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miRNAs can both promote and repress tumorigenesis, and directly control epithelial–mesenchymal transition (EMT). miR-9 (which is upregulated in breast cancer cells) is activated by MYC and MYCN, and regulates EMT and metastasis through direct control of E-cadherin. In contrast, tumour angiogenesis is controlled indirectly through effects on vascular endothelial growth factor (VEGF) expression. MicroRNAs (miRNAs) are increasingly implicated in regulating the malignant progression of cancer. Here we show that miR-9, which is upregulated in breast cancer cells, directly targets CDH1 , the E-cadherin-encoding messenger RNA, leading to increased cell motility and invasiveness. miR-9-mediated E-cadherin downregulation results in the activation of β -catenin signalling, which contributes to upregulated expression of the gene encoding vascular endothelial growth factor (VEGF); this leads, in turn, to increased tumour angiogenesis. Overexpression of miR-9 in otherwise non-metastatic breast tumour cells enables these cells to form pulmonary micrometastases in mice. Conversely, inhibiting miR-9 by using a 'miRNA sponge' in highly malignant cells inhibits metastasis formation. Expression of miR-9 is activated by MYC and MYCN, both of which directly bind to the mir-9-3 locus. Significantly, in human cancers, miR-9 levels correlate with MYCN amplification, tumour grade and metastatic status. These findings uncover a regulatory and signalling pathway involving a metastasis-promoting miRNA that is predicted to directly target expression of the key metastasis-suppressing protein E-cadherin.

Much interest is currently focused on the emerging role of tumor-stroma interactions essential for supporting tumor progression. Carcinoma-associated fibroblasts (CAFs), frequently present in the stroma of human breast carcinomas, include a large number of myofibroblasts, a hallmark of activated fibroblasts. These fibroblasts have an ability to substantially promote tumorigenesis. However, the precise cellular origins of CAFs and the molecular mechanisms by which these cells evolve into tumor-promoting myofibroblasts remain unclear. Using a coimplantation breast tumor xenograft model, we show that resident human mammary fibroblasts progressively convert into CAF myofibroblasts during the course of tumor progression. These cells increasingly acquire two autocrine signaling loops, mediated by TGF-β and SDF-1 cytokines, which both act in autostimulatory and cross-communicating fashions. These autocrine-signaling loops initiate and maintain the differentiation of fibroblasts into myofibroblasts and the concurrent tumor-promoting phenotype. Collectively, these findings indicate that the establishment of the self-sustaining TGF-β and SDF-1 autocrine signaling gives rise to tumor-promoting CAF myofibroblasts during tumor progression. This autocrine-signaling mechanism may prove to be an attractive therapeutic target to block the evolution of tumor-promoting CAFs.

Inhibition of DOT1L, the H3K79 histone methyltransferase, increases cell reprogramming and substituted for KLF4 and c-Myc, showing that chromatin-modifying enzymes act not only as facilitators but also as barriers to reprogramming. Chromatin in iPS-cell formation A study of the role of chromatin-modifying enzymes in the reprogramming of human fibroblasts to induced pluripotent stem (iPS) cells suggests that such enzymes can act as facilitators — but also as barriers — to epigenetic remodelling. By knocking down 22 selected genes involved in DNA and histone methylation pathways, George Daley and colleagues identified both positive and negative regulators of iPS-cell generation. In particular, inhibition of DOT1L, the H3K79 histone methyltransferase, increased reprogramming and substituted for KLF4 and c-Myc, two of the factors needed in the reprogramming cocktail. The effect of DOT1L inhibition seems to be associated with increase in the reprogramming factors NANOG and LIN28. This work demonstrates that specific chromatin modifiers can be modulated to generate iPS cells more efficiently and with fewer exogenously introduced transcription factors. Generation of induced pluripotent stem cells (iPSCs) by somatic cell reprogramming involves global epigenetic remodelling 1 . Whereas several proteins are known to regulate chromatin marks associated with the distinct epigenetic states of cells before and after reprogramming 2 , 3 , the role of specific chromatin-modifying enzymes in reprogramming remains to be determined. To address how chromatin-modifying proteins influence reprogramming, we used short hairpin RNAs (shRNAs) to target genes in DNA and histone methylation pathways, and identified positive and negative modulators of iPSC generation. Whereas inhibition of the core components of the polycomb repressive complex 1 and 2, including the histone 3 lysine 27 methyltransferase EZH2, reduced reprogramming efficiency, suppression of SUV39H1, YY1 and DOT1L enhanced reprogramming. Specifically, inhibition of the H3K79 histone methyltransferase DOT1L by shRNA or a small molecule accelerated reprogramming, significantly increased the yield of iPSC colonies, and substituted for KLF4 and c-Myc (also known as MYC). Inhibition of DOT1L early in the reprogramming process is associated with a marked increase in two alternative factors, NANOG and LIN28, which play essential functional roles in the enhancement of reprogramming. Genome-wide analysis of H3K79me2 distribution revealed that fibroblast-specific genes associated with the epithelial to mesenchymal transition lose H3K79me2 in the initial phases of reprogramming. DOT1L inhibition facilitates the loss of this mark from genes that are fated to be repressed in the pluripotent state. These findings implicate specific chromatin-modifying enzymes as barriers to or facilitators of reprogramming, and demonstrate how modulation of chromatin-modifying enzymes can be exploited to more efficiently generate iPSCs with fewer exogenous transcription factors.

The epithelial-to-mesenchymal transition (EMT) produces cancer cells that are invasive, migratory, and exhibit stem cell characteristics, hallmarks of cells that have the potential to generate metastases. Inducers of the EMT include several transcription factors (TFs), such as Goosecoid, Snail, and Twist, as well as the secreted TGF-β1. Each of these factors is capable, on its own, of inducing an EMT in the human mammary epithelial (HMLE) cell line. However, the interactions between these regulators are poorly understood. Overexpression of each of the above EMT inducers up-regulates a subset of other EMT-inducing TFs, with Twist, Zeb1, Zeb2, TGF-β1, and FOXC2 being commonly induced. Up-regulation of Slug and FOXC2 by either Snail or Twist does not depend on TGF-β1 signaling. Gene expression signatures (GESs) derived by overexpressing EMT-inducing TFs reveal that the Twist GES and Snail GES are the most similar, although the Goosecoid GES is the least similar to the others. An EMT core signature was derived from the changes in gene expression shared by up-regulation of Gsc, Snail, Twist, and TGF-β1 and by down-regulation of E-cadherin, loss of which can also trigger an EMT in certain cell types. The EMT core signature associates closely with the claudin-low and metaplastic breast cancer subtypes and correlates negatively with pathological complete response. Additionally, the expression level of FOXC1, another EMT inducer, correlates strongly with poor survival of breast cancer patients.

Triple-negative breast cancer (TNBC) is a particularly aggressive and frequently recurring form of breast cancer, where chemotherapy is the primary treatment approach. Unfortunately, the development of resistance to chemotherapy poses a considerable challenge, restricting the already limited therapeutic alternatives for recurrent cases. Here, we generated two Taxol-resistant TNBC cell lines with a dose-escalation method to mimic chemotherapy resistance in vitro. These cells exhibited reduced growth rates, altered morphology and evasion of apoptosis. Transcriptome analysis uncovered elevated ABCB1 expression and multidrug-resistant phenotype in these resistant cells. To comprehensively investigate the key epigenetic regulators of Taxol resistance, we conducted chromatin-focused genetic and chemical screens and pinpointed Bromodomain and PHD Finger Containing 1 (BRPF1) as a novel regulator of Taxol resistance. Knockout of BRPF1, the reader protein in the MOZ–MORF histone acetyltransferase complex, but not the other complex members, sensitized resistant cells to Taxol. In addition, BRPF1 inhibitors, PFI-4 and OF-1, in combination with Taxol significantly reduced cell viability. Transcriptome analysis upon BRPF1 loss or inhibition revealed a negative impact on ribosome biogenesis-related gene sets, resulting in a global decrease in protein translation in Taxol-resistant cells. CUT&RUN-qPCR analysis demonstrated that BRPF1 directly binds to the ABCB1 promoter, enhancing its expression toward inducing a multidrug-resistant phenotype. Conversely, knockout or inhibition of BRPF1 leads to decreased ABCB1 expression. Our findings uncover a comprehensive molecular framework, highlighting the pivotal role of epigenetic reader protein BRPF1 in Taxol resistance and providing potential avenues for therapeutic intervention in TNBC. Targeting BRPF1 offers hope against Taxol resistance Triple-negative breast cancer (TNBC) is a challenging type of breast cancer that lacks targeted treatment options. Researchers aimed to understand why some TNBC cells become resistant to the chemotherapy drug Taxol and focused on the role of a protein called BRPF1 in this resistance. They created Taxol-resistant TNBC cells in the laboratory and found that these cells had high levels of a protein called ABCB1, which helps to pump drugs out of cells. They used advanced techniques to identify key proteins involved in this resistance and discovered that BRPF1, part of a complex that modifies DNA structure, plays a crucial role. By using genetic tools and chemical inhibitors to reduce BRPF1 activity, they found that the resistant cells became more sensitive to Taxol. This suggests that targeting BRPF1 could be a new way to treat resistant TNBC. This summary was initially drafted using artificial intelligence, then revised and fact-checked by the author.

The midbody is a singular organelle formed between daughter cells during cytokinesis and required for their final separation. Midbodies persist in cells long after division as midbody derivatives (MB d s), but their fate is unclear. Here we show that MB d s are inherited asymmetrically by the daughter cell with the older centrosome. They selectively accumulate in stem cells, induced pluripotent stem cells and potential cancer ‘stem cells’ in vivo and in vitro . MB d loss accompanies stem-cell differentiation, and involves autophagic degradation mediated by binding of the autophagic receptor NBR1 to the midbody protein CEP55. Differentiating cells and normal dividing cells do not accumulate MB d s and possess high autophagic activity. Stem cells and cancer cells accumulate MB d s by evading autophagosome encapsulation and exhibit low autophagic activity. MB d enrichment enhances reprogramming to induced pluripotent stem cells and increases the in vitro tumorigenicity of cancer cells. These results indicate unexpected roles for MB d s in stem cells and cancer ‘stem cells’. Doxsey and colleagues report that midbodies accumulate in stem cells, including induced pluripotent stem cells and potential cancer-initiating cells. Loss of midbodies accompanies stem-cell differentiation and is mediated through binding of the autophagy receptor NBR1 to the midbody protein CEP55. Downregulation of NBR1 is associated with enrichment of midbodies, enhanced reprogramming and increased tumorigenicity in cancer cells.

Reactive oxygen species (ROS) appear to play a role in limiting both cellular and organismic lifespan. However, because of their pleiotropic effects, it has been difficult to ascribe a specific role to ROS in initiating the process of cellular senescence. We have studied the effects of oxidative DNA damage on cell proliferation, believing that such damage is of central importance to triggering senescence. To do so, we devised a strategy to decouple levels of 8-oxoguanine, a major oxidative DNA lesion, from ROS levels. Suppression of MTH1 expression, which hydrolyzes 8-oxo-dGTP, was accompanied by increased total cellular 8-oxoguanine levels and caused early-passage primary and telomerase-immortalized human skin fibroblasts to rapidly undergo senescence, doing so without altering cellular ROS levels. This senescent phenotype recapitulated several salient features of replicative senescence, notably the presence of senescence-associated beta-galactosidase (SA beta-gal) activity, apparently irreparable genomic DNA breaks, and elevation of $p21^{Cip1} $, p53, and $p16^{INK4A} $ tumor suppressor protein levels. Culturing cells under low oxygen tension (3%) largely prevented the shMTH1-dependent senescent phenotype. These results indicate that the nucleotide pool is a critical target of intracellular ROS and that oxidized nucleotides, unless continuously eliminated, can rapidly induce cell senescence through signaling pathways very similar to those activated during replicative senescence.

Dysregulation of the epigenome due to alterations in chromatin modifier proteins commonly contribute to malignant transformation. To interrogate the roles of epigenetic modifiers in cancer cells, we generated an epigenome-wide CRISPR-Cas9 knockout library (EPIKOL) that targets a wide-range of epigenetic modifiers and their cofactors. We conducted eight screens in two different cancer types and showed that EPIKOL performs with high efficiency in terms of sgRNA distribution and depletion of essential genes. We discovered novel epigenetic modifiers that regulate triple-negative breast cancer (TNBC) and prostate cancer cell fitness. We confirmed the growth-regulatory functions of individual candidates, including SS18L2 and members of the NSL complex (KANSL2, KANSL3, KAT8) in TNBC cells. Overall, we show that EPIKOL, a focused sgRNA library targeting ~800 genes, can reveal epigenetic modifiers that are essential for cancer cell fitness under in vitro and in vivo conditions and enable the identification of novel anti-cancer targets. Due to its comprehensive epigenome-wide targets and relatively high number of sgRNAs per gene, EPIKOL will facilitate studies examining functional roles of epigenetic modifiers in a wide range of contexts, such as screens in primary cells, patient-derived xenografts as well as in vivo models.

Background Glioblastoma is the most common and aggressive primary brain tumor with extremely poor prognosis, highlighting an urgent need for developing novel treatment options. Identifying epigenetic vulnerabilities of cancer cells can provide excellent therapeutic intervention points for various types of cancers. Method In this study, we investigated epigenetic regulators of glioblastoma cell survival through CRISPR/Cas9 based genetic ablation screens using a customized sgRNA library EpiDoKOL, which targets critical functional domains of chromatin modifiers. Results Screens conducted in multiple cell lines revealed ASH2L , a histone lysine methyltransferase complex subunit, as a major regulator of glioblastoma cell viability. ASH2L depletion led to cell cycle arrest and apoptosis. RNA sequencing and greenCUT&RUN together identified a set of cell cycle regulatory genes, such as TRA2B, BARD1, KIF20B, ARID4A and SMARCC1 that were downregulated upon ASH2L depletion. Mass spectrometry analysis revealed the interaction partners of ASH2L in glioblastoma cell lines as SET1/MLL family members including SETD1A, SETD1B, MLL1 and MLL2. We further showed that glioblastoma cells had a differential dependency on expression of SET1/MLL family members for survival. The growth of ASH2L -depleted glioblastoma cells was markedly slower than controls in orthotopic in vivo models. TCGA analysis showed high ASH2L expression in glioblastoma compared to low grade gliomas and immunohistochemical analysis revealed significant ASH2L expression in glioblastoma tissues, attesting to its clinical relevance. Therefore, high throughput, robust and affordable screens with focused libraries, such as EpiDoKOL, holds great promise to enable rapid discovery of novel epigenetic regulators of cancer cell survival, such as ASH2L . Conclusion Together, we suggest that targeting ASH2L could serve as a new therapeutic opportunity for glioblastoma. BDzH7XPE9o9JSJXVEAFWyz Video Abstract

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) can selectively kill tumor cells. TRAIL resistance in cancers is associated with aberrant expression of the key components of the apoptotic program. However, how these components are regulated at the epigenetic level is not understood. In this study, we investigated novel epigenetic mechanisms regulating TRAIL response in glioblastoma multiforme (GBM) cells by a short-hairpin RNA loss-of-function screen. We interrogated 48 genes in DNA and histone modification pathways and identified KDM2B, an H3K36-specific demethylase, as a novel regulator of TRAIL response. Accordingly, silencing of KDM2B significantly enhanced TRAIL sensitivity, the activation of caspase-8, -3 and -7 and PARP cleavage. KDM2B knockdown also accelerated the apoptosis, as revealed by live-cell imaging experiments. To decipher the downstream molecular pathways regulated by KDM2B, levels of apoptosis-related genes were examined by RNA-sequencing upon KDM2B loss, which revealed derepression of proapoptotic genes Harakiri ( HRK ), caspase-7 and death receptor 4 ( DR4 ) and repression of antiapoptotic genes. The apoptosis phenotype was partly dependent on HRK upregulation, as HRK knockdown significantly abrogated the sensitization. KDM2B-silenced tumors exhibited slower growth in vivo. Taken together, our findings suggest a novel mechanism, where the key apoptosis components are under epigenetic control of KDM2B in GBM cells.