YGYNO-976640; No of pages: 8; 4C: 5, 6, 7, Gynecologic Oncology xxx (2017) xxx–xxx Contents lists available at ScienceDirect Gynecologic Oncology journal homepage: www.elsevier.com/locate/ygyno MELK expression in ovarian cancer correlates with poor outcome and its inhibition by OTSSP167 abrogates proliferation and viability of ovarian cancer cells Reto S Kohler a,1, Henriette Kettelhack a,1, Alexandra M Knipprath-Mészaros b, André Fedier a, Andreas Schoetzau a, Francis Jacob a,c,⁎, Viola Heinzelmann-Schwarz a,b,⁎⁎ a b c Ovarian Cancer Research, Department of Biomedicine, University Hospital Basel, University of Basel, Switzerland Hospital for Women, Department of Gynecology and Gynecological Oncology, University Hospital Basel, University of Basel, Switzerland Glyco-Oncology, Ovarian Cancer Research, Department of Biomedicine, University Hospital Basel, University of Basel, Switzerland H I G H L I G H T S • Elevated MELK in serous ovarian cancer associates with poor progression-free survival • Silencing and inhibition of MELK results in apoptosis in serous ovarian cancer cell lines • Drug-resistant cells retain sensitivity to MELK-inhibitor OTSSP167 a r t i c l e i n f o Article history: Received 12 November 2016 Received in revised form February 2017 Accepted February 2017 Available online xxxx Keywords: TCGA Apoptosis Disease outcome Oncogenic growth Drug resistance a b s t r a c t Objective Maternal embryonic leucine-zipper kinase (MELK) shows oncogenic properties in basal-like breast cancer, a cancer subtype sharing common molecular features with high-grade serous ovarian cancer We examined the potential of MELK as a molecular and pharmacological target for treatment of epithelial ovarian cancer (EOC) Methods/materials Bioinformatic analysis was performed on nine OC transcriptomic data sets totaling 1241 patients Effects of MELK depletion by shRNA or inhibition by OTSSP167 in cell lines were assessed by colony formation and MTT (proliferation) assays, Western blotting (apoptosis), and flow cytometry (cell cycle analysis) Results Elevated MELK expression was correlated with histological grading (n = data sets, p b 0.05) and progression-free survival (HR 5.73, p b 0.01) in OC patients and elevated MELK expression in other cancers with disease-free survival (n = 3495, HR 1.071, p b 0.001) Inhibition or depletion of MELK reduced cell proliferation and anchorage-dependent and -independent growth in various OC cell lines through a G2/M cell cycle arrest, eventually resulting in apoptosis OTSSP167 retained its cytotoxicity in Cisplatin- and Paclitaxel-resistant IGROV1 and TYK-nu OC cells and sensitized OVCAR8 cells to Carboplatin but not Paclitaxel Conclusion MELK inhibition by OTSSP167 may thus present a strategy to treat patients with aggressive, progressive, and recurrent ovarian cancer © 2017 The Authors Published by Elsevier Inc This is an open access article under the CC BY-NC-ND license (http:// creativecommons.org/licenses/by-nc-nd/4.0/) Introduction Epithelial ovarian cancer (EOC) is the leading cause of death from a gynecologic cancer and patients with high-grade serous ovarian cancer ⁎ Corresponding author at: Department of Biomedicine, University Hospital Basel, Hebelstrasse 20, 4031 Basel, Switzerland ⁎⁎ Correspondence to: V Heinzelmann-Schwarz, University Hospital Basel, Spitalstrasse 21, 4031 Basel, Switzerland E-mail addresses: francis.jacob@unibas.ch (F Jacob), viola.heinzelmann@usb.ch (V Heinzelmann-Schwarz) Contributed equally (HGSOC), the most common and aggressive type, have a 5-year survival of b20% Effective screening or early detection is limited and hence patients commonly present at an advanced stage Overall survival has, despite improved surgical techniques and drug regimens, not changed significantly for several decades, in part owing the considerable risk for disease recurrence even after full remission following cytoreductive surgery and platinum- and taxane-based chemotherapy [1–3] Hence novel drugs or additional therapeutic strategies are required to improve patient survival MELK (maternal embryonic leucine zipper kinase) is a highly conserved serine/threonine kinase initially found in a wide range of early http://dx.doi.org/10.1016/j.ygyno.2017.02.016 0090-8258/© 2017 The Authors Published by Elsevier Inc This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/) Please cite this article as: R.S Kohler, et al., MELK expression in ovarian cancer correlates with poor outcome and its inhibition by OTSSP167 abrogates proliferation and viabilit , Gynecol Oncol (2017), http://dx.doi.org/10.1016/j.ygyno.2017.02.016 R.S Kohler et al / Gynecologic Oncology xxx (2017) xxx–xxx embryonic cellular stages It is proposed to play prominent roles in cell cycle control, cell proliferation, apoptosis, drug resistance, cell renewal, embryogenesis, and oncogenesis [4] MELK is therefore believed to have an oncogenic driver function This may be attributed to its capacity to disable critical cell-cycle checkpoints, reduce DNA replication stress, and stimulating proliferation by its capacity to increase the threshold for DNA-damage tolerance [5] Recent studies indicate that MELK is highly expressed in several human cancers including breast, gastric, and lung cancers [6] However, there seems to be some subtype specificity within cancer types as MELK is overexpressed in basal-like (BBC) but not in luminal breast cancer and normal breast tissue, correlating with poor prognosis and tumor aggressiveness [7] MELK silencing by shRNA reduced oncogenic colony formation and induced apoptosis in BBC cells but not in other types of breast cancer cells and pharmacological MELK inhibition by OTSSP167 reduced BBC tumor but not luminal cancer growth in mice [7] OTSSP167 is a selective small molecule inhibitor of MELK developed in 2012 and was shown to inhibit proliferation in several cancer cells of different origin [8,9] OTSSP167 has been examined in an Australian clinical phase I study for its safety in healthy volunteers and is at present being evaluated in a clinical phase I study on various solid tumors but not including ovarian tumors [4] Owing the lack of respective data in ovarian cancer and owing the fact that BBC and HGSOC share striking similarities regarding molecular and genetic profiles [10,11], i.e that N90% of BBC carry TP53 mutations, which is also a hallmark in HGSOC [1], that frequencies of RB1 and CMYC pathway mutations are also significantly similar in BBC and HGSOC [11, 12], that transcriptomic profiles are shared by both cancer types [13,14] and that BRCA1/2 mutations are frequently found in BBC [12] and HGSOC [15–17], we investigated the putative role of MELK in EOC Specifically, we evaluated MELK gene and protein expression in transcriptomic data sets as well as in vitro in ovarian cancer, normal ovarian surface epithelial, and fallopian tube cell lines, respectively; compared the transcriptomics data to patient outcome parameters; and evaluated the effect of shRNA and OTSSP167 on proliferation, oncogenic growth, cell cycle progression, and apoptosis in various (including drug-resistant) ovarian cancer cell lines CDK4-R24C), and FT246 (hTERT + p53 shRNA + CDK4-R24C) (kind gifts by Dr Drapkin) were cultured in DMEM F12/50 without HEPES supplemented with 2% Ultroser (USG, Pall Corporation, USA) and penicillin/streptomycin [18] TYK-nu and TYK-nu(R) cells were obtained from JCRB cell bank, Japan and were cultured in MEM Eagle supplemented with 10% FBS and penicillin/streptomycin [19] Cell lines were characterized by STR profiles and routinely tested for mycoplasma infection MELK-inhibitor OTSSP167 (hydrochloride) was purchased from MedchemExpress (Monmouth Junction, NJ, USA) 2.3 Western blot analysis Western blot analysis was used to detect expression of MELK and MDR1 in cell lines and assess apoptosis in OTSSP167-treated or shMELK-treated cells Cells were harvested and lysed in cell lysis buffer (9803, Cell Signaling Technology, Bioconcept, Allschwil, Switzerland) supplemented with 0.1% SDS, 0.5% sodium deoxycholate and mM PMSF Protein concentrations were determined using Pierce™ BCA assay kit (Pierce, Life Technologies Europe BV, Zug, Switzerland) A total of 30 μg of protein was separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), followed by blotting onto PVDF membrane (Bio-Rad, Cressier, Switzerland) according to standard protocols Proteins were detected by specific primary antibodies and the respective secondary horseradish peroxidase-conjugated antibodies The primary antibodies were: anti-MELK (EPR 3981, Source Bioscience, Berlin, Germany), anti-MDR1 (sc-13,131, Santa Cruz Biotechnology, Labforce AG, Muttenz, Switzerland), anti-cleaved poly(ADP-ribose) polymerase (PARP-1) (9541; Cell Signaling Technology), anti-Tubulin (2148, Cell Signaling Technology), and anti-actin (A5441, Sigma, Buchs, Switzerland) The secondary antibodies were: anti-rabbit IgGHRP (7074, Cell Signaling Technology) and anti-mouse IgG-HRP (7076, Cell Signaling Technology) Complexes were visualized by enhanced chemiluminescence (SuperSignal West Dura, Pierce) according to the manufacturer's instructions using a chemiluminescent imaging system (ChemiDoc XR, Bio-Rad) 2.4 shRNA-mediated knockdown of MELK expression and effects on apoptosis and colony formation Materials and methods 2.1 Data acquisition and bioinformatical analysis MELK expression was initially evaluated in our oligonucleotide array data published in Heinzelmann et al 2004 [10] Publicly available transcriptomic data sets were downloaded from Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo/) Information including gene expression accession numbers of the data sets are listed in Supplementary Table S1 All publicly available data sets were searched for the presence of the following criteria: 1) serous ovarian cancer, 2) histopathological grade, 3) and normal versus ovarian cancer Statistical analysis and figures were obtained through the use of the software R version 3.1.3 (www.R-project.org) Gene expression data for the “Cancer Cell Line Encyclopedia were accessed through the cbioportal using R (www.cbioportal.org) Following R packages were therefore applied: “cgdsr”, “catspec”, “compareGroups”, “party”, “ggplot2”, and “Rcpp” 2.2 Cell culture and MELK-inhibitor HOSE6-3, OVCAR3, OVCAR4, OVCAR5, OVCAR8, SKOV3, A2780, BG1, IGROV1, OAW42, T47D, MCF7, and MDA-MB-468 cells were cultured in RPMI supplemented with 10% fetal bovine serum (FBS) and penicillin/ streptomycin TOV112D was cultured in DMEM supplemented with 10% FBS and penicillin/streptomycin EFO27 were cultured in RPMI containing 20% FBS including mM sodium pyruvate and penicillin/streptomycin FT33-Tag (hTERT + SV40 large T), FT190 (hTERT + SV40 TAg), FT194 (hTERT + SV40 Tag), FT237 (hTERT + p53 shRNA + This included the construction of the plasmids, the production of the lentivirus, and the generation of stable cell lines To obtain pLKO-TetshMELK vector that expresses doxycycline-inducible short-hairpin RNA against human MELK, the following oligonucleotide pairs [7] were inserted into Tet-pLKO-puro (21915, Addgene, Cambridge, MA, USA) using AgeI and EcoRI restriction sites: 5′-ccg ggc ctg aaa gaa act cca att act cga gta att gga gtt tct ttc agg ctt ttt g −3′ and 5′-aat tca aaa agc ctg aaa gaa act cca att act cga gta att gga gtt tct ttc agg c − 3′ Targeting sequences are underlined Constructs were verified by Sanger DNA sequencing (Source Biosciences, Berlin, Germany) HEK293T cells were transfected with pLKO-Tet-shMELK together with packaging vectors pMD2.G (12,259, Addgene) and pCMV8.74 (22,036, Addgene) using JetPEI transfection reagent (Chemie Brunschwig, Basel, Switzerland) Medium was replaced 24 h after transfection and after another 48 h supernatant was harvested, filtered through a 0.45 μm filter, aliquoted and stored at −80 °C To generate stable inducible cell lines, HOSE6-3 and OVCAR8 cells were infected with viral supernatant supplemented with μg/ml polybrene After 24 h medium was changed and μg/ml puromycin was added after another 24 h Stable cell lines were maintained in RPMI containing 0.5 μg/ ml puromycin shMELK expression was induced by doxycycline addition (0.5 μg/ml) for 72 h Effects of MELK-knockdown on apoptosis and colony formation in soft-agar were determined For apoptosis analysis, cells (300′000) were seeded, fresh medium with or without doxycycline (0.5 μg/ml) was added for cells expressing pLKO-Tet-shMELK on the next day, lysed after 48 h, and stored for Western blot analysis For colony Please cite this article as: R.S Kohler, et al., MELK expression in ovarian cancer correlates with poor outcome and its inhibition by OTSSP167 abrogates proliferation and viabilit , Gynecol Oncol (2017), http://dx.doi.org/10.1016/j.ygyno.2017.02.016 R.S Kohler et al / Gynecologic Oncology xxx (2017) xxx–xxx formation, 5′000 cells were suspended in 0.4% low melt agarose and plated onto a layer of 0.6% low melt agarose in 12-well plates On the next day, fresh medium with or without doxycycline (0.5 μg/ml) was added to the wells for cells expressing pLKO-Tet-shMELK After 14 days colonies were fixed (4% formaldehyde) and stained (0.05% crystal violet) 2.5 OTSSP167-mediated colony formation and anchorage-independent growth inhibition and apoptosis induction For the colony formation assay, 200 cells were seeded into 12-well plates and treated with 100 nM OTSSP167 for 14 days Colonies were fixed (4% formaldehyde) and stained (0.05% crystal violet) for h For the anchorage-independent growth assay, 5′000 cells suspended in 0.4% low melt agarose were plated onto a layer of 0.6% low melt agarose in 12-well plates On the next day, fresh medium with or without 100 nM OTSSP167 was added Colonies were fixed and stained as described For apoptosis analysis, 300′000 cells seeded into 12-well plates were treated with 100 nM OTSSP167 for 48 h and harvested for Western blot analysis (PARP-1 cleavage) as described above 2.6 Cell cycle analysis Cell cycle analysis was performed by flow cytometry Cells (300′000) were seeded on 6-well plates and treated with various concentrations of OTSSP167 for 24 h On the next day, adherent and floating cells were harvested and resuspended in PBS, fixed by adding 500 μl −20 °C 70% ethanol and stored on ice for at least 30 Cells were then rehydrated with cold PBS and stained overnight at °C with Hoechst 33367 (BD Bioscience, Allschwil, Switzerland) The DNA content was analyzed using a FACS Fortessa flow cytometer (BD Biosciences) 2.7 Confocal fluorescence microscopy Cells were grown on polylysine glass slides attached to a 8-well chamber, fixed with 4% para-formaldehyde, permeabilized with 0.3% Triton X-100 and blocked with 5% (w/v) BSA fraction V (Sigma) dissolved in PBS Cells were stained with anti-tubulin (2148, Cell Signaling Technology), Alexa Fluor 647 Phalloidin (8940, Cell Signaling Technology) and counterstained with ProLong Gold Antifade Reagent with DAPI (8961, Cell Signaling Technology) according to manufacturers' protocol Fluorescence images were taken with a LSM 780 confocal microscope (Zeiss, Feldbach, Switzerland) 2.8 MTT cell viability assay This assay was performed to determine the effect of OTSSP167 alone or a combination of OTSSP167 with either Carboplatin or Paclitaxel on proliferation and cell viability Cells were seeded into 96-well plates and treated on the next day with various concentrations of OTSSP167 or a combination with Carboplatin or Paclitaxel for 72 h Cells were then incubated with 500 μg/ml (final concentration) MTT dye (in PBS) for h, followed by removal of the medium, dissolution of the violet crystals with 200 μl DMSO, and optical density measurement (OD, absorbance at 540 nm) (Synergy H1 Hybrid Reader, BioTek, Luzern, Switzerland) Data are presented as relative cell viability (OD as percentage of untreated control) as a function of drug concentration IC50 values were calculated by linear extrapolation to compare drug sensitivity Each experiment was performed independently at least three times in quadruplicates 2.9 Generation of drug-resistant cells In addition to the Cisplatin-resistant TYK-nu(R) cells and the respective sensitive parental TYK-nu cells generated previously [19], IGROV1 and OVCAR8 cells were step-wise exposed to increasing concentrations of Paclitaxel or Carboplatin (IGROV1) or OTSSP167 (OVCAR8) in order to generate drug-resistant sublines by a protocol described before [20] The principle of selection was the clonal growth in the presence of increasing drug concentrations, assuming that cells acquire new features in an irreversible fashion by chronic drug exposure Briefly, 100′ 000 cells are seeded into 12-well plates and exposed to IC50 concentration of Paclitaxel, Carboplatin or OTSSP167 for 48 h Remaining viable cells were allowed to recover in drug-free culture medium until reaching confluency This cycle was repeated with incrementing drug concentrations until a stable resistance was reached or no viable cells were left Stable resistance was verified by MTT assay performed over several passages of this resistant cell population and arbitrarily defined by a resistance factor (IC50 value ratio of resistant to parental cells) of 2.0 or above 2.10 Statistical analysis Comparisons between cancer and control or the various cancer subtypes were examined using one-way ANOVA Survival was investigated using Cox proportional hazard regression model, conditional inference trees, and Kaplan-Meier analysis Comparisons of IC50-values in drugresistant and -sensitive cells were done by the two-tailed Student ttest A p-value b 0.05 was considered statistically significant All statistical evaluations were done using the statistical software R version 3.1.3 (www.R-project.org) including following packages: ‘ctree’ in R package party [21] and survival [22] Results 3.1 Increased MELK expression in EOC correlates with histological grading and shorter disease-free survival Two recent studies reported elevated MELK expression in several human cancers including epithelial ovarian, breast, gastric, and lung cancer [6] and a correlation between elevated MELK expression and poor prognosis and tumor aggressiveness in basal-like breast cancer (BBC) [7] In order to expand on these studies, we reviewed the TCGA data base (PANCAN12) and its bioinformatical analysis revealed that MELK expression was also a predictor of poorer disease-free survival in cancers in general (n = 3495, HR 1.071, CI [1.031 to 1.112], p-value based on Cox regression 0.000364), i.e regardless of the cancer subtype (Fig 1A) and that elevated MELK expression was present also in cancers (n = 3599) of the head and neck regions, bladder, colon, rectum, and ovary (Fig 1B) MELK expression in ovarian cancer, our cancer of interest, ranked between cancers with the origin rectum and endometrium Worthwhile mentioning that our previously performed gene expression profiling on various ovarian tumors and healthy ovarian surface epithelium already in 2004 demonstrated an interesting profile for KIAA0175, later to be named MELK [10,11]: KIAA0175 was generally low expressed in normal ovarian surface epithelium, slightly elevated in mucinous and endometrioid borderline (low-malignant) tumors, and at higher but varying levels primary and metastatic ovarian cancer samples of different histotypes (serous, endometrioid, and mucinous) (Fig 2A) In the present study we determined and validated MELK expression in ovarian cancer searching the publicly accessible GEO Gene Expression Omnibus database for the respective transcriptomic data sets and compared MELK expression with clinico-pathological characteristics and outcome data provided along with the transcriptomic data Nine transcriptomic data sets were identified comprising 1241 patients subdivided into serous ovarian carcinomas (SOC, n = 1144), serous benign tumors (SBenign, n = 20), serous borderline tumors (SBL, n = 43), and tissue from normal ovarian surface epithelium (HOSE, n = 34) (Supplementary Table S1) The comparison of HOSE versus SOC revealed a statistically significant (p b 0.01; five data sets) increase in MELK expression in SOC (Supplementary Fig S1A) One data set showed that MELK expression Please cite this article as: R.S Kohler, et al., MELK expression in ovarian cancer correlates with poor outcome and its inhibition by OTSSP167 abrogates proliferation and viabilit , Gynecol Oncol (2017), http://dx.doi.org/10.1016/j.ygyno.2017.02.016 R.S Kohler et al / Gynecologic Oncology xxx (2017) xxx–xxx Fig MELK is elevated in various cancers and is a predictor disease-free survival in all TCGA cancer samples regardless of the cancer subtype (A) Kaplan-Meier curve for disease-free survival for low and high MELK expression in all TCGA cancer samples (B) Transcriptomic data set analysis Box-whisker plots (data log transformed) showing MELK gene expression in the various cancers compared to “normal” kidney (ovarian cancer indicated by arrow) progressively increased from benign to borderline to SOC (Supplementary Fig S1B) MELK expression positively correlated with the histological grade, i.e lesser histopathological differentiation (grade) the higher MELK expression (Supplementary Fig S1C) TCGA data set analysis separated high-grade SOC (HGSOC) into “low” and “high” MELK and identified high MELK expression as a predictor for poor progression-free survival (HR 5.73, p = 0.0036) (Fig 2B) These results indicate that MELK is elevated in SOC compared to normal ovarian tissue and that MELK expression increases with the histological grading, i.e with aggressiveness of SOC, and correlates with shorter progression-free survival and HOSE6-3 cells (Fig 3B) Both normal controls have been recently suggested to be the origin of EOC and their subtypes [23,24] 3.3 Depletion of MELK by shRNA induces apoptosis and inhibits colony formation We determined whether depletion of MELK protein by shRNA-mediated knockdown induces apoptosis and abrogates oncogenic growth in EOC cells The results show that doxycycline-induced MELK silencing induced PARP-1 cleavage (measure for ongoing apoptosis) in OVCAR8 cells but not HOSE6-3 cells (Fig 4A: top panel) and inhibited colony formation in soft-agar (representing oncogenic growth) in OVCAR8 cells (bottom) 3.2 Elevated MELK in HGSOC cell lines To study MELK expression in ovarian cancer cell lines we accessed the Cell Line Encyclopedia (CCLE) for MELK expression in a large set of ovarian cancer cell lines Twenty-eight cell lines with an MELK expression level between MDA-MB-468 (BBC and positive for MELK-associated oncogenesis [7]) and T47D (luminal breast cancer and negative for MELK-associated oncogenesis [7]) were identified Those with high (e.g SKOV3), intermediate (e.g OVCAR8, IGROV1), and moderate (e.g OVCAR4) MELK gene expression (Fig 3A) were chosen for Western blot analysis (Fig 3B) Cell lines representing “normal” ovary surface epithelium (HOSE6-3) and fallopian tube epithelium (FT33-tag, FT190, FT194, FT237, FT246) were also included MELK expression was highest in EOC cells, low or poorly detectable in almost all fallopian tube cells 3.4 MELK-inhibitor OTSSP167 induces G2/M cell cycle arrest, inhibits proliferation and colony formation, and activates apoptosis preferentially in HGSOC cell lines The observation that shRNA-mediated depletion of MELK induced apoptosis and inhibited proliferation prompted us to determine the effects of pharmacological inhibition of MELK by OTSSP167 on proliferation and survival OTSSP167 has been reported to inhibit proliferation in several cancer cells of different origin in vitro and in vivo [8,9] by inducing a cell cycle arrest in G2/M [25] We therefore determined the effects of OTSSP167 on cell cycle transition and cell morphology, on colony formation, apoptosis, and proliferation in a panel of selected cell lines The results demonstrate that OTSSP167 induced a marked Fig MELK expression is associated with aggressive ovarian cancer subtypes Bar chart showing MELK expression in the different subtypes as published in Heinzelmann et al 2004 [10,11] (A) (B) Kaplan-Meier curve for low and high MELK expression (threshold determined by conditional inference trees) and progression-free survival (TCGA data set for high-grade SOC) Please cite this article as: R.S Kohler, et al., MELK expression in ovarian cancer correlates with poor outcome and its inhibition by OTSSP167 abrogates proliferation and viabilit , Gynecol Oncol (2017), http://dx.doi.org/10.1016/j.ygyno.2017.02.016 R.S Kohler et al / Gynecologic Oncology xxx (2017) xxx–xxx Fig (A) Differential MELK expression in ovarian cancer cell lines as retrieved from the “Cancer Cell Line Encyclopedia (CCLE)” Broad Institute data base Data are normalized and cell lines are sorted by descending MELK expression MELK expression in MDA-MB-468 basal-like breast cancer cells (indicated in red) and T47D (blue) and MCF-7 (green) luminal breast cancer cells are shown as references (B) Western blot displaying MELK expression in cell lines representing the ovarian surface epithelium, fallopian tube epithelium, and HGSOC Whole cell protein extracts were produced, separated, and analyzed as described in “Materials and methods” Tubulin is the sample loading control arrest at the G2/M transition of the cell cycle in BG1 and OVCAR8 cells (Supplementary Fig S2A) In addition, OTSSP167-treated cells displayed enlarged nuclei and altered cell morphology and tubulin and F-actin distribution, typically seen in cells arrested at the G2/M transition (Supplementary Fig S2B) OTSSP167 inhibited anchorage-independent growth in soft agar (Fig 4B) and anchorage-dependent colony formation (Fig 4C) Western blotting demonstrated that 100 nM OTSSP167 for 48 h produced marked PARP-1 cleavage in HGSOC cell lines whereas no or weak PARP-1 cleavage was observed in normal ovarian surface epithelial cells (HOSE6-3) and in the fallopian tubal cells (FT33-tag, FT190, FT237) (Fig 4D) Breast cancer cell lines MDA-MB-468 and T47D were used as positive and negative apoptosis controls, respectively MTTassay results (Fig 4E) showed that OTSSP167 inhibited proliferation Fig Effects of MELK depletion and pharmacological inhibition of MELK activity on cell cycle transition, morphology, colony formation, apoptosis, and proliferation (A) Apoptosis induction (top panel) and oncogenic growth inhibition in soft-agar (bottom) in shRNA-mediated MELK-depleted cells (B) OTSSP167 inhibits anchorage-independent growth in soft agar (C) OTSSP167 reduces colony growth formation (anchorage-dependent) (D) Western blot for PARP1-cleavage in cell lines representing the ovarian surface epithelium (HOSE), the fallopian tube epithelium (FT), and ovarian cancer cell lines of various histotypes Whole cell protein extracts of cell cultures treated with 100 nM OTSSP167 for 48 h were produced, separated, and analyzed Tubulin, sample loading control; luminal (T47D) and basal (MDA-MB-468) breast cancer cell lines: negative and positive MELK controls, respectively (E) MTT-assay results for the cell lines indicated presented as IC50 concentrations sorted by the histotypes (fallopian tubes, ovary surface epithelium, HGSOC) Cells were exposed to OTSSP1167 for 72 h Mean ± SD of at least four independent experiments performed in quadruples For further details, see “Material and methods” Please cite this article as: R.S Kohler, et al., MELK expression in ovarian cancer correlates with poor outcome and its inhibition by OTSSP167 abrogates proliferation and viabilit , Gynecol Oncol (2017), http://dx.doi.org/10.1016/j.ygyno.2017.02.016 R.S Kohler et al / Gynecologic Oncology xxx (2017) xxx–xxx Fig OTSSP167 and drug resistance Sensitivity to OTSSP167 of Cisplatin-resistant TYK-nu(R) and -sensitive TYK-nu (A) and Paclitaxel-resistant IGROV1-PXL and -sensitive IGROV1 cells (B) (C) MDR1 expression (Western blot) in parental IGROV1 and TYK-nu cells and in their respective resistant counterparts (pxl5 and nu(R)) Tubulin is sample loading control and Pos is positive MDR1 control (D) Sensitivity to OTSSP167 of OVCAR8 and OVCAR8-OTS cells (obtained by four cycles of step-wise exposure to increasing OTSSP167 concentrations: details in “Materials and methods”) and their cross-sensitivity to Carboplatin, Paclitaxel, and Doxorubicin Effect of co-treatment of OVCAR8 cells with OTSSP167 and Carboplatin (E) or Paclitaxel (F) on cell viability: white bars (Carboplatin or Paclitaxel alone), grey bars (Carboplatin or Paclitaxel plus 50 nM OTSSP167), black bars (Carboplatin or Paclitaxel plus 70 nM OTSSP167) MTT-assay results presented as mean ± SD of at least independent experiments performed in quadruples 3-times more efficiently in EOC (IC50 range: 24 to 82 nM) than in FT cells (IC50 range: 134 to 160 nM) BG1 and OVCAR3 cells were to most sensitive among the EOC cells (IC50: 24 nM and 36 nM) HOSE6-3 cells were as sensitive as the two least sensitive EOC cells (TYK-nu and IGROV1) 3.5 OTSSP167 retains efficiency in drug-resistant HGSOC cells and does not produce drug resistance acquisition Drug resistance (both intrinsic and acquired) is one major and critical obstacle in cancer management and the development of novel anticancer compounds which are highly effective also in recurrent tumors is of great importance We therefore evaluated whether ovarian cancer cell lines resistant to platinum compounds and Paclitaxel (both currently used in ovarian cancer treatment) remain sensitive to OTSSP167 MTT-assay results demonstrate that 4-fold (p = 0.011, n = 4) Cisplatin-resistant TYK-nu(R) cells (Fig 5A) and 9.3-fold (p = 0.002, n = 3) Paclitaxel-resistant IGROV1-PXL cells (Fig 5B) were as sensitive to OTSSP167 as their parental counterparts The IGROV1-PXL cells were generated in our laboratory by step-wise exposing IGROV1 cells to increasing Paclitaxel concentrations, starting with μM Paclitaxel (details see “Materials and methods”) and, unlike the Cisplatin-resistant TYKnu(R) cells, overexpressed multidrug resistance protein (MDR1) (Fig 5C) In contrast, four cycles of Carboplatin exposure in IGROV1 cells (starting concentration: 20 μM) failed to yield a stable and significantly Carboplatin-resistant IGROV1 subpopulation (1.22-fold, p = 0.338, n = 3) We also determined whether OTSSP167 causes resistance acquisition We repeatedly exposed OVCAR8 cells to OTSSP167 (starting at 75 nM) and were able to produce a subline with reduced OTSSP167 sensitivity (1.45-fold (p = 0.123, n = 3; Fig 5D) after four cycles: no viable cells were found when OTSSP167 concentration was increased to 120 nM or higher in two independent attempts This OVCAR8-OTS subline did not show cross-resistance to Carboplatin, Paclitaxel or Doxorubicin (Fig 5D) Taken together, these results indicate that OTSSP167 remains effective in drug-resistant ovarian cancer cells and suggest that OTSSP167 is unlikely to cause cross-resistance acquisition in these cells 3.6 OTSSP167 sensitizes OVCAR8 cells to Carboplatin but not Paclitaxel Addressing the chemo-sensitizing property of OTTSSP167 we determined whether sub-lethal concentrations of OTSSP167 sensitize OVCAR8 cells to Carboplatin and Paclitaxel MTT-assay results showed that the combination of Carboplatin and OTSSP167 was more cytotoxic than each compound itself (Fig 5E), whereas the combination of Paclitaxel with OTSSP167 was less cytotoxic than each compound itself (Fig 5F) In contrast to a narrow additive effect for Carboplatin a clear-cut antagonistic effect was observed with Paclitaxel Discussion Unlike for other cancers MELK and its inhibitor OTSSP167 have not yet been investigated in ovarian cancer Here we (i) compared MELK expression, clinico-pathological outcome parameters in nine independent transcriptomic data sets; (ii) investigated the effects of genetic and pharmacological MELK inhibition on proliferation, oncogenic growth, and apoptosis; and (iii) evaluated the efficiency of MELK-inhibitor OTSSP167 in drug-resistant ovarian cancer cell lines, its possibility to cause resistance acquisition, and its chemosensitizing potential in these cells Four major findings emerge from this study: Firstly, MELK was elevated in EOC and in particular increased towards aggressiveness of SOC; secondly, elevated MELK expression correlated with poorer disease outcome (progression-free survival); Thirdly, MELK inhibition/depletion abrogated proliferation and oncogenic growth by arresting cells at the G2/M transition of the cell cycle and induced apoptosis in EOC Please cite this article as: R.S Kohler, et al., MELK expression in ovarian cancer correlates with poor outcome and its inhibition by OTSSP167 abrogates proliferation and viabilit , Gynecol Oncol (2017), http://dx.doi.org/10.1016/j.ygyno.2017.02.016 R.S Kohler et al / Gynecologic Oncology xxx (2017) xxx–xxx cells Fourthly, MELK-inhibitor OTSSP167 retained its effectivity in Paclitaxel-resistant (IGROV1) and Cisplatin-resistant (TYK-nu) cells and sensitized OVCAR8 cells to Carboplatin but not to Paclitaxel From this we may conclude that elevated MELK in EOC is a possible therapeutic target and that OTSSP167 is a potential novel compound against (drug-resistant) aggressive ovarian cancers, in particular EOC, undifferentiated cancers, and MMMT Although elevated MELK expression has been previously reported in various cancers [6], we were one of the first group to identify MELK as an interesting target in epithelial ovarian cancer [10,11]: its gene expression profile was outstanding in our results published in 2004, when MELK was still only known as KIAA0175 Since the development of the small-molecule MELK-inhibitor OTSSP167 in 2012 [8,9], investigations into this gene have continuously grown almost exponentially In the present study we addressed MELK expression particularly in HGSOC, the most common and lethal gynecological cancer in women with a poor overall survival [1] in more detail and clearly demonstrated elevated MELK in ovarian cancer on the gene expression level, on protein level in tissues, and in cell lines In contrast, MELK was poorly expressed in normal surface epithelial, fallopian tube cells, and in benign tumors, indicating a clear-cut and significant preference of MELK expression for ovarian cancer Intriguingly, MELK expression correlated with increasing histopathological grades and with a shorter progression-free survival in the TCGA data set Our finding is therefore clinically relevant as it proposes MELK expression as marker for tumor aggressiveness and progression and as prognostic predictor for poor outcome and expands a recent study showing that MELK expression correlated with early recurrence and poor prognosis in hepatocellular carcinoma [26] These characteristics of MELK have been reported for BBC, where (unlike in luminal breast cancer and normal breast) MELK was highly expressed and correlated with poor prognosis [7] The comparability of our results with HGSOC and those reported for BBC may be not surprising and may be attributed to the similarities between these two cancers [11,12] Despite its specificity towards aggressive cancer types, it remains to be elucidated whether MELK can drive oncogenic transformation Not less important is our finding that OTSSP167 significantly reduced proliferation and oncogenic growth at low nanomolar concentrations in EOC cells, associated with a marked arrest at the G2/M transition of the cell cycle and apoptosis OTSSP167 also inhibited proliferation and induced apoptosis in fallopian tube epithelial cells but at a considerable lesser extent Accordingly, the anti-proliferative and proapoptotic effect of OTSSP167 has also been reported with other cancer cells including BBC cells and myeloma cells [7,27–30] MELK phosphorylates CDC25B which is required for the entry in mitosis [31]: this event is abrogated by MELK inhibition, resulting in the G2/M arrest [25] MELK seems to disable critical cell-cycle checkpoints, reduce replication stress, and stimulate proliferation by its capacity to increase the threshold for DNA-damage tolerance [5] Roles for FOXM1, a transcription factor regulated by MELK via direct phosphorylation [4,27,32], and for TP53 [33, 34] have been suggested, and OTSSP167.has been shown to decrease MELK and FOXM1 [32] We observed abrogation of proliferation and oncogenic growth and apoptosis induction also in EOC cells where MELK expression was silenced by doxycycline-inducible shRNA This is consistent with the BBC cell study [7] and indicates that not only the enzymatic inhibition of MELK but also its loss results in growth inhibition and apoptosis This view is supported by a recent study [5] showing that the structurally unrelated MELK inhibitor MELK-T1 provoked the rapid proteasome-mediated degradation of MELK: this was associated with a rapid and long-lasting ataxia telangiectasia-mutated (ATM) activation, phosphorylation of checkpoint kinase (CHK2), a strong phosphorylation of TP53, a prolonged up-regulation of p21, and a down-regulation of FOXM1 target genes The authors conclude that MELK is a key stimulator of proliferation by its ability to increase the threshold for DNA-damage tolerance and propose that targeting MELK by the inhibition of both its catalytic activity and its protein stability might sensitize tumors to DNA-damaging agents or radiation therapy The latter has indeed been shown recently as genetic and pharmacological knockdown/inhibition of MELK radio- and chemo-sensitized rectal cancer [35] and breast cancer cells [36] Another intriguing finding is related to the issue of OTSSP167 and drug resistance in EOC cells Our data indicate that OTSSP167 retains its effectivity in Paclitaxel- or Cisplatin-resistant cells and that overexpression of MDR1 in Paclitaxel-resistant OVCAR8 cells does not impair OTSSP167 sensitivity The latter suggests that OTSSP167 efficacy is MDR1-independent, but is opposed to a recent study reporting OTSSP167 resistance in ABCB1 transporter-overexpressing myeloma cells [7,27–30] Our data also indicate that repeated exposure to OTSSP167 cause drug resistance acquisition in OVCAR8 cells, at least to some extent which may be considered minor (1.45-fold) when compared to those obtained for Paclitaxel (9.3-fold) and Cisplatin (4.0-fold) Regardless of whether or not considered minor the observed resistance to OTSSP167 is not associated with cross-resistance to Paclitaxel, Carboplatin, and Doxorubicin Interestingly, our results confirm for Carboplatin the reported sensitizing property of OTSSP167 to DNA damaging agents [35] but also provide an opposing example with Paclitaxel: as a microtubule-poison Paclitaxel kills cells by inducing mitotic arrest and/or interferes with the interphase [37], but it is unknown how MELK inhibition encounters the cytotoxic effect of Paclitaxel This finding, though deriving from one cell line only, may be of clinical interest, in particular for the design of combinational clinical trial These characteristics of OTSSP167 together with its radio and (at least for some drugs) chemosensitizing property are clinically significant and may underline the intriguing advantages of OTSSP167 Interestingly, a recent study suggests that OTSSP167 has “off-MELK” effects, i.e that OTSSP167 has other target kinases and may have additional mechanisms of action for cancer cell killing [38] For instance, in addition to MELK, OTSSP167 inhibits Aurora B and BUB1, both of which compromise mitotic checkpoint regulation and hence contribute to cell killing in a MELK-independent manner Whether and to which extent these kinases can be accounted for the apoptotic effect of OTSSP167 observed in ovarian cancer cells remains open, especially since shRNA against MELK recapitulated the effects seen with OTSSP167 in our study Our results collectively propose MELK as an attractive drugable target in patients with (refractory) ovarian cancer This warrants the evaluation of OTSSP167 in clinical trials, either as single compound or in combination with chemotherapeutic drugs (e.g Carboplatin) or radiation OTSSP167 may not only present an interesting alternative to the current treatment of the aggressive forms of ovarian cancer, the survival of which has, despite improved surgical techniques and drug regimens, only changed marginally [1,39,40] but may also provide new hope to improve the poor survival rate of this disease Conflict of interest statement All authors declare no conflict of interest Funding and acknowledgements This work was supported by Swiss National Science Foundation (310030_156982, 310030_143619 and 32 to VHS); OncoSuisse Grant (KFS_3013-08-2012 to VHS), Krebsliga Beider Basel (06-2013 to VHS) We thank Prof R Drapkin (Penn Ovarian Cancer Research Center, University of Pennsylvania, Philadelphia, PA, USA) for generously providing FTSEC cells We thank Danny Labes and Emmanuel Traunecker (Flow Cytometry Facility) and Michael Abanto and Beat Erne (Microscopy Facility) for all necessary support We also thank Monica Nunez Lopez for her experimental contributions to this manuscript Please cite this article as: R.S Kohler, et al., MELK expression in ovarian cancer correlates with poor outcome and its inhibition by OTSSP167 abrogates proliferation and viabilit , Gynecol Oncol (2017), http://dx.doi.org/10.1016/j.ygyno.2017.02.016 R.S Kohler et al / Gynecologic Oncology xxx (2017) xxx–xxx Appendix A Supplementary data Supplementary data to this article can be found online at http://dx doi.org/10.1016/j.ygyno.2017.02.016 References [1] D.D Bowtell, S Bohm, A.A Ahmed, P.J Aspuria, R.C Bast Jr., V Beral, et al., Rethinking ovarian cancer II: reducing mortality from high-grade serous ovarian cancer, Nat Rev Cancer 15 (2015) 668–679 [2] R.E Bristow, R.S Tomacruz, D.K Armstrong, E.L Trimble, F.J Montz, 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growth in mice [7] OTSSP167. .. this article as: R.S Kohler, et al., MELK expression in ovarian cancer correlates with poor outcome and its inhibition by OTSSP167 abrogates proliferation and viabilit , Gynecol Oncol (2017),... this article as: R.S Kohler, et al., MELK expression in ovarian cancer correlates with poor outcome and its inhibition by OTSSP167 abrogates proliferation and viabilit , Gynecol Oncol (2017),