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Caveolin-1 (Cav-1) has been extensively characterized in cancer biological research. However, the role of Cav-1 in the interaction between tumor and stromal cells remains unclear. In the present study, we examined Cav-1 expression in tumor cells and stromal cells in breast cancer tissue by immunohistochemical analysis and evaluated its prognostic value in a training cohort. Immunohistochemical analysis of Cav-1 expression was scored as (++), (+) or (−) according to the proportion of positively stained tumor cells (T) and stromal cells (S). Correlation analysis between tumor/stromal Cav-1 expression and clinicopathological parameters revealed that only T(++) Cav-1 status was positively associated with tumor size and histological nodal status (P = 0.019 and 0.021, respectively). Univariate analysis revealed that combined T(++)/S(−) status was significantly correlated with unfavorable prognostic outcomes (P < 0.001). Multivariate analysis demonstrated that this combined status is an independent prognostic factor for primary breast cancer (P = 0.002). Clinical outcomes in different subgroups of breast cancer patients were also strictly dependent on this combined status (P < 0.05). The prognostic value of T(++)/S(−) Cav-1 status was also validated in the testing cohort. Collectively, our data indicate that high Cav-1 expression in tumor cells and lack of this expression in stromal cells could help identify a particular subgroup of breast cancer patients with potentially poor survival. Further studies are required to understand the regulatory mechanism of Cav-1 in the tumor microenvironment. (Cancer Sci 2011; 102: 1590–1596)
Breast cancer is the most common female cancer. Late-onset diagnosis, axillary lymph node metastases, tumor size, pathological type and resistance to antitumor therapy indicate a poor prognosis for breast cancer patients. Although treatment strategies for breast cancer have recently made great progress, recurrence and death rates remain unacceptably high.(1) Therefore, molecular biomarkers for recurrence and progression of breast cancer must be explored to help clinicians identify new diagnostic and therapeutic techniques to detect and treat breast cancer.(2)
Caveolins (Cav) are a family of scaffolding proteins that coat 50–100 nm plasma membrane invaginations. The Cav family is composed of three isoforms: Cav-1, Cav-2 and Cav-3. The Cav-1 gene is located on chromosome 7 (locus 7q31.1) and includes three exons (30, 165 and 342 bp) and two introns (1.5 and 32 kb).(3) Cav-1 expression depends on the type of tumor and its expression is downregulated in several human cancers such as sarcoma and lung cancer and might function as a tumor suppressor.(4,5) However, upregulation of Cav-1 expression has been reported in esophageal and pancreatic cancers and is also correlated with histopathological grade and poor prognosis.(6,7)
Cav-1 is mainly involved in vesicular transport, cholesterol homeostasis and signal transduction.(8) Furthermore, it might facilitate DNA repair and stabilize the insulin receptor against degradation. Cav-1 also plays a negative role in cell movement,(9) cellular senescence(10) and cell growth.(11) Endothelial cells from Cav-1−/− mice exhibit a diminished response to angiogenic growth factors.(12) Furthermore, Cav-1 overexpression is sufficient to induce premature cellular senescence in fibroblasts.(13,14) Cancer-associated fibroblasts (CAFs), which are derived from malignant or normal epithelial cells, promote tumor growth.(15)In vitro studies have shown that both stromal and epithelial Cav-1 play a protective role against mammary hyperplasia and tumorigenesis in breast cancer.(11,16,17) In addition, clinical studies have indicated that stromal loss of Cav-1 is a single independent predictor of early breast cancer recurrence and progression.(18,19) However, the value of combined tumor/stromal Cav-1 expression on the outcome of breast cancer patients is largely unknown.
In the present study, we investigated the clinical significance of Cav-1 expression (including tumor and stromal expression) in a training cohort and the correlation between tumor/stromal Cav-1 expression and clinicopathological characteristics. In addition, effects of combined tumor/stromal Cav-1 expression on outcomes in breast cancer patients were investigated. In addition, the prognostic value of combined tumor/stromal Cav-1 expression was also clarified in a testing cohort. Intriguingly, our results indicated that a counter balance of Cav-1 levels in the tumor microenvironment and epithelial compartment were the most strongly influenced clinical outcomes.
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- Materials and Methods
- Disclosure Statement
- Supporting Information
The Cav-1 gene is colocalized at the D7S522 locus on human chromosome 7q31.1 and is commonly deleted in breast, colon, kidney, prostate, ovary, head and neck cancers. Thus, it seems feasible to propose that the Cav-1 gene might serve as a candidate tumor suppressor gene.(21) In the present study, we focused on breast cancer patients to determine the correlation of tumor/stromal Cav-1 expression with clinicopathological parameters and survival. Cav-1 expression was evaluated semi-quantitatively based on the proportion of positively stained tumor and stromal cells. We found that tumor Cav-1 demonstrated a prevalent membrane pattern associated with cytoplasm positive and that T(−) Cav-1 expression was noted in 16% of cases, which is consistent with previous reports.(22) The stromal Cav-1 expression rate and pattern in the present study are similar to a previous report.(23)
According to previous reports, total tumor Cav-1 expression has no prognostic value in primary breast cancer patients.(18,24) In the present study, we examined stromal hot-spot, total stromal and tumor hot-spot Cav-1 expression. First, we analyzed correlations between stromal hot-spot and total stromal and tumor Cav-1 expression and found a weak but significant correlation between stromal and tumor expression, indicating that Cav-1 expression could be regulated differently between tumor and stromal cells and that Cav-1 might influence different functions in those cells.(25,26) T(++) Cav-1 expression was positively associated with tumor size and histological nodal status. Previous reports revealed that tumor Cav-1expression was negatively associated with HER2 status.(27–29) Several studies have indicated that Cav-1 might function as a negative signal transduction regulator to HER2/neu and that it might play a negative regulatory role in mammary tumor development. In addition, activation of HER2/neu might downregulate Cav-1 expression in vitro.(30,31) However, this finding was not supported by our results. Stromal Cav-1 expression showed no significant correlation with any of the clinicopathological parameters, which was inconsistent with a previous report,(18) and therefore we focused on combined tumor/stromal Cav-1 expression. T(++)/S(−) was observed frequently in large-size tumors and histological node+ cases, indicating that tumor/stromal Cav-1 expression is involved in breast cancer progression. Interestingly, survival analyses revealed that patients with T(++)/S(−) Cav-1 expression had the shortest disease-free survival among various Cav-1 expression subgroups. Multivariate analysis confirmed an independent prognostic value of the combined status. Consistent with these results, T(++)/S(−) Cav-1 expression was significantly related to tumor size, histological nodal status and disease-free survival in the testing cohort. Besides, the positive correlation between T(++)/S(−) Cav-1 expression and tumor stage was also indicated in the testing cohort. A possible explanation for the discrepancy could be due to the difference in sample size. Furthermore, T(++)/S(−) Cav-1 expression also impacted the clinical outcomes stratified by ER status, PgR status, HER2 status, tumor size, age, histological nodal status and grade in both the training and testing cohorts. Therefore, these results indicate that combined Cav-1 status has a more potent prognostic value than either stromal or tumor hot-spot alone. We believe that these results are important when considering breast cancer biology. Given the limited number of cases, prospective studies with long-term follow-up data are warranted.(32)
Tumor Cav-1 expression with respect to tumorigenesis seems more complex than originally believed. Cav-1 loss-of-function induces ligand-independent hyperactivation of Ras-p42/44 MAPK and Smad signaling pathways as well as enhanced matrix metalloproteinase-2/9 secretion. Each of these pathways is likely to contribute to cell cycle progression, growth factor independence, cell invasiveness and epithelial–mesenchymal transition.(33) Despite extensive evidence supporting the role of Cav-1 as a tumor suppressor, several studies have suggested an alternative view of Cav-1 expression in tumors. In breast cancer, Cav-1 protects tumor cells from anoikis, promotes tumor cell survival and abrogates detachment-induced p53 activation.(34,35) Furthermore, Cav-1 expression is upregulated in multidrug-resistant MCF-7 cells.(36,37) A hypothesis has been proposed to explain the divergent roles of Cav-1; even if an initial loss of Cav-1 is observed in breast cancer, re-expression of Cav-1 at later stages might correlate with more malignant characteristics.(35,38)
Stromal Cav-1 plays a vital role in tumorigenesis. Loss of stromal Cav-1 is an independent predictor for therapeutic resistance and poor prognosis in primary breast cancers.(12,18,19,39) Woodman et al.(12) reported that endothelial cells from Cav-1−/− mice exhibit a disrupted response to angiogenic growth factors. Senescent human diploid fibroblasts exhibit increased levels of the Cav-1 protein.(10) In addition, loss of Cav-1 in stromal cells of various organs directly leads to disorganised stromal compartments and dysfunctional organ systems.(40)
Furthermore, recent studies have revealed a role played by Cav-1 in the interaction between tumor and stromal cells in breast cancer. During tumor formation, cancer cells and adjacent fibroblasts are metabolically coupled. A new model has been proposed in which glycolytic CAF promote tumor growth by secreting energy-rich metabolites that can be taken up by adjacent tumor cells.(41) Loss of Cav-1 in vitro induces metabolic coupling between CAF and tumor cells and leads to the formation of a host–parasite relationship. Martinez-Outschoorn et al.(42) showed that Cav-1 expression is downregulated in fibroblasts co-cultured with MCF-7 cells and that it mediates autophagic/lysosomal degradation. Furthermore, autophagy induced by loss of Cav-1 in fibroblasts provides cancer cells with essential chemical building blocks.(42,43) Loss of stromal Cav-1 fibroblasts protects adjacent cancer cells via decreased apoptosis, increased TP53-induced glycolysis and apoptosis regulator expression.(44) Furthermore, loss of Cav-1 induces oxidative stress in CAF, which is the root cause of mitochondrial dysfunction in CAF and promotes DNA damage. In the present study, the predictive value of T (++)/S(−) was demonstrated in luminal-type cancers and HER2+ cancers. Its value was stratified by an intrinsic subtype and warrants an examination with a greater number of cases.
The regulatory mechanism of Cav-1 expression in breast cancer remains to be elucidated. Pro-autophagic stimuli such as hypoxia, oxidative stress and nuclear factor κB activation might cause the loss of Cav-1.(45) Conversely, multiple factors are present during Cav-1 upregulation.
In conclusion, we provide evidence that T (++)/S(−) Cav-1 expression is closely associated with unfavorable prognostic outcomes in primary breast cancer patients. This particular subgroup seems to be engaged in rapid disease progression. Further studies involving analysis of molecular mechanisms of Cav-1 expression are required. The interaction between tumor and stromal cells and Cav-1 in the tumor microenvironment is also a key issue to investigate. Moreover, new therapies targeting Cav-1 expression might be a novel therapeutic approach, particularly for patients with T (++)/S(−) Cav-1 status.