An Absence of Stromal Caveolin-1 Expression Predicts Early Tumor Recurrence and Poor Clinical Outcome in Human Breast Cancers

Previously, we showed that caveolin-1 (Cav-1) expression is down-regulated in human breast cancer-associated fibroblasts. However, it remains unknown whether loss of Cav-1 occurs in the breast tumor stroma in vivo. Here, we immunostained a well-annotated breast cancer tissue microarray with antibodies against Cav-1 and scored its stromal expression. An absence of stromal Cav-1 was associated with early disease recurrence, advanced tumor stage, and lymph node metastasis, resulting in a 3.6-fold reduction in progression-free survival. When tamoxifen-treated patients were selected, an absence of stromal Cav-1 was a strong predictor of poor clinical outcome, suggestive of tamoxifen resistance. Interestingly, in lymph node-positive patients, an absence of stromal Cav-1 predicted an 11.5-fold reduction in 5-year progression-free survival. Clinical outcomes among patients positive for HER2, and patients triple-negative for estrogen receptor, progesterone receptor and HER2, were also strictly dependent on stromal Cav-1 levels. When our results were adjusted for tumor and nodal staging, an absence of stromal Cav-1 remained an independent predictor of poor outcome. Thus, stromal Cav-1 expression can be used to stratify human breast cancer patients into low-risk and high-risk groups, and to predict their risk of early disease recurrence at diagnosis. Based on related mechanistic studies, we suggest that breast cancer patients lacking stromal Cav-1 might benefit from anti-angiogenic therapy in addition to standard regimens. We conclude that Cav-1 functions as a tumor suppressor in the stromal microenvironment.Carcinoma cells grow in a complex tumor microenvironment composed of (i) non-epithelial cells (including fibroblasts, pericytes, endothelial, and inflammatory cells), (ii) extracellular matrix, and (iii) secreted diffusible growth factors/cytokines.^1,2,3 Although under normal physiological conditions the stroma serves as an important barrier to malignant transformation, its role changes during neoplastic transformation. Instead, the stroma plays a key role in driving cancer cell invasiveness and progression.⁴ Recently, it was demonstrated that fibroblasts isolated from tumor stroma can promote tumor growth.^1,2,3,5 This population of tissue fibroblasts termed “cancer associated fibroblasts” (CAFs) is characterized by a hyperproliferative phenotype, and these cells secrete increased amounts of growth factors, extracellular matrix components, and matrix metalloproteinases.^5,6 CAFs also show an ability to prevent cancer cell apoptosis, induce cancer cell proliferation, and stimulate tumor angiogenesis.⁷ In vitro studies of breast carcinomas showed that CAFs mixed with epithelial carcinoma cells are more proficient than normal fibroblasts at enhancing tumor growth and give rise to highly vascularized tumors.⁸ To date, the mechanisms that govern the conversion of benign mammary stromal fibroblasts to tumor-associated fibroblasts are poorly understood, and their relationship to disease outcome has not been addressed.Down-regulation of caveolin-1 (Cav-1) is one of the mechanisms implicated in the oncogenic transformation of fibroblasts. Caveolins are the principal protein component of caveolae, which are located at the cell surface in most cell types.⁹ One of the caveolins, Cav-1, plays a major role in tumorigenesis through its various functions such as lipid transport, membrane trafficking, gene regulation, and signal transduction.¹⁰ In cell culture, the transformation of NIH-3T3 fibroblasts with various activated oncogenes, such as H-Ras (G12V), Bcr-Abl, or v-Abl, causes dramatic reductions in Cav-1 protein expression.^11,12Furthermore, knock-down of endogenous Cav-1 in NIH-3T3 fibroblasts promotes anchorage-independent growth in soft agar and tumor formation in nude mice, which could be reversed by Cav-1 re-expression.¹³ Finally, Cav-1^−/− null fibroblasts have a hyperproliferative phenotype (similar to CAFs) and Cav-1 re-expression drives their arrest in the G0/G1 phase of the cell cycle.¹⁴ Taken together, these data suggest that loss of Cav-1 leads to the oncogenic transformation of fibroblasts, where Cav-1 normally functions as a transformation suppressor that prevents cell cycle progression.Using primary cell cultures established from surgically excised breast tumors, we recently demonstrated that Cav-1 is down-regulated in human breast CAFs when compared with matching normal fibroblasts isolated from the same patient.¹⁵ In addition, orthotopic transplantation of Cav-1^+/+ tumor tissue into the mammary stroma of Cav-1^−/− null mice results in up to a ∼twofold increase in tumor mass, functionally demonstrating that the mammary stroma of Cav-1^−/− mice behaves as a tumor promoter.¹⁶ However, to date, there is no study addressing the clinical significance of stromal Cav-1 expression in invasive carcinoma of the breast in vivo.The aim of this study was to evaluate the in vivo stromal expression of Cav-1 in a large series of invasive breast carcinomas and to examine the association between stromal Cav-1 expression, clinicopathological variables, and patient outcome. Our results indicate that loss of stromal caveolin-1 is a novel breast cancer biomarker that predicts early disease recurrence, metastasis, survival, and tamoxifen-resistance. Clinical outcome in HER2(+) and triple-negative (estrogen receptor [ER]−/progesterone receptor [PR]−/HER2−) patients was also strictly dependent on stromal Cav-1 levels. Remarkably, in lymph node-positive [LN(+)] patients, an absence of stromal Cav-1 was associated with an ∼11.5-fold reduction in 5-year progression-free survival. As such, Cav-1 may function as a critical tumor/metastasis suppressor in the mammary stromal compartment.