MIR-183/182/96 CLUSTER IN EARLY BREAST CANCER EVENTS: DOES MIR-183 TRIGGER EPITHELIAL DEDIFFERENTIATION?

Abstract

Breast Cancer (BC) is the most commonly occurring cancer worldwide and the leading cause of cancer related deaths in women. BC arises due to an accumulation in epigenetic changes and genetic alterations. The deregulation of microRNAs – which are short noncoding RNA molecules – is implicated in the development of a number of diseases such as BC.

MiR-183 is overexpressed in numerous cancer types, and there have been several reports on its upregulation in BC and its involvement in BC progression. Previous work from our lab has shown that in Cx43 knockout ductular mammary epithelial HMT-3522 S1 cells, which are pre-carcinogenic in vitro models, microRNAs 183 and 182 are overexpressed. Additionally, we have previously reported that the overexpression of miR183 in non-neoplastic ductular mammary epithelial S1 cells led to the disruption of lumen formation, disrupted polarity, and increased invasion and proliferation. These phenotypic changes are indicative of a loss of a differentiated phenotype in ductular epithelial cells. As such, the aim of this study was to assess whether miR183 would play a similar role in alveolar mammary epithelial cells.

For that, we used alveolar mammary epithelial SCp2 cells that functionally differentiate as marked by the production of β-casein when supplemented with prolactin and grown either on a basement membrane (BM) or in coculture with the SCg6 myoepithelial cell strain. Our results show that miR-183-5p levels are low when SCp2 cells are grown on an exogenously provided BM either in the presence or absence of prolactin, or when they are in coculture with SCg6 in the presence of prolactin and presumably deposit their own BM. As such, we used in silico tools to investigate possible miR-183-5p targets that are involved in the secretory differentiation of alveolar cells. Interestingly, we found that β1-integrin and prolactin receptor, which are essential key regulators of β-casein production, are experimentally validated targets of miR-183-5p. However, the upregulation of miR-183-5p by the single transient transfection of SCp2 cells differentiated by EHS-Drip or the double transfection of SCp2 cells grown in coculture had no effect on the functional differentiation of SCP2 cells as determined by unchanged β-casein expression levels. Our results also show no effect for miR-183-5p on the proliferation rate of SCp2 cells differentiated by EHS-Drip. Additionally, preliminary data indicate that miR-183-5p has no apparent effect on β1-integrin and β-catenin protein levels. Although miR183’s role is not manifested through the reduction of β1-integrin and consequent loss of β-casein production, we sought to investigate alternative modes of action through which it could be acting. Additionally, since miR-182 was also overexpressed in Cx43 knockouts, and because miRs 183, 182, and 96 are polycistronic, we aimed to investigate the cluster’s involvement in early-stage breast cancer.

To do that, we performed in silico work to assess what pathways are mainly targeted by the miR-183 cluster and found that the miR-183 cluster targets are enriched for genes involved in the adherens junction pathway. Additionally, using TCGA-BRCA data from stage I breast cancer patients, we found that WASF3, EGFR, MET, and CTNNA3 are downregulated adherens junction genes which are targets of hsa-miR-182 but not hsa-miR-183 or 96. Additionally, these genes are negatively correlated with miR-182’s expression. However, we could not establish that the low expression of these genes is indeed due to the expression of miR-183/182/96. Since the relationship between microRNA 182 and these genes is not linear, we propose that other microRNAs, specifically hsa-miR-200a, is also potentially influencing these genes’ expression levels, except that of CTNNA3. Our data show that CTNNA3 is potentially also influenced by hsa-miR-3065.

In short, our results suggest that miR-183 does not have an effect on the functional differentiation of SCp2 cells, nor is it directly linked to the downregulation in adherens junction signaling in early-stage breast cancer. We suggest that miR-183 could be acting by a different mechanism, or that its potential effect is not evident by transient transfection. Additionally, hsa-miR-182 is associated with the downregulation of adherens junction signaling in early-stage BC and we anticipate that through a domino effect, this can impact the assembly of other junctional complexes. Nonetheless, the relationship between the miRs and gene expression is not perfectly reflected by mRNA sequencing data, and more studies incorporating proteomics data are necessary for a better understanding of the impact of the microRNA cluster on cell-cell signaling during early stages of breast cancer development.