Stromal Cell Expression of Caveolin-1 Predicts Outcome in Breast Cancer
2009; Elsevier BV; Volume: 174; Issue: 6 Linguagem: Inglês
10.2353/ajpath.2009.080924
ISSN1525-2191
AutoresErica K. Sloan, Daniel R. Ciocca, Normand Pouliot, Anthony Natoli, Christina M. Restall, Michael A. Henderson, Mariel A. Fanelli, F. Darío Cuello-Carrión, Francisco E. Gago, Robin L. Anderson,
Tópico(s)Medicinal Plant Pharmacodynamics Research
ResumoCaveolin-1 has been linked to tumor progression and clinical outcome in breast cancer, but a clear resolution of its role as a prognostic marker is lacking. We assessed caveolin-1 levels in normal breast tissue and two breast cancer cohorts for which outcome data were available. We found that caveolin-1 was not expressed in normal breast luminal epithelium but was present in the epithelial compartment of some tumors. We found no association between caveolin-1 expression in the epithelial compartment and clinical outcome. However, high levels of caveolin-1 in the stromal tissue surrounding the tumor, rather than within tumor cells, associated strongly with reduced metastasis and improved survival (P < 0.0001). The onset of mammary tumors driven by Her2/neu overexpression was accelerated in mice lacking caveolin-1, thereby supporting the observation that the presence of caveolin-1 in the tumor microenvironment modulates tumor development. These studies suggest that stromal caveolin-1 expression may be a potential therapeutic target and a valuable prognostic indicator of breast cancer progression. Caveolin-1 has been linked to tumor progression and clinical outcome in breast cancer, but a clear resolution of its role as a prognostic marker is lacking. We assessed caveolin-1 levels in normal breast tissue and two breast cancer cohorts for which outcome data were available. We found that caveolin-1 was not expressed in normal breast luminal epithelium but was present in the epithelial compartment of some tumors. We found no association between caveolin-1 expression in the epithelial compartment and clinical outcome. However, high levels of caveolin-1 in the stromal tissue surrounding the tumor, rather than within tumor cells, associated strongly with reduced metastasis and improved survival (P < 0.0001). The onset of mammary tumors driven by Her2/neu overexpression was accelerated in mice lacking caveolin-1, thereby supporting the observation that the presence of caveolin-1 in the tumor microenvironment modulates tumor development. These studies suggest that stromal caveolin-1 expression may be a potential therapeutic target and a valuable prognostic indicator of breast cancer progression. In recent years, it has become increasing apparent that stromal components in the tumor microenvironment, including the extracellular matrix and cell types including fibroblasts, vascular endothelial cells, immune and in-flammatory cells, have a profound influence on the growth and metastasis of tumors. The molecular cross talk between tumor cells and these stromal elements plays an important role in defining the phenotype of a tumor.1Bissell MJ Radisky D Putting tumors in context.Nature Reviews (Cancer). 2001; 1: 1-19Crossref Google Scholar Tumor cells can trigger the deposition of a reactive stroma or desmoplasia containing activated fibroblasts, connective tissue, immune and inflammatory cells that may favor invasion and metastasis of the cancer.2Mueller MM Fusenig NE Friends or foes—bipolar effects of the tumour stroma in cancer.Nat Rev Cancer. 2004; 4: 839-849Crossref PubMed Scopus (1455) Google Scholar Many cell types in the mammary stroma express caveolin-1. However the role of this protein in molecular cross talk between tumor and stromal cells remains unknown. Caveolin-1 is an integral plasma membrane protein that resides in specialized lipid rafts called caveolae in terminally differentiated mesenchymal cells including adipocytes, endothelial cells, and fibroblasts.3Parton RG Hanzal-Bayer M Hancock JF Biogenesis of caveolae: a structural model for caveolin-induced domain formation.J Cell Sci. 2006; 119: 787-796Crossref PubMed Scopus (229) Google Scholar Within caveolae, the bilayer of cholesterol and sphingolipids is in an ordered state that restricts the movement of lipids. 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Implications for human breast cancer.J Biol Chem. 1998; 273: 20448-20455Crossref PubMed Scopus (191) Google Scholar, 12Lee 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 (399) Google Scholar are contradicted by more recent studies that found that its expression is associated with the myoepithelium and other tissues of mesenchymal origin and not with normal luminal epithelium.13Hurlstone AF Reid G Reeves JR Fraser J Strathdee G Rahilly M Parkinson EK Black DM Analysis of the Caveolin-1 gene at human chromosome 7q31.1 in primary tumours and tumour-derived cell lines.Oncogene. 1999; 18: 1881-1890Crossref PubMed Scopus (120) Google Scholar, 14Sagara Y Mimori K Yoshinaga K Tanaka F Nishida K Ohno S Inoue H Mori M Clinical significance of Caveolin-1. Caveolin-2 and HER2/neu mRNA expression in human breast cancer.Br J Cancer. 2004; 91: 959-965Crossref PubMed Scopus (84) Google Scholar, 15Savage K Lambros MB Robertson D Jones RL Jones C Mackay A James M Hornick JL Pereira EM Milanezi F Fletcher CD Schmitt FC Ashworth A Reis-Filho JS Caveolin 1 is overexpressed and amplified in a subset of basal-like and metaplastic breast carcinomas: a morphologic, ultrastructural, immunohistochemical, and in situ hybridization analysis.Clin Cancer Res. 2007; 13: 90-101Crossref PubMed Scopus (187) Google Scholar, 16Pinilla SM Honrado E Hardisson D Benitez J Palacios J Caveolin-1 expression is associated with a basal-like phenotype in sporadic and hereditary breast cancer.Breast Cancer Res Treat. 2006; 99: 85-90Crossref PubMed Scopus (117) Google Scholar A number of studies have reported that caveolin-1 is down-regulated in breast cancers compared with normal mammary tissue.14Sagara Y Mimori K Yoshinaga K Tanaka F Nishida K Ohno S Inoue H Mori M Clinical significance of Caveolin-1. Caveolin-2 and HER2/neu mRNA expression in human breast cancer.Br J Cancer. 2004; 91: 959-965Crossref PubMed Scopus (84) Google Scholar, 17Yang G Truong LD Timme TL Ren C Wheeler TM Park SH Nasu Y Bangma CH Kattan MW Scardino PT Thompson TC Elevated expression of caveolin is associated with prostate and breast cancer.Clin Cancer Res. 1998; 4: 1873-1880PubMed Google Scholar, 18Chen ST Lin SY Yeh KT Kuo SJ Chan WL Chu YP Chang JG Mutational, epigenetic and expressional analyses of caveolin-1 gene in breast cancers.Int J Mol Med. 2004; 14: 577-582PubMed Google Scholar, 19Park SS Kim JE Kim YA Kim YC Kim SW Caveolin-1 is down-regulated and inversely correlated with HER2 and EGFR expression status in invasive ductal carcinoma of the breast.Histopathology. 2005; 47: 625-630Crossref PubMed Scopus (60) Google Scholar Similarly, caveolin-1 was absent in 10 invasive breast carcinomas, but present in two breast tumors of myoepithelial origin (basal-like cancers).13Hurlstone AF Reid G Reeves JR Fraser J Strathdee G Rahilly M Parkinson EK Black DM Analysis of the Caveolin-1 gene at human chromosome 7q31.1 in primary tumours and tumour-derived cell lines.Oncogene. 1999; 18: 1881-1890Crossref PubMed Scopus (120) Google Scholar Down-regulation of caveolin-1 may be due to inactivating mutations in the gene, as reported previously,20Hayashi 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, 21Li 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 (95) Google Scholar but not confirmed in another study.18Chen ST Lin SY Yeh KT Kuo SJ Chan WL Chu YP Chang JG Mutational, epigenetic and expressional analyses of caveolin-1 gene in breast cancers.Int J Mol Med. 2004; 14: 577-582PubMed Google Scholar Caveolin-1 gene expression may also be regulated in breast cancer through methylation.18Chen ST Lin SY Yeh KT Kuo SJ Chan WL Chu YP Chang JG Mutational, epigenetic and expressional analyses of caveolin-1 gene in breast cancers.Int J Mol Med. 2004; 14: 577-582PubMed Google Scholar New data demonstrate that high caveolin-1 expression in tumor epithelium is associated with basal, metaplastic, and triple negative breast cancers (negative for estrogen receptor [ER], progesterone receptor [PR], and Her2),15Savage K Lambros MB Robertson D Jones RL Jones C Mackay A James M Hornick JL Pereira EM Milanezi F Fletcher CD Schmitt FC Ashworth A Reis-Filho JS Caveolin 1 is overexpressed and amplified in a subset of basal-like and metaplastic breast carcinomas: a morphologic, ultrastructural, immunohistochemical, and in situ hybridization analysis.Clin Cancer Res. 2007; 13: 90-101Crossref PubMed Scopus (187) Google Scholar, 22Elsheikh SE Green AR Rakha EA Samaka RM Ammar AA Powe D Reis-Filho JS Ellis IO Caveolin 1 and caveolin 2 are associated with breast cancer basal-like and triple-negative immunophenotype.Br J Cancer. 2008; 99: 327-334Crossref PubMed Scopus (122) Google Scholar as well as with inflammatory breast cancers.23Van den Eynden GG Van Laere SJ Van der Auwera I Merajver SD Van Marck EA van Dam P Vermeulen PB Dirix LY van Golen KL Overexpression of caveolin-1 and -2 in cell lines and in human samples of inflammatory breast cancer.Breast Cancer Res Treat. 2006; 95: 219-228Crossref PubMed Scopus (77) Google Scholar The role of caveolin-1 in other tumor types appears to be varied, with expression associated with cancer suppression in ovarian, small cell lung, and colon cancers,24Wiechen K Diatchenko L Agoulnik A Scharff KM Schober H Arlt K Zhumabayeva B Siebert PD Dietel M Schafer R Sers C Caveolin-1 is down-regulated in human ovarian carcinoma and acts as a candidate tumor suppressor gene.Am J Pathol. 2001; 159: 1635-1643Abstract Full Text Full Text PDF PubMed Scopus (260) Google Scholar, 25Sunaga N Miyajima K Suzuki M Sato M White MA Ramirez RD Shay JW Gazdar AF Minna JD Different roles for caveolin-1 in the development of non-small cell lung cancer versus small cell lung cancer.Cancer Res. 2004; 64: 4277-4285Crossref PubMed Scopus (166) Google Scholar, 26Bender FC Reymond MA Bron C Quest AF Caveolin-1 levels are down-regulated in human colon tumors, and ectopic expression of caveolin-1 in colon carcinoma cell lines reduces cell tumorigenicity.Cancer Res. 2000; 60: 5870-5878PubMed Google Scholar but with cancer progression in prostate, non-small cell lung, esophageal, and colon cancers.25Sunaga N Miyajima K Suzuki M Sato M White MA Ramirez RD Shay JW Gazdar AF Minna JD Different roles for caveolin-1 in the development of non-small cell lung cancer versus small cell lung cancer.Cancer Res. 2004; 64: 4277-4285Crossref PubMed Scopus (166) Google Scholar, 27Karam JA Lotan Y Roehrborn CG Ashfaq R Karakiewicz PI Shariat SF Caveolin-1 overexpression is associated with aggressive prostate cancer recurrence.Prostate. 2007; 67: 614-622Crossref PubMed Scopus (123) Google Scholar, 28Fine SW Lisanti MP Galbiati F Li M Elevated expression of caveolin-1 in adenocarcinoma of the colon.Am J Clin Pathol. 2001; 115: 719-724Crossref PubMed Scopus (111) Google Scholar, 29Kato K Hida Y Miyamoto M Hashida H Shinohara T Itoh T Okushiba S Kondo S Katoh H Overexpression of caveolin-1 in esophageal squamous cell carcinoma correlates with lymph node metastasis and pathologic stage.Cancer. 2002; 94: 929-933Crossref PubMed Scopus (219) Google Scholar To clarify the relationship between caveolin-1 and breast cancer progression or suppression, we have analyzed tissue sections specifically for stromal and tumor epithelial cell expression of caveolin-1 from two cohorts of breast cancer patients. Here we report that caveolin-1 levels in the tumor microenvironment rather than tumor epithelial compartment correlate strongly with clinical outcome. This observation is supported by our studies of Her2/neu driven tumor development in caveolin-1 null mice, where tumor onset is accelerated in the absence of stromal caveolin-1. These findings indicate that stromal caveolin-1 has a potential role as a new prognostic marker for breast cancer progression as well as being a possible therapeutic target. Normal human breast tissue was collected by the Peter Mac Tissue Bank from consenting women undergoing breast reduction surgery. The Breast Prognosis tissue microarray, kindly provided by Dr. O. Kallioniemi, contains one 0.6-mm biopsy from the periphery of each of 612 human primary breast carcinomas with extensive clinical follow-up data.30Barlund M Forozan F Kononen J Bubendorf L Chen Y Bittner ML Torhorst J Haas P Bucher C Sauter G Kallioniemi OP Kallioniemi A Detecting activation of ribosomal protein S6 kinase by complementary DNA and tissue microarray analysis.J Natl Cancer Inst. 2000; 92: 1252-1259Crossref PubMed Scopus (249) Google Scholar The analysis reported here includes 429 samples with interpretable data. The remaining samples did not contain useable information for a number of reasons: (i) samples were not present on the glass slide or were lost from the slide during the immunostaining procedure, (ii) no tumor tissue was present in the sample, (iii) the quality of the sample was too poor to interpret expression, or (iv) survival data were not available. The mean age at diagnosis was 61 years (SD = 12.3; range 33 to 97 years). The stage distribution was TNM stage I, 22%; stage II, 53%; stage III, 17%; and stage IV, 2% of the patients. TNM stage unavailable for 6% of the patients. The samples included 78% ductal, 9% lobular, 3% medullary, and 10% with other tumor subtypes. The median tumor diameter was 23 mm (range, 4 to 130 mm). The 5-year cancer-specific survival rate for all patients was 78% (95% CI: 74% to 83%). Whole sections were analyzed from archival breast cancer tissues from consecutive patients with stage 1 to 3 operable breast cancer who presented at a single clinic (Dr. F. E. Gago, Mendoza, Argentina) between June 1985 and June 1999. For 173 patients there was sufficient tissue for analysis of both epithelial and stromal caveolin-1 immunostaining, and complete pathology information was available. Informed consent to obtain and analyze tissue was given before surgery, and tissue blocks and clinical data were stored and managed following standard international procedures. The median age of this cohort was 54 years (range, 57 to 81). The median tumor size was 25 mm (range 5 mm to 90 mm). The majority of patients (54%) were lymph node negative. AJCC stage presentation was stage 1, 27%; stage 2a, 38%; stage 2b, 17%; stage 3a, 13%; and stage 3b, 5%. All patients were followed until death or until June 2004 with a median follow up of 11.5 years. Patients were all treated according to approved protocols active at the time of diagnosis, with 28% receiving a mastectomy, 72% receiving chemotherapy, 76% receiving radiotherapy, and 72% being treated with tamoxifen. The diagnosis of metastasis was based on pathological confirmation or on other investigations that confirmed the presence of metastasis. Apart from three, all patients received surgery as the first treatment, and samples for analysis were obtained before chemotherapy. The remaining three samples were obtained after chemotherapy. Parallel analysis that excluded these three patients found substantially similar results to analysis of the entire cohort. Caveolin-1 immunostaining was completed on 3.5-micron sections of individual tissues or on the tissue microarray using a standard protocol with antigen retrieval in 10 mmol/L sodium citrate, pH 6.0, at 98°C for 15 minutes.31Chia J Kusuma N Anderson R Parker B Bidwell B Zamurs L Nice E Pouliot N Evidence for a role of tumor-derived laminin-511 in the metastatic progression of breast cancer.Am J Pathol. 2007; 170: 2135-2148Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar All tissues were fixed in 10% buffered formalin and paraffin-embedded. Mouse monoclonal and rabbit polyclonal anti-caveolin-1 antibodies (BD Transduction Laboratories, Lexington, KY), were used at 2.5 and 0.5 μg/ml respectively. Non-specific mouse IgG1 antibody and purified rabbit pre-immune serum (DAKO, Kingsgrove, NSW, Australia) were used as isotype controls. Secondary biotin-conjugated goat anti-mouse/rabbit antibodies (Vector Laboratories Homebush, NSW, Australia) were used at 1:250 dilution. Specific primary-secondary antibody complexes were detected using ABC reagent (Vector) and visualized using a diaminobenzidine peroxidase substrate kit (Vector). For the whole sections, the extent and intensity of caveolin-1 immunostaining was assessed independently by two experienced breast pathologists and in the 22% of the samples where there was some disagreement (often relating to the level of staining intensity), the matter was resolved by majority consensus with a third experienced breast pathologist. The tissue microarray was assessed by one pathologist and two other independent scorers and the majority opinion taken. Caveolin-1 immunostaining in tumor cells or stroma was scored as 0 (no expression), 1 (weak expression), or 2 (strong expression). Analysis of ER, PR, c-erbB2, proliferating nuclear cell antigen, p53, and p170 on the whole tissue sections has been published previously.32Gago FE Tello OM Diblasi AM Ciocca DR Integration of estrogen and progesterone receptors with pathological and molecular prognostic factors in breast cancer patients.J Steroid Biochem Mol Biol. 1998; 67: 431-437Crossref PubMed Scopus (25) Google Scholar, 33Gago FE Fanelli MA Ciocca DR Co-expression of steroid hormone receptors (estrogen receptor alpha and/or progesterone receptors) and Her2/neu (c-erbB-2) in breast cancer: clinical outcome following tamoxifen-based adjuvant therapy.J Steroid Biochem Mol Biol. 2006; 98: 36-40Crossref PubMed Scopus (24) Google Scholar Frequency data were analyzed with the Fisher Exact test and continuous data by the Mann-Whitney U-test. The Kaplan-Meier method was used to estimate overall survival, and differences in outcome for each variable were compared with the log-rank test. Multivariate analysis of factors affecting survival was performed using a Cox proportional hazard model. All statistical tests were two-sided and P < 0.05 was considered to be statistically significant. SPSS software (version 16, Chicago, IL) was used for the statistical analysis of the human data and Graph Pad Prism (version 5.01) for the mouse outcome data. Mice lacking stromal caveolin-1 and with mammary-specific expression of Her-2/neu were bred from caveolin-1 null mice (129/Sv/C57Bl/6) obtained from Dr. T. Kurchalia34Drab M Verkade P Elger M Kasper M Lohn M Lauterbach B Menne J Lindschau C Mende F Luft FC Schedl A Haller H Kurzchalia TV Loss of caveolae, vascular dysfunction, and pulmonary defects in caveolin-1 gene-disrupted mice.Science. 2001; 293: 2449-2452Crossref PubMed Scopus (1305) Google Scholar through Dr. R. Parton (University of Queensland, Australia) and from mice transgenic for the MMTV-neu oncogene (FVB/N, obtained from Dr. W. Muller through Dr. J. Visrader).35Guy CT Webster MA Schaller M Parsons TJ Cardiff RD Muller WJ Expression of the neu protooncogene in the mammary epithelium of transgenic mice induces metastatic disease.Proc Natl Acad Sci USA. 1992; 89: 10578-10582Crossref PubMed Scopus (1011) Google Scholar Matings were performed with male MMTV-neu+/−:caveolin-1+/−, and female caveolin-1+/− mice. Six genotypes, either heterozygote or null for MMTV-neu and with either two, one, or no alleles of caveolin-1, with a minimum of 15 female mice from each genotype were obtained from the same generation of MMTV-neu+/−:caveolin-1+/− male and female breeders. At 8 to 10 weeks and again at 16 to 20 weeks, the mice in each genotype were subjected to pregnancy and 10 days of lactation and were monitored weekly for development of mammary tumors. Once tumors became palpable they were measured weekly using electronic calipers and mice were culled when the primary tumor reached a volume of 1500 mm3. All procedures were performed in a barrier facility under protocols approved by the Peter MacCallum Animal Experimentation Ethics Committee. Primary tumors and lungs were removed at autopsy, weighed, fixed in 10% buffered formalin, and processed for paraffin embedding. Primary tumors were sectioned and stained with H&E for general morphology or immunostained to determine caveolin-1 levels as described above. The lungs were ribbon cut into 5-micron sections and every 20th section was H&E-stained and assessed by microscopy for metastatic nodules. The localization of caveolin-1 in normal breast tissue was assessed by immunostaining of tissue samples from ten healthy individuals using either a monoclonal antibody or a rabbit polyclonal antibody. The same expression pattern was seen with both antibodies. The specificity of the antibodies for caveolin-1 was confirmed previously by western analysis.36Sloan EK Stanley KL Anderson RL Caveolin-1 inhibits breast cancer growth and metastasis.Oncogene. 2004; 23: 7893-7897Crossref PubMed Scopus (138) Google Scholar Caveolin-1 immunoreactivity was observed in stromal fibroblasts and in myoepithelial cells underlying the luminal epithelial cells (Figure 1, A, B, D, and E). Adipocytes and vascular endothelial cells were also positive for caveolin-1 (Figure 1, C and F). However, no evidence for caveolin-1 expression was found in luminal epithelial cells. Consistent with our findings, a number of other reports describe the specific localization of caveolin-1 to breast myoepithelial and stromal cells, but not luminal epithelial cells13Hurlstone AF Reid G Reeves JR Fraser J Strathdee G Rahilly M Parkinson EK Black DM Analysis of the Caveolin-1 gene at human chromosome 7q31.1 in primary tumours and tumour-derived cell lines.Oncogene. 1999; 18: 1881-1890Crossref PubMed Scopus (120) Google Scholar, 14Sagara Y Mimori K Yoshinaga K Tanaka F Nishida K Ohno S Inoue H Mori M Clinical significance of Caveolin-1. Caveolin-2 and HER2/neu mRNA expression in human breast cancer.Br J Cancer. 2004; 91: 959-965Crossref PubMed Scopus (84) Google Scholar, 15Savage K Lambros MB Robertson D Jones RL Jones C Mackay A James M Hornick JL Pereira EM Milanezi F Fletcher CD Schmitt FC Ashworth A Reis-Filho JS Caveolin 1 is overexpressed and amplified in a subset of basal-like and metaplastic breast carcinomas: a morphologic, ultrastructural, immunohistochemical, and in situ hybridization analysis.Clin Cancer Res. 2007; 13: 90-101Crossref PubMed Scopus (187) Google Scholar although epithelial cell expression has also been reported.11Engelman 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 human breast cancer.J Biol Chem. 1998; 273: 20448-20455Crossref PubMed Scopus (191) Google Scholar, 12Lee 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 (399) Google Scholar To assess the association between caveolin-1 and clinical outcome in breast cancer, we used a monoclonal anti-caveolin-1 antibody to immunostain 429 arrayed primary human breast cancer samples. Details of the tissue array have been published previously.30Barlund M Forozan F Kononen J Bubendorf L Chen Y Bittner ML Torhorst J Haas P Bucher C Sauter G Kallioniemi OP Kallioniemi A Detecting activation of ribosomal protein S6 kinase by complementary DNA and tissue microarray analysis.J Natl Cancer Inst. 2000; 92: 1252-1259Crossref PubMed Scopus (249) Google Scholar Breast tumor cells in each sample were scored for the intensity of caveolin-1 staining; 268 (63%) of breast tumors were negative for caveolin-1, 130 (30%) had weak caveolin-1 immunoreactivity, and 31 (7%) were strongly positive for caveolin-1. Representative examples are shown in Figure 2A. The level of caveolin-1 expression in primary breast tumor cells was assessed in relation to other factors known to be associated with tumor progression. The presence of caveolin-1 in epithelial tumor cells was positively associated with lower TNM tumor stage (P = 0.05). However, caveolin-1 levels in tumor epithelium did not predict cancer-specific survival over 5 years, 77% (95% CI: 71% to 83%) for patients with caveolin-1 negative primary tumors, 79% (95% CI: 71% to 87%) for patients with weak caveolin-1 immunostaining and 88% (95% CI: 76% to 100%) for patients with strong caveolin-1 immunostaining (P = 0.24) (Figure 2B). Consistent with these findings, caveolin-1 expression in tumor cells was not associated with any of the other standard prognostic factors investigated (data not shown). During screening of caveolin-1 levels in the arrayed breast tumor samples, we noted significant variation of caveolin-1 levels in stromal tissue in the tumor microenvironment in the few samples where significant amounts of stroma were present. However, further analysis of stromal caveolin-1 levels in the arrayed tumor samples was prevented by insufficient samples containing adequate amounts of stromal tissue. The samples were very small (0.6 mm) and the biopsy location was chosen to maximize tumor cell density. The heterogeneity of breast cancer confounds the analysis of samples with very small amounts of stroma. To explore further the relationship between caveolin-1 levels in the tumor microenvironment and breast cancer progression, we immunostained for caveolin-1 in a consecutive series of 173 primary breast tumors and specifically analyzed the amount of caveolin-1 protein either in tumor cells or in stromal cells. There were 103 of 173 patients (60%) who had unambiguous staining of the stromal compartment of the tumor. As in normal breast tissue, caveolin-1 was detected in vascular endothelial cells in these tumor sections, but expression in other stromal elements, often fibroblast-rich desmoplastic tissue, was variable. Representative examples of tumors with caveolin-1 positive and negative stromal tissue are shown in Figure 2C. The relationship between standard prognostic factors and other molecular markers with caveolin-1 expression in the stroma is shown in Table 1. Caveolin-1 stromal staining was associated with smaller tumor size (T stage) (p = 0.03) and grade (P = 0.001). There was a trend toward patients expressing stromal caveolin-1 being younger (P = 0.08), having lower AJCC stage tumors at presentation (P = 0.07) and less proliferative tumors (proliferating nuclear cell antigen) (P = 0.07). Caveolin-1 stromal staining was not related to caveolin-1 staining in tumor epithelium.Table 1Correlations between Clinicopathologic Factors and Specific Protein Markers with Caveolin-1 Expression in the Stromal Compartment of 173 Breast CancersAll patients n = 173 (%)Caveolin-1 positive stroma n = 103 (60%)Caveolin-1 negative stroma n = 70 (40%)P valueMedian age (27–81 years), n = 1625451570.08Median tumor size (5–90 mm), n = 1642525250.7Tumor stage, n = 1650.03 T1 (0–20 mm)64 (39)41 (42)23 (34) T2 (20–50 mm)94 (57)54 (56)40 (59) T3 (>50 mm)/T47 (4)2 (2)5 (7)Lymph node status, n = 1690.1 N0 (0 nodes)92 (54)58 (57)34 (50) N1 (1–3 nodes)47 (28)30 (30)17 (25) N2 (4–9 nodes)20 (12)10 (10)10 (15) N3 (>9 nodes)10 (6)3 (3)7 (10)AJCC stage0.07 I45 (27)29 (29)17 (25) IIa62 (38)40 (39)34 (23) IIb28 (17)18 (19)10 (1
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