Exploratory genome-wide analysis of the tumor microenvironment in breast cancer has been limited to date. Using serial analysis of gene expression coupled with antibody-based ex vivo
tissue fractionation, Allinen and colleagues identified a limited set of 417 cell-type-specific genes among the most prominent cell types in breast cancer (epithelial, myoepithelial, and endothelial cells, fibroblasts, and leukocytes) [7
]. Finak and colleagues more recently obtained gene expression profiles of both epithelial and stromal compartments from the same tumor biopsy via LCM [25
]. These workers only analyzed the morphologically normal epithelium and normal stroma, however, leaving the gene expression changes in the tumor-activated stroma unexplored. Our work therefore provides the first comprehensive comparative analysis of in vivo
gene expression changes in the tumor epithelium and its stromal microenvironment during breast cancer progression from normal to DCIS to IDC.
We observed extensive gene expression changes in the stroma associated with DCIS and IDC, suggesting that tumor-adjacent stroma coevolves with the tumor epithelium, even before tumor invasion occurs. These alterations included many components of the extracellular matrix and the extracellular-matrix-remodeling matrix metalloproteases. Increased mitotic gene expression occurred both in the malignant epithelium and adjacent stroma, which may reflect the often observed desmoplastic reaction around the tumor cells. Expression of cytoplasmic ribosomal proteins was generally decreased in both compartments during cancer progression. While this result may seem paradoxical in that increased protein synthesis is considered a hallmark of cancer, it is supported by several different lines of studies. First, decreased expression of many ribosomal proteins has also been observed in colorectal cancer compared with normal mucosal epithelium [26
]. Secondly, many ribosomal protein genes have been found to be haploinsufficient tumor suppressors in zebrafish [27
]. Thirdly, the oncogenic activity of c-Myc is inhibited by the ribosomal protein L11, and inactivation of the L11 gene by small interfering RNA increases c-Myc-induced transcription and cell proliferation [28
The mechanism by which ribosomal proteins contribute to tumorigenesis is unknown. Decreased expression of ribosomal proteins in cancer may reflect a qualitative change in ribosomal structure, which may allow differential translation of gene products required for rapid tumor growth. Alternatively, it may reflect some unknown nonribosomal functions by these proteins. In contrast to the decreased expression of these cytoplasmic ribosomal protein genes, we observed increased expression of a number of mitochondrial ribosomal protein genes in both the tumor epithelium and the stroma. The human mitochondrial ribosomes are responsible for the production of several key proteins in bioenergetics including subunits of the ATP synthase. Given the importance of mitochondria in cancer [29
], our novel finding suggests that the mitochondrial ribosome may be a potential therapeutic target and thus warrants further study.
The top differentially expressed genes between tumor-associated stroma and the adjacent normal stroma included several signaling molecules known to be important for tumorigenesis. Two antagonists of WNT receptor signaling, WIF1 and SFRP1, were consistently downregulated both in the tumor epithelium and stroma. The WNT signaling pathway plays an important role in development and tissue homeostasis, and its aberrant activation by loss of expression WIF1 or SFRP1 has been shown to be an important early event in breast cancer progression [31
]. Two transforming growth factor beta superfamily members (GREM1 and INHBA) are strongly induced in the tumor-associated stroma. GREM1 is a bone morphogenetic protein antagonist, and it is overexpressed in cancer-associated stromal cells in many solid tumors [34
]. It has been hypothesized that bone morphogenetic proteins and bone morphogenetic protein antagonists may play opposing roles in the maintenance of a niche of self-renewing stem cells, with bone morphogenetic protein antagonists such as GREM1 blocking cell differentiation [34
]. WNT3A was recently demonstrated in human fibroblasts to markedly increase the expression of GREM2, a close paralog of GREM1 – raising the possibility that the significant downregulation of WNT antagonists (WIF1 and SFRP1) and upregulation of GREM1 in the stroma [35
] we observed here may be functionally linked.
INHBA is the gene for the beta A subunit of inhibin and activin, which are pleiotropic growth factors regulating the growth and differentiation of many cell types via autocrine and paracrine mechanisms [36
]. Although its role in breast cancer remains unclear, circulating levels of INHBA has been shown to be higher in breast cancer patients with bone metastasis [37
]. These signaling molecules could serve as key messengers between the tumor and its microenvironment, as shown for CXCL12 and CXCL14, which are overexpressed in tumor-associated myoepithelial cells and myofibroblasts [6
]. We note that in our dataset, however, CXCL12 and CXCL14 were also expressed in normal stroma. This discrepancy could be due to the fact that Allinen and colleagues used purified stromal cell types [7
] and we used the whole stroma compartment in our study.
A watershed event in breast cancer progression is the invasion of tumor cells into the stromal compartment. The only morphological diagnostic criterion distinguishing DCIS from IDC is the association of DCIS with a complete basement membrane. Understanding the molecular events that drive the DCIS-IDC transition has been of great interest. We have previously shown [9
], and confirm in the present study, that the malignant epithelium of DCIS and IDC are very similar without significant differences at the transcriptome level. This conclusion is supported by the recent demonstration that MCFDCIS cells, a cell line model for DCIS, make the DCIS-IDC transition spontaneously without further molecular changes in the malignant epithelial cells themselves [39
]. Instead, this transition is driven by fibroblasts and blocked by myoepithelial cells.
In the present article we demonstrated that the stromal compartment is associated with a relatively small number of significant changes accompanying the DCIS-IDC transition. In particular, several matrix metalloproteases (MMP2, MMP11 and MMP14) showed significantly increased expression in IDC-associated stroma. MMP14, a membrane-type matrix metalloprotease, can activate MMP2 protease activity, which degrades type IV collagen, the major structural component of the basement membrane [40
]. MMP11 has recently been shown to exhibit protease activity towards type VI collagen and to promote tumor progression [42
]. MMP11 has been shown to be differentially expressed in IDC relative to DCIS in two other studies. Schuetz and colleagues conducted a study similar to ours, using LCM and microarrays to profile the epithelium of patient-matched DCIS and IDC, and found MMP11 to be upregulated in IDC relative to DCIS [43
]. Their result differs from ours, however, in that we observed upregulation of MMP11 in the IDC-associated stroma but not in the epithelium. A stromal origin of MMP11 expression had been established previously [44
]. The result by Schuetz and coworkers might be due to contaminating nonepithelial cells in their LCM samples, a possibility acknowledged by these authors [43
]. In another study, Hannemann and colleagues identified a gene expression signature including MMP11 to be able to distinguish IDC from DCIS [45
]. Since no microdissection was performed in that study, the gene expression profiles they obtained were from mixtures of tumor epithelium and stroma. Nevertheless, our results together with these other studies support the notion that stroma-produced matrix metalloproteases may be key players driving the DCIS-IDC transition.
Finally, we showed that – like the epithelial compartment [9
] – tumor stroma also exhibited a robust gene expression signature correlating with the histological tumor grade. These genes are primarily involved in immune response and cell-cycle progression. The association of an immune response signature with the more aggressive high-grade tumors is seemingly paradoxical. The interactions between tumor cells and the various immune cells are complex, however, ranging from tumor growth-suppressing effects to tumor growth-promoting effects [46
]. Perhaps the immune response signature associated with high-grade tumors represents the escape phase [48
], when the cancer cells become resistant to immune attack and hijack the abundant cytokines and chemokines made by the immune cells to grow, invade and spread to distant organs.