Structural basis for distinct roles of SMAD2 and SMAD3 in FOXH1 pioneer-directed TGF-ß signaling.

TGF-ß receptors phosphorylate SMAD2 and SMAD3 transcription factors, which then form heterotrimeric complexes with SMAD4 and cooperate with context-specific transcription factors to activate target genes. Here we provide biochemical and structural evidence showing that binding of SMAD2 to DNA depends on the conformation of the E3 insert, a structural element unique to SMAD2 and previously thought to render SMAD2 unable to bind DNA. Based on this finding, we further delineate TGF-ß signal transduction by defining distinct roles for SMAD2 and SMAD3 with the forkhead pioneer factor FOXH1 as a partner in the regulation of differentiation genes in mouse mesendoderm precursors. FOXH1 is prebound to target sites in these loci and recruits SMAD3 independently of TGF-ß signals, whereas SMAD2 remains predominantly cytoplasmic in the basal state and set to bind SMAD4 and join SMAD3:FOXH1 at target promoters in response to Nodal TGF-ß signals. The results support a model in which signal-independent binding of SMAD3 and FOXH1 prime mesendoderm differentiation gene promoters for activation, and signal-driven SMAD2:SMAD4 binds to promoters that are preloaded with SMAD3:FOXH1 to activate transcription.

Publisher Correction: TGF-ß orchestrates fibrogenic and developmental EMTs via the RAS effector RREB1.

An Amendment to this paper has been published and can be accessed via a link at the top of the paper.

TGF-ß orchestrates fibrogenic and developmental EMTs via the RAS effector RREB1.

Epithelial-to-mesenchymal transitions (EMTs) are phenotypic plasticity processes that confer migratory and invasive properties to epithelial cells during development, wound-healing, fibrosis and cancer1-4. EMTs are driven by SNAIL, ZEB and TWIST transcription factors5,6 together with microRNAs that balance this regulatory network7,8. Transforming growth factor ß (TGF-ß) is a potent inducer of developmental and fibrogenic EMTs4,9,10. Aberrant TGF-ß signalling and EMT are implicated in the pathogenesis of renal fibrosis, alcoholic liver disease, non-alcoholic steatohepatitis, pulmonary fibrosis and cancer4,11. TGF-ß depends on RAS and mitogen-activated protein kinase (MAPK) pathway inputs for the induction of EMTs12-19. Here we show how these signals coordinately trigger EMTs and integrate them with broader pathophysiological processes. We identify RAS-responsive element binding protein 1 (RREB1), a RAS transcriptional effector20,21, as a key partner of TGF-ß-activated SMAD transcription factors in EMT. MAPK-activated RREB1 recruits TGF-ß-activated SMAD factors to SNAIL. Context-dependent chromatin accessibility dictates the ability of RREB1 and SMAD to activate additional genes that determine the nature of the resulting EMT. In carcinoma cells, TGF-ß-SMAD and RREB1 directly drive expression of SNAIL and fibrogenic factors stimulating myofibroblasts, promoting intratumoral fibrosis and supporting tumour growth. In mouse epiblast progenitors, Nodal-SMAD and RREB1 combine to induce expression of SNAIL and mesendoderm-differentiation genes that drive gastrulation. Thus, RREB1 provides a molecular link between RAS and TGF-ß pathways for coordinated induction of developmental and fibrogenic EMTs. These insights increase our understanding of the regulation of epithelial plasticity and its pathophysiological consequences in development, fibrosis and cancer.

Therapy-induced tumour secretomes promote resistance and tumour progression.

Drug resistance invariably limits the clinical efficacy of targeted therapy with kinase inhibitors against cancer. Here we show that targeted therapy with BRAF, ALK or EGFR kinase inhibitors induces a complex network of secreted signals in drug-stressed human and mouse melanoma and human lung adenocarcinoma cells. This therapy-induced secretome stimulates the outgrowth, dissemination and metastasis of drug-resistant cancer cell clones and supports the survival of drug-sensitive cancer cells, contributing to incomplete tumour regression. The tumour-promoting secretome of melanoma cells treated with the kinase inhibitor vemurafenib is driven by downregulation of the transcription factor FRA1. In situ transcriptome analysis of drug-resistant melanoma cells responding to the regressing tumour microenvironment revealed hyperactivation of several signalling pathways, most prominently the AKT pathway. Dual inhibition of RAF and the PI(3)K/AKT/mTOR intracellular signalling pathways blunted the outgrowth of the drug-resistant cell population in BRAF mutant human melanoma, suggesting this combination therapy as a strategy against tumour relapse. Thus, therapeutic inhibition of oncogenic drivers induces vast secretome changes in drug-sensitive cancer cells, paradoxically establishing a tumour microenvironment that supports the expansion of drug-resistant clones, but is susceptible to combination therapy.

MicroRNA-335 inhibits tumor reinitiation and is silenced through genetic and epigenetic mechanisms in human breast cancer.

Post-transcriptional regulators have emerged as robust effectors of metastasis and display deregulated expression through unknown mechanisms. Here, we reveal that the human microRNA-335 locus undergoes genetic deletion and epigenetic promoter hypermethylation in every metastatic derivative obtained from independent patients' malignant cell populations. Genetic deletion of miR-335 is a common event in human breast cancer, is enriched for in breast cancer metastases, and also correlates with ovarian cancer recurrence. We furthermore identify miR-335 as a robust inhibitor of tumor reinitiation. We thus implicate the miR-335 locus on 7q32.2 as the first selective metastasis suppressor and tumor initiation suppressor locus in human breast cancer.

Genes that mediate breast cancer metastasis to the brain.

The molecular basis for breast cancer metastasis to the brain is largely unknown. Brain relapse typically occurs years after the removal of a breast tumour, suggesting that disseminated cancer cells must acquire specialized functions to take over this organ. Here we show that breast cancer metastasis to the brain involves mediators of extravasation through non-fenestrated capillaries, complemented by specific enhancers of blood-brain barrier crossing and brain colonization. We isolated cells that preferentially infiltrate the brain from patients with advanced disease. Gene expression analysis of these cells and of clinical samples, coupled with functional analysis, identified the cyclooxygenase COX2 (also known as PTGS2), the epidermal growth factor receptor (EGFR) ligand HBEGF, and the alpha2,6-sialyltransferase ST6GALNAC5 as mediators of cancer cell passage through the blood-brain barrier. EGFR ligands and COX2 were previously linked to breast cancer infiltration of the lungs, but not the bones or liver, suggesting a sharing of these mediators in cerebral and pulmonary metastases. In contrast, ST6GALNAC5 specifically mediates brain metastasis. Normally restricted to the brain, the expression of ST6GALNAC5 in breast cancer cells enhances their adhesion to brain endothelial cells and their passage through the blood-brain barrier. This co-option of a brain sialyltransferase highlights the role of cell-surface glycosylation in organ-specific metastatic interactions.

A multigenic program mediating breast cancer metastasis to bone.

We investigated the molecular basis for osteolytic bone metastasis by selecting human breast cancer cell line subpopulations with elevated metastatic activity and functionally validating genes that are overexpressed in these cells. These genes act cooperatively to cause osteolytic metastasis, and most of them encode secreted and cell surface proteins. Two of these genes, interleukin-11 and CTGF, encode osteolytic and angiogenic factors whose expression is further increased by the prometastatic cytokine TGF beta. Overexpression of this bone metastasis gene set is superimposed on a poor-prognosis gene expression signature already present in the parental breast cancer population, suggesting that metastasis requires a set of functions beyond those underlying the emergence of the primary tumor.

Genes that mediate breast cancer metastasis to lung.

By means of in vivo selection, transcriptomic analysis, functional verification and clinical validation, here we identify a set of genes that marks and mediates breast cancer metastasis to the lungs. Some of these genes serve dual functions, providing growth advantages both in the primary tumour and in the lung microenvironment. Others contribute to aggressive growth selectively in the lung. Many encode extracellular proteins and are of previously unknown relevance to cancer metastasis.

The p53 Family Coordinates Wnt and Nodal Inputs in Mesendodermal Differentiation of Embryonic Stem Cells.

In this study, we outline a regulatory network that involves the p53 tumor suppressor family and the Wnt pathway acting together with the TGF-ß pathway in mesendodermal differentiation of mouse and human embryonic stem cells. Knockout of all three members, p53, p63, and p73, shows that the p53 family is essential for mesendoderm specification during exit from pluripotency in embryos and in culture. Wnt3 and its receptor Fzd1 are direct p53 family target genes in this context, and induction of Wnt signaling by p53 is critical for activation of mesendodermal differentiation genes. Globally, Wnt3-activated Tcf3 and nodal-activated Smad2/3 transcription factors depend on each other for co-occupancy of target enhancers associated with key differentiation loci. Our results therefore highlight an unanticipated role for p53 family proteins in a regulatory network that integrates essential Wnt-Tcf and nodal-Smad inputs in a selective and interdependent way to drive mesendodermal differentiation of pluripotent cells.

Matrix metalloproteinase-7 expression in colorectal cancer liver metastases: evidence for involvement of MMP-7 activation in human cancer metastases.

PURPOSE: Matrix metalloproteinase-7 (MMP-7) is a member of the MMP family, which is overexpressed by some tumor cells and is thought to enhance the tumor metastatic potential. The aim of this study is to examine the MMP-7 expression in the human colorectal cancer (CRC) liver metastases and normal liver tissue using multiple techniques and to determine its association with liver metastases formation. EXPERIMENTAL DESIGN: MMP-7 mRNA, protein, and enzymatic levels were determined by reverse transcription-PCR, Western blot analysis, and casein zymography in the specimens of human CRC liver metastases and paired normal liver tissue from 44 patients. The cellular localization of MMP-7 was analyzed by immunohistochemistry. RESULTS: Our data reveal that all of the investigated liver metastases samples overexpressed MMP-7 mRNA and protein compared with the normal liver tissue. By zymogram, higher levels of the latent form of MMP-7 were found in 88.6% (39 of 44) liver metastases samples, whereas normal liver tissue exhibited only trace amounts. The activated form of MMP-7 was only found in those in which the pro-MMP-7 was present (n = 39); in contrast, it was not detected in the normal liver tissues. Immunohistochemically, MMP-7 is localized to the cytoplasm of tumor cells, and the strong signal is concentrated in the tumor front areas. CONCLUSIONS: Our observations emphasize the important role of MMP-7 production and activation in human CRC liver metastases formation.

Loss of the multifunctional RNA-binding protein RBM47 as a source of selectable metastatic traits in breast cancer.

The mechanisms through which cancer cells lock in altered transcriptional programs in support of metastasis remain largely unknown. Through integrative analysis of clinical breast cancer gene expression datasets, cell line models of breast cancer progression, and mutation data from cancer genome resequencing studies, we identified RNA binding motif protein 47 (RBM47) as a suppressor of breast cancer progression and metastasis. RBM47 inhibited breast cancer re-initiation and growth in experimental models. Transcriptome-wide HITS-CLIP analysis revealed widespread RBM47 binding to mRNAs, most prominently in introns and 3'UTRs. RBM47 altered splicing and abundance of a subset of its target mRNAs. Some of the mRNAs stabilized by RBM47, as exemplified by dickkopf WNT signaling pathway inhibitor 1, inhibit tumor progression downstream of RBM47. Our work identifies RBM47 as an RNA-binding protein that can suppress breast cancer progression and demonstrates how the inactivation of a broadly targeted RNA chaperone enables selection of a pro-metastatic state.

Epigenetic expansion of VHL-HIF signal output drives multiorgan metastasis in renal cancer.

Inactivation of the von Hippel-Lindau tumor suppressor gene, VHL, is an archetypical tumor-initiating event in clear cell renal carcinoma (ccRCC) that leads to the activation of hypoxia-inducible transcription factors (HIFs). However, VHL mutation status in ccRCC is not correlated with clinical outcome. Here we show that during ccRCC progression, cancer cells exploit diverse epigenetic alterations to empower a branch of the VHL-HIF pathway for metastasis, and the strength of this activation is associated with poor clinical outcome. By analyzing metastatic subpopulations of VHL-deficient ccRCC cells, we discovered an epigenetically altered VHL-HIF response that is specific to metastatic ccRCC. Focusing on the two most prominent pro-metastatic VHL-HIF target genes, we show that loss of Polycomb repressive complex 2 (PRC2)-dependent histone H3 Lys27 trimethylation (H3K27me3) activates HIF-driven chemokine (C-X-C motif) receptor 4 (CXCR4) expression in support of chemotactic cell invasion, whereas loss of DNA methylation enables HIF-driven cytohesin 1 interacting protein (CYTIP) expression to protect cancer cells from death cytokine signals. Thus, metastasis in ccRCC is based on an epigenetically expanded output of the tumor-initiating pathway.

Mad upregulation and Id2 repression accompany transforming growth factor (TGF)-beta-mediated epithelial cell growth suppression.

The growth inhibitory cytokine TGF-beta enforces homeostasis of epithelia by activating processes such as cell cycle arrest and apoptosis. Id2 expression is often highest in proliferating epithelial cells and declines during differentiation. Recently, Id2 expression has been found to depend on Myc-Max transcriptional complexes. We observed that TGF-beta signaling inhibits Id2 expression in human and mouse epithelial cell lines from different tissue origins. Furthermore, the observed Id2 down-regulation by TGF-beta in mouse mammary epithelial cells occurs without a concurrent drop in c-Myc levels. However, sustained Id2 repression in these cells and in human keratinocytes coincides with induction of the Myc antagonistic repressors Mad2 and Mad4, decreased formation of Myc-Max heterodimers and the replacement of Myc-Max complexes with Mad-Max complexes on the Id2 promoter. These results argue that induction of Mad expression and Id2 down-regulation are important events during the TGF-beta cytostatic program in epithelial cells.

Transforming growth factor beta signaling impairs Neu-induced mammary tumorigenesis while promoting pulmonary metastasis.

The influence of transforming growth factor beta (TGF-beta) signaling on Neu-induced mammary tumorigenesis and metastasis was examined with transgenic mouse models. We generated mice expressing an activated TGF-beta type I receptor or dominant negative TGF-beta type II receptor under control of the mouse mammary tumor virus promoter. When crossed with mice expressing activated forms of the Neu receptor tyrosine kinase that selectively couple to the Grb2 or Shc signaling pathways the activated type I receptor increased the latency of mammary tumor formation but also enhanced the frequency of extravascular lung metastasis. Conversely, expression of the dominant negative type II receptor decreased the latency of Neu-induced mammary tumor formation while significantly reducing the incidence of extravascular lung metastases. These observations argue that TGF-beta can promote the formation of lung metastases while impairing Neu-induced tumor growth and suggest that extravasation of breast cancer cells from pulmonary vessels is a point of action of TGF-beta in the metastatic process.