Complex regulatory networks control epithelial-to-mesenchymal transition (EMT) but the underlying epigenetic control is poorly understood. Lysine-specific demethylase 1 (LSD1) is a key histone demethylase that alters the epigenetic landscape. Here we explored the role of LSD1 in global epigenetic regulation of EMT, cancer stem cells (CSCs), the tumour microenvironment, and therapeutic resistance in breast cancer. LSD1 induced pan-genomic gene expression in networks implicated in EMT and selectively elicits gene expression programs in CSCs whilst repressing non-CSC programs. LSD1 phosphorylation at serine-111 (LSD1-s111p) by chromatin anchored protein kinase C-theta (PKC-θ), is critical for its demethylase and EMT promoting activity and LSD1-s111p is enriched in chemoresistant cells in vivo. LSD1 couples to PKC-θ on the mesenchymal gene epigenetic template promotes LSD1-mediated gene induction. In vivo, chemotherapy reduced tumour volume, and when combined with an LSD1 inhibitor, abrogated the mesenchymal signature and promoted an innate, M1 macrophage-like tumouricidal immune response. Circulating tumour cells (CTCs) from metastatic breast cancer (MBC) patients were enriched with LSD1 and pharmacological blockade of LSD1 suppressed the mesenchymal and stem-like signature in these patient-derived CTCs. Overall, LSD1 inhibition may serve as a promising epigenetic adjuvant therapy to subvert its pleiotropic roles in breast cancer progression and treatment resistance.

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g Epithelial to mesenchymal transition (EMT) is activated during cancer invasion and metastasis, enriches for cancer stem cells (CSCs), and contributes to therapeutic resistance and disease recurrence. Signal transduction kinases play a pivotal role as chromatin-anchored proteins in eukaryotes. Here we report for the first time that protein kinase C-theta (PKC-) promotes EMT by acting as a critical chromatin-anchored switch for inducible genes via transforming growth factor ␤ (TGF-␤) and the key inflammatory regulatory protein NF-B. Chromatinized PKC-exists as an active transcription complex and is required to establish a permissive chromatin state at signature EMT genes. Genome-wide analysis identifies a unique cohort of inducible PKC--sensitive genes that are directly tethered to PKC-in the mesenchymal state. Collectively, we show that cross talk between signaling kinases and chromatin is critical for eliciting inducible transcriptional programs that drive mesenchymal differentiation and CSC formation, providing novel mechanisms to target using epigenetic therapy in breast cancer. Epithelial to mesenchymal transition (EMT) is a key step in cancer progression and the process of metastasis that creates a reservoir for cancer stem cells (CSCs) and is associated with highly aggressive traits (1-4). As well as driving metastasis, these CSCs, or "precursor" metastatic cells, play a pivotal role in therapeutic resistance and relapse in breast cancer patients (5, 6). Breast CSCs are a distinct subpopulation of mesenchymal cells that possess several important features, namely, expression of key surface markers (CD44 high and CD24 low ) (7), a distinct transcriptome (3), the ability to form spherical colonies in suspension cultures (termed mammospheres) (8), and enhanced resistance to chemotherapy (9, 10) and ionizing radiation (11)(12)(13)(14).Eukaryotes utilize the chromatin landscape as its epigenetic template within the nucleus of living cells in order to promote inducible gene transcription in response to environmental signals. Highly compacted chromatin structures are enriched in nucleosomes and are transcriptionally silent, and a net loss of nucleosomes from gene-specific regulatory regions increases chromatin accessibility and initiates context-specific transcriptional programs. In addition to ATP-dependent chromatin remodeling and exchange of histone variants with canonical histones, histone modifications are thought to alter gene expression, by changing chromatin structure and/or by providing a platform that promotes binding of transcriptional regulators (15-23).Novel classes of chromatin-associated enzymes that play critical roles in modulating chromatin structure within the human genome have recently been discovered. In particular, signaling kinases can act as chromatin regulators of inducible gene transcription in both higher and lower eukaryotes by two distinct mechanisms: relaying signals from the cytoplasm to the nucleus and direct association with chromatin-bound transcription complexes at activated tar...

Senescence, the molecular program that limits the finite proliferative potential of a cell, acts as an important barrier to protect the body from cancer. Techniques for measuring transcriptome changes and for modulating their expression suggest that it may be possible to dissect the transcriptional networks underlying complex cellular processes. HMF3A cells are conditionally immortalized human mammary fibroblasts that can be induced to undergo coordinated senescence. Here, we used these cells in conjunction with microarrays, RNA interference, and in silico promoter analysis to promote the dissection of the transcriptional networks responsible for regulating cellular senescence. We first identified changes in the transcriptome when HMF3A cells undergo senescence and then compared them with those observed upon replicative senescence in primary human mammary fibroblasts. In addition to DUSP1 and known p53 and E2F targets, a number of genes such as PHLDA1, NR4A3, and a novel splice variant of STAC were implicated in senescence. Their role in senescence was then analyzed by RNA silencing followed by microarray analysis. In silico promoter analysis of all differential genes predicted that nuclear factor-kappaB and C/EBP transcription factors are activated upon senescence, and we confirmed this by electrophoretic mobility shift assay. The results suggest a putative signaling network for cellular senescence.

Experimental autoimmune encephalomyelitis (EAE) is a T cell-mediated autoimmune disease involving effector Th subsets such as Th1 and Th17. In this study, we demonstrate that mice lacking the NF-κB transcription factor family member c-Rel (rel−/−), which are known to be resistant to EAE, show impaired Th17 development. Mixed bone marrow chimeras and EAE adoptive transfer experiments show that the deficiency of effector Th17 cells in rel−/− mice is T cell intrinsic. Consistent with this finding, c-Rel was activated in response to TCR signaling in the early stages of Th17 development and controlled the expression of Rorc, which encodes the Th17 transcription factor retinoic acid-related orphan receptor γt. CD28, but not IL-2, repression of Th17 development was dependent on c-Rel, implicating a dual role for c-Rel in modulating Th17 development. Adoptive transfer experiments also suggested that c-Rel control of regulatory T cell differentiation and homeostasis influences EAE development and severity by influencing the balance between Th17 and regulatory T cells. Collectively, our findings indicate that in addition to promoting Th1 differentiation, c-Rel regulates the development and severity of EAE via multiple mechanisms that impact on the generation of Th17 cells.