In the last decade, several tyrosine kinase inhibitors (TKIs), which disrupt pathways involved in the proliferation and tumorigenesis of thyroid cancer, have been extensively studied. Two different TKIs, lenvatinib and sorafenib, were recently approved by both the US FDA and European Medicine Agency. Until date, the duration of the TKI response is not sufficient and resistance eventually occurs. The goal of this study was to investigate a new treatment protocol, SoLAT, using sorafenib and lenvatinib alternatively on refractory thyroid cancer.Patient-derived aggressive papillary thyroid cancer (PTC) cell lines from patients with biochemical and histologically proven aggressive RAI-refractory papillary thyroid cancer were exposed to sorafenib and lenvatinib alternatively. Human thyroid cancer cell xenografts were obtained by injecting patient-derived aggressive PTC cell lines into the flank of female BALB/c nude mice. Tumor-bearing mice were treated with sorafenib and lenvatinib alternatively. Cell viability assay, immunofluorescence analysis, confocal imaging, immunoblot analysis, flow cytometry analysis of cell cycle and a tube formation assay were performed.0/G1 population was observed in the SoLAT group, leading to apoptosis, cell cycle arrest, and strong inhibition of advanced PTC cell viability. SoLAT reduced the level of EMT markers such as vimentin, E-cadherin, Snail and Zeb1 by FGFR inhibition. In the xenograft model, individual treatment with sorafenib or lenvatinib did not markedly suppress patient-derived aggressive PTC cell xenograft tumors, whereas SoLAT significantly suppressed the proliferation of these tumors.SoLAT was more effective for advanced PTC cell lines than individual treatment. Immunoblot analysis showed that SoLAT markedly increased levels of cell cycle inhibitors (p53 and p21), and pro-apoptotic factors (Apaf-1 and cleaved caspase 3) and decreased levels of positive cell cycle regulators  and anti-apoptotic factors . Increased sub-GSoLAT was more effective than individual treatment with sorafenib or lenvatinib in inhibiting PTC progression by inducing cell cycle arrest. Studies using both in vitro cell culture and an in vivo xenograft model provided evidence of tumor shrinkage with SoLAT. We suggest that these effects may be due to reduced EMT-mediated drug resistance in the aggressive PTC model. Thyroid cancer accounts for more than 90% of all endocrine cancers and is the most common endocrine malignancy, as its incidence has increased over the last three decades . ThyroidSorafenib was the first tyrosine kinase inhibitor tested in a phase III trial and was approved for the treatment of metastatic DTC in 2013. Patients with progressive RAI-refractory DTC treated with oral sorafenib showed improved progression-free survival compared to patients receiving placebo , 15. TheIn this study, we delineated the mechanism of drug resistance of cancer cells by inhibiting FGFR signaling and EMT in response to current treatments and discussed how these problems are being addressed.Fresh tumors were gained from patients with biochemical and histologically proven aggressive RAI-refractory papillary thyroid cancer who were treated at the Thyroid Cancer Center, Gangnam Severance Hospital, Yonsei University College of Medicine, Seoul, Korea. Further protocol and details are described in our previous articles , 21.After resection, the tumors were transported to the laboratory. Normal tissue and fat were eliminated and rinsed with 1× Hank’s balanced salt solution (HBSS). Additional protocol and details are indicated in our previous article . The resThe patient-derived aggressive papillary thyroid cancer (PTC) cell lines were grown in RPMI-1640 medium with 10~ 15% FBS. Authentication of the cell lines were carried out by with Cell ID system  comparing their profiles with those published on the DMSZ database. Mycoplasma contamination was invariably checked with the Lookout Mycoplasma PCR Detection Kit .Cell proliferation was measured using the 3--2, 5-diphenyl tetrazolium bromide (MTT) assay. Additional protocol is described in our previous article .At first, the combination treatment of the Sorafenib and Lenvatinib for 5 days, after Sorafenib and Lenvatinib alternating treatment for 5 days.At first, the combination treatment of the Sorafenib and Lenvatinib for 10 days, after Sorafenib and Lenvatinib alternating treatment for 10 days.β-catenin expression was analyzed with immunofluorescent staining. Further protocol and data analysis details are described in our previous article .Antibodies against Ki-67 (Abcam), cyclin D1 , CDK4 (Santa Cruz Biotechnology), p21 (Santa Cruz Biotechnology), p53 (Santa Cruz Biotechnology), p-ERK 1/2 (Santa Cruz Biotechnology), ERK 1/2 (Santa Cruz Biotechnology), Apaf-1 (Abcam), p-NFκB (Santa Cruz Biotechnology), Bcl-2 (Santa Cruz Biotechnology), caspase 3 (Santa Cruz Biotechnology), vimentin (Abcam), E-cadherin (Abcam), Snail (Abcam), Zeb1 (Abcam), and β-actin (Santa Cruz Biotechnology) overnight at 4 °C.Cells were treated with sorafenib and lenvatinib alone in an alternating regimen (SoLAT) in RPMI-1640 medium containing 10% FBS for 40 h, harvested by trypsinization, and fixed with 70% ethanol. Further protocol and data analysis details are described in our previous articles , 21.4) were cultured with growth factor-reduced Matrigel  in MV1 for 1 h for cell attachment, following which the endothelial growth basal medium-2 (EBM-2) was replaced with conditioned medium and cell culture was continued for 24 h. Tube length was quantified after 8 h by measuring the total cumulative tube length in 3 random microscopic fields with a computer-assisted microscope using Image J software. The original magnification used was × 100.Human umbilical vein endothelial cells (HUVECs) (7 × 106 cells/mouse) were cultured in vitro and then injected subcutaneously into the upper left flank region of female BALB/c nude mice. After 11 days, tumor-bearing mice were grouped randomly (n = 10/group) and 10 mg/kg lenvatinib (administered p.o.) and 40 mg/kg sorafenib (administered p.o.), or lenvatinib or sorafenib alone (administered p.o.) were administered once every two days. Tumor size was measured on alternative days using calipers.. All experiments were approved by the Animal Experiment Committee of the Yonsei University.The patient-derived aggressive PTC cells (3.5 × 10Primary Antibodies against p21 (Santa Cruz Biotechnology) and Bcl-2 (Abcam) were diluted with PBS at a ratio of 1:100 and incubated overnight at 4 °C. All tissue sections were counterstained with hematoxylin, dehydrated, and mounted.P < 0.05 indicated statistical significance.Statistical analyses were performed using GraphPad Prism software . Immunohistochemistry results were subjected to ANOVA followed by a Bonferroni post hoc test. Values are expressed as the mean ± SD. 50 of sorafenib and lenvatinib in GSP2 and GSP3 cells  on advanced PTC. Individual treatments with sorafenib or lenvatinib did not significantly inhibit advanced PTC. SoLAT was more effective than the individual treatments Fig.  and d. W0/G1 population (P < 0.05) and induced cell death in advanced PTC, GSP2 and GSP3  was more effective than individual treatment with sorafenib or lenvatinib in inhibiting PTC tumor progression by inducing cell cycle arrest. There have been studies testing alternating chemoradiotherapy instead of concurrent chemoradiation for nasopharyngeal cancer and studies which showed alternating treatment for mantle cell lymphoma , 23. SeqStudies using both in vitro cell culture and in vivo xenograft model provided evidence of tumor shrinkage in the alternative switching group. We suggest that these effects may be due to reduced EMT-mediated drug resistance in the aggressive PTC model.ZEB1 could restore drug sensitivity [EMT is induced in aggressive forms of thyroid cancer with elevated ZEB1 levels, which can promote drug resistance through EMT-dependent and EMT-independent mechanisms –26. Studsitivity , 28. Sorsitivity . EMT revsitivity .50 is reduced when the drugs are used alternately compared to when used individually, which suggests that this can be an option for decreasing drug toxicity. In contrast to combination therapy, the drugs may be effective before being washed out of the body in the case of interval treatment; however, this requires further in vivo evidence. These findings may assist in developing a treatment protocol with reduced toxicity and enhanced drug efficacy.The frequency and nature of adverse side effects differ between sorafenib and lenvatinib. Hand foot skin reaction is the most common side effect of sorafenib, whereas hypertension is the most common adverse effect of lenvatinib. Our results show that the ICTKIs are recommended for the treatment of RAI-refractory DTC patients with metastatic, rapidly progressive, symptomatic, and/or imminently threatening disease, which is not otherwise amenable to local control using alternative approaches. The benefits of systemic therapeutics have been demonstrated in the form of improved progression-free survival in three randomized, double-blinded, placebo-controlled clinical trials for vandetanib, sorafenib, and lenvatinib , 14, 15.Mechanisms for TKI resistance include receptor autophosphorylation, autophagy, involvement of hypoxia-inducing factor, epigenetic regulation, and EMT , 35. FurIt is necessary to investigate the mechanism underlying the success of the alternating treatment with lenvatinib and sorafenib compared to that of individual lenvatinib treatment in inhibiting the growth of aggressive PTC both in vitro and in vivo. Further studies are required to compare the efficiencies and toxicities of combination therapy and SoLAT. In addition, the effect of reusing one drug after development of resistance to both drugs  has to be determined. Similarly, the effectiveness of reusing a therapeutic agent after development of resistance versus alternative interval treatment for prolonging disease-free survival has to be evaluated; in addition, the appropriate interval should be determined if interval treatment is found to be more efficient.The current study suggests that SoLAT was more effective than individual treatment with sorafenib or lenvatinib in inhibiting PTC progression by inducing cell cycle arrest and reducing EMT-mediated drug resistance.


We observed a significant increase in the expression of ACAT-1 and CE levels in a panel of ovarian cancer cell lines  compared to primary ovarian epithelial cells . To confirm the tumor promoting capacity of ACAT-1, we inhibited ACAT-1 expression and activity by treating our cell lines with an ACAT inhibitor, avasimibe, or by stable transfection with ACAT-1 specific short hairpin RNA (shRNA). We observed significant suppression of cell proliferation, migration and invasion in ACAT-1 knockdown ovarian cancer cell lines compared to their respective controls (cell lines transfected with scrambled shRNA). ACAT-1 inhibition enhanced apoptosis with a concurrent increase in caspases 3/7 activity and decreased mitochondrial membrane potential. Increased generation of reactive oxygen species (ROS) coupled with increased expression of p53 may be the mechanism(s) underlying pro-apoptotic action of ACAT-1 inhibition. Additionally, ACAT-1 inhibited ovarian cancer cell lines displayed enhanced chemosensitivity to cisplatin treatment. These results suggest ACAT-1 may be a potential new target for the treatment of ovarian cancer.Abnormal accumulation of acyl-CoA cholesterol acyltransferase-1 (ACAT-1) mediated cholesterol ester has been shown to contribute to cancer progression in various cancers including leukemia, glioma, breast, pancreatic and prostate cancers. However, the significance of ACAT-1 and cholesterol esters (CE) is relatively understudied in ovarian cancer. In this  Epithelial ovarian cancer has the highest mortality rate among all gynecologic cancers with no curative treatment and poor survival , 2. Althde novo lipid synthesis, inducing lipid raft formation and finally altering cell signaling [in vitro. These observations suggest that strategies targeting the prevention of CE accumulation may prove beneficial for treatment of ovarian cancer.Numerous studies are being carried out to decode the altered lipid metabolic profiles of cancer cells to formulate cancer specific therapeutic strategies. Altered lipid metabolism leads to increased cancer cell proliferation, migration and invasion resulting in metastasis –9. Identignaling –20. Loweignaling  lymphocyignaling  and glioignaling  cell linin vitro study, we determined the expression levels and contribution of ACAT-1 in ovarian cancer progression utilizing a panel of ovarian cancer cell lines. The role of ACAT-1 in tumor cell aggression was studied by blocking ACAT-1 expression/activity in OC-314, SKOV-3 and IGROV-1 cell lines using ACAT-1 specific short hairpin RNA (shRNA). Important tumor associated activities, such as cell migration, invasion and proliferation capabilities, were compared between ACAT-1 inhibited cell lines and their respective scrambled control cell lines. Furthermore, to investigate the molecular mechanism(s) underlying ACAT-1 mediated cancer progression, we studied the effect of ACAT-1 inhibition on cell cycle, apoptosis and mitochondrial membrane potential. Additionally, we evaluated the possible involvement of reactive oxygen species (ROS) and tumor suppressor p53 in ACAT- 1 mediated effects. Finally, we studied the effect of ACAT-1 inhibition on chemosensitivity towards cisplatin as previous reports have linked cholesterol/CE to drug resistance [Free excess cholesterol is esterified to CE by acyl-CoA cholesterol acyltransferase (ACAT)  which exsistance , 29.2. SKOV-3, IGROV-1 and OC314 cell lines were authenticated by the ATCC using STR profiling technique. All cells tested negative for mycoplasma. Avasimibe used in the experiments was purchased from Selleckchem, TX, USA.Human primary ovarian epithelial cells (H-6036) were obtained from Cell Biologics, . Human ovarian carcinoma cell lines, OC-314 and SKOV-3 were obtained from Dr. McAsey’s laboratory . Isogenic ovarian cancer cell line pairs, e.g., A2780 / A2780-CDDP and IGROV-1 / IGROV-1CDDP were obtained from Dr. Brodsky . As previously reported , all celAntibodies for ACAT-1, ACAT-2 and Phosphatase and tensin homolog (PTEN) were obtained from abcam . Antibodies for p53, p21, p27, Bax, bcl-2 and β-actin were purchased from Cell Signaling Technology, Inc .™ Ambion™ Cells-to-CT™ Power S 1-Step Power SYBR Green Kit. The kit’s instructions were followed with the exception that extracted RNA was used instead of cells directly. The ACAT-1 specific primers were purchased from Integrated DNA Technologies, Inc. . The sequences of primers used for ACAT-1 qRT-PCR are listed as below:Total RNA was extracted from cells using RNeasy mini-kit  following manufacturer’s instructions. After RNA yield and quality were assessed, samples were stored at −80°C until use. 50 ng of extracted RNA was used to perform qRT-PCR analysis of ACAT-1 gene expression using Invitrogen5′-CAAGGCGCTCTCTCTTAGATGAAC-3′;Forward: forward primer, 5′-GATAAAGAGAATGAGGAGGGCAATAA-3′Reverse: reverse primer, 18srRNA was used as a reference housekeeping gene. The threshold cycle (Ct) values were normalized to the housekeeping gene to quantify ACAT-1 mRNA (Comparative Ct method). Data were expressed as mean ± SD of triplicate experiments.Western blotting was performed to evaluate expression of ACAT-1, ACAT-2 and other important regulators of cell cycle and apoptosis. Cell seeding, cell lysis and western blotting were done as described previously with slight modifications . In brie5 cells were seeded into a six well plate and incubated for 48 hours at 37°C in a humidified atmosphere with 5% CO2. After the stipulated time, media were collected from each well and centrifuged to remove any particulates. The assay was performed immediately with the supernatant according to the kits instructions. The absorbance was measured at 450 nm using synergy H1 microplate reader . Data were expressed as mean ± SD of triplicate experiments. Ovarian cancer cell lines were compared against primary ovarian epithelial cells .ACAT-1 protein from cell supernatants was quantified using the ACAT-1 ELISA Kit (Human) from Aviva Systems Biology . Briefly, 4 × 10Cells grown on glass coverslips were fixed with 4% paraformaldehyde and permeabilized using 0.1% Triton X-100 in PBS. Cells were subsequently incubated overnight with ACAT-1 primary antibody (1:50 dilution) at 4°C, probed with FITC labeled secondary antibody and observed under Olympus DP73 fluorescence microscope. Cell were counterstained with DAPI for nuclei staining (blue).6 cells were processed for lipid extraction by adding 200 μL of chloroform: Isopropanol: NP-40 (7:11:0.1) in a micro homogenizer and centrifuged at 15,000 x g. All the liquid phase (organic phase) was transferred to a new tube and air dried at 50ºC to remove the chloroform. The samples were kept under vacuum to remove organic traces. The dried samples were then dissolved in 200 μL of cholesterol assay buffer by sonication and vortexing until homogeneous. 50 μL of the sample was used for assay of CE, free cholesterol (FC) and total cholesterol (TC) concentrations. Data was expressed as mean ± SD of against primary ovarian epithelial cells .Quantification of CE from ovarian cancer/normal cells was done using Total Cholesterol and Cholesteryl Ester Colorimetric Assay Kit  following the manufacturer’s instructions. This kit detects total cholesterol (cholesterol and CE) in the presence of cholesterol esterase in the reaction. Briefly, 1 × 10Cell viability was determined by Presto Blue cell viability reagent  as described previously . In brie50 values, cells plated in 96-well plates (5000 cells/well) overnight were treated with cisplatin at concentrations ranging from 1.57–400 μM for a total of 48 hours and cell viability was assessed as described above. IC50 value was obtained by fitting the data with a sigmoidal dose response model using GraphPad software. Data were expressed as mean ± SD of triplicate experiments.For assessment of cisplatin ICStable ACAT-1 knockdown was done using ACAT-1 shRNA lentiviral particle purchased from Santa Cruz Biotechnology, Inc. . ACAT-1 specific shRNA was transfected into the cells  according to the manufacturer’s protocols using polybrene transfection reagent. Stable transfected cells were selected under 2 μg/mL puromycin treatment. To assess nonspecific effects of transfection, cells were transfected with scrambled shRNA lentiviral particles  and are referred to as “scrambled controls” for comparative purposes.Cell proliferation studies were performed using CyQuant cell proliferation assay kit  which accurately estimates cell number based on the DNA content. Cells were seeded into a 96-well plate at a density of 1000 cells / 100 μL per well and incubated for 1, 2, 3, 4, 6, and 14 days. At the end of each time point, 100 μL CyQuant solution was added to the wells and fluorescence was measured (excitation 485/emission 530 nm) using synergy H1 microplate reader . ACAT-1 inhibited cell lines were compared against their respective scrambled controls for each cell line. Data were expressed as mean ± SD of triplicate experiments.Colony formation assay was performed following a standard protocol . In brie™ cell culture inserts with 8 μm pore-size PET membrane. Cell culture inserts with BD matrigel coated PET membrane were used for invasion assays whereas uncoated inserts were used for migration assays. Briefly, 1 ×105 cells were seeded in the upper chamber of the trans-wells in 0.5 ml serum-free media. The lower chamber contains 750 μL of media with 20% FBS. Cells were allowed to migrate for 22 hours at 37°C, 5% CO2. Quantitation of cell migration and invasion was done by labeling the cells with a fluorescent dye calcein AM and measuring the fluorescence of migrated/invaded cells using a synergy H1 microplate reader (BioTek). As BD FluoroBlok membrane blocks the light from 490–700 nm with >99% efficiency, only migrated/invaded cells are detected by the bottom-reading fluorescence plate reader. Data were expressed as mean ± SD of triplicate experiments.Migration and Invasion assays were performed using FluoroBlok® Cell Cycle Assay Kit (MCH100106) following the manufacturer’s instructions. Briefly, 70% ethanol fixed cells were washed and stained with a pre-mixed reagent containing nuclear DNA stain propidium iodide and RNAse A. Then, cells were categorized using Muse cell analyzer . The Muse Cell Cycle Software module performs calculations automatically and categorizes the cells into different cell cycle phases based on propidium staining of nuclei.Cell cycle analysis was carried out using Muse5 cells) were seeded in 60 mm2 culture dishes and incubated for 4 days. At the end of the stipulated time, cells were washed and stained with Hoechst stain (2 drops/mL of media) for 15 minutes at 25°C and observed under a fluorescent microscope. Representative images were taken with an inverted microscope  and 20× objective. Further quantification of apoptosis was done using Muse’s Annexin V & Dead Cell Assay Kit  following the manufacturer’s instructions. Briefly, cells were incubated with Muse Annexin V & Dead Cell reagent at room temperature in the dark and analyzed using Muse® Cell Analyzer . The Muse Software Module performs calculations automatically.Nuclear condensation indicative of apoptosis was assessed using NucBlue Live Cell Stain Hoechst 33342 . This qualitative test was performed as described previously , 32. In 6) were seeded into a 100 mm2 culture dishes and incubated for 3 days. Cells were harvested, and stained with Muse™ MitoPotential Dye for 30 min at 37°C followed by addition of 7-AAD. The Data was acquired using Muse® Cell Analyzer . The Muse Software Module performs calculations automatically.Mitochondrial transmembrane potential was determined Muse’s MitoPotential Assay Kit , which simultaneously measures changes in mitochondrial potential  and cell death using MitoPotential Dye and 7-AAD. Ovarian cancer cell lines . Data were expressed as mean ± SD of triplicate experiments.Caspase 3/7 activity was measured using Caspase-Glo 3/7 assay kit  following the manufacturer's recommendations. Briefly, 1 × 106 cells were seeded in a petri dish and incubated for 3 days after which the cells were washed and re-suspended in fresh PBS and incubated with 5 μM 5,6-carboxy-2′,7′-dichlorodihydrofluorescein diacetate  for 30 min at 37°C. The cells were washed twice with DPBS, re-suspended in an equal volume of DPBS and fluorescence measured with flow cytometry. Data was acquired on a BD Accuri C6 flow cytometer and analyzed using Accuri C6 software . Twenty thousand cells were analyzed for each sample.ROS were detected via carboxy-H2DCFDA using flow cytometry as described previously . BrieflyAll experiments were performed at least in triplicate. Data are expressed as the means ± standard deviation (SD). Statistical analyses were performed using Student’s t-test. Differences were considered statistically significant at P<0.05.Using qRT-PCR, we found significant expression of ACAT-1 mRNA in a panel of ovarian cancer cell lines compared to primary ovarian epithelial cells, H-6036  . SignifiSince ACAT exists predominantly in two isoforms (ACAT-1 and ACAT-2), it is essential to understand the expression levels of both isoforms in ovarian cancer cell lines. Western blot analysis show significant expression of ACAT-1 protein in cancer cell lines compared to normal control cells . This reACAT-1 mediates esterification of cholesterol to CE for its storage in LDs; thus, we assessed the levels of TC, FC and CE in ovarian cancer cell lines. In positive correlation with ACAT-1 data, CE levels in ovarian cancer cell lines were significantly higher (5–7 fold) than normal control cells . BesidesTo further understand the role of ACAT-1 in tumor progression, we performed cell viability assays using three ovarian cancer cell lines SKOV-3, OC-314 and IGROV-1. Initially, we inhibited ACAT using the non-specific inhibitor, avasimibe. Cells treated with avasimibe for 3 days exhibited significantly reduced cell viability (PrestoBlue assay) compared to untreated controls. As shown in ACAT-1 inhibition by stable transfection with shRNA resulted in significant decrease in cell viability  in OC-314, SKOV-3 and IGROV-1 cell lines, respectively, compared to their respective scrambled controls, when assessed 6 days post-seeding .To assess the effect of ACAT-1 inhibition on cell proliferation, we performed time course studies ranging from 1 to 14 days post-seeding using CyQuant cell proliferation assay kit that accurately measures cell number based upon the DNA content. As shown in Unlimited proliferation is the predominant feature of all cancer cells. In order to understand the effect of ACAT-1 inhibition on proliferation, we performed a colony formation assay that measures cells ability to undergo sufficient proliferation to form a colony. Our results clearly show that in all three cell lines, ACAT-1 inhibited cells formed less number of colonies compared to their respective scrambled controls . These rTo evaluate whether ACAT-1 inhibition affects tumor aggressiveness, we measured cell invasion and migration abilities of ACAT-1 inhibited and control cell lines. We observed that both migration and invasion capabilities of OC-314, SKOV-3 and IGROV-1 cell lines were markedly suppressed upon ACAT-1 inhibition. Quantitation of results indicated that ACAT-1 inhibition reduced cell migration by 29 ± 7%, 35 ± 5% and 41 ± 7`% in ACAT-1 inhibited (shRNA) cell lines of OC-314, SKOV-3 and IGROV-1 respectively compared to their respective scrambled controls . SimilarIn order to understand the mechanisms underlying the suppressive effects of ACAT-1 inhibition on cancer cell growth, we performed cell cycle analysis. Flow cytometry analysis revealed that in comparison to scrambled controls, ACAT-1 inhibited cell lines exhibited a significantly higher number of cells in sub-G1 (apoptotic) phase with the corresponding decrease in G1 phase of cell cycle . A signiSince cell cycle data show significant population of cells in sub-G1 (apoptotic) phase in ACAT-1 inhibited cell lines, we performed qualitative assessment of apoptosis using nuclear stain Hoechst 33342. As shown in In this study, we mainly focused on apoptosis related changes associated with ACAT-1 inhibition to understand the underlying mechanism(s). These include mitochondrial membrane potential change, activities of pro-apoptotic markers such as caspases 3/7 and expression of proteins associated with tumor suppression and ROS generation.Mitochondrial membrane potential changes were assessed using Muse MitoPotential Assay Kit. As shown in To assess caspase3/7 activities, we utilized Promega’s Caspase-Glo3/7 assay kit that measures caspase-3 and -7 activities luminometrically. In general, tumor suppression is associated with cell cycle arrest and apoptosis. In this study, we focused on the important tumor suppressor genes p53 and PTEN. Densitometric analysis of western blots showed expression of PTEN in all the three cell lines. There was no significant difference in PTEN expression between ACAT-1 inhibited cell lines of OC-314, SKOV-3 and IGROV-1 and their respective scrambled controls . Howeverp53 restricts cellular growth by inducing apoptosis, cell cycle arrest or senescence. Therefore, we analyzed the expression levels cell cycle regulators p21, p27 and important bcl-2 family members, Bax and bcl-2. As shown in As ROS generation is commonly associated with mitochondrial depolarization and apoptosis , we inve50) values were assessed and compared between ACAT-1 inhibited and scrambled controls of OC-314, SKOV-3 and IGROV-1. Cells were treated with cisplatin at concentrations ranging from 0.78 μM to 400 μM for 48 hours. We observed a significant decrease in cisplatin IC50 values in ACAT-1 inhibited cell lines compared to their respective scrambled controls , 20.28 ± 2.32 (vs. 10.77 ± 2.11 μM in the control) and 16.14 ± 2.45 (vs. 10.27 ± 1.64 μM in the control) respectively. Data were expressed as mean ± SD of triplicate experiments.Cisplatin half maximal inhibitory concentration  phase was observed. These results suggest that ACAT-1 inhibition causes the cells to undergo predominantly apoptosis rather than cell cycle arrest. Additionally, we observed increased generation of ROS in ACAT-1 inhibited cell lines compared to their respective scrambled controls. This is consistent with the fact that ROS generation is commonly associated with mitochondrial membrane depolarization , 39.Ohmoto T et al. showed that the selective ACAT-1 inhibitor K604 effectively suppressed the proliferation of U251-MG glioblastoma cells via reduced phosphorylation of Akt and ERK1/2 [Excess CE may alter tumor cell signaling to promote tumor proliferation and survival –20 and dd ERK1/2 . CollectAmong various tumor suppressor proteins, p53 tumor-suppressor protein encoded by the TP53 is considered the “guardian of the genome”. TP53 also plays an important role in the regulation of cholesterol pathways . MutatioIncreased levels of p53 have been observed upon a variety of cellular stresses including genotoxic damages, oncogene activation and hypoxia –46. p53 Kokontis et al. reported that wild type p53 induces cell cycle arrest in a p21 dependent manner, without apoptosis, whereas a p53 mutant (defective in trans-activation) induces apoptosis without cell cycle arrest [et al. reported that, similar to wild type p53, the pP53gly (281) mutant was able to induce apoptosis despite the lack of p53-specific transactivation function [A number of p53 mutants were shown to display normal cell cycle arrest and apoptotic functions, while others lacked both activities. e arrest . He et afunction . In thisOvarian cancer recurrence has been mainly attributed to drug resistance even after initial successful response to platinum-based chemotherapy , 4. CancIn summary, our results show increased expression of ACAT-1 in ovarian cancer cell lines compared to the primary ovarian epithelial cells ; thus, confirming that ACAT-1 mediated CE accumulation is a cancer specific event. Inhibiting ACAT-1 and depleting CE levels seems to have an anti-tumor effect in terms of regulation of apoptosis, cell proliferation, migration and invasion properties in addition to enhancing the sensitivity to cisplatin. Therefore, therapeutic strategies to regulate ACAT-1/CE levels may prove beneficial for ovarian cancer treatments. It is essential to understand that multiple lipidogenic and cholesterogenic pathways are affected in ovarian cancer. Therefore, it is critical to target multiple lipid/cholesterol metabolism routes for effective ovarian cancer treatement as multiple feedback loops exist for compensating any single blockage.


H-tetrazolium (MTS) assay, Annexin V/propidium iodide apoptosis assay and scratch wound assay. Tube formation of all the cell lines was inhibited by AqB013, AqB050 and the combination of the two compounds. The inhibition of 2H-11 and 3B-11 was frequently accompanied by impaired migration, whereas that of HUVEC treated with AqB050 and the combination was associated with reduced cell viability due to apoptosis. AqB013 and AqB050 exhibited an anti-tubulogenic effect through inhibition of AQP1-mediated cell migration and induction of apoptosis. Together with previously reported anti-tumour cell effect of AqB013 and AqB050, our findings support further evaluation of these compounds as potential cancer therapeutics.AqB013 and AqB050 compounds inhibit aquaporin 1 (AQP1), a dual water and ion channel implicated in tumour angiogenesis. We tested AqB013 and AqB050 either as monotherapy or in combination on tube formation of murine endothelial cells (2H-11 and 3B-11) and human umbilical vascular endothelial cells (HUVECs). The mechanism underlying their anti-tubulogenic effect was explored by examining cell viability, induction of apoptosis and migration using 3--5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2 While mortality of infectious disease and vascular disease has declined in many countries, cancer has now become one of the leading causes of death and its incidence and mortality are rising worldwide. The World Health Organisation estimates that 18.1 million new cancer cases and 9.6 million cancer deaths occurred in 2018 [Xenopus laevis oocytes injected with AQP1 cRNA, AQP1 was demonstrated to serve as a cyclic nucleotide-gated cation channel [Aquaporin 1 (AQP1), a small hydrophobic integral transmembrane protein, which spans the plasma membrane as a tetramer, was first characterised in erythrocytes and renal tubules by Denker et al. in the late 1980s and is reported to function as a water channel ,4. More  channel ,6. While channel ,8. Over- channel ,15,16,17One of the proposed mechanisms underlying AQP1-mediated cancer development and growth is enhanced tumour angiogenesis. Angiogenesis is a fundamental property of cancer as it provides tumour cells with essential nutrients and oxygen required to sustain growth and an entry to systemic circulation for distant metastasis . SaadounAqB013 and AqB050 are synthetic small molecules that were developed based on a loop diuretic bumetanide . AqB013 p = 0.0182) and 58% (p < 0.0001) at 40 µM, respectively, and 57% (p < 0.0001) and 86% (p < 0.0001) at 80 µM, respectively, compared to the vehicle control. Combining the two compounds achieved equivalent inhibition to AqB050 alone; tube formation was reduced by 61% for AqB013 plus AqB050 at 40 µM each (p = 0.0001), designated 40/40 µM. For 3B-11, tube formation was reduced similarly by AqB013 and AqB050: 65% (p < 0.0001) and 78% (p < 0.0001) inhibition was observed, respectively, at 80 µM compared to the vehicle control. Combination treatment at 40/40 µM resulted in reduction by 84% (p < 0.0001). The HUVEC line was more sensitive to AqB013 than AqB050 and no tube formation was observed for AqB013 at 80 µM (p < 0.0001), while AqB050 at 80 µM caused 64% inhibition (p < 0.0001). Fifty-five per cent inhibition was achieved by the combination treatment at 40/40 µM. The effect of AqB013 and AqB050 either as monotherapy or in combination was assessed in tube formation assays using 2H-11, 3B-11 and HUVEC lines . For 2H-p = 0.0006). 3B-11 line demonstrated no inhibition of cell viability with any of the treatments tested. In contrast, cell viability of HUVEC line was reduced by 21% for AqB050 at 40 µM (p = 0.0004), 48% for AqB050 at 80 µM (p < 0.0001), 17% for AqB013 plus AqB050 at 20/20 µM (p = 0.0041) and 31% for AqB013 plus AqB050 at 40/40 µM (p < 0.0001).To examine the mechanism underlying reduced tube formation of endothelial cells when treated with AqB013 and AqB050 either as monotherapy or in combination, cell viability was assessed using MTS assay . For 2H-To further elucidate the cause of impaired cell viability induced by the compounds, induction of apoptosis was assessed in the HUVEC line treated with AqB050 either as monotherapy or in combination with AqB013. A reduction in cell viability was demonstrated. An increase in apoptosis was observed with treatment containing AqB050 at 40 µM or higher doses. Compared to the vehicle control, the proportion of cells in apoptosis increased by 25% with 40 µM AqB050, 32% with 80 µM AqB050, and 29% with AqB013 plus AqB050 at 40/40 µM .p = 0.0002), AqB050 at 40 µM  and AqB013 plus AqB050 at 20/20 µM . Wound closure in the 3B-11 line was similarly inhibited by the compounds; however, the magnitude of inhibition was smaller than that for the 2H-11 line. Closure rate was reduced by 23% for AqB013 at 80 µM (p = 0.0035), 23% and 26% for AqB050 at 40 µM (p = 0.0037) and 80 µM (p = 0.001), respectively, and 33% for AqB013 plus AqB050 at 40/40 µM (p < 0.0001), compared to the vehicle control. The most prominent inhibition of wound closure was observed in the HUVEC line. Unlike the murine endothelial cell lines, AqB013 did not reduce wound closure of HUVEC line even at 80 µM and no statistically significant difference was observed at AqB013 at 40 and 80 µM compared to the vehicle control. AqB050 at 40 and 80 µM reduced wound closure by 89% (p < 0.0001) and 94% (p < 0.0001), respectively, and AqB013 plus AqB050 at 20/20 and 40/40 µM reduced wound closure by 34% (p = 0.0045) and 91% (p < 0.0001), respectively, compared to the vehicle control. Anti-migratory actions of AqB013 and AqB050 either as monotherapy or in combination were assessed on scratch wound assay using 2H-11, 3B-11 and HUVEC lines . The lowThis is the first report of the effect of AqB013 and AqB050 combined, showing anti-tubulogenic properties, extending our previous report of the effect of AqB013 in inhibiting HUVEC tube formation . In the The anti-tubulogenic effect of AqB050 was accompanied by impaired cell viability. The reduced tube formation induced by AqB050 at 80 µM in the 2H-11 and HUVEC lines was accompanied by a significant decrease in cell viability with HUVEC line being particularly susceptible to the effect. No impaired cell viability was observed for 3B-11, while AqB050 at 80 µM inhibited its tube formation. This difference in the sensitivity to cytotoxicity of AqB050 between the HUVEC primary cell line and 2H-11 and 3B-11 cell lines may relate to the simian virus 40-transformed nature of 2H-11 and 3B-11 cell lines, to make them immortal. SV40 transformation is mediated largely by the interaction of its T-antigen with tumour suppressor proteins p53 and pRb of transformed cells . BlockinThe increase in annexin-V staining following treatment of HUVECs with AqB050 at doses which inhibited tube formation suggests apoptosis was at least partly involved in the anti-tubulogenic action of AqB050 on this cell line. A link between AQP1 and apoptosis has been suggested previously. Wu et al. reported shRNA-mediated knockdown of AQP1 in two osteosarcoma lines U2OS and MG63 resulted in promotion of apoptosis with reduction in the level of antiapoptotic protein BCL-2 and increase in the levels of proapoptotic protein Bax and cleaved caspase 3 . AdditioTreatment of HUVECs with the lower dose combination of AqB013 plus AqB050 at 20/20 µM, where tube formation was inhibited by 28%, was not associated with an increase in the annexin-V staining. As this combination treatment resulted in 17% inhibition of cell viability, the impaired tube formation observed can be explained by its anti-proliferative effect through mechanisms other than induction of apoptosis. Additionally, AqB013 plus AqB050 at 20/20 µM reduced wound closure of HUVECs by 34% compared to the vehicle control, suggesting inhibition of two-dimensional migration by this combination treatment partly contributed to the reduced tube formation. In fact, for the murine endothelial cell lines, inhibition of tube formation seen with AqB013 and AqB050 either as monotherapy or in combination was frequently accompanied by impaired wound closure in the absence of reduced cell viability. Treatment of 2H-11 line with AqB013 at 80 µM, AqB050 at 40 µM and 80 µM, and AqB013 plus AqB050 at 40/40 µM, which inhibited tube formation by more than 50%, all reduced its two-dimensional migration. Similar observations were made with 3B-11; treatment with AqB013 at 80 µM, AqB050 at 80 µM and AqB013 plus AqB050 at 40/40 µM was all associated with greater than 50% inhibition of tube formation and reduced two-dimensional migration. It is plausible that AqB013 and AqB050 exhibit their anti-tubulogenic effect through inhibition of cell motility for these cell lines. This is supported by the finding that AQP1-deficient endothelial cells generated from the aorta of AQP1-null mice demonstrated impaired cell migration and invasion, compared to those from AQP1-wild type mice . FurtherIn contrast to the murine cell lines, HUVEC line treated with AqB013 at 80 µM showed complete inhibition of tube formation in the absence of impaired two-dimensional migration or cell viability, supporting an alternative underlying mechanism. As extracellular matrix was present in the tube formation assay, but not in the scratch wound assay, we postulate this unusual discordance between the effect of AqB013 on cell motility and tube formation derives from its effect on extracellular matrix (ECM) degradation. Using a syngeneic melanoma mouse model, Simone et al. found intra-tumoural injection of AQP1 siRNA reduced expression of matrix metallopeptidase-2 (MMP-2), one of the key regulators of tumour angiogenesis . TreatmeIn the current study, AqB050, the ion channel inhibitor, appeared more effective in impairing two-dimensional migration of endothelial cells than the water channel inhibitor AqB013. AqB050 at 40 and 80 µM inhibited wound closure of all the endothelial cell lines tested, whereas AqB013-mediated impaired wound closure was only observed for murine cell lines at 80 µM with the magnitude of inhibition by AqB050 at 80 µM being larger than that of AqB013 at 80 µM. Similarly, we observed treatment with AqB013 plus AqB050 at 40/40 µM resulted in the equivalent inhibition of wound closure to AqB050 at 40 µM for all the endothelial cell lines. Both water and ion channel functions of AQP1 have been suggested to play a part in cell motility. Jiang demonstrated reduced water permeability induced by AQP1 RNA-interference was associated with decelerated wound closure of HT-20 human colon cancer cell line . AqB011,There have been two publications describing the anti-cancer efficacy of AqB013 and AqB050 in vitro. Our group previously reported that wound closure and invasion of moderately AQP1-expressing colon cancer cell line HT-29 was inhibited by AqB013 . SimilarTM; Life Technologies) and 50 U/mL-50 µg/mL penicillin–streptomycin (Life Technologies). HUVEC line was maintained in Vascular Cell Basal Medium supplemented with Endothelial Cell Growth Kit-VEGF (ATCC). Cultures were maintained in 5% CO2 at 37 °C.Human umbilical vein endothelial cell (HUVEC) and two murine endothelial cell lines (2H-11 and 3B-11) were purchased from American Type Culture Collection . 2H-11 and 3B-11 lines were maintained in Dulbecco’s Modified Eagle Medium , supplemented with 10% foetal bovine serum , 2 mM L-alanyl-L-glutamine dipeptide  were prepared by resuspending each compound in dimethyl sulphoxide  at 40 mM and this was diluted in the respective complete medium to achieve the indicated concentrations for the experiment. Vehicle control was prepared by adding DMSO to the complete medium at the dilution equivalent to that for the highest dose of compounds assessed. 5 cells per well on 6-well plates and incubated for 48 h in 5% CO2 at 37 °C. Apoptosis controls were treated with Bacopaside II (Sigma-Aldrich) at 20 µM for 24 h prior to harvest. Necrosis controls were prepared by heating harvested cells to 63 °C for 30 min. Cells were treated with endothelial growth medium, vehicle (DMSO), AqB050 at 40 and 80 µM or AqB013 plus AqB050 at 20/20 and 40/40 µM for 24 h. Harvested cells were stained with Annexin-V-FLUOS staining kit  as per the manufacturer’s instruction and fluorescence was read on BD FACSCanto II cell analyser , and analysed using FlowJo software  v10.4 as described previously [HUVECs were seeded at 1 × 104 cells per well on 96-well plate in a respective complete medium and incubated overnight in 5% CO2 at 37 °C. The medium was replaced by fresh complete medium containing various concentrations of the compounds with DMSO as the vehicle control. Following further incubation for 24 h, viability was assessed using CellTiter 96® AQueous Non-Radioactive Cell Proliferation Assay  according to the manufacturer’s instruction. The absorbance was read at 492 nm and the results were expressed as absorbance normalised to the vehicle control.Cells were seeded at 1 × 104, 4 × 104 and 3.5 × 104 cells per well for 2H-11, 3B-11 and HUVEC lines, respectively. After 6 h of incubation in 5% CO2 at 37 °C, the medium was replaced with DMEM-based media supplemented with 2% FBS for 2H-11 and 3B-11 lines and HUVEC growth medium for HUVEC line, both containing 1 µg/mL mitomycin C (Sigma-Aldrich). These media are referred to as “mitomedia”. A scratch wound was made on the cell monolayer and cells were incubated for 1 h in the fresh mitomedia. The medium was then changed to mitomedia containing various concentrations of compounds with DMSO as a vehicle. Area surface of the wound was serially monitored and images were obtained for the next 16 h on Eclipse TE2000-U light microscope  at 40× magnification. The wound area was quantified using NIS-Elements BR software (Nikon) and the result was expressed as relative wound closure (%) compared to time point 0.Cells were plated on 96-well plate in the respective complete medium at 1.2 × 10Cells were plated on a 96-well angiogenesis µ-plate  coated with Matrigel (Corning) according to the supplied protocol in the respective medium containing various concentration of compounds with DMSO as a vehicle. The number of loops formed was counted at 2 h.p < 0.05.Graph Pad Prism was used for statistical analysis. A one-way ANOVA with Tukey’s multiple comparisons test was carried out. Statistical significance was accepted at The current study demonstrated two bumetanide-derived inhibitors of AQP1, AqB013 and AqB050, inhibit tube formation in vitro using murine and human endothelial cell lines known to express AQP1, supporting their potential utility as cancer therapeutics. Their anti-angiogenic efficacy was suggested to be at least partly driven by inhibition of cell migration and induction of apoptosis where AQP1 has been reported to play a role. Additionally, we postulate inhibition of AQP1 water channel by AqB013 resulted in impaired AQP1-mediated degradation of ECM, possibly explaining the inhibited tube formation of HUVEC line observed with this compound, where two-dimensional migration measured using scratch wound assay was preserved. Further research is required to continue characterising the compounds with respect to their mechanisms of actions and their in vivo efficacy.


P16 (CDKN2Aink4a) gene is an endogenous CDK4/6 inhibitor. Palbociclib (PD0332991) is an anti-CDK4/6 chemical for cancer treatment. P16 is most frequently inactivated by copy number deletion and DNA methylation in cancers. It is well known that cancer cells with P16 deletion are more sensitive to palbociclib than those without. However, whether P16 methylation is related to palbociclib sensitivity is not known. By analyzing public pharmacogenomic datasets, we found that the IC50 of palbociclib in cancer cell lines (n = 522) was positively correlated with both the P16 expression level and P16 gene copy number. Our experimental results further showed that cancer cell lines with P16 methylation were more sensitive to palbociclib than those without. To determine whether P16 methylation directly increased the sensitivity of cancer cells to palbociclib, we induced P16 methylation in the lung cancer cell lines H661 and HCC827 and the gastric cancer cell line BGC823 via an engineered P16-specific DNA methyltransferase (P16-Dnmt) and found that the sensitivity of these cells to palbociclib was significantly increased. The survival rate of P16-Dnmt cells was significantly lower than that of vector control cells 48 hrs post treatment with palbociclib (10 μM). Notably, palbociclib treatment also selectively inhibited the proliferation of the P16-methylated subpopulation of P16-Dnmt cells, further indicating that P16 methylation can increase the sensitivity of cells to this CDK4/6 inhibitor. These results were confirmed in an animal experiment. In conclusion, inactivation of the P16 gene by DNA methylation can increase the sensitivity of cancer cells to palbociclib.The  The ability to sustain uncontrolled cell proliferation is one of the hallmarks of cancer cells . The norThe clinical implementation of first-generation nonselective CDK inhibitors was originally hampered by the high toxicity and low efficacy of these agents , 8. SecoCDKN2Aink4a gene, is an endogenous cellular CDK4/6 inhibitor that controls the G1-S phase transition of the cell cycle. This gene is one of the most frequently inactivated genes in cancer genomes; it is inactivated mainly by DNA methylation  [https://www.cancerrxgene.org/translation/Drug/1054#t_IC50] [rg/ccle] . The hal#t_IC50] .2.Human lung cancer cell lines H1975, H1395, H661, H596, H460, H358, H292 and HCC827, as well as human breast cancer cell lines HCC1937, MDA-MD-468 and MDA-MD-157, were purchased from the National Infrastructure of Cell Line Resource . Human lung cancer cell lines H1299 and A549; human breast cancer cell lines MDA-MB-231 and MCF7; human gastric cancer cell lines BGC823, MGC803 and AGS; human colon cancer cell lines HCT116, RKO and SW480; and human liver cancer cell line HepG2 were obtained from laboratories at Peking University Cancer Hospital and Institute. The H596 and MCF7 cell lines were cultured in DMEM  containing 10% FBS (Gibco) and 1% penicillin-streptomycin (Gibco), the AGS cell line was cultured in F12 medium (Gibco), and the remaining cell lines were cultured in RPMI 1640 medium (Gibco). All cells were maintained at 37°C in humidified air with 5% CO2O as a stock solution at concentration of 10 mM. Cells were seeded in 96-well plates at 4,000 cells per well (4 wells/group). Forty-eight hrs post treatment with various concentrations of palbociclib, cell confluence was evaluated and analyzed using the IncuCyte ZOOM system . The half-maximal inhibitory concentration (IC50) was calculated using GraphPad Prism 6 software.The CDK4/6 inhibitor palbociclib (PD0332991) was purchased from Selleck Chemicals  and dissolved in double-distilled HP16 transcription start site, an engineered P16 promoter-specific seven zinc finger protein (7ZFP) was fused with the catalytic domain of mouse Dnmt3a (approximately 608–908 aa) and integrated into the pTRIPZ vector, which contained a “Tet-On” switch . The lung cancer cell lines H661 and HCC827 and the gastric cancer cell line BGC823 were infected with lentiviral particles containing the P16-Dnmt or control vector and incubated for 48 hrs. Then, puromycin  was added to the medium  to kill nontransfected cells. The pooled cells treated with puromycin for two weeks were considered stably transfected cells. Then, these cells were treated with 0.25 μg/mL doxycycline  for 14 days to induce P16-Dnmt expression.The established P16-Dnmt expression vector and pTRIPZ empty control vector were prepared and used to transfect cells as previously described . BrieflyGenomic DNA was extracted from cells or tumor tissues and subjected to bisulfite treatment using an EZ DNA Methylation-Gold Kit  according to the manufacturer’s instructions. The modified DNA was stored at -20°C until use.P16 CpG islands was assessed by a 150/151-bp methylation-specific PCR (MSP) or 115-bp quantitative MethyLight assay as previously described .The methylation status of escribed –34. Brieht assay . All PCRP16 mRNA was performed using primer sets shown in GAPDH was used as the reference gene. The amplification was performed with FastStart Universal SYBR Green Master (ROX)  in the Applied Biosystems 7500 Real-Time PCR System, as previously described  via oral gavage (i.g.) daily for 3 weeks. Then, mice were sacrificed by spine dislocation, and the tumors were weighed. Tumor samples were processed for formalin-fixed and paraffin-embedded (FFPE) sections or Western blot analysis.HCC827 cells  sections were incubated with primary antibody against Ki-67 (1:300) overnight at 4°C. The remaining IHC steps were the same as previously described .P16 (CDKN2Aink4a) mRNA level or copy number in various cancer cell lines was assessed using the nonparametric Spearman correlation test and a linear regression model. The Mann–Whitney test was used for nonnormally distributed data. Student’s t-test was used for normally distributed data. Statistical significance was accepted at p<0.05.Statistical analyses were performed using SPSS 23 software or GraphPad Prism 6 software. The Kolmogorov–Smirnov test and Shapiro–Wilk test were used to estimate the normality of distributions. The relationship between the IC50 value of palbociclib and the relative P16 methylation and the sensitivity of cancer cells to palbociclib, we first analyzed the correlation between the IC50 levels of palbociclib in 522 cell lines and the transcription level and copy number of the CDKN2A/P16 gene using public pharmacogenomics datasets [CDKN2A mRNA level , P16-methylated (P16-M), and P16-deleted (P16-D) groups according to the results of MSP (P16-U cell lines (n = 12) were consistently higher than those in the P16-M cell lines (n = 5) and P16-D cell lines (n = 5) [18.88 vs. 4.61 and 3.99 (μM), p<0.01; P16-M and P16-D cell lines. These results indicate that similar to copy number deletion, P16 methylation is positively associated with high sensitivity of cancer cells to palbociclib.Then, we validated the correlation in 22 cancer cell lines, which were divided into s of MSP . We founP16 methylation directly increases the sensitivity of cancer cells to palbociclib, we stably transfected three P16-U cell lines  with the P16-Dnmt vector and induced P16-Dnmt expression in these cells by doxycycline treatment for 2 weeks. P16 methylation was successfully induced in these P16-Dnmt cells, as evidenced by MSP  cells is selectively inhibited by palbociclib treatment  or control buffer for three weeks. No adverse event was observed in these mice. Compared with control buffer, palbociclib significantly inhibited the growth of P16-Dnmt tumors  did not improve outcomes of the administration of palbociclib plus letrozole in HR-positive breast cancer patients [CCND1 amplification vs. CDKN2A and CDKN2B codeletion trended toward a longer vs. shorter treatment duration [P16 methylation could increase the resistance of lung (and stomach) cancers to paclitaxel treatment in a clinical trial [P16 methylation can increase the sensitivity of lung and stomach cancers to palbociclib. It is worth performing clinical trials to assess whether P16 methylation could be used to predict the therapeutic efficacy of CDK4/6 inhibitors such as palbociclib in patients with lung and stomach cancers.In the phase II clinical study PALOMA-1, it was found that patient selection based on patients . In the patients , 40. In duration . We receal trial . In contP16 methylation can increase the sensitivity of cancer cells to the CDK4/6 inhibitor palbociclib. P16 methylation may be used as a predictive marker for sensitivity to CDK4/6 inhibitors.This study demonstrates that S1 FigThe results of immunofluorescence and confocal microscopy analyses to directly detect alterations in the subpopulation of cells staining positive for P16 protein within HCC827 lung cancer cells stably transfected with P16-Dnmt. Scale bar, 400 μm. Student’s t-test, **p<0.01.(TIF)Click here for additional data file.S2 Figp<0.01.The results of immunofluorescence and confocal microscopy analysis to directly detect alterations of the subpopulation of cells staining positive for P16 protein within the BGC823 gastric cancer cells stably transfected with P16-Dnmt. Scale bar, 400 μm. Student’s t-test, **(TIF)Click here for additional data file.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file.


Oligonucleotides with an antiproliferative activity for human cancer cells have attracted attention over the past decades; many of them have a G-quadruplex structure (GQ), and a cryptic target. In particular, DNA oligonucleotide HD1, a minimal GQ, could inhibit proliferation of some cancer cell lines. The HD1 is a 15-nucleotide DNA oligonucleotide that folds into a minimal chair-like monomolecular antiparallel GQ structure. In this study, for eight human cancer cell lines, we have analyzed the antiproliferative activities of minimal bimodular DNA oligonucleotide, biHD1, which has two HD1 modules covalently linked via single T-nucleotide residue. Oligonucleotide biHD1 exhibits a dose-dependent antiproliferative activity for lung cancer cell line RL-67 and cell line of central nervous system cancer U87 by MTT-test and Ki-67 immunoassay. The study of derivatives of biHD1 for the RL-67 and U87 cell lines revealed a structure-activity correlation of GQ folding and antiproliferative activity. Therefore, a covalent joining of two putative GQ modules within biHD1 molecule provides the antiproliferative activity of initial HD1, opening a possibility to design further GQ multimodular nanoconstructs with antiproliferative activity—either as themselves or as carriers. G-quadruplex (GQ) folded oligonucleotides are capable of inhibiting proliferation of cancer cells, both as themselves and as carriers . De15-nucleotide DNA aptamer HD1 was originally selected as thrombin binding aptamer , and in In the study of Bates, antiproliferative activity of HD1 was discovered . This ac-/-p53 harboring a mutation of the p53 gene, treatment with 10 and 50 μM HD1—even for one day—has a notable effect, as the cell viability decreases to 80% and 25%, respectively [Treatment of HeLa cells with 10 μM HD1 for seven days has decreased the cell viability to 35% to 60% ,16. Antiectively . For theThere are a few examples when molecular constructs assembled from separate functional modules retain activity corresponding to the original modules ,20. The Therefore, because HD1 has definite antiproliferative activity, then it is of value to use this GQ module to build bimodular constructs. A minimal version of this construct is a ‘tween’, made by covalently linking two GQs via a single thymidine residue, producing 31-mer biHD1. Coming back to the story with thrombin, this ‘tween’ molecule had been thoroughly tested for anti-thrombin functioning under the name RA-36 , which hIn addition to biHD1 alone, a series of biHD1 derivatives were tested to find a structure-activity relationship: Whether any GQ alterations would affect antiproliferative activity.For studies regarding antiproliferative activity of some GQs and HD1, only a few cell lines were applied. Generally, an antiproliferative effect depends on both GQ structure and cell line. Therefore, for the particular study of biHD1, a selection of the proper cell line is required. In addition, a lack of standard conditions for testing has hindered the comparison on published data. Therefore, this study is dedicated to the screening of antiproliferative activity of the bimodular DNA oligonucleotide, biHD1 and derivatives for eight human cancer cell lines—including non-malignant human embryonic fibroblasts as a control—to establish an appropriate model for further studies.The nucleotide sequences of HD1, biHD1 and its derivatives are shown in The oligonucleotide biHD1 is a 31-mer, and it was designed by covalently joining two 15-meric HD1s via single thymidine residue. biHD1 was originally proposed as an anti-thrombin aptamer under the name RA-36 . To studInteractions of two GQs with each other within biHD1 molecule has been indirectly demonstrated previously .Circular dichroism spectroscopy is a straightforward conventional approach to monitor both GQ folding and its orientations because of highly characteristic spectra of GQ. The CD spectra of biHD1 and its derivatives have shown a topology of antiparallel GQ similar to single-modular HD1—except for biHD1+Ba . The dat+ cation is 1.5 times more than that for HD1, and it has proved the existence of two unequal GQs [The molar circular dichroism for biHD1 with Kqual GQs ,26.Replacement of T-nucleotide as a linker with more flexible three-methylene bridge led to a 1.3-time decrease of maximum 294 nm, showing some deviations from the initial conformation of HD1. A decrease of the maximum for two other derivatives correlates with expected distortions of the GQs of biHD1. A decrease of 1.3 times or slightly more of the maximum happened when two base-paired thymines of lateral TT-loops had been replaced with two more bulky adenines in biHD1-T4A,T20A. Several other structural distortions were observed for biHD1-5’-Δ2G with a 1.7 times decrease of the maximum when the first GQ had been destroyed.2+ [+ cation. An increase in the amplitude of the maximum of the CD spectrum and better proportion could be a result of conformational freezing of GQs due to stronger chelation of the guanine bases with barium cations then that with potassium one [biHD1+Ba shows both an increased maximum of the molar circular dichroism and bathochromic shift; its spectrum correlates well with spectra of single-modular HD1 with Ba2+ . The molsium one .To reveal the dose-dependent cellular response, three concentrations of biHD1 were tested: 0.1, 1.0 and 10 μM. Unsurprisingly, the responses for different cell lines turned out to be different, which suggests that the mechanisms of biHD1 action are not uniform but depend on the cell line nature .The most pronounced effect of the bimodular biHD1 on the cell viability was for lung cancer cells RL-67 A. The efThe U87 cell line showed approximately the same dose-dependent response when treated with biHD1 B. All otThe antiproliferative activity of biHD1 was independently tested by assessing the change in the number of proliferating cells for U87 in particular, and MCF7 as a control, treated with solutions of oligonucleotide biHD1 of 0.1, 1 and 10 μM for 72 h. To test the hypothesis that GQ structure is responsible for the antiproliferative activity, a series of biHD1 derivatives were created . In SectIf the more flexible biHD1-C3 molecule has decreased antiproliferative potential, then it is worth to directly compare its activity with properties of single-modular HD1.p53-/- [Until now, an antiproliferative effect of single-modular GQ HD1 has been shown on the limited number of cell lines such as HeLa, Calu-6 and mutant HCT116p53-/- ,16,17,18To introduce possible minor deviations in the GQ structure of biHD1, TT-loops structure/interactions had been changed. In each GQ TT-loops could interact via TT base pair T4-T13 in the 5′-terminal GQ and T20-T29 in the 3′-terminal GQ. In biHD1-T4A,T20A derivative, T4 and T20 are replaced with adenine residues to change the nature of the base pair partner. These replacements eliminate the antiproliferative activity of biHD1-T4A,T20A for all cell lines—except for the most sensitive lung cancer RL-67 cell line with 50% cell viability . For allOligonucleotide biHD1-5’-Δ2G could only have a single 3’-terminal GQ, because 5′-terminal GQ could not be folded due to the deletion of two 5′-terminal critical G-residues. BiHD1-5’-Δ2G does not exhibit antiproliferative activity for all cell lines—except for U87 .The complex of biHD1 with barium cation, biHD1+Ba, has an unusual conformation of both GQs . Interes50 has been determined [Antiproliferative activity of DNA oligonucleotide with G-quadruplex structure (GQ) is well known for more than decades. Bates et al. developed an antiproliferative GQ oligonucleotide AS1411 and made a thorough study aiming to translate AS1411 into the clinic . Then, Htermined . NeverthSeveral strategies for further molecular design could be proceeded either by irrational random selection or rational design. A simple way for rational design is to reveal some minimal functional structures and manipulate them; for example, to assemble them covalently into multi-functional constructs. The creation of multi-modular structures combining several structural or functional modules within a single molecule appears to be a promising approach for drug development.A simple example of antiproliferative GQ is a chair-like monomolecular antiparallel GQ, 15-mer HD1 . In someThis construct has already been made for other purposes , but itsCircular dichroism spectroscopy is an ideal method to monitor GQ folding, because every particular topology of GQ has a characteristic spectrum, and the value of molar circular dichroism is proportional to a percentage of GQ fold in the sample. As was shown before in , CD specAfter GQ structure of biHD1 had been proved, the next question is whether HD1 retains its antiproliferative activity. Antiproliferative activities of GQs with different topology vary; there is no molecular mechanism of its activity. Therefore, the very first step to check an antiproliferative potential of biHD1 is to screen the sensitivity of different human cancer cell lines and select suitable ones for further research. Different human cancer cell lines: Melanoma, glioblastoma; lung, breast, intestines and prostate cancer were used in this screening . Some ceThe antiproliferative effect depends on the concentration of GQ, the duration and frequency of treatment. In many cases, the most pronounced effect has been found at high GQ concentration of 50 μM ,17,18 anIt is generally accepted that the cell viability correlates well with proliferating potential, and that the conventional approach is the MTT test . The MTTAntigen KI-67  is a nuclear protein that is strictly associated with cell proliferation, but is absent in resting  cells; and hence is a classical cellular marker for proliferation ,33. Ki-6According to the MTT data, biHD1 has antiproliferative activity for all tested human cancer cell lines. It is the most active for two cell lines: Lung cancer cells RL-67 and CNS cancer cells U87, with a cell viability of less than 50%, and it is important that the response is dose-dependent , Table 2Two examples illustrate the idea that there is no uniform mechanism of action of different GQs for different cell lines. Firstly, the cell viability of human embryonic fibroblast cells has decreased to 70% after treatment with 10 µM biHD1-C3 B, and thThe MTT assay is a colorimetric assay to measure cell metabolic activity, and not proliferation itself. Nevertheless, the assay is widely applicable to estimate cytotoxicity (loss of viable cells) or cytostatic activity (shift from proliferation to quiescence) of potential anti-cancer agents, because all these parameters are interlinked. Therefore, it could be more informative to evaluate the antiproliferative activity of biHD1 by an independent method, for example, by direct counting of proliferating cells before and after treatment with biHD1. We focused on the sensitive U87 cell line and compared with the less sensitive MCF7 cell line. Immunohistochemistry results indicated that the number of Ki67-positive cells, indicating proliferating cells, was decreased in the case of U87 cell line  treated To test a hypothesis, which is that the GQ structure could be responsible for the antiproliferative activity, a series of biHD1 derivatives were created .The two GQs within the original biHD1 are linked via a single thymidine residue, providing a rather compact structure that unfolds in a rather cooperative manner . On the The most striking example of unusual activity of biHD1-C3 is its effect for the PC3 cell line, where proliferation is even increased by 30% after treatment with 10 μM biHD1-C3. This activity is dose-dependent . SimilarThe properties of biHD1-C3—having more independent GQs—led us to directly compare the properties of bimodular biHD1-C3 and single-modular HD1.p53-/- [The antiproliferative effect of HD1 is described in the literature just for several types of cell lines, for example, MCF7 ,12, HeLap53-/- ,18. We hThe single-module of HD1 has T4-T13 noncanonical base pair, which fixes a mutual conformation of two lateral TT-loops. This T4-T13 pair stacks on the adjacent G-quartet and stabilizes GQ . In biHDTo check the hypothesis that the antiproliferative activity requires a GQ structure of biHD1, biHD1-5’-Δ2G molecule was designed. The 5’-terminal GQ could not fold due to the deletion of critical 5′-terminal two Gs forming GQ. For the single-module of HD1, the lack of the 5′-terminal two Gs could yield an alternative structure under certain conditions , but the stability of this conformation is low . Indeed,2+ is strongly chelated into the central cavity of GQ and stiffens it [Finally, what happened when both GQs of biHD1 would be fixed by a strong chelating cation, similar to barium in the ‘biHD1+Ba’ molecule? It is well known that Baffens it . Moreoveffens it . The CD spectrum of biHD1+Ba differs significantly from the spectra of other derivatives of biHD1 . SimilarFor almost all cell lines, there is no antiproliferative effect at all concentrations of biHD1+Ba . This obDNA oligonucleotides were synthesized and HPLC-purified by Evrogen Ltd. .2PO4/Na2HPO4, 10 mM KCl and 140 mM NaCl with pH of 7.5 (PBS). The ODN solutions were subjected to annealing procedure: Heating to 95 °C, then slow cooling to 25 °C. A two-fold molar excess of barium chloride was added to biHD1 to prepare ‘biHD1+Ba’ samples. The nucleotide sequences of ODNs are provided in 2PO4/Na2HPO4, 10 mM KCl, 140 mM NaCl, pH 7.5, PBS). CD spectra were registered on CD spectrometer CHIRASCAN  and dichrograph MARK-5 . Quartz cuvettes with an optical path length of 1 cm were used. CD spectra were registered in the wavelength range from 220 to 340 nm at the scan rate of 120 nm min−1, with a response of 2 s at 2.0 nm bandwidth, and normalized by subtraction of the background scan with buffer only. The temperature was kept constant at 20 °C with a thermoelectrically controlled cell holder. The CD signal was recalculated as molar circular dichroism, Δɛ (cm−1 M−1).Circular dichroism spectroscopy (CD) samples of oligonucleotides were prepared with 2.5 μM ODN (5 μM for HD1) in phosphate-buffered saline .2 at 37 °C.Cell lines are listed in 3 cells per well and incubated for 24 h at 37 °C. Then, the medium was replaced with a 50% solution of medium with ODN at various concentrations  or phosphate buffer only. The cells were incubated for three days. Then, 20 μL of 5 mg/mL MTT reagent -2,5-diphenyltetrazolium bromide)  was added to each well, and the incubation was continued for 2 h at 37 °C. The medium was removed from the wells and 60 μL/well of DMSO  was added, stirred for 15 min, and the absorbance at 540 nm was measured using Tecan plate reader . The data were normalized to MTT results for the cells without ODN; the results represent the mean ± SEM of n = 5 repeats.The cell viability was measured by MTT test using colorimetric measurements of metabolic activity of viable cells. The cells were seeded into 96-well plates with a density of 3 × 105 cells per well. Cells were incubated with biHD1 solutions with final concentrations of 0.10, 1.0 and 10 μM in PBS or PBS without ODN in 50% cell medium for 72 h at 37 °C and 5% CO2. Cells were washed three times with PBS, and the solution of 3.7% formaldehyde in phosphate buffer was added to each well for fixation, the cells were washed three times with PBS. The primary antibody to the Ki-67 protein proliferation marker  with 0.1% Triton×100 was added to the cells for 1 h at 20 °C. Cells were washed with PBS three times, then secondary antibody conjugated with the fluorescent dye Cy2  was added to the cells for 1 h. After washing three times with PBS, the cells were tinted with a bisbenzimide nuclear fluorescent dye . The cells were photographed using an Olympus IX81 inverted fluorescence microscope  equipped with a D-70 digital camera  applying two modes to register the fluorescence of the Cy2 dye and bisbenzimide. For each well, six representative images were randomly captured. The percentages of Ki-67 positive cells were quantified by a masked researcher. For every captured area, the amount of Ki-67 positive cells (stained with Cy2) and the total amount of cells (stained with bisbenzimide) were counted. Ki-67 negative cells were not stained with Cy2. In total, for each dose of biHD1 more than 600 cells were analyzed.U87 and MCF7 cells were grown in a 4-well plate, 1 × 10p < 0.05.Statistica 13.0 software  was used for data analysis. The data are shown as ± SD. Analysis of variance was used to evaluate statistical differences between groups. The χ2 test was applied to evaluate the difference between the enumeration data of different groups. The Mann–Whitney test was used to determine the mean number of Ki-67-positive cells per field. A statistically significant difference was assumed for Building a novel molecular construct by combining several functional units (modules) is a perspective direction of a molecular design, and oligonucleotides are very suitable molecules to perform these tasks. Development of nucleic acids therapeutics requires intensification of this direction as well. Here, a feasibility of building of antiproliferating bimodular construct by covalently linking two antiproliferative DNA antiparallel G-quadruplex (GQ) 15-mers (HD1) have been demonstrated. Antiproliferative activities of bimodular GQ biHD1 and its derivatives were tested using eight different human cancer cell lines: Lung cancer RL-67, central nervous system cancer U87, epithelioid cervix carcinoma HeLa, colon cancer HCT116, breast adenocarcinoma MCF7, melanoma mS, prostate cancer PC3, and a control non-malignant human embryonic fibroblast—hEF. Antiproliferative effects of biHD1 vary for different cell lines; therefore, there is a lack of unique mechanism. BiHD1 does not inhibit proliferation of control non-cancerous hEF. BiHD1 exhibits a dose-dependent antiproliferative activity for RL-67 and U87. The study of derivatives of biHD1 for RL-67 and U87 revealed a strict structure-activity correlation of GQ folding and antiproliferative activity.These initial results are bases from where to further explore the antiproliferative properties of GQs and to study the following topics: How does GQ penetrate into the cells; molecular and cellular mechanisms; what is a target; and others. This research provides an experimental support for the development of new GQ-based antiproliferative agents either as GQ itself or as a carrier.


We previously showed that extracts from in vitro cell suspension cultures of strawberry reduced murine melanoma cell proliferation, as shown for fruit extracts. In this work, chromatographic, mass spectrometric, and spectrophotometric analyses were carried out to identify the bioactive compound exerting the detected cytotoxic activity. Moreover, aiming to confirm the anti-proliferative activity of the extracts against both paediatric and adult human tumors, cytotoxic experiments were performed on neuroblastoma, colon, and cervix carcinoma cell lines. Extracts from in vitro cell suspension cultures of strawberry induced a statistically significant reduction of cell growth in all the tumor cell lines tested. Interestingly, human fibroblasts from healthy donors were not subjected to this cytotoxic effect, highlighting the importance of further preclinical investigations. The accurate mass measurement, fragmentation patterns, and characteristic mass spectra and mass losses, together with the differences in chromatographic retention times and absorbance spectra, led us to hypothesize that the compound acting as an anti-proliferative agent could be a novel acetal dihydrofurofuran derivative (C Several natural compounds have been isolated from plant sources, and their use in the clinic leads to estimate that approximately two-thirds of the active ingredients present in anticancer drugs and in drugs for infectious diseases are of plant origin . Many exOn the other hand, many natural products used for cancer treatment occur at very low levels in plant tissues and, due to their complex structure, a completely synthetic production would be too expensive. As a consequence, the challenge to produce natural compounds in a sustainable way implies that improved methods of supply of anticancer agents would be desirable. The use of plant cell cultures for the production of metabolites is an interesting area of science , especiaFragaria x ananassa Duch (cv. Don) reduced murine melanoma cell proliferation The comparison of orthogonal information from samples and standards was used for putative metabolite identification and molecular structure elucidation .®. Chemical nomenclature was generated by ACD/ChemSketch® CCL23™), colon adenocarcinoma HT-29 cell line (ATCC® HTB-38™), and colorectal adenocarcinoma Caco-2 (ATCC® HTB-37™) cell line were grown in DMEM supplemented with 10% fetal bovine serum , 5 mM lL-glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin. The cells were tested for mycoplasma contamination and characterized in terms of cell proliferation and morphology.MYCN-amplified (HTLA-230 and IMR-32) and MYCN-single-copy (SH-SY5Y) human NB cell lines, representing the genomic and phenotypic variability of NB, and human fibroblasts from healthy donors (Fibro 2/93) were grown in complete Dulbecco’s Modified Eagle Medium (DMEM) or RPMI-1640 medium, as previously described [5, 1.5 × 105, and 2 × 105 cells/cm2 for NB, Fibro 2/93, and adult tumor cell lines, respectively, and, 24 h after, exposed for 48 h to the strawberry extracts, dissolved in EtOH 70%. EDHFF final concentration corresponded to 0.075 mM, 0.19 mM, and 0.29 mM . Viable cells were then counted on the basis of Trypan blue exclusion.In the cytotoxic experiments, the cells were seeded at a density of 2.5–3 × 10In the carboxyfluorescein succinimidyl ester (CFSE) proliferation assay, NB cells were incubated the day before with CellTrace™ CFSE staining solution (2 µM) for 15 min at 37 °C degrees, following the manufacturer’s instruction (Thermo Fisher Scientific). After 24 h, the labeled cells were treated with 0.075 mM  EDHFF . CFSE dilution was evaluated by FACS analysis. Cancer cell lines treated with pure HMF were included as a control, at HMF concentration of 15 µg/mL, which represents the highest HMF concentration detected in the sample extracts.Each treatment was performed on three samples and repeated at least three times.p-value ranges: * = p < 0.05, ** = p < 0.01, *** = p < 0.001.The standard error (SE) was calculated on the data collected, and significant differences among mean values were assessed with Prism 5 software . One-way analysis of variance (ANOVA) with Tukey’s Multiple Comparison Test was used to evaluate differences within treatments. Asterisks indicate the following Each analysis was performed on three samples per extract and repeated at least three times. Fragaria x ananassa Duch), cv. Don, as a prerequisite for in vitro studies of the production of bioactive metabolites. Extracts from the in vitro cell suspension cultures induced a statistically significant reduction of proliferation in all cancer cell lines tested. Strawberry healthy effects have been mainly attributed to polyphenols, though this conclusion is still controversial. In fact, the evidence supporting the potential anti-proliferative effects of strawberry polyphenols in several types of cancer in humans is still very limited when compared with that obtained in preclinical studies.An efficient cell suspension culture was established with leaves-derived friable calli of micropropagated strawberry . The data provided by the current study warrant NMR approaches to confirm the identity of the proposed molecule, and further studies are needed to investigate the mechanisms of the anti-proliferative effects of the putative compound and to optimize its productive process and purification. Our results confirm the potential of in vitro cultures as drug factories for the production of novel compounds for pharmaceutical studies. The discovery of compounds that are, at present, unidentified suggests that bioactive chemicals derived from plants can be detected via the proposed in vitro culture strategy, which represents a simple approach to finding novel useful metabolites.


In 481 primary BCP-ALL cases, 28 samples from 19 unique relapsed BCP-ALL cases, and twelve BCP-ALL cell lines we found that mutations are rare  and do not affect codons which are frequently mutated in other malignancies. However, recombinant ligand FGF2 reduced the response to prednisolone in several BCP-ALL cell lines in vitro. We therefore conclude that FGFR signaling can contribute to prednisolone resistance in BCP-ALL cells, but that activating mutations in this receptor tyrosine kinase family are very rare.The FGF receptor signaling pathway is recurrently involved in the leukemogenic processes. Oncogenic fusions of FGFR1 with various fusion partners were described in myeloid proliferative neoplasms, and overexpression and mutations of FGFR3 are common in multiple myeloma. In addition, fibroblast growth factors are abundant in the bone marrow, and they were shown to enhance the survival of acute myeloid leukemia cells. Here we investigate the effect of FGFR stimulation on pediatric BCP-ALL cells  Despite generally excellent outcome for children with pediatric B-cell precursor acute lymphoblastic leukemia (BCP-ALL), therapeutic options are limited for refractory or relapsed cases. Small molecule inhibitors of oncogenic pathways, such as those targeting ABL-, MEK, and JAK kinase families, have shown (pre-)clinical efficacy and may be added to chemotherapy in the future. Here we studied the role of fibroblast growth factor receptor (FGFR) signaling in pediatric BCP-ALL.2. FGF2 is expressed in the hematopoietic and stromal compartments of the bone marrow1. FGFR fusions have been described in atypical myeloproliferative disorders which may involve lymphoma or present as BCP-ALL3. High FGFR3 expression is a hallmark of t-positive multiple myeloma, and activating mutations are recurrently detected in this context6. Furthermore, FGFR signaling has been shown to contribute to drug resistance in chronic myeloid leukemia7. Thus, oncogenic FGFR signaling contributes to hematologic malignancies, but so far information on its role in pediatric BCP-ALL is limited. We therefore applied a customized targeted sequencing approach for mutational hotspots of FGFR1, FGFR2, and FGFR3 and evaluated the effect of recombinant FGF2 on in vitro prednisolone sensitivity in BCP-ALL cell lines.FGFs are not only essential in hematopoietic development, but also influence bone formation by regulating expansion and stemness of mesenchymal stromal cells8. All samples contained at least 90% leukemic blasts. Cytogenetic subtypes were determined by karyotype, fluorescence in-situ hybridization (FISH), and/or fusion-gene specific PCR. BCR-ABL1-like cases were identified using microarray gene expression profiling by the means of a 110 probeset classifier9. Leukemic cell lines  were obtained from the German Collection of Microorganisms and Cell Cultures  and routinely validated by DNA fingerprinting. Unless indicated otherwise, all cell lines were cultured in RPMI-1640 supplemented with 10 to 20% fetal calf serum and penicillin, streptomycin and fungizone .The study comprised children with BCP-ALL with age at diagnosis ranging from 0 to 18 years. Use of excess materials for research purposes has been approved by the Institutional Review Board of the University Medical Center Rotterdam on August 2nd, 2004, IRB approval file number MEC 2004-203. Written informed consent was obtained from parents or legal guardians and studies were conducted in accordance with the Declaration of Helsinki. Clinical characteristics were kindly provided by the Dutch Childhood Oncology Group . Mononuclear cells were isolated using density gradient centrifugation with Lymphoprep  as previously described50 of prednisolone was determined in an MTS assay for cell lines and an MTT assay for primary ALL cells based on a six-step range from 250 to 0.008 µg/mL  or 1 to 0.003 µg/mL . For these assays, cells were tested for viability in the prednisolone concentration range with or without 50 ng/mL recombinant human FGF2 (Bio-Rad) and with or without 1 µM AZD4547 (Selleckchem). After four days incubation at 37 °C and 5% CO2, cell viability was evaluated using MTS/MTT. IC50-values were calculated as the prednisolone concentration at which 50% of cells survived relative to the respective prednisolone-free control of each condition. To avoid redundancies with abundant cytokines in fetal calf serum (FCS), these assays were performed under reduced serum conditions . Sensitivity towards AZD4547 was measured similarly, but at normal serum concentrations (10 µM–0.3 nM).The IC10 and GATK indel realigner v.3.3-0. Single nucleotide variants were called with Freebayes v0.9.18–2411, Varscan v.2.3.712, Bcftools v1.013, and GATK v3.3-014. The resulting variant call format files were annotated using snpEff and snpSift v.4.1a15 and dbNSFP v.2.716. For reliable detection of high-confidence mutations, variants were filtered based on several criteria: For each sample, variants were excluded if they were reported by only one caller, coverage was <100 reads, or <20 reads supported the variant allele. Also variants likely to result from amplification or sequencing errors were excluded, i.e. those that occur repeatedly (n ≥10) but in a low fraction of reads within all samples  within each sample <2%) and those with an unequal distribution between runs. Furthermore, variants were only considered if they were non-synonymous, unlikely to be germline variants and not known SNPs. Mutational hotspots are indicated in Fig. th November 2016)6. All data and analysis code can be made available upon request.DNA was isolated using Trizol reagent (Life Technologies), or in case of three cell lines using the DNeasy Kit (Qiagen). For TruSeq Custom Amplicon sequencing , sequencing libraries were prepared from 100–250 ng genomic DNA. Successful library preparation was confirmed using the Labchip GX genomic analyzer . Samples were then pooled equimolarly and sequenced on an Illumina MiSeq in paired-end reads of 250 bp each. Targeted regions and the analysis script can be provided upon request. Next, sequence reads were aligned to the 1000 genomes human reference sequences  using BWA v0.7.107. We therefore evaluated the resistance-inducing effect of FGF2 on prednisolone cytotoxicity, a core component of ALL chemotherapy, using in vitro drug sensitivity assays. To avoid redundancies with abundant cytokines in fetal calf serum (FCS), these assays were performed under reduced serum conditions . The IC50 of prednisolone was determined in an MTS assay for eight BCP-ALL cell lines see also Supplemental Fig. 50-values for prednisolone , we screened for mutations in 481 primary BCP-ALL cases at initial diagnosis, in 28 BCP-ALL relapse samples (representing 19 unique patients) and in twelve BCP-ALL cell lines. The regions covered by the custom amplicons are indicated in Fig. FGFR1, FGFR2, and FGFR3  varied, with 8.4% for the FGFR2 D522N variant, 28.5% for the FGFR1 D129A variant, 40.8% for the FGFR2 V274I variant, and 52.9% for the FGFR3 F384I variant. None of these mutations represents a frequently observed pathogenic mutation, such as those reported in multiple myeloma, according to literature and the COSMIC database .Using the TruSeq Custom Amplicon technique as previously described  was found in two out of four cases, high minimal residual disease (percentage blasts in bone marrow on day 33 of therapy ≥0.1%) in one out of three cases  domain and the tyrosine kinase domain (TKD), respectively. They might therefore alter ligand-binding and kinase activation. Last, the FGFR3 F384I mutation is located in the transmembrane domain (TMD), which is frequently affected by mutations that cause aberrant receptor dimerization (e.g. by insertion of a cysteine). Of note, a rare F384L SNP at the same location has been reported and was also found in our screen. No functional consequences have been reported for this SNP, and the high similarity between leucine and isoleucine suggests no effect of the F384I variant on FGFR3 function22.The observed mutations are located within functional domains of the FGFR proteins: The FGFR1 D129A mutation affects the so-called acid box of the receptor, which has been shown to be essential for auto-inhibition and interaction with adhesion moleculesin vitro7. Our data indicate that the ligand FGF2 induced resistance to glucocorticoids which could be reversed by inhibition of FGFR. Traer et al. suggested that reactivation of the MAPK pathway by FGFR3 accounts for FGF-mediated resistance of BCR-ABL1-rearranged chronic myeloid leukemia cells to imatinib. In line with this observation, we and others have previously shown that MAPK pathway mediated signaling can also increase resistance towards prednisolone25. Downstream-located MAPK signaling may therefore be the mediator of FGF-induced glucocorticoid resistance, and could serve as therapeutic target for sensitization towards prednisolone in resistant cases.Given the previous implications of FGFR signaling in resistance, we evaluated its effect on the efficacy of prednisolone 19. As FGFR activating mutations were virtually absent, FGF2 ligand-mediated activation of FGFR-signaling may be more relevant for the sensitivity of leukemic cells to prednisolone.Taken together, our data demonstrates that FGFR-activating lesions are rare in newly diagnosed pediatric BCP-ALL, and were not enriched at the time of relapse. Activating mutations which are frequently observed in multiple myeloma or solid cancers were not observed in 481 newly diagnosed nor in 19 relapsed BCP-ALL casesSupplementary Information