Caveolin-1 Mutations in Human Breast Cancer
2006; Elsevier BV; Volume: 168; Issue: 6 Linguagem: Inglês
10.2353/ajpath.2006.051089
ISSN1525-2191
AutoresTianhong Li, Federica Sotgia, Magalis Vuolo, Maomi Li, Wancai Yang, Richard G. Pestell, Joseph A. Sparano, Michael P. Lisanti,
Tópico(s)Medicinal Plant Pharmacodynamics Research
ResumoA Japanese study reported that up to 16% of breast cancer samples harbor a sporadic mutation within the human Cav-1 gene, namely P132L. To date, however, no studies have examined the United States' population. Here, we developed a novel allele-specific real-time PCR assay to detect the Cav-1 P132L mutation in mammary tumor cells isolated by laser capture microdissection from formalin-fixed paraffin-embedded breast cancer samples. We report that the Cav-1 P132L mutation is present in ∼19% of estrogen receptor α (ERα)-positive breast cancers but not in ERα-negative breast cancers. This is the first demonstration that the P132L mutation is exclusively associated with ERα-positive mammary tumors. We also identified six novel Cav-1 mutations associated with ERα-positive breast cancers (W128Stop, Y118H, S136R, I141T, Y148H, and Y148S). Thus, the overall incidence of Cav-1 mutations in ERα-positive breast cancers approaches 35% (greater than one-third). To mechanistically dissect the functional relationship between Cav-1 gene inactivation and ERα expression, we isolated primary mammary epithelial cells from wild-type and Cav-1−/− mice and cultured them in a three-dimensional system, allowing them to form mammary acinar-like structures. Under conditions of growth factor deprivation, Cav-1-deficient mammary acini displayed increased ERα levels and enhanced sensitivity toward estrogen-stimulated growth, with specific up-regulation of cyclin D1. Finally, we discuss the possibility that sporadic Cav-1 mutations may act as an initiating event in human breast cancer pathogenesis. A Japanese study reported that up to 16% of breast cancer samples harbor a sporadic mutation within the human Cav-1 gene, namely P132L. To date, however, no studies have examined the United States' population. Here, we developed a novel allele-specific real-time PCR assay to detect the Cav-1 P132L mutation in mammary tumor cells isolated by laser capture microdissection from formalin-fixed paraffin-embedded breast cancer samples. We report that the Cav-1 P132L mutation is present in ∼19% of estrogen receptor α (ERα)-positive breast cancers but not in ERα-negative breast cancers. This is the first demonstration that the P132L mutation is exclusively associated with ERα-positive mammary tumors. We also identified six novel Cav-1 mutations associated with ERα-positive breast cancers (W128Stop, Y118H, S136R, I141T, Y148H, and Y148S). Thus, the overall incidence of Cav-1 mutations in ERα-positive breast cancers approaches 35% (greater than one-third). To mechanistically dissect the functional relationship between Cav-1 gene inactivation and ERα expression, we isolated primary mammary epithelial cells from wild-type and Cav-1−/− mice and cultured them in a three-dimensional system, allowing them to form mammary acinar-like structures. Under conditions of growth factor deprivation, Cav-1-deficient mammary acini displayed increased ERα levels and enhanced sensitivity toward estrogen-stimulated growth, with specific up-regulation of cyclin D1. Finally, we discuss the possibility that sporadic Cav-1 mutations may act as an initiating event in human breast cancer pathogenesis. Multiple independent lines of experimental evidence suggest that Cav-1 functions as a mammary gland tumor suppressor gene.1Williams TM Lisanti MP Caveolin-1 in oncogenic transformation, cancer, and metastasis.Am J Physiol Cell Physiol. 2005; 288: C494-C506Crossref PubMed Scopus (462) Google Scholar, 2Bouras T Lisanti MP Pestell RG Caveolin-1 in breast cancer.Cancer Biol Ther. 2004; 3: 82-92Crossref PubMed Scopus (58) Google Scholar First, Cav-1 mRNA and protein levels are down-regulated in oncogene-transformed NIH 3T3 cells, in many human and mouse breast cancer cell lines, in primary human mammary gland tumors, and in transgenic breast cancer mouse models.3Koleske AJ Baltimore D Lisanti MP Reduction of caveolin and caveolae in oncogenically transformed cells.Proc Natl Acad Sci USA. 1995; 92: 1381-1385Crossref PubMed Scopus (474) Google Scholar, 4Engelman JA Lee RJ Karnezis A Bearss DJ Webster M Siegel P Muller WJ Windle JJ Pestell RG Lisanti MP Reciprocal regulation of Neu tyrosine kinase activity and caveolin-1 protein expression in vitro and in vivo. Implications for mammary tumorigenesis.J Biol Chem. 1998; 273: 20448-20455Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar, 5Lee SW Reimer CL Oh P Campbell DB Schnitzer JE Tumor cell growth inhibition by caveolin re-expression in human breast cancer cells.Oncogene. 1998; 16: 1391-1397Crossref PubMed Scopus (400) Google Scholar, 6Park DS Razani B Lasorella A Schreiber-Agus N Pestell RG Iavarone A Lisanti MP Evidence that Myc isoforms transcriptionally repress caveolin-1 gene expression via an INR-dependent mechanism.Biochemistry. 2001; 40: 3354-3362Crossref PubMed Scopus (55) Google Scholar Conversely, Cav-1 re-expression in breast cancer cell lines inhibits anchorage-dependent growth in soft agar and decreases their invasive potential.5Lee SW Reimer CL Oh P Campbell DB Schnitzer JE Tumor cell growth inhibition by caveolin re-expression in human breast cancer cells.Oncogene. 1998; 16: 1391-1397Crossref PubMed Scopus (400) Google Scholar, 7Fiucci G Ravid D Reich R Liscovitch M Caveolin-1 inhibits anchorage-independent growth, anoikis and invasiveness in MCF-7 human breast cancer cells.Oncogene. 2002; 21: 2365-2375Crossref PubMed Scopus (251) Google Scholar Cav-1 expression also reduces the migratory and invasive potential of MTLn3 cells, a metastatic mammary carcinoma line, by preventing epidermal growth factor (EGF)-induced lamellipodia formation and reducing cell migration.8Zhang W Razani B Altschuler Y Bouzahzah B Mostov KE Pestell RG Lisanti MP Caveolin-1 inhibits epidermal growth factor-stimulated lamellipod extension and cell migration in metastatic mammary adenocarcinoma cells (MTLn3). 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For example, Cav-1−/− mammary glands exhibit signs of premalignant lesions, ie, ductal hyperplasia with wall thickening to three to four cell layers.10Lee 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 Simultaneous loss of Cav-1 and of another tumor suppressor gene, INK4a, further perturbs mammary gland morphology, with increased ductal hyperplasia and lateral branching and the presence of fibrosis.11Williams 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: Role of INK4a/CAV-1 in mammary epithelial cell hyperplasia.J Biol Chem. 2004; 279: 24745-24756Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar Moreover, in the context of a mammary gland tumor-prone mouse model (MMTV-PyMT), genetic ablation of Cav-1 expression accelerates the appearance and growth of dysplastic lesions at the very early stages of mammary gland development, greatly facilitates mammary tumor formation at 14 weeks of age, and augments metastasis to distant sites, such as the lung.12Williams 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, 13Williams 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: 51630-51646Abstract Full Text Full Text PDF PubMed Scopus (267) Google Scholar Genetic validation of the idea that Cav-1 functions as a tumor suppressor gene emerged from the observation that the human Cav-1 gene maps to the long arm of chromosome 7, in very close proximity to the D7S522 locus.14Engelman JA Zhang XL Lisanti MP Genes encoding human caveolin-1 and -2 are co-localized to the D7S522 locus (7q31.1), a known fragile site (FRA7G) that is frequently deleted in human cancers.FEBS Lett. 1998; 436: 403-410Crossref PubMed Scopus (199) Google Scholar This region includes a known fragile site (FRA7G) and is often associated with loss of heterozygosity in various human cancers, including breast, ovarian, and renal cell carcinomas.15Zenklusen JC Bieche I Lidereau R Conti CJ (C-A)n microsatellite repeat D7S522 is the most commonly deleted region in human primary breast cancer.Proc Natl Acad Sci USA. 1994; 91: 12155-12158Crossref PubMed Scopus (127) Google Scholar, 16Zenklusen JC Thompson JC Troncoso P Kagan J Conti CJ Loss of heterozygosity in human primary prostate carcinomas: a possible tumor suppressor gene at 7q31.1.Cancer Res. 1994; 54: 6370-6373PubMed Google Scholar, 17Zenklusen JC Thompson JC Klein-Szanto AJ Conti CJ Frequent loss of heterozygosity in human primary squamous cell and colon carcinomas at 7q31.1: evidence for a broad range tumor suppressor gene.Cancer Res. 1995; 55: 1347-1350PubMed Google Scholar, 18Zenklusen JC Weitzel JN Ball HG Conti CJ Allelic loss at 7q31.1 in human primary ovarian carcinomas suggests the existence of a tumor suppressor gene.Oncogene. 1995; 11: 359-363PubMed Google Scholar, 19Achille A Biasi MO Zamboni G Bogina G Magalini AR Pederzoli P Perucho M Scarpa A Chromosome 7q allelic losses in pancreatic carcinoma.Cancer Res. 1996; 56: 3808-3813PubMed Google Scholar, 20Koike M Takeuchi S Yokota J Park S Hatta Y Miller CW Tsuruoka N Koeffler HP Frequent loss of heterozygosity in the region of the D7S523 locus in advanced ovarian cancer.Genes Chromosomes Cancer. 1997; 19: 1-5Crossref PubMed Scopus (57) Google Scholar, 21Shridhar V Sun QC Miller OJ Kalemkerian GP Petros J Smith DI Loss of heterozygosity on the long arm of human chromosome 7 in sporadic renal cell carcinomas.Oncogene. 1997; 15: 2727-2733Crossref PubMed Scopus (65) Google Scholar, 22Huang H Qian C Jenkins RB Smith DI Fish mapping of YAC clones at human chromosomal band 7q31.2: identification of YACS spanning FRA7G within the common region of LOH in breast and prostate cancer.Genes Chromosomes Cancer. 1998; 21: 152-159Crossref PubMed Scopus (69) Google Scholar, 23Jenkins RB Qian J Lee HK Huang H Hirasawa K Bostwick DG Proffitt J Wilber K Lieber MM Liu W Smith DI A molecular cytogenetic analysis of 7q31 in prostate cancer.Cancer Res. 1998; 58: 759-766PubMed Google Scholar As such, a putative tumor suppressor gene is thought to be located within this chromosomal region. In support of this notion, a Japanese study detected a sporadic mutation in the Cav-1 gene, leading to a proline-to-leucine substitution at position 132 (P132L) in up to 16% of patients with primary breast tumors.24Hayashi 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 Recombinant expression of the Cav-1 P132L mutant in NIH 3T3 cells induced cellular transformation, activation of the p42/44 mitogen-activated protein kinase signaling cascade, and promoted cellular invasion.24Hayashi 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 Moreover, the Cav-1 P132L mutant was shown to act in a dominant-negative fashion, causing the mislocalization and intracellular retention of wild-type endogenous Cav-1 in a nontransformed human mammary epithelial cell line.10Lee 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 As such, this heterozygous mutation leads to complete functional inactivation of the Cav-1 protein in the context of mammary epithelial cells. However, it remains unknown whether the Cav-1 P132L mutation or any other Cav-1 mutations are associated with human breast cancers in the United States. It is believed that estrogen increases the proliferation rate of mammary epithelial cells and, thus, that estrogen exposure increases the risk of developing breast cancer. Estrogen binds to the estrogen receptor (ER), which belongs to a large family of nuclear receptors. ER functions as a transcription factor that, upon estrogen-induced ligand-activation, binds DNA and regulates the expression of estrogen-responsive genes. ERα is the primary mediator of estrogen responses during cell proliferation in the breast, whereas ERβ possesses antiproliferative properties.25Koehler KF Helguero LA Haldosen LA Warner M Gustafsson JA Reflections on the discovery and significance of estrogen receptor beta.Endocr Rev. 2005; 26: 465-478Crossref PubMed Scopus (333) Google Scholar ERα is essential for mammary ductal growth, and ERα knockout (KO) mice lack duct formation. Despite this, in the normal adult mammary gland, ERα is found only in a small percentage (∼10 to 20%) of luminal epithelial cells. Interestingly, normal epithelial cells exhibit mutual exclusion of ERα expression and cell proliferation, as assessed by a lack of double immunostaining of ERα and the Ki-67 proliferation marker.26Clarke RB Howell A Potten CS Anderson E Dissociation between steroid receptor expression and cell proliferation in the human breast.Cancer Res. 1997; 57: 4987-4991PubMed Google Scholar However, ERα expression is elevated at the earliest stages of mammary tumorigenesis, such as ductal hyperplasia, and increases even further with increasing atypia.27Shoker BS Jarvis C Sibson DR Walker C Sloane JP Oestrogen receptor expression in the normal and pre-cancerous breast.J Pathol. 1999; 188: 237-244Crossref PubMed Scopus (157) Google Scholar, 28Shoker BS Jarvis C Clarke RB Anderson E Hewlett J Davies MP Sibson DR Sloane JP Estrogen receptor-positive proliferating cells in the normal and precancerous breast.Am J Pathol. 1999; 155: 1811-1815Abstract Full Text Full Text PDF PubMed Scopus (232) Google Scholar As such, the inverse correlation between ERα expression and proliferation is lost in some breast cancers, in which a large percentage of proliferating cells become ERα-positive. However, the molecular mechanisms for initiating increased steroid receptor expression in breast cancer cells remain largely unknown. The aim of the present study was to evaluate the incidence of Cav-1 mutations in human breast cancers within the United States' population. We found that ∼20% of primary breast cancers carry a Cav-1 mutation. Remarkably, Cav-1 mutations were exclusively found in ERα-positive breast tumors, with a relative incidence of 35%. Importantly, this is the first demonstration that Cav-1 mutations are associated with ERα-positive breast cancers. As such, we propose that Cav-1 loss-of-function may be one of the initiating mechanisms underlying ERα overexpression during early mammary tumorigenesis. To test this hypothesis directly, we reconstituted mammary acini formation in vitro using primary cultures of mammary epithelial cells derived from wild-type (WT) and Cav-1−/− mice. Interestingly, we demonstrate that, when cultured in the absence of a growth factor stimulus, Cav-1-null acini displayed ∼4-fold increased levels of ERα. In addition, in the absence of EGF, estrogen-stimulated Cav-1-deficient acini demonstrated enhanced growth rates and up-regulation of cyclin D1 levels. Antibodies and their sources were as follows: ERα (H-184 and MC-20) and Cav-1 (N-20) from Santa Cruz Biotechnology (Santa Cruz, CA); E-cadherin was from BD Pharmingen (San Diego, CA); and cyclin D1 (Ab-3) from NeoMarkers (Fremont, CA). Other reagents were as follows: hydrocortisone, cholera toxin, insulin, β-estradiol, and gentamicin (from Sigma, St. Louis, MO), Collagenase type I, phenol red-free Dulbecco's modified Eagle's medium-F12 (from Gibco, Grand Island, NY); phenol-free reduced growth factor Matrigel (from Trevigen, Gaithersburg, MD); Lab-Tek II 8-well chamber slides (from Nalgene Nunc, Rochester, NY); and charcoal-stripped horse serum from Bioreclamation, Inc. (Hicksville, NY). All patients included in the study were female, with the histopathological diagnosis of invasive ductal carcinoma of the breast, under an Institutional Review Board-approved protocol at Montefiore Medical Center. No subpopulations were excluded. The clinical and pathological information regarding age at diagnosis, histology, stage according to the fifth version of the American Joint Committee on Cancer, status of the ER, time to first relapse or time to progression, and overall survival were summarized and recorded in a breast cancer database. Of the formalin-fixed, paraffin-embedded tissue blocks from >150 patients examined, analyzable DNA was obtained from only 55 patients who were included in this study. The study materials were coded to protect confidentiality. The tumor areas for microdissection were identified by two expert surgical pathologists using hematoxylin and eosin-stained slides. The quality of the resulting genomic DNA was stringently assessed by low percentage agarose gel electrophoresis and by conventional polymerase chain reaction (PCR) using primer set 1 to amplify the sequence of Cav-1. Only the 55 Cav-1 PCR-positive patient samples were selected for further mutational analysis. Sections (5-μm thickness) from formalin-fixed, paraffin-embedded human breast cancer blocks were placed onto standard glass slides (Fisher Scientific, Pittsburgh, PA), deparaffinized, rehydrated, and stained with hematoxylin and eosin according to standard procedures. A PixCell IIe LCM system (Arcturus, Mountain View, CA) was used to isolate breast cancer cell areas from normal cells and place them onto a thin polymer film (CapSure LCM Caps, Arcturus), using a laser beam of 7.5-μm diameter. About 3,000 to 10,000 laser shots were needed to obtain analyzable DNA from each tissue specimen. As normal controls, normal mammary epithelial cells from the same sample were isolated either by LCM or by macrodissection (if normal tissue was predominant in the sample). After lysis at 55°C overnight, genomic DNA was extracted from LCM-isolated cells using a DNeasy tissue kit (Qiagen, Valencia, CA), according to the manufacturer's recommendations, and eluted with 20 to 30 μl of distilled water. Genomic DNA (5 to 10 μl) was used for each conventional or allele-specific PCR analysis. For conventional PCR, the forward primer (5′-CCAGCTTCACCACCTTCACT-3′) and reverse primer (5′-CACAGACGGTGTGGACGTAG-3′) were used to amplify a 210-bp DNA fragment corresponding to a 70-amino acid region (amino acids 88 to 156), which includes the entire transmembrane domain (amino acids 102 to 134) of the Cav-1 gene (GenBank accession number: NM001753, See also Table 1). Each PCR reaction was performed in a 50-μl final volume containing ∼20 to 100 ng of genomic DNA, 10 mmol/L Tris-HCl, 50 mmol/L KCl, 1.5 mmol/L MgCl2, 10 mg of gelatin, 10 pmol/L of each primer, 200 μmol/L of each dNTP, and 0.2 U of TaqDNA polymerase (Promega, Madison, WI). PCR was performed in a thermal cycler (model 9600; PerkinElmer-Cetus, Boston, MA) using the following program: denaturation at 95°C for 5 minutes, followed by 35–40 amplification cycles (denaturation at 95°C for 60 seconds, annealing at 56°C for 60 seconds, and extension at 72°C for 60 seconds), and final extension at 72°C for 10 minutes. Both positive and negative controls were performed in parallel for each PCR reaction. The template for the positive control was genomic DNA extracted from the human breast cancer cell line MCF-7. Negative control reactions were performed without DNA template to exclude nonspecific amplification.Table 1Regular and Allele-Specific Primer Sets for Cav-1 WT and CAV-1 (P132L)SpecificityForward primerReverse primerPCR product size (bp)Membrane spanning domain and flanking sequences5′-ccagcttcaccaccttcact-3′5′-cacagacggtgtggacgtag-3′210 (Primer set 1)WT allele-specific (P132)5′-cacatctgggcagttgtacc-3′5′-cacagacggtgtggacgtag-3′93 (Primer set 2)Mutant allele-specific (P132L)5′-cacatctgggcagttgtact-3′5′-cacagacggtgtggacgtag-3′93 (Primer set 3)Mutant allele-specific (P132L), with degeneracy5′-cacatctgggcagttgtrct-3′5′-cacagacggtgtggacgtag-3′93 (Primer set 4)Four sets of primers are shown. Primer set 1 was designed to amplify the entire region (amino acids 87 to 156), and to subject genomic DNA to direct sequencing. Note that primers sets 2, 3, and 4 are allele-specific, recognizing the WT or P132L allele, and were employed in real-time PCR screening. The reverse primers are identical in all four sets. Also, primer set 4 was designed with degeneracy, due to a polymorphism at valine 131. Sequence differences are indicated in bold and are underlined. r = a or g degeneracy. Open table in a new tab Four sets of primers are shown. Primer set 1 was designed to amplify the entire region (amino acids 87 to 156), and to subject genomic DNA to direct sequencing. Note that primers sets 2, 3, and 4 are allele-specific, recognizing the WT or P132L allele, and were employed in real-time PCR screening. The reverse primers are identical in all four sets. Also, primer set 4 was designed with degeneracy, due to a polymorphism at valine 131. Sequence differences are indicated in bold and are underlined. r = a or g degeneracy. PCR products were separated by electrophoresis on 1.6 to 1.8% agarose gels before visualization via UV light. The PCR products were gel-extracted using a Gel Extraction Kit (Qiagen). To detect Cav-1 mutations, direct automated sequencing of PCR products was performed using the forward PCR primer by the dye-terminator fluorescence sequencing method on a fluorescent sequencer (model 3700; Applied Biosystems, Foster City, CA) at the DNA Sequencing Facility (Albert Einstein College of Medicine). All Cav-1 mutations were confirmed by direct sequencing using the reverse PCR primer. In addition, Cav-1 mutations were confirmed on independently LCM-isolated normal and breast tumor cells from the same tissue block or from different tissue blocks from the same patient, if available. A strategy to quickly detect the P132L mutation was designed using allele-specific real-time PCR. The template for allele-specific real-time PCR was either genomic DNA (50 to 100 ng) or the 210-bp PCR product from the conventional PCR (1 to 10 ng), described above. The allele-specific primers were designed to distinguish the P132L mutant from its wild-type counterpart. Because of a naturally occurring polymorphism in the third nucleotide of amino acid codon 131, the forward primer was designed with degeneracy (see Table 1). Amplification was performed using the allele-specific forward primer and a common reverse primer (50 to 900 nmol/L), using a SYBR Green master mixture (containing heat-activated AmpliTaq Gold DNA polymerase, dNTPs, buffer, SYBR Green (Applied Biosystems, Foster City, CA), and a reference dye). An ABI PRISM 7900HT (Applied Biosystems) was used for real-time PCR amplification and fluorescence melting curve analysis. Amplification consisted of a 2-minute AmpErase UNG incubation at 50°C, a 10-minute preincubation at 95°C to activate the TaqDNA polymerase, followed by 35 to 45 cycles (denaturation at 95°C for 15 seconds, primer annealing, and extension for 1 minute at 60°C) in 96-well plates. The fluorescence melting curve was analyzed immediately after amplification by measuring the fluorescence intensity of the PCR product from 60 to 95°C at a slope of 2%. The maximum rate of fluorescence change occurred at the Tm of the PCR product. The relative quantification of the target gene was acquired and analyzed using SDS 2.0 software (Applied Biosystems). The size of the expected PCR products was confirmed by agarose gel electrophoresis, and the candidate mutation was validated by direct sequencing. We checked our primer sequences very carefully, and they do not co-amplify other caveolins, such as Cav-2 or Cav-3. The DNA and protein sequences of the caveolins are actually quite divergent. If Cav-2 or Cav-3 sequences were co-amplified, we would have detected them, because they are easily distinguished based on their divergent DNA sequences. Several synonymous nucleotide polymorphisms in the Cav-1 gene were identified in our study, eg, the third nucleotide of P132P (CCA → CCA/G) and S136S (AGC → AGT/C). We discussed the chromatogram sequencing results with the Director of the Sequencing Facility at our institution: Although the A and G of the P132 were not completely lined up, its location and surrounding nucleotide sequences exclude the possibility of an insertion, and it thus should be considered as a polymorphism. This was further supported by its absence in the normal tissues from the same archival tissue blocks and in the tumors that did not have P132L mutations. All of the caveolin-1 mutations and polymorphisms found in the genomic DNA of tumor cells were not detected in the genomic DNA of corresponding normal cells. Interestingly, Lièvre et al29Lievre A Landi B Cote JF Veyrie N Zucman-Rossi J Berger A Laurent-Puig P Absence of mutation in the putative tumor-suppressor gene KLF6 in colorectal cancers.Oncogene. 2005; 24: 7253-7256Crossref PubMed Scopus (38) Google Scholar also observed the presence of three synonymous polymorphisms in tumor tissue DNA but not in matched normal tissue DNA. We do not understand the biological significance of these synonymous polymorphisms in the tumor cells. The results were reproducible by repeated PCR and sequencing analyses using genomic DNA isolated from different LCM isolations of the same tissue block or using a different tumor block whenever it was available. The P values for age, stage, and time to first relapse were calculated using the paired or unpaired Student's t-test. Frequency comparisons were analyzed using Fisher's exact test. The 95% confidence interval was calculated using the relevant 2 × 2 contingency tables. Differences with P < 0.05 were considered statistically significant. Sections (5-μm thickness) from archived paraffin-embedded human breast tissues were deparaffinized, rehydrated, and quenched with 1.5% H2O2. For ER staining, slides were treated with DakoCytomation Target Retrieval Solution (Dako, Carpinteria, CA) in a steam bath at 95°C for 45 minutes. After equilibration in phosphate-buffered saline for 15 minutes, slides were placed in an autostainer apparatus (Dako) and stained with antibodies to ERα (1:50 dilution; monoclonal antibody clone 1D5; Dako). Immunoreactivity was detected using the Dako EnVision method, according to the manufacturer's recommended procedures. It is important to note that the antibody used initially for the clinical screening of ERα positivity (by the Department of Pathology) was a mouse monoclonal. However, all further immunohistochemistry experiments performed in the Lisanti laboratory used rabbit polyclonal antibodies directed against ERα (H-184 and MC-20, from Santa Cruz Biotechnology). This approach provided independent validation of the ER positivity of a given clinical sample. Similarly, sections were also immunostained with a rabbit polyclonal antibody directed against cyclin D1 (Ab-3, from NeoMarkers). For negative controls, slides were subjected to the same procedures, including antigen retrieval, except for 1) omitting the primary antibody or 2) treating samples with nonimmune rabbit IgG. Both of these critical negative controls clearly demonstrated the specificity of the immunostaining that we observed. All animals were housed and maintained in a pathogen-free environment/barrier facility at the Institute for Animal Studies at the Albert Einstein College of Medicine under National Institute of Health guidelines. Mice were kept on a 12-hour light/dark cycle with ad libitum access to chow and water. Cav-1 KO mice were generated as previously described.30Razani B Engelman JA Wang XB Schubert W Zhang XL Marks CB Macaluso F Russell RG Li M Pestell RG Di Vizio D Hou Jr, H Kneitz B Lagaud G Christ GJ Edelmann W Lisanti MP Caveolin-1 null mice are viable but show evidence of hyperproliferative and vascular abnormalities.J Biol Chem. 2001; 276: 38121-38138Abstract Full Text Full Text PDF PubMed Scopus (258) Google Scholar All WT and Cav-1 KO mice used in this study were in the FVB/N genetic background.12Williams 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, 13Williams TM Medina F Badano I Hazan RB Hutchinson J Muller WJ Chopra NG Scherer PE Pest
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