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. (Am J Pathol

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Heat shock proteins (Hsps) are induced in vitro by several cytotoxic drugs; in human breast cancer cells these proteins appear to be involved in anti-cancer drug resistance. The present report was designed to analyze whether chemotherapy affects in vivo the expression of Hsp27, Hsp70, Hsc70 and Hsp90 in breast cancer patients treated with induction chemotherapy and whether these proteins may be determinants of tumor resistance to drug administration. We have analyzed 35 biopsies from breast cancer patients treated with induction chemotherapy. Expression of the Hsps in the tumors was compared with (i) histological and clinical responses to chemotherapy, (ii) tumor cell proliferation measured by proliferating cell nuclear antigen (PCNA) immunostaining and nucleolar organizer regions (AgNORs) staining and (iii) the expression of estrogen and progesterone receptors. We also compared disease-free survival (DFS) and overall survival (OS) with the expression of the Hsps studied. After chemotherapy, nuclear Hsp27 and Hsp70 expression was increased and Hsp70 and Hsc70 cytoplasmic expression was decreased. A high nuclear proportion of Hsp70 in tumor cells (G10%) correlated significantly with drug resistance. We also observed that patients whose tumors expressed nuclear or a high cytoplasmic proportion (G66%) of Hsp27 had shorter DFS. The combination of Hsp27 and Hsp70 levels showed a strong correlation with DFS. Neither the cellular proliferation nor the levels of steroid receptors showed any significant difference before or after drug administration or during follow-up of patients. Our results suggest that Hsp27 and Hsp70 are involved in drug resistance in breast cancer patients treated with combination chemotherapies.

The cadherin-catenin proteins have in common with heat shock proteins (HSP) the capacity to bind/interact proteins of other classes. Moreover, there are common molecular pathways that connect the HSP response and the cadherin-catenin protein system. In the present study, we have explored whether in breast cancer the HSP might interact functionally with the cadherin-catenin cell adhesion system. Beta-catenin was immunoprecipitated from breast cancer biopsy samples, and the protein complexes isolated in this way were probed with antibodies against HSP family members. We are thus the first to demonstrate a specific interaction between beta-catenin and Hsp27. However, beta-catenin did not bind Hsp60, Hsp70, Hsp90, gp96, or the endoplasmic reticulum stress response protein CHOP. To confirm the finding of Hsp27-beta-catenin interaction, the 27-kDa immunoprecipitated band was excised from one-dimensional polyacrylamide gel electrophoresis gels and submitted to liquid chromatography-tandem mass spectrometry with electrospray ionization, confirming a role for Hsp27. In addition, beta-catenin interacted with other proteins including heat shock transcription factor 1, P-cadherin, and caveolin-1. In human breast cancer biopsy samples, beta-catenin was coexpressed in the same tumor areas and in the same tumor cells that expressed Hsp27. However, this coexpression was strong when beta-catenin was present in the cytoplasm of the tumor cells and not when beta-catenin was expressed at the cell surface only. Furthermore, murine breast cancer cells transfected with hsp25 showed a redistribution of beta-catenin from the cell membrane to the cytoplasm. When the prognostic significance of cadherin-catenin expression was examined by immunohistochemistry in breast cancer patients (n = 215, follow-up = >10 years), we found that the disease-free survival and overall survival were significantly shorter for patients expressing P-cadherin and for patients showing expression of beta-catenin in the cytoplasm only (not at the cell surface). The interactions of beta-catenin with Hsp27 and with HSF1 may explain some of the molecular pathways that influence tumor cell survival and the clinical significance in the prognosis of the breast cancer patients.

Using the clinically relevant 4T1-derived syngeneic murine model of spontaneous mammary metastasis to bone, we have identified the cysteine cathepsin inhibitor Stefin A as a gene differentially expressed in primary and metastatic mammary tumours. In primary tumours, Stefin A expression correlated inversely with metastatic potential in 4T1-derived lines and was not detected in tumour cells in culture, indicating induction only within the tumour microenvironment. Enforced expression of Stefin A in the highly metastatic 4T1.2 cell line significantly reduced spontaneous bone metastasis following orthotopic injection into the mammary gland. Consistent with the mouse data, Stefin A expression correlated with disease-free survival (absence of distant metastasis) in a cohort of 142 primary tumours from breast cancer patients. This was most significant for patients with invasive ductal carcinoma expressing Stefin A, who were less likely to develop distant metastases (log rank test, p = 0.0075). In a multivariate disease-free survival analysis (Cox proportional hazards model), Stefin A expression remained a significant independent prognostic factor in patients with invasive ductal carcinoma (p = 0.0014), along with grade and progesterone receptor (PR) status. In human lung and bone metastases, we detected irregular Stefin A staining patterns, with expression often localizing to micrometastases (<0.2 mm) in direct contact with the stroma. We propose that Stefin A, as a cysteine cathepsin inhibitor, may be a marker of increased cathepsin activity in metastases. Using immunohistology, the cathepsin inhibitor was detected co-expressed with cathepsin B in lung and bone metastases in both the murine model and human tissues. We conclude that Stefin A expression reduces distant metastasis in breast cancer and propose that this may be due to the inhibition of cysteine cathepsins, such as cathepsin B.

In human primary breast cancer, anthracycline treatment causes an early increase in apoptosis and a decrease in proliferation. In this pilot study, higher apoptosis and topo IIalphaa levels in primary tumors were associated with greater responsiveness to anthracyclines, and topo IIalpha levels declined in responsive tumors.

Breast carcinogenesis is a multistep process that involves both genetic and epigenetic alterations. Identification of aberrantly methylated genes in breast tumors and their relation to clinical parameters can contribute to improved diagnostic, prognostic, and therapeutic decision making. Our objective in the present study was to identify the methylation status of 34 cancer-involved genes in invasive ductal carcinomas (IDC). Each of the 70 IDC cases analyzed had a unique methylation profile. The highest methylation frequency was detected in the WT1 (95.7%) and RASSF1 (71.4%) genes. Hierarchical cluster analysis revealed three clusters with different distribution of the prognostic factors tumor grade, lymph node metastasis, and proliferation rate. Methylation of TP73 was associated with high histological grade and high proliferation rate; methylation of RARB was associated with lymph node metastasis. Concurrent methylation of TP73 and RARB was associated with high histological grade, high proliferation rate, increased tumor size, and lymph node metastasis. Patients with more than six methylated genes had higher rates of relapse events and cancer deaths. In multivariate analysis, TP73 methylation and the methylation index were associated with disease outcome. Our results indicate that methylation index and methylation of TP73 and/or RARB are related to unfavorable prognostic factors in patients with IDC. These epigenetic markers should be validated in further studies to improve breast cancer management.

Breast cancer is a group of clinically, histopathologically and molecularly heterogeneous diseases, with different outcomes and responses to treatment. Triple-negative (TN) breast cancers are defined as tumors that lack the expression of estrogen receptor, progesterone receptor and epidermal growth factor receptor 2. This subgroup accounts for 15% of all types of breast cancer and its prevalence is higher among young African, African-American and Latino women. The hypermethylation of CpG islands (CpGI) is a common epigenetic alteration for suppressing gene expression in breast cancer and has been shown to be a key factor in breast carcinogenesis. In this study we analyzed the hypermethylation of 110 CpGI within 69 cancer-related genes in TN tumors. For the methylation analysis, we used the methyl-specific multiplex-ligation probe amplification assay. We found that the number of methylated CpGI is similar between TN and non-TN tumors, but the methylated genes between the groups are different. The methylation profile of TN tumors is defined by the methylation of five genes (that is, CDKN2B, CD44, MGMT, RB and p73) plus the non-methylation of 11 genes (that is, GSTP1, PMS2, MSH2, MLH1, MSH3, MSH6, DLC1, CACNA1A, CACNA1G, TWIST1 and ID4). We conclude that TN tumors have a specific methylation profile. Our findings give new information for better understanding tumor etiology and encourage future studies on potential drug targets for triple-negative breast tumors, which now lack a specific treatment.