Robust evidence is now available that underscores the role of CAFs in tumor progression
]. Previous gene expression profile analyses comparing CAFs and fibroblasts derived from matched normal adjacent breast tissues have demonstrated significant differences between the CAF and their normal counterparts but, to the best of our knowledge, no prior studies have addressed whether CAFs derived from various breast cancer subtypes harbor subtype specific gene expression signatures. In this study we demonstrate for the first time that CAFs from several breast cancer subtypes exhibit subtype-specific gene expression profiles. Specifically, we show that the gene expression profile of CAFs derived from Her2+ breast cancers are significantly different from CAFs derived from ER
or TNBC breast cancers.
Heterogeneity among fibroblasts has been described in various organ sites including lung, skin, sclera and orbit
]. Furthermore, Sugimoto and coworkers demonstrated that the expression of various fibroblast markers are heterogeneous within the tumor stroma in mouse breast and pancreatic tumor models using immunohistochemical analyses
]. Several studies have generated gene expression profiles from breast cancer-associated fibroblasts but none of these studies have stratified their results based on tumor subtypes. Work by Allinen and coworkers evaluated gene expression profiles of breast cancer stromal cells which were isolated by negatively selecting out epithelial cells, lymphocytes and endothelial cells
]. Work described by Singer et al. compared gene expression profiles of stromal fibroblasts derived from 10 invasive breast cancers with stromal fibroblasts derived from normal breast tissues of 10 women undergoing breast reduction surgery
]. Their results demonstrated increased expression of tumor promotion-associated genes in the pooled CAFs. Work by Bauer et al. (2010) evaluated gene expression profiles of fibroblasts derived from 6 matched breast cancers and adjacent normal breast tissues
] and found distinct differences between CAFs and normal fibroblasts, specifically in genes related to paracrine or intracellular signaling, transcriptional regulation, extracellular matrix and cell adhesion/migration. However, all of the above studies were not designed to test subtype specific differences in CAFs due to these studies’ relatively small sample size. In addition, when tumor subtype data were reported, the less common breast cancer subtypes, i.e., Her2+ or TNBC cancer, were underrepresented.
Our results showed that CAFs derived from Her2+ breast cancers significantly up-regulated pathways associated with actin cytoskeleton and integrin signaling (Table
). Integrins mediate cell attachment with extracellular matrix (ECM) to provide traction necessary for cell motility and invasion. These upregulated signaling pathways may have contributed to the elevated migratory phenotype of breast cancer cells (T47D) in our in vitro
transwell assays (Figure
The extracellular matrix and integrins collaborate to regulate gene expression associated with cell growth, differentiation and survival; all of which are deregulated during cancer progression and metastasis. A recent study using a three-dimensional squamous cell carcinoma (SCC)/fibroblast co-culture model elegantly demonstrated the role of three genes, integrin α3, integrin α5 and Rho, in promoting a fibroblast-led collective invasion of SCC cells into the extracellular matrix
]. Interestingly, all three genes were significantly up-regulated in CAFs derived from Her2+ breast cancer with integrin signaling as the second most enriched pathway (Table
). Moreover, many of the genes and pathways downstream of integrin signaling are also significantly upregulated in Her2+ CAFs. These include focal adhesion kinase (FAK), Rac and Rho signaling pathways as well as several members of the mitogen-activated protein kinases (MAPKs), further underscoring the importance of integrin signaling in CAF. In addition to the well-established role of integrins in migration and invasion, integrins can also regulate cell proliferation, including mammary gland proliferation
] through integrin-linked kinase (ILK)
], which was also noted to be significantly upregulated in HER2+ derived CAFs. These characteristic differences in CAFs derived from Her2+ breast cancer may contribute to the aggressiveness of this particular breast cancer subtype which is known to have an increased propensity for local and distant recurrence
]. In addition, the sites of distant metastasis appear to differ according to breast cancer subtype with Her2+ breast cancer having a higher rate of brain, liver, and lung metastases than ER
]. The role of CAF in contributing to a subtype-specific trophism for the various distant metastatic sites is unknown.
Gene expression profile differences between CAFs derived from ER
and TNBC breast cancer were less pronounced and we were unable to confirm them with independent validation set using the limited sample numbers (Figure
B). While it is possible that true differences may exist among these two subtypes, a larger number of samples would be required to find those differences with an acceptable false discovery rate.