Combined Wnt/ß-catenin, Met, and CXCL12/CXCR4 signals characterize basal breast cancer and predict disease outcome.

Prognosis for patients with estrogen-receptor (ER)-negative basal breast cancer is poor, and chemotherapy is currently the best therapeutic option. We have generated a compound-mutant mouse model combining the activation of ß-catenin and HGF (Wnt-Met signaling), which produced rapidly growing basal mammary gland tumors. We identified the chemokine system CXCL12/CXCR4 as a crucial driver of Wnt-Met tumors, given that compound-mutant mice also deficient in the CXCR4 gene were tumor resistant. Wnt-Met activation rapidly expanded a population of cancer-propagating cells, in which the two signaling systems control different functions, self-renewal and differentiation. Molecular therapy targeting Wnt, Met, and CXCR4 in mice significantly delayed tumor development. The expression of a Wnt-Met 322 gene signature was found to be predictive of poor survival of human patients with ER-negative breast cancers. Thus, targeting CXCR4 and its upstream activators, Wnt and Met, might provide an efficient strategy for breast cancer treatment.

Wnt/ß-catenin signalling induces MLL to create epigenetic changes in salivary gland tumours.

We show that activation of Wnt/ß-catenin and attenuation of Bmp signals, by combined gain- and loss-of-function mutations of ß-catenin and Bmpr1a, respectively, results in rapidly growing, aggressive squamous cell carcinomas (SCC) in the salivary glands of mice. Tumours contain transplantable and hyperproliferative tumour propagating cells, which can be enriched by fluorescence activated cell sorting (FACS). Single mutations stimulate stem cells, but tumours are not formed. We show that ß-catenin, CBP and Mll promote self-renewal and H3K4 tri-methylation in tumour propagating cells. Blocking ß-catenin-CBP interaction with the small molecule ICG-001 and small-interfering RNAs against ß-catenin, CBP or Mll abrogate hyperproliferation and H3K4 tri-methylation, and induce differentiation of cultured tumour propagating cells into acini-like structures. ICG-001 decreases H3K4me3 at promoters of stem cell-associated genes in vitro and reduces tumour growth in vivo. Remarkably, high Wnt/ß-catenin and low Bmp signalling also characterize human salivary gland SCC and head and neck SCC in general. Our work defines mechanisms by which ß-catenin signals remodel chromatin and control induction and maintenance of tumour propagating cells. Further, it supports new strategies for the therapy of solid tumours.

Circular RNAs are a large class of animal RNAs with regulatory potency.

Circular RNAs (circRNAs) in animals are an enigmatic class of RNA with unknown function. To explore circRNAs systematically, we sequenced and computationally analysed human, mouse and nematode RNA. We detected thousands of well-expressed, stable circRNAs, often showing tissue/developmental-stage-specific expression. Sequence analysis indicated important regulatory functions for circRNAs. We found that a human circRNA, antisense to the cerebellar degeneration-related protein 1 transcript (CDR1as), is densely bound by microRNA (miRNA) effector complexes and harbours 63 conserved binding sites for the ancient miRNA miR-7. Further analyses indicated that CDR1as functions to bind miR-7 in neuronal tissues. Human CDR1as expression in zebrafish impaired midbrain development, similar to knocking down miR-7, suggesting that CDR1as is a miRNA antagonist with a miRNA-binding capacity ten times higher than any other known transcript. Together, our data provide evidence that circRNAs form a large class of post-transcriptional regulators. Numerous circRNAs form by head-to-tail splicing of exons, suggesting previously unrecognized regulatory potential of coding sequences.

Genes involved in the metastatic cascade of medullary thyroid tumours.

The process of how a benign tumour turns invasive and capable to survive in distant organs remains poorly understood, despite the evidence that metastasis formation is the primary cause of cancer patient mortality. This ignorance is partly due to the lack of appropriate animal models from which to investigate this complex process. The retinoblastoma (Rb) tumour suppressor pathway (pRb/E2F) is mutated in almost all human tumours, and a number of laboratories have now established pRb- or E2F-deficient mouse models. Consistent with the role of mutation in retinoblastoma in cancer biology, Rb heterozygous mice are prone to develop tumours. Among the ensuing tumours, the medullary thyroid carcinomas (MTCs) have a lessened tendency to form secondary cancers and metastases. Intriguingly, if an E2f3 mutation is introduced in this genetic background, more aggressive MTCs develop, which metastasize more frequently. Gene chip microarrays, however, provide an unbiased approach for examining the genome-wide expression levels and enable identification of a large set of metastasis-enriched gene sets. The identified genes may simply represent putative markers of the disease stage. Alternatively, genes may be identified that causally determine a link to the onset of metastasis. We describe the use of gene chip microarrays for identification of putative markers enriched in metastatic mouse MTCs. The chapter details how the most promising candidates are verified using additional methods, such as quantitative real-time PCR. In this case, co-transfection of the E2F-transcription factor using a heterologous reporter gene system is suggestive of E2Fs directly regulating putative metastasis markers.

Gene expression of pluripotency determinants is conserved between mammalian and planarian stem cells.

Freshwater planaria possess extreme regeneration capabilities mediated by abundant, pluripotent stem cells (neoblasts) in adult animals. Although planaria emerged as an attractive in vivo model system for stem cell biology, gene expression in neoblasts has not been profiled comprehensively and it is unknown how molecular mechanisms for pluripotency in neoblasts relate to those in mammalian embryonic stem cells (ESCs). We purified neoblasts and quantified mRNA and protein expression by sequencing and shotgun proteomics. We identified ∼4000 genes specifically expressed in neoblasts, including all ∼30 known neoblast markers. Genes important for pluripotency in ESCs, including regulators as well as targets of OCT4, were well conserved and upregulated in neoblasts. We found conserved expression of epigenetic regulators and demonstrated their requirement for planarian regeneration by knockdown experiments. Post-transcriptional regulatory genes characteristic for germ cells were also enriched in neoblasts, suggesting the existence of a common ancestral state of germ cells and ESCs. We conclude that molecular determinants of pluripotency are conserved throughout evolution and that planaria are an informative model system for human stem cell biology.

The SNF2-like helicase HELLS mediates E2F3-dependent transcription and cellular transformation.

The activating E2F-transcription factors are best known for their dependence on the Retinoblastoma protein and their role in cellular proliferation. E2F3 is uniquely amplified in specific human tumours where its expression is inversely correlated with the survival of patients. Here, E2F3B interaction partners were identified by mass spectrometric analysis. We show that the SNF2-like helicase HELLS interacts with E2F3A in vivo and cooperates with its oncogenic functions. Depletion of HELLS severely perturbs the induction of E2F-target genes, hinders cell-cycle re-entry and growth. Using chromatin immmunoprecipitation coupled to sequencing, we identified genome-wide targets of HELLS and E2F3A/B. HELLS binds promoters of active genes, including the trithorax-related MLL1, and co-regulates E2F3-dependent genes. Strikingly, just as E2F3, HELLS is overexpressed in human tumours including prostate cancer, indicating that either factor may contribute to the malignant progression of tumours. Our work reveals that HELLS is important for E2F3 in tumour cell proliferation.

Wnt signaling in stem and cancer stem cells.

Canonical Wnt signaling supports the formation and maintenance of stem and cancer stem cells. Recent studies have elucidated epigenetic mechanisms that control pluripotency and stemness, and allow a first assessment how embryonic and tissue stem cells are generated and maintained, and how Wnt signaling might be involved. The core of this review highlights the roles of Wnt signaling in stem and cancer stem cells of tissues such as skin, intestine and mammary gland. Lastly, we refer to the characterization of novel and powerful inhibitors of canonical Wnt signaling and describe attempts to bring these compounds into preclinical and clinical studies.

Ectopic expression of E2F1 stimulates beta-cell proliferation and function.

OBJECTIVE: Generating functional beta-cells by inducing their proliferation may provide new perspectives for cell therapy in diabetes. Transcription factor E2F1 controls G(1)- to S-phase transition during the cycling of many cell types and is required for pancreatic beta-cell growth and function. However, the consequences of overexpression of E2F1 in beta-cells are unknown. RESEARCH DESIGN AND METHODS: The effects of E2F1 overexpression on beta-cell proliferation and function were analyzed in isolated rat beta-cells and in transgenic mice. RESULTS: Adenovirus AdE2F1-mediated overexpression of E2F1 increased the proliferation of isolated primary rat beta-cells 20-fold but also enhanced beta-cell death. Coinfection with adenovirus AdAkt expressing a constitutively active form of Akt (protein kinase B) suppressed beta-cell death to control levels. At 48 h after infection, the total beta-cell number and insulin content were, respectively, 46 and 79% higher in AdE2F1+AdAkt-infected cultures compared with untreated. Conditional overexpression of E2F1 in mice resulted in a twofold increase of beta-cell proliferation and a 70% increase of pancreatic insulin content, but did not increase beta-cell mass. Glucose-challenged insulin release was increased, and the mice showed protection against toxin-induced diabetes. CONCLUSIONS: Overexpression of E2F1, either in vitro or in vivo, can stimulate beta-cell proliferation activity. In vivo E2F1 expression significantly increases the insulin content and function of adult beta-cells, making it a strategic target for therapeutic manipulation of beta-cell function.

E2F3 loss has opposing effects on different pRB-deficient tumors, resulting in suppression of pituitary tumors but metastasis of medullary thyroid carcinomas.

The E2F transcription factors are key downstream targets of the retinoblastoma protein (pRB) tumor suppressor. We have previously shown that E2F3 plays a critical role in mediating the mitogen-induced activation of E2F-responsive genes and contributes to both the inappropriate proliferation and the p53-dependent apoptosis that arise in pRB-deficient embryos. Here we show that E2F3 also has a significant effect on the phenotype of tumor-prone Rb(+/-) mice. The absence of E2F3 results in a significant expansion in the life spans of these animals that correlates with a dramatic alteration in the tumor spectrum. E2F3 loss suppresses the development of the pituitary tumors that normally account for the death of Rb(+/-) mice. However, it also promotes the development of medullary thyroid carcinomas yielding metastases at a high frequency. This increased aggressiveness does not seem to result from any change in p53 levels or activity in these tumors. We show that, instead, E2F3 loss leads to an increase in the rate of tumor initiation. Finally, analysis of Rb(+/-); E2f3(+/-) mice shows that this tumor-suppressive function of E2F3 is dose dependent.

E2F4 loss suppresses tumorigenesis in Rb mutant mice.

The E2F transcription factors mediate the activation or repression of key cell cycle regulatory genes under the control of the retinoblastoma protein (pRB) tumor suppressor and its relatives, p107 and p130. Here we investigate how E2F4, the major "repressive" E2F, contributes to pRB's tumor-suppressive properties. Remarkably, E2F4 loss suppresses the development of both pituitary and thyroid tumors in Rb(+/-) mice. Importantly, E2F4 loss also suppresses the inappropriate gene expression and proliferation of pRB-deficient cells. Biochemical analyses suggest that this tumor suppression occurs via a novel mechanism: E2F4 loss allows p107 and p130 to regulate the pRB-specific, activator E2Fs. We also detect these novel E2F complexes in pRB-deficient cells, suggesting that they play a significant role in the regulation of tumorigenesis in vivo.

Mutant mouse models reveal the relative roles of E2F1 and E2F3 in vivo.

The E2F1, -2, and -3 transcription factors are key downstream targets of the retinoblastoma protein (pRB) tumor suppressor that drive expression of proliferation-associated genes. Here we use mutant mouse strains to investigate E2F3's role in vivo. We show that E2F3 is essential for embryonic viability in the pure 129/Sv background but the presence of C57BL/6 alleles yields some adult survivors. Although growth retarded, surviving E2f3(-/-) animals are initially healthy. However, they die prematurely, exhibiting no obvious tumor phenotype but with the typical signs of congestive heart failure. The defects are completely distinct from those arising in E2f1 mutant mice (S. J. Field et al., Cell 85:549-561; 1996; L. Yamasaki et al., Cell 85:537-548, 1996), supporting the prevailing view that these E2Fs must have some unique biological functions in vivo. To test this model, we examined the phenotypes of E2f1 E2f3 compound mutant mice. Almost all of the developmental and age-related defects arising in the individual E2f1 or E2f3 mice were exacerbated by the mutation of the other E2f. Thus, E2F1 and E2F3 appear to play critical, overlapping roles in the development and maintenance of a variety of tissues. Importantly, this study did identify one major difference in the properties of E2F1 and E2F3: either alone or in combination with E2F1 loss, E2f3 mutation did not increase the incidence of tumor formation. These data strongly suggest that tumor suppression is a specific property of E2F1 and not E2F3.