Epithelial ovarian cancer (EOC) is a spectrum of different diseases, which makes their treatment a challenge. Forkhead box M1 (FOXM1) is an oncogene aberrantly expressed in many solid cancers including serous EOC, but its role in non-serous EOCs remains undefined. We examined FOXM1 expression and its correlation to prognosis across the three major EOC subtypes, and its role in tumorigenesis and chemo-resistance in vitro.Gene signatures were generated by microarray for 14 clear-cell and 26 endometrioid EOCs, and 15 normal endometrium snap-frozen biopsies. Validation of FOXM1 expression was performed by RT–qPCR and immunohistochemistry in the same samples and additionally in 50 high-grade serous EOCs and in their most adequate normal controls . Correlations of FOXM1 expression to clinic-pathological parameters and patients’ prognosis were evaluated by Kaplan-Meier and Cox proportional-hazards analyses. OVCAR-3 and two novel deeply characterized EOC cell lines  were employed for in vitro studies. Effects of FOXM1 inhibition by transient siRNA transfection were evaluated on cell-proliferation, cell-cycle, colony formation, invasion, and response to conventional first- and second-line anticancer agents, and to the PARP-inhibitor olaparib. Gene signatures of FOXM1-silenced cell lines were generated by microarray and confirmed by RT-qPCR.p = 0.001) and advanced FIGO stage (p = 0.004). Multivariate analyses confirmed FOXM1 protein overexpression as an independent indicator of worse disease specific survival in non-serous EOCs, and of shorter time to progression in platinum-resistant cases. FOXM1 downregulation in EOC cell lines inhibited cell growth and clonogenicity, and promoted the cytotoxic effects of platinum compounds, doxorubicin hydrochloride and olaparib. Upon FOXM1 knock-down in EOC-CC1 and OSPC2 cells, microarray and RT-qPCR analyses revealed the deregulation of several common and other unique subtype-specific FOXM1 putative targets involved in cell cycle, metastasis, DNA repair and drug response.A significant FOXM1 mRNA up-regulation was found in EOCs compared to normal controls. FOXM1 protein overexpression significantly correlated to serous histology  contains supplementary material, which is available to authorized users. BRCA1 and BRCA2 mutations, who have received three or more prior lines of chemotherapy [Epithelial ovarian cancer (EOC) represents 90% of ovarian cancers, being also associated to very heterogeneous clinical presentation, histological features and therapeutic response . The higotherapy . MoreoveNonetheless, further advancements in the management of recurrent or persistent disease are still required, especially for drug-resistant EOCs that generally show poor responsiveness to additional cytotoxic therapy. Transcriptional profiling represents a useful tool to identify tissue-specific therapeutic targets that impact on clinical outcome.Several studies show that the transcription factor Forkhead box M1 (FOXM1) is widely expressed in solid tumors , acting In the present investigation, we generated the mRNA signatures of tumor specimens belonging to two EOC patients’ cohorts representing the less common cancer subtypes (endometrioid and clear-cell), and of their normal counterpart (endometrium samples). Next, we explored the trend in FOXM1 expression at mRNA and protein level across clear-cell, endometrioid and HGS EOCs compared to their supposed normal tissue of origin . We investigated the association between FOXM1 protein and patients’ survival in relation to their tumor histology and sensitivity to platinum-based therapy. We determined the functional activity of FOXM1 in EOC cells in terms of cell proliferation, cell-cycle regulation, colony formation, and invasion ability by transient FOXM1 knockdown, using two in-house derived EOC cell lines expressing FOXM1, as in vitro models that accurately represent a metastatic HGSC and a primary clear-cell EOC. HGSC is the most frequent, lethal and extensively studied EOC subtype , whereasSince FOXM1 is an emerging master regulator of response to DNA damage , in thisThis study was performed on 90 cases of EOCs diagnosed and treated at the Division of Gynecologic Oncology, Department of Obstetrics and Gynecology, University of Brescia, Italy, between November 2001 and September 2013, following the Declaration of Helsinki set of principles, and with approval by the Research Review Board- the Ethics Committee- of the ASST Spedali Civili, Brescia, Italy (study reference number: NP1284). Normal control tissue samples were obtained from 45 patients undergoing surgery for benign pathologies. Written informed consent was obtained from all patients enrolled.Patients’ details are reported in Table Disease Specific survival (DSS) was defined as the time from surgery to death from disease or the last follow-up. Progression Free Survival (PFS) was calculated from the time of surgery until the first clinical recurrence/progression. Cancer progression was defined according to RECIST 1.1 . The PFINinety EOC specimens were collected at the time of primary debulking and immediately frozen as previously described . Normal Ten normal luminal fallopian tube epithelial cells (TEC) and 6 pools of ovarian surface epithelial-cell (OSE) brushings obtained from 20 patients, were separately collected by scraping in 1 ml of physiological saline solution immediately after surgery, as previously described .Two new EOC cell lines (named OSPC2 and EOC-CC1) were derived in our laboratory from fresh clinical samples. Source-patient characteristics and full method of cell line characterization  DNA profiling, BRCA1/2 sequencing and growth rate analysis) are described in Additional file The following Silencer® select pre-designed siRNAs  were employed for in vitro transient gene knockdown: FOXM1 (ID # s5248), negative control no.1 (AM4636) and KIF11 (Eg5) (ID # AM4639). Based on cell lines’ growth-rate analysis  previously verified to be free of any neoplastic pathology, and from EOC cell lines after siRNA transfection. For both tissues and cells, TRIzolⓇ Reagent (ThermoFisher) was used according to manufacturer’s instructions. Total RNA extraction and quality control were performed as previously reported .N = 26) and clear-cell histology (N = 14), as well as NE samples (N = 15), were labelled and hybridized to GeneChip® Human Genome U133 Plus 2.0 oligonucleotide microarray chips  following the manufacturer’s protocols. Microarray experiments on scramble- (siControl) and FOXM1-siRNA transfected (siFOXM1) OSPC2 and EOC-CC1 cell lines were performed using the commercially available G4851B human whole GE Microarray kit (SurePrint G3 Human Gene Expression 8 × 60 K v2 Microarray Kit Agilent Technologies) according to manufacturer’s instructions.EOC samples of endometrioid  EOC tissues , stored in the Department of Pathology at the University of Brescia, Italy. As controls, specimens obtained from normal ovaries, fallopian tubes and endometria were used. FOXM1 immunostaining was performed using a primary antibody  diluted at 1:160 after pretreatment with microwave in citrate buffer at pH 6.0 (3 cycles of 5 min at 750 Watt). Immunoreactivity for FOXM1 was considered positive in tumor cells showing a nuclear staining, with or without cytoplasmic staining. FOXM1-immunostained slides were digitalized using an Aperio ScanScope CS Slide Scanner  at 40× magnification, and analyzed using Tissue Studio™ 2.0 workstation . Subsequently, a quantitative scoring algorithm was customized for FOXM1 using commercially available templates from Aperio Technologies and Definiens, and applied in order to quantify the percentage of FOXM1-positive tumor cells and to assess FOXM1 nuclear-staining intensity according to mean brown chromogen intensity. The overall FOXM1 expression “positive index” was determined by multiplying the percentage of the positive cells and the intensity score, then a weighted sum was calculated for each tumor samples. A final immunoreactive score (IRS) from 2 to 90 was calculated.One μg of extracted RNA was reverse-transcribed using random hexamers according to the SuperScript TM II protocol (Invitrogen). The qPCR reactions were performed on CFX96 Touch™ Real-Time PCR Detection System  using the TaqMan Universal PCR master mix and the following Taqman gene expression assays: Hs01073586_m1 (FOXM1), HS99999905_m1 (GAPDH), Hs00188166_m1 (SDHA), Hs00259126_m1 (CCNB1), HS00244740_m1 (CDC25B), Hs00411505_m1 (ASPM), Hs01118845_m1 (CENPF), Hs00921424_m1 (FOXO3), Hs00959834_m1 (XRCC1), Hs00254718_m1 (XRCC4), Hs00998500_g1 (CYR61), Hs00172214_m1 (TOP2A), Hs01037414_m1 (BRCA2), Hs00947967_m1 (RAD51), Hs00766186_m1 (MACC1), Hs01070181_m1 (CEP55), Hs01026371_m1 (CDK6), Hs01548894_m1 (CDK2), Hs00976734_m1 (CXCR4), Hs01548727_m1 (MMP2). Reaction and thermal cycling conditions were performed as previously reported [FOXM1 isoform-specific RT-qPCR was performed using iTaq Universal SYBR Green Supermix (BIO-RAD) and the following primer pairs: FOXM1c F caattgcccgagcacttggaatca, FOXM1c R tccttcagctagcagcaccttg, FOXM1b F ccaggtgtttaagcagcaga, FOXM1b R tccttcagctagcagcaccttg . An anneWhole protein extracts from cells at 24 h following siRNA transfection or untransfection were lysed in NaCl, 1% Nonidet‑40, 50 mM Tris‑HCl (pH 7.5) and Halt Protease Inhibitor Cocktail (Thermo Fisher Scientific), and protein concentrations were determined using a Bio-Rad protein assay system (Bio-Rad). Equivalent amounts of proteins were separated by SDS-PAGE, and then transferred to polyvinylidene difluoride membranes (Bio-Rad). After being blocked in Tris-buffered saline (TBS) containing 5% non-fat milk, the blots were incubated with the following primary antibodies: anti-FOXM1 , and anti-β-actin ((20–33), 1:200 dilution) , at 4 °C for 12 h, followed by incubation with horseradish peroxidase-conjugated secondary (anti-mouse or anti-rabbit) IgGs at room temperature for 1 h. Signals were detected on BioSpectrum® Imaging System  with the LiteAblot® EXTEND (EuroClone). Images were processed with VisionWorks® LS Image Acquisition and Analysis software .Fixed cells were stained with propidium iodide (PI)  at times 0, 24, 48, and 72 h after transfection with scramble siRNA or specific siRNA targeted for FOXM1 and KIF11. Tests were performed in triplicate, and cell-cycle percentages were measured .For accurate cell-cycle analysis, FOXM1- or scramble-treated cells up to 72 h were exposed to 30 μM bromodeoxyuridine (BrdU) and fixed in 70% ethanol into Matrix™ small tubes. Nuclei were isolated using pepsin/0.1 M HCl mixture and subsequently treated with 2 N HCl for 20 min; BrdU detection was performed after DNA denaturation using FITC Anti-BrdU mAb (BD Biosciences) for 1 h and DNA-content staining using PI (2 μg/ml) for 30 min at room temperature . BrdU anFollowing 24 h siRNA transfection, cells were seeded in 6-well plates in 2 ml medium and grown for 7–9 days. Colonies were fixed and stained with 20% ethanol, 1% crystal violet solution and automated counted using the Entry Level image analysis system . A background correction was performed and the control-cell colony size (≥50 cells) was established as the minimum for setting the cut-off point .4 (OVCAR-3), 1 × 105 (OSPC2), 1.4 × 105 (EOC-CC1) cells/well in serum-free medium. RPMI 10% FBS was applied to the lower chamber as chemoattractant. Following 72 h incubation, the invading cells on the lower surface of the membrane were fixed with methanol, stained with 0.5% crystal violet, photographed and counted in 3 randomly selected fields per well, and the mean number of cells was recorded. The experiments were done in triplicate and repeated four times.After 24 h siRNA transfection, cells were seeded on the upper chamber of the BioCoat Matrigel Invasion Chamber (BD Biosciences) at a density of 2 × 10) was dissolved in DMSO (Sigma).Carboplatin, Cisplatin and Doxorubicin hydrochloride (Sigma) were diluted in culture medium immediately before addition to cell lines. Olaparib  , according to manufacturer’s instructions. Absorbance was measured by a 96-well plate reader  at a reference wavelength of 490 nm. Each experiment was repeated a minimum of three times. The effect of drugs on cell growth inhibition was assessed as percent cell viability, where vehicle-treated cells were taken as 100% viable.The prognostic value of FOXM1 was evaluated on serous and non-serous EOC patients’ subgroups. The survival analyses were performed using Cox’s proportional-hazards models  with FOXProliferation data were modelled as normalized counts to a reference and transformed to a logarithm scale. The model was fit by a linear mixed model to account for the variation of technical replicates between independent experiments. Time was expressed on a 24 h scale.All gene chip analyses are explained in detail in Additional file From differentially expressed genes, network reconstruction was carried out in siFOXM1 EOC-CC1 and OSPC2 cell lines, respectively, with the Ingenuity Pathway Analysis software .The differentially expressed genes in each cell line were assessed for “enrichment” or over-representation in a set of 111 invasion-related genes  or SAS v9.4 software , and employed significance levels of alpha = 0.05 except where previously indicated.The correlation between microarray and RT-qPCR data for FOXM1 gene expression was evaluated by Spearman rank correlation. The statistical significance of differences between two groups was evaluated by Mann–Whitney Comprehensive gene expression profiles of 40 EOC specimens of two different histological patterns (26 endometrioid and 14 clear-cell EOCs), and of their relative normal counterpart  were generated using high-density oligonucleotide microarrays, with the aim to identify subtype-specific biomarkers.A total of 1462 genes (468 up-regulated and 994 down-regulated) were found significantly expressed in 26 endometrioid EOCs when compared to 15 NEs , and the 12th-most up-regulated gene in clear-cell EOCs compared to NEs .FOXM1 was the second-most up-regulated gene in endometrioid EOCs compared to NEs , endometrioid EOCs vs NE . The negative values of the correlation coefficients come from the fact that higher Ct means lower expression.FOXM1 expression measured by genechip platform was highly correlated with FOXM1 expression measured on the same samples via confirmatory RT-qPCR: clear-cell EOCs vs NE , and advanced FIGO stage (p = 0.004), as shown in Table FOXM1 protein overexpression was significantly correlated to serous histology  factor and patients were categorized into the high and the low FOXM1 expression groups, respectively. Thresholds for FOXM1 categorization were computed both for DSS and PFS based on time-dependent ROC curves using 24 and 12 months, respectively, as time points that correspond approximately to 75% survival in each outcome. The estimated best thresholds for grouping FOXM1 expression as high versus low were 10.7 for DSS and 13.3 for PFS. DSS was regressed on FOXM1 expression groups via Cox regression with additional covariates being the FIGO stage, platinum resistance, and histotype dichotomized as serous versus non-serous. Given a significant departure from the proportional-hazards assumption, platinum response was entered as a stratification variable. Moreover, an interaction term for FOXM1 and histotype was entered in the model.The effect of FOXM1 expression group on PFS was similarly modelled via Cox regression, with additional covariates again being the FIGO stage, platinum resistance and serous histotype. An interaction term was added for FOXM1 and platinum resistance.p-value = 0.042) in non-serous histotypes   Fig. .Fig. 3Kap-value < 0.001) in partially sensitive/resistant subjects   Fig. .BRCA status and growth rate analyses is reported in Additional file We derived two new cell lines from clinical samples collected from either solid tumor (EOC-CC1) at first surgery or ascites (OSPC2) at disease progression, respectively. A detailed description of their immunocytochemical features, genetic fingerprint, Since it is well known that alternative splicing of the Va (A1) and VIIa (A2) exons gives rise to three distinct FOXM1 variants, FOXM1a, FOXM1b, and FOXM1c , we firsp < 0.001). Similarly, we estimated a FC = 0.78  and FC = 0.89  for OVCAR-3 and OSPC2 cell lines, respectively  and fold difference (2-fold) in each cell line  software. Functional analysis and annotation revealed a unique highly significant network in each cell line, respectively .By enrichment analysis on a gene set of 111 invasion-related genes, we found that 4 genes (3.6%) were differentially expressed in siFOXM1 OSPC2 cells, namely, CXCR4, EPHB2, TIMP3, and TNFSF10. This constituted statistically significant enrichment compared to OSPC2’s background rate of 1.1% (387 out of 36.226) differential expression in genes unrelated to invasion  and clear-cell (EOC-CC1) histology, to evaluate and compare its functional role in two different EOC subtypes. First of all, we demonstrated that both newly established cell lines retained the histopathologic characteristics of the original clinical samples, thus representing a new platform to study the role of FOXM1 in a case of progressive HGSC, the most common and lethal form of EOC, and in a rarer clear-cell EOC. In agreement with FOXM1 isoforms’ expression in a panel of commercially available HGSC cell lines , we founIn agreement with previously published studies –19, our A cell line-specific FOXM1-dependent gene-expression pattern was successfully validated by RT-qPCR for each cell line, respectively. Of interest, in FOXM1-depleted EOC-CC1 cells, we confirmed the down-regulation of cyclin-dependent kinase 2 (CDK2) gene, known as an ideal target to suppress in order to reduce the peritoneal spread of clear-cell EOC in xenograft mice and thereby to prolong their survival , 43. OthThe molecular profile of FOXM1-depleted OSPC2 cells revealed some common and other unique altered genes when compared to the profile of EOC-CC1. Despite the unexpected findings of invasion assay, we assessed by RT-qPCR that some FOXM1 downstream targets involved in metastasis, cancer progression, and cisplatin resistance, such as MMP2  and MACCBeyond altering tumor phenotype, we demonstrated that FOXM1 silencing conferred a greater sensitivity to standard first-line chemotherapeutic drugs, carboplatin and cisplatin, in our novel cellular models. Our data are consistent with those previously reported for commercially available HGSC cell lines , 18 and BRCA1 and BRCA2, since their absence results in unrepaired lesions, cell-cycle arrest and cell death in cancer cells exposed to cytotoxic chemotherapy [BRCA1/2 status is not the only useful biomarker to predict PARP-inhibitor effectiveness [BRCA1/2 genes, it is likely that their responsiveness to this PARP inhibitor might be associated to other mild genetic, epigenetic or molecular HR defects, and we verified that FOXM1 inhibition enhanced olaparib cytotoxicity in both cell lines.The efficacy of PARP inhibitors has been ascertained in HR-deficient breast and ovarian cancers as single agents or in combination with cytotoxic chemotherapy or ionizing radiation . Essentiotherapy . Howevertiveness . In the tiveness , as a sitiveness . Since oFinally, we demonstrated by RT-qPCR the significant overexpression of FOXO3 mRNA in FOXM1-depleted EOC cells of both subtypes. FOXO3, like FOXM1, is a forkhead transcription factor, a typical tumor suppressor and a functional antagonist of the oncogene FOXM1 . Since FThis is the first report proving evidence of a consistent overexpression of FOXM1 among the three major EOC subtypes, and demonstrating its prognostic value in non-serous EOC patients and in platinum-resistant cases, regardless of their histotype. A full description of the tumorigenic properties of FOXM1 has been reported for a clear-cell and a metastatic HGS EOC cell lines, accurately representing their tumors of origin. Importantly, our data suggest that FOXM1 might be considered a valid target for combinatorial anticancer therapy, despite molecular and histological tumor heterogeneity. Consistent with this view, FOXM1 small-molecule inhibitors  and otheAdditional file 1:Table S1: Clinical features of patients and tumor characteristics of samples used to derive cell lines; Table S2: Antibodies features and detailed staining protocols; description of short tandem repeat (STR) DNA profiling; description of BRCA1/2 sequencing; determination of cell lines’ growth rate. (DOCX 23 kb)Detailed protocols for the establishment and the characterization of the EOC cell lines. Additional file 2:Table S3: Genes related to tumor invasion-metastasis used for the Gene-Sets Enrichment Analysis (GSEA). (DOCX 18 kb)Description of microarray analysis on EOC tissue specimens and cell lines. Additional file 3: Table S4.Differentially expressed genes in endometrioid EOCs compared to normal endometrium. (XLSX 327 kb)Additional file 4: Table S5.Differentially expressed genes in clear-cell EOCs compared to normal endometrium. (XLSX 337 kb)Additional file 5:Table S6: Panel of immunocytochemical stains in EOC cell cultures; Table S7: STR profiles of EOC-CC1 and OSPC2 biopsies and derived cell lines; Table S8: List of BRCA1 and BRCA2 sequence variants in OSPC2 cell line; Table S9: List of BRCA1 and BRCA2 sequence variants in EOC-CC1 cell line; Figure S1: Immunohistochemical stain for FOXM1 in original clinical tumor samples and in derived cell lines. Figure S2: EOC cell lines growth curves; Table S10: Optimal cell densities for seeding different cell lines in culture. (DOCX 552 kb)Results of the characterization of OSPC2 and EOC-CC1 cell lines; Additional file 6: Figure S3.Flow cytometric and BrdU analyses of DNA content in siFOXM1 EOC cells. (DOCX 1136 kb)Additional file 7:Figure S4: Volcano plot displaying differential expressed genes between siFOXM1 and siControl EOC-CC1 cells. Table S11: List of down-regulated genes in siFOXM1 EOC-CC1 cells. Table S12: List of up-regulated genes in siFOXM1 EOC-CC1 cells. (DOCX 160 kb)Additional file 8:Figure S5: Volcano plot displaying differential expressed genes between siFOXM1 and siControl OSPC2 cells. Table S13: List of down-regulated genes in siFOXM1 OSPC2 cells. Table S14: List of up-regulated genes in siFOXM1 OSPC2 cells. (DOCX 260 kb)Additional file 9:Figure S6: Volcano plot displaying differential expressed genes between siFOXM1 and siControl OVCAR-3 cells. Table S15: List of down-regulated genes in siFOXM1 OVCAR-3 cells. Table S16: List of up-regulated genes in siFOXM1 OVCAR-3 cells. (DOCX 345 kb)Additional file 10: Table S17.Differentially expressed genes in siFOXM1 EOC cells compared to siControl. (XLSX 100 kb)Additional file 11: Figure S7.Functional analysis of the genome-wide transcriptional response in FOXM1-silenced EOC cells. Putative network reconstruction on EOC-CC1 and OSPC2 cells. (PDF 178 kb)


CD105 was postulated as a renal cell carcinoma (RCC) stem cell marker, and CD133 as a putative RCC progenitor. Hypoxia, a natural microenvironment that prevails in tumors, was also incorporated into the study, especially in terms of the promotion of hypothetical stem-like cell properties.Within this study, we verify the existence of CD105+ and CD133+ populations in selected papillary subtype RCC (pRCC) cell lines. Both populations were analyzed for correlation with stem-like cell properties, such as stemness gene expression, and sphere and colony formation. For the preliminary analysis, several RCC cell lines were chosen  and the control was human kidney cancer stem cells (HKCSC) and renal cells of embryonic origin (ASE-5063). Four cell lines were chosen for further investigation: Caki-2 , ACHN , HKCSC (putative positive control), and ASE-5063 .CD105 and/or CD133 expression with the enhancement of stem-like properties.In 769-P and RCC6, we could not detect a CD105+ population. Hypoxia variously affects pRCC cell growth, and mainly diminishes the stem-like properties of cells. Furthermore, we could not observe the correlation of Based on this analysis, CD105/CD133 cannot be validated as cancer stem cell markers of pRCC cell lines. Renal cell carcinoma (RCC) is the seventh most common tumor and is associated with high mortality . SeveralIn the case of RCC, putative CSCs markers were proposed, including CD105 , CXCR-4 2) /cell-free area (0 h) × 100. Each scratch was performed in triplicate.After passage, the cells were collected as described above and seeded at 500 cells per 15 μL drop of appropriate medium on the inner side of a 100 mm Petri dish lid. The lid was turned upside down and placed on top of the dish filled with 10 mL of PBS. Cells were observed daily for 10 days to detect the creation of aggregates. Photos were taken using an inverted light microscope . For quantitative analysis, areas of formed aggregates were counted from three representative microscope fields (drops) by two independent experimenters using CellSens software .2 cell culture flasks as described above. After 72 h incubation, cells with approximately 80% confluence were collected with Accutase, and suspended in PBS. Then cells were centrifuged and re-suspended in 4% PFA for 10 min at room temperature. PFA was removed by centrifugation, and cells were placed in ddH2O onto ICC SuperFrost microscope slides  and allowed to dry. The staining was performed in a Flex Autostainer instrument  and with the use of EnVisionFlex kits . Antigen retrieval was performed with 5 min proteinase incubation for CD105 or 1 h incubation in a target retrieval solution (pH = 6) for CD133. After the blockade of endogenous peroxidase activity, the slides were incubated for 1 h with primary antibodies: mouse anti-human CD105 , or rabbit anti-human CD133 . After washing, the slides were incubated with appropriate secondary antibodies labelled with HRP. For CD105 staining, HRP activity was amplified with the FLEX+ Mouse Linker. Visualization was performed with Flex DAB+ chromogen. Slides were counterstained with hematoxylin , coverslipped with Pertex  and observed in a light microscope .Cells were cultured in 75 cmP-value was less than 0.05 in the Student’s t-Test calculated in MS Excel (Microsoft), and were designated with an asterisk on graphs (P < 0.1 is designated with a double asterisk).Data are presented as the mean values ± SD or ratios (hypoxia/normoxia). Each experiment was performed at least three times with three technical replicates. The differences between the groups studied were considered significant when the Among an evaluated panel of established RCC cell lines, including primary tumor derived and metastasis derived Table , the CD1For further analyses, HKCSCs, ASE, Caki-2 (high CD105 expression), and ACHN (low expression) cell lines were selected. Caki-2 and ACHN cell lines were recently evaluated as derivatives of papillary RCC –34; therA high number of CD105+ cells in Caki-2 were confirmed in ICC staining—one-third of the cells were positive for this marker Fig. —and CD10The CD133 receptor as the RCC progenitor cell’s putative marker –37 was aoct4 and sox2 genes—markers of embryonic, adult, and CSC—was evaluated, but only oct4 expression was significantly higher in CD105-high cells (Caki-2). The expression of oct4 and sox2 was also detected in ASE cells, but not in HKCSCs , only ACHN and Caki-2 cells were viable Fig. . ACHN ceCell–cell cohesion properties were screened in a hanging drop assay, and Caki-2 and ASE cells formed the aggregates, while HKSCSs created a compact structure with firm edges Fig. . Through2 would promote stemness features. Both pRCC cell lines Caki-2 and ACHN overexpressed hif1, in comparison to tested normal renal ASE cells, but the level of the hif2 gene was lower than in the ASE cells  subpopulations were previously effectively isolated from established cell lines including RCC, mostly with the side-population isolation approach  , 59, 60.2 tension develops in tumors in vivo [oct-4 and sox2). TF expression was observed both in ACHN cells where growth was suppressed by low O2, and in Caki-2 that proliferate more extensively in hypoxia. The above mentioned TFs are known to be up-regulated by HIF2 [hif2 expression simultaneously with stem-related TFs in hypoxia. This is in accordance with pRCC showing lower hif2 expression than ccRCC [111. Cancer cell movement in the wound healing assay corresponds to the cell’s ability to exit a primary tumor, to penetrate into blood vessels, and to finally spread in vivo. In many cancer types, it has been proven that internal tumor hypoxia induces cancer migration potential both in vivo [2. On the other hand, Caki-2 cells, which proliferate more rapidly in hypoxia, simultaneously exerted more stem-like features and grew in large spheres, but not in semi-soft agar while remaining in aggregates in a hanging drop culture. Cumulatively, in vitro assays performed to test cell line aggressiveness and tumorigenicity [2 tension, promotes vascularization, and protects cells from hypoxia-induced apoptosis [vhl status [tp53/vhl wild-type.The next aim of the study was to unravel the role of hypoxia in pRCC invasiveness and the promotion of stem-like features and marker expression. It is well described that low O in vivo , 65 and  in vivo . Our stu by HIF2 ; howeveran ccRCC . As a rean ccRCC , 69, 70.an ccRCC . Cancer  in vivo  and in v in vivo , 74, altgenicity  suggest poptosis . Howeverpoptosis  nor on vl status , as bothWe did not observe a positive correlation of CD105 level and colony formation and clonogenicity in tested ACHN and Caki-2 cell lines, neither in normoxic nor hypoxic conditions. The Caki-2 pRCC cell line, characterized as CD105high, had weaker stem features when compared to the CD105low ACHN cell line with a more aggressive phenotype. The differences in stem potential of those cells describe the characteristics of primary and metastatic cells rather than the contribution of CD105+. Also, in contrast to other studies, we have shown that in the case of pRCC, hypoxic conditions in vitro diminish the stem-like properties of cell lines –85.


Medulloblastoma is comprised of at least four molecular subgroups with distinct clinical outcome (WHO classification 2016). SHH-TP53-mutated as well as MYC-amplified Non-WNT/Non-SHH medulloblastoma show the worst prognosis.in vivo for the treatment of most aggressive medulloblastoma variants.Here we present evidence that single application of the multi-kinase inhibitor Vandetanib displays anti-neoplastic efficacy against cell lines derived from high-risk SHH-TP53-mutated and MYC-amplified Non-WNT/Non-SHH medulloblastoma. The narrow target spectrum of Vandetanib along with a favourable toxicity profile renders this drug ideal for multimodal treatment approaches. In this context our investigation documents that Vandetanib in combination with the clinically available PI3K inhibitor GDC-0941 leads to enhanced cytotoxicity against MYC-amplified and SHH-TP53-mutated medulloblastoma. In line with these findings we show for MYC-amplified medulloblastoma a profound reduction in activity of the oncogenes STAT3 and AKT. Furthermore, we document that Vandetanib and the standard chemotherapeutic Etoposide display additive anti-neoplastic efficacy in the investigated medulloblastoma cell lines that could be further enhanced by PI3K inhibition. Of note, the combination of Vandetanib, GDC-0941 and Etoposide results in MYC-amplified and SHH-TP53-mutated cell lines in complete loss of cell viability. Our findings therefore provide a rational to further evaluate Vandetanib in combination with PI3K inhibitors as well as standard chemotherapeutics  Angiogenesis is critical for tumor growth and metastasis, therefore receptor tyrosine kinase inhibitors targeting tumor blood vessel formation by VEGFR inhibition increasingly gain access to multimodal therapeutic approaches against brain tumors , 2. ReceThe efficacy of Vandetanib has been suggested to rely not solely on inhibition of angiogenesis but also on direct attenuation of tumor growth by blockade of its target receptors namely vascular, endothelial growth factor receptor 2 and 3, epidermal growth factor receptor (EGFR) and c-RET expressed on the cancer cells themselves. –11. VEGFHere we present first evidence that Vandetanib has profound cytotoxic activity in pediatric medulloblastoma cell lines derived from high-risk SHH-TP53-mutated and MYC-amplified Non-WNT/Non-SHH medulloblastoma –25. In kin vitro and in vivo potential for the clinically available PI3K inhibitor GDC-0941 for medulloblastoma therapy [In our latest study we already documented promising  therapy . In thisIn a dose-response analysis we evaluated the anti-carcinogenic potency of Vandetanib in Daoy, a cell line modeling SHH-TP53-mutated medulloblastoma, and in the MYC-amplified Non-WNT/Non-SHH medulloblastoma derived cell lines MEB-Med8-A, D283 Med and D341 Med,  in comparison to control (CN: 164±23 and ACS: 1028±29p2). At the same concentration in Daoy we only documented a reduction in colony size (1618±900p2) compared to control (3723±1000p2) but not in colony numbers. Increasing the dose to 4 μM enhanced the observed effects in both cell lines and additionally resulted for Daoy in a profound decline in the number of colonies (173±45) in comparison to control (300±35).At 2 μM, a concentration typically achieved in patient plasma, MEB-Med-8A showed significant reduction of colony numbers and colony size  or patient plasma (10 μM) levels results in enhanced anti-neoplastic efficacy , 36. ForIn the SHH-TP53-mutated cell line Daoy and the MYC-amplified cell lines D283 Med and D341 Med single application of Etoposide at cerebral spinal fluid levels of 1 μM displayed anti-neoplastic efficacy with 30-60% residual cell viability. In contrast MEB-Med-8A, which also models MYC-amplified tumors, was resistant at 1 μM of Etoposide and displayed only moderate susceptibility to 10 μM Etoposide corresponding to patient plasma levels with 74±4% residual cell viability.We have previously show additive anti-neoplastic efficacy for the concomitant application of Etoposide and the PI3K-antagonist GDC-0941 in medulloblastoma cell lines . Here weWith regards to concomitant application of Vandetanib and GDC-0941 in the SHH-TP53-mutated cell line Daoy we determined in our previous experiments a trend of higher anti-neoplastic efficacy in comparison to single drug application. Here in a cumulative assay determining anti-proliferative and cytotoxic drug effects by means of the metabolic state of the residual cell population this trend could be confirmed. Furthermore, we posed the question, whether inhibition of receptor tyrosine kinases and their downstream signaling molecules could enhance medulloblastoma standard treatment regimes. We treated the investigated cell lines therefore concomitantly with Vandetanib and GDC-0941 in the presence of the chemotherapeutic Etoposide. Indeed, in the SHH-TP53-mutated cell line Daoy as well as in the MYC-amplified cell lines D283 Med and D341 Med the application of the Vandetanib-GDC-Etoposide combination augmented the cytotoxic effect regardless of the applied concentration of Etoposide in comparison to single or dual drug application. In detail, in Daoy and D283 Med at 1 and 5 μM of Etoposide respectively the concomitant application of Vandetanib, GDC-0941 and Etoposide resulted in complete loss of viable cells while in D341 Med at Etoposide concentrations of 1 and 5 μM residual cell viability plateaued 13.6±6 and 15.3±4% respectively. Applying higher Etoposide concentrations in combination with the applied kinase inhibitors did not lead to further enhancement of anti-neoplastic efficacy. In line with above described Etoposide resistancy of MEB-Med-8A concomitant application of Vandetanib and GDC-0941 was sufficient to reduce cell viability completely.In this study we report that Vandetanib exerts anti-neoplastic activity against a panel of four pediatric medulloblastoma cell lines. MEB-Med-8A, D283 Med and D341 Med exhibit distinct characteristics such as MYC-amplification and occurrence of isochromosome 17 of the most aggressive group of tumors within the Non-WNT/Non-SHH group of medulloblastoma –23. In c0/G1-Phase of the cell cycle , was still highly sensitive to single treatment with Vandetanib and GDC-0941 respectively. Moreover, concomitant application of Vandetanib and GDC-0941 resulted in de facto complete loss of cell viability. Whether this finding, although promising, can be translated to refractory tumors therapy in patients needs further evaluation.The development of rational and personalized cancer therapy depends on the identification of valid biomarkers. Expression of target structures might serve as markers for the integration of TKI such as Vandetanib into standard treatment regimes of medulloblastoma. However, in other cancers microRNA expression patters are discussed as promising indicators of eligibility for patient treatment with Vandetanib .In general, our findings underscore the notion that combined inhibition of specific oncogenic tyrosine kinases together with important downstream signaling nodes - such as the PI3K that integrates signals of multiple oncogenic kinases - can enhance anti-neoplastic efficacy in combination with standard medulloblastoma chemotherapeutics.In conclusion, our study demonstrates that Vandetanib, a clinically available angiogenesis inhibitor, displays direct anti-neoplastic activity against pediatric medulloblastoma cell lines modeling the clinically most aggressive SHH-TP53-mutated and MYC-amplified tumor variants. Furthermore, our study documents that combined application of Vandetanib and GDC-0941, a clinically available PI3K/AKT pathway inhibitor, results in fortified anti-neoplastic responses against SHH-TP53-mutated and MYC-amplified medulloblastoma. Furthermore, we delineate that Vandetanib in combination with the standard chemotherapeutic Etoposid displays additive anti-neoplastic efficacy that can be further enhanced by PI3K inhibition. Therefore, our findings provide a rational to further investigate Vandetanib alone, in combination with PI3K/AKT pathway inhibitors and standard chemotherapeutics for the treatment of medulloblastoma variants that despite of intensive multi-modality treatment are currently still associated with poor prognosis.Vandetanib and GDC-0941 were obtained from LC Laboratories. The primary antibody pSTAT3 , STAT3 (124H6), pAKT (Ser473), AKT (11E7) and GAPDH (D18H11) were purchased from Cell Signaling while secondary antibodies were purchased from Dianova. Carboxyfluoreszein-Succinimidyl Ester (CFSE) was purchased from Invitrogen, while Hoechst 33258 was provided by Sigma.The human medulloblastoma cell lines, Daoy (HTB 186), D283 Med (HTB-185) and D341 Med (HTB-187) were obtained from American Type Culture Collection (ATCC). The medulloblastoma cell line, MEB-Med-8A, was generated by Prof. T. Pietsch. The medulloblastoma cell lines Daoy, D283 Med and MEB-Med-8A were maintained in complete medium, namely Dulbecco's Modified Eagle Medium  with L-glutamine supplemented with 1 mM sodium pyruvate (PAA), 1% penicilline/streptomycine (Invitrogen) and 10% fetal bovine serum . The medulloblastoma cell line D341 Med was maintained in DMEM with L-glutamine supplemented with 1 mM sodium pyruvate, 1 % penicilline/streptomycine and 10 % Human Serum .3 Daoy, 6×103 MEB-Med-8A, 104 D283 Med and 104 D341 Med cells respectively. After overnight culture in complete medium, the cells were treated with 1, 2, 4 and 10 μM of Vandetanib alone or in combination with 0.1, 0.5, 1, 5 and 10 μM of etoposide and 1 μM of GDC-0941. The vehicle Dimethylsulfoxid (DMSO) served as control. After 48h of MKI treatment, MTS was added according to the supplier's protocol and the absorbance was measured at 490 nm using an ELISA plate reader . Cell viability was calculated in percent of control.Cell viability was assessed with CellTiter 96 Aqueous One Solution Cell proliferation Assay (Promega) that contains 3--5-(3- carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS). To ensure a linear growth curve over 48h for assessment of MKI-effects, each well of 96-well plates was seeded with 2.5×10Medulloblastoma cells were stained with CFSE according to the instructions of the supplier. Daoy, MEB-Med-8A, D283 Med and D341 Med cells were seeded in 6-well cell culture dishes in complete medium. After overnight culture, the cells were treated with 2 μM of Vandetanib for 48h. Thereafter floating and attached cells were collected, resuspended in 200 μl, stained with Hoechst33258 and analysed by flow cytometry. The number of viable cells was assessed for 120 sec in same volume and at constant speed. Proliferation was traced by CFSE staining and normalized to the control DMSO, while cell death was determined by Hoechst33258 staining.in vitro scratch assay was performed as described by Liang et al. [The g et al. . BrieflyA total protein concentration of 25 μg derived from medulloblastoma cell lines was separated by SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes (Bio-Rad). The membranes were blocked for 1h at RT in 1x Tris-buffered saline containing 0.1% tween-20 (TBST) supplemented with 5% BSA. Thereafter, the membranes were incubated with the primary antibodies overnight at 4°C and subsequently with the respective secondary antibody for 1h at room temperature. Immunoreactivity was detected by chemiluminescence and quantified by means of a ChemiDoc XRS Imaging System (Bio-Rad).2 were excluded from the analysis since these represented stain artefacts, cell detritus or non-proliferating single cells.The cell lines Daoy (200 cells/well) and MEB-Med-8A (1000 cells/well) were plated in six well cell culture dishes. The cells were allowed to adhere and spread properly for 12h at 37 °C. Thereafter the cells were exposed to 1, 2 and 4 μM of Vandetanib. After 48h of exposure the cells were washed with standard medium to remove any trace of the inhibitor and cultured for another week. Colony numbers, and colony size was assessed by IMAGEJ. Particles smaller than 40 pixelt-test was applied to determine statistical significance between groups. p<0.05 (*), was considered as statistically significant. Values stated within text and figures represent mean ± standard deviation.The two-sided Student's


EGFR, HER1) is a therapeutic target in head and neck squamous cell carcinoma (HNSCC). After initial promising results with EGFR‐targeted therapies such as cetuximab, therapeutic resistance has become a major clinical problem, and new treatment options are therefore necessary. Moreover, the relationship between HER receptors, anti‐EGFR therapies, and the human papillomavirus (HPV) status in HNSCC is not fully understood. In contrast to first‐generation EGFR inhibitors, afatinib irreversibly inhibits multiple HER receptors simultaneously. Therefore, treatment with afatinib might result in a more pronounced therapeutic benefit, even in patients experiencing cetuximab resistance. In this study, the cytotoxic effect of afatinib as single agent and in combination with cisplatin was investigated in cetuximab‐sensitive, intrinsically cetuximab‐resistant, and acquired cetuximab‐resistant HNSCC cell lines with different HPV status under normoxia and hypoxia. Furthermore, the influence of cetuximab resistance, HPV, and hypoxia on the expression of HER receptors was investigated. Our results demonstrated that afatinib was able to establish cytotoxicity in cetuximab‐sensitive, intrinsically cetuximab‐resistant, and acquired cetuximab‐resistant HNSCC cell lines, independent of the HPV status. However, cross‐resistance between cetuximab and afatinib might be possible. Treatment with afatinib caused a G0/G1 cell cycle arrest as well as induction of apoptotic cell death. Additive to antagonistic interactions between afatinib and cisplatin could be observed. Neither cetuximab resistance nor HPV status significantly influenced the expression of HER receptors in HNSCC cell lines. In contrast, the expression of EGFR, HER2, and HER3 was significantly altered under hypoxia. Oxygen deficiency is a common characteristic of HNSCC tumors, and these hypoxic tumor regions often contain cells that are more resistant to treatment. However, we observed that afatinib maintained its cytotoxic effect under hypoxia. In conclusion, our preclinical data support the hypothesis that afatinib might be a promising therapeutic strategy to treat patients with HNSCC experiencing intrinsic or acquired cetuximab resistance.The epidermal growth factor receptor ( AnnVannexin V‐FITCCIcombination indexEGFRepidermal growth factor receptorHIFhypoxia‐inducible factorHNSCChead and neck squamous cell carcinomaHPVhuman papillomavirusmAbmonoclonal antibodyMFImean fluorescence intensityPIpropidium iodideR/Mrecurrent/metastaticSRBsulforhodamine BTKItyrosine kinase inhibitor1et al., Targeted therapies are at the forefront of personalized medicine in cancer treatment. Thanks to our rapidly expanding understanding of the molecular biology of cancer, an increasing number of patients are currently considered as candidates for treatment with molecular targeted pharmaceuticals. Interestingly, the epidermal growth factor receptor  plays an integral role in the tumorigenesis and is richly expressed in a wide range of malignancies, including head and neck squamous cell carcinoma (HNSCC). Increased or sustained activation of the EGFR signaling pathway can convert a normal cell to a malignant cell by providing sustained signals for cell proliferation, anti‐apoptotic signaling, angiogenesis, and metastasis), thus making it a compelling drug target  inhibiting cell proliferation, cell repopulation, DNA repair, and tumor oxygenation; (b) modulating cell cycle perturbation and cell accumulation in radiosensitive phases; and (c) inducing apoptosis and necrosis  or with platinum‐based drugs plus 5‐fluorouracil in the recurrent/metastatic (R/M) disease setting   is a diagnostic marker for a separate entity of HNSCC with enhanced overall and disease‐free survival, but its use as a predictive marker has not been proven yet . Cells were grown as monolayers and maintained in exponential growth in 5% CO2/95% air in a humidified incubator at 37 °C. All cell lines were confirmed free of mycoplasma infection through regular testing .Eleven human HNSCC cancer cell lines with different cetuximab sensitivity and HPV status were included in this study. Cal‐27 and UM‐SCC‐104 were obtained from American Type Culture Collection  and Merck Millipore , respectively. SC263 and SQD9 were kindly provided by Sandra Nuyts , and LICR‐HN1 and SCC22b were kindly provided by Olivier De Wever . 93‐VU147‐T was provided by Josephine Dorsman . All cell lines were HPV negative, with the exception of 93‐VU147‐T and UM‐SCC‐104. All cell lines were cultured in Dulbecco's modified Eagle's medium, supplemented with 10% fetal bovine serum, 1% penicillin/streptomycin, and 1% 2.22) was achieved in a Bactron IV anaerobic chamber , as described previously , afatinib , or afatinib in combination with cisplatin . Two sequential combination schedules were tested:Cell survival was assessed using the colorimetric sulforhodamine B (SRB) assay, as previously described  were calculated using winnonlin software . Possible synergism between afatinib and cisplatin was determined by calculation of the combination index (CI) using the additive model as described by others  and cisplatin , were diluted in sterile PBS. Afatinib  was diluted in DMSO (Merck Millipore SA/NV), and further dilutions were made in cell culture medium. IC2.4et al., 50 concentration of cetuximab. In parallel, controlled parental cells were exposed to the vehicle control (suffix PBS). After 10 dose doublings, dose–response studies were determined for each resistant cell line (suffix R). To examine whether acquired resistance was a transient or permanent effect, dose–response curves of cetuximab were re‐assessed in the resistant cell lines after 6 weeks in culture without cetuximab.Generation of resistant cell clones was performed as described previously . These receptor‐specific antibodies recognize and bind to the extracellular domain of the receptor. Corresponding isotype controls  were included for all samples and served as negative controls. Dead cells were excluded from the analysis by staining with Live/Dead Fixable Far‐Red Dead Cell Stain Kit . All samples were measured on a FACScan flow cytometer . Flow cytometric data were analyzed using flowjo v10.1 . The percentage of EGFR‐, HER2‐, HER3‐ and HER4‐positive cells (overton) was determined in comparison with the corresponding isotype control. Furthermore, the signal for aspecific binding was subtracted from the mean fluorescence intensities (=ΔMFI). This parameter indicates the amount of cellular membrane expression of EGFR, HER2, HER3, and HER4 on individual cells.The cellular membrane expression level of EGFR, HER2, HER3, and HER4 under both normoxia and hypoxia was assessed using flow cytometry. Cells were fixed in 4% formaldehyde (10 min) under normoxia or hypoxia after EGFR, HER2, HER3, and HER4 PE‐conjugated antibody incubation . Induction of apoptotic cell death was investigated flow cytometrically using the annexin V‐FITC (AnnV)/propidium iodide (PI) assay (BD Biosciences). Both assays were performed on a FACScan flow cytometer and analyzed with flowjo v10.1.After overnight incubation, cells were treated for 72 h with afatinib. As the sensitivity to afatinib strongly varied between the cell lines, afatinib concentrations were based on the outcome of the monotherapy experiments and corresponded with the ICm. This caspase‐3/7 apoptosis reagent is cleaved by activated caspase‐3/7, which results in the release of a DNA dye and green fluorescent staining of nuclear DNA. Images were taken every 2 h from the start of treatment. Kinetic activation of caspase‐3/7 was monitored morphologically using live‐cell imaging and quantified using the IncuCyte basic analyzer (Essen BioScience).Alternatively, the induction of apoptotic cell death was examined by real‐time measurements of caspase‐3/7 activity using the IncuCyte ZOOM live‐cell analysis instrument . After overnight incubation, cells were treated with afatinib. The IncuCyte caspase‐3/7 green apoptosis reagent (Essen BioScience) was added at the start of treatment at a final concentration of 2.5 μ2.7post hoc analysis with Tukey's correction for multiple testing was performed.We performed all experiments at least three times independently, unless otherwise stated. In cytotoxicity experiments and IncuCyte Caspase‐3/7 Green Apoptosis Assays, each condition was tested in triplicate in each of the three experiments. Flow cytometry experiments were independently performed three times with one sample for each condition. Results are presented as mean ± standard deviation. The effects of various conditions and treatments were studied using linear regression or linear mixed models in case of nonindependent observations. All models were fitted using a stepwise backward strategy, starting from a model with all fixed effects and their interaction. If the interaction term was not significant, a model with only the main was fitted. If one of these terms was significant, effect sizes were estimated. If the treatment effect was significant, a 0/G1 cells as well as AnnV+/PI− and AnnV+/PI+ cells. Regarding the combination experiments, differences in IC50 values were tested for significance with the Mann–Whitney U‐test.Effects of oxygen and resistance status on the expression of HER family members and afatinib's cytotoxic effect were modeled using a linear mixed model with oxygen status, resistance status, and their interaction as fixed effects. A random intercept for cell line was added to account for the dependence between observations within the same cell line. Furthermore, a separate analysis was performed to test for differences in HER status between HPV‐positive and HPV‐negative HNSCC cell lines. Linear regression models were fitted to study the effect of treatment, oxygen condition, and their interaction on the percentage of Ggraphpad prism 7  was used for data comparison and artwork. All statistical analyses were performed in r version 3.3.2 . P‐values below 0.050 were considered significant.33.1et al., et al., 50 values, five of seven HNSCC cell lines  were considered as intrinsically resistant to cetuximab as the percentage of viable cells in these cell lines did not decrease below 50%. SC263 and SCC22b were identified as cetuximab sensitive .We previously identified intrinsic resistance to cetuximab in several HNSCC cell lines such as LICR‐HN1 and Cal‐27  and the ΔMFI of receptor‐positive cells (P ≥ 0.170) were observed between cetuximab‐sensitive, intrinsically cetuximab‐resistant, and acquired cetuximab‐resistant HNSCC cell lines. In addition, no significant effect of HPV status was detected for the percentage of EGFR‐, HER2‐, HER3‐, and HER4‐positive cells (P ≥ 0.302) as well as ΔMFI of receptor‐positive cells (P ≥ 0.110). Hence, the percentage EGFR‐, HER2‐, HER3‐, and HER4‐positive cells and ΔMFI of receptor‐positive cells seems to be cell line specific and independent of HPV status.No significant differences in the percentages of EGFR‐, HER2‐, HER3‐, and HER4‐positive cells (P = 0.006) and ΔMFI of EGFR‐positive cells (P < 0.001) under hypoxic conditions in all HNSCC cell lines (Table P = 0.016)   (Table P = 0.024) . This significant increase under hypoxia was mainly noticed in intrinsically cetuximab‐resistant as well as acquired cetuximab‐resistant and PBS‐treated control HNSCC cell lines (P ≤ 0.050)  (Table P = 0.425). However, taking the difference in cetuximab resistance status between cell lines into account, a significant decrease in ΔMFI under hypoxia was observed for HER3 in acquired cetuximab‐resistant and PBS‐treated control HNSCC cell lines (P ≤ 0.010)  (data not shown).Next, the effect of reduced oxygen levels on the expression of EGFR, HER2, HER3, and HER4 was evaluated. No significant interaction between oxygen condition and cetuximab resistance status on EGFR, HER2, HER3, and HER4 expression was found. When looking at the effect of oxygen availability, there was a significant increase in the percentage of EGFR‐positive cells  after 72 h of treatment was observed in all HNSCC cell lines  nor the HPV status (P = 0.800) seems to influence the inhibitory potential of afatinib. However, certain intrinsically cetuximab‐resistant and acquired cetuximab‐resistant HNSCC cell lines demonstrated higher IC50 values compared to other intrinsically cetuximab‐resistant and acquired cetuximab‐resistant HNSCC cell lines used in this study. For instance, the intrinsically cetuximab‐resistant HNSCC cell lines Cal27 and UM‐SCC‐104 demonstrated considerably lower IC50 values compared to LICR‐HN1 and 93‐VU‐147T. Despite that statistical analysis did not reveal a significant influence of cetuximab resistance on the cytotoxicity of afatinib, these results indicate the possibility of cross‐resistance between cetuximab and afatinib.The cytotoxic effect of the irreversible HER family blocker afatinib was studied in cetuximab‐sensitive, intrinsically cetuximab‐resistant as well as acquired cetuximab‐resistant and PBS‐treated control HNSCC cell lines under both normoxia and hypoxia. A clear concentration‐dependent cytotoxic effect of afatinib  and HPV status (P = 0.630) were not associated with afatinib's cytotoxicity under hypoxic conditions. However, statistical analysis showed a significant interaction between resistance status and oxygen condition (P < 0.001). This implies that the difference in IC50 value for afatinib between the normoxia and hypoxia group varies across the different resistance statuses. As such, we analyzed the difference in IC50 value of afatinib between normoxia and hypoxia for cetuximab‐sensitive, intrinsically cetuximab‐resistant, and acquired cetuximab‐resistant HNSCC cell lines. The estimated effect sizes, showing the difference in mean log(IC50) between normoxia and hypoxia, are displayed in Fig. 50) values were observed under normoxia compared to log(IC50) values under hypoxia in cetuximab‐sensitive, intrinsically cetuximab‐resistant, and acquired cetuximab‐resistant HNSCC cell lines (P < 0.001). This means that afatinib demonstrated an increased cytotoxicity under hypoxic conditions. However, this effect of hypoxia on afatinib's cytotoxic activity was less pronounced in intrinsically cetuximab‐resistant cell lines.The cytotoxic effect of afatinib was maintained under reduced oxygen conditions Table . CetuximOverall, afatinib showed a clear concentration‐dependent cytotoxic effect in cetuximab‐sensitive, intrinsically cetuximab‐resistant, and acquired cetuximab‐resistant HNSCC cell lines. Furthermore, our results demonstrate that therapeutic resistance to afatinib is not associated with HPV infection or prolonged exposure to hypoxia in our panel of HNSCC cell lines with different sensitivities to cetuximab.3.43.4.10/G1 cells, accompanied by a decrease in the percentage of cells in the S and G2/M phases, in the majority of the HNSCC cell lines, irrespective of their sensitivity to cetuximab.As presented in Fig. 0/G1 cells were analyzed using linear regression analyses  or late (AnnV+/PI+) phases of apoptosis. The majority of intrinsically cetuximab‐resistant HNSCC cell lines demonstrated a dose‐dependent increase in AnnV+/PI− and AnnV+/PI+ cells as well as a corresponding decrease in viable AnnV−/PI− cells after 72 h of treatment with afatinib under normal and reduced oxygen conditions . In contrast, treatment with cisplatin followed by afatinib resulted generally in a significant increase in IC50 compared to the IC50 of cisplatin monotherapy (0.050 ≤ P ≤ 0.825). Furthermore, CI ranged from 1.01 ± 0.06 to 1.96 ± 0.40. Thus, sequential exposure to afatinib followed by cisplatin or the inverse regimen  revealed additive or subadditive to antagonistic, yet no synergistic interactions.To investigate the potential interaction between afatinib and cisplatin, tumor cells were incubated with fixed doses of afatinib for 72 h combined with sequential treatment of 0–10 μ4Therapeutic resistance to EGFR‐targeted therapies remains a major clinical problem. In order to overcome resistance to these EGFR‐targeted therapies, new treatment options are urgently needed. Due to extensive crosstalk among HER receptors, blockade of one HER receptor can be partially compensated by other HER family members, which therefore must be targeted by new therapeutic regimens. In contrast to the first‐generation EGFR inhibitors, afatinib blocks irreversibly EGFR, HER2, and HER4. As a result, we hypothesized that treatment with afatinib might result in a distinct and more pronounced therapeutic benefit. To test this hypothesis, we investigated the cytotoxicity of afatinib in a panel of two HPV‐positive and nine HPV‐negative HNSCC cell lines that are either sensitive or intrinsically/acquired resistant to cetuximab.et al., et al., et al., We observed that the majority of HNSCC cell lines used in this study demonstrated high expression of EGFR, HER2, and HER3, but rather low HER4 expression under both normal and reduced oxygen conditions. It has already been established that EGFR is a key survival factor under hypoxic conditions, as EGFR stimulates HIF signaling to improve cellular survival . Furthermore, afatinib displayed a radiosensitizing effect in preclinical studies (Huguet 50/G1 cell cycle arrest and induces apoptotic cell death. Sequential combinations of afatinib with cisplatin demonstrated additive to antagonistic effects. In contrast to cetuximab, afatinib seems not to be the ideal combination partner with platinum‐based drugs for the treatment of HNSCC. In this study, neither cetuximab resistance nor HPV status significantly influenced the expression of HER family members in HNSCC cell lines. In contrast, the expression of EGFR, HER2, and HER3 was significantly altered under reduced oxygen conditions. Furthermore, the cytotoxic effect of afatinib was increased under hypoxia. Overall, these data support the hypothesis that afatinib might be a promising therapeutic strategy to treat patients with HNSCC experiencing intrinsic or acquired cetuximab resistance. Nevertheless, further identification of predictive biomarkers is necessary.Our results suggest that afatinib has the potential to overcome intrinsic and acquired cetuximab resistance in both HPV‐positive and HPV‐negative HNSCC tumors, as it was able to establish a cytotoxic effect in HNSCC cell lines with different cetuximab sensitivity and HPV status. However, cross‐resistance between cetuximab and afatinib might be possible. Therefore, further research is required to identify predictive biomarkers to optimize patient selection. Treatment with afatinib causes a Gin vitro experiments, performed statistical analysis, and drafted the manuscript. FL obtained funding for the study, participated in the design, and helped to draft the manuscript. JVB assisted in the in vitro experiments, participated in its design, and contributed to draft the manuscript. HB assisted in the in vitro experiments toward his Master thesis in Biomedical Sciences. EF performed statistical analysis. VD and PP participated in the design of the study. MP obtained funding for the study. JBV participated in the design of the study and helped to draft the manuscript. AW conceived of the study, participated in its design and coordination, and assisted to draft the manuscript. All authors read and approved the final manuscript.IDP participated in the design of the study, carried out the JBV participated in advisory boards of Boehringer‐Ingelheim. The other authors declare that they have no conflict of interest.


BAP1 loss-of-function mutations, which are frequent in MM, as a potential genomic stratification tool for TRAIL sensitivity with immediate and actionable therapeutic implications.Malignant mesothelioma (MM) is poorly responsive to systemic cytotoxic chemotherapy and invariably fatal. Here we describe a screen of 94 drugs in 15 exome-sequenced MM lines and the discovery of a subset defined by loss of function of the nuclear deubiquitinase BRCA associated protein-1 (BAP1) that demonstrate heightened sensitivity to TRAIL (tumour necrosis factor-related apoptosis-inducing ligand). This association is observed across human early passage MM cultures, mouse xenografts and human tumour explants. We demonstrate that BAP1 deubiquitinase activity and its association with ASXL1 to form the Polycomb repressive deubiquitinase complex (PR-DUB) impacts TRAIL sensitivity implicating transcriptional modulation as an underlying mechanism. Death receptor agonists are well-tolerated anti-cancer agents demonstrating limited therapeutic benefit in trials without a targeting biomarker. We identify  Two patients with the same disease who receive the same treatment may respond in different ways. This variation often arises from differences in each patient’s genetic code. Genes encode proteins, and proteins are the targets of most medical drugs and thus determine the patient’s response to treatment.st century is that doctors recognise that patients can respond differently to the same treatment and now try to predict which patients will respond best to which drug – an approach known as personalised medicine. Cancer treatment has been at the forefront of personalised medicine because mutations in different genes underlie each different cancer. By analysing which mutations are present in a cancer, doctors can thus predict which drug (or combination of drugs) will be most effective. This approach has been used successfully in several cancers, including breast and lung cancer, leading to fewer patients being exposed to ineffective treatments and their associated side effects and costs.A major advance in the 21Mesothelioma is a cancer of the lining of the lung that is associated with exposure to the mineral asbestos. Current treatment options for mesothelioma are unfortunately limited and not very effective. No personalised treatments are currently in use and new treatment approaches are desperately needed.Kolluri, Alifrangis, Kumar, Ishii et al. set out to determine if any of the mutations commonly seen in mesothelioma affected how the cancer would respond to 94 anticancer drugs that are either in use or in development. In the laboratory, mesothelioma cells that have mutations in the gene that codes for a protein known as BRCA associated protein-1 (or BAP1 for short) were killed much more effectively by a drug known as TNF-related apoptosis-inducing ligand (TRAIL). The same link was seen in experiments with tumours of mesothelioma cells that had been transplanted into mice, and for fragments of mesothelioma tumours taken from patients. When Kolluri et al. studied why these tumours might be killed more effectively with TRAIL, they found that mutations in the gene for BAP1 result in a change in the levels of proteins that transmit the signal from the receptors targeted by the TRAIL drug.These findings may one day result in a new approach to treating patients with mesothelioma. But first, the next step would be to conduct a clinical trial of TRAIL in patients with mesothelioma and assess if those with tumours that have mutations in the gene for BAP1 do indeed respond better. If this proves to be the case, this would result in a new personalised treatment option for patients that suffer from this disease. BCR-ABL fusion product in chronic myeloid leukaemia (CML) and the receptor products of HER2 mutations in breast cancer have transformed the prognosis of these cancers  developed against the  cancers . Maligna cancers . A pleth mutated  and ther mutated .BAP1) that demonstrated heightened sensitivity to the death receptor agonist recombinant tumour necrosis factor (TNF)-related apoptosis-inducing ligand (rTRAIL). We validated this finding using in vitro, in vivo and ex vivo models supporting the use of BAP1 as a genomic biomarker to identify TRAIL-sensitive MM tumours and a novel stratified approach to treat MM.We aimed to determine if the mutational status of these tumour driver genes could predict response to a range of existing anti-cancer compounds with a view to identifying genomic biomarkers for responsive subsets of MM. We have previously reported on the ability of such unbiased high-throughput chemical screens in cancer cell lines to identify drug-sensitising mutations in other cancer types . To thisEGFR mutations and EGFR TKIs  and treatment with recombinant TRAIL  (BAP1 wild-type (wt BAP1) lines when treated with rTRAIL. We subsequently confirmed this association in a larger panel of MM cell lines  harbouring wild-type BAP1 were resistant. BAP1 LOF mutations correlated with a loss of BAP1 protein expression in the majority of cell lines  . No signll lines . Strikinll lines . No sensnt of MM . A margiMEDI3039 . Thus, wBAP1 expression in four wt BAP1 MM cell lines using a lentiviral shRNA construct. Knockdown of BAP1 resulted in increased cell death following rTRAIL treatment compared with empty vector (EV) control shRNA and the parental cell line in all four MM cell lines (BAP1 (http://cancer.sanger.ac.uk/cancergenome/projects/cell_lines/). These include CCRC, bladder and breast cancer lines. Treatment of cancer cell lines harbouring nonsense mutations in BAP1 with rTRAIL resulted in markedly reduced cell viability compared with cancer cell lines harbouring missense mutations  , a wild-type BAP1 expression vector or one of these five mutant BAP1 expression vectors. rTRAIL sensitivity of the parental BAP1-null H226 MM line was significantly diminished following expression of wild-type BAP1 and each of the mutant constructs except those with an inactive deubiquitinating or ASXL protein-binding site (BAP1 is a nuclear deubiquitinase that forms multi-protein complexes with transcription factors to regulate gene transcription . To elucic site) , ΔHBM (dic site) , T493A (ic site) , ΔASXL (ic site)  and ΔCTDic site) . H226 MMing site , implicaing site . Transduing site .ASXL1, but not ASXL2, induced sensitivity to MEDI3039 and rTRAIL in the BAP1/ASXL1/ASXL2-wild-type MM line MPP-89  ((http://www.genome.jp/dbget-bin/www_bget?path:map04210). Among those pathways significantly altered when comparing wild-type versus C91A BAP1 (FDR < 0.2) was that of apoptosis. In particular, there was altered mRNA expression of components of the extrinsic death pathway  and vehicle-treated BAP1-mutant tumours (p=0.019)  as assessed by longitudinal bioluminescence intensity (To test the  of mice . On day p=0.019) . BAP1-wintensity .Malignant mesothelioma remains a devastating disease with limited systemic treatment options . BiomarkBAP1 mutation rates in the order of 20–30% .The genomic data is available in the COSMIC database    . The reshttp://www.sanger.ac.uk/genetics/CGP/Software/PICNIC/). All genes were mapped onto this segmentation data to give a gene level copy number analysis. For genes to be classified as amplified the complete coding footprint of the gene had to map onto segment(s) present in eight or more copies. For genes to be classed as homozygously deleted a minimum of 1 bp of coding sequence had to be present within a segment of copy number ‘0’.Genome-wide copy number data were obtained for the cell lines using the Affymetrix SNP6 microarray analysed using the ‘PICNIC’ algorithm, which segments the genome into integer value copy number segments  . Adherent cell lines in the screens were plated 1 day prior to treatment with each compound using liquid handling robotics and assayed after 6 days of treatment with either the single agent or in combination with rTRAIL. Cells were fixed in 4% formaldehyde for 30 min and then stained with 1 μM of the fluorescent nucleic acid stain Syto 60  for 1 hr. Quantitation of fluorescent signal intensity was performed using a fluorescent plate reader at excitation and emission wavelengths of 630/695 nm. The sensitivity of each cell line to various concentrations of compound was calculated as the fraction of viable cells relative to DMSO-treated cells following a 6 day exposure. All screening plates were subjected to stringent quality control measures and a Z-factor score comparing negative and positive control wells was calculated across all screening plates .We derived the area under the curve (AUC) parameter from the 6 day cell line viability data to identify cell lines that are sensitive to a specific compound, with decreasing AUC associated with increasing sensitivity. The AUCs were computed using a trapezoid integration below the five measured viability of the dose-response curve and scaled so that a constant viability of 1 gives AUC of 1.BAP1, TAOK1, NF2, TP53 and CDKN2A). We defined groups of cell lines based on mutations and copy number alterations (homozygous deletions or amplifications) in these genes. This resulted in a set of input features of 4 genes altered in at least 2 of the cell lines . For the association of gene mutations with sensitivity to each compound we restricted the set of drugs to test to those with ≥2 cell lines with AUC <0.7. This resulted in 45 drugs being suitable for analysis .We used 15 mesothelioma cell lines with molecular and drug response data: H2369, H2373, H2461, H2591, H2722, H2731, H2803, H2804, H2810, H2818, H2869, H513, MPP-89, NCI-H2452 and NCI-H28. We selected five genes for inclusion in the analysis  and Sequenom profiling of a panel of 92 single nucleotide polymorphisms for each cell line to ensure non-synonymous cell lines were not used. As a cell line classified as mesothelioma, H513 (on the list of commonly misidentified cell lines) was included in the drug screen of 15 mesothelioma cell lines conducted. Use of this cell line however was not carried forward to further experiments in the paper. The 25 early passage MM cultures were purchased from MesobanK . All cel2.Cell lines were cultured in RPMI-1640 or Dulbecco's modified Eagle's medium and nutrient mix 12 medium (DMEM:F12) supplemented with 10% fetal bovine serum (FBS), penicillin/streptavidin and sodium pyruvate. Early passage human mesothelioma cultures were cultured in RPMI-1640 medium supplemented with 5% FBS, 25 mM HEPES, penicillin/streptavidin and sodium pyruvate. 293 T cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 2 mM L-glutamine. All cells were maintained in a humidified environment at 37°C and 5% COAB_626723), caspase 8  Cat# 9746, RRID:AB_2275120), c-FLIP  Cat# ALX-804-961-0100 RRID:AB_2713915), cIAP1 , cIAP2 , FADD , XIAP , survivin , α-tubulin , H2AK119Ub , H2A , anti-mouse HRP  and anti-rabbit HRP . To detect the ubiquitination status of the histones, the cells were lysed with TBS buffer containing 1% SDS, protease and phosphatase inhibitors. The cell extract was denatured by heating up at 95°C for 10 min and centrifuged at 13000 rpm for 10 min. The supernatant was collected and immunoblotted as described above.Cells were lysed in radioimmunoprecipitation assay (RIPA) buffer  with protease inhibitors  on ice to extract protein. 20 μg of protein samples were separated by SDS–PAGE and transferred onto nitrocellulose membranes. Membranes were incubated with specific primary antibodies, washed, incubated with secondary antibodies and visualised using an ImageQuant LAS 4000 imaging system . Antibodies used include BAP1  Cat# sc-28383, RRID:Cells were seeded in 96-well plates in 100 μl media per well at a density of 40,000 cells/ml 1 day prior to treatment with soluble recombinant TRAIL  or MEDI3039 . XTT  or MTT  reagent was added on day 3. The absorbance was measured with a spectrophotometer at a wavelength of 490 nm or 560 nm for XTT or MTT respectively. Relative cell viability was calculated as a fraction of viable cells relative to untreated cells.BAP1 cDNA was amplified by PCR from pCMV6-AC BAP1 plasmid  and cloned into the lentiviral plasmid pCCL-CMV-flT vector previously described (BAP1 vector designated pCCL-CMV-BAP1. Vectors expressing mutant BAP1 constructs were generated by site-directed mutagenesis (New England Biolabs) of the pCCL-CMV-BAP1 vector. The primers used are listed below. All mutations were confirmed by sequencing.Full-length escribed  in placeBAP1-F CGTGGATCCGCCACCATGAATAAGGGCTGGCTGGABAP1-R GTCGGTCGACTCACTGGCGCTTGGCCTTGTAC91A-F ATACCCAACTCTGCTGCAACTCATGCCTTGCTGC91A-R CAGCTGGTGGGCAAAGAACATGTTATTCACAATATCATCHBM-F CGCTGCTGCCAAGTCCCCCATGCAGGAGGAHBM-R GCAGCGTCTAGAAAGGCCGGCAGCCGCTCTD-F CGTGGATCCGCCACCATGAATAAGGGCTGGCTGGACTD-R GTCGTTCGAATCAGTCAGGCTTCCGCTGCTTGTGGT493A-F GCAGACACGGCCTCTGAGATCGGCAGTGCTT493A-R ACTCTCATTGCTGGGGGTGGGTGAASXL-F AACTACGATGAGTTCATCTGCACCTASXL-R CTGGTCATCAATCTTGAACTTCTTCCTCThe ZS-green luciferase plasmid, pHIV-Luc-ZsGreen  was used for generating ZS-Green luciferase-expressing lentivirus to transduce the H226 cells used in animal experiments.Short hairpin RNAs (shRNAs) were expressed as part of a mir30-based GIPZ lentiviral vector . The clones used in this study include BAP1 (V2LHS_41473), ASXL1 (V2LHS_78829), ASXL2 (V3LHS_313940) and the empty GIPZ control vector.Lentiviral vectors were produced by co-transfection of 293 T cells with construct plasmids together with the packaging plasmids pCMV-dR8.74 and pMD2.G  in the presence of a DNA transfection reagent jetPEI (Source Bioscience UK Ltd). Lentiviruses were concentrated by ultracentrifugation at 17,000 rpm  for 2 hr at 4°C. To determine the titres of prepared lentiviruses 293 T cells were transduced with serial dilutions of viruses in the presence of 8 μg/ml Polybrene (Sigma-Aldrich) and BAP1 expression was assessed by flow cytometry. shRNA- and luciferase-expressing vectors were assessed by analysis of GFP expression. Cell lines were transduced in the presence of 8 μg/ml Polybrene at a range of MOIs and transduction efficacy was assessed by flow cytometry for BAP1 expression.BAP1 construct. SPIA pathway analysis as described in Tarca et al  method . We discca et al  was perfAB_626723; 1:50) and then with an AlexaFluor 488-conjugated anti-mouse antibody . For analysis of apoptosis and cell death all floating and adherent cells were harvested and stained with an Annexin V AlexaFluor 647-conjugated antibody  and 4’, 6-diamidino-2-phenylindole . For analysis of DR4 and DR5 expression on cell surface cells were stained with PE-conjugated antibody  Cat# 307205, RRID:AB_314669, DR5 - BioLegend Cat# 307405, RRID:AB_314677, Isotype control - Biolegend #400112; 1:100). FlowJo software was used to analyse all data.All flow cytometry analysis was performed on a LSR Fortessa analyser . For analysis of BAP1 expression cells were stained with primary antibody to BAP1  primary antibody  was incubated overnight at 4°C, before incubating for 1 hr at room temperature with Alexafluor 488-conjugated anti-rabbit secondary antibody. Nuclei were stained with Hoechst 33342 (Thermo Fisher Scientific Cat# 62249). Images were acquired (n = 3) with a BioTek Cytation3 Multimode reader. Using a 10x objective 4 fields of view were acquired per well (n = 3) and the level of nuclear ubiquityl-histone H2A intensity was determined within the primary nuclear mask and normalised to total cell number.H226 cells were seeded at 2.5 × 10AB_626723; 1:150) for 15 min at room temperature. Epitope retrieval was completed using HIER using Leica Bond ER2 (high pH) for 30 min and a Leica Bond Polymer Refine with DAB chromogen detections system used.BAP1 immunohistochemistry of human early passage cell lines was conducted on sections of cell pellets mounted on slides. Automated staining on a Leica Bond III staining platform was used. Slides were incubated with BAP1 primary antibody , EGF (20 ng/ml) and FGF (10 ng/ml). After 24 hr the explants were treated with rTRAIL  for a further 24 hr, following which explants were either fixed for PARP immunohistochemistry. The explants were fixed in 10% neutral-buffered formalin (NBF) for 24 hr and then transferred into 70% ethanol followed by paraffin embedding. Subsequently, 5 μm sections were used for immunohistochemistry, as previously described . Informed consent to conduct research on samples collected and to publish results was obtained from patients. The diagnosis of mesothelioma was confirmed histologically for all patients prior to consent and surgery. Patients underwent pleurectomy, following which primary pleural tissue was sectioned into fragments measuring approximately 2 mmescribed .AB_777102) was used at a 1:6000 dilution and the rabbit-specific HRP/DAB (ABC) detection IHC kit (Abcam) was used for immunohistochemistry, according to the manufacturer’s instructions. Sections were counterstained with haematoxylin and mounted using Vectamount permanent mounting media . Images were taken at 40x magnification on a Hamamatsu Nanozoomer Digital slide scanner. Cleaved PARP-positive cells were scored as the percentage of cells with nuclear staining.Cleaved PARP primary antibody  Act 1986 . Mice were purchased from Charles River, kept in individually ventilated cages under specific pathogen-free conditions and had access to sterile irradiated food and autoclaved water IMSR_CRL:394) were injected with 1 × 106 H226 cells transduced with a plasmid containing wild-type BAP1 and luciferase on the right flank and with a plasmid containing a catalytically inactive BAP1-mutant (C91A) and luciferase on the left flank in a 1:1 mixture of Matrigel  and medium. Tumour size was assessed by bioluminescence in vivo imaging system  15 min following intraperitoneal injection of 0.2 ml (2 mg) luciferin. Tumours were allowed to establish for 2 weeks prior to baseline assessment of size at day 13. Mice were then divided into two groups each of which received either 600 μg TRAIL or vehicle 6 days a week from day 14 until day 40. Bioluminescence was measured on days 0, 13, 19, 26 and 41. Mice were sacrificed on day 42 and tumours harvested for measurement. TRAIL used in the mouse experiment was made in Henning Walczak’s laboratory as per the established protocol (12 8 week old NOD.CB17-Prkdcscid/NcrCrl (NOD SCID) mice . t-test was used to analyse differences between two groups whilst the analysis of variance (ANOVA) test with a Tukey post-hoc analysis was used to analyse differences between three groups. For multiple groups measured over multiple time points repeated measures ANOVA was used. All  In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Charles Sawyers as the Senior Editor. The following individuals involved in review of your submission have agreed to reveal their identity: Andrew Thorburn (Reviewer #1); Dan Longley (Reviewer #3).Thank you for submitting your article "Loss of functional BAP1 is a biomarker for TRAIL sensitivity in cancer" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.Summary:Kolluri et.al. present convincing data arising from a drug screen in a panel of mesothelioma cell lines that the nuclear deubiquitinase BAP1 regulates TRAIL sensitivity in mesothelioma cells (and some other tumor types too.) Loss of BAP1 expression, which occurs in some cancers, could potentially serve as a biomarker for sensitivity to agonists that target TRAIL receptors (TRAIL R). As noted by the authors TRAIL R-targeted therapeutics have been quite widely tested and usually shown to be safe, however the lack of an ability to identify patients who are likely to benefit from these treatments has stymied development of this class of drugs. Therefore, the area that the authors are addressing is potentially quite important. Reviewers found the data presented to be quite convincing and to support the authors' case that BAP1 can affect TRAIL sensitivity most likely through epigenetic regulation of gene expression by a mechanism involving the AXLS1 protein. However, a number of concerns were raised about the ultimate power of BAP1 status to serve as a biomarker in the clinic, as well as the minimal mechanistic insight provided in the report.After thorough discussion, the reviewers agreed to invite a resubmission of a revised manuscript addressing the following major points:1) A main concern with the paper in its current form is that it doesn't represent a major advance for the field, in the sense that there are many genes and processes that have been shown to confer TRAIL sensitivity and proposed as potential biomarkers, yet none of them so far have worked out to be useful. Reviewers agreed that more evidence is needed in this regard. First, it's not clear whether the effects are really specific to TRAIL. Many perturbations that make tumor cells more sensitive to apoptosis will appear to confer sensitivity on a canonical apoptosis inducer like TRAIL. Therefore, reviewers request a series of experiments addressing this issue, to define whether or not BAP1 mutation/loss is a general sensitizer to death ligands and/or general apoptotic stimuli. Reviewers suggest testing a different death ligand, such as a Fas agonist (e.g. CH-11 antibody) or, alternatively, a TNF/IAP antagonist combination. For the general apoptotic stimulus, the reviewers propose disease-relevant genotoxic agents such as pemetrexed or cisplatin.2) Second, reviewers agreed that more work should be performed to establish the notion of BAP1 status as a biomarker for TRAIL efficacy. Although the pattern presented is very strong, many BAP1 positive cells are sensitive to TRAIL and vice versa, indicating that other factors in addition to BAP1 define sensitivity to TRAIL. The report will be significantly strengthened by performing cause-effect relationship experiments in a much larger number of cell lines. More specifically, reviewers would like to see the BAP1 depletion experiments done in a larger panel of cell lines. The report currently shows the effect of BAP1 knockdown in only two cell lines: H226 and MDA-MB-231. All other cause-effect experiments involve artifact-prone overexpression experiments of BAP1 (wt or various mutants). Given the ready access to cell lines and proven shRNAs for BAP1, the conclusions would be significantly strengthened by testing the effect of BAP1 depletion in two additional MM cell lines that are BAP1 positive and TRAIL-resistant. Additionally, BAP1 mutations are most frequent among kidney clear cell carcinomas (CRCC) estimated at 10% by tumorportal.org. The impact of the report would be significantly increased by measuring TRAIL sensitivity upon BAP1 depletion in two CCRC cell lines expressing wild type BAP1.3) Third, the clinical relevance would be increased by testing a large number of patient samples in the ex vivo analyses in 4) Another major issue was the minimal mechanistic insight as to how BAP1 status regulates sensitivity to TRAIL. What is the impact of BAP1 on known regulators and effectors of the TRAIL receptor pathway? Some data and discussion on the relationship between BAP1 mutation and gene expression of TRAIL-R1/2, FADD, FLIP, caspase-8, etc. is needed. What happens to these genes when WT versus mymutant BAP1 is overexpressed in BAP1 negative cell line?5) Additionally, insight into how the ASXL1 connection translates into increased cell death should be provided, such as additional measures of key cell death signaling nodes as described in #4 above in experiments where ASXL1 activity/expression has been manipulated.6) In the absence of true validation of BAP1 status as a biomarker in the clinical setting, the title was deemed overambitious, and reviewers request a more accurate title and some attenuation of some of the conclusions describing BAP1 as a biomarker. […] After thorough discussion, the reviewers agreed to invite a resubmission of a revised manuscript addressing the following major points:1) A main concern with the paper in its current form is that it doesn't represent a major advance for the field, in the sense that there are many genes and processes that have been shown to confer TRAIL sensitivity and proposed as potential biomarkers, yet none of them so far have worked out to be useful. Reviewers agreed that more evidence is needed in this regard. First, it's not clear whether the effects are really specific to TRAIL. Many perturbations that make tumor cells more sensitive to apoptosis will appear to confer sensitivity on a canonical apoptosis inducer like TRAIL. Therefore, reviewers request a series of experiments addressing this issue, to define whether or not BAP1 mutation/loss is a general sensitizer to death ligands and/or general apoptotic stimuli. Reviewers suggest testing a different death ligand, such as a Fas agonist (e.g. CH-11 antibody) or, alternatively, a TNF/IAP antagonist combination. For the general apoptotic stimulus, the reviewers propose disease-relevant genotoxic agents such as pemetrexed or cisplatin.We agree and highlight in our manuscript that to date a key limiting factor of TRAIL therapeutics has been the lack of a validated biomarker for sensitivity. However, no approach thus far has included the search for a sensitising mutation using an unbiased drug screen in sequenced cell lines. Importantly the retrospective identification of drug sensitising mutations has re-directed the use of other anticancer therapies with initially disappointing results in unselected populations.BAP1 as a sensitising mutation to TRAIL, and other death receptor agonists, we have treated a panel of BAP1 mutant and wild-type MM lines with additional apoptotic stimuli as suggested. No sensitising association with BAP1 was observed for pemetrexed or cisplatin, current first line agents for the treatment of MM  Second, reviewers agreed that more work should be performed to establish the notion of BAP1 status as a biomarker for TRAIL efficacy. Although the pattern presented is very strong, many BAP1 positive cells are sensitive to TRAIL and vice versa, indicating that other factors in addition to BAP1 define sensitivity to TRAIL. The report will be significantly strengthened by performing cause-effect relationship experiments in a much larger number of cell lines. More specifically, reviewers would like to see the BAP1 depletion experiments done in a larger panel of cell lines. The report currently shows the effect of BAP1 knockdown in only two cell lines: H226 and MDA-MB-231. All other cause-effect experiments involve artifact-prone overexpression experiments of BAP1 (wt or various mutants). Given the ready access to cell lines and proven shRNAs for BAP1, the conclusions would be significantly strengthened by testing the effect of BAP1 depletion in two additional MM cell lines that are BAP1 positive and TRAIL-resistant. Additionally, BAP1 mutations are most frequent among kidney clear cell carcinomas (CRCC) estimated at 10% by tumorportal.org. The impact of the report would be significantly increased by measuring TRAIL sensitivity upon BAP1 depletion in two CCRC cell lines expressing wild type BAP1.BAP1 can affect TRAIL sensitivity as evidenced in our panel of MM lines  Third, the clinical relevance would be increased by testing a large number of patient samples in the ex vivo analyses in Since review we have obtained 7 video assisted thoracoscopic surgery (VATS) pleural biopsy samples from treatment naïve patients with a suspected diagnosis of MM. Four of these proved to comprise benign tissue only. We attempted to assess apoptosis in response to MEDI3039 treatment in explants from the remaining three. However, as these were VATS rather than pleurectomy biopsies, the tissue volume was extremely small precluding appropriate immunohistochemical analysis. Successful explant data is ultimately subject to the frequency of pleurectomy cases performed at our local surgical centre and within the suggested time period for resubmission there were none. We agree however that an expanded cohort would strengthen the clinical relevance and continue with our explant programme to this end.4) Another major issue was the minimal mechanistic insight as to how BAP1 status regulates sensitivity to TRAIL. What is the impact of BAP1 on known regulators and effectors of the TRAIL receptor pathway? Some data and discussion on the relationship between BAP1 mutation and gene expression of TRAIL-R1/2, FADD, FLIP, caspase-8, etc. is needed. What happens to these genes when WT versus mymutant BAP1 is overexpressed in BAP1 negative cell line?BAP1 null MM line with overexpression of BAP1 wild-type or the catalytically inactive BAP1 C91A. Signaling pathway impact analysis highlighted apoptosis as being significantly altered by BAP1 function. Thus we proceeded as suggested to specifically assess the impact of BAP1 function on the death receptor pathway at a gene expression and protein level. This data supports a role for BAP1 in the modulation of transcription of death receptor pathway proteins.We have extended our mechanistic data as suggested. We have compared gene expression data from a We have added the following text to the Results section:BAP1-null H226 cells transduced with the C91A BAP1 mutant or with wild-type BAP1, and carried out a signalling pathway impact analysis SPIA  Additionally, insight into how the ASXL1 connection translates into increased cell death should be provided, such as additional measures of key cell death signaling nodes as described in #4 above in experiments where ASXL1 activity/expression has been manipulated.BAP1 null MM cells transduced with the ASXL1/2 binding mutant ΔASXL BAP1. The changes observed in this analysis are consistent with that of those cells transduced with the catalytically inactive C91A BAP1 mutant. This supports the role of both functions, deubiquitinase activity and ASXL binding, in the regulation of expression of components of the death receptor pathway.We have conducted immunoblot analysis of the death receptor pathway on These changes to the manuscript are detailed above in point 4.6) In the absence of true validation of BAP1 status as a biomarker in the clinical setting, the title was deemed overambitious, and reviewers request a more accurate title and some attenuation of some of the conclusions describing BAP1 as a biomarker.We have amended the title to:“Loss of functional BAP1 augments TRAIL sensitivity in cancer cells.”We have minimised the reference of BAP1 as a biomarker replacing such instances with reference to its use as a ‘genomic stratification tool’ or ‘potential biomarker’.