Asunción Fernández-Barral

EFNA3 long noncoding RNAs induced by hypoxia promote metastatic dissemination

The presence of hypoxic regions in solid tumors is an adverse prognostic factor for patient outcome. Here, we show that hypoxia induces the expression of Ephrin-A3 through a novel hypoxia-inducible factor (HIF)-mediated mechanism. In response to hypoxia, the coding EFNA3 mRNA levels remained relatively stable, but HIFs drove the expression of previously unknown long noncoding (lnc) RNAs from EFNA3 locus and these lncRNA caused Ephrin-A3 protein accumulation. Ephrins are cell surface proteins that regulate diverse biological processes by modulating cellular adhesion and repulsion. Mounting evidence implicates deregulated ephrin function in multiple aspects of tumor biology. We demonstrate that sustained expression of both Ephrin-A3 and novel EFNA3 lncRNAs increased the metastatic potential of human breast cancer cells, possibly by increasing the ability of tumor cells to extravasate from the blood vessels into surrounding tissue. In agreement, we found a strong correlation between high EFNA3 expression and shorter metastasis-free survival in breast cancer patients. Taken together, our results suggest that hypoxia could contribute to metastatic spread of breast cancer via HIF-mediated induction of EFNA3 lncRNAs and subsequent Ephrin-A3 protein accumulation.

Hypoxia negatively regulates antimetastatic PEDF in melanoma cells by a hypoxia inducible factor-independent, autophagy dependent mechanism

Pigment epithelium-derived factor (PEDF), a member of the serine protease inhibitor (SERPIN) superfamily, displays a potent antiangiogenic and antimetastatic activity in a broad range of tumor types. Melanocytes and low aggressive melanoma cells secrete high levels of PEDF, while its expression is lost in highly aggressive melanomas. PEDF efficiently abrogates a number of functional properties critical for the acquisition of metastatic ability by melanoma cells, such as neovascularization, proliferation, migration, invasiveness and extravasation. In this study, we identify hypoxia as a relevant negative regulator of PEDF in melanocytes and low aggressive melanoma cells. PEDF was regulated at the protein level. Importantly, although downregulation of PEDF was induced by inhibition of 2-oxoglutarate-dependent dioxygenases, it was independent of the hypoxia inducible factor (HIF), a key mediator of the adaptation to hypoxia. Decreased PEDF protein was not mediated by inhibition of translation through untranslated regions (UTRs) in melanoma cells. Degradation by metalloproteinases, implicated on PEDF degradation in retinal pigment epithelial cells, or by the proteasome, was also excluded as regulatory mechanism in melanoma cells. Instead, we found that degradation by autophagy was critical for PEDF downregulation under hypoxia in human melanoma cells. Our findings show that hypoxic conditions encountered during primary melanoma growth downregulate antiangiogenic and antimetastasic PEDF by a posttranslational mechanism involving degradation by autophagy and could therefore contribute to the acquisition of highly metastatic potential characteristic of aggressive melanoma cells.

The endocrine vitamin D system in the gut.

The active vitamin D metabolite 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3) has important regulatory actions in the gut through endocrine and probably also intracrine, autocrine and paracrine mechanisms. By activating the vitamin D receptor (VDR), which is expressed at a high level in the small intestine and colon, 1,25(OH)2D3 regulates numerous genes that control gut physiology and homeostasis. 1,25(OH)2D3 is a major responsible for epithelial barrier function and calcium and phosphate absorption, and the hosts defense against pathogens and the inflammatory response by several types of secretory and immune cells. Moreover, recent data suggest that 1,25(OH)2D3 has a regulatory effect on the gut microbiota and stromal fibroblasts. Many studies have linked vitamin D deficiency to inflammatory bowel diseases (ulcerative colitis and Crohns disease) and to an increased risk of colorectal cancer, and the possible use of VDR agonists to prevent or treat these diseases is receiving increasing interest.

Vitamin D receptor expression and associated gene signature in tumour stromal fibroblasts predict clinical outcome in colorectal cancer

Objective Colorectal cancer (CRC) is a major health concern. Vitamin D deficiency is associated with high CRC incidence and mortality, suggesting a protective effect of vitamin D against this disease. Given the strong influence of tumour stroma on cancer progression, we investigated the potential effects of the active vitamin D metabolite 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3) on CRC stroma. Design Expression of vitamin D receptor (VDR) and two 1,25(OH)2D3 target genes was analysed in 658 patients with CRC with prolonged clinical follow-up. 1,25(OH)2D3 effects on primary cultures of patient-derived colon normal fibroblasts (NFs) and cancer-associated fibroblasts (CAFs) were studied using collagen gel contraction and migration assays and global gene expression analyses. Publicly available data sets (n=877) were used to correlate the 1,25(OH)2D3-associated gene signature in CAFs with CRC outcome. Results High VDR expression in tumour stromal fibroblasts was associated with better overall survival (OS) and progression-free survival in CRC, independently of its expression in carcinoma cells. 1,25(OH)2D3 inhibited the protumoural activation of NFs and CAFs and imposed in CAFs a 1,25(OH)2D3-associated gene signature that correlated with longer OS and disease-free survival in CRC. Furthermore, expression of two genes from the signature, CD82 and S100A4, correlated with stromal VDR expression and clinical outcome in our cohort of patients with CRC. Conclusions 1,25(OH)2D3 has protective effects against CRC through the regulation of stromal fibroblasts. Accordingly, expression of VDR and 1,25(OH)2D3-associated gene signature in stromal fibroblasts predicts a favourable clinical outcome in CRC. Therefore, treatment of patients with CRC with VDR agonists could be explored even in the absence of VDR expression in carcinoma cells.

Regulatory and functional connection of microphthalmia-associated transcription factor and anti-metastatic pigment epithelium derived factor in melanoma.

Pigment epithelium-derived factor (PEDF), a member of the serine protease inhibitor superfamily, has potent anti-metastatic effects in cutaneous melanoma through its direct actions on endothelial and melanoma cells. Here we show that PEDF expression positively correlates with microphthalmia-associated transcription factor (MITF) in melanoma cell lines and human samples. High PEDF and MITF expression is characteristic of low aggressive melanomas classified according to molecular and pathological criteria, whereas both factors are decreased in senescent melanocytes and naevi. Importantly, MITF silencing down-regulates PEDF expression in melanoma cell lines and primary melanocytes, suggesting that the correlation in the expression reflects a causal relationship. In agreement, analysis of Chromatin immunoprecipitation coupled to high throughput sequencing (ChIP-seq) data sets revealed three MITF binding regions within the first intron of SERPINF1, and reporter assays demonstrated that the binding of MITF to these regions is sufficient to drive transcription. Finally, we demonstrate that exogenous PEDF expression efficiently halts in vitro migration and invasion, as well as in vivo dissemination of melanoma cells induced by MITF silencing. In summary, these results identify PEDF as a novel transcriptional target of MITF and support a relevant functional role for the MITF-PEDF axis in the biology of melanoma.

Microphthalmia-Associated Transcription Factor: A Central Regulator of Pigment Epithelium-Derived Factor Controlling Human Melanoma Progression.

To the Editor-in-Chief: We read with great interest the article by Dadras et al in The American Journal of Pathology describing microphthalmiaassociated transcription factor (MITF) as a regulator of pigment epithelium-derived factor (PEDF) in human melanoma. Although we are pleased to see that the authors have confirmed our recent results, we feel that the authors overlooked a careful review of the concurrent literature on the biological role of PEDF during melanoma progression and the identification of MITF as a transcriptional regulator of PEDF. First, Dadras et al state in their abstract that “PEDF expression and/or regulation during melanoma development have not been investigated previously.” This statement is inaccurate, as our group and another have previously addressed this point. Specifically, by performing a highthroughput analysis of the data from molecular profiling studies of human melanoma cell lines, we demonstrated that PEDF is highly expressed in melanocytes and low aggressive melanoma cells but is lost in highly aggressive melanomas. Furthermore, we studied paired cell lines isolated from human metastatic lesions displaying extreme phenotypes and demonstrated that PEDF expression is restricted to the less aggressive counterparts. In mouse models of human melanoma metastases, we established that PEDF levels critically impact melanoma progression by gain and loss of function experiments (PEDF overexpression or silencing, respectively). The mention of a subsequent study identifying the mechanisms by which PEDF exerts its antimetastatic actions is also missing from the article by Dadras et al. Importantly, this study showed that PEDF is able to efficiently block two modes of melanoma invasion by suppressing ameboid morphology and mesenchymal proteolysis. Second, Dadras et al show that PEDF is a direct transcriptional target of MITF in human melanocytes and melanoma cells but overstate the novelty of their discoveries. We have recently demonstrated that PEDF expression positively correlates with MITF during human melanoma progression. Of note, we showed that oncogene-induced senescence promotes down-regulation of PEDF and MITF in primary melanocytes, providing a likely explanation to the lack of PEDF and MITF expression in nevi described in our article and confirmed by Dadras et al. Furthermore, we identified three MITF regulatory sites in the first intron of SERPINF1 based on published ChIP-seq data that were further validated by an independent ChIP-seq experiment and reporter assays that revealed that at least two of the identified MITF-binding sites have functional activity. Importantly, our study also provided a functional connection between MITF and PEDF, which is critical for melanoma dissemination. Using an established zebrafish model of metastasis, we demonstrated that in vivo dissemination of human melanoma cells induced by MITF silencing is halted by PEDF sustained expression, providing solid evidence for the role of the MITFePEDF axis in the control of melanoma biology. In summary, we believe that the article published by Dadras et al lacks a complete description of the previous findings describing the biological role of PEDF and its regulation by MITF during human melanoma progression. We consider it important to highlight these references to the readers of the Journal.

Increased melanoma formation and dissemination in TyrNRas mice deficient in the tumor suppressor Ing1

Reference EPFL-ARTICLE-197894doi:10.1111/pcmr.12241View record in Web of Science Record created on 2014-03-26, modified on 2016-08-09

Urothelial organoids originate from Cd49f-High stem cells and display Notch-dependent differentiation capacity

The urothelium is a specialized stratified epithelium with unique structural and functional features. Understanding the mechanisms involved in urothelial stem cell biology and differentiation has been limited by the lack of methods for unlimited propagation. Here, we establish normal mouse urothelial organoid (NMU-o) cultures that can be maintained uninterruptedly for >1 year. Organoid growth is dependent on EGF and Wnt activators. High CD49f/ITGA6 expression features a subpopulation of organoid-forming urothelial stem cells expressing basal markers. On induction of differentiation, multilayered organoids show reduced layer number, acquire barrier function, and activate the urothelial program, including expression of uroplakins and tight junction components. Combined pharmacological modulation of PPARγ and EGFR was most potent driving cell differentiation. Transcriptome analysis of organoids widely validates the system, highlights the transcriptional networks involved, and reveals NOTCH signaling as a novel pathway required for normal urothelial organoid differentiation.

Pigment epithelium-derived factor - An angiostatic factor with a broader function in melanoma

Metastatic spread is achieved through changes in the tissue microenvironment driven by tumor cells that allow the formation of various dissemination routes using a variety of mechanisms; such as angiogenesis and vasculogenesis (hematogeneous routes), lymphangiogenesis (lymphatic routes), and in some particular cases like melanoma, vasculogenic mimicry (vasculogenic channels lined by melanoma cells) (Carmeliet, 2005; Hendrix et al., 2003; Kopp et al., 2006; Tammela and Alitalo, 2010). Building of dissemination routes has to be coordinated with the acquisition of new capabilities by tumor cells that enable them to locally invade, intravasate into dissemination channels, survive in the circulation, extravasate, and ultimately adapt to a foreign territory. All this complex cascade of events is orchestrated by multiple cell types and diverse families of factors and signaling circuits controlling intracellular as well as intercellular key communication events (Nguyen et al., 2009b). Interestingly, a particular subset of extracellular factors have the dual capacity to simultaneously impinge on the formation of the dissemination routes and to modulate many of the properties that the tumor cells themselves have to acquire in order to fulfill all steps required to successfully colonize a foreign territory starting from a primary lesion in a drastically different environment. This chapter focuses on an angiostatic factor, pigment epithelium derived factor (PEDF), with a broader function in melanoma that allows it to dually impinge on destroying some of the more relevant dissemination routes and on counteracting key tumor cell properties that enable the metastatic spread of melanoma cells. Understanding of the molecular and cellular mechanisms controlling melanoma progression has become an active field of research over the last five years unveiling a complex intertwined relationship between melanoma cells and the diverse cell types present in the tumor microenvironment, as well as a number of key molecular mediators (Shackleton and Quintana, 2010; Villanueva and Herlyn, 2008). Plasticity of melanoma cells allows them for appropriate reprogramming underlying the decision making process that arbitrates