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Summary: Transcription factors and chromatin-remodeling complexes are key determinants of embryonic stem cell (ESC) identity. Here, we demonstrate that BRD4, a member of the bromodomain and extraterminal domain (BET) family of epigenetic readers, regulates the self-renewal ability and pluripotency of ESCs. BRD4 inhibition resulted in induction of epithelial-to-mesenchymal transition (EMT) markers and commitment to the neuroectodermal lineage while reducing the ESC multidifferentiation capacity in teratoma assays. BRD4 maintains transcription of core stem cell genes such as OCT4 and PRDM14 by occupying their super-enhancers (SEs), large clusters of regulatory elements, and recruiting to them Mediator and CDK9, the catalytic subunit of the positive transcription elongation factor b (P-TEFb), to allow Pol-II-dependent productive elongation. Our study describes a mechanism of regulation of ESC identity that could be applied to improve the efficiency of ESC differentiation. : Di Micco et al. now dissect the mechanisms by which BRD4 regulates embryonic stem cell (ESC) identity by binding to super-enhancers of core pluripotency genes and recruiting active transcription complexes. BRD4 inhibition results in defective elongation of super-enhancer-associated gene transcripts, loss of ESC self-renewal/pluripotency, and commitment to the neuroectodermal lineage.

The Ras-extracellular signal-regulated kinase (ERK) cascade is an important signaling module in cells. One regulator of the Ras-ERK cascade is phosphatidic acid (PA) generated by phospholipase D (PLD) and diacylglycerol kinase (DGK). Using a newly developed PA biosensor, PASS (phosphatidic acid biosensor with superior sensitivity), we found that PA was generated sequentially by PLD and DGK in epidermal growth factor (EGF)-stimulated HCC1806 breast cancer cells. Inhibition of PLD2, one of the two PLD members, was sufficient to eliminate most of the PA production, whereas inhibition of DGK decreased PA production only at the later stages of EGF stimulation, suggesting that PLD2 precedes DGK activation. The temporal production of PA by PLD2 is important for the nuclear activation of ERK. While inhibition of both PLD and DGK had no effect on the overall ERK activity, inhibition of PLD2 but not PLD1 or DGK blocked the nuclear ERK activity in several cancer cell lines. The decrease of active ERK in the nucleus inhibited the activation of Elk1, c-fos, and Fra1, the ERK nuclear targets, leading to decreased proliferation of HCC1806 cells. Together, these findings reveal that PA production by PLD2 determines the output of ERK in cancer cell growth factor signaling.

Signal transduction is regulated by the lateral segregation of proteins into nanodomains on the plasma membrane. However, the molecular mechanisms that regulate the lateral segregation of cell surface receptors, such as receptor tyrosine kinases, upon ligand binding are unresolved. Here we used high-resolution spatial mapping to investigate the plasma membrane nanoscale organization of the epidermal growth factor (EGF) receptor (EGFR). Our data demonstrate that in serum-starved cells, the EGFR exists in preformed, cholesterol-dependent, actin-independent nanoclusters. Following stimulation with EGF, the number and size of EGFR nanoclusters increase in a time-dependent manner. Our data show that the formation of EGFR nanoclusters requires receptor tyrosine kinase activity. Critically, we show for the first time that production of phosphatidic acid by phospholipase D2 (PLD2) is essential for ligand-induced EGFR nanocluster formation. In accordance with its crucial role in regulating EGFR nanocluster formation, we demonstrate that modulating PLD2 activity tunes the degree of EGFR nanocluster formation and mitogen-activated protein kinase signal output. Together, these data show that EGFR activation drives the formation of signaling domains by regulating the production of critical second-messenger lipids and modifying the local membrane lipid environment.

Squamous cell carcinomas (SCCs) are heterogeneous tumors that are sustained by tumor propagating cancer cells (TPCs). SCCs frequently resist chemotherapy through mechanisms that are still unknown. Here, we combine H2BGFP based pulse-chasing with cell surface markers to distinguish quiescent from proliferative TPCs within SCCs. We find that quiescent TPCs resist DNA damage and exhibit increased tumorigenic potential in response to chemotherapy, whereas proliferative TPCs undergo apoptosis. Quiescence is regulated by TGFβ/SMAD signaling, which directly regulates cell cycle gene transcription to control a reversible G1 cell cycle arrest, independent of p21CIP function. Indeed, genetic or pharmacological TGFβ inhibition increases the susceptibility of TPCs to chemotherapy as it prevents entry into a quiescent state. These findings provide direct evidence that TPCs can reversibly enter a quiescent, chemoresistant state which underscores the need for combinatorial approaches to improve treatment of chemotherapy-resistant SCCs.

Several recycling regulatory proteins are overexpressed in breast cancer patients and promote the progression of breast cancer. Increased receptor recycling leads to elevated receptors on the plasma membrane, thereby augments signaling cascades important in cancer progression and drives tumor aggressiveness. Inhibition of the altered recycling pathway in breast cancer cells would thus dampen oncogenic signaling and increase the efficacy of therapy. To screen druggable proteins regulating receptor recycling, we proposed to monitor EGFR trafficking using a newly developed AP-tag system. We found that while the AP-tag labeling worked for receptor endocytosis studies as described in the original literatures, it could not efficiently label the recycled EGFR. Therefore, this technique is not suitable for studying EGFR recycling as originally proposed.

Breast cancer is the most common form of cancer among women. Compared with other serum polypeptides, autoantibodies have many appealing features as biomarkers including sensitivity, stability, and easy detection. Anti-lipid autoantibodies are routinely used in the diagnosis of autoimmune diseases, but their potential for cancer diagnosis has not been explored. Dysregulation of cellular signaling in cancer cells would be expected to lead to irregular metabolism of many lipids, which could be sensed by the immune system and cause the production of autoantibodies. Discovery of anti-lipid antibodies could be used as biomarkers for early breast cancer diagnosis. We describe here a more sensitive and accurate method for lipid microarray detection using dual fluorescent labeling, and used it to examine global anti-lipid profiles in the MMTV-Neu transgenic breast cancer model. We conclude that, at the current technology, lipid microarray is not a preferred method for anti-lipid antibody detection in breast cancer animal models. Our result will help the future application of lipid microarrays in identifying anti-lipid autoantibodies in breast cancer and other human diseases.

Although the principles that balance stem cell self-renewal and differentiation in normal tissue homeostasis are beginning to emerge, it is still unclear whether cancer cells with tumour initiating potential are similarly governed, or whether they have acquired distinct mechanisms to sustain self-renewal and long-term tumour growth. Here we show that the transcription factor Sox2, which is not expressed in normal skin epithelium and is dispensable for epidermal homeostasis, marks tumour initiating cells (TICs) in cutaneous squamous cell carcinomas (SCCs). We demonstrate that Sox2 is required for SCC growth in mouse and human, where it enhances Nrp1/Vegf signalling to promote the expansion of TICs along the tumour–stroma interface. Our findings suggest that distinct transcriptional programmes govern self-renewal and long-term growth of TICs and normal skin epithelial stem and progenitor cells. These programmes present promising diagnostic markers and targets for cancer-specific therapies. The mechanisms that sustain the self-renewal and expansion of cancer cells with tumour initiating potential are not completely understood. Here the authors show that the transcription factor, Sox2, marks tumour initiating cells in cutaneous squamous cell carcinomas and is required for their expansion.