Background MicroRNAs are a class of non-coding RNAs that regulate gene expression through binding to mRNAs and preventing their translation. One family of microRNAs known as the miR-200 family is an important regulator of epithelial identity. The miR-200 family consists of five members expressed in two distinct clusters; the miR-200c/141 cluster and the miR-200b/200a/429 cluster. We have found that murine and human mammary tumor cells with claudin-low characteristics are associated with very low levels of all five miR-200s. Methods To determine the impact of miR-200s on claudin-low mammary tumor cells, the miR-200c/141 cluster and the miR-200b/200a/429 cluster were stably re-expressed in murine (RJ423) and human (MDA-MB-231) claudin-low mammary tumor cells. Cell proliferation and migration were assessed using BrdU incorporation and transwell migration across Matrigel coated inserts, respectively. miRNA sequencing and RNA sequencing were performed to explore miRNAs and mRNAs regulated by miR-200 re-expression while Enrichr-based pathway analysis was utilized to identify cellular functions modified by miR-200s. Results Re-expression of the miR-200s in murine and human claudin-low mammary tumor cells partially restored an epithelial cell morphology and significantly inhibited proliferation and cell invasion in vitro. miRNA sequencing and mRNA sequencing revealed that re-expression of miR-200s altered the expression of other microRNAs and genes regulated by SUZ12 providing insight into the complexity of miR-200 function. SUZ12 is a member of the polycomb repressor complex 2 that suppresses gene expression through methylating histone H3 at lysine 27. Flow cytometry confirmed that re-expression of miR-200s increased histone H3 methylation at lysine 27. Conclusions Re-expression of miR-200s in claudin-low mammary tumor cells alters cell morphology and reduces proliferation and invasion, an effect potentially mediated by SUZ12-regulated genes and other microRNAs.

The miR-200 family of microRNAs plays a significant role in inhibiting mammary tumor growth and progression, and its members are being investigated as therapeutic targets. Additionally, if future studies can prove that miR-200s prevent mammary tumor initiation, the microRNA family could also offer a preventative strategy. Before utilizing miR-200s in a therapeutic setting, understanding how they regulate normal mammary development is necessary. No studies investigating the role of miR-200s in embryonic ductal development could be found, and only two studies examined the impact of miR-200s on pubertal ductal morphogenesis. These studies showed that miR-200s are expressed at low levels in virgin mammary glands, and elevated expression of miR-200s have the potential to impair ductal morphogenesis. In contrast to virgin mammary glands, miR-200s are expressed at high levels in mammary glands during late pregnancy and lactation. miR-200s are also found in the milk of several mammalian species, including humans. However, the relevance of miR-200s in milk remains unclear. The increase in miR-200 expression in late pregnancy and lactation suggests a role for miR-200s in the development of alveoli and/or regulating milk production. Therefore, studies investigating the consequence of miR-200 overexpression or knockdown are needed to identify the function of miR-200s in alveolar development and lactation.

MicroRNAs (miRNAs) are small, noncoding RNAs that regulate mRNA translation through inhibiting the translation of mRNAs into proteins or through degrading mRNAs. Each miRNA potentially targets hundreds of mRNAs, and thus an individual miRNA can regulate entire gene networks. The miR-200 family consists of 5 miRNAs (miR-141, miR-200a, miR-200b, miR-200c, miR-429) organized into two clusters: the miR-200c/141 cluster and the miR-200b/200a/429 cluster. The miR-200s regulate a variety of cellular functions, with the best characterized being their ability to target mesenchymal transcription factors and thus maintain an epithelial phenotype. Given that miR-200s maintain an epithelial phenotype, we evaluated their role in mesenchymal mammary tumors. All five miR-200s were expressed at significantly lower levels in the murine mesenchymal mammary tumor cell line RJ423 and the human mesenchymal breast cancer cell line MDA-MB-231 compared to epithelial tumor cell lines. To evaluate the function of miR-200s, the miR-200c/141 and miR-200b/200a/429 clusters were re-expressed in the mesenchymal mammary tumors. Re-expression of the miR-200c/141 cluster partially restored an epithelial phenotype in MDA-MB-231 cells while the miR-200b/200a/429 cluster partially restored an epithelial phenotype in RJ423 cells. Re-expression of the miR-200c/141 cluster in MDA-MB-231 cells and re-expression of the miR-200b/200a/429 cluster in RJ423 cells significantly suppressed tumor growth following intramammary injection and altered the tumor microenvironment. To identify genes regulated by miR-200 expression, RNA sequencing was performed on control and miR-200 re-expressing cells grown in 2D culture and following intramammary injection. Interestingly, the in vivo environment dramatically altered the gene repertoire regulated by the miR-200s. Hierarchical clustering revealed that alterations in gene expression induced by miR-200 re-expression in vitro poorly correlated with the alterations in gene expression induced by miR-200 in vivo. Since miR-200 re-expression significantly decreased tumor growth in vivo, a transgenic model was used to determine whether miR-200s could also impact tumor initiation. Using doxycycline-inducible miR-200 expression in combination with the mammary tumor oncogene, IGF-IR, we found that overexpression of the miR-200b/200a/429 cluster dramatically reduced mammary tumor incidence (100% vs. 19%). These data show the potent impact miR-200s have on mammary tumorigenesis and that gene expression profiling of 2D cultures is a poor predictor of genes regulated by miR-200s in vivo. Citation Format: Katrina L. Watson, Kaitlyn Simpson, Majesta Roth, Courtney Martin, Garret Conquer-Van Heumen, Roger A. Moorehead. Modeling the impact of miR-200s on mammary tumor initiation and progression in vitro and in vivo [abstract]. In: Proceedings of the AACR Special Conference on the Evolving Landscape of Cancer Modeling; 2020 Mar 2-5; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2020;80(11 Suppl):Abstract nr B27.