Genome-wide miRNA expression analyses and functional studies have revealed important roles for these small regulatory molecules in breast cancer biology. This study of miRNA expression in relevant GEM models of human breast cancer provides the opportunity to distinguish miRNA expression patterns in a supervised manner according to the known molecular alterations that induce tumor formation and characteristics of the tumor phenotype. The miRNA expression patterns can be further interpreted based upon our previous studies that have delineated gene expression patterns for these same GEM models [13
]. This is the first large-scale miRNA gene expression study across a variety of GEM models of human breast cancer and strongly suggests that a primary determinant of miRNA expression is the lineage of the tumor (that is, basal versus luminal), supporting the previous report that altered miRNA expression is confined to specific epithelial cell subpopulations in human breast cancer [36
We chose to analyze these eight GEM mammary tumor models since they have been designed to initiate tumorigenesis through different molecular pathways that are quite relevant to human breast cancer. We identified miRNAs that are associated with specific models or that are commonly deregulated in all of the mammary tumors models. Unlike similar studies involving human patient samples, genomic analyses of GEM models may be performed in defined genetic backgrounds, which greatly reduces variability in expression due to genetic variation as is often the case in human studies. The results of this study have demonstrated that miRNA expression profiling can classify GEM models according to luminal or basal subtypes and that relatively few miRNAs are expressed in a model-specific manner despite different initiating oncogenic drivers used in the design of the models. Although these results strongly suggest that the miRNA expression patterns primarily reflect the state of tumor cell differentiation (luminal versus basal), more subtle distinctions in miRNA expression can be identified in the different models.
The differential expression of miRNAs among the eight murine models resulted in their segregation into several clusters. One major cluster included the p53-/-
transplant and C3(1)/SV40 T/t
-antigen GEM models. These two models both develop mammary tumors with basal features, suggesting that the associated miRNAs reflect the phenotype of the basal tumor lineage. Both of these model systems share mechanistic similarities through the loss of p53
function. SV40 Tag sequesters p53
by forming a Tag-p53
complex, thus inactivating p53
tumor suppressor function leading to abnormalities in cell cycle regulation, apoptotic response, genome instability and tumorigenesis [37
]. These findings suggest that a common mechanism of miRNA deregulation may be involved in p53
-mediated tumorigenesis. Although clustered within the basal group of tumors, the BRCA1-/-p53+/-
model forms an independent cluster, which may indicate that these tumors express distinct molecular features as has been suggested previously [13
Another major cluster of tumors includes four of the MMTV-promoter driven GEM models - MMTV-H-Ras
, and MMTV-Wnt1
- that develop mammary tumors with more luminal features. Interestingly, there was some overlap between the miRNA expression patterns between these mouse mammary tumors with luminal features and the normal mammary gland, further suggesting that the miRNA expression pattern of these tumors is related to a luminal phenotype. This is consistent with a previous report that a cluster of luminal breast cancer miRNAs may be involved in the control of normal mammary gland development and become deregulated in breast cancer [38
]. Nevertheless, our findings that the MMTV-driven tumors cluster with normal mammary glands also suggest that the MMTV LTR may target expression to a mammary cell lineage with luminal characteristics. Mammary epithelial cells in the pregnant mammary gland are in a state of increased proliferation and differentiation. This may also contribute to the clustering of the normal pregnant glands with the MMTV promoter-driven tumors.
We identified a signature of 122 miRNAs that are associated with the basal-like mammary tumors, and a signature of 73 miRNAs associated with the luminal-type mammary tumors. Blenkiron et al
] reported 38 miRNAs that are differentially expressed among human basal-like, HER2+, luminal A, luminal B or normal-like tumor subtypes, and these miRNAs have been shown to be involved in mammary gland development [38
]. Importantly, we find that several of these miRNAs are consistent with our findings in the GEM models. Three miRNAs associated with human basal-type tumors (miR-135b, miR-505 and miR-155), and seven miRNAs associated with human luminal type tumors (let-7a, let-7f, miR-100, miR-130a, miR-152, miR-214 and miR-29b) are similarly expressed in mouse basal-like and luminal-type tumors, respectively. This suggests that the expression of these miRNAs may be evolutionarily conserved during mammary tumor differentiation. Therefore, the mouse models described may prove useful for understanding tumor lineage specification and how miRNAs play a role in this process.
Many of the miRNAs that we have identified to be associated with luminal type GEM tumors have been shown to be expressed at various stages of normal murine mammary gland development. Avril-Sassen et al
] identified seven miRNA clusters with distinct patterns of expression during mouse mammary gland development. Many of the miRNAs we have identified as being primarily expressed in luminal type GEM mammary tumors are found in two of these miRNA clusters. miR-193, -30b, -30c, -26a, and -26b are highly expressed during early development, gestation and late involution; miR-141, -200a, -148a, and -146b are highly expressed during gestation, lactation, and early and late involution. These results suggest that the various mouse luminal-type tumors induced by the MMTV LTR-targeted expression of oncogenes maintain specific luminal miRNA expression patterns, although the cells have become tumorigenic. Interestingly, the mRNA expression patterns of several oncogene-induced GEM tumor models driven by the MMTV LTR also cluster together despite utilizing oncogenes that function in different oncogenic pathways. This suggests that the MMTV LTR in these models may be targeting a particular mammary luminal epithelial cellular compartment at a specific stage of differentiation, resulting in tumors that share many similarities in miRNA and mRNA expression.
Several of the miRNAs that we have identified as being specific for the luminal-type GEM tumors (miR-141, -200a and -200b) have been shown to repress an EMT [39
]. miR-141 inhibits EMT in part through targeting of transforming growth factor-β2. miR-200a has been shown to repress EMT through targeting of β-catenin. The miR-200 family has also been shown to target SIP1 and ZEB1, which are mediators of EMT. Thus, expression of miR-141, -200a and -200b in luminal tumors is in keeping with maintenance of the luminal phenotype.
Comparison of miRNA expression of normal mammary epithelium from glands harvested at day 17.5 of gestation to the GEM tumors identified several miRNAs that were primarily expressed only in the normal epithelium. Interestingly, we identified five miRNAs - miR-10b, -148a, -150, -199a and -486 - that are down-regulated in all of the mammary tumors compared to normal mammary gland tissue irrespective of the initiating genetic lesion. Four of these miRNAs - miR-10b, -148a, -150, -199a - have been implicated in mouse mammary gland development [38
]. One of these, miR-10b, has been shown to be down-regulated in human breast carcinoma compared to normal breast tissue. miR-10b, which targets HOXD10
, was additionally shown to be down-regulated in all the breast carcinomas from metastasis-free patients [42
]. miR-199a functions as an onco-suppressor targeting the oncogene Met
, therefore impairing Met-mediated invasive growth of cells [43
]. miR-150 has been shown to negatively regulate the expression of the Myb
]. These findings suggest that the loss of some or all of these miRNAs may be important for tumor development. miR-486, also expressed in normal epithelium, has been shown to be down-regulated in mammary cancer. Together, these data suggest that these miRNAs might function as tumor suppressors or regulate cellular differentiation and become deregulated during mammary tumor development.
Interestingly, although we identified many miRNAs whose expression was observed in basal type tumors, few of these miRNAs have been previously characterized. Thus, these basal GEM mammary tumor models may offer an important opportunity to delineate the functions of these less well studied basal-associated miRNAs.
Relatively few miRNAs were identified as being specifically expressed in particular GEM models. miR-22 was found to be primarily expressed in MMTV-Wnt1
tumors. miR-22 has previously been shown to be over-expressed in progenitor cells [45
]. This would be in keeping with earlier studies that have suggested that MMTV-Wnt1
tumors are enriched for cells with stem cell characteristics [46
]. Three miRNAs were found to be highly expressed in the c-Myc
model, including miR-494, miR-699 and miR-685. Among them, miR-494 is highly associated with the luminal-type of mammary tumors, suggesting a potential role for miR-494 in c-Myc
-mediated oncogenic signaling and in mammary tumor differentiation. miR-494 is highly expressed in human retinoblastoma [50
]. It also negatively regulates PTEN
gene expression at the translational level in human bronchial epithelial cells induced by anti-benzo(a)pyrene-trans-7,8-dihydrodiol-9,10-epoxide (anti-BPDE) and functions as a micro-oncogene in carcinogenesis [51
Furthermore, by using an integrated miRNA and mRNA gene expression analysis, we demonstrated in vivo
that the expression of miRNAs can be associated with the inverse expression of a subset of predicted target mRNAs in mammary gland tumors, leading to a more focused set of miRNAs to functionally validate. Since computational prediction of miRNA targets is inconsistent across different algorithms and usually identifies hundreds of potential targets, our approach of identifying an inverse correlation between miRNA and mRNA significantly reduces the number of potential candidates. However, it must be remembered that this analysis does not consider inhibition of protein translation by miRNA, which has been considered the primary mode of action of miRNAs. Therefore, additional miRNA targets need to be considered at the protein level. However, whether miRNA works primarily through inhibition of translation or transcription remains controversial [52
Real-time RT-PCR demonstrated that the expression of Birc4
was reduced in mammary tumor epithelial cells that over-expressed miR-494. However, further analyses will confirm that miR-494 targets the putative mRNA sequence in the 3' UTR of Birc4
. miR-412 was the only miRNA associated specifically with the C3(1)Tag model, and is also highly associated with the basal-like mammary tumors. Real-time RT-PCR demonstrated that overexpression of miR-412 reduces expression of Bmpr1a
. Identification of the mRNA target in the 3' UTR of Bmpr1
will validate this finding. Bmpr1a is a type 1A bone morphogenetic protein receptor, but its functional role in breast cancer has not been defined. Decreased expression of Bmpr1b predicts poor prognosis in breast cancer patients and leads to increased cell proliferation of breast cancer cells in vitro
, suggesting the tumor suppressor role of the Bmpr family in breast cancer carcinogenesis [53
]. Therefore, inhibition of Bmpr1a
expression by miR-412 could be involved in tumor initiation or progression of the C3(1)Tag and basal models.