YES1 Is a Druggable Oncogenic Target in SCLC.

INTRODUCTION: SCLC is an extremely aggressive subtype of lung cancer without approved targeted therapies. Here we identified YES1 as a novel targetable oncogene driving SCLC maintenance and metastasis. METHODS: Association between YES1 levels and prognosis was evaluated in SCLC clinical samples. In vitro functional experiments for proliferation, apoptosis, cell cycle, and cytotoxicity were performed. Genetic and pharmacologic inhibition of YES1 was evaluated in vivo in cell- and patient-derived xenografts and metastasis. YES1 levels were evaluated in mouse and patient plasma-derived exosomes. RESULTS: Overexpression or gain/amplification of YES1 was identified in 31% and 26% of cases, respectively, across molecular subgroups, and was found as an independent predictor of poor prognosis. Genetic depletion of YES1 dramatically reduced cell proliferation, three-dimensional organoid formation, tumor growth, and distant metastasis, leading to extensive apoptosis and tumor regressions. Mechanistically, YES1-inhibited cells revealed alterations in the replisome and DNA repair processes, that conferred sensitivity to irradiation. Pharmacologic blockade with the novel YES1 inhibitor CH6953755 or dasatinib induced marked antitumor activity in organoid models and cell- and patient-derived xenografts. YES1 protein was detected in plasma exosomes from patients and mouse models, with levels matching those of tumors, suggesting that circulating YES1 could represent a biomarker for patient selection/monitoring. CONCLUSIONS: Our results provide evidence that YES1 is a new druggable oncogenic target and biomarker to advance the clinical management of a subpopulation of patients with SCLC.

Stratification of radiosensitive brain metastases based on an actionable S100A9/RAGE resistance mechanism.

Whole-brain radiotherapy (WBRT) is the treatment backbone for many patients with brain metastasis; however, its efficacy in preventing disease progression and the associated toxicity have questioned the clinical impact of this approach and emphasized the need for alternative treatments. Given the limited therapeutic options available for these patients and the poor understanding of the molecular mechanisms underlying the resistance of metastatic lesions to WBRT, we sought to uncover actionable targets and biomarkers that could help to refine patient selection. Through an unbiased analysis of experimental in vivo models of brain metastasis resistant to WBRT, we identified activation of the S100A9-RAGE-NF-κB-JunB pathway in brain metastases as a potential mediator of resistance in this organ. Targeting this pathway genetically or pharmacologically was sufficient to revert the WBRT resistance and increase therapeutic benefits in vivo at lower doses of radiation. In patients with primary melanoma, lung or breast adenocarcinoma developing brain metastasis, endogenous S100A9 levels in brain lesions correlated with clinical response to WBRT and underscored the potential of S100A9 levels in the blood as a noninvasive biomarker. Collectively, we provide a molecular framework to personalize WBRT and improve its efficacy through combination with a radiosensitizer that balances therapeutic benefit and toxicity.

Comprehensive Characterization of Human Lung Large Cell Carcinoma Identifies Transcriptomic Signatures with Potential Implications in Response to Immunotherapy.

Lung cancer is the leading cause of cancer mortality worldwide, with non-small cell lung cancer (NSCLC) being the most prevalent histology. While immunotherapy with checkpoint inhibitors has shown outstanding results in NSCLC, the precise identification of responders remains a major challenge. Most studies attempting to overcome this handicap have focused on adenocarcinomas or squamous cell carcinomas. Among NSCLC subtypes, the molecular and immune characteristics of lung large cell carcinoma (LCC), which represents 10% of NSCLC cases, are not well defined. We hypothesized that specific molecular aberrations may impact the immune microenvironment in LCC and, consequently, the response to immunotherapy. To that end, it is particularly relevant to thoroughly describe the molecular genotype-immunophenotype association in LCC-to identify robust predictive biomarkers and improve potential benefits from immunotherapy. We established a cohort of 18 early-stage, clinically annotated, LCC cases. Their molecular and immune features were comprehensively characterized by genomic and immune-targeted sequencing panels along with immunohistochemistry of immune cell populations. Unbiased clustering defined two novel subgroups of LCC. Pro-immunogenic tumors accumulated certain molecular alterations, showed higher immune infiltration and upregulated genes involved in potentiating immune responses when compared to pro-tumorigenic samples, which favored tumoral progression. This classification identified a set of biomarkers that could potentially predict response to immunotherapy. These results could improve patient selection and expand potential benefits from immunotherapy.

Tumor mutational burden assessment in non-small-cell lung cancer samples: results from the TMB2 harmonization project comparing three NGS panels.

BACKGROUND: Tumor mutational burden (TMB) is a recently proposed predictive biomarker for immunotherapy in solid tumors, including non-small cell lung cancer (NSCLC). Available assays for TMB determination differ in horizontal coverage, gene content and algorithms, leading to discrepancies in results, impacting patient selection. A harmonization study of TMB assessment with available assays in a cohort of patients with NSCLC is urgently needed. METHODS: We evaluated the TMB assessment obtained with two marketed next generation sequencing panels: TruSight Oncology 500 (TSO500) and Oncomine Tumor Mutation Load (OTML) versus a reference assay (Foundation One, FO) in 96 NSCLC samples. Additionally, we studied the level of agreement among the three methods with respect to PD-L1 expression in tumors, checked the level of different immune infiltrates versus TMB, and performed an inter-laboratory reproducibility study. Finally, adjusted cut-off values were determined. RESULTS: Both panels showed strong agreement with FO, with concordance correlation coefficients (CCC) of 0.933 (95% CI 0.908 to 0.959) for TSO500 and 0.881 (95% CI 0.840 to 0.922) for OTML. The corresponding CCCs were 0.951 (TSO500-FO) and 0.919 (OTML-FO) in tumors with <1% of cells expressing PD-L1 (PD-L1<1%; N=55), and 0.861 (TSO500-FO) and 0.722 (OTML-FO) in tumors with PD-L1≥1% (N=41). Inter-laboratory reproducibility analyses showed higher reproducibility with TSO500. No significant differences were found in terms of immune infiltration versus TMB. Adjusted cut-off values corresponding to 10 muts/Mb with FO needed to be lowered to 7.847 muts/Mb (TSO500) and 8.380 muts/Mb (OTML) to ensure a sensitivity >88%. With these cut-offs, the positive predictive value was 78.57% (95% CI 67.82 to 89.32) and the negative predictive value was 87.50% (95% CI 77.25 to 97.75) for TSO500, while for OTML they were 73.33% (95% CI 62.14 to 84.52) and 86.11% (95% CI 74.81 to 97.41), respectively. CONCLUSIONS: Both panels exhibited robust analytical performances for TMB assessment, with stronger concordances in patients with negative PD-L1 expression. TSO500 showed a higher inter-laboratory reproducibility. The cut-offs for each assay were lowered to optimal overlap with FO.

An Fc-free EGFR-specific 4-1BB-agonistic Trimerbody Displays Broad Antitumor Activity in Humanized Murine Cancer Models without Toxicity.

PURPOSE: The induction of 4-1BB signaling by agonistic antibodies can drive the activation and proliferation of effector T cells and thereby enhance a T-cell-mediated antitumor response. Systemic administration of anti-4-1BB-agonistic IgGs, although effective preclinically, has not advanced in clinical development due to their severe hepatotoxicity. EXPERIMENTAL DESIGN: Here, we generated a humanized EGFR-specific 4-1BB-agonistic trimerbody, which replaces the IgG Fc region with a human collagen homotrimerization domain. It was characterized by structural analysis and in vitro functional studies. We also assessed pharmacokinetics, antitumor efficacy, and toxicity in vivo. RESULTS: In the presence of a T-cell receptor signal, the trimerbody provided potent T-cell costimulation that was strictly dependent on 4-1BB hyperclustering at the point of contact with a tumor antigen-displaying cell surface. It exhibits significant antitumor activity in vivo, without hepatotoxicity, in a wide range of human tumors including colorectal and breast cancer cell-derived xenografts, and non-small cell lung cancer patient-derived xenografts associated with increased tumor-infiltrating CD8+ T cells. The combination of the trimerbody with a PD-L1 blocker led to increased IFNγ secretion in vitro and resulted in tumor regression in humanized mice bearing aggressive triple-negative breast cancer. CONCLUSIONS: These results demonstrate the nontoxic broad antitumor activity of humanized Fc-free tumor-specific 4-1BB-agonistic trimerbodies and their synergy with checkpoint blockers, which may provide a way to elicit responses in most patients with cancer while avoiding Fc-mediated adverse reactions.

Human Intestinal Macrophages Are Involved in the Pathology of Both Ulcerative Colitis and Crohn Disease.

BACKGROUND: Intestinal macrophages are key immune cells in the maintenance of intestinal immune homeostasis and have a role in the pathogenesis of inflammatory bowel disease (IBD). However, the mechanisms by which macrophages exert a pathological influence in both ulcerative colitis (UC) and Crohn disease (CD) are not yet well understood. METHODS: We purified intestinal macrophages from gastrointestinal mucosal biopsies (patients with UC, patients with CD, and healthy donors) and analyzed their transcriptome by RNA sequencing and bioinformatics, confirming results with quantitative polymerase chain reaction and immunohistochemistry. RESULTS: Compared with those of healthy donors, intestinal macrophages in patients with UC and with CD showed cellular reprograming of 1287 and 840 dysregulated genes, respectively (false discovery rate ≤ 0.1). The UC and CD intestinal macrophages showed an activated M1 inflammatory phenotype and the downregulation of genes engaged in drug/xenobiotic metabolism. Only macrophages from CD showed, concomitant to an M1 phenotype, a significant enrichment in the expression of M2 and fibrotic and granuloma-related genes. For the first time, we showed (and validated by quantitative polymerase chain reaction and immunohistochemistry) that intestinal macrophages in patients with IBD present both M1 and M2 features, as recently described for tumor-associated macrophages, that affect key pathways for IBD pathology, represented by key markers such as MMP12 (fibrosis), CXCL9 (T-cell attraction), and CD40 (T-cell activation). CONCLUSIONS: Our data support the therapeutic targeting of macrophages to maintain remission in IBD but also indicate that a shift toward an M2 program-as proposed by some reports-may not limit the recruitment and activation of T cells because M2 features do not preclude M1 activation in patients with UC or CD and could exacerbate M2-related CD-specific features such as fibrosis and the formation of granulomas.

M1hot tumor-associated macrophages boost tissue-resident memory T cells infiltration and survival in human lung cancer.

BACKGROUND: The role of tumor-associated macrophages (TAMs) in determining the outcome between the antitumor effects of the adaptive immune system and the tumor's anti-immunity stratagems, is controversial. Macrophages modulate their activities and phenotypes by integration of signals in the tumor microenvironment. Depending on how macrophages are activated, they may adopt so-called M1-like, antitumor or M2-like, protumor profiles. In many solid tumors, a dominance of M2-like macrophages is associated with poor outcomes but in some tumor types, strong M1-like profiles are linked to better outcomes. We aimed to investigate the interrelationship of these TAM populations to establish how they modulate the efficacy of the adaptive immune system in early lung cancer. METHODS: Macrophages from matched lung (non-tumor-associated macrophages (NTAMs)) and tumor samples (TAMs) from resected lung cancers were assessed by bulk and single-cell transcriptomic analysis. Protein expression of genes characteristic of M1-like (chemokine (C-X-C motif) ligand 9) or M2-like (matrix metallopeptidase 12) functions was confirmed by confocal microscopy. Immunohistochemistry related the distribution of TAM transcriptomic signatures to density of CD8+ tissue-resident memory T cells (TRM) in tumors and survival data from an independent cohort of 393 patients with lung cancer. RESULTS: TAMs have significantly different transcriptomic profiles from NTAMs with >1000 differentially expressed genes. TAMs displayed a strong M2-like signature with no significant variation between patients. However, single-cell RNA-sequencing supported by immuno-stained cells revealed that additionally, in 25% of patients the M2-like TAMs also co-expressed a strong/hot M1-like signature (M1hot). Importantly, there was a strong association between the density of M1hot TAMs and TRM cells in tumors that was in turn linked to better survival. Our data suggest a mechanism by which M1hot TAMs may recruit TRM cells via CXCL9 expression and sustain them by making available more of the essential fatty acids on which TRM depend. CONCLUSIONS: We showed that in early lung cancer, expression of M1-like and M2-like gene signatures are not mutually exclusive since the same TAMs can simultaneously display both gene-expression profiles. The presence of M1hot TAMs was associated with a strong TRM tumor-infiltrate and better outcomes. Thus, therapeutic approaches to re-program TAMs to an M1hot phenotype are likely to augment the adaptive antitumor responses.

Evaluation of a Hybrid Capture-Based Pan-Cancer Panel for Analysis of Treatment Stratifying Oncogenic Aberrations and Processes.

Stratification of patients for targeted and immune-based therapies requires extensive genomic profiling that enables sensitive detection of clinically relevant variants and interrogation of biomarkers, such as tumor mutational burden (TMB) and microsatellite instability (MSI). Detection of single and multiple nucleotide variants, copy number variants, MSI, and TMB was evaluated using a commercially available next-generation sequencing panel containing 523 cancer-related genes (1.94 megabases). Analysis of formalin-fixed, paraffin-embedded tissue sections and cytologic material from 45 tumor samples showed that all previously known MSI-positive samples (n = 7), amplifications (n = 9), and pathogenic variants (n = 59) could be detected. TMB and MSI scores showed high intralaboratory and interlaboratory reproducibility (eight samples tested in 11 laboratories). For reliable TMB analysis, 20 ng DNA was shown to be sufficient, even for relatively poor-quality samples. A minimum of 20% neoplastic cells was required to minimize variations in TMB values induced by chromosomal instability or tumor heterogeneity. Subsequent analysis of 58 consecutive lung cancer samples in a diagnostic setting was successful and revealed sufficient somatic mutations to generate mutational signatures in 14 cases. In conclusion, the 523-gene assay can be applied for evaluation of multiple DNA-based biomarkers relevant for treatment selection.

CXCL12-CXCR4 signalling plays an essential role in proper patterning of aortic arch and pulmonary arteries.

AIMS: Chemokine CXCL12 (stromal derived factor 1: SDF1) has been shown to play important roles in various processes of cardiovascular development. In recent avian studies, CXCL12 signalling has been implicated in guidance of cardiac neural crest cells for their participation in the development of outflow tract and cardiac septum. The goal of this study is to investigate the extent to which CXCL12 signalling contribute to the development of aortic arch and pulmonary arteries in mammals. METHODS AND RESULTS: Novel Cxcl12-LacZ reporter and conditional alleles were generated. Using whole mount X-gal staining with the reporter allele and vascular casting techniques, we show that the domain branching pattern of pulmonary arteries in Cxcl12-null mice is completely disrupted and discordant with that of pulmonary veins and airways. Cxcl12-null mice also displayed abnormal and superfluous arterial branches from the aortic arch. The early steps of pharyngeal arch remodelling in Cxcl12-null mice appeared to be unaffected, but vertebral arteries were often missing and prominent aberrant arteries were present parallel to carotid arteries or trachea, similar to aberrant vertebral artery or thyroid ima artery, respectively. Analysis with computed tomography not only confirmed the results from vascular casting studies but also identified abnormal systemic arterial supply to lungs in the Cxcl12-null mice. Tie2-Cre mediated Cxcr4 deletion phenocopied the Cxcl12-null phenotypes, indicating that CXCR4 is the primary receptor for arterial patterning, whereas Cxcl12 or Cxcr4 deletion by Wnt1-Cre did not affect aortic arch patterning. CONCLUSION: CXCL12-CXCR4 signalling is essential for the correct patterning of aortic arches and pulmonary arteries during development. Superfluous arteries in Cxcl12-null lungs and the aortic arch infer a role of CXCL12 in protecting arteries from uncontrolled sprouting during development of the arterial system.

Tissue-resident memory features are linked to the magnitude of cytotoxic T cell responses in human lung cancer.

Therapies that boost the anti-tumor responses of cytotoxic T lymphocytes (CTLs) have shown promise; however, clinical responses to the immunotherapeutic agents currently available vary considerably, and the molecular basis of this is unclear. We performed transcriptomic profiling of tumor-infiltrating CTLs from treatment-naive patients with lung cancer to define the molecular features associated with the robustness of anti-tumor immune responses. We observed considerable heterogeneity in the expression of molecules associated with activation of the T cell antigen receptor (TCR) and of immunological-checkpoint molecules such as 4-1BB, PD-1 and TIM-3. Tumors with a high density of CTLs showed enrichment for transcripts linked to tissue-resident memory cells (TRM cells), such as CD103, and CTLs from CD103hi tumors displayed features of enhanced cytotoxicity. A greater density of TRM cells in tumors was predictive of a better survival outcome in lung cancer, and this effect was independent of that conferred by CTL density. Here we define the 'molecular fingerprint' of tumor-infiltrating CTLs and identify potentially new targets for immunotherapy.

Enhanced responses to angiogenic cues underlie the pathogenesis of hereditary hemorrhagic telangiectasia 2.

Hereditary Hemorrhagic Telangiectasia (HHT) is a genetic vascular disease in which arteriovenous malformations (AVMs) manifest in skin and multiple visceral organs. HHT is caused by heterozygous mutations in endoglin (ENG), activin receptor-like kinase 1 (ALK1), or SMAD4. ALK1 regulates angiogenesis, but the precise function of ALK1 in endothelial cells (ECs) remains elusive. Since most blood vessels of HHT patients do not produce pathological vascular lesions, ALK1 heterozygous ECs may be normal unless additional genetic or environmental stresses are imposed. To investigate the cellular and biochemical phenotypes of Alk1-null versus Alk1-heterozygous ECs, we have generated pulmonary EC lines in which a genotype switch from the Alk1-conditional allele (Alk1 (2f)) to the Alk1-null allele (Alk1 (1f)) can be induced by tamoxifen treatment. Alk1-null (1 f/1 f) ECs displayed increased migratory properties in vitro in response to bFGF compared with Alk1-het (2 f/1 f) ECs. The 1 f/1 f-ECs formed a denser and more persistent tubular network as compared with their parental 2 f/1 f-ECs. Interestingly, the response to BMP-9 on SMAD1/5 phosphorylation was impaired in both 2 f/1 f- and 1 f/1 f-ECs at a comparable manner, suggesting that other factors in addition to SMADs may play a crucial role for enhanced angiogenic activity in 1 f/1 f-ECs. We also demonstrated in vivo that Alk1-deficient ECs exhibited high migratory and invasive properties. Taken together, these data suggest that enhanced responses to angiogenic cues in ALK1-deficient ECs underlie the pathogenesis of HHT2.

Vascular injury triggers Krüppel-like factor 6 mobilization and cooperation with specificity protein 1 to promote endothelial activation through upregulation of the activin receptor-like kinase 1 gene.

RATIONALE: Activin receptor-like kinase-1 (ALK1) is an endothelial transforming growth factor ß receptor involved in angiogenesis. ALK1 expression is high in the embryo vasculature, becoming less detectable in the quiescent endothelium of adult stages. However, ALK1 expression becomes rapidly increased after angiogenic stimuli such as vascular injury. OBJECTIVE: To characterize the molecular mechanisms underlying the regulation of ALK1 on vascular injury. METHODS AND RESULTS: Alk1 becomes strongly upregulated in endothelial (EC) and vascular smooth muscle cells of mouse femoral arteries after wire-induced endothelial denudation. In vitro denudation of monolayers of human umbilical vein ECs also leads to an increase in ALK1. Interestingly, a key factor in tissue remodeling, Krüppel-like factor 6 (KLF6) translocates to the cell nucleus during wound healing, concomitantly with an increase in the ALK1 gene transcriptional rate. KLF6 knock down in human umbilical vein ECs promotes ALK1 mRNA downregulation. Moreover, Klf6(+/-) mice have lower levels of Alk1 in their vasculature compared with their wild-type siblings. Chromatin immunoprecipitation assays show that KLF6 interacts with ALK1 promoter in ECs, and this interaction is enhanced during wound healing. We demonstrate that KLF6 is transactivating ALK1 gene, and this transactivation occurs by a synergistic cooperative mechanism with specificity protein 1. Finally, Alk1 levels in vascular smooth muscle cells are not directly upregulated in response to damage, but in response to soluble factors, such as interleukin 6, released from ECs after injury. CONCLUSIONS: ALK1 is upregulated in ECs during vascular injury by a synergistic cooperative mechanism between KLF6 and specificity protein 1, and in vascular smooth muscle cells by an EC-vascular smooth muscle cell paracrine communication during vascular remodeling.

Transcription factors Sp1 and p73 control the expression of the proapoptotic protein NOXA in the response of testicular embryonal carcinoma cells to cisplatin.

Testicular germ cell tumors (TGCTs) are highly responsive to and curable by cisplatin-based chemotherapy even in advanced stages. We have studied the molecular mechanisms involved in the induction of apoptosis in response to cisplatin, and found that proapoptotic Noxa is transcriptionally up-regulated following cisplatin exposure, even in the absence of p53, in NTERA2 cisplatin-sensitive cells but not in 1411HP-resistant cells. Blockade of Noxa reduced the apoptotic response of embryonal carcinoma (EC) NTERA2 cells to cisplatin. A detailed analysis of the Noxa promoter revealed that p73 and Sp1-like factors, Sp1 and KLF6, played key roles in the transcriptional control of this gene. Overexpression of TAp73 induced Noxa whereas the dominant negative isoform ΔNp73, reduced the levels of Noxa after cisplatin exposure in NTERA2 and 2102EP. Interestingly, down-regulation of Sp1 increased Noxa expression in response to cisplatin. However, blockade of KLF6 decreased cisplatin-induced up-regulation of Noxa in EC cell lines. In addition, tissue microarray analyses of TGCTs revealed that expression of Noxa correlates with good clinical prognosis in patients with embryonal carcinoma. Thus, our data show the transcriptional network that regulates Noxa in EC cells, which is key for their apoptotic response to cisplatin-based chemotherapy, and propose Noxa as a predictive factor of therapeutic response.

The TGF-beta co-receptor endoglin modulates the expression and transforming potential of H-Ras.

Endoglin is a coreceptor for transforming growth factor-ß (TGF-ß) that acts as a suppressor of malignancy during mouse skin carcinogenesis. Because in this model system H-Ras activation drives tumor initiation and progression, we have assessed the effects of endoglin on the expression of H-Ras in transformed keratinocytes. We found that TGF-ß1 increases the expression of H-Ras at both messenger RNA and protein levels. The TGF-ß1-induced H-Ras promoter transactivation was Smad4 independent but mediated by the activation of the TGF-ß type I receptor ALK5 and the Ras-mitogen-activated protein kinase (MAPK) pathway. Endoglin attenuated stimulation by TGF-ß1 of both MAPK signaling activity and H-Ras gene expression. Moreover, endoglin inhibited the Ras/MAPK pathway in transformed epidermal cells containing an H-Ras oncogene, as evidenced by the levels of Ras-guanosine triphosphate, phospho-MAPK kinase (MEK) and phospho-extracellular signal-regulated kinase (ERK) as well as the expression of c-fos, a MAPK downstream target gene. Interestingly, in spindle carcinoma cells, that have a hyperactivated Ras/MAPK pathway, endoglin inhibited ERK phosphorylation without affecting MEK or Ras activity. The mechanism for this effect is unknown but strongly depends on the endoglin extracellular domain. Because the MAPK pathway is a downstream mediator of the transforming potential of Ras, the effect of endoglin on the oncogenic function of H-Ras was assessed. Endoglin inhibited the transforming capacity of H-Ras(Q61K) and H-Ras(G12V) oncogenes in a NIH3T3 focus formation assay. The ability to interfere with the expression and oncogenic potential of H-Ras provides a new face of the suppressor role exhibited by endoglin in H-Ras-driven carcinogenesis.

Characterization of the human Activin-A receptor type II-like kinase 1 (ACVRL1) promoter and its regulation by Sp1.

BACKGROUND: Activin receptor-like kinase 1 (ALK1) is a Transforming Growth Factor-beta (TGF-beta) receptor type I, mainly expressed in endothelial cells that plays a pivotal role in vascular remodelling and angiogenesis. Mutations in the ALK1 gene (ACVRL1) give rise to Hereditary Haemorrhagic Telangiectasia, a dominant autosomal vascular dysplasia caused by a haploinsufficiency mechanism. In spite of its patho-physiological relevance, little is known about the transcriptional regulation of ACVRL1. Here, we have studied the different origins of ACVRL1 transcription, we have analyzed in silico its 5'-proximal promoter sequence and we have characterized the role of Sp1 in the transcriptional regulation of ACVRL1. RESULTS: We have performed a 5'Rapid Amplification of cDNA Ends (5'RACE) of ACVRL1 transcripts, finding two new transcriptional origins, upstream of the one previously described, that give rise to a new exon undiscovered to date. The 5'-proximal promoter region of ACVRL1 (-1,035/+210) was analyzed in silico, finding that it lacks TATA/CAAT boxes, but contains a remarkably high number of GC-rich Sp1 consensus sites. In cells lacking Sp1, ACVRL1 promoter reporters did not present any significant transcriptional activity, whereas increasing concentrations of Sp1 triggered a dose-dependent stimulation of its transcription. Moreover, silencing Sp1 in HEK293T cells resulted in a marked decrease of ACVRL1 transcriptional activity. Chromatin immunoprecipitation assays demonstrated multiple Sp1 binding sites along the proximal promoter region of ACVRL1 in endothelial cells. Furthermore, demethylation of CpG islands, led to an increase in ACVRL1 transcription, whereas in vitro hypermethylation resulted in the abolishment of Sp1-dependent transcriptional activation of ACVRL1. CONCLUSIONS: Our results describe two new transcriptional start sites in ACVRL1 gene, and indicate that Sp1 is a key regulator of ACVRL1 transcription, providing new insights into the molecular mechanisms that contribute to the expression of ACVRL1 gene. Moreover, our data show that the methylation status of CpG islands markedly modulates the Sp1 regulation of ACVRL1 gene transcriptional activity.

TGF-beta regulates the expression of transcription factor KLF6 and its splice variants and promotes co-operative transactivation of common target genes through a Smad3-Sp1-KLF6 interaction.

KLF6 (Krüppel-like factor 6) is a transcription factor and tumour suppressor with a growing range of biological activities and transcriptional targets. Among these, KLF6 suppresses growth through transactivation of TGF-beta1 (transforming growth factor-beta1). KLF6 can be alternatively spliced, generating lower-molecular-mass isoforms that antagonize the full-length WT (wild-type) protein and promote growth. A key target gene of full-length KLF6 is endoglin, which is induced in vascular injury. Endoglin, a homodimeric cell membrane glycoprotein and TGF-beta auxiliary receptor, has a pro-angiogenic role in endothelial cells and is also involved in malignant progression. The aim of the present work was to explore the effect of TGF-beta on KLF6 expression and splicing, and to define the contribution of TGF-beta on promoters regulated by co-operation between KLF6 and Sp1 (specificity protein 1). Using co-transfection, co-immunoprecipitation and fluorescence resonance energy transfer, our data demonstrate that KLF6 co-operates with Sp1 in transcriptionally regulating KLF6-responsive genes and that this co-operation is further enhanced by TGF-beta1 through at least two mechanisms. First, in specific cell types, TGF-beta1 may decrease KLF6 alternative splicing, resulting in a net increase in full-length, growth-suppressive KLF6 activity. Secondly, KLF6-Sp1 co-operation is further enhanced by the TGF-beta-Smad (similar to mothers against decapentaplegic) pathway via the likely formation of a tripartite KLF6-Sp1-Smad3 complex in which KLF6 interacts indirectly with Smad3 through Sp1, which may serve as a bridging molecule to co-ordinate this interaction. These findings unveil a finely tuned network of interactions between KLF6, Sp1 and TGF-beta to regulate target genes.

Caveolin-1 interacts and cooperates with the transforming growth factor-beta type I receptor ALK1 in endothelial caveolae.

AIMS: Activin receptor-like kinase (ALK)1 is a transforming growth factor (TGF)-beta type I membrane receptor restricted almost entirely to endothelial cells (ECs) and involved in vascular remodelling and angiogenesis. Previous reports have shown that the ubiquitous TGF-beta type I receptor ALK5 and the type II receptor are located in cholesterol-rich membrane microdomains named caveolae. The aim of this work was to assess the location of ALK1 in endothelial caveolae as well as to study the role of caveolin-1 on the TGF-beta/ALK1 signalling pathway. METHODS AND RESULTS: The subcellular distribution of ALK1 was analysed by confocal microscopy and co-fractionation experiments in human ECs. The association between human ALK1 and caveolin-1 was studied in caveolin-1-deficient human epithelial cells by co-immunoprecipitation. The functional role of caveolin-1 on the ALK1-mediated TGF-beta signalling was elucidated using ALK1-specific luciferase reporters in human ECs, caveolin-1(-/-)mouse embryonic fibroblasts, and rat myoblasts. Confocal microscopy analyses, as well as cholesterol depletion experiments in the presence of cholesterol-depleting agents such as nystatin or methyl-beta-cyclodextrin, demonstrated that ALK1 is located in endothelial caveolae. Also, co-immunoprecipitation assays showed that ALK1 associates with the main caveolae component caveolin-1. Mapping of the ALK1/caveolin-1 interaction revealed that the caveolin-1 scaffolding domain and the caveolin-1 binding motif in ALK1 are responsible for this association. Moreover, this hitherto not reported interaction had a functional consequence for the ALK1-dependent signalling. In contrast with the previously published ALK5/caveolin-1 interaction, caveolin-1 enhances the TGF-beta/ALK1 signalling pathway, promoting the activity of the ALK1-specific reporters. Conversely, specific suppression of caveolin-1 abrogated the ALK1 signalling pathway. CONCLUSION: ALK1 is located in endothelial caveolae where it functionally interacts with caveolin-1 through its scaffolding domain, suggesting a joint contribution of ALK1 and caveolin-1 as key mediators of the TGF-beta pathway in angiogenesis.

Gene expression fingerprinting for human hereditary hemorrhagic telangiectasia.

Hereditary hemorrhagic telangiectasia (HHT) or Osler-Weber-Rendu syndrome is an autosomal dominant vascular disorder characterized by telangiectases and internal arteriovenous malformations. It is caused by mutations in elements of the transforming growth factor-beta (TGF-beta) receptor complex: endoglin, a co-receptor, responsible for HHT1, or ALK1 (activin receptor-like kinase 1), a type I receptor leading to HHT2. Recently, we have established cultures of HHT endothelial cells, primary targets of the disease. These cells showed deficient TGF-beta signaling and angiogenesis, representing a useful human model to study the molecular mechanism of this disease. To understand the pathogenic mechanism underlying HHT, we have used total RNA probes to compare HHT versus non-HHT cells by expression microarrays. This work represents a systematic study to identify target genes affected in HHT cells. Given the similarity of symptoms in HHT1 and HHT2, special interest has been put on the identification of common targets for both HHT types. As a result, 277 downregulated and 63 upregulated genes were identified in HHT versus control cells. These genes are involved in biological processes relevant to the HHT pathology, such as angiogenesis, cytoskeleton, cell migration, proliferation and NO synthesis. The type of misregulated genes found in HHT endothelial cells lead us to propose a model of HHT pathogenesis, opening new perspectives to understand this disorder. Moreover, as the disease is originated by mutations in proteins of the TGF-beta receptor complex, these results may be useful to find out targets of the TGF-beta pathway in endothelium.

Therapeutic action of tranexamic acid in hereditary haemorrhagic telangiectasia (HHT): regulation of ALK-1/endoglin pathway in endothelial cells.

Recurrent epistaxis is the most frequent clinical manifestation of hereditary haemorrhagic telangiectasia (HHT). Its treatment is difficult. Our objective was to assess the use of tranexamic acid (TA), an antifibrinolytic drug, for the treatment of epistaxis in HHT patients and to investigate in vitro the effects of TA over endoglin and ALK-1 expression and activity in endothelial cells. A prospective study was carried out on patients with epistaxis treated with oral TA in the HHT Unit of Sierrallana Hospital (Cantabria, Spain). Primary cultures of endothelial cells were treated with TA to measure the levels of endoglin and ALK-1 at the cell surface by flow cytometry. RNA levels were also measured by real-time PCR, and the transcriptional effects of TA on reporters for endoglin, ALK-1 and the endoglin/ALK-1 TGF-beta pathway were assessed. The results showed that the fourteen HHT patients treated orally with TA improved, and the frequency and severity of their epistaxis were decreased. No complications derived from the treatment were observed. Cultured endothelial cells incubated with TA exhibited increased levels of endoglin and ALK-1 at the protein and mRNA levels, enhanced TGF-beta signaling, and improved endothelial cell functions like tubulogenesis and migration. In summary, oral administration of TA proved beneficial for epistaxis treatment in selected patients with HHT. In addition to its already reported antifibrinolytic effects, TA stimulates the expression ofALK-1 and endoglin, as well as the activity of the ALK-1/endoglin pathway.