Introduction
The mammalian breast is a dynamic organ, with major morphological changes occurring during organogenesis, puberty, pregnancy, lactation, and involution [
1]. Underlying these mammary gland changes is a complex cell hierarchy that supports these processes [
2‐
4]. The simplest model places the multipotent mammary stem cell (MaSC) at the base of this hierarchy, having extensive, self-regenerative potential [
5]. During mammary development, the MaSC has been proposed to divide asymmetrically to produce basal/myoepithelial cells as well as luminal progenitors (LumProg), which have more restricted proliferative and differentiation capabilities [
5]. LumProg cells are capable of further differentiation into mature luminal (MatureLum) cells, such as estrogen receptor (ER)-positive ductal epithelium, which have an even more limited proliferative potential and some of which are terminally differentiated [
5].
Breast tumors may originate from several, if not all, of the cell types within this complex mammary hierarchy. These various cellular foundations for tumor initiation may help explain the heterogeneous nature of human breast tumors [
6], which consist of multiple histological and genomic subtypes; these genomic groups, which are defined by their gene expression profiles, have become known as the intrinsic subtypes of breast cancer and are referred to as basal-like, claudin-low, HER2-enriched, luminal A, and luminal B [
7‐
10]. A simple etiological explanation for these different subtypes involves a one-to-one relationship between each intrinsic subtype and a distinct cell type of origin that largely maintains its phenotypic identity after oncogenic transformation; however, both normal and neoplastic non-stem cells can acquire stem-like properties, suggesting that the normal cell hierarchy model could also include an element of reversibility [
11]. This also raises the possibility that molecular features defining tumor subtypes, may be acquired during tumorigenesis [
12].
Genetically engineered mouse models (GEMMs) of breast carcinoma develop heterogeneous tumors [
13,
14], but the extent to which they represent human disease is an area of active investigation. We previously showed that murine mammary tumors comprise at least 17 distinct intrinsic subtypes/classes, with eight classes being identified as strong human subtype counterparts by gene expression similarity [
14]. As with human breast cancer, the degree to which murine models reflect normal mammary epithelial subpopulations requires further analysis. Characterization of the cellular features of these murine classes is also needed to better determine their preclinical utility, to shed light on trans-species associations [
14], and to help interpret preclinical study observations [
15‐
18].
Several studies have independently profiled fluorescence-activated cell sorted (FACS) purified normal mammary cell types from both human [
19‐
21] and murine [
22,
23] mammary tissues. Here, we use a meta-analysis approach to compare the transcriptomic profiles from FACS-enriched mammary cell populations with each other and with primary tumors. These data not only identify a number of clinically relevant biomarkers that may be useful for predicting chemotherapy benefit, but also suggest a cell type of origin for many tumor subtypes.
Discussion
Normal mammary gland physiology is supported by an underlying, complex cell hierarchy [
2‐
5]. The simplest model treats differentiation from mammary stem cells to progenitor cells to mature cells as unidirectional, but recent observations indicate that bidirectional processes are also possible for normal and neoplastic cells [
11]. This differentiation plasticity may allow tumors to acquire cell features foreign to the initial cell-of-origin or to lose native features through the accumulation of specific genetic aberrations [
40].
Regardless of how different cellular traits are acquired, it is critical to identify the ‘current’ normal cellular features within a tumor, and therefore, we first analyzed the expression profiles of normal human and mouse mammary epithelial cell subpopulations [
19‐
23]. We chose to use nomenclature that maintains continuity with the literature. However, these terms should be considered provisional as the complete biological profiles of these FACS fractions are investigated [
4]. Recent work by Prater et al. [
41] found that mouse ‘LumProg’ cells (CD49f
+, EpCAM
+) have complete mammary gland repopulating potential, indicating that ‘LumProg’ may be a misnomer. Importantly, even if our understanding and naming of these cell subpopulations change, only the retrospective interpretation of the data presented here will be affected, not the data itself.
Using a meta-analysis approach, FACS-purified mammary epithelial cell subpopulation ‘enriched’ gene signatures were derived and a nearest centroid predictor was developed to identify which normal mammary subpopulation each human and mouse tumor most represented using over three thousand human patients and 27 mouse models of mammary carcinoma [
14]. While these analyses imply a cell-of-origin for a given tumor, additional experiments (e.g., lineage tracing) will be required to unequivocally determine this. Nevertheless, these associations at the very least identify which normal mammary subpopulation a given tumor most represents in its current state.
With this in mind, several associations between both the human and mouse intrinsic subtypes and specific normal cell subpopulations were observed. First, human basal-like tumors have been referred to as ‘undifferentiated’, which is consistent with their exhibiting LumProg [
19] and fetal MaSC features [
23]. Three mouse classes have been identified to be human basal-like counterparts: Myc
Ex, p53null-Basal
Ex, and C3-Tag
Ex [
14]. Myc
Ex tumors were the most similar to the LumProg cell profile. By contrast, both p53null-Basal
Ex and C3-Tag
Ex tumors had adult MaSC features. These results indicate that Myc
Ex tumors share similar cell features as their human basal-like counterpart, making it an attractive mouse model for studying basal-like tumors with aberrant Myc signaling [
10,
42]. Interestingly, neither p53null-Basal
Ex nor C3-Tag
Ex tumors had strong LumProgs features, indicating that their association with human basal-like tumors is more likely driven by their underlying genetics [
10].
Human claudin-low tumors had heterogeneous normal cell features. While most were similar to LumProg cells, the claudin-low subtype also had the largest percentage of tumors classified as adult MaSC. Given that claudin-low tumors are enriched with epithelial-to-mesenchymal transition features [
9,
43,
44], our results suggest that these tumors may originate from the LumProg population prior to acquiring adult MaSC and/or mesenchymal features. Similarly, mouse Claudin-low
Ex tumors were also strongly associated with the adult MaSC population, indicating that such tumors may be the closest analogs of the subset of human claudin-low tumors with adult MaSC features.
Human HER2-enriched tumors were the most similar to the LumProg subpopulation. This is a novel finding and may explain why both human basal-like and HER2-enriched subtype tumors show high TP53 mutation frequencies (>70 %) and widespread chromosomal instability [
10]. These data could suggest that the normal LumProg cell is somehow extremely dependent on TP53 function. The murine Erbb2-like
Ex class has been identified as a mouse counterpart for human HER2-enriched tumors [
14] and was shown here to also have LumProg features.
When analyzing the human luminal A and B subtypes, a clear association with normal MatureLum cells was observed. The murine Neu
Ex class is a proposed counterpart for human luminal A tumors [
14], yet these mouse tumors were most similar to normal mouse LumProg cells. The Myc
Ex class was also identified to resemble human luminal B tumors [
14]. As discussed, Myc
Ex tumors have LumProg features; therefore, most mouse luminal A/B tumor models do not share the same normal cell features as their human tumor counterparts. These differences may reflect limitations of model system design, as tumors within these mouse classes are primarily driven by either the WAP or MMTV promoter. These differences in cell features, however, indicate that the trans-species associations observed previously [
14] are possibly driven by the genetics of each mouse model. Nevertheless, broad molecular features are conserved between these human–murine counterparts [
14]. Therefore, we propose that these mouse models retain significant preclinical utility provided that shared versus distinct molecular features are taken into account.
Neoadjuvant chemotherapy is a common approach for treating breast tumors, but only a relatively low percentage of patients have a pCR (~20 % overall). We tested the clinical significance of normal cellular features for predicting pCR using a combination of UVA and MVA logistic regression analyses. Human LumProg and mouse fetal MaSC expression features were identified as predictive of pCR sensitivity across all breast cancer patients. More specifically, LumProg-HsEnriched-feature1 and fMaSC-MmEnriched-feature1 were highly expressed in basal-like tumors. This is consistent with the clinical observation that basal-like tumors have better neoadjuvant chemotherapy response rates since higher expression of these normal cell signatures was associated with a higher likelihood of pCR. Distinct from these signatures, tumors with high expression of fMaSC-MmEnriched-feature2 were more resistant to neoadjuvant chemotherapy. Not surprisingly, this signature was most highly expressed in luminal A and B tumors, consistent with the clinical observation that these subtypes have lower chemotherapy response rates. Importantly, these signatures remained significant even after controlling for intrinsic subtype, proliferation, and clinical variables in the MVA analysis; thus these normal cell signatures add information even when tumor subtype and clinical features are known. It is presently unknown whether tumors with these features arise from a LumProg or fetal MaSC cell-of-origin or acquire these features during tumorigenesis. Whether these features are acquired or inherent, the ‘current’ cellular traits of a tumor are likely most important as these appear to be a major determinant of chemotherapy sensitivity. The biological explanation for why LumProg and fetal MaSC expression features predict tumor responsiveness to neoadjuvant chemotherapy will need to be explored further, but it is likely linked to the common genetic features of TP53 loss [
45], RB-pathway loss [
46], and high proliferation status [
47], as well as other inherent characteristics of these cellular states. This work highlights the efficacy of studying the normal mammary gland cell hierarchy and development to provide insights into human tumor therapy responsiveness.
Acknowledgments
We would like to thank J.S. Parker, J.C. Harrell, and the Perou lab for helpful suggestions. This study was supported by funds from the following sources: NCI P50-CA58223 Breast SPORE program (CMP), RO1-CA138255 (CMP), RO1-CA148761 (CMP), Department of Defense W81XWH-12-1-0106 and W81XWH-12-1-0107 (CMP and GMW), NIEHS T32-ES007017-35S1 (ADP), and the Breast Cancer Research Foundation (CMP and GMW).