p73 is required for vessel integrity controlling endothelial junctional dynamics through Angiomotin.

Preservation of blood vessel integrity, which is critical for normal physiology and organ function, is controlled at multiple levels, including endothelial junctions. However, the mechanism that controls the adequate assembly of endothelial cell junctions is not fully defined. Here, we uncover TAp73 transcription factor as a vascular architect that orchestrates transcriptional programs involved in cell junction establishment and developmental blood vessel morphogenesis and identify Angiomotin (AMOT) as a TAp73 direct transcriptional target. Knockdown of p73 in endothelial cells not only results in decreased Angiomotin expression and localization at intercellular junctions, but also affects its downstream function regarding Yes-associated protein (YAP) cytoplasmic sequestration upon cell-cell contact. Analysis of adherens junctional morphology after p73-knockdown in human endothelial cells revealed striking alterations, particularly a sharp increase in serrated junctions and actin bundles appearing as stress fibers, both features associated with enhanced barrier permeability. In turn, stabilization of Angiomotin levels rescued those junctional defects, confirming that TAp73 controls endothelial junction dynamics, at least in part, through the regulation of Angiomotin. The observed defects in monolayer integrity were linked to hyperpermeability and reduced transendothelial electric resistance. Moreover, p73-knockout retinas showed a defective sprout morphology coupled with hemorrhages, highlighting the physiological relevance of p73 regulation in the maintenance of vessel integrity in vivo. We propose a new model in which TAp73 acts as a vascular architect integrating transcriptional programs that will impinge with Angiomotin/YAP signaling to maintain junctional dynamics and integrity, while balancing endothelial cell rearrangements in angiogenic vessels.

Metabolic labeling of RNA uncovers the contribution of transcription and decay rates on hypoxia-induced changes in RNA levels.

Cells adapt to environmental changes, including fluctuations in oxygen levels, through the induction of specific gene expression programs. However, most transcriptomic studies do not distinguish the relative contribution of transcription, RNA processing, and RNA degradation processes to cellular homeostasis. Here we used metabolic labeling followed by massive parallel sequencing of newly transcribed and preexisting RNA fractions to simultaneously analyze RNA synthesis and decay in primary endothelial cells exposed to low oxygen tension. We found that changes in transcription rates induced by hypoxia are the major determinant of changes in RNA levels. However, degradation rates also had a significant contribution, accounting for 24% of the observed variability in total mRNA. In addition, our results indicated that hypoxia led to a reduction of the overall mRNA stability from a median half-life in normoxia of 8.7 h, to 5.7 h in hypoxia. Analysis of RNA content per cell confirmed a decrease of both mRNA and total RNA in hypoxic samples and that this effect is dependent on the EGLN/HIF/TSC2 axis. This effect could potentially contribute to fundamental global responses such as inhibition of translation in hypoxia. In summary, our study provides a quantitative analysis of the contribution of RNA synthesis and stability to the transcriptional response to hypoxia and uncovers an unexpected effect on the latter.

Hypoxia compensates cell cycle arrest with progenitor differentiation during angiogenesis.

Angiogenesis, the main mechanism that allows vascular expansion for tissue regeneration or disease progression, is often triggered by an imbalance between oxygen consumption and demand. Here, by analyzing changes in the transcriptomic profile of endothelial cells (ECs) under hypoxia we uncovered that the repression of cell cycle entry and DNA replication stand as central responses in the early adaptation of ECs to low oxygen tension. Accordingly, hypoxia imposed a restriction in S-phase in ECs that is mediated by Hypoxia-Inducible Factors. Our results indicate that the induction of angiogenesis by hypoxia in Embryoid Bodies generated from murine Stem Cells is accomplished by the compensation of decreased S-phase entry in mature ECs and differentiation of progenitor cells. This conditioning most likely allows an optimum remodeling of the vascular network. Identification of the molecular underpinnings of cell cycle arrest by hypoxia would be relevant for the design of improved strategies aimed to suppress angiogenesis in pathological contexts where hypoxia is a driver of neovascularization.

The SIN3A histone deacetylase complex is required for a complete transcriptional response to hypoxia.

Cells adapt to environmental changes, including fluctuations in oxygen levels, through the induction of specific gene expression programs. To identify genes regulated by hypoxia at the transcriptional level, we pulse-labeled HUVEC cells with 4-thiouridine and sequenced nascent transcripts. Then, we searched genome-wide binding profiles from the ENCODE project for factors that correlated with changes in transcription and identified binding of several components of the Sin3A co-repressor complex, including SIN3A, SAP30 and HDAC1/2, proximal to genes repressed by hypoxia. SIN3A interference revealed that it participates in the downregulation of 75% of the hypoxia-repressed genes in endothelial cells. Unexpectedly, it also blunted the induction of 47% of the upregulated genes, suggesting a role for this corepressor in gene induction. In agreement, ChIP-seq experiments showed that SIN3A preferentially localizes to the promoter region of actively transcribed genes and that SIN3A signal was enriched in hypoxia-repressed genes, prior exposure to the stimulus. Importantly, SINA3 occupancy was not altered by hypoxia in spite of changes in H3K27ac signal. In summary, our results reveal a prominent role for SIN3A in the transcriptional response to hypoxia and suggest a model where modulation of the associated histone deacetylase activity, rather than its recruitment, determines the transcriptional output.

Identification of non-coding genetic variants in samples from hypoxemic respiratory disease patients that affect the transcriptional response to hypoxia.

A wide range of diseases course with an unbalance between the consumption of oxygen by tissues and its supply. This situation triggers a transcriptional response, mediated by the hypoxia inducible factors (HIFs), that aims to restore oxygen homeostasis. Little is known about the inter-individual variation in this response and its role in the progression of disease. Herein, we sought to identify common genetic variants mapping to hypoxia response elements (HREs) and characterize their effect on transcription. To this end, we constructed a list of genome-wide HIF-binding regions from publicly available experimental datasets and studied the genetic variability in these regions by targeted re-sequencing of genomic samples from 96 chronic obstructive pulmonary disease and 144 obstructive sleep apnea patients. This study identified 14 frequent variants disrupting potential HREs. The analysis of the genomic regions containing these variants by means of reporter assays revealed that variants rs1009329, rs6593210 and rs150921338 impaired the transcriptional response to hypoxia. Finally, using genome editing we confirmed the functional role of rs6593210 in the transcriptional regulation of EGFR. In summary, we found that inter-individual variability in non-coding regions affect the response to hypoxia and could potentially impact on the progression of pulmonary diseases.

Melanoma Cells Block PEDF Production in Fibroblasts to Induce the Tumor-Promoting Phenotype of Cancer-Associated Fibroblasts.

Loss of pigment epithelium-derived factor (PEDF, SERPINF1) in cancer cells is associated with poor prognosis and metastasis, but the contribution of stromal PEDF to cancer evolution is poorly understood. Therefore, we investigated the role of fibroblast-derived PEDF in melanoma progression. We demonstrate that normal dermal fibroblasts expressing high PEDF levels attenuated melanoma growth and angiogenesis in vivo, whereas PEDF-depleted fibroblasts exerted tumor-promoting effects. Accordingly, mice with global PEDF knockout were more susceptible to melanoma metastasis. We also demonstrate that normal fibroblasts in close contact with PEDF-null melanoma cells lost PEDF expression and tumor-suppressive properties. Further mechanistic investigations underlying the crosstalk between tumor and stromal cells revealed that melanoma cells produced PDGF-BB and TGFß, which blocked PEDF production in fibroblasts. Notably, cancer-associated fibroblasts (CAF) isolated from patient-derived tumors expressed markedly low levels of PEDF. Treatment of patient CAF and TGFß-treated normal fibroblasts with exogenous PEDF decreased the expression of CAF markers and restored PEDF expression. Finally, expression profiling of PEDF-depleted fibroblasts revealed induction of IL8, SERPINB2, hyaluronan synthase-2, and other genes associated with tumor promotion and metastasis. Collectively, our results demonstrate that PEDF maintains tumor-suppressive functions in fibroblasts to prevent CAF conversion and illustrate the mechanisms by which melanoma cells silence stromal PEDF to promote malignancy. Cancer Res; 76(8); 2265-76. ©2016 AACR.

Nuclear DICKKOPF-1 as a biomarker of chemoresistance and poor clinical outcome in colorectal cancer.

Sporadic colorectal cancer (CRC) insurgence and progression depend on the activation of Wnt/ß-catenin signaling. Dickkopf (DKK)-1 is an extracellular inhibitor of Wnt/ß-catenin signaling that also has undefined ß-catenin-independent actions. Here we report for the first time that a proportion of DKK-1 locates within the nucleus of healthy small intestine and colon mucosa, and of CRC cells at specific chromatin sites of active transcription. Moreover, we show that DKK-1 regulates several cancer-related genes including the cancer stem cell marker aldehyde dehydrogenase 1A1 (ALDH1A1) and Ral-binding protein 1-associated Eps domain-containing 2 (REPS2), which are involved in detoxification of chemotherapeutic agents. Nuclear DKK-1 expression is lost along CRC progression; however, it remains high in a subset (15%) of CRC patients (n = 699) and associates with decreased progression-free survival (PFS) after chemotherapy administration and overall survival (OS) [adjusted HR, 1.65; 95% confidence interval (CI), 1.23-2.21; P = 0.002)]. Overexpression of ALDH1A1 and REPS2 associates with nuclear DKK-1 expression in tumors and correlates with decreased OS (P = 0.001 and 0.014) and PFS. In summary, our findings demonstrate a novel location of DKK-1 within the cell nucleus and support a role of nuclear DKK-1 as a predictive biomarker of chemoresistance in colorectal cancer.

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.

(V600E)BRAF promotes invasiveness of thyroid cancer cells by decreasing E-cadherin expression through a Snail-dependent mechanism.

BRAF is a main oncogene in human thyroid cancer. Here, we show that BRAF depletion by siRNA or inhibition of its activity by treatment with BRAF inhibitor PLX4720 decreases migration and invasion in thyroid cancer cells expressing oncogenic (V600E)BRAF through a MEK/ERK-dependent mechanism, since treatment with the MEK inhibitor U0126 exerts the same effect. Moreover, over-expression of (V600E)BRAF increases migration and invasion of wild-type BRAF thyroid cells. Using the same strategies, we demonstrate that these effects are mediated by upregulation of the transcriptional repressor Snail with a concomitant decrease of its target E-cadherin, both hallmarks of EMT. These results reveal a novel (V600E)BRAF-induced mechanism in thyroid tumours progression and provides a rationale for using the PLX4720 inhibitor to target (V600E)BRAF signalling to effectively control progression of thyroid cancer.

Autocrine stimulation of clear-cell renal carcinoma cell migration in hypoxia via HIF-independent suppression of thrombospondin-1.

Thrombospondin-1 is a matricellular protein with potent antitumour activities, the levels of which determine the fate of many different tumours, including renal carcinomas. However, the factors that regulate this protein remain unclear. In renal carcinomas, hypoxic conditions enhance the expression of angiogenic factors that help adapt tumour cells to their hostile environment. Therefore, we hypothesized that anti-angiogenic factors should correspondingly be dampened. Indeed, we found that hypoxia decreased the thrombospondin-1 protein in several clear cell renal carcinoma cell lines (ccRCC), although no transcriptional regulation was observed. Furthermore, we proved that hypoxia stimulates multiple signals that independently contribute to diminish thrombospondin-1 in ccRCC, which include a decrease in the activity of oxygen-dependent prolylhydroxylases (PHDs) and activation of the PI3K/Akt signalling pathway. In addition, thrombospondin-1 regulation in hypoxia proved to be important for ccRCC cell migration and invasion.

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.

Pigment epithelium-derived factor blocks tumor extravasation by suppressing amoeboid morphology and mesenchymal proteolysis.

Metastatic melanoma cells are highly adaptable to their in vivo microenvironment and can switch between protease-dependent mesenchymal and protease-independent amoeboid invasion to facilitate metastasis. Such adaptability can be visualized in vitro, when cells are cultured in conditions that recapitulate three-dimensional microenvironments. Using thick collagen layers in cell culture and in vivo extravasation assays, we found that pigment epithelium-derived factor (PEDF) suppressed lung extravasation of aggressive melanoma by coordinated regulation of cell shape and proteolysis. In cells grown on a thick collagen bed, PEDF overexpression and exogenous PEDF blocked the rapidly invasive, rounded morphology, and promoted an elongated, mesenchymal-like phenotype associated with reduced invasion. These changes in cell shape depended on decreased RhoA and increased Rac1 activation and were mediated by the up-regulation of Rac1-GEF, DOCK3 and down-regulation of Rac1-GAP, ARHGAP22. Surprisingly, we found that PEDF overexpression also blocked the trafficking of membrane-tethered, MT1-MMP to the cell surface through RhoA inhibition and Rac1 activation. In vivo, knockdown of Rac1 and DOCK3 or overexpression of MT1-MMP was sufficient to reverse the inhibitory effect of PEDF on extravasation. Using functional studies, we demonstrated that PEDF suppressed the rounded morphology and MT1-MMP surface localization through its antiangiongenic, 34-mer epitope and the recently identified PEDF receptor candidate, PNPLA2. Our findings unveil the coordinated regulation of cell shape and proteolysis and identify an unknown mechanism for PEDF's antimetastatic activity.

Changes in the gene expression profile of A375 human melanoma cells induced by overexpression of multifunctional pigment epithelium-derived factor.

Pigment epithelium-derived factor (PEDF) is a broad-spectrum angiogenesis inhibitor that displays potent antimetastatic activity in multiple tumor types. We have previously shown that PEDF prevents primary tumor growth and metastatic spread of human melanoma in mouse experimental models. Consistent with these observations, PEDF expression is lost at the late stages of melanoma progression, allowing melanoma cells to become angiogenic, migratory, and invasive. PEDF's ability to modify the interplay between the host and tumor tissues strongly supports its use as a therapeutic agent for the treatment of metastatic melanoma. However, transition to the clinic requires a more detailed knowledge of the molecular mechanisms underpinning PEDF's activity. In this study, we describe changes in the gene expression profile of A375 human melanoma cells induced by PEDF overexpression. PEDF modulated diverse categories of genes known to be involved in angiogenesis and migration. It downregulated cytokines such as interleukin-8 and extracellular matrix proteins such as collagen IV, while it upregulated fibronectin. Multiple transcripts previously described as contributing to the acquisition of malignant phenotype by melanoma were also diminished by PEDF overexpression, among which we validated galectin 3 and jagged 1. In addition, PEDF downregulated S100ß and melanoma inhibitory activity, which are widely used in the pathological diagnosis of melanoma. Interestingly, PEDF increased the expression of melanophilin and decreased rab27A, which are relevant targets for melanosome transport; suggesting that PEDF could directly impinge on melanocytic lineage-specific processes. Our study identifies new molecular targets and signaling pathways that may potentially contribute to determine PEDF's ability to restrict the aggressiveness of A375 human melanoma cells.

Functional impact of cancer-associated mutations in the tumor suppressor protein ING4.

Inhibitor of growth 4 (ING4) is a member of the ING family of tumor suppressor proteins. In this study, we have analyzed the impact of two mutations in ING4 associated with human tumors (Y121N and N214D), testing their behavior in a series of functional, biochemical and structural analyses. We report that the N214D mutation dramatically dampened the ability of ING4 to inhibit proliferation, anchorage-independent growth or cell migration or to sensitize to cell death. In turn, the Y121N mutant did not differ significantly from wild-type ING4 in our assays. Neither of the mutations altered the normal subcellular localization of ING4, showing predominantly nuclear accumulation. We investigated the molecular basis of the defect in the activity of the N214D mutant. The folding and ability to bind histone marks of ING4 was not significantly altered by this mutation. Instead, we found that the functional impairment of the N214D mutant correlates with reduced protein stability due to increased proteasome-mediated degradation. In summary, our data demonstrates that a point mutation of ING4 associated to human tumors leads to the loss of several essential functions of ING4 pertinent to tumor protection and highlight the importance of ING4 function to prevent tumorigenesis.

Following up tumour angiogenesis: from the basic laboratory to the clinic.

Preceded by three decades of intense basic research on tumour angiogenesis, we are assisting to the translation of anti-antiangiogenic therapies as medical oncologists are increasingly using pioneering anti-angiogenic drugs in combination with standard treatments. While basic knowledge in the field of angiogenesis is reaching maturity and our level of understanding of the complex process of vessel development and growth in health and disease has been enriched at the molecular and cellular levels, the translation of this knowledge to the clinic is still in its infancy. Identifying the most suitable drugs, and the optimal dosage and schedule, as well as monitoring patients' responses to anti-angiogenic therapy, remain challenging issues that currently limit the benefit of this new therapeutic approach in cancer. This review will focus on a comprehensive description of the experimental assays in which angiogenesis research has been founded and how the different assays complement and provide relevant information for the task of characterising the angiogenic properties of diverse tumours, giving us a variety of tools to follow up tumour angiogenesis in research models. Following up tumour angiogenesis in patients and their response to antiangiogenic therapy is a more challenging task that will benefit in the near future from the use of non-invasive imaging methods as well as molecular and cellular biomarkers of angiogenesis suitable for clinical oncology. As both the design of the anti-angiogenic therapies and monitoring of the response are improved in the coming years to properly tailor them to the angiogenic profile of every patient, we hope to achieve increasing response and benefit of including antiangiogenic drugs as standard in cancer therapy.

1alpha,25-Dihydroxyvitamin D3 regulates the expression of Id1 and Id2 genes and the angiogenic phenotype of human colon carcinoma cells.

1alpha,25-Dihydroxyvitamin D3 (1alpha,25(OH)2D3) has antitumor activity in addition to its classical action on calcium metabolism and bone tissue biology. It is thought to regulate the expression of multiple target genes and thus modulate processes critical for tumor growth and metastases. Here we show that 1alpha,25(OH)2D3 differentially regulates the expression of Id1 and Id2 genes, members of a family of transcriptional regulators of cell proliferation and differentiation. 1alpha,25(OH)2D3 induced epithelial differentiation in SW480-ADH human colon carcinoma cell line by promoting expression of the proteins implicated in adherent junction formation, including E-cadherin, and by inhibiting beta-catenin transcriptional activity. 1alpha,25(OH)2D3 activated the human Id1 gene promoter and rapidly induced Id1 RNA and protein. Ectopic overexpression of Id1 was not sufficient to induce E-cadherin, which was critical for the morphological changes induced by 1alpha,25(OH)2D3 in SW480-ADH cells. Conversely, Id2 transcription rate, RNA and protein levels were decreased by 1alpha,25(OH)2D3. Id2 downregulation by 1alpha,25(OH)2D3 mediated the antiproliferative effect of 1alpha,25(OH)2D3 on SW480-ADH cells. In addition, we showed that 1alpha,25(OH)2D3 changed the levels of the inducer of angiogenesis, vascular endothelial growth factor and the potent antiangiogenic factor thrombospondin-1, leading to a balanced change in the angiogenic potential of SW480-ADH human colon carcinoma cells.

Inhibition of xenografted human melanoma growth and prevention of metastasis development by dual antiangiogenic/antitumor activities of pigment epithelium-derived factor.

Human melanoma mortality is associated with the growth of metastasis in selected organs including the lungs, liver, and brain. In this study, we examined the consequences of overexpression of pigment epithelium-derived factor (PEDF), a neurotrophic factor and potent angiogenesis inhibitor, on both melanoma primary tumor growth and metastasis development. PEDF overexpression by melanoma cells greatly inhibited subcutaneous tumor formation and completely prevented lung and liver metastasis in immunocompromised mice after tail vein injection of metastatic human melanoma cell lines. Whereas the effects of PEDF on primary tumor xenografts appear mostly associated with inhibition of the angiogenic tumor response, abrogation of melanoma metastasis appears to depend on direct PEDF effects on both migration and survival of melanoma cells. PEDF-mediated inhibition of melanoma metastases could thus have a major impact on existing therapies for melanoma.

Inhibition of tumor growth by systemic treatment with thrombospondin-1 peptide mimetics.

Many normal human cells produce thrombospondin-1 (TSP-1), a potent antiangiogenic protein that promotes vascular quiescence. In various organ systems, including the brain, breast and bladder and in fibroblasts, TSP-1 secretion is reduced during tumorigenesis, thereby allowing induction of the vigorous neovascularization required for tumor growth and metastasis. Full-length and short TSP-1-derived peptides inhibit angiogenesis by inducing endothelial cell apoptosis and thus disrupting the vasculature of the growing tumor. CD36 expressed on the surface of endothelial cells functions as the primary antiangiogenic receptor for TSP-1. A D-isoleucyl enantiomer of a TSP-1 heptapeptide specifically inhibits the proliferation and migration of capillary endothelial cells. DI-TSP, an approximately 1 kDa capped version of this peptide, is also antiangiogenic in vitro, with a specific activity approaching that of the 450 kDa parental molecule. Here, we show that DI-TSP delivered systemically dose-dependently inhibits the growth of murine melanoma metastases in syngeneic animals and that its more soluble isomer, DI-TSPa, similarly blocks the progression of primary human bladder tumors in an orthotopic model in immune-deficient mice. Like intact TSP-1, these peptide mimetics had no effect on cancer cells growing in vitro but markedly suppressed the growth of endothelial cells by inducing receptor-dependent apoptosis. Antibodies raised against CD36 blocked the ability of peptides to induce apoptosis in endothelial cells but had no effect on tumor necrosis factor-alpha-induced apoptosis. In vivo, the peptide mimetics were associated with a significantly reduced microvessel density and increased apoptotic indices in both the endothelial and tumor cell compartments. Such short peptides targeted to a specific antiangiogenic receptor, potent and easy to synthesize, show great promise as lead compounds in clinical antiangiogenic strategies.