The Atonal Proneural Transcription Factor Links Differentiation and Tumor Formation in Drosophila

The acquisition of terminal cell fate and onset of differentiation are instructed by cell type–specific master control genes. Loss of differentiation is frequently observed during cancer progression, but the underlying causes and mechanisms remain poorly understood. We tested the hypothesis that master regulators of differentiation may be key regulators of tumor formation. Using loss- and gain-of-function analyses in Drosophila, we describe a critical anti-oncogenic function for the atonal transcription factor in the fly retina, where atonal instructs tissue differentiation. In the tumor context, atonal acts by regulating cell proliferation and death via the JNK stress response pathway. Combined with evidence that atonal's mammalian homolog, ATOH1, is a tumor suppressor gene, our data support a critical, evolutionarily conserved, function for ato in oncogenesis

Imaging strategies for detection of urgent conditions in patients with acute abdominal pain: diagnostic accuracy study

Objective To identify an optimal imaging strategy for the accurate detection of urgent conditions in patients with acute abdominal pain

Atonal homolog 1 Is a Tumor Suppressor Gene

Colon cancer accounts for more than 10% of all cancer deaths annually. Our genetic evidence from Drosophila and previous in vitro studies of mammalian Atonal homolog 1 (Atoh1, also called Math1 or Hath1) suggest an anti-oncogenic function for the Atonal group of proneural basic helix-loop-helix transcription factors. We asked whether mouse Atoh1 and human ATOH1 act as tumor suppressor genes in vivo. Genetic knockouts in mouse and molecular analyses in the mouse and in human cancer cell lines support a tumor suppressor function for ATOH1. ATOH1 antagonizes tumor formation and growth by regulating proliferation and apoptosis, likely via activation of the Jun N-terminal kinase signaling pathway. Furthermore, colorectal cancer and Merkel cell carcinoma patients show genetic and epigenetic ATOH1 loss-of-function mutations. Our data indicate that ATOH1 may be an early target for oncogenic mutations in tissues where it instructs cellular differentiation

A comparison of the Accuracy of Ultrasound and Computed Tomography in common diagnoses causing acute abdominal pain

Head-to-head comparison of ultrasound and CT accuracy in common diagnoses causing acute abdominal pain. Consecutive patients with abdominal pain for > 2 h and <5 days referred for imaging underwent both US and CT by different radiologists/radiological residents. An expert panel assigned a final diagnosis. Ultrasound and CT sensitivity and predictive values were calculated for frequent final diagnoses. Effect of patient characteristics and observer experience on ultrasound sensitivity was studied. Frequent final diagnoses in the 1,021 patients (mean age 47; 55% female) were appendicitis (284; 28%), diverticulitis (118; 12%) and cholecystitis (52; 5%). The sensitivity of CT in detecting appendicitis and diverticulitis was significantly higher than that of ultrasound: 94% versus 76% (p <0.01) and 81% versus 61% (p = 0.048), respectively. For cholecystitis, the sensitivity of both was 73% (p = 1.00). Positive predictive values did not differ significantly between ultrasound and CT for these conditions. Ultrasound sensitivity in detecting appendicitis and diverticulitis was not significantly negatively affected by patient characteristics or reader experience. CT misses fewer cases than ultrasound, but both ultrasound and CT can reliably detect common diagnoses causing acute abdominal pain. Ultrasound sensitivity was largely not influenced by patient characteristics and reader experience

The FOAM study : Is Hysterosalpingo foam sonography (HyFoSy) a cost-effective alternative for hysterosalpingography (HSG) in assessing tubal patency in subfertile women? Study protocol for a randomized controlled trial

This is an investigator initiated trial, VU medical center Amsterdam is the sponsor, contact information: prof. CJM de Groot, Department of Obstetrics and Gynaecology, De Boelelaan 1117, 1081 HV Amsterdam, The Netherlands, Tel: + 31-204444444. This study is funded by ZonMw, a Dutch organization for Health Research and Development, project number 837001504. ZonMW gives financial support for the whole project. IQ Medical Ventures provides the ExEm FOAM® kits. The funding bodies have no role in the design of the study; collection, analysis, and interpretation of data; and in writing the manuscript.Peer reviewedPublisher PD

Can hysterosalpingo-foam sonography replace hysterosalpingography as first-choice tubal patency test? A randomized non-inferiority trial

Funding Information: The FOAM study was an investigator-initiated study funded by ZonMw, The Netherlands organization for Health Research and Development (project number 837001504). ZonMw funded the whole project. IQ Medical Ventures provided the ExEm-foamVR kits free of charge. The funders had no role in study design, collection, analysis and interpretation of the data. The corresponding author had full access to all the data in the study and had final responsibility for the decision to submit for publication.Peer reviewedPublisher PD

Abortive Autophagy Induces Endoplasmic Reticulum Stress and Cell Death in Cancer Cells

Autophagic cell death or abortive autophagy has been proposed to eliminate damaged as well as cancer cells, but there remains a critical gap in our knowledge in how this process is regulated. The goal of this study was to identify modulators of the autophagic cell death pathway and elucidate their effects on cellular signaling and function. The result of our siRNA library screenings show that an intact coatomer complex I (COPI) is obligatory for productive autophagy. Depletion of COPI complex members decreased cell survival and impaired productive autophagy which preceded endoplasmic reticulum stress. Further, abortive autophagy provoked by COPI depletion significantly altered growth factor signaling in multiple cancer cell lines. Finally, we show that COPI complex members are overexpressed in an array of cancer cell lines and several types of cancer tissues as compared to normal cell lines or tissues. In cancer tissues, overexpression of COPI members is associated with poor prognosis. Our results demonstrate that the coatomer complex is essential for productive autophagy and cellular survival, and thus inhibition of COPI members may promote cell death of cancer cells when apoptosis is compromised

Hysterosalpingo-foam sonography versus hysterosalpingography during fertility work-up: an economic evaluation alongside a randomized controlled trial

STUDY QUESTION: What are the costs and effects of tubal patency testing by hysterosalpingo-foam sonography (HyFoSy) compared to hysterosalpingography (HSG) in infertile women during the fertility work-up? SUMMARY ANSWER: During the fertility work-up, clinical management based on the test results of HyFoSy leads to slightly lower, though not statistically significant, live birth rates, at lower costs, compared to management based on HSG results. WHAT IS KNOWN ALREADY: Traditionally, tubal patency testing during the fertility work-up is performed by HSG. The FOAM trial, formally a non-inferiority study, showed that management decisions based on the results of HyFoSy resulted in a comparable live birth rate at 12 months compared to HSG (46% versus 47%; difference −1.2%, 95% CI: −3.4% to 1.5%; P¼ 0.27). Compared to HSG, HyFoSy is associated with significantly less pain, it lacks ionizing radiation and exposure to iodinated contrast medium. Moreover, HyFoSy can be performed by a gynaecologist during a one-stop fertility work-up. To our knowledge, the costs of both strategies have never been compared. STUDY DESIGN, SIZE, DURATION: We performed an economic evaluation alongside the FOAM trial, a randomized multicenter study conducted in the Netherlands. Participating infertile women underwent, both HyFoSy and HSG, in a randomized order. The results of both tests were compared and women with discordant test results were randomly allocated to management based on the results of one of the tests. The follow-up period was twelve months. PARTICIPANTS/MATERIALS, SETTING, METHODS: We studied 1160 infertile women (18–41 years) scheduled for tubal patency testing. The primary outcome was ongoing pregnancy leading to live birth. The economic evaluation compared costs and effects of management based on either test within 12 months. We calculated incremental cost-effectiveness ratios (ICERs): the difference in total costs and chance of live birth. Data were analyzed using the intention to treat principle. MAIN RESULTS AND THE ROLE OF CHANCE: Between May 2015 and January 2019, 1026 of the 1160 women underwent both tubal tests and had data available: 747 women with concordant results (48% live births), 136 with inconclusive results (40% live births), and 143 with discordant results (41% had a live birth after management based on HyFoSy results versus 49% with live birth after management based on HSG results). When comparing the two strategies—management based on HyfoSy results versus HSG results—the estimated chance of live birth was 46% after HyFoSy versus 47% after HSG (difference −1.2%; 95% CI: −3.4% to 1.5%). For the procedures itself, HyFoSy cost e136 and HSG e280. When costs of additional fertility treatments were incorporated, the mean total costs per couple were e3307 for the HyFoSy strategy and e3427 for the HSG strategy (mean difference e−119; 95% CI: e−125 to e−114). So, while HyFoSy led to lower costs per couple, live birth rates were also slightly lower. The ICER was e10 042, meaning that by using HyFoSy instead of HSG we would save e10 042 per each additional live birth lost. LIMITATIONS, REASONS FOR CAUTION: When interpreting the results of this study, it needs to be considered that there was a considerable uncertainty around the ICER, and that the direct fertility enhancing effect of both tubal patency tests was not incorporated as women underwent both tubal patency tests in this study. WIDER IMPLICATION OF THE FINDINGS: Compared to clinical management based on HSG results, management guided by HyFoSy leads to slightly lower live birth rates (though not statistically significant) at lower costs, less pain, without ionizing radiation and iodinated contrast exposure. Further research on the comparison of the direct fertility-enhancing effect of both tubal patency tests is needed. STUDY FUNDING/COMPETING INTEREST(S): FOAM trial was an investigator-initiated study, funded by ZonMw, a Dutch organization for Health Research and Development (project number 837001504). IQ Medical Ventures provided the ExEm®-FOAM kits free of charge. The funders had no role in study design, collection, analysis, and interpretation of the data. K.D. reports travel-and speakers fees from Guerbet and her department received research grants from Guerbet outside the submitted work. H.R.V. received consulting—and travel fee from Ferring. A.M.v.P. reports received consulting fee from DEKRA and fee for an expert meeting from Ferring, both outside the submitted work. C.H.d.K. received travel fee from Merck. F.J.M.B. received a grant from Merck and speakers fee from Besins Healthcare. F.J.M.B. is a member of the advisory board of Merck and Ferring. J.v.D. reported speakers fee from Ferring. J.S. reports a research agreement with Takeda and consultancy for Sanofi on MR of motility outside the submitted work. M.v.W. received a travel grant from Oxford Press in the role of deputy editor for Human Reproduction and participates in a DSMB as independent methodologist in obstetrics studies in which she has no other role. B.W.M. received an investigator grant from NHMRC GNT1176437. B.W.M. reports consultancy for ObsEva, Merck, Guerbet, iGenomix, and Merck KGaA and travel support from Merck KGaA. V.M. received research grants from Guerbet, Merck, and Ferring and travel and speakers fees from Guerbet. The other authors do not report conflicts of interest. TRIAL REGISTRATION NUMBER: International Clinical Trials Registry Platform No. NTR4746

Cancer and Differentiation: a tumour suppressor function for atonal-group genes

Loss of differentiation during oncogenesisis generally thought to be a coincidental, rather than a causal, aspectoftumour progression. We tested whether master regulators of differentiationcould have more direct roles in tumour formation by acting as tumour suppressorgenes. In this work we demonstrate that the transcription factor atonal, which is necessary fordifferentiation in the Drosophilaeye, and ATOH1, which is essential forthe differentiation of Merkel cells in the skin and secretory cells in theintestine, shows all the hallmarks of a tumour suppressor gene. To investigatethe possible tumour suppressor function, we looked in tumours derived fromtissues which depend on ato-orthologuesto differentiate, namely eye tumours in Drosophila,two mouse models for colon adenocarcinoma (CAC), Merkel cell carcinoma (MCC) and CAC samples from patients and in celllines derived from MCC and CAC patients. Gain of atonalfunction leads to strong suppression of tumour formation in Drosophila.Conversely, loss of atonal (ato) in the Drosophila cancermodel enhances tumour progression and denovo tumour formation. We show that atofunctions by enhancing apoptosis and by the expression of cell cycle inhibitors.The effects of ato depend on JNKsignalling pathway. In addition, the mouse orthologue Atoh1 shows all the hallmarks of atumour suppressor gene. To this end, we conditionally ablated Atoh1 in the gut in two mouse models of CAC.The first model uses chemical carcinogenesis, the second sensitizes themice bymutations in the APC gene. In both cases, a significant increase in theincidence, the number as well as the size of the tumours is observed. Knock-outof Atoh1 in pre-neoplastic colonepithelium leads to more proliferation but no more apoptosis. In agreement with the functional data inthe Drosophila and the mouse model, humanCAC and MCC tumours show a marked decrease in the expression of ATOH1 opposed to control tissue. Moreaggressive stages of CAC have less ATOH1expression than less malignant tumours. This is due to genetic and epigeneticmutations in the form of deletions and methylation of the ATOH1 locus. Finally, gain and loss of function of ATOH1 in cell lines derived from CAC andMCC lead to a decrease and increase in growth, respectively. The decrease ingrowth upon Atoh1 activation ismediated by an increase in apoptosis and a slowing of the cell cycle. ATOH1 functions by activation ofreceptor tyrosine kinase signalling leading to phosphorylation of JNK.Similarly like in Drosophila, theeffects of ATOH1 are mediated by JNK.ABSTRACT III SAMENVATTING V ACKNOWLEDGEMENTS VII LIST OF ABBREVIATIONS IX TABLE OF CONTENTS - 1 - 1 INTRODUCTION - 5 - 1.1 ORIGIN OF CANCER - 5 - 1.1.1 Stem cells in oncogenesis - 6 - 1.1.2 Stem cell biology - 6 - 1.2 DIFFERENTIATION AND CANCER - 9 - 1.2.1 Differentiation and the cell cycle: establishing a point-of-no-return - 10 - 1.2.1.1 Transcriptional regulation - 11 - 1.2.1.2 Degrade towards differentiation - 13 - 1.2.1.3 Maintain your inhibition - 14 - 1.2.2 Differentiation factors implicated in cancer - 15 - 1.3 ATONAL, ATOH1, ATOH1: MANY NAMES, EVEN MORE FUNCTIONS. - 19 - 1.3.1 Regulation of downstream genes by atonal - 20 - 1.3.2 A progenitor-specifying function for atonal orthologues - 22 - 1.3.3 Terminal differentiation function for atonal orthologues - 24 - 1.3.4 ATOH1 implicated in tumours - 27 - 1.4 DROSOPHILA AS A MODEL FOR CANCER - 29 - 1.5 MERKEL CELL CARCINOMA - 35 - 1.6 COLON ADENOCARCINOMA - 37 - 2 AIMS - 41 - 3 METHODOLOGY - 43 - 3.1 DROSOPHILA HUSBANDRY - 43 - 3.2 IMMUNOHISTOCHEMISTRY - 43 - 3.3 GENERATION OF UAS- ATOERD TRANSGENIC FLIES - 43 - 3.4 QRT-PCR ON DROSOPHILA LARVAL EYE-ANTENNAL DISCS - 44 - 3.5 CLONING OF ATOH1ERD EXPRESSION CONSTRUCT - 44 - 3.6 ANIMALS AND TREATMENTS - 44 - 3.7 IMMUNOHISTOCHEMISTRY AND MICROSCOPIC ANALYSIS - 45 - 3.8 CELL CULTURE CONDITIONS - 46 - 3.9 DOUBLING TIME - 47 - 3.10 LENTIVIRAL VECTORS - 47 - 3.11 COLONY FORMATION IN SOFT AGAR - 48 - 3.12 CELL CYCLE DISTRIBUTION - 49 - 3.13 APOPTOSIS DETECTION - 49 - 3.14 WESTERN BLOTTING - 49 - 3.15 TYROSINE KINASE INHIBITOR, SAPK INHIBITOR II AND GAMMA-SECRETASE INHIBITOR - 50 - 3.16 BRDU STAINING - 50 - 3.17 RT-PCR AND QPCR - 51 - 3.18 90 KINASE RT-PCR ASSAY - 52 - 3.19 METHYLATION DETECTION - 52 - 3.20 ARRAY CGH - 53 - 3.21 SEQUENCING - 54 - 4 RESULTS - 55 - 4.1 ATONAL ACTS AS A TUMOUR SUPPRESSOR IN A DROSOPHILA CANCER PARADIGM - 55 - 4.1.1 Gain and loss of function of atonal, respectively repress and facilitate cancer progression - 55 - 4.1.2 ato functions through JNK-dependent inhibition of cell cycle and induction of apoptosis - 58 - 4.2 ATOH1 FUNCTIONS AS A TUMOUR SUPPRESSOR GENE IN TWO MOUSE MODELS OF CAC. - 61 - 4.3 ATOH1 IS GENETICALLY AND EPIGENETICALLY MUTATED IN HUMAN CAC AND MCC TUMOURS - 65 - 4.3.1 Frequent deletions in the ATOH1 locus - 65 - 4.3.2 ATOH1 locus is methylated during oncogenesis - 66 - 4.4 ATOH1 ACTS BY SIMILAR MECHANISMS IN HUMAN CANCER CELLS AS IN DROSOPHILA - 71 - 4.4.1 High levels of ATOH1 correlate with higher doubling times and decreased malignancy - 71 - 4.4.2 ATOH1 is not dependent on Notch signalling - 75 - 4.4.3 ATOH1 tumour suppressor function is mediated by RTK’s - 76 - 5 DISCUSSION - 81 - 5.1 ATOH1 IS A TUMOUR SUPPRESSOR: THREE PREDICTIONS - 81 - 5.2 ATONAL’S MODE OF ACTION - 82 - 5.3 THE ATOH1 LOCUS: A SPECIAL CASE - 85 - 5.4 ATOH1 STATUS MIGHT BE LINKED TO DISEASE STAGE AND OUTCOME - 88 - 5.5 LOSS OF DIFFERENTIATION AND CANCER: A “CONDITIO SINE QUA NON”? - 89 - 6 REFERENCES - 93 - 7 CURRICULUM VITAE - 105 -status: publishe