validity of this model, we also show that the Cav-1 (P132L) mutant induces ER-α protein expression (as seen by Western blotting) and activates ER-α signaling (as seen by gene expression profiling), consistent with the association of this mutation with ER positivity in human breast cancers. Antibodies and their sources were as follows: anti-Cav-1 (N-20) rabbit polyclonal antibody (pAb) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), anti-β-actin mouse monoclonal antibody (mAb) AC-15 (Sigma, St. Louis, MO), anti-Ki-67 rabbit pAb (Abcam, Inc., Cambridge, MA), anti-cyclin D1 rabbit pAb (Lab Vision, Inc., Fremont, CA), anti-ER-α rabbit pAbs (MC-20 and H-184) (Santa Cruz Biotechnology, Inc.), anti-Cyr61 rabbit pAb (H-78, Santa Cruz Biotechnology, Inc), anti-Foxf1a rabbit pAb (Abcam), anti-Krt14 rabbit pAb (Covance, Princeton, NJ), anti-Wnt10a rat mAb (R&D Systems, Inc., Minneapolis, MN), anti-Gab2 rabbit pAb (26B6; Cell Signaling Technology, Beverly, MA), and anti-Usp34 mouse mAb (clone 2E2; Abnova Corp., Ann Arbor, MI). Rabbit polyclonal antibodies to MLN64 were from Abcam (for Western blotting) or were as previously described (for immunohistochemistry).16Alpy F Boulay A Moog-Lutz C Andarawewa KL Degot S Stoll I Rio MC Tomasetto C Metastatic lymph node 64 (MLN64), a gene overexpressed in breast cancers, is regulated by Sp/KLF transcription factors.Oncogene. 2003; 22: 3770-3780Crossref PubMed Scopus (26) Google Scholar, 17Alpy F Latchumanan VK Kedinger V Janoshazi A Thiele C Wendling C Rio MC Tomasetto C Functional characterization of the MENTAL domain.J Biol Chem. 2005; 280: 17945-17952Crossref PubMed Scopus (56) Google Scholar, 18Alpy F Stoeckel ME Dierich A Escola JM Wendling C Chenard MP Vanier MT Gruenberg J Tomasetto C Rio MC The steroidogenic acute regulatory protein homolog MLN64, a late endosomal cholesterol-binding protein.J Biol Chem. 2001; 276: 4261-4269Crossref PubMed Scopus (145) Google Scholar, 19Alpy F Tomasetto C Give lipids a START: the StAR-related lipid transfer (START) domain in mammals.J Cell Sci. 2005; 118: 2791-2801Crossref PubMed Scopus (298) Google Scholar, 20Alpy F Wendling C Rio MC Tomasetto C MENTHO, a MLN64 homologue devoid of the START domain.J Biol Chem. 2002; 277: 50780-50787Crossref PubMed Scopus (49) Google Scholar, 21Degot S Le Hir H Alpy F Kedinger V Stoll I Wendling C Seraphin B Rio MC Tomasetto C Association of the breast cancer protein MLN51 with the exon junction complex via its speckle localizer and RNA binding module.J Biol Chem. 2004; 279: 33702-33715Crossref PubMed Scopus (91) Google Scholar, 22Kedinger V Alpy F Tomasetto C Thisse C Thisse B Rio MC Spatial and temporal distribution of the traf4 genes during zebrafish development.Gene Expr Patterns. 2005; 5: 545-552Crossref PubMed Scopus (17) Google Scholar Antibodies to Cab45, an established Golgi marker protein,23Scherer PE Lederkremer GZ Williams S Fogliano M Baldini G Lodish HF Cab45, a novel (Ca2+)-binding protein localized to the Golgi lumen.J Cell Biol. 1996; 133: 257-268Crossref PubMed Scopus (129) Google Scholar were the generous gift of Dr. Philipp E. Scherer (University of Texas Southwestern, Dallas, TX). Inhibitors of EGF-R/ErbB2 (GW-583340), the c-MET receptor tyrosine kinase (PHA-665752), and the transforming growth factor (TGF)-β type I receptor (LY-364947) were purchased from Tocris Biosciences, Ellisville, MO. Met-1 cells were the generous gift of Dr. Robert D. Cardiff (University of California–Davis); it is important to note that in the current studies we used an earlier passage (less aggressive/less metastatic) version of the cell line. Our previously published studies, which did not examine the effects of the Cav-1 (P132L) mutant, used the later passage/more metastatic Met-1 cells.7Williams TM Medina F Badano I Hazan RB Hutchinson J Muller WJ Chopra NG Scherer PE Pestell RG Lisanti MP Caveolin-1 gene disruption promotes mammary tumorigenesis and dramatically enhances lung metastasis in vivo: role of Cav-1 in cell invasiveness and matrix metalloproteinase (MMP-2/9) secretion.J Biol Chem. 2004; 279: 51630-51646Crossref PubMed Scopus (269) Google Scholar Met-1 stable cell lines were produced by retroviral-mediated transduction (using the vector pBABE-puro), essentially as we previously described.7Williams TM Medina F Badano I Hazan RB Hutchinson J Muller WJ Chopra NG Scherer PE Pestell RG Lisanti MP Caveolin-1 gene disruption promotes mammary tumorigenesis and dramatically enhances lung metastasis in vivo: role of Cav-1 in cell invasiveness and matrix metalloproteinase (MMP-2/9) secretion.J Biol Chem. 2004; 279: 51630-51646Crossref PubMed Scopus (269) Google Scholar Briefly, C-terminally Myc-tagged cDNAs encoding WT Cav-1 and Cav-1 (P132L) were generated by polymerase chain reaction (PCR) and subcloned into the pBABE vector using the BamH1/EcoR1 restriction site. The correctness of intended base substitutions and the absence of unwanted mutations were verified by DNA sequencing. After retroviral transduction, Met-1 cells were selected for 5 to 7 days in 10 μg/ml of puromycin. Met-1 cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 2 mmol/L glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin. Met-1 cells were lysed in 800 μl of lysis buffer (10 mmol/L Tris, pH 7.5, 150 mmol/L NaCl, 1% Triton X-100, 60 mmol/L octyl glucoside), containing protease (Roche Applied Science, Indianapolis, IN) and phosphatase inhibitors (Sigma). Cell lysates were then centrifuged at 12,000 × g for 10 minutes to remove insoluble debris. Protein concentrations were analyzed using the BCA reagent (Pierce, Rockford, IL) and the volume required for 50 μg of protein was determined. Cell lysates were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (8 to 10% acrylamide) and transferred to nitrocellulose. The nitrocellulose membranes were stained with Ponceau S (to visualize protein bands), followed by immunoblot analysis. Subsequent wash buffers contained 10 mmol/L Tris, pH 8.0, 150 mmol/L NaCl, 0.05% Tween-20 (TBS-Tween), which was supplemented with 1% bovine serum albumin (BSA) and 4% nonfat dry milk (Carnation, Wilkes-Barre, PA) for the blocking solution and 1% BSA for the antibody diluent. For phospho-antibody analysis, the blocking solution contained only 5% BSA in TBS-Tween (without nonfat milk). Primary antibodies were used at a 1:100 to 1:500 dilution. Horseradish peroxidase-conjugated secondary antibodies [anti-mouse, 1:6000 dilution (Pierce) or anti-rabbit 1:5000 (BD Pharmingen, San Diego, CA)] were used to visualize bound primary antibodies, with the Supersignal chemiluminescence substrate (Pierce). Met-1 cells were grown on sterile glass coverslips, washed three times in phosphate-buffered saline (PBS), and fixed for 30 minutes at room temperature with 2% paraformaldehyde in PBS. After fixation, cells were permeabilized with 0.1% Triton X-100/0.2% BSA/PBS for 10 minutes. Cells were then treated with 25 mmol/L NH4Cl in PBS for 10 minutes at room temperature to quench free aldehyde groups. After rinsing with PBS, cells were incubated with primary antibody diluted in 0.1% Triton X-100/0.2% BSA/PBS, overnight at 4°C. The day after, three washes with PBS for 5 minutes each were done before the secondary antibody incubation (with a rhodamine-conjugated anti-mouse or anti-rabbit antibody) for 30 minutes at room temperature. Finally, cells were washed three times with PBS (10 minutes each wash), and mounted on a glass slide with slow-fade anti-fade reagent (Molecular Probes, Eugene, OR). All animals were housed and maintained in a barrier facility at the Kimmel Cancer Center at Thomas Jefferson University. All WT mice used in this study were virgin female in the FVB/N genetic background. Animal protocols used for this study were pre-approved by the institutional animal care and use committee. For orthotopic implantation, 0.5 × 105 cells were resuspended in 5 μl of PBS and injected through the nipple of the inguinal (no. 4) mammary gland into 2-month-old FVB/N female mice using a Hamilton syringe with a 26-gauge needle.24Williams TM Sotgia F Lee H Hassan G Di Vizio D Bonuccelli G Capozza F Mercier I Rui H Pestell RG Lisanti MP Stromal and epithelial caveolin-1 both confer a protective effect against mammary hyperplasia and tumorigenesis: caveolin-1 antagonizes cyclin d1 function in mammary epithelial cells.Am J Pathol. 2006; 169: 1784-1801Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar Met-1 cells are syngeneic to the FVB/N strain. At 6 weeks after injection, mice were sacrificed, and the tumors were carefully excised and weighed. Immunostaining of slides containing deparaffinized formalin-fixed mammary tumor sections was performed essentially as we described.2Li T Sotgia F Vuolo MA Li M Yang WC Pestell RG Sparano JA Lisanti MP Caveolin-1 mutations in human breast cancer: functional association with estrogen receptor alpha-positive status.Am J Pathol. 2006; 168: 1998-2013Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar, 7Williams TM Medina F Badano I Hazan RB Hutchinson J Muller WJ Chopra NG Scherer PE Pestell RG Lisanti MP Caveolin-1 gene disruption promotes mammary tumorigenesis and dramatically enhances lung metastasis in vivo: role of Cav-1 in cell invasiveness and matrix metalloproteinase (MMP-2/9) secretion.J Biol Chem. 2004; 279: 51630-51646Crossref PubMed Scopus (269) Google Scholar Briefly, paraffin-embedded tumors were sectioned at 5 μm. Sections were then deparaffinized first by treatment with xylene and rehydrated by passage through a graded series of ethanol. Antigen retrieval was performed by microwaving the slides in 100 mmol/L sodium citrate buffer for 15 minutes. Endogenous peroxide activity was quenched by incubating the slides for 10 minutes in 3% H2O2. Slides were then washed in phosphate-buffered saline (PBS) and blocked with a solution containing 10% goat serum in PBS for 1 hour at room temperature. Samples were washed with PBS and incubated with the primary antibody in blocking solution for 12 to 16 hours at 4°C. Slides were then washed with PBS (three washes, 5 minutes each) and incubated with a biotinylated secondary antibody in blocking solution for 30 minutes at room temperature. Slides were further washed in PBS (three washes, 5 minutes each) and incubated with the avidin/biotin-horseradish peroxidase reagent for 30 minutes at room temperature. Next, samples were washed in PBS and incubated with the 3,3′-diaminobenzidine reagent until color production developed. Finally, the slides were washed in PBS to remove excess diaminobenzidine, counterstained with hematoxylin, dehydrated, and mounted with coverslips. TUNEL-positive cells were identified using the ApopTag peroxidase in situ apoptosis detection kit (Millipore, Temecula, CA) per kit instructions. Briefly, paraffin sections were deparaffinized in xylene, rehydrated in ethanol, and washed with PBS. The tissue was treated with 20 μg/ml of proteinase K (Roche) diluted in PBS for 15 minutes at room temperature, washed, and blocked with 3% hydrogen peroxide for 5 minutes. The sections were then incubated with equilibration buffer briefly, followed by working strength TdT enzyme for 1 hour at 37°C. After washing, the sections were incubated with anti-digoxigenin horseradish peroxidase conjugated antibody for 30 minutes at room temperature, washed, and TUNEL-positive cells were detected using 3,3′-diaminobenzidine. TUNEL-positive cells were enumerated in 8 to 12 random ×40 fields from each group and the mean number of TUNEL-positive cells per field was calculated. For quantitation purposes, focal areas of necrosis were omitted and random viable areas were used. To study cell invasive capacity in vivo, 5 × 105 cells suspended in 0.1 ml of PBS were injected through the tail vein of six WT FVB/N female mice for each cell line. After 4 weeks, the lungs were removed and insufflated with 2 ml of 15% India Ink, washed in water for 5 minutes, and bleached in Fekete's solution (70% ethanol, 3.7% paraformaldehyde, 0.75 mol/L glacial acetic acid).7Williams TM Medina F Badano I Hazan RB Hutchinson J Muller WJ Chopra NG Scherer PE Pestell RG Lisanti MP Caveolin-1 gene disruption promotes mammary tumorigenesis and dramatically enhances lung metastasis in vivo: role of Cav-1 in cell invasiveness and matrix metalloproteinase (MMP-2/9) secretion.J Biol Chem. 2004; 279: 51630-51646Crossref PubMed Scopus (269) Google Scholar Surface lung colonies were counted in a blinded manner under low power using a Nikon (Tokyo, Japan) SMZ-1500 stereomicroscope. P values were determined by applying the Mann-Whitney statistical analysis parameters, which does not assume a Gaussian distribution (nonparametric test). The invasive potential of Met-1 cell lines was measured via an in vitro modified Boyden chamber assay.25Albini A Iwamoto Y Kleinman HK Martin GR Aaronson SA Kozlowski JM McEwan RN A rapid in vitro assay for quantitating the invasive potential of tumor cells.Cancer Res. 1987; 47: 3239-3245PubMed Google Scholar, 26Hulit J Suyama K Chung S Keren R Agiostratidou G Shan W Dong X Williams TM Lisanti MP Knudsen K Hazan RB N-cadherin signaling potentiates mammary tumor metastasis via enhanced extracellular signal-regulated kinase activation.Cancer Res. 2007; 67: 3106-3116Crossref PubMed Scopus (166) Google Scholar Briefly, Met-1 cells in 0.5 ml of serum-free Dulbecco's modified Eagle's medium were added to the wells of 8-μm-pore membrane modified Boyden chambers, either coated with (for invasion assays; catalog no. 354483) or without (for migration assays; catalog no. 354578) Matrigel (Transwells; BD Biosciences, San Jose, CA). The lower chambers contained 10% fetal bovine serum in Dulbecco's modified Eagle's medium to serve as a chemoattractant. Cells were incubated at 37°C and allowed to migrate or invade, respectively, throughout the course of 6 and 18 hours. Noninvasive cells were removed from the upper surface of the membrane by scrubbing with cotton swabs. Chambers were stained in 0.5% crystal violet diluted in 100% methanol for 30 to 60 minutes, rinsed in water, and examined under a bright-field microscope. Values for invasion and migration were obtained by counting five fields per membrane (×20 objective) and represent the average of three independent experiments performed throughout multiple days. For inhibitor studies, the inhibitor was placed in both the lower and upper chambers. Total RNA (5 μg) was reverse transcribed using the Superscript III first-strand synthesis system (Invitrogen, Carlsbad, CA) using a HPLC purified T7-dT24 primer (Sigma Genosys, St. Louis, MO) which contains the T7 polymerase promoter sequence. The single-stranded cDNA was converted to double-stranded cDNA using DNA polymerase I (Promega, Madison, WI) and purified by cDNA spin column purification using GeneChip Sample Cleanup Module (Affymetrix, Santa Clara, CA). The double-stranded cDNA was used as a template to generate biotinylated cRNA using the Bioarray high-yield RNA transcription labeling kit (Enzo, New York, NY) and the labeled cRNA purified by GeneChip Sample Cleanup Module (Affymetrix). Fifteen μg of cRNA was fractionated to produce fragments of between 35 to 200 bp using 5× fragmentation buffer provided in the Cleanup Module. The sample was hybridized to mouse 430 2.0 microarray (Affymetrix) representing more than 39,000 transcripts. The hyb" @default.
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• W2045010127 title "Caveolin-1 (P132L), a Common Breast Cancer Mutation, Confers Mammary Cell Invasiveness and Defines a Novel Stem Cell/Metastasis-Associated Gene Signature" @default.
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