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Caveolin-1 Gene Disruption Promotes Mammary Tumorigenesis and Dramatically Enhances Lung Metastasis in Vivo

2004; Elsevier BV; Volume: 279; Issue: 49 Linguagem: Inglês

10.1074/jbc.m409214200

ISSN

1083-351X

Autores

Terence M. Williams, Freddy A. Medina, Inés Badano, Rachel B. Hazan, John N. Hutchinson, William J. Muller, Neeru Chopra, Philipp E. Scherer, Richard G. Pestell, Michael P. Lisanti,

Tópico(s)

RNA Research and Splicing

Resumo

Caveolin-1 (Cav-1) is the principal structural component of caveolae membrane domains in non-muscle cells, including mammary epithelia. There is now clear evidence that caveolin-1 influences the development of human cancers. For example, a dominant-negative mutation (P132L) in the Cav-1 gene has been detected in up to 16% of human breast cancer samples. However, the exact functional role of caveolin-1 remains controversial. Mechanistically, in cultured cell models, Cav-1 is known to function as a negative regulator of the Rasp42/44 MAP kinase cascade and as a transcriptional repressor of cyclin D1 gene expression, possibly explaining its in vitro transformation suppressor activity. Genetic validation of this hypothesis at the in vivo and whole organismal level has been prevented by the lack of a Cav-1 (-/-)-null mouse model. Here, we examined the role of caveolin-1 in mammary tumorigenesis and lung metastasis using a molecular genetic approach. We interbred a well characterized transgenic mouse model of breast cancer, MMTV-PyMT (mouse mammary tumor virus-polyoma middle T antigen), with Cav-1 (-/-)-null mice. Then, we followed the onset and progression of mammary tumors and lung metastases in female mice over a 14-week period. Interestingly, PyMT/Cav-1 (-/-) mice showed an accelerated onset of mammary tumors, with increased multiplicity and tumor burden (∼2-fold). No significant differences were detected between PyMT/Cav-1 (+/+) and PyMT/Cav-1 (+/-) mice, indicating that complete loss of caveolin-1 is required to accelerate both tumorigenesis and metastasis. Molecularly, mammary tumor samples derived from PyMT/Cav-1 (-/-) mice showed ERK-1/2 hyperactivation, cyclin D1 up-regulation, and Rb hyperphosphorylation, consistent with dys-regulated cell proliferation. PyMT/Cav-1 (-/-) mice also developed markedly advanced metastatic lung disease. Conversely, recombinant expression of Cav-1 in a highly metastatic PyMT mammary carcinoma-derived cell line, namely Met-1 cells, suppressed lung metastasis by ∼4.5-fold. In vitro, these Cav-1-expressing Met-1 cells (Met-1/Cav-1) demonstrated a ∼4.8-fold reduction in invasion through Matrigel-coated membranes. Interestingly, delivery of a cell permeable peptide encoding the caveolin-1 scaffolding domain (residues 82-101) into Met-1 cells was sufficient to inhibit invasion. Coincident with this decreased invasive index, Met-1/Cav-1 cells exhibited marked reductions in MMP-9 and MMP-2 secretion and associated gelatinolytic activity, as well as diminished ERK-1/2 signaling in response to growth factor stimulation. These results demonstrate, for the first time, that caveolin-1 is a potent suppressor of mammary tumor growth and metastasis using novel in vivo animal model approaches. Caveolin-1 (Cav-1) is the principal structural component of caveolae membrane domains in non-muscle cells, including mammary epithelia. There is now clear evidence that caveolin-1 influences the development of human cancers. For example, a dominant-negative mutation (P132L) in the Cav-1 gene has been detected in up to 16% of human breast cancer samples. However, the exact functional role of caveolin-1 remains controversial. Mechanistically, in cultured cell models, Cav-1 is known to function as a negative regulator of the Rasp42/44 MAP kinase cascade and as a transcriptional repressor of cyclin D1 gene expression, possibly explaining its in vitro transformation suppressor activity. Genetic validation of this hypothesis at the in vivo and whole organismal level has been prevented by the lack of a Cav-1 (-/-)-null mouse model. Here, we examined the role of caveolin-1 in mammary tumorigenesis and lung metastasis using a molecular genetic approach. We interbred a well characterized transgenic mouse model of breast cancer, MMTV-PyMT (mouse mammary tumor virus-polyoma middle T antigen), with Cav-1 (-/-)-null mice. Then, we followed the onset and progression of mammary tumors and lung metastases in female mice over a 14-week period. Interestingly, PyMT/Cav-1 (-/-) mice showed an accelerated onset of mammary tumors, with increased multiplicity and tumor burden (∼2-fold). No significant differences were detected between PyMT/Cav-1 (+/+) and PyMT/Cav-1 (+/-) mice, indicating that complete loss of caveolin-1 is required to accelerate both tumorigenesis and metastasis. Molecularly, mammary tumor samples derived from PyMT/Cav-1 (-/-) mice showed ERK-1/2 hyperactivation, cyclin D1 up-regulation, and Rb hyperphosphorylation, consistent with dys-regulated cell proliferation. PyMT/Cav-1 (-/-) mice also developed markedly advanced metastatic lung disease. Conversely, recombinant expression of Cav-1 in a highly metastatic PyMT mammary carcinoma-derived cell line, namely Met-1 cells, suppressed lung metastasis by ∼4.5-fold. In vitro, these Cav-1-expressing Met-1 cells (Met-1/Cav-1) demonstrated a ∼4.8-fold reduction in invasion through Matrigel-coated membranes. Interestingly, delivery of a cell permeable peptide encoding the caveolin-1 scaffolding domain (residues 82-101) into Met-1 cells was sufficient to inhibit invasion. Coincident with this decreased invasive index, Met-1/Cav-1 cells exhibited marked reductions in MMP-9 and MMP-2 secretion and associated gelatinolytic activity, as well as diminished ERK-1/2 signaling in response to growth factor stimulation. These results demonstrate, for the first time, that caveolin-1 is a potent suppressor of mammary tumor growth and metastasis using novel in vivo animal model approaches. Caveolin-1 (Cav-1) 1The abbreviations used are: Cav-1, caveolin-1; Rb, retinoblastoma; MAP, mitogen-activated protein; MMTV, mouse mammary tumor virus; FBS, fetal bovine serum; MMP, matrix metalloproteinase; ER, estrogen receptor; PR, progesterone receptor; PI, phosphatidylinositol; mAb, monoclonal antibody; DMEM, Dulbecco's modified Eagle's medium; PyMT, polyoma middle T antigen; PBS, phosphate-buffered saline; BSA, bovine serum albumin; ERK, extracellular signal-regulated kinase; MEF, mouse embryonic fibroblasts. was first discovered as a tyrosine-phosphorylated target in Rous sarcoma virus (RSV)-transformed avian fibroblasts, suggesting a possible role for this protein in cellular transformation (1Glenney Jr., J.R. J. Biol. Chem. 1989; 264: 20163-20166Abstract Full Text PDF PubMed Google Scholar). Subsequent studies identified caveolin-1 as a component of plasma membrane caveolae, small 50-100-nm omega-shaped invaginations involved in vesicular trafficking and cholesterol homeostasis (2Glenney Jr., J.R. Soppet D. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10517-10521Crossref PubMed Scopus (343) Google Scholar, 3Kurzchalia T. Dupree P. Parton R.G. Kellner R. Virta H. Lehnert M. Simons K. J. Cell Biol. 1992; 118: 1003-1014Crossref PubMed Scopus (465) Google Scholar). Analysis of its protein expression pattern revealed that Cav-1 is found in a diverse range of cell types, including adipocytes, fibroblasts, endothelial cells, smooth muscle cells, and mammary epithelial cells (4Lisanti M.P. Scherer P. Tang Z.-L. Sargiacomo M. Trends Cell Biol. 1994; 4: 231-235Abstract Full Text PDF PubMed Scopus (590) Google Scholar, 5Scherer P.E. Tang Z.-L. Chun M.C. Sargiacomo M. Lodish H.F. Lisanti M.P. J. Biol. Chem. 1995; 270: 16395-16401Abstract Full Text Full Text PDF PubMed Scopus (323) Google Scholar, 6Engelman J.A. Lee R.J. Karnezis A. Bearss D.J. Webster M. Siegel P. Muller W.J. Windle J.J. Pestell R.G. Lisanti M.P. J. Biol. Chem. 1998; 273: 20448-20455Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar, 7Lee S.W. Reimer C.L. Oh P. Campbell D.B. Schnitzer J.E. Oncogene. 1998; 16: 1391-1397Crossref PubMed Scopus (400) Google Scholar). It is now clear that the majority of caveolae require caveolin-1 for proper formation, indicating that Cav-1 is a requisite caveolar structural protein. For example, Cav-1 (-/-)-null mice lack morphologically identifiable caveolae in all cell types where Cav-1 is normally expressed (8Razani B. Engelman J.A. Wang X.B. Schubert W. Zhang X.L. Marks C.B. Macaluso F. Russell R.G. Li M. Pestell R.G. Di Vizio D. Hou Jr., H. Kneitz B. Lagaud G. Christ G.J. Edelmann W. Lisanti M.P. J. Biol. Chem. 2001; 276: 38121-38138Abstract Full Text Full Text PDF PubMed Scopus (258) Google Scholar, 9Schubert W. Frank P.G. Woodman S.E. Hyogo H. Cohen D.E. Chow C.W. Lisanti M.P. J. Biol. Chem. 2002; 277: 40091-40098Abstract Full Text Full Text PDF PubMed Scopus (277) Google Scholar, 10Drab M. Verkade P. Elger M. Kasper M. Lohn M. Lauterbach B. Menne J. Lindschau C. Mende F. Luft F.C. Schedl A. Haller H. Kurzchalia T.V. Science. 2001; 293: 2449-2452Crossref PubMed Scopus (1318) Google Scholar). Since its initial discovery more than a decade ago, a variety of novel functions are now attributed to Cav-1, including a role in signal transduction. Caveolae, a type of membrane subdomain akin to lipid rafts, serve to compartmentalize many components of diverse signaling pathways that include Src family tyrosine kinases, eNOS, H-Ras, epidermal growth factor receptor, and G-proteins (11Okamoto T. Schlegel A. Scherer P.E. Lisanti M.P. J. Biol. Chem. 1998; 273: 5419-5422Abstract Full Text Full Text PDF PubMed Scopus (1347) Google Scholar, 12Galbiati F. Volonte D. Engelman J.A. Watanabe G. Burk R. Pestell R.G. Lisanti M.P. EMBO J. 1998; 17: 6633-6648Crossref PubMed Scopus (432) Google Scholar). Cav-1 negatively regulates the activation state of many of these signaling molecules (13Li S. Okamoto T. Chun M. Sargiacomo M. Casanova J.E. Hansen S.H. Nishimoto I. Lisanti M.P. J. Biol. Chem. 1995; 270: 15693-15701Abstract Full Text Full Text PDF PubMed Scopus (559) Google Scholar, 14Li S. Couet J. Lisanti M.P. J. Biol. Chem. 1996; 271: 29182-29190Abstract Full Text Full Text PDF PubMed Scopus (675) Google Scholar, 15Couet J. Li. S. Okamoto T. Ikezu T. Lisanti M.P. J. Biol. Chem. 1997; 272: 6525-6533Abstract Full Text Full Text PDF PubMed Scopus (811) Google Scholar, 16Couet J. Sargiacomo M. Lisanti M.P. J. Biol. Chem. 1997; 272: 30429-30438Abstract Full Text Full Text PDF PubMed Scopus (549) Google Scholar, 17Michel J.B. Feron O. Sacks D. Michel T. J. Biol. Chem. 1997; 272: 15583-15586Abstract Full Text Full Text PDF PubMed Scopus (512) Google Scholar, 18Ju H. Zou R. Venema V.J. Venema R.C. J. Biol. Chem. 1997; 272: 18522-18525Abstract Full Text Full Text PDF PubMed Scopus (526) Google Scholar), including ERK-1/2 and MEK-1/2, by direct binding to Cav-1 through the caveolin-scaffolding domain. From in vitro biochemical studies, it appears that the caveolin-scaffolding domain (residues 82-101 in Cav-1) is a modular protein domain that acts as a protein kinase inhibitor, by recognizing and binding to a specific aromatic amino acid motif located within the catalytic domain of many known serine/threonine and tyrosine kinases (15Couet J. Li. S. Okamoto T. Ikezu T. Lisanti M.P. J. Biol. Chem. 1997; 272: 6525-6533Abstract Full Text Full Text PDF PubMed Scopus (811) Google Scholar, 16Couet J. Sargiacomo M. Lisanti M.P. J. Biol. Chem. 1997; 272: 30429-30438Abstract Full Text Full Text PDF PubMed Scopus (549) Google Scholar). Thus, Cav-1 may function as a natural endogenous inhibitor of a variety of signaling cascades, such as the Ras-p42/44 MAP kinase cascade (12Galbiati F. Volonte D. Engelman J.A. Watanabe G. Burk R. Pestell R.G. Lisanti M.P. EMBO J. 1998; 17: 6633-6648Crossref PubMed Scopus (432) Google Scholar, 19Engelman J.A. Chu C. Lin A. Jo H. Ikezu T. Okamoto T. Kohtz D.S. Lisanti M.P. FEBS Lett. 1998; 428: 205-211Crossref PubMed Scopus (347) Google Scholar). One prediction of this hypothesis is that loss of Cav-1 expression in vivo may lead to hyperactivation of p42/44 MAP kinase signaling. Potentially, this dysregulation could drive increases in tumorigenesis and/or metastasis in Cav-1 (-/-)-deficient mice, if they are provided with an oncogenic stimulus. Here, we test this hypothesis experimentally, using the mouse mammary gland as a model system. Elucidation of a role for Cav-1 in cellular transformation began with the observation that Cav-1 is down-regulated in oncogenically transformed cells (20Koleske A.J. Baltimore D. Lisanti M.P. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 1381-1385Crossref PubMed Scopus (472) Google Scholar). Additional research demonstrated that the Cav-1 protein is down-regulated in human cancers, tumors derived from transgenic mice, and in human or rodent cancer cell lines, including breast tumors or breast cancer cell lines. Furthermore, Lee et al. (7Lee S.W. Reimer C.L. Oh P. Campbell D.B. Schnitzer J.E. Oncogene. 1998; 16: 1391-1397Crossref PubMed Scopus (400) Google Scholar) demonstrated that Cav-1 possesses a transformation suppressor function, as Cav-1 overexpression in human mammary cancer cells (T47D) results in tumor cell growth inhibition. Genetically, the CAV-1 gene is localized to a region on human chromosome 7q31.1 near microsatellite marker D7S522, a known fragile site (FRA7G) that is commonly deleted in a number of human cancers, including squamous cell carcinomas, ovarian carcinomas, colon carcinomas, and breast carcinomas (reviewed in Ref. 21Razani B. Schlegel A. Liu J. Lisanti M.P. Biochem. Soc. Trans. 2001; 29: 494-499Crossref PubMed Scopus (114) Google Scholar). In terms of human breast cancer, one particular study demonstrated that up to 16% of patients with primary mammary tumors harbor a sporadic mutation at codon 132 (P132L) in CAV-1 (22Hayashi K. Matsuda S. Machida K. Yamamoto T. Fukuda Y. Nimura Y. Hayakawa T. Hamaguchi M. Cancer Res. 2001; 61: 2361-2364PubMed Google Scholar). Interestingly, the CAV-1 (P132L) mutation behaves in a dominant-negative fashion, causing the intracellular retention of wild-type CAV-1 at the level of the Golgi complex (27Lee H. Park D.S. Razani B. Russell R.G. Pestell R.G. Lisanti M.P. Am. J. Pathol. 2002; 161: 1357-1369Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar). Recently, Han et al. (23Han S.E. Park K.H. Lee G. Huh Y.J. Min B.M. Int. J. Oncol. 2004; 24: 435-440PubMed Google Scholar) have also identified novel CAV-1 mutations in human oral squamous cell carcinomas. Mechanistically, in cultured cell models, Cav-1 is known to function as a negative regulator of the Ras-p42/44 MAP kinase cascade and as a transcriptional repressor of cyclin D1 gene expression, possibly explaining its in vitro transformation suppressor activity. However, genetic validation of this hypothesis at the in vivo and whole organismal level has been prevented by the lack of a Cav-1 (-/-)-null mouse model. In this report, we have employed MMTV-PyMT transgenic mice to directly explore the role of Cav-1 in tumor development and metastasis, using an autochthonous animal model. PyMT transgenic mice express high levels of this transforming oncogene under the control of the MMTV-LTR promoter, which specifically directs expression to the mammary epithelium (24Guy C.T. Cardiff R.D. Muller W.J. Mol. Cell. Biol. 1992; 12: 954-961Crossref PubMed Scopus (1268) Google Scholar). All female virgin PyMT transgenic mice rapidly develop multifocal mammary adenocarcinomas that are palpable, as early as 6-7 weeks of age. The benefit of this mouse tumor model over others is that female PyMT mice also develop pulmonary metastases by ∼3-4 months of age, with an extremely high penetrance (∼90-100%). Importantly, this mouse tumor model has been shown to recapitulate human breast cancer progression, from early hyperplasia to malignant breast carcinoma, including the up-regulation of ErbB2/Neu and cyclin D1 expression (25Maglione J.E. Moghanaki D. Young L.J. Manner C.K. Ellies L.G. Joseph S.O. Nicholson B. Cardiff R.D. MacLeod C.L. Cancer Res. 2001; 61: 8298-8305PubMed Google Scholar, 26Lin E.Y. Jones J.G. Li P. Zhu L. Whitney K.D. Muller W.J. Pollard J.W. Am. J. Pathol. 2003; 163: 2113-2126Abstract Full Text Full Text PDF PubMed Scopus (780) Google Scholar). We interbred PyMT transgenic mice with Cav-1 null (-/-) mice to generate three genetically distinct cohorts of female mice that are all hemizygous for the PyMT transgene: 1) PyMT/Cav-1 (+/+); 2) PyMT/Cav-1 (+/-); and 3) PyMT/Cav-1 (-/-) mice. During a 14-week observation period, mice were evaluated for tumor onset, incidence, burden, and metastasis. We now demonstrate that genetic disruption of the caveolin-1 gene markedly increases tumorigenesis by reducing tumor latency, and increasing tumor multiplicity/burden, without altering histopathological progression. Mammary tumor samples derived from PyMT/Cav-1 (-/-) mice showed ERK-1/2 hyperactivation, cyclin D1 up-regulation, and Rb hyperphosphorylation, consistent with dysregulated cell proliferation. However, (i) no differences in PyMT expression or kinase-associated activity were detected in the absence of Cav-1; (ii) no alterations in the expression levels or phosphoactivation states among PyMT-associated proteins (Src, Shc, PI 3-kinase) were observed; and (iii) the genetic disruption of Cav-1 had no effect on the estrogen receptor or progesterone receptor status of these tumors. In addition, we find that an absence of caveolin-1 results in striking increases in the frequency of distant lung metastases from the primary site of origin. Finally, metastatic mammary tumor cells engineered to recombinantly express Cav-1 show significant reductions in Matrigel invasion and dramatically reduced MMP-9/MMP-2 activities. As such, this is the first demonstration that loss of caveolin-1 promotes mammary tumorigenesis and lung metastasis in an in vivo animal model. Materials and Expression Vectors—Mouse monoclonal antibodies to caveolin-1 (clones 2297 and 2234) were the generous gift of Dr. Roberto Campos-Gonzalez (BD Pharmingen, Inc.). Rabbit polyclonal antibodies directed against total ERK-1/2, activated phospho-ERK-1/2, phospho-Rb (Ser-780), and phospho-Src (Tyr-416) were obtained from Cell Signaling, Inc. The anti-Rb rabbit polyclonal antibody was obtained from Santa Cruz Biotechnology, Inc., as well as rabbit polyclonal antibodies to ER, PR, c-Src, and MMP-2. Monoclonal antibodies directed against Shc, phospho-Shc, Akt, phospho-Akt (Ser-472/473), and PI 3-kinase were obtained from BD Pharmingen, Inc. A rabbit polyclonal antibody specific for mouse MMP-9 was obtained from Triple Point Biologics, Inc. (Forest Grove, OR). Additional antibodies and their sources include anti-cyclin D1 rabbit pAb (NeoMarkers, Fremont, CA), anti-β-actin mAb AC-15 (Sigma), anti-β-tubulin mAb (Sigma), and anti-MT1-MMP mAb (Chemicon). Dr. William J. Muller generously provided a mouse monoclonal antibody (PAB 762) directed against PyMT. We have previously reported on the use of the retroviral vector pBABE for the delivery of cloned cDNAs (27Lee H. Park D.S. Razani B. Russell R.G. Pestell R.G. Lisanti M.P. Am. J. Pathol. 2002; 161: 1357-1369Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar). A construct bearing the PyMT cDNA was generously provided by Dr. Robert Freund. Murine EGF and FGF-2 were purchased from PeproTech, Inc. (Rocky Hill, NJ). Heparin, 4-aminophenylmercuric acetate (APMA), gelatin, and crystal violet were obtained from Sigma. GM6001 and N-sulfonylamino acid MMP-2/MMP-9-specific inhibitors I and II were all obtained from Calbiochem/EMD Biosciences, Inc. (San Diego, CA). Cell Culture—All cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, 2 mm glutamine, 100 units/ml penicillin, and 100 μg/ml streptomycin. Animal Studies—All animals were housed and maintained in a barrier facility at the Institute for Animal Studies, Albert Einstein College of Medicine (AECOM). Mice were kept on a 12-hour light/dark cycle with ad libitum access to chow (Picolab 20, PMI Nutrition International) and water. Animal protocols used for this study were approved by the AECOM Institute for Animal Studies. Cav-1-null mice and MMTV-PyMT transgenic mice (strain 634) were generated as previously described (8Razani B. Engelman J.A. Wang X.B. Schubert W. Zhang X.L. Marks C.B. Macaluso F. Russell R.G. Li M. Pestell R.G. Di Vizio D. Hou Jr., H. Kneitz B. Lagaud G. Christ G.J. Edelmann W. Lisanti M.P. J. Biol. Chem. 2001; 276: 38121-38138Abstract Full Text Full Text PDF PubMed Scopus (258) Google Scholar, 28Williams T.M. Cheung M.W. Park D.S. Razani B. Cohen A.W. Muller W.J. Di Vizio D. Chopra N.G. Pestell R.G. Lisanti M.P. Mol. Biol. Cell. 2003; 14: 1027-1042Crossref PubMed Scopus (135) Google Scholar). All the mice used in this study were in the FVB/N background. Matings were performed with PyMT male hemizygous mice. PyMT/Cav-1 (+/+) and PyMT/Cav-1 (+/-) male mice were interbred with Cav-1 (+/+) or Cav-1 (-/-) female mice to generate a cohort of Cav-1 (+/+), Cav-1 (+/-), or Cav-1 (-/-) female mice, all hemizygous for the PyMT transgene. None of the PyMT transgene-negative control mice developed tumors (n = 40). For the female tumor study, all the mice analyzed were virgin. Tumor Palpation and Excision—Female mice were palpated twice weekly beginning at 6 weeks of age for the development of tumors in their mammary glands. Mice were examined in a genotype-blinded fashion and palpated in each of the ten mammary glands up until 12 weeks of age. Beginning at 12 weeks, female mice were sacrificed, and all mammary tumors were carefully excised and weighed. Portions of the tumors were also frozen in liquid nitrogen or stored in formalin for fixation purposes. Identical procedures were performed on mice sacrificed at 13 and 14 weeks of age. For the male tumor study, mice were sacrificed at 23 weeks of age, and all tumors were excised, weighed, and fixed in formalin. Histological Analysis of Mammary Tumors—Mammary tumors were excised, cut into smaller portions, and fixed with 10% neutral buffered formalin for over 24 h before embedding in paraffin. Sections were cut at 5 microns, stained with hematoxylin and eosin, and evaluated by an experienced histopathologist (Dr. Neeru G. Chopra). Analyses, and descriptions were performed in accordance with the guidelines put forth by the mouse mammary gland pathology consensus meeting in Annapolis (29Cardiff R.D. Anver M.R. Gusterson B.A. Hennighausen L. Jensen R.A. Merino M.J. Rehm S. Russo J. Tavassoli F.A. Wakefield L.M. Ward J.M. Green J.E. Oncogene. 2000; 19: 968-988Crossref PubMed Scopus (415) Google Scholar). Lung Metastasis Analysis—Female PyMT mice at 13 and 14 weeks of age were sacrificed and the lungs exposed by thoracic and tracheal dissection. Before removal, lungs were injected with 2 ml of 10% neutral buffered formalin by tracheal cannulation in order to fix the inner airspaces and inflate the lung lobes. Lungs were then excised and placed into formalin for 48 h. Lung lobes were separated in order to visualize all surfaces of each lobe. During counting, an equal number of lobes were matched for size and location in the thorax. Representative lungs were also paraffin-embedded and processed for histological analysis. For the Met-1 cell metastasis study, 1 × 105 cells suspended in 0.1 ml of PBS were injected through the tail vein of female athymic nude mice for each cell line. After 3 weeks, the lungs were removed and insuflated with 2 ml of 15% India Ink dye, washed in water for 5 min, and bleached in Fekete's solution (70% ethanol, 3.7% paraformaldehyde, 0.75 m glacial acetic acid). For the Met-1 metastasis study in wild-type and Cav-1 (-/-) FVB/N mice, 5 × 105 Met-1 cells were injected in 0.1 ml of PBS, and after 4 weeks, the lungs were removed and stained with India Ink. Surface lung metastases were scored in a genotype-blinded fashion under low power using a Nikon SMZ-1500 stereomicroscope. p values were determined by applying Mann-Whitney statistical analysis parameters, which does not assume a Gaussian distribution (non-parametric test). Immunoblot Analysis—150 to 200 mg portions of tumor tissue frozen in liquid nitrogen were homogenized in 2 ml of boiling lysis buffer (10 mm Tris, pH 7.5, 150 mm NaCl, 1% Triton X-100, 60 mm octyl glucoside), containing protease inhibitors (Roche Applied Science), using a Polytron tissue grinder. Tissue lysates were then centrifuged at 12,000 × g for 10 min to remove insoluble debris. For analyzing PyMT expression, tissue lysates were also precleared with protein G following centrifugation. Protein concentrations were analyzed using the BCA reagent (Pierce) and the volume required for 20-100 μg of protein was determined. Tissue or cell lysates were separated by SDS-PAGE (8-12% 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 mm Tris pH 8.0, 150 mm NaCl, 0.05% Tween-20 (TBS-Tween), which was supplemented with 1% bovine serum albumin (BSA) and 4% nonfat dry milk (Carnation) for the blocking solution and 1% BSA for the antibody diluent. For phosphoantibody analysis, the blocking solution contained only 5% BSA in TBS-Tween (without nonfat milk). Primary antibodies were used at a 1:500 dilution. Horseradish peroxidase-conjugated secondary antibodies (anti-mouse 1:10,000 dilution (Pierce) or anti-rabbit 1:5,000 (BD Pharmingen) were used to visualize bound primary antibodies, with the Supersignal chemiluminescence substrate (Pierce). Retroviral Vectors—PCR primers engineered with 5′-BamHI and 3′-EcoRI restriction sites were used to amplify the PyMT cDNA (generously provided by Dr. Robert Freund) for subcloning into the BamHI and EcoRI sites of the pBABE retroviral vector, yielding pBABE-PyMT. Individual pBABE-PyMT clones were confirmed by restriction digestion and sequencing. The cloning of the murine Cav-1 cDNA into the pBABE retroviral vector was achieved through a similar strategy (pBABE-Cav-1). Retroviral Transduction—The retroviral pBABE constructs were transfected into Phoenix cells by a calcium phosphate precipitation method. About 6-8 h post-tranfection, cells were washed once with PBS, and the media was changed to complete growth medium (DMEM, 10% FBS, glutamine, penicillin/streptomycin). Approximately 48 h post-transfection, the supernatant from transfected Phoenix cells was harvested, passed through a 0.45-μm filter to remove cellular debris, and mixed with polybrene (5 μg/ml). 2 × 105 cells of each cell line were infected with a 1:1 mixture of viral-containing supernatant and complete growth medium. Aspiration and infection was repeated every 12 h for a total of 3 cycles. To generate stable pools of pBABE-transfected cell lines, the cells were placed in complete growth media supplemented with puromycin (2.5 μg/ml) for 1 week, thus avoiding clonal variability. Nude Mouse Flank Injections—Female athymic immunodeficient (nude) mice at 6-8 weeks of age were obtained from the National Cancer Institute (NCI). Briefly, immortalized mouse embryonic fibroblasts (MEFs) or Db7 cells (a generous gift of Dr. Robert D. Cardiff) were suspended in sterile PBS at a concentration of 107 cells/ml, and 100 μl were injected subcutaneously into each flank of the nude mice. Mice were sacrificed at 6-weeks (MEFs) or 3-weeks (Db7) post-injection, at which point tumors were carefully excised to determine their weight. At least 10 injections were performed for each stable cell line, with the same cell line injected into both flanks of the same animal. Immunofluorescence Microscopy—Met-1 cells (generous gift of Dr. Robert D. Cardiff, University of California-Davis) were grown on sterile glass coverslips, washed three times in PBS, and fixed for 30 min 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 min. Cells were then treated with 25 mm NH4Cl in PBS for 10 min at room temperature to quench free aldehyde groups. After rinsing with PBS, cells were incubated with primary antibody (anti-Cav-1 IgG mAb) diluted in 0.1% Triton X-100/0.2% BSA/PBS for 1 h at room temperature, washed three times with PBS for 10 min each, then incubated with a rhodamine-conjugated anti-mouse secondary antibody for 1 h at room temperature. Finally, cells were washed three times with PBS (10 min each wash), and mounted on a glass slide with slow-Fade anti-fade reagent (Molecular Probes, Eugene, OR). A cooled charge-coupled device (CCD) camera on an Olympus microscope was used for the detection of bound secondary antibodies. In Vitro Kinase Assay—Cells were lysed in TNE buffer (1% Nonidet P-40, 50 mm Tris, pH 8.0, 150 mm NaCl, 2.5 mm EDTA, 10 mm NaF, 1 mm sodium orthovanadate, protease inhibitors), and 1 mg of lysate was immunoprecipitated with 2 μg of a PyMT mouse monoclonal antibody bound to protein G-Sepharose (Amersham Biosciences). As a control, pull-downs were performed in parallel with Sepharose beads without PyMT mAb pre-bound to them (beads alone). Sepharose beads were washed four times with TNE buffer and then resuspended in 10 μl of 2× kinase buffer (50 mm HEPES pH 7.0, 10 mm MgCl2, 10 mm MnCl2, 150 mm NaCl). Reactions were initiated by adding 10 μCi of [γ-32P]ATP and 10 μg of acid-denatured enolase. Reactions were inbuated at RT for 10 min before stopping with 20 μl of 2× SDS-PAGE sample buffer and boiling for 2 min. Samples were separated on 10% SDS-PAGE gels, transferred to nitrocellulose after cutting away the leading edge of the gel (containing free [γ-32P]ATP), and exposed with an intensifying screen for 24 h at -80 °C. Cell Migration and Invasion Assays—The invasive potential of the tumor-derived cell lines was measured by an in vitro Boyden chamber assay (30Albini A. Iwamoto Y. Kleinman H.K. Martin G.R. Aaronson S.A. Kozlowski J.M. McEwan R.N. Cancer Res. 1987; 47: 3239-3245PubMed Google Scholar). Briefly, 105 cells in 0.5 ml of serum-free DMEM were added to the wells of 8 μm pore membrane Boyden chambers, either coated with (for invasion assays) or without (for migration assays) Matrigel (Transwells; BD Biosciences). The lower chambers contained either 0

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