Abstract
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 ( approximately 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 approximately 4.5-fold. In vitro, these Cav-1-expressing Met-1 cells (Met-1/Cav-1) demonstrated a approximately 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.
Highlights
Caveolin-1 (Cav-1) is the principal structural component of caveolae membrane domains in non-muscle cells, including mammary epithelia
Caveolin-1 Expression Potently Inhibits Matrigel Invasion by Metastatic Mammary Tumor Cells—In order to investigate the potential mechanisms for the observed decreases in experimental metastasis, we examined whether the expression of Cav-1 in Met-1 cells altered their invasive potential
Treatment of Met1/Cav-1 cells with these same Matrix metalloproteinases (MMPs) inhibitors only slightly reduced invasion, and the data was not statistically significant (Fig. 11D, bottom panel). These findings suggest that the metastasisand invasion-inhibiting effects of Cav-1 expression in metastatic mammary tumor cells appears to be mediated in part through inhibition of MMP-2 and MMP-9 activities
Summary
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. The benefit of this mouse tumor model over others is that female PyMT mice develop pulmonary metastases by ϳ3– 4 months of age, with an extremely high penetrance (ϳ90 –100%) 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 [25, 26]. 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
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