Loss of Slug Compromises DNA Damage Repair and Accelerates Stem Cell Aging in Mammary Epithelium.

DNA damage activates checkpoints that limit the replicative potential of stem cells, including differentiation. These checkpoints protect against cancer development but also promote tissue aging. Because mice lacking Slug/Snai2 exhibit limited stem cell activity, including luminobasal differentiation, and are protected from mammary cancer, we reasoned that Slug might regulate DNA damage checkpoints in mammary epithelial cells. Here, we show that Slug facilitates efficient execution of RPA32-mediated DNA damage response (DDR) signaling. Slug deficiency leads to delayed phosphorylation of ataxia telangiectasia mutated and Rad3-related protein (ATR) and its effectors RPA32 and CHK1. This leads to impaired RAD51 recruitment to DNA damage sites and persistence of unresolved DNA damage. In vivo, Slug/Snai2 loss leads to increased DNA damage and premature aging of mammary epithelium. Collectively, our work demonstrates that the mammary stem cell regulator Slug controls DDR checkpoints by dually inhibiting differentiation and facilitating DDR repair, and its loss causes unresolved DNA damage and accelerated aging.

Epigenetic Reprogramming of Lineage-Committed Human Mammary Epithelial Cells Requires DNMT3A and Loss of DOT1L.

Organogenesis and tissue development occur through sequential stepwise processes leading to increased lineage restriction and loss of pluripotency. An exception to this appears in the adult human breast, where rare variant epithelial cells exhibit pluripotency and multilineage differentiation potential when removed from the signals of their native microenvironment. This phenomenon provides a unique opportunity to study mechanisms that lead to cellular reprogramming and lineage plasticity in real time. Here, we show that primary human mammary epithelial cells (HMECs) lose expression of differentiated mammary epithelial markers in a manner dependent on paracrine factors and epigenetic regulation. Furthermore, we demonstrate that HMEC reprogramming is dependent on gene silencing by the DNA methyltransferase DNMT3A and loss of histone transcriptional marks following downregulation of the methyltransferase DOT1L. These results demonstrate that lineage commitment in adult tissues is context dependent and highlight the plasticity of somatic cells when removed from their native tissue microenvironment.

The Hippo transducer TAZ interacts with the SWI/SNF complex to regulate breast epithelial lineage commitment.

Lineage-committed cells of many tissues exhibit substantial plasticity in contexts such as wound healing and tumorigenesis, but the regulation of this process is not well understood. We identified the Hippo transducer WWTR1/TAZ in a screen of transcription factors that are able to prompt lineage switching of mammary epithelial cells. Forced expression of TAZ in luminal cells induces them to adopt basal characteristics, and depletion of TAZ in basal and/or myoepithelial cells leads to luminal differentiation. In human and mouse tissues, TAZ is active only in basal cells and is critical for basal cell maintenance during homeostasis. Accordingly, loss of TAZ affects mammary gland development, leading to an imbalance of luminal and basal populations as well as branching defects. Mechanistically, TAZ interacts with components of the SWI/SNF complex to modulate lineage-specific gene expression. Collectively, these findings uncover a new role for Hippo signaling in the determination of lineage identity through recruitment of chromatin-remodeling complexes.

EGF receptor activates MET through MAPK to enhance non-small cell lung carcinoma invasion and brain metastasis.

MET amplification as a mechanism of acquired resistance to EGF receptor (EGFR)-targeted therapies in non-small cell lung carcinoma (NSCLC) led to investigation of novel combinations of EGFR and MET kinase inhibitors. However, promiscuous interactions between MET and ERBB family members have made it difficult to evaluate the effects of MET on EGFR signaling, both independent of drug treatment and in the context of drug resistance. We addressed this issue by establishing a 32D model cell system wherein ERBBs or MET are expressed alone and in combination. Using this model, we determined that EGFR signaling is sufficient to induce MET phosphorylation, although MET activation is enhanced by coexpression of ERBB3. EGFR-MET cross-talk was not direct, but occurred by a combined regulation of MET levels and intermediary signaling through mitogen-activated protein kinases (MAPK). In NSCLCs harboring either wild-type or mutant EGFR, inhibiting EGFR or MAPK reduced MET activation and protein levels. Furthermore, MET signaling promoted EGFR-driven migration and invasion. Finally, EGFR-MET signaling was enhanced in a highly metastatic EGFR-mutant cell subpopulation, compared with the indolent parental line, and MET attenuation decreased the incidence of brain metastasis. Overall, our results establish that EGFR-MET signaling is critical for aggressive behavior of NSCLCs and rationalize its continued investigation as a therapeutic target for tumors harboring both wild-type and mutant EGFR at early stages of progression.

ErbB3 is required for ductal morphogenesis in the mouse mammary gland.

INTRODUCTION: The receptor ErbB3/HER3 is often over-expressed in human breast cancers, frequently in conjunction with over-expression of the proto-oncogene ERBB2/HER2/NEU. Although the prognostic/predictive value of ErbB3 expression in breast cancer is unclear, ErbB3 is known to contribute to therapeutic resistance. Understanding ErbB3 functions in the normal mammary gland will help to explain its role in cancer etiology and as a modulator of signaling responses to the mammary oncogene ERBB2. METHODS: To investigate the roles of ErbB3 in mouse mammary gland development, we transplanted mammary buds from ErbB3-/- embryos into the cleared mammary fat pads of wild-type immunocompromised mice. Effects on ductal outgrowth were analyzed at 4 weeks, 7 weeks and 20 weeks after transplantation for total ductal outgrowth, branch density, and number and area of terminal end buds. Sections of glands containing terminal end buds were analyzed for number and epithelial area of terminal end buds. Terminal end buds were also analyzed for presence of mitotic figures, apoptotic figures, BrdU incorporation, and expression of E-cadherin, P-cadherin, alpha-smooth muscle actin, and cleaved caspase-3. RESULTS: The mammary ductal trees developed from ErbB3-/- buds only partly filled the mammary fat pad. In contrast to similar experiments with ErbB2-/- mammary buds, this phenotype was maintained through adulthood, pregnancy, and parturition. In addition, and in contrast to similar work with ErbB4-/- mammary buds, lobuloalveolar development of ErbB3-/- transplanted glands was normal. The ErbB3-/- mammary outgrowth defect was associated with a decrease in the size of the terminal end buds, and with increases in branch density, in the number of terminal end buds, and in the number of luminal spaces. Proliferation rates were not affected by the lack of ErbB3, but there was an increase in apoptosis in ErbB3-/- terminal end buds. CONCLUSIONS: Endogenous ErbB3 regulates morphogenesis of mammary epithelium.