Amy N. Shore

Pregnancy-induced noncoding RNA (PINC) associates with polycomb repressive complex 2 and regulates mammary epithelial differentiation.

Pregnancy-induced noncoding RNA (PINC) and retinoblastoma-associated protein 46 (RbAp46) are upregulated in alveolar cells of the mammary gland during pregnancy and persist in alveolar cells that remain in the regressed lobules following involution. The cells that survive involution are thought to function as alveolar progenitor cells that rapidly differentiate into milk-producing cells in subsequent pregnancies, but it is unknown whether PINC and RbAp46 are involved in maintaining this progenitor population. Here, we show that, in the post-pubertal mouse mammary gland, mPINC is enriched in luminal and alveolar progenitors. mPINC levels increase throughout pregnancy and then decline in early lactation, when alveolar cells undergo terminal differentiation. Accordingly, mPINC expression is significantly decreased when HC11 mammary epithelial cells are induced to differentiate and produce milk proteins. This reduction in mPINC levels may be necessary for lactation, as overexpression of mPINC in HC11 cells blocks lactogenic differentiation, while knockdown of mPINC enhances differentiation. Finally, we demonstrate that mPINC interacts with RbAp46, as well as other members of the polycomb repressive complex 2 (PRC2), and identify potential targets of mPINC that are differentially expressed following modulation of mPINC expression levels. Taken together, our data suggest that mPINC inhibits terminal differentiation of alveolar cells during pregnancy to prevent abundant milk production and secretion until parturition. Additionally, a PRC2 complex that includes mPINC and RbAp46 may confer epigenetic modifications that maintain a population of mammary epithelial cells committed to the alveolar fate in the involuted gland.

Ror2 regulates branching, differentiation, and actin-cytoskeletal dynamics within the mammary epithelium

Intricate cross-talk between classical and alternative Wnt signaling pathways includes an essential role for Ror2 in mammary epithelial development and differentiation.

Regulation of mammary epithelial cell homeostasis by lncRNAs

The epithelial cells of the mammary gland develop primarily after birth and undergo surges of hormonally regulated proliferation, differentiation, and apoptosis during both puberty and pregnancy. Thus, the mammary gland is a useful model to study fundamental processes of development and adult tissue homeostasis, such as stem and progenitor cell regulation, cell fate commitment, and differentiation. Long noncoding RNAs (lncRNAs) are emerging as prominent regulators of these essential processes, as their extraordinary versatility allows them to modulate gene expression via diverse mechanisms at both transcriptional and post-transcriptional levels. Not surprisingly, lncRNAs are also aberrantly expressed in cancer and promote tumorigenesis by disrupting vital cellular functions, such as cell cycle, survival, and migration. In this review, we first broadly summarize the functions of lncRNAs in mammalian development and cancer. Then we focus on what is currently known about the role of lncRNAs in mammary gland development and breast cancer. This article is part of a Directed Issue entitled: The Non-coding RNA Revolution.

Targeting CreER T2 expression to keratin 8-expressing murine simple epithelia using bacterial artificial chromosome transgenesis

Keratin 8 (K8) is a type II keratin that is associated with the type I keratins K18 or K19 in single layered epithelia. We generated a bacterial artificial chromosome (BAC) transgenic mouse line that expresses the tamoxifen inducible CreERT2 inserted into the endogenous murine K8 gene. The transgenic mouse line contains two copies of the BAC transgene. To determine the expression specificity and inducibility of CreERT2, the K8–CreERT2 mice were bred with a Gt(ROSA26)ACTB–tdTomato–EGFP fluorescent protein-based reporter transgenic mouse line. We demonstrated that CreERT2 and the endogenous K8 gene share the same patterns of expression and that the enzymatic activity of CreERT2 can be efficiently induced by tamoxifen in all K8-expressing tissues. This mouse line will be useful for studying gene function in development and homeostasis of simple epithelia, and investigating both tissue lineage hierarchy and the identity of the cells of origin for epithelial cancers.

Plk2 regulates mitotic spindle orientation and mammary gland development

Disruptions in polarity and mitotic spindle orientation contribute to the progression and evolution of tumorigenesis. However, little is known about the molecular mechanisms regulating these processes in vivo. Here, we demonstrate that Polo-like kinase 2 (Plk2) regulates mitotic spindle orientation in the mammary gland and that this might account for its suggested role as a tumor suppressor. Plk2 is highly expressed in the mammary gland and is required for proper mammary gland development. Loss of Plk2 leads to increased mammary epithelial cell proliferation and ductal hyperbranching. Additionally, a novel role for Plk2 in regulating the orientation of the mitotic spindle and maintaining proper cell polarity in the ductal epithelium was discovered. In support of a tumor suppressor function for Plk2, loss of Plk2 increased the formation of lesions in multiparous glands. Collectively, these results demonstrate a novel role for Plk2 in regulating mammary gland development.

PTEN is required to maintain luminal epithelial homeostasis and integrity in the adult mammary gland.

In the mammary gland, PTEN loss in luminal and basal epithelial cells results in differentiation defects and enhanced proliferation, leading to the formation of tumors with basal epithelial characteristics. In breast cancer, PTEN loss is associated with a hormone receptor-negative, basal-like subtype that is thought to originate in a luminal epithelial cell. Here, we show that luminal-specific PTEN loss results in distinct effects on epithelial homeostasis and mammary tumor formation. Luminal PTEN loss increased proliferation of hormone receptor-negative cells, thereby decreasing the percentage of hormone receptor-positive cells. Moreover, luminal PTEN loss led to misoriented cell divisions and mislocalization of cells to the intraluminal space of mammary ducts. Despite their elevated levels of activated AKT, Pten-null intraluminal cells showed increased levels of apoptosis. One year after Pten deletion, the ducts had cleared and no palpable mammary tumors were detected. These data establish PTEN as a critical regulator of luminal epithelial homeostasis and integrity in the adult mammary gland, and further show that luminal PTEN loss alone is not sufficient to promote the progression of mammary tumorigenesis.

The chromatin landscape of the casein gene locus

Abstract For several decades, the regulation of casein gene expression by the lactogenic hormones, prolactin and glucocorticoids, has provided an excellent model system in which to study how steroid and peptide hormones regulate gene expression. Early studies of casein gene regulation defined conserved sequence elements in the 5′ flanking region of these genes, including one of which was identified as a γ-interferon activation sequence (GAS). Although this site was thought to interact with a mammary gland-specific factor, purification and cloning of this factor by Bernd Groner and his colleagues revealed it was instead a new member of the signal transducers and activators of transcription family, Stat5, which was expressed in many tissues. The exquisite tissue-specific expression of the casein genes was subsequently shown to depend not on a single transcription factor but on composite response elements that interacted with a number of ubiquitous transcription factors in response to the combinatorial effects of peptide and steroid hormone signaling. More recent studies have defined cooperative effects of prolactin and glucocorticoids as well as antagonistic effects of progesterone on the chromatin structure of both the casein gene proximal promoter region as well as a distal enhancer. Local chromatin modifications as well as long-range interactions facilitated by DNA looping are required for the hormonal regulation of β-casein gene expression. The casein genes are part of a large gene cluster, and the chromatin landscape of the entire cluster is regulated in a tissue-specific and developmental manner. Finally, newly discovered large non coding RNAs, such as the pregnancy-induced non coding RNA (PINC) may play an important role in the epigenetic regulation of mammary gland differentiation.