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Colorectal cancer (CRC) is a leading cause of cancer-related deaths globally, with the majority of cases initiated by inactivation of the APC tumor suppressor. This results in the constitutive transcriptional activation of the canonical WNT signal transduction pathway effector β-Catenin, along with induction of WNT feedback inhibitors, including the extracellular palmitoleoyl-protein carboxylesterase NOTUM. Here, we show that NOTUM retains cell-autonomous tumor suppressive activity in APC-null adenomatous lesions despite constitutive β-Catenin activation. Strikingly, we find that NOTUM becomes an obligate oncogene upon subsequent P53 inactivation during the adenoma-adenocarcinoma transition, and that these phenotypes are WNT-independent, resulting from differential activity of NOTUM upon its enzymatic targets Glypican 1 and 4 in early vs. late-stage disease, respectively. Ultimately, preclinical mouse models of CRC and human tumoroid cultures demonstrate that pharmacological inhibition of NOTUM is highly effective in arresting primary adenocarcinoma growth and inhibiting metastatic colonization of distal organs. The finding that a single agent targeting an extracellular enzyme is effective in treating highly aggressive tumors make NOTUM a novel therapeutic vulnerability in advanced colorectal adenocarcinomas.

Development of chemoresistance in breast cancer patients greatly increases mortality. Thus, understanding mechanisms underlying breast cancer resistance to chemotherapy is of paramount importance to overcome this clinical challenge. Although activated Notch receptors have been associated with chemoresistance in cancer, the specific Notch ligands and their molecular mechanisms leading to chemoresistance in breast cancer remain elusive. Using conditional knockout and reporter mouse models, we demonstrate that tumor cells expressing the Notch ligand Dll1 is important for tumor growth and metastasis and bear similarities to tumor-initiating cancer cells (TICs) in breast cancer. RNA-seq and ATAC-seq using reporter models and patient data demonstrated that NF-κB activation is downstream of Dll1 and is associated with a chemoresistant phenotype. Finally, pharmacological blocking of Dll1 or NF-κB pathway completely sensitizes Dll1+ tumors to chemotherapy, highlighting therapeutic avenues for chemotherapy resistant breast cancer patients in the near future., Although activated Notch receptors have been associated with chemoresistance in cancer, the role of specific Notch ligands remain elusive. Here, the authors show that in breast cells the Notch ligand DLL1 is expressed in cells with a cancer stem cell phenotype and promote doxorubicin resistance in part through NF-kB, as well as metastasis.

Triple-negative breast cancer (TNBC) is characterized by a high degree of immune infiltrate in the tumour microenvironment, which may influence the fate of TNBC cells. We reveal that loss of the tumour suppressive transcription factor Elf5 in TNBC cells activates intrinsic interferon-γ (IFN-γ) signalling, promoting tumour progression and metastasis. Mechanistically, we find that loss of the Elf5-regulated ubiquitin ligase FBXW7 ensures stabilization of its putative protein substrate IFN-γ receptor 1 (IFNGR1) at the protein level in TNBC. Elf5(low) tumours show enhanced IFN-γ signalling accompanied by an increase of immunosuppressive neutrophils within the tumour microenvironment and increased programmed death ligand 1 expression. Inactivation of either programmed death ligand 1 or IFNGR1 elicited a robust anti-tumour and/or anti-metastatic effect. A positive correlation between ELF5 and FBXW7 expression and a negative correlation between ELF5, FBXW7 and IFNGR1 expression in the tumours of patients with TNBC strongly suggest that this signalling axis could be exploited for patient stratification and immunotherapeutic treatment strategies for Elf5(low) patients with TNBC.

The Dnmt3a DNA methyltransferase is essential for mammalian development and is responsible for the generation of genomic methylation patterns, which lead to transcriptional silencing. Here, we show that Dnmt3a associates with RP58, a DNA-binding transcriptional repressor protein found at transcriptionally silent heterochromatin. Dnmt3a acts as a co-repressor for RP58 in a manner that does not require its de novo methyltransferase activity. Like other characterized co-repressors, Dnmt3a associates with the histone deacetylase HDAC1 using its ATRX-homology domain. This domain of Dnmt3a represents an independent transcriptional repressor domain whose silencing functions require HDAC activity. These results identify Dnmt3a as a co-repressor protein carrying deacetylase activity and show that Dnmt3a can be targeted to specific regulatory foci via its association with DNA-binding transcription factors.

Summary H3K27M mutations resulting in epigenetic dysfunction are frequently observed in diffuse intrinsic pontine glioma (DIPGs), an incurable pediatric cancer. We conduct a CRISPR screen revealing that knockout of KDM1A encoding lysine-specific demethylase 1 (LSD1) sensitizes DIPG cells to histone deacetylase (HDAC) inhibitors. Consistently, Corin, a bifunctional inhibitor of HDACs and LSD1, potently inhibits DIPG growth in vitro and in xenografts. Mechanistically, Corin increases H3K27me3 levels suppressed by H3K27M histones, and simultaneously increases HDAC-targeted H3K27ac and LSD1-targeted H3K4me1 at differentiation-associated genes. Corin treatment induces cell death, cell-cycle arrest, and a cellular differentiation phenotype and drives transcriptional changes correlating with increased survival time in DIPG patients. These data suggest a strategy for treating DIPG by simultaneously inhibiting LSD1 and HDACs.

Triple-negative breast cancer (TNBC) is particularly aggressive, with enhanced incidence of tumor relapse, resistance to chemotherapy, and metastases. As the mechanistic basis for this aggressive phenotype is unclear, treatment options are limited. Here, we showed an increased population of myeloid-derived immunosuppressor cells (MDSCs) in TNBC patients compared with non-TNBC patients. We found that high levels of the transcription factor ΔNp63 correlate with an increased number of MDSCs in basal TNBC patients, and that ΔNp63 promotes tumor growth, progression, and metastasis in human and mouse TNBC cells. Furthermore, we showed that MDSC recruitment to the primary tumor and metastatic sites occurs via direct ΔNp63-dependent activation of the chemokines CXCL2 and CCL22. CXCR2/CCR4 inhibitors reduced MDSC recruitment, angiogenesis, and metastasis, highlighting a novel treatment option for this subset of TNBC patients. Finally, we found that MDSCs secrete prometastatic factors such as MMP9 and chitinase 3–like 1 to promote TNBC cancer stem cell function, thereby identifying a nonimmunologic role for MDSCs in promoting TNBC progression. These findings identify a unique crosstalk between ΔNp63+ TNBC cells and MDSCs that promotes tumor progression and metastasis, which could be exploited in future combined immunotherapy/chemotherapy strategies for TNBC patients.

SummaryIn mammalian cells, DNA methylation on the fifth position of cytosine (5mC) plays an important role as an epigenetic mark. However, DNA methylation was considered to be absent in C. elegans because of the lack of detectable 5mC, as well as homologs of the cytosine DNA methyltransferases. Here, using multiple approaches, we demonstrate the presence of adenine N6-methylation (6mA) in C. elegans DNA. We further demonstrate that this modification increases trans-generationally in a paradigm of epigenetic inheritance. Importantly, we identify a DNA demethylase, NMAD-1, and a potential DNA methyltransferase, DAMT-1, which regulate 6mA levels and crosstalk between methylations of histone H3K4 and adenines and control the epigenetic inheritance of phenotypes associated with the loss of the H3K4me2 demethylase spr-5. Together, these data identify a DNA modification in C. elegans and raise the exciting possibility that 6mA may be a carrier of heritable epigenetic information in eukaryotes.

Emerging evidence suggests that cancer is populated and maintained by tumour-initiating cells (TICs) with stem-like properties similar to those of adult tissue stem cells. Despite recent advances, the molecular regulatory mechanisms that may be shared between normal and malignant stem cells remain poorly understood. Here we show that the ΔNp63 isoform of the Trp63 transcription factor promotes normal mammary stem cell (MaSC) activity by increasing the expression of the Wnt receptor Fzd7, thereby enhancing Wnt signalling. Importantly, Fzd7-dependent enhancement of Wnt signalling by ΔNp63 also governs tumour-initiating activity of the basal subtype of breast cancer. These findings establish ΔNp63 as a key regulator of stem cells in both normal and malignant mammary tissues and provide direct evidence that breast cancer TICs and normal MaSCs share common regulatory mechanisms.

SummaryMetastatic dissemination is often initiated by the reactivation of an embryonic development program referred to as epithelial-mesenchymal transition (EMT). The transcription factor SNAIL promotes EMT and elicits associated pathological characteristics such as invasion, metastasis, and stemness. To better understand the posttranslational regulation of SNAIL, we performed a luciferase-based, genome-wide E3 ligase siRNA library screen and identified SCF-FBXO11 as an important E3 that targets SNAIL for ubiquitylation and degradation. Furthermore, we discovered that SNAIL degradation by FBXO11 is dependent on Ser-11 phosphorylation of SNAIL by protein kinase D1 (PKD1). FBXO11 blocks SNAIL-induced EMT, tumor initiation, and metastasis in multiple breast cancer models. These findings establish the PKD1-FBXO11-SNAIL axis as a mechanism of posttranslational regulation of EMT and cancer metastasis.

Epithelial–mesenchymal transition (EMT) is a complex process which occurs during organogenesis and in cancer metastasis. Despite recent progress, the molecular pathways connecting the physiological and pathological functions of EMT need to be better defined. Here we show that the transcription factor Elf5, a key regulator of mammary gland alveologenesis, controls EMT in both mammary gland development and metastasis. We uncovered this role of Elf5 through analyses of Elf5 conditional knockout animals, various in vitro and in vivo models of EMT and metastasis, an MMTV-neu transgenic model of mammary tumor progression, and clinical breast cancer samples. Furthermore, we demonstrate that Elf5 suppresses EMT by directly repressing the transcription of Snail2/Slug, a master regulator of mammary stem cells and a known inducer of EMT. These findings establish Elf5 not only as a key cell lineage regulator during normal mammary gland development, but also as a suppressor of EMT and metastasis in breast cancer.

El pdf del artículo es el manuscrito de autor.-- et al., Although the role of miR-200s in regulating E-cadherin expression and epithelial-to-mesenchymal transition is well established, their influence on metastatic colonization remains controversial. Here we have used clinical and experimental models of breast cancer metastasis to discover a pro-metastatic role of miR-200s that goes beyond their regulation of E-cadherin and epithelial phenotype. Overexpression of miR-200s is associated with increased risk of metastasis in breast cancer and promotes metastatic colonization in mouse models, phenotypes that cannot be recapitulated by E-cadherin expression alone. Genomic and proteomic analyses revealed global shifts in gene expression upon miR-200 overexpression toward that of highly metastatic cells. miR-200s promote metastatic colonization partly through direct targeting of Sec23a, which mediates secretion of metastasis-suppressive proteins, including Igfbp4 and Tinagl1, as validated by functional and clinical correlation studies. Overall, these findings suggest a pleiotropic role of miR-200s in promoting metastatic colonization by influencing E-cadherin-dependent epithelial traits and Sec23a-mediated tumor cell secretome. © 2011 Nature America, Inc. All rights reserved., This research was supported by grants from the US National Institutes of Health (1R01-CA141062) and the Brewster Foundation to Y.K., and from Cancer Research UK, Oxford NHS Biomedical Research Centre and Friends of Kennington Cancer Fund to A.L.H. M.K. and Y.H. are recipients of Department of Defense pre-doctoral fellowships.

Bone is the one of the most common sites of distant metastasis of solid tumors. Secreted proteins are known to influence pathological interactions between metastatic cancer cells and the bone stroma. To comprehensively profile secreted proteins associated with bone metastasis, we used quantitative and non-quantitative mass spectrometry to globally analyze the secretomes of nine cell lines of varying bone metastatic ability from multiple species and cancer types. By comparing the secretomes of parental cells and their bone metastatic derivatives, we identified the secreted proteins that were uniquely associated with bone metastasis in these cell lines. We then incorporated bioinformatic analyses of large clinical metastasis datasets to obtain a list of candidate novel bone metastasis proteins of several functional classes that were strongly associated with both clinical and experimental bone metastasis. Functional validation of selected proteins indicated that in vivo bone metastasis can be promoted by high expression of (1) the salivary cystatins CST1, CST2, and CST4; (2) the plasminogen activators PLAT and PLAU; or (3) the collagen functionality proteins PLOD2 and COL6A1. Overall, our study has uncovered several new secreted mediators of bone metastasis and therefore demonstrated that secretome analysis is a powerful method for identification of novel biomarkers and candidate therapeutic targets.

Metastasis is the deadliest and most poorly understood feature of malignant diseases. Recent work has shown that Metadherin (MTDH) is overexpressed in over 40% of breast cancer patients and promotes metastasis and chemoresistance in experimental models of breast cancer progression. Here we applied mass spectrometry-based screen to identify staphylococcal nuclease domain-containing 1 (SND1) as a candidate MTDH-interacting protein. After confirming the interaction between SND1 and MTDH, we tested the role of SND1 in breast cancer and found that it strongly promotes lung metastasis. SND1 was further shown to promote resistance to apoptosis and to regulate the expression of genes associated with metastasis and chemoresistance. Analyses of breast cancer clinical microarray data indicated that high expression of SND1 in primary tumors is strongly associated with reduced metastasis-free survival in multiple large scale data sets. Thus, we have uncovered SND1 as a novel MTDH-interacting protein and shown that it is a functionally and clinically significant mediator of metastasis.

Background Histone post-translational modifications (PTMs) constitute a branch of epigenetic mechanisms that can control the expression of eukaryotic genes in a heritable manner. Recent studies have identified several PTM-binding proteins containing diverse specialized domains whose recognition of specific PTM sites leads to gene activation or repression. Here, we present a high-throughput proteogenomic platform designed to characterize the nucleosomal make-up of chromatin enriched with a set of histone PTM binding proteins known as histone PTM readers. We support our findings with gene expression data correlating to PTM distribution. Results We isolated human mononucleosomes bound by the bromodomain-containing proteins Brd2, Brd3 and Brd4, and by the chromodomain-containing heterochromatin proteins HP1β and HP1α. Histone PTMs were quantified by mass spectrometry (ChIP-qMS), and their associated DNAs were mapped using deep sequencing. Our results reveal that Brd- and HP1-bound nucleosomes are enriched in histone PTMs consistent with actively transcribed euchromatin and silent heterochromatin, respectively. Data collected using RNA-Seq show that Brd-bound sites correlate with highly expressed genes. In particular, Brd3 and Brd4 are most enriched on nucleosomes located within HOX gene clusters, whose expression is reduced upon Brd4 depletion by short hairpin RNA. Conclusions Proteogenomic mapping of histone PTM readers, alongside the characterization of their local chromatin environments and transcriptional information, should prove useful for determining how histone PTMs are bound by these readers and how they contribute to distinct transcriptional states.

The advent of genomic profiling technology has brought about revolutionary changes in our understanding of breast cancer metastasis. Gene expression analyses of primary tumors have been used to predict metastatic propensity with high accuracy. Animal models of metastasis additionally offer a platform to experimentally dissect components of the metastasis genetic program. Recent integrated studies have synergized clinical bioinformatic analyses with advanced experimental methodology and begun to uncover the identities and dynamics of signaling programs driving breast cancer metastasis. Such functional genomics studies hold great promise for understanding the genetic basis of metastasis and improving therapeutics for advanced diseases.

Secreted tumor proteins are critical for establishing the requisite tumor-stromal interactions in the process of metastasis and are superior therapeutic targets. Here we present a proteome-scale analysis to identify novel secreted proteins associated with bone metastasis. To characterize the complete set of secreted proteins involved in tissue-specific metastasis, we analyzed the secreted protein fractions from four sublines of the MDA-MB-231 breast cancer cell line which differ in bone metastasis ability. Using LC-MS/MS mass spectrometry technology, we identified 1153 total unique proteins from these fractions and filtered out those proteins predicted to be non-secreted using SignalP 3.0. This analysis yielded 206 unique secretory-predicted proteins, thus providing the first view of the MDA-MB-231 secretome. To determine potential therapeutic targets, we looked at which secreted proteins were found only in the highly bone metastatic cell lines. This 32-protein bone metastasis “secretome signature” contained several proteins already known to play a role in promoting bone metastasis (eg. MMP1, CTGF, and TGF-) as well as many novel proteins with no prior link to metastasis or cancer. GO categories significantly enriched in this signature included “extracellular region,” “calcium ion binding,” and “ IGF-binding,” further confirming its biological relevance. Oncomine database analysis indicated that 6 of 32 secretome signature proteins are strongly upregulated in multiple human cancers as compared to matched normal tissue. To narrow the secretome signature biologically, we repeated the LC-MS/MS analysis on two additional sets of matched parental vs. highly bone metastatic derivative cell lines. From the three total secretome analyses, 5 secreted proteins were found to be unique to bone metastatic derivatives in two of three cell types, and one protein was found in every bone metastatic derivative (and in none of the weakly bone metastatic lines). Oncomine analysis indicated that three of these five proteins were also strongly upregulated in multiple human cancers as compared to normal tissue. This analysis of the metastasis secretome and its associated bone metastasis-specific changes has identified multiple novel secreted proteins strongly implicated in clinical malignant progression. Following experimental and clinical validation, any could be considered for therapeutic targeting via monoclonal antibody. Citation Information: Clin Cancer Res 2010;16(7 Suppl):B7

SummaryThe Metadherin gene (MTDH) is prevalently amplified in breast cancer and associated with poor prognosis; however, its functional contribution to tumorigenesis is poorly understood. Using mouse models representing different subtypes of breast cancer, we demonstrated that MTDH plays a critical role in mammary tumorigenesis by regulating oncogene-induced expansion and activities of tumor-initiating cells (TICs), whereas it is largely dispensable for normal development. Mechanistically, MTDH supports the survival of mammary epithelial cells under oncogenic/stress conditions by interacting with and stabilizing Staphylococcal nuclease domain-containing 1 (SND1). Silencing MTDH or SND1 individually or disrupting their interaction compromises tumorigenenic potential of TICs in vivo. This functional significance of MTDH-SND1 interaction is further supported by clinical analysis of human breast cancer samples.

SummaryTargeted therapy for metastatic diseases relies on the identification of functionally important metastasis genes from a large number of random genetic alterations. Here we use a computational algorithm to map minimal recurrent genomic alterations associated with poor-prognosis breast cancer. 8q22 genomic gain was identified by this approach and validated in an extensive collection of breast tumor samples. Regional gain of 8q22 elevates expression of the metastasis gene metadherin (MTDH), which is overexpressed in more than 40% of breast cancers and is associated with poor clinical outcomes. Functional characterization of MTDH revealed its dual role in promoting metastatic seeding and enhancing chemoresistance. These findings establish MTDH as an important therapeutic target for simultaneously enhancing chemotherapy efficacy and reducing metastasis risk.