A YAP-centered mechanotransduction loop drives collective breast cancer cell invasion.

Dense and aligned Collagen I fibers are associated with collective cancer invasion led by protrusive tumor cells, leader cells. In some breast tumors, a population of cancer cells (basal-like cells) maintain several epithelial characteristics and express the myoepithelial/basal cell marker Keratin 14 (K14). Emergence of leader cells and K14 expression are regarded as interconnected events triggered by Collagen I, however the underlying mechanisms remain unknown. Using breast carcinoma organoids, we show that Collagen I drives a force-dependent loop, specifically in basal-like cancer cells. The feed-forward loop is centered around the mechanotransducer Yap and independent of K14 expression. Yap promotes a transcriptional program that enhances Collagen I alignment and tension, which further activates Yap. Active Yap is detected in invading breast cancer cells in patients and required for collective invasion in 3D Collagen I and in the mammary fat pad of mice. Our work uncovers an essential function for Yap in leader cell selection during collective cancer invasion.

Regulation of a progenitor gene program by SOX4 is essential for mammary tumor proliferation.

In breast cancer the transcription factor SOX4 has been shown to be associated with poor survival, increased tumor size and metastasis formation. This has mostly been attributed to the ability of SOX4 to regulate Epithelial-to-Mesenchymal-Transition (EMT). However, SOX4 regulates target gene transcription in a context-dependent manner that is determined by the cellular and epigenetic state. In this study we have investigated the loss of SOX4 in mammary tumor development utilizing organoids derived from a PyMT genetic mouse model of breast cancer. Using CRISPR/Cas9 to abrogate SOX4 expression, we found that SOX4 is required for inhibiting differentiation by regulating a subset of genes that are highly activated in fetal mammary stem cells (fMaSC). In this way, SOX4 re-activates an oncogenic transcriptional program that is regulated in many progenitor cell-types during embryonic development. SOX4-knockout organoids are characterized by the presence of more differentiated cells that exhibit luminal or basal gene expression patterns, but lower expression of cell cycle genes. In agreement, primary tumor growth and metastatic outgrowth in the lungs are impaired in SOX4KO tumors. Finally, SOX4KO tumors show a severe loss in competitive capacity to grow out compared to SOX4-proficient cells in primary tumors. Our study identifies a novel role for SOX4 in maintaining mammary tumors in an undifferentiated and proliferative state. Therapeutic manipulation of SOX4 function could provide a novel strategy for cancer differentiation therapy, which would promote differentiation and inhibit cycling of tumor cells.

C/EBPɑ is crucial determinant of epithelial maintenance by preventing epithelial-to-mesenchymal transition.

Extracellular signals such as TGF-ß can induce epithelial-to-mesenchymal transition (EMT) in cancers of epithelial origin, promoting molecular and phenotypical changes resulting in pro-metastatic characteristics. We identified C/EBPα as one of the most TGF-ß-mediated downregulated transcription factors in human mammary epithelial cells. C/EBPα expression prevents TGF-ß-driven EMT by inhibiting expression of known EMT factors. Depletion of C/EBPα is sufficient to induce mesenchymal-like morphology and molecular features, while cells that had undergone TGF-ß-induced EMT reverted to an epithelial-like state upon C/EBPα re-expression. In vivo, mice injected with C/EBPα-expressing breast tumor organoids display a dramatic reduction of metastatic lesions. Collectively, our results show that C/EBPα is required for maintaining epithelial homeostasis by repressing the expression of key mesenchymal markers, thereby preventing EMT-mediated tumorigenesis. These data suggest that C/EBPα is a master epithelial "gatekeeper" whose expression is required to prevent unwarranted mesenchymal transition, supporting an important role for EMT in mediating breast cancer metastasis.

Global transcriptional analysis identifies a novel role for SOX4 in tumor-induced angiogenesis.

The expression of the transcription factor SOX4 is increased in many human cancers, however, the pro-oncogenic capacity of SOX4 can vary greatly depending on the type of tumor. Both the contextual nature and the mechanisms underlying the pro-oncogenic SOX4 response remain unexplored. Here, we demonstrate that in mammary tumorigenesis, the SOX4 transcriptional network is dictated by the epigenome and is enriched for pro-angiogenic processes. We show that SOX4 directly regulates endothelin-1 (ET-1) expression and can thereby promote tumor-induced angiogenesis both in vitro and in vivo. Furthermore, in breast tumors, SOX4 expression correlates with blood vessel density and size, and predicts poor-prognosis in patients with breast cancer. Our data provide novel mechanistic insights into context-dependent SOX4 target gene selection, and uncover a novel pro-oncogenic role for this transcription factor in promoting tumor-induced angiogenesis. These findings establish a key role for SOX4 in promoting metastasis through exploiting diverse pro-tumorigenic pathways.

Functional Dissection of the CCBE1 Protein: A Crucial Requirement for the Collagen Repeat Domain.

RATIONALE: Collagen- and calcium-binding EGF domain-containing protein 1 (CCBE1) is essential for lymphangiogenesis in vertebrates and has been associated with Hennekam syndrome. Recently, CCBE1 has emerged as a crucial regulator of vascular endothelial growth factor-C (VEGFC) signaling. OBJECTIVE: CCBE1 is a secreted protein characterized by 2 EGF domains and 2 collagen repeats. The functional role of the different CCBE1 protein domains is completely unknown. Here, we analyzed the functional role of the different CCBE1 domains in vivo and in vitro. METHODS AND RESULTS: We analyzed the functionality of several CCBE1 deletion mutants by generating knock-in mice expressing these mutants, by analyzing their ability to enhance Vegfc signaling in vivo in zebrafish, and by testing their ability to induce VEGFC processing in vitro. We found that deleting the collagen domains of CCBE1 has a much stronger effect on CCBE1 activity than deleting the EGF domains. First, although CCBE1ΔCollagen mice fully phenocopy CCBE1 knock-out mice, CCBE1ΔEGF knock-in embryos still form rudimentary lymphatics. Second, Ccbe1ΔEGF, but not Ccbe1ΔCollagen, could partially substitute for Ccbe1 to enhance Vegfc signaling in zebrafish. Third, CCBE1ΔEGF, similarly to CCBE1, but not CCBE1ΔCollagen could activate VEGFC processing in vitro. Furthermore, a Hennekam syndrome mutation within the collagen domain has a stronger effect than a Hennekam syndrome mutation within the EGF domain. CONCLUSIONS: We propose that the collagen domains of CCBE1 are crucial for the activation of VEGFC in vitro and in vivo. The EGF domains of CCBE1 are dispensable for regulation of VEGFC processing in vitro, however, they are necessary for full lymphangiogenic activity of CCBE1 in vivo.

Divergence of zebrafish and mouse lymphatic cell fate specification pathways.

In mammals, the homeodomain transcription factor Prox1 acts as the central regulator of lymphatic cell fate. Its restricted expression in a subset of cardinal vein cells leads to a switch towards lymphatic specification and hence represents a prerequisite for the initiation of lymphangiogenesis. Murine Prox1-null embryos lack lymphatic structures, and sustained expression of Prox1 is indispensable for the maintenance of lymphatic cell fate even at adult stages, highlighting the unique importance of this gene for the lymphatic lineage. Whether this pre-eminent role of Prox1 within the lymphatic vasculature is conserved in other vertebrate classes has remained unresolved, mainly owing to the lack of availability of loss-of-function mutants. Here, we re-examine the role of Prox1a in zebrafish lymphangiogenesis. First, using a transgenic reporter line, we show that prox1a is initially expressed in different endothelial compartments, becoming restricted to lymphatic endothelial cells only at later stages. Second, using targeted mutagenesis, we show that Prox1a is dispensable for lymphatic specification and subsequent lymphangiogenesis in zebrafish. In line with this result, we found that the functionally related transcription factors Coup-TFII and Sox18 are also dispensable for lymphangiogenesis. Together, these findings suggest that lymphatic commitment in zebrafish and mice is controlled in fundamentally different ways.

Mutation in vascular endothelial growth factor-C, a ligand for vascular endothelial growth factor receptor-3, is associated with autosomal dominant milroy-like primary lymphedema.

RATIONALE: Mutations in vascular endothelial growth factor (VEGF) receptor-3 (VEGFR3 or FLT4) cause Milroy disease, an autosomal dominant condition that presents with congenital lymphedema. Mutations in VEGFR3 are identified in only 70% of patients with classic Milroy disease, suggesting genetic heterogeneity. OBJECTIVE: To investigate the underlying cause in patients with clinical signs resembling Milroy disease in whom sequencing of the coding region of VEGFR3 did not reveal any pathogenic variation. METHODS AND RESULTS: Exome sequencing of 5 such patients was performed, and a novel frameshift variant, c.571_572insTT in VEGFC, a ligand for VEGFR3, was identified in 1 proband. The variant cosegregated with the affected status in the family. An assay to assess the biological function of VEGFC activity in vivo, by expressing human VEGFC in the zebrafish floorplate was established. Forced expression of wild-type human VEGFC in the floorplate of zebrafish embryos leads to excessive sprouting in neighboring vessels. However, when overexpressing the human c.571_572insTT variant in the floorplate, no sprouting of vessels was observed, indicating that the base changes have a marked effect on the activity of VEGFC. CONCLUSIONS: We propose that the mutation in VEGFC is causative for the Milroy disease-like phenotype seen in this family. This is the first time a mutation in one of the ligands of VEGFR3 has been reported to cause primary lymphedema.

A similar approach in bracing of adolescent scoliosis and kyphosis with the use of growth itself in Thoracolumbar Lordotic Intervention (TLI).

Using older and new concepts on aetiology a new brace technique with thoracolumbar lordotic intervention in adolescent scoliotic and kyphotic deformities was created. The technique with symmetrical forces in the sagittal plane only on the thoracolumbar joint and its effect on the formative function of the extending muscles is explained.

Intensification of antibiotic use within acute care hospitals in the Netherlands.

OBJECTIVES: To report and analyse trends in antibiotic use in Dutch university hospitals, large teaching hospitals and general hospitals over the period 2003 to 2009. METHODS: Data on the use of antibiotics and hospital resource indicators were obtained by distributing a questionnaire to all Dutch hospital pharmacies. Antibiotic use was expressed as the number of defined daily doses (DDDs) per 100 patient-days, per 100 admissions and per 1000 inhabitants per day. The latter was achieved by extrapolating sample data by means of imputation and up-scaling. RESULTS: From 2003 to 2009, the mean length of hospital stay decreased from 6.27 to 4.50 days (-28%). Total systemic antibiotic use significantly increased from 52.3 to 69.8 DDDs per 100 patient-days (P < 0.001). Despite the overall constant use when expressed in DDDs per 100 admissions, we found a significant increase in the total use of piperacillin/tazobactam, cefazolin, ceftriaxone, meropenem, azithromycin, gentamicin, ciprofloxacin and vancomycin. Mean total systemic use expressed in DDDs per 1000 inhabitants per day gradually increased by 38% from 0.73 in 2003 to 1.01 in 2009. CONCLUSIONS: Total hospital antibiotic consumption is still low in the Netherlands compared with other European countries. Also, between 2003 and 2009 the use of antibiotics in individual hospitalized patients remained stable. However, since they remained in the hospital for a shorter period of time, the number of DDDs per 100 patient-days increased. This results in an intensification of antibiotic treatment per hospital bed, leading to a possible increase in selection pressure towards resistance. This may create a problem for future patients. To limit the emergence and transmission of antimicrobial-resistant bacteria, effective antibiotic stewardship is essential.

Control of endothelial sprouting by a Tel-CtBP complex.

We show that the transcriptional repressor Tel plays an evolutionarily conserved role in angiogenesis: it is indispensable for the sprouting of human endothelial cells and for normal development of the Danio rerio blood circulatory system. Tel orchestrates endothelial sprouting by binding to the generic co-repressor, CtBP. The Tel-CtBP complex temporally restricts a VEGF (vascular endothelial growth factor)-mediated pulse of dll4 expression and thereby directly links VEGF receptor intracellular signalling and intercellular Notch-Dll4 signalling. It further controls branching by regulating expression of other factors that constrain angiogenesis such as sprouty family members and ve-cadherin. Thus, the Tel-CtBP complex conditions endothelial cells for angiogenesis by controlling the balance between stimulatory and antagonistic sprouting cues. Tel control of branching seems to be a refinement of invertebrate tracheae morphogenesis that requires Yan, the invertebrate orthologue of Tel. This work highlights Tel and its associated networks as potential targets for the development of therapeutic strategies to inhibit pathological angiogenesis.

Downregulation of vertebrate Tel (ETV6) and Drosophila Yan is facilitated by an evolutionarily conserved mechanism of F-box-mediated ubiquitination.

The vertebrate Ets transcriptional repressor Tel (ETV6) and its invertebrate orthologue, Yan, are both indispensable for development, and they orchestrate cell growth and differentiation by binding to DNA, thus inhibiting gene expression. To trigger cell differentiation, these barriers to transcriptional activation must be relieved, and it is established that posttranslational modifications, such as phosphorylation and sumoylation, can specifically impair the repressive functions of Tel and Yan and are crucial for modulating their transcriptional activity. To date, however, relatively little is known about the control of Tel and Yan protein degradation. In recent years, there has been a concentrated effort to assign functions to the large number of F-box proteins encoded by both vertebrate and invertebrate genomes. Here, we report the identification and characterization of a previously unreported, evolutionarily conserved F-box protein named Fbl6. We isolated both human and Drosophila melanogaster fbl6 cDNA and show that the encoded Fbl6 protein binds to both Tel and Yan via their SAM domains. We demonstrate that both Tel and Yan are ubiquitinated, a process which is stimulated by Fbl6 and leads to proteasomal degradation. We recently established that the sumoylation of Tel on lysine 11 negatively regulates its repressive function and that the sumoylation of Tel monomers, but not that of Tel oligomers, may sensitize Tel for proteasomal degradation. Here, we found that Fbl6 regulates Tel/Yan protein stability and allows appropriate spatiotemporal control of gene expression by these repressors.

Identification of a new site of sumoylation on Tel (ETV6) uncovers a PIAS-dependent mode of regulating Tel function.

Cell proliferation and differentiation are governed by a finely controlled balance between repression and activation of gene expression. The vertebrate Ets transcriptional repressor Tel (ETV6) and its invertebrate orthologue Yan, play pivotal roles in cell fate determination although the precise mechanisms by which repression of gene expression by these factors is achieved are not clearly defined. Here, we report the identification and characterization of the primary site of sumoylation of Tel, lysine 11 (K11), which is highly conserved in vertebrates (except Danio rerio). We demonstrate that in cells PIAS3 binds to Tel and stimulates sumoylation of K11 in the nucleus. Both Tel monomers and oligomers are efficiently sumoylated on K11 in vitro; but in cells only Tel oligomers are found conjugated with SUMO, whereas sumoylation of Tel monomers is transitory and appears to sensitize them for proteasomal degradation. Mechanistically, sumoylation of K11 inhibits repression of gene expression by full-length Tel. In accordance with this observation, we found that sumoylation impedes Tel association with DNA. By contrast, a Tel isoform lacking K11 (TelM43) is strongly repressive. This isoform results from translation from an alternative initiation codon (M43) that is common to all Tel proteins that also contain the K11 sumoylation consensus site. We find that PIAS3 may have a dual, context-dependent influence on Tel; it mediates Tel sumoylation, but it also augments Tel's repressive function in a sumoylation-independent fashion. Our data support a model that suggests that PIAS-mediated sumoylation of K11 and the emergence of TelM43 in early vertebrates are linked and that this serves to refine spatiotemporal control of gene expression by Tel by establishing a pool of Tel molecules that are available either to be recycled to reinforce repression of gene expression or are degraded in a regulated fashion.

Reliable gastric tonometry after coronary artery surgery: need for acid secretion suppression despite transient failure of acid secretion.

OBJECTIVE: To study the need for suppression of gastric acid secretion for reliable intragastric partial pressure of carbon dioxide (PCO2) tonometry by evaluating the effect of an oral dose of sodium bicarbonate before and after administration of the H2-blocker ranitidine to mimic CO2 generation following the buffering of acid by bicarbonate in patients after cardiac surgery. DESIGN: Prospective, open, non-randomized clinical study. SETTING: Cardiothoracic intensive care unit at a university hospital. PATIENTS: 10 patients after elective coronary artery bypass surgery. INTERVENTIONS: An oral dose of 500 mg sodium bicarbonate before and after acid secretion suppression by 100 mg ranitidine as an intravenous bolus given at approximately 3 h after surgery (day 0) and on the first postoperative day (day 1). MEASUREMENTS AND RESULTS: Intragastric PCO2 (iPCO2; tonometry), gastric juice pH (aspirate) and arterial blood gas values were measured. On day 0, the iPCO2 was 25 +/- 5 mmHg before and 31 +/- 5 mmHg after the bicarbonate dose, 29 +/- 5 mmHg after ranitidine infusion, and 31 +/- 5 mmHg after the bicarbonate dose following the ranitidine infusion (NS). On day 1, the basal iPCO2 was 32 +/- 4 mmHg and it increased to 56 +/- 25 mmHg following bicarbonate (p < 0.01). After ranitidine, the iPCO2 was 33 +/- 4 mmHg before and 40 +/- 14 mmHg after bicarbonate (NS). Basal gastric juice pH was > 4 in nine of ten patients on day 0 and > 4 in seven of ten patients on day 1. CONCLUSIONS: Pharmacological suppression of gastric acid secretion is mandatory for reliable iPCO2 tonometry after cardiopulmonary bypass surgery, even when gastric acid secretion is transiently inhibited. In fact, gastric acid secretion was inhibited immediately after surgery, but returned on the first postoperative day in most patients, as judged from the bicarbonate back titration of gastric acid, even when gastric juice pH was relatively high.

Clinical presentation and prognosis of left main coronary artery disease in the 1980s.

Clinical presentation and course were studied in 127 consecutive patients with angiographically proven left main coronary artery disease. Mean age was 62 (37-79) years. Thirteen patients (10%) had no history of chest pain, seven (5%) had atypical chest pain, and the remaining 107 (85%) typical angina pectoris. Eighty-two patients (65%) had unstable angina, 73 had suffered a myocardial infarction (MI) in the past, and 50 (68%) had post MI angina pectoris. The electrocardiogram was analysed in 102/125 patients during an episode of chest pain and also when they were without chest pain. Outside an episode of chest pain the ST segment was normal in 42 patients (32%), the T wave was normal in 50 patients (38%) and both the ST and T were normal in 33 patients (25%). During chest pain all patients had an abnormal ECG, the most frequent pattern being ST segment depression in leads V3, V4 and V5 (with maximal depression in V4), and ST segment elevation in leads V1 and aVR. The average number of leads with ST-T abnormalities was 6.4. A symptom-limited exercise test on a treadmill with 12-lead ECG monitoring was performed in 89 patients. The exercise test was abnormal in 88 patients (99%), most of whom (74 patients) were already in the first or second stage of the Bruce protocol. The most frequently observed abnormality was ST segment depression of 2 mm or more in leads V4, V5, and V6, and ST segment elevation in leads V1 and aVR. The systolic blood pressure during exercise fell or remained at the same level in 38 patients (43%).(ABSTRACT TRUNCATED AT 250 WORDS)

Clinical and electrophysiologic characteristics of patients with antidromic circus movement tachycardia in the Wolff-Parkinson-White syndrome.

Antidromic circus movement tachycardia was documented in 36 of 345 consecutive patients with Wolff-Parkinson-White syndrome undergoing detailed electrophysiologic evaluation. Twenty-six patients were men and 10 were women (mean age +/- standard deviation 26 +/- 12 years [range 12 to 45]). Multiple accessory pathways were identified in 12 of these 36 patients (33%). Ten of the patients (67%) with clinically documented antidromic tachycardia had multiple accessory pathways. Dizziness and syncope occurred in 61 and 50% of patients with antidromic circus movement tachycardia. Six patients had clinical documentation of atrial fibrillation, and 4 patients (11%) were resuscitated from ventricular fibrillation. In the 36 patients, 56 distinct antidromic tachycardias were recorded and several different pathways were observed. Orthodromic tachycardia was the most frequently associated arrhythmia (72%). Dual atrioventricular nodal pathways were present in 12 patients (33%); however, atrioventricular nodal tachycardia could be initiated in only 2 of them. Interruption of the accessory pathway was successfully performed in all 20 patients undergoing surgery.