miRNA expression profiling of 51 human breast cancer cell lines reveals subtype and driver mutation-specific miRNAs

Numerous lines of evidence indicate that breast cancer is genetically and epigenetically not just one disease but a diverse group of diseases with diverse clinically relevant biological and phenotypical features. Recent technological advances in molecular profiling have led to the identification of an increasing number of molecular subtypes in breast cancer, each with distinct co-regulated and anti-regulated genes. However, the biology of these molecular subtypes and their underlying genetic drivers may be affected by numerous biological factors, including miRNAs.

miRNAs are a class of small nonprotein-coding genes that regulate the expression of genes post-transcriptionally via sequence-specific interaction with the 3' UTR of target mRNAs, resulting in inhibition of translation and/or mRNA degradation [1, 2]. A large number of studies have established that miRNAs play essential roles in biological processes, such as development [3, 4], cell proliferation [5], apoptosis [6], stress response, and tumorigenesis [7, 8]. Aberrant expression levels of miRNAs have been observed in many solid cancers including breast cancer. In breast cancer, the expression levels of several miRNAs are significantly different between normal and cancerous tissues, between breast cancers of different molecular subtypes [9‐11] with a different prognosis [12‐14], and between breast cancers showing different responses to endocrine therapy [15, 16]. Despite significant progress in the last few years on miRNA biology, the exact biological functions and the genetic factors driving their expression have been revealed for only a limited number of miRNAs in breast cancer.

Human breast cancer cell lines are excellent experimental models and renewable resources to investigate biological functions of clinically important miRNAs both in in vitro cultured conditions and in vivo when raised as xenografts [6, 17‐20]. Here, using microarrays we analyzed miRNA expression levels in 51 molecularly well-characterized human breast cancer cell lines. We explored the association of individual miRNA expression levels with intrinsic subtypes and the most common recurrent genetic aberrations. Through this analysis, we provide a catalog of miRNAs in human breast cancer cell lines, which can be used to understand underlying biology of clinically relevant miRNAs and to reveal the genetic factors that may be involved in their regulation.

Human breast cancer cell lines are renewable resources that are extensively utilized as reliable workhorses to explore biological functions of clinically relevant molecules in breast cancer. Extensive molecular characterization and gene mutation analysis by us and other researchers have suggested that breast cancer cell lines have retained significant molecular features that are commonly observed in clinical breast tumors [36, 37]. This suggestion prompted us to use 51 human breast cancer cell lines as a discovery cohort to identify differentially expressed miRNAs between known intrinsic subtypes of breast cancer as well as those associated with common genetic aberrations present in the cell lines.

In this study, we demonstrated that global miRNA expression profiling can assign our cell line collection into two clusters. These two clusters predominantly mirror the ER-based dichotomy present in human breast cancer cell lines, which may point to the fact that, like mRNA, miRNA expression profiling allows the discrimination between ER-positive and ER-negative cell lines. This suggests that a significant number of miRNAs may be under the control of ER regulation. In line with this, we observed a trend of finding ER binding sites closer to the significantly differentially expressed miRNAs between ER-positive and ER-negative cell lines than nonsignificant miRNAs (see Table S19 in Additional file 1 and Figure S4 in Additional file 2). Interestingly, not all miRNAs discriminating ER-positive and ER-negative cell lines are associated with ER status and therefore the division of the cell lines into two clusters may not be purely dictated by the expression of ER-related miRNAs, but these miRNAs are probably also related to luminal versus basal cellular differentiation.

Our supervised analysis revealed a signature of 79 differentially expressed miRNAs between ER-positive and ER-negative cell lines. Six of these miRNAs were also found similarly differentially expressed between ER-positive and ER-negative primary tumor specimens as they were in cell lines [29] (see Table S4B in Additional file 1). This overlap may seem small but we have observed discrepancies between miRNA profiling platforms (unpublished observations), and our study used LNA™ technology-based microarrays to quantify miRNA expression in the cell lines whereas Cimino and colleagues used Agilent microarrays to measure miRNAs in tumors. Also not all miRNAs are present on both platforms, which already explains one-half of the discrepancies. Furthermore, tumor heterogeneity and stromal contribution (such as connective tissues, blood vessels and immune infiltrates) and a likely selection bias in the cell lines - which are more ER-negative, mesenchymal, and TP53 mutant than primary tumors - could explain the observed discrepancies. A perfect correlation would thus not have been anticipated. Reassuringly, however, we found miRNAs previously reported to be ER-regulated miRNAs, such as a cluster of hsa-miR-221/222 [12, 38]. In line with this, we also found a significant inverse correlation between the expression levels of these ER-regulated miRNAs and mRNA expression levels of their functionally validated target genes - for instance, hsa-miR-221and hsa-miR-222 have been shown to target the CDKN1B and ESR1genes (see Table S20 in Additional file 1). Such miRNAs have been shown to interact directly with ER and cause a phenotypic shift from ER-positive to ER-negative tumor cells [38]. Our miRNA analyses in this cohort of cell lines therefore confirms previous findings but also revealed new miRNAs (discussed below), which may have potentially interesting biological roles in ER-driven cancer.

Among these potentially novel ER-related miRNAs, four miRNAs (hsa-miR-26a, hsa-miR-92b, hsa-miR-191, hsa-miR-492) appear to show consistently higher expression across all the ER-positive cell lines (fold change ≥ 1.5), and as yet only hsa-miR-26a has been implicated in breast carcinogenesis whereas hsa-miR-26a and hsa-miR-92b have also been implicated in brain tumors [39, 40]. In breast cancer, high expression of the hsa-miR-26a miRNA downregulates EZH2 and is therefore related to a favorable outcome on tamoxifen in metastatic breast cancer [41], and also interacts with CDK4 and CENTG1 oncogenes and forms an integrated oncomir/oncogene DNA cluster, which promotes glioblastoma tumor growth via RB1, PI3K/AKT, and JNK pathways [39]. On the other hand, hsa-miR-92b has been found to be exclusively overexpressed in primary brain tumors but serves as a biomarker to discriminate brain primary cancer from metastasis [40]. The other two miRNAs (hsa-miR-191, hsa-miR-492) have been linked to hepatic cancer. The increased expression of hsa-miR-191 stimulates proliferation in hepatocellular carcinoma cell lines and its therapeutic targeting suppresses tumor masses in vivo [42]. The hsa-miR-492 miRNA has been shown to be processed from the keratin 19 gene and upregulated in metastatic hepatoblastoma [43]. How these miRNAs play their biological roles in the context of breast cancer remains unknown and demands clinical and functional validation studies.

Another major contribution to the overall miRNA profiling of this cell line cohort is the identification of a signature of 42 differentially expressed miRNAs, which discriminates between basal-like and normal-like/claudin-low breast cancer cell lines. These miRNAs together with the ER-associated miRNAs are the major determinants of the overall clustering of the cell lines. Importantly, this signature includes all four members of the hsa-miR-200 family (hsa-miR-200a, hsa-miR-200b, hsa-miR-200c, hsa-miR-141), hsa-miR-155, and hsa-miR-622 miRNAs. Several studies have implicated these miRNAs to be involved in epithelial-mesenchymal transition (EMT), to be related to the stem-cell-like phenotype, and to be associated with a switch in paclitaxel responsiveness [44‐47]. In breast cancer, these miRNAs are known to regulate the EMT process by targeting the ZEB family of the transcription factors through an active negative feedback loop [48, 49]. Importantly, the ZEB family of transcription factors was reported to be a repressor of E-cadherin expression in several epithelial carcinomas including breast carcinoma.

Interestingly, we observed a significant inverse correlation between the expression levels of all miRNAs of the hsa-miR-200 family and the mRNA expression level of the ZEB1 transcription factor, which is a well-known target of this miRNA family (see Table S20 in Additional file 1). Additionally, our finding that the hsa-miR-200 cluster showed lower expression in normal-like/claudin-low breast cancer cell lines fits with the fundamental literature that these miRNAs are indeed involved in EMT, because the majority of the normal-like/claudin-low cell lines lack E-cadherin protein expression, exhibit low claudin expression, and generally display EMT-like features as well as the breast cancer stem cell phenotype (CD44^high/CD24^low) [50‐53]. Other known significant miRNAs of this signature are hsa-miR-155 and hsa-miR-622, which were also linked to enhanced tumorigenesis in various cancer types besides breast cancer [54, 55]. In concordance with this, we recently found that the normal-like/claudin-low cell lines with low expression of these EMT-related miRNAs show highly aggressive growth characteristics in vivo when raised as xenografts in nude mice (M Riaz and colleagues, unpublished data). Importantly, this signature includes three previously unknown miRNAs (hsa-miR-492, hsa-miR-26b, hsa-miR-617; fold change ≥ 1.5) found to be associated with cell lines frequently showing EMT-like characteristics.

We observed that the miRNA signatures associated with E-cadherin mutation and promoter hypermethylation include distinct miRNAs. This may point to an existence of unique biology at the miRNA level in tumors that show E-cadherin inactivation due to gene mutation rather than those that lose E-cadherin expression due to promoter hypermethylation. Important to note is that E-cadherin promoter hypermethylated cell lines include all normal-like/claudin-low cell lines, and most EMT-related miRNAs were also associated with E-cadherin promoter hypermethylation.

With respect to common genetic aberrations present in breast cancer, our study reveals differentially expressed miRNA signatures associated with commonly mutated tumor suppressors and oncogenes. Most significantly, a signature of 30 miRNAs associated with p16 ^INK4a mutant cell lines can strongly discriminate between mutant and wild-type ER-negative cell lines. P16 ^INK4a is a tumor suppressor gene located on chromosomal band 9p21 that has been frequently altered in many human cancers [56]. P16 ^INK4a regulates cell cycle progression by targeting CDK4/6 through the pRb signaling pathway. Interestingly, this signature includes some miRNAs that are involved in cell cycle regulation. For instance, hsa-miR-100, which is highly expressed in the p16 ^INK4a wild-type cell lines, targets the RBSP3 gene that in acute myeloid leukemia regulates the cell cycle through partial modulation of pRB/E2F1 [57]. hsa-miR-34a has been reported as a suppressor of cell proliferation and migration in colon cancer [58] and its mechanism of growth inhibition also involves cell cycle arrest followed by apoptosis [59]. Besides this, we found a few miRNAs to be only associated with mutation in BRCA1, TP53 and PIK3CA/PTEN; these observed associations require further independent validation in breast cancer specimens.

Our finding that the differential expression of miRNAs is associated with ERBB2-overexpressing luminal-type cell lines is also intriguing since the miRNAs showing elevated expression in ERBB2-positive cell lines (fold change ≥ 1.5) are not located on the ERBB2 amplicon (17q12) and thus may be regulated indirectly by genes in the ERBB2 amplicon. It is also important to mention that a majority of these miRNAs have already been implicated in various types of epithelial carcinoma including breast cancer [60‐64]. One should, however, note that our study is associative and does not necessarily reveal causality. We therefore propose functional studies on these miRNAs to reveal more biological insights into their role regarding ERBB2 overexpression in breast cancer.

Finally, we also identified 12 miRNAs to be associated with CNVs in breast cancer cell lines (see Table S17 in Additional file 1). The majority of these miRNAs (hsa-miR-130a, hsa-miR-93, hsa-miR-383, hsa-miR29c, hsa-miR-382, hsa-miR-31) were already found to be located in regions that exhibited DNA copy number abnormalities in breast cancer tumors [65]. Importantly, this repertoire of miRNAs includes hsa-miR-22 previously shown to be regulated by ER [66] and we provide evidence that it can also be regulated by the loss of the locus containing this miRNA. Six miRNAs of this repertoire are located in the genomic regions containing known protein coding genes. All miRNAs of this repertoire have also been implicated in various cancers but a thorough validation of these miRNAs with respect to DNA copy number changes in clinical specimen is imperative.

In summary, our analyses show that the dichotomy in breast cancer cell lines is, in line with the general consensus in the field, related to ER status. However, part of this dichotomy may be related to ER-independent luminal versus basal differentiation status of cell lines. Secondly, another prominent dichotomy observed among breast cancer cell lines discriminating basal-like cell lines from normal-like/claudin-low cell lines involves miRNAs related to EMT and/or stemness. Furthermore, we reveal sets of miRNAs associated with genes frequently amplified (ERBB2) or mutated (p16 ^INK4a , PIK3CA and/or PTEN, E-cadherin, BRCA1) in breast cancer cell lines and thus these miRNAs may contribute to the function of these oncogenes/tumor suppressors. Finally, certain miRNAs themselves are located in genomic regions, which frequently show genetic aberrations, implying that these miRNAs themselves may have a potential driver role or contribute to known drivers in these genomic regions. Our current findings call for further validation of these signatures in clinical specimens. Importantly, our study provides a unique molecular miRNA portrait of human breast cancer cell lines, which can be exploited for functional studies of clinically important miRNAs.