Specific upregulation of RHOA and RAC1 in cancer-associated fibroblasts found at primary tumor and lymph node metastatic sites in breast cancer
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The importance of tumor–stromal cell interactions in breast tumor progression and invasion is well established. Here, an evaluation of differential genomic profiles of carcinoma-associated fibroblasts (CAFs) compared to fibroblasts derived from tissues adjacent to fibroadenomas (NAFs) revealed altered focal adhesion pathways. These data were validated through confocal assays. To verify the possible role of fibroblasts in lymph node invasion, we constructed a tissue microarray consisting of primary breast cancer samples and corresponding lymph node metastasis and compared the expression of adhesion markers RhoA and Rac1 in fibroblasts located at these different locations. Two distinct tissue microarrays were constructed from the stromal component of 43 primary tumors and matched lymph node samples, respectively. Fibroblasts were characterized for their expression of α-smooth muscle actin (α-SMA) and vimentin. Moreover, we verified the level of these proteins in the stromal compartment from normal adjacent tissue and in non-compromised lymph nodes. Our immunohistochemistry revealed that 59 % of fibroblasts associated with primary tumors and 41 % of the respective metastatic lymph nodes (p = 0.271) displayed positive staining for RhoA. In line with this, 57.1 % of fibroblasts associated with primary tumors presented Rac1-positive staining, and the frequency of co-positivity within the lymph nodes was 42.9 % (p = 0.16). Expression of RhoA and Rac1 was absent in fibroblasts of adjacent normal tissue and in compromised lymph nodes. Based on our findings that no significant changes were observed between primary and metastatic lymph nodes, we suggest that fibroblasts are active participants in the invasion of cancer cells to lymph nodes and support the hypothesis that metastatic tumor cells continue to depend on their microenvironment.
KeywordsBreast cancer Tumor microenvironment Carcinoma-associated fibroblasts Rho GTPases Metastasis
The authors are grateful to Ana Lúcia Garippo for her technical assistance in confocal microscopy. This research was supported by Fundação de Amparo à Pesquisa no Estado de São Paulo (FAPESP) 01/13513-1, 05/51593-5, 04/04607-8, 05/60333-7, 2014/03090-3 and 09/10088-7 and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq).
Conflicts of interest
All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.
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