Introduction
The vast majority of deaths due to breast cancer for nearly half a million people annually worldwide are due to distant metastases in the lung, liver, and brain [
1]. Numerous studies have focused on breast cancer metastases and how they might differ from primary breast tumors; however, controversy remains regarding (A) the predisposition of specific classes of breast tumors to spread to distant sites and (B) the degree of similarity between primary breast tumors and their associated metastases.
Estrogen receptor (ER) status is known to be associated with breast cancer relapse in specific organs [
2]. In 2008, this organ selectivity was refined by contrasting relapse patterns in 344 patients who had their tumors genomically subtyped as luminal A or B, HER2-enriched, basal-like, or normal-like [
3]. In general, bone metastases were associated with the luminal subtypes, whereas basal-like and HER2-enriched tumors were significantly associated with brain and lung relapse. Similar results were also observed in an immunohistochemical-based study on 3,726 patients [
4]. Recently, a new breast cancer subtype was identified, named claudin-low [
5‐
7]. This subtype exhibits aggressive characteristics including expression of mesenchymal markers and low expression of genes involved in tight junctions and cell–cell adhesion. The lack of epithelial cell features and expression of mesenchymal traits is reminiscent of features associated with breast stem cells [
8]. Since breast cancer stem cells are relatively resistant to both chemotherapy and radiation [
9,
10], and because metastases frequently progress despite treatment, it is important to determine if these claudin-low/mesenchymal cells are associated with metastatic potential.
To better understand the biology driving breast cancer metastases, 1,319 human gene expression microarrays from primary tumors, metastases, and cancer cell lines were analyzed here. Tumors and their associated metastases, on average, were much more similar to each other than they were different. By including the recently defined claudin-low subtype we extend previous findings [
3,
4] and better define the metastatic predilections of each intrinsic subtype. Increasingly “undifferentiated” breast cancer cells [as quantitatively measured by a Differentiation Score predictor (DS)] tend to express stem cell signatures and preferentially metastasize to the brain and lung. These results identify that breast cancer intrinsic subtype is maintained throughout disease progression, and that a combination of several genomic signatures can add prognostic value and therefore direct where disease monitoring should be focused.
Discussion
Metastases are the main cause of death for breast cancer patients and predicting a tumor’s likelihood to spread, and organ of relapse, is clinically important information. Analysis of 265 breast tumors and 85 metastases found that a breast tumor’s overall gene expression phenotype is largely maintained in its metastases. The gene expression differences that do occur may be due to a combination of different amounts of epithelial/stromal cells (Fig.
1, Supplemental Table 1), and/or clonal expansion of a more aggressive subclone of a tumor [
4,
29]. The microenvironment also effects gene expression and response to therapeutics [
30], therefore, targeting the host organ cells, vascular cells, as well as tumor cell specific targets may be the best approach to inhibit disease progression [
31]. This overall similarity, however, does suggest that important information about metastatic potential can be revealed by studying primary tumors.
Basal-like and claudin-low breast cancers both exhibit a high probability to metastasize to the brain and lung while HER2-enriched subtype tumors preferentially colonize the liver (Fig.
2; Table
1). The basal-like and claudin-low tumor types are genomically related [
6], exhibit similar treatment response characteristics, and as shown here, have similar metastasis patterns. The CD49f
+/Epcam
−/low fraction of the SUM149PT cell line (which is enriched for claudin-low tumor features) was significantly more migratory than the more differentiated basal-like component cells. Interestingly over time, the SUM149PT cells with claudin-low characteristics asymmetrically divide into two distinct populations of more (i.e., basal-like) and less-differentiated cells, whereas the more differentiated fraction produces similarly differentiated cells [
6]. Since the less-differentiated claudin-low-like cells contain higher levels of genes that facilitate cellular movement (Supplemental Figs. 4, 5), we hypothesize that these cells may initiate the metastatic cascade; after seeding a host organ, they asymmetrically divide, spawning both more and less differentiated cells. Precisely why these cells show predilection for the brain and lung requires further investigation, however, the cell line studies of Massagué and colleagues using the claudin-low MDA-MB-231 cells are providing for some initial candidates. These studies have shown that the cells that are relatively more capable of spreading to the CNS express genes that function to increase cellular extravasation and blood brain barrier penetration [
16], while also upregulating glycolytic pathways and increasing vascularization [
28].
Our re-analyses of the data presented by Bos et al. [
16] find that the DS of brain-tropic breast cancer cells is significantly lower than the parental cell line (Fig.
4); correspondingly, low DS was also found to associate with brain relapse in patients (Fig.
5). While basal-like and claudin-low breast tumors can relapse in bone, recurrence in vital organs, such as the brain and lung is more symptomatic. Thus, first site of recorded relapse for basal-like and claudin-low tumors is typically not bone. DS, however, is not the only factor that determines metastagenicity. For example, luminal A and B tumors have similar DS, yet luminal B tumors are much more likely to relapse. Perhaps all luminal tumors can effectively seed certain organs, however, the faster proliferation rate inherent to luminal B tumors accounts for the differential relapse frequency. Correspondingly, 58% of luminal B tumors present with multiple organs as first relapse events, compared to only 21% from luminal A.
After observing the metastasis patterns of the less-differentiated basal-like and claudin-low breast tumors, it was not surprising that the BrMS and LMS signatures associate with subtype and DS. The BoMS was not strongly expressed in any subtype, a finding which may reflect the fact that bone was the most common site of metastasis in our study. These findings complement analyses by Culhane and Quackenbush [
32] who found that a different lung metastasis signature [
33] was a surrogate for the basal-like subtype. This does not argue, however, that these signatures are not biologically important. In fact, the BrMS identifies some of the least differentiated tumors within the claudin-low and basal-like subtypes and these data support continued investigation of select genes within the BrMS as targeting these genes, along with others that function to increase cellular differentiation, may serve to slow metastatic progression.
To gain a mechanistic understanding for site-specific tumor colonization, we tested a compendium of 298 expression signatures as individual predictors of site of relapse. These analyses showed enrichment for stem cell signatures in brain/lung relapse (Supplemental Table 4). The majority of these signatures provide information that is encoded within DS; however, some of the signatures further divide ER-negative tumors into two distinct groups that are more or less likely to metastasize to the brain/lung. As an example, one such signature is the MM_WapINT3, which is a signature derived from a transgenic mouse mammary tumor model that over-expresses Notch4 and aggressively spreads to the lung [
34]. This is a clinically relevant finding in that half of patients with advanced triple negative breast cancer relapse within the brain [
35], and survival following CNS relapse is less than 4 months [
36], regardless of receipt of systemic therapy.
Overall, the results from Table
2 reveal shared and unique features predicting relapse to distinct sites. For example, intrinsic subtype (as represented by individual subtypes or the ROR-S score) make every final MVA model, but then each site of relapse shows individual characteristics. For brain, the BrMS signature and HER2 status add important information, while for lung the VEGF/hypoxia and LMS signature add information, for bone the DS score was valuable, and for liver, most information was carried by the HER2-enriched subtype; thus for the most accurate site of metastasis predictions, multiple signatures and/or clinical variables are needed. Our ability to predict patients at the highest risk for CNS relapse may impact the manner in which we approach CNS screening and future prevention strategies. The data presented herein provides clinically useful information that could be used to identify patients most likely to experience site-specific breast cancer relapse.
Acknowledgments
This study was supported by funds from the NCI Breast SPORE program (P50-CA58223), by RO1-CA138255, by the Breast Cancer Research Foundation, the V Foundation for Cancer Research, and a 2008 Department of Defense Era of Hope Postdoc Award (BC085270) to JCH. We thank Olga Karginova (UNC) for FACS and Xiang Zhang (MSKCC) for the 855 tumor database information. A. Prat is affiliated to the Internal Medicine PhD program of the Autonomous University of Barcelona, Spain.
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