The combination of different analysis techniques allowed us to describe intermediate/hybrid and complete EMT phenotypes respectively in HCC827- and HCC4006-derived drug-resistant human cancer cell lines. Interestingly, intermediate/hybrid EMT phenotypes, a collective cell migration and increased stem-like ability associate to resistance to the epidermal growth factor receptor inhibitor, erlotinib, in HCC827 derived cell lines. Moreover, the use of three complementary approaches for gene expression analysis supported the identification of a small EMT-related gene list, which may have otherwise been overlooked by standard stand-alone methods for gene expression analysis.Increasing evidence points to a key role played by epithelial-mesenchymal transition (EMT) in cancer progression and drug resistance. In this study, we used  Severtastasis , 4. Moretastasis , 6. The  studies , 8. In t studies , 8.Multiple signaling pathways and complex genetic and epigenetic mechanisms regulate the EMT program in normal and neoplastic epithelial tissues , 9–12. ILung cancer is the most frequent cause of cancer-related mortality worldwide leading to over a million deaths each year . Based oIn order to investigate the mechanisms of resistance to TKI, we have recently reported the establishment and characterization of NSCLC cell lines resistant to the EGFR inhibitor erlotinib .wet and in silico approaches to investigate whether E/M phenotypes are associated to erlotinib-resistance in our cellular model system. The combination of different analysis techniques allowed us to describe intermediate and complete EMT phenotypes in HCC827- and HCC4006-derived erlotinib-resistant cell lines respectively. Interestingly, EMT intermediate phenotypes, collective cell migration and increased stem-like ability associate to resistance to target therapy in the erlotinib-resistant HCC827-derived cell lines. Moreover, the use of three complementary approaches for gene expression analysis supported the identification of a small EMT-related gene list, which may have otherwise been overlooked by standard stand-alone methods for gene expression analysis.The effect of TKI target therapy on the selection of intermediate E/M phenotypes in cancer cells is still poorly investigated. Therefore, in this study, we used in vitro models of acquired resistance to the EGFR TKI erlotinib [50 > 10 μM) [Recently, in order to investigate mechanisms leading to resistance to EGFR-targeted therapy, two NSCLC cell lines (HCC827 and HCC4006) have been used to derive rlotinib . Both pa> 10 μM) . Charact> 10 μM)  and is sInterestingly, morphological analysis of the erlotinib-resistant NSCLC cells showed the presence of cells with a fibroblast-like cell shape reminiscent of EMT, especially in the RA1, RB1, RB2 and RC2.2 cell lines . Indeed,ZEB [Vimentin and Cadherin-1 despite generally used to characterize the EMT phenotype, could be not sufficient to identify and describe complex EMT features. Therefore, the expression levels of other EMT-related markers, such as Cadherin-2 , Claudin-1 (CLDN1), SNAI1 , SNAI2  and ZEB1, were also investigated by western blotting Figure  and qPCRZEB –35, we aA more complex picture was found for some EMT markers. In particular Cadherin-2 and Claudin-1 are observed respectively upregulated and downregulated in the HCC4006-derived RC2.2 cell line while in the HCC827-derived cell lines, Cadherin-2 appears to be upregulated only in RA1, RA2 and RB1 cell lines Figure  and ClauOverall the data suggest that the total population of the RC2.2 cell line, that does not express Cadherin-1 and expresses high levels of Vimentin, is in a mesenchymal state while the majority of HCC827-derived erlotinib-resistant cell lines might likely contain subpopulations with intermediate states of EMT.We used wound-healing assays in the erlotinib-resistant cell lines in order to investigate the effect of the observed EMT phenotypes on cell migration. The RC2.2 (HCC4006-derived) and the RA2 (HCC827-derived) erlotinib-resistant cell lines show an increased rate of wound closure, while for all other erlotinib-resistant cell lines quantitative differences in terms of wound closure capacity were not observed by comparison with their parental cell line Figure . ImportaMany cancer cells are known to exhibit collective invasion potential wherein cell–cell adhesions remain intact. Accordingly, in EMT phenotypes, marked by an heterogeneous cancer cell population with epithelial, E/M and mesenchymal features, the cells with intermediate E/M phenotypes could potentially contribute to a collective cell migration due to their ability to interact with epithelial cells by cell-cell contacts while also being migration-prone for their mesenchymal features. Indeed, in the wound healing assays of the HCC827-derived erlotinib-resistant cell lines, E/M Vimentin and Cadherin-1 double positive cells are detected at the wound leading edge in cell-cell contact with Cadherin-1 positive and Vimentin negative cells Figure . In partErlotinib-resistance and EMT phenotypes can associate with more aggressive behaviours, such as increased cell invasion and stem-like features. Therefore to further study the functional phenotype of the erlotinib-resistant NSCLC cell lines, matrigel-coated transwell invasion and sphere-forming assays were performed. The matrigel assays showed an increased cell invasion of the RA2 and RC2.2 NSCLC cell lines when compared to their parental erlotinib-sensitive cell lines Figure . InsteadRegarding the sphere-forming assays, we observed that the number of spheroids of the RA1, RB1, RB1.1 and RB2 cell lines with a diameter ≥ 50 μm was 2-3 fold higher than of the HCC827 parental cell line Figure  while thin vitro cell migration and cell invasion features of the RA2 and RC2.2 erlotinib-resistant cell lines do not associate with an increased stem-like ability. On the contrary the majority of HCC827-derived erlotinib resistant NSCLC cell lines with intermediated E/M phenotypes that do not show an increase in cell migration or invasion are more prone to form spheroids in vitro when compared to their parental erlotinib-sensitive cell line.Overall the results show that the increased To identify genes and pathways involved in erlotinib-resistance, microarray gene expression analysis was performed. Thousands of genes differentially expressed in the erlotinib-resistant cell lines were identified Table . InteresIn order to analyze the EMT phenotypes associated to erlotinib-resistance, we collected five lists of genes whose expression has been previously associated to EMT Table  Byers e. The firCO_59, SVM_85, and PROF_58 according to the related selection method  and the number of selected genes  (http://www.proteinatlas.org/ENSG00000104892-KLC3/cancer). KLC3 while donwregulated in the majority of our erlotinib-resistant cell lines is upregulated in the RA2 cell line  cut-off on gene expression (CO); (ii.) support vector machine (SVM); (iii.) expression profile analysis (PROF). Specifically, we used these supervised methods to identify subsets of genes having similar expression levels within the epithelial cell lines (HCC827 and HCC4006) while exhibiting different expression in the mesenchymal (RC2.2) and the cell lines with E/M features  (see the methods section for details). The gene subsets resulting by application of the above three methods, hereafter also referred to as ctively) , were exctively) . More inctively) . Similarctively)  as shownctively)  and the ctively) . Remarkactively) . Five ouctively) . CTSL2 octively) . KLC3 ore Figure . Interese Figure . STEAP1,e Figure . Finallyin NSCLC , is stroAnalysis of enriched functional annotations for the three subsets of EMT-related genes highlighted pathways associated to the EMT process, including cell adhesion molecules, tight junction, Cadherin and TGF β signaling pathway. Interestingly the TGF β pathway was found enriched in all three gene lists Tables -7.Prompted by the functional enrichment analysis, we decided to further investigate the TGF β signaling pathway. Indeed, it has been previously reported that NSCLC cells undergo EMT phenotypic changes upon short exposure to TGF β  or long Finally, growth inhibition assays indicated that LY-364947, alone or in combination with erlotinib, does not impair the viability of HCC827-derived erlotinib-resistant cell lines .Overall these results suggest that the observed EMT stable phenotypes associated to erlotinib-resistance likely appear independent or only partially mediated by TGF β-1 activation.Increasing evidence points to a key role played by EMT in cancer progression and drug resistance. Here we concentrated our study on the intermediate E/M phenotypes and their association to target therapy resistance in NSCLC cell lines. As observed in both physiology and pathology, a cell population can be highly heterogeneous with respect to its EMT phenotype. Indeed, we consistently observed distinct subpopulations of cells representing the three phenotypes previously described as epithelial, mesenchymal and intermediate E/M in all HCC827-derived erlotinib-resistant cell lines. Notably, the HCC827-derived cell lines studied in this work include cell subpopulations co-expressing Cadherin-1 and Vimentin, common markers for epithelial and mesenchymal phenotypes, respectively. The percentage of E/M cells was remarkably stable within each cell line, suggesting the existence of regulatory networks that preserve the cell phenotypes. It is also interesting to note that these intermediate E/M phenotypes may be selected by erlotinib treatment because the number of E/M cells was found increased in the erlotinib-resistant cell lines derived from the HCC827 cell line. On the contrary, the only established HCC4006-derived erlotinib-resistant cell line (ie: RC2.2) was found to be in a complete and stable mesenchymal state, with essentially all cells negative for Cadherin-1 and positive for Vimentin. Interestingly, only the RA2 and RC2.2 cell lines showed an increase in cell migration and cell invasion rate, suggesting that a qualitative rather than a quantitative variation of cell migration and eventually invasion may deserve further investigation in the other erlotinib-resistant cell lines. Remarkably HER3 a receptor suggested to be a strong epithelial marker and regulator of EGFR TKI sensitivity , was fouin vitro cell migration and cell invasion of the RA2 and RC2.2 erlotinib-resistant cell lines do not associate with increased stem-like features. On the contrary the majority of HCC827-derived erlotinib-resistant NSCLC cell lines, with intermediated E/M phenotypes, have similar or decreased cell migration and cell invasion rate when compared with their parental cell line but show an increased sphere-forming ability. Importantly, the mRNA expression profile of some largely used stemness markers could not predict such stem-like behavior  genes  genes . ImportaFurthermore, wound healing cell migration assays demonstrated a collective cell migration (reviewed in ) propertNext, we used our NSCLC cell lines microarray data to evaluate the expression of publicly available EMT-related gene signatures and applied different supervised analytical methods for selecting novel gene sets able to partition our cancer cell lines according to their observed EMT phenotypes. Notably, despite the heterogeneous composition, all the selected gene sets showed significant enrichment of the TGF β signaling pathway, confirming the relevance of this pathway with respect to the EMT phenotypes –55.Finally we investigated the effect of the modulation of the TGF β1 signaling pathway on cell survival and EMT phenotypes. We observed that the inhibition of TGFR-1, alone or in combination with EGFR inhibition, did not affect NSCLC cell lines survival, but it was able to partially decrease the number of Vimentin positive cells in some HCC827-derived erlotinib-resistant cell lines. Further investigation of the TGF β1 and of other enriched signaling pathways (i.e. the Wnt pathways) will be required to understand their contribution to the erlotinib resistance and the intermediate E/M phenotypes in our cellular model system.Overall these results confirmed cellular heterogeneity during EMT in cancer cell lines upon drug treatment. Importantly, our findings highlighted the association an intermediate E/M phenotypes to resistance to EGFR TKI in NSCLC cell lines. Treatment of NSCLC patients sensitive or resistant to EGFR inhibitors should attempt to impact not only oncogene-driven cancer cell growth but also the EMT phenotype in early NSCLC development stages.2 humidified incubator. For the treatment with human transforming growth factor beta-1 (TGF β-1) (Sigma-Aldrich) (2.5 ng/ml) and LY-364947 (Sigma-Aldrich), a TGF β receptor type-1 (TGFR-1) inhibitor (1 μM), cells were cultured in Opti-MEM reduced serum media (Thermo Fisher Scientific) for 48 hours. For the sphere assays the cells were cultured in DMEM/F12  supplemented with commercial hormone mix B27 (Gibco), EGF , FGF (20 ng/mL), purchased from ThermoFisher Scientific, and heparin (10U/mL) (Sigma-Aldrich). The primary antibodies used Vimentin (D21H3), E-cadherin (24E10), N-cadherin (D4R1H), Claudin-1 (D5H1D), Snail (C15D3), Slug (C19G7), ZEB1 , GAPDH (D16H11) were from Cell Signaling Technology; Vimentin (V9 and V9Cy3), α-Tubulin (DM1A), β-actin (AC15) and horseradish peroxidase (HRP)-β-actin (AC15) were purchased from Sigma-Aldrich; GAPDH (clone 1D4) was obtained from Novus Biologicals; Phalloidin Alexa Fluor-488 and E-cadherin (HECD-1) were from Thermo Fisher Scientific; N-Cadherin (32/N-Cadherin), E-cadherin (CD324)-Alexa Fluor-647, Vimentin Alexa Fluor-488 and isotype control antibodies: Alexa Fluor-647 Mouse IgG1k (Clone MOPC-21), Alexa Fluor-488 Mouse IgG1k (Clone MOPC-21) were from BD Biosciences; STEAP (B-4) and KLC3 (E-7) were from Santa Cruz Biotechnology. Secondary antibodies goat anti-rabbit IgG (H+L)-HRP and goat anti-mouse IgG (H+L)-HRP were purchased from Bio-Rad; goat anti-mouse Alexa Fluor-488 and 546 and Hoechst 33258 were obtained from Thermo Fisher Scientific. Erlotinib hydrochloride salt, an EGFR tyrosine kinase inhibitor, was from LC Laboratories, USA; Corning matrigel basement membrane matrix growth factor reduced was from BD Biosciences. Stock solutions (10 mM) for all inhibitors were prepared in DMSO and stored at -20°C or -80°C as suggested by the product datasheet. MTT, 3--2,5-diphenyltetrazolium bromide and bovine serum albumin were from Sigma-Aldrich. MTT stock solution  was stored at 4°C for 1 month.The human cell lines: HCC827 (ATCC CRL-2868) and HCC4006 (ATCC CRL-2871), kindly provided by Oreste Segatto, and the erlotinib-resistant cell lines , previously described  were culPower SYBR Green PCR master mix was from Applied Biosystems. Trizol reagent was from Life Technologies, the GoScript reverse transcription system kit was from Promega.4 cells/well) plated in 96-well plates at day 0 were treated at day 1 with increasing concentrations of erlotinib or LY-364947 (from 64 pM to 10 μM) or their combination in the ratio (1:3) in complete tissue culture medium and cultured for 72 hours at 37°C in 5% CO2. Next, cells were gently washed with 1x PBS, incubated for 4 hours with MTT and processed for color detection with DMSO. The resulting purple solution was spectrophotometrically measured at 570 nm and 630 nm [MTT assay was performed as previously described . Brieflyd 630 nm , 63.t0) and then cells were allowed to migrate from the edge of the scratch at 37°C/5% CO2. The monolayer was imaged at different time points to record the size of the wound closure at 5-24 (time t) hours with an Olympus BX41 microscope using a 4-10x lens and 8-10 images were recorded with an Olympus SP-350 camera. The area of wound coverage was calculated using the ImageJ software [The HCC827 and HCC4006 parental cell lines and the erlotinib-resistant derived cell lines were plated onto 35 mm plastic petri dishes and grown until confluence. A cell-free area was introduced in a monolayer of cells using a sterile yellow tip (time software . The deg35mm with Culture Insert (Ibidi) and an equal number of cells (0.5-1 × 105) in 70 μl of culture medium was seeded into the two reservoirs of the same insert and incubated at 37°C/5% CO2. After appropriate cell attachment (24 h) the culture insert was gently removed using sterile tweezers, and a 500 μm wide cell-free gap (wound) was generated. The dish was then filled with 2 mL of FBS containing medium and incubated for 8-16 hours. Then cells were processed for immunofluorescence and confocal microscopy analysis.For immunofluorescence and confocal microscopy analysis the cells were also subjected to wound healing assay with the μ-Dish5), HCC4006 (2.5x104) or RC2.2 (2.5x104) were seeded onto the upper surface of a filter with a volume of 100 μl RPMI 1640 medium containing 0.5% BSA and placed into the lower wells containing 600 μl of complete medium with 10% FBS or 0.1% BSA (negative control) to induce cell migration. The migration transwell chambers were incubated for 16h at 37°C in a humidified atmosphere containing 5% CO2. Following incubation, cells were fixed in 3.7% PFA for 10 min at room temperature, stained with 0.1% crystal violet (20% methanol solution) for 10 min, then washed 3 times with PBS. Next, the cells on the upper side of the membrane were removed by cotton swabs, and the crystal violet staining on the bottom surface of the membrane was eluted in 10% acetic acid and read at an optical density of 590 nm with a Varioskan Lux instrument.Assays were performed using a transwell  containing a polycarbonate membrane filter (8μm pore size) for 24-well plates according to the manufacturer’s instructions. HCC827 and RA2 (1x105/w) in RPMI 1640 containing 0.5% BSA were seeded in the upper chamber, and 500 μl RPMI 1640 containing 10% FBS was added to the lower chamber. After 24 h incubation at 37°C in a 5% CO2 atmosphere, cells that remained in the upper chamber were removed by cotton swabs and penetrating cells were stained with 1% crystal violet in 20% methanol. Then, the membranes were rinsed several times with water and images were recorded using a stereomicroscope Leica MZ12 served by an Olympus SP-350 camera. Next images were processed with the Image software in order to calculate the area of the membrane filter occupied by invasive cells.Matrigel invasion assays using transwell chambers  containing a polycarbonate membrane filter (8μm pore size) for 24-well plates coated with matrigel was used to determine NSCLC cell invasion properties. In brief, 1 mg/ml matrigel (50 μl) was added to each insert and solidified in a 37°C incubator for 5 h to form a thin gel layer. Next, 24 h serum-starved cells , FGF (20 ng/mL), and heparin (10 U/mL), and seeded into 6-well ultra- low attachment plates . Culture medium was replaced every 3 days; the number and size of the spheroids were recorded at 6-7 days after the start of the culture period using a stereomicroscope Leica MZ12 served by an Olympus SP-350 camera. The number of spheroids exceeding a diameter of 50 μm and their surface area were evaluated by the ImageJ software [The assay was performed essentially as previously described . Brieflysoftware .50 values were determined using a sigmoidal dose response inhibition variable slope method. The synergy of erlotinib and LY-364947 was evaluated by the Chou-Talalay method [Data were analysed by GraphPad Prism software v6.0c. The optical density values of cells treated with drugs, obtained by MTT assay reading, were expressed as percentage of cell survival and normalized with the value of cells treated with vehicle (DMSO). The regression trendline were fitted using a non-linear regression method and ICy method  using Coy method  as previy method .HCC827 parental and erlotinib-resistant cells, grown on poly-L-lysine coated coverslips or on 35 mm tissue culture plates or on the Ibidi culture dish and HCC4006 and RC2.2 cells, on 35 mm tissue culture plates, were fixed with 3.7% PFA for 10 minutes at room temperature. Next cells were permeabilized  for 6 minutes at room temperature, treated with blocking solution  and stained with primary antibodies in blocking solution, for 1 hour, washed with 1x PBS for 10 times and stained with fluorochrome-labeled secondary antibodies for 45 min at room temperature and Hoechst 33258 for 5 min. HCC827 samples were analysed with laser scanning confocal microscope, TCS SP5  using a 63x (NA=1.4) or 40x (NA=1.25) oil-immersion lens with optical pinhole at 1AU. For fluorescence images Argon ion laser at 488 nm, HeNe ion laser at 543 nm and blue diode laser at 405 nm were used as excitation sources. Confocal Z-stacks were collected at 0.4 μm intervals to a total optical depth of 8-12 μm. Confocal images (at least 10 images for each sample in three independent experiments) were processed with Volocity  and Adobe Photoshop CS4 software for image rendering and representation of x/y view. Images for direct comparison were all collected under same parameters and representative images were chosen. HCC4006 and RC2.2 cells, stained in tissue culture plates, were analyzed with an Olympus AX70 fluorescence microscope using a 20x or 40x lens and images were recorded with an Olympus XM10 camera and processed using Olympus CellSens Standard 1.8.1 software. The following setting were used for fluorescence microscopy images acquisition: exposure time at 500 ms, gain at 4 dB, and thresholds of the fluorescence intensity on dynamic range in order to include the overall histogram area. Next, the immunoreactivity for Vimentin, was independently evaluated by two investigators and Vimentin positive cells were independently counted. When discrepancies in counting occurred, a consensus interpretation was reached after reexamination. All cell counts were carried out using the ImageJ software . All ima6 cells were stained in 5ml round bottom tubes  with Alexa Fluor-conjugated antibodies for 45 min at room temperature. Cells were then washed twice with PBS+1% FBS. Finally samples were analyzed using a BD FACS Calibur flow cytometer (Becton Dickinson). Unspecific background of individual channels was determined with isotype matched controls. Plots were generated using FlowJo v10.2 software.Adherent cells were detached from tissue cell culture plates by treatment with a cell dissociation buffer enzyme-free PBS (Thermo Fisher) at 37°C. Cells were collected in 15 ml tubes, washed with ice-cold RPMI 1640 medium supplemented with 10% FBS and centrifuged at 400g for 5 min at 4°C. Next cell pellets were first washed with ice-cold PBS supplemented with 1% FBS to block unspecific antibody binding and fixed with PFA (3.7%) for 15 min on ice. After a wash with ice-cold PBS supplemented with 1% FBS, cells were permeabilized with 0.1% Triton X-100 in PBS for 15 min at room temperature and washed with PBS+1%FBS. Then 0.5-1x10Cells were lysed in ice-cold RIPA buffer  containing a combination of protease and phosphatase inhibitor cocktails or in SDS Laemmli sample buffer. Cell lysates (RIPA buffer) were quantified for proteins content with the Bio-Rad DC Protein Assay kit. Proteins (25-50 μg) were then separated by 8-12% SDS-PAGE and transferred to nitrocellulose membranes 0.45 μm . The membranes were blocked with 5% non-fat milk (EuroClone) or 2% BSA (Sigma-Aldrich) in 1x TBS pH 7.6-8.0 containing 0.1 or 0.2% Tween 20 (TBST) for 2 hour at room temperature (RT) and subsequently probed with primary antibodies in 5% non-fat milk or 2-5% BSA in TBST overnight at 4°C. Then membranes were washed 10 minutes for 3 times with TBST and probed with horseradish peroxidase-conjugated secondary antibodies in 5% non-fat milk in TBST for 1 hour at RT. Chemidoc XRS Bio-Rad was used for images acquisition with a chemi-luminescent camera, band signals were quantified using ImageLab 4.0 Bio-Rad software.Ribosomal protein L31 (rp-L31) was used as a reference gene to normalize quantitation of target genes for differences in the amount of total RNA in each sample.For mRNA analysis, total RNA was extracted using the TRIzol (Life Techologies) reagent and retro-transcribed with the GoScript Reverse Transcription System (Promega) using oligo (dT) and random primers. qPCR analysis was performed with specific primers Table  and analmicroRNA levels were analyzed from total RNA using the following TaqMan MicroRNA Assays: miR-200a ID:000502, miR-200b ID:002251, miR-200c ID:002300 and RNU24 ID:001001. Relative expression was calculated using the comparative Ct method. The small nucleolar RNA U24 (RNU24) was used as a reference gene.Both for mRNA and microRNA analysis, total RNA of HCC827 and HCC4006 parental cell lines were used as calibrator samples, relative to which differences in the RNA amount of resistant cell lines have been calculated. The data were analyzed using SDS (Ver. 1.4) software (Applied Biosystems).For microarray gene expression analysis, total RNA was isolated using a total RNA purification kit . RNA quantity was determined by absorbance at 260 nm using a NanoDrop ND-1000 UV-VIS spectrophotometer . RNA quality was assessed with Agilent Bioanalyzer RNA 6000 Nano kit; 200 ng of RNA were labeled with LowInput QuickAmp Labeling Kit One-Color (Agilent Technologies), purified and hybridized overnight onto an Agilent SurePrint G3 Human Gene Expression Array (8x60K v2 G4851B - 039494) before detection, according to the manufacturer’s instructions. Agilent DNA Microarray scanner was used for slide acquisition and spot analysis was performed with Feature Extraction software (Agilent Technologies).limma package [Microarray raw data, provided by the European Brain Research Institute  were background corrected, log2 transformed and quantile normalised by using  package . A totalhttp://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE80344.The whole Agilent microarray dataset used in this study is publicly available from the NCBI's Gene Expression Omnibus (GEO) database  under acPCA of cell lines was performed using gene expression values as variables that were scaled to have unit variance before the analysis took place. Unsupervised hierarchical clustering was performed using the complete linkage method and Euclidean distances applied to the normalized expression data matrix .The expression values of biological sample duplicates were averaged and mean expression differences were calculated as positive and negative fold-changes of gene abundance between erlotinib-resistant and -sensitive cell lines. We used -1 and 1 log fold-change cutoffs in order to select genes with relevant down- and up-regulation, respectively. Recurrence in gene expression modulation of erlotinib-resistant cell lines were evaluated by selecting genes with down- or up-regulation in at least five of the six derived vs parental cell lines comparisons. The recurrent differentially expressed genes were ranked according their log fold-change mean in the 5/6 erlotinib-resistant cell lines.Five gene lists previously associated to EMT . This method selects genes differentially expressed between epithelial and mesenchymal cell lines (fold-change ≥ 2) and with no modulation between the subset of epithelial cell lines . This method selected a list of 59 genes .Support Vector Machines classification (SVM). A supervised classification of cell lines was performed by using the SVM implemented by the Weka software . Based oExpression profile analysis (PROF). A hierarchical clustering was firstly performed to group genes of the COMBO_270 list according to their expression profiles. The resulting cluster dendrogram was cut at N=38 clusters and expression trends of each cluster separately evaluated. Next, genes populating clusters showing modulation between epithelial and mesenchymal cell lines and/or between E/M phenotype cell lines and both epithelial and mesenchymal cell lines were selected (hereafter the PROF_58 list).Finally, intersection of the above subsets of selected genes yielded a shared list of 25 genes related to EMT (hereafter INT_25 list).Differentially expressed genes between erlotinib-resistant and -sensitive cell lines and EMT-related gene sets were submitted to the DAVID tool suite  to analyStatistical analysis was performed using Microsoft Excel and GraphPad Prism software 6.0c. Two-tailed t-test was used for all indicated comparisons if not otherwise stated and p-values lower than 0.05 were considered significant. In particular, the effects of treatment with LY-364947, TGF β1 and their combination were evaluated by analyzing the fold-changes of Vimentin positive cells in vehicle (control) vs LY-364947, vehicle vs TGF β1, and TGF β1 vs LY-364947+ TGF β1 comparisons. In the wound healing assay, differences between leading edge and inner side were evaluated by comparing the counts of total Vimentin positive, Vimentin/Cadherin-1 double positive, and Vimentin positive/Cadherin-1 negative cells. In qPCR experiments resistant cell lines were compared with their corresponding parental cell lines. Statistical analysis of the invasion and sphere-forming assays data was performed using the non-parametric Mann-Whitney U test.


Taken together, the combination of CHKi with cetuximab plus irradiation displayed significant antitumor effects in HNSCCs both in vitro and in vivo, suggesting that this combination therapy may increase clinical benefit. A clinical trial to test this treatment for patients with head and neck cancer is currently ongoing (NCT02555644).EGFR inhibition and radiotherapy are potent inducers of DNA damage. Checkpoint kinases 1 and 2 (Chk1/2) are critical regulators of the DNA-damage response, controlling cell-cycle checkpoints that may permit recovery from therapy-associated genomic stress. We hypothesized that Chk1/2 inhibition (CHKi) with prexasertib may enhance cytotoxicity from EGFR inhibition plus radiotherapy in head and neck squamous cell carcinoma (HNSCC). In this study, we found that the addition of CHKi to the EGFR inhibitor cetuximab with and without radiotherapy significantly decreased cell proliferation and survival fraction in human papillomavirus virus (HPV)-positive and HPV-negative HNSCC cell lines. Reduced proliferation was accompanied by decreased checkpoint activation, induced S-phase accumulation, persistent DNA damage, and increased caspase cleavage and apoptosis. Importantly, a significant tumor growth delay was observed  Head and neck squamous cell carcinomas (HNSCC) are aggressive tumors with high recurrence rates and poor 5-year survival. Although HNSCCs account for only 3% of all cancers in the United States, the incidence of oropharyngeal squamous cell carcinoma (OPSCC) specifically has been increasing over the past 20 years . This inOne effect of treatment with cetuximab and irradiation is the induction of replication stress and DNA damage with simultaneous suppression of DNA repair . These eA new class of targeted anticancer agents has been developed that inhibits Chk1/2 (CHKi), blocking cell-cycle checkpoint activation, and permitting cell-cycle progression despite unrepaired DNA damage . Specifiin vitro and in vivo analysis of combination therapy with cetuximab, prexasertib, and irradiation (IR) in HNSCC cell lines. The combination of prexasertib and cetuximab with or without IR inhibited cell proliferation greater than single-agent treatment alone in both HPV-positive and HPV-negative HNSCC cell lines in vitro. This effect was associated with induction of apoptosis, persistent DNA damage, and alteration of cell-cycle distribution. On the basis of these in vitro results, we designed in vivo studies using xenograft models to test the potential antitumor effects of the combination therapy of prexasertib, cetuximab, and IR. Importantly, triple combination treatment significantly delayed tumor growth in vivo in HNSCC cell line xenografts. These results suggest that prexasertib has activity against head and neck cancer cells, and combining prexasertib, cetuximab, and IR in HNSCC may provide additional clinical benefit and offer a potential therapeutic strategy for this disease.On the basis of these observations, we hypothesized that prexasertib may increase the efficacy of cetuximab plus radiotherapy in HNSCCs. We conducted an The HPV-negative UM-SCC1, UM-SCC2, and UM-SCC6 cell lines were obtained courtesy of Dr. Thomas E. Carey . HPV-positive UM-SCC47 and UPCI:SCC090 cells were a gift from Dr. Susan Golin  and Dr. John H. Lee . UM-SCC1-luciferase was obtained from Dr. Eben Rosenthal . These cell lines have been previously described –14. The Cell proliferation assays were performed as described previously . BrieflyClonogenic survival was assessed by colony formation assay as described previously . Cells wApoptosis was analyzed using the Annexin V-FITC Apoptosis Detection kit  according to the manufacturer's instructions and was previously described .Protein was analyzed by SDS-PAGE as previously described . The fol2 dishes and treated accordingly. 48 and 72 hours after treatments, cells were collected, fixed, treated with RNAse , stained with propidium iodide (PI), and read on FACS Calibur using Cell Quest. Data were analyzed using ModFit LT (Verity Software Inc).Cell-cycle distribution was measured as previously described . Cells w6 cells were injected into the right flank, and tumors were measured by caliper biweekly starting at day 4 after injection. Mice bearing HNSCC cell line xenografts were subjected to 3 weekly cycles of prexasertib (Mondays and Thursdays), cetuximab (Mondays), and 2 Gy irradiation (Mondays and Thursdays). Prexasertib was injected subcutaneously at 4 mg/kg twice a day. Cetuximab was given at 0.1 mg/injection intraperitoneally. Twenty percent Captisol was used as a vehicle control.All animal procedures were approved and in accordance with the UAB Institutional Animal Care and Use Committee guidelines. Four-week-old, 20 g, female athymic nude mice (Charles River Laboratories) were allowed to acclimatize for 1 week before experiments. For the orthotopic UM-SCC1-luc model, 100,000 cells were injected into the oral tongue, and tumors were imaged biweekly using a luciferase bioluminescence assay starting at day 4 after injection. Mice received intraperitoneal injections of D-luciferin substrate (150 mg/kg) 15 minutes before imaging, and luminescence was measured in photons per second. For the heterotopic UM-SCC47 model, 3 × 10Data were analyzed by ANOVA followed by Bonferroni post-test using GraphPad Prism version 4.02 (GraphPad Software). Data are presented as average ± SE.To evaluate the antitumor effects of prexasertib with cetuximab and IR, we first assessed cell proliferation following various combinations of prexasertib, cetuximab (C225), and IR in HNSCC cell lines. In both HPV-positive and -negative cell lines, treatment with C225 alone, prexasertib alone, or combination of C225 plus prexasertib with or without IR significantly decreased proliferation compared with control at all timepoints . Also, iNext, we also assessed cell survival with different doses of IR using the colony formation assay in the UM-SCC1 and UM-SCC47 cells treated with prexasertib and C225. Similar to the cell proliferation data, combined prexasertib with C225 significantly reduced cell survival fraction compared with either agent alone in both cell lines . In the To investigate the mechanism of cytotoxicity of combining prexasertib, C225, and IR, we first examined cells for Annexin V, an early cell surface marker of apoptosis, 48 hours after treatment. In both HPV-negative and HPV-positive cell lines, treatment with C225 alone, prexasertib alone, or C225 and prexasertib all increased apoptosis compared with control . In bothTo confirm the increased apoptotic signaling, we also investigated caspase-3 cleavage in treated cells. In the HPV-negative cells, there is minimal caspase-3 cleavage observed at 48 hours following low dose (2 Gy) of IR. In contrast, a robust increase in cleaved caspase-3 was observed following treatment with prexasertib alone or in combination with C225 or IR or both C225 and IR . A similAs apoptosis can be activated by the presence of persistent DNA damage, a known effect of CHKi treatment, we assessed phosphorylation of H2AX (γH2AX), a well-accepted marker of DNA double strand breaks. In all cell lines, γH2AX remained detectable up to 48 hours after treatment with prexasertib or combination C225 and prexasertib with and without IR . In UM-S1 phase accumulation due to serum starvation . As the order and timing of dosing are thought to influence the efficacy of combination treatment regimens, especially those including EGFR inhibition, we also used the pilot study to explore four different dosing schedules based on possible mechanisms of synergy between prexasertib, C225, and IR ; ref. 19A similar experiment was also performed using the HPV-positive UM-SCC47 heterotopic flank model. In these cell line xenografts, we saw a substantial tumor growth delay in all treatment groups with IR as expected. Interestingly, the combination of prexasertib, C225, and IR significantly inhibited tumor growth as compared with other treatment groups, as shown in the tumor growth delay graph  and reprAlthough targeted therapies against EGFR, such as C225, have been developed for use in HNSCC, resistance is a common occurrence and survival rates remain poor. Therefore, effective alternative treatments are greatly needed to improve clinical outcomes in this disease. In this study, we demonstrate that prexasertib, an inhibitor of Chk1/2, attenuates checkpoint activation induced by C225 and IR, leading to persistent DNA-damage and increased apoptotic cell death in both HPV-positive and HPV-negative HNSCC cell lines. Moreover, combining prexasertib with C225 and IR led to a significant tumor growth delay in mice bearing orthotopic or heterotopic HNSCC xenografts. Thus, combining prexasertib with C225 and IR may be an innovative treatment strategy for both HPV-positive and HPV-negative HNSCC patients.We found that prexasertib treatment in HNSCC cells resulted in S-phase accumulation and induction of persistent γ-H2AX, suggesting that the induction of replication stress may lead to the cell death observed in treated cells. These results are similar to those reported in recent studies from Martinelli and colleagues  and KingCombination treatment with prexasertib, C225, and IR was also sufficient to overcome the underlying variability in cell-cycle checkpoint pathways , 21, leaDecreased phosphorylation of checkpoint proteins in response to CHKi was somewhat expected. P-Chk1(Ser296) detects autophosphorylation, which should be directly inhibited by prexasertib, and P-Chk2(Thr68) detects phosphorylation by ATM/ATR, which may be decreased because altered checkpoints affect the ability of cells to activate the DNA damage response. Consistent with our findings, it has been shown that radiotherapy combined with CHKi reduces homologous DNA repair in pancreatic and breast cancer models , 11. HowCS and Metnase, have a secondary role in checkpoint stabilization, and decreased recruitment may repress this positive feedback loop  in some of the tested cell lines. However, in the in vivo studies, the triple combination exhibited greater antitumor effects compared with the double combination of prexasertib and C225. This may be related to the inherent shortcomings of the in vitro model that demonstrates the short-term effects of the tested therapies, because the in vitro models does not account for the accumulated long-term effects of the combination therapy observed in the in vivo models. Even modest changes in the rate of cytotoxicity may over time contribute to significant reductions in tumor volumes in vivo. Nevertheless, the combination of prexasertib and C225 may be an interesting therapeutic strategy, which is currently being tested in a clinical trial (NCT02124148) for patients with recurrent head and neck cancer.The current non-surgical standard therapies for locally advanced HNSCC are concurrent C225 with IR and cisplatin with IR. Cisplatin induces DNA damage by forming DNA adducts, which therefore activate the cell cycle checkpoint response. It is interesting to test whether combining prexasertib with cisplatin-IR will also enhance cytotoxicity in HNSCC. Overall, our findings from this study support further clinical investigation of prexasertib in locally advanced HNSCC to improve response and reduce acquired resistance in patients treated with C225 and IR. A phase Ib clinical trial to test prexasertib in combination with C225 and irradiation in patients with locally advanced HNSCC is currently ongoing (NCT02555644).Supplemental Fig 1Supplemental Fig 2Supplemental Fig 3Supplemental Figure Legend


These cells are all derived from human tissue samples, thus there is a possibility of virus infection. Virus tests are routinely performed in clinical practice, but are limited in cell lines. In this study, we investigated 15 kinds of viruses in 844 human cell lines registered at the Japanese Collection of Research Bioresources (JCRB) Cell Bank. Our real-time PCR analysis revealed that six viruses, EBV, HTLV-1, HBV, B19V, HHV-6 and HHV-7, were detected in 43 cell lines. Of them, 20 cell lines were transformed by intentional infection in vitro with EBV or HTLV-1. Viruses in the other 23 cell lines and one EBV transformed cell line are derived from an in vivo infection, including five de novo identifications of EBV, B19V or HHV-7 carriers. Among them, 17 cell lines were established from patients diagnosed with virus-associated diseases. However, the other seven cell lines originated from in vivo cells unrelated to disease or cellular tropism. Our approach to screen for a set of 15 viruses in each cell line has worked efficiently to identify these rare cases. Virus tests in cell lines contribute not only to safety assessments but also to investigation of in vivo viral infection which can be a characteristic feature of cell lines.Human cell lines have been used in a variety of research fields as an  These resources have been used in a broad research area, including as an experimental model for drug development and to gain a deeper understanding of molecular pathogenesis. General guidelines for the use of cell lines are provided to obtain reliable data from experiments using cultured cells ,2. AutheA diverse range of viruses have been identified within specific tissues and reported in relation to disease pathogenesis . Human tWhen viruses enter the human body, they proliferate in susceptible cells and establish an infection. This can cause illness with clinical symptoms such as fever, rash or headache, leading to a certain type of infectious disease unique to infected tissues. Alternatively, viruses are often observed in a latent state, which is sometimes related to reactivation . Virusesin vitro, some viruses originated from in vivo infection are detected in certain cell lines. It is reported that HHV-8 was detected in human cell lines specifically derived from primary effusion lymphomas [Certain types of viruses are employed to induce cellular oncogenesis and immortalization , represeymphomas . Screeniymphomas . This imBacterial contamination is visually found in cell cultures through a conventional light microscope, and can be prevented using antibiotics. However, virus detection requires molecular analysis, and, once cells have become infected the viruses cannot be easily removed from the cells. Datasets from virus examination in cell lines contribute to the safe management of cell culture and characterization of cell lines. In this report, we have screened for 15 viruses in 844 human cell lines registered with the Japanese Collection of Research Bioresources (JCRB) Cell Bank and have detected six different viruses in 43 cell lines.2.2.1.Cell lines examined in this study are registered with the JCRB Cell Bank and are listed in the electronic supplementary material, table S1. These cells had been authenticated by short tandem repeat (STR) analysis and had been confirmed as mycoplasma-negative in our routine quality control. Certain cell lines carrying the same name appear with different registration numbers. These cell lines were deposited by various depositors through different routes, and would normally have the same characteristics, but there is the possibility of subtle strain differences.2.2.The residual cells from every freezing lot were inoculated into two tubes and one agar plate  for a sterility test. These tubes and plates were incubated at 35°C for 2 weeks to examine bacterial and fungal contamination.2.3.Total DNA and RNA samples were extracted from each cell line with the AllPrep DNA/RNA Mini kit . Some DNA was prepared by a conventional protocol using phenol–chloroform. When extracting RNA for copy number titration, DNA contamination was eliminated by digestion with RNase-free DNase I  at room temperature.2.4.The viral DNA test was targeted to nine and four kinds of DNA and RNA viruses, respectively , CMV, EB2.5.Viral RNA was examined for six kinds of RNA viruses , HAV, HC2.6.g for 10 min at 4°C to remove cell debris. The supernatant was concentrated by centrifugation (22 000g) for 2 h at 4°C, then the pellets were analysed by the Reverse Transcriptase Assay  as described by the manufacturer.Some cell lines were transfected with the luciferase expressing gene using retrovirus or lentivirus vectors. To examine lentivirus replication, the cultured medium of these cell lines was centrifuged at 2503.Our virus test detected six viruses, EBV, HTLV-1, HBV, B19V, HHV-6 and HHV-7, in 43 cell lines . One of 3.1.in vivo.The most common virus we detected in human cell lines was EBV, found in 30 cell lines, all of which are derived from blood cells .1. Two oin vivo infection, 16 lymphoblastoid cell lines were established from in vitro transformation of peripheral blood mononuclear cells using EBV. They were obtained from patients with retinoblastoma and their parents, or patients with a rare disease such as Bloom syndrome, ataxia telangiectasia or xeroderma pigmentosum. Different from these cell lines related to inherited diseases, TK6(IVGT) is characterized by being heterozygous for thymidine kinase and has been used for in vitro mutagenesis assays [Compared with these 14 EBV-positive cell lines originated from s assays . EBV-tra3.2.in vivo infection. MT-2, MT-3 and MT-4 were established by co-culture with lymphoid cells from ATL patients and were infected with HTLV-1 in vitro [HTLV-1 was detected in six cell lines, one lung cancer and four leucocytes .2. KHM-3in vitro . The virin vitro . It is rin vitro .3.3.HBV was detected from three hepatoma cell lines, huH-1, JHH-7 and PLC/PRF/5 .3. PLC/P3.4.in vivo infection. These cell lines were fibroblast derived from the skin of patients with hereditary diseases, carrying a high cancer risk. Different from the EBV, HTLV-1 and HBV detected mostly in cancer cell lines, the two B19V-positive cell lines have no association with diseases. Their copy numbers were low, which would be related to their primary cells. Although B19V can propagate in a leukaemia cell line, UT-7 [B19V was detected in two cell lines, FPC5JTO and XP2SA .4, whichne, UT-7 , the pos3.5.in vivo infection. This is distinct from other HHV-6-positive cell lines generated by in vitro transfection [HHV-6 was detected in a human vascular endothelial cell line, HUV-EC-C, derived from normal umbilical cord tissue .5. We hasfection .3.6.in vivo infection which remains detectable in cell culture.HHV-7 was detected in one cell line, AT(L)7KY, which was not previously reported to be positive. This cell line is a B-cell line transformed by EBV, indicating that both EBV and HHV-7 can be detected in this cell line .6. It wa3.7.A very small copy number of DNA fragments targeting the gag gene of HIV-1 and LTR of HIV-2 were detected in some of the luciferase-expressing cell lines . However, these viral RNAs were not detected in these cell lines, and reverse transcriptase activity was below the detection limit. The absence of the RNA and reverse transcriptase in these cell lines confirmed that the cell lines are negative for HIV-1 and HIV-2. The luciferase gene was introduced into these cell lines by the lentiviral vector , which m4.in vivo samples, our virus tests for cell lines detect viral DNA or RNA in the cytoplasm or the nuclei. The presence of these viral components in cells can be a molecular characterization of the cell line.Virus infection is one of the major public health concerns and is related to disease pathogenesis . Virus tin vivo, leading to establishment of the immortalized cell line. These examples show that further investigation of virus-positive cell lines could lead to an understanding of viral dynamics and that such cell lines can serve as invaluable research materials. Virus tests contribute to the characterization of cell lines and elucidation of cellular origins. Because each cell line is derived from a specific tissue and established from a certain cell population in the sample, cell lines that show negative on virus tests are not necessarily derived from uninfected individuals.Our previous study demonstrated that chromosomally integrated HHV-6B has been identified in a human vascular endothelial cell line, HUV-EC-C (IFO50271) . The anain vitro always have a risk of viral infection in the culture environment because, by themselves, they lack a defence system against infection. Assessment of virus contamination in cells is undertaken for the purpose of risk management [in vivo infection characterizing cell lines. The use of human cell lines is accompanied by a potential biohazard, because virus tests are limited to certain species and do not cover all pathogens. It is recommended that human cell lines are primarily handled at a biosafety level 2 laboratory, regardless of results from virus tests. Especially, cell lines which produce virus particles need careful handling and their application should be restricted. Each virus has distinct host cell tropisms, implying that negative results would be expected from the majority of cell lines. Because it is not clear that viral tropism is identical between in vivo and in vitro environments, we applied the whole set of virus tests to all cell types. Apart from the cell lines transformed using EBV or HTLV-1 by in vitro infection, 22 cell lines had been infected with virus in vivo before their establishment. Of them, two cell lines are inconsistent with the cellular tropism, showing unexpected results.Cells nagement . When cenagement . Examinanagement . Anothernagement . This imIt is reported that induced pluripotent stem cells can be established from EBV-transformed lymphoblastoid cell lines, and that the reprogrammed cells have been shown to be EBV free ,43. This


Hypoxia is a negative prognostic factor and this study investigated the relationship between hypoxia, hypoxia inducible factor 1 (HIF-1) and AKT signaling in head and neck squamous cell carcinoma (HNSCC) and non-small-cell lung cancer (NSCLC).2) in a part of HNSCC (3/4) and squamous (2/3) and adenocarcinoma (1/3) NSCLS lines. AKT-inhibitor MK-2206 reduced hypoxic HIF-1 signaling in most HNSCC cell lines. This reduction did not correlate with hypoxic induction of pAKT or with sensitivity to MK-2206 under hypoxia. Patient biopsies revealed a hypoxia-induced expression pattern of pAKT in HNSCC (n = 16), which was not observed in squamous cell (n = 34) or adenocarcinoma (n = 41) NSCLC.pAKT was induced by hypoxia  and is, therefore, an inherent negative factor for outcome. In this study, we show that the interaction between hypoxia and these pathways varies widely between tumor types and histologies. This variation could significantly affect response to treatments targeting hypoxia or these signaling pathways. Hypoxia is a common feature of solid tumors and is an inherent negative factor for treatment outcome . The hypMultiple studies indicate that the interaction between the AKT pathway and HIF-1 signaling is important for hypoxic cell survival and hypoxia-induced treatment resistance . The AKTin vitro relations between AKT activation and hypoxia are also observed in the clinic.In this study, we investigated the relationship between hypoxia, AKT activation and HIF-1 signaling in a head and neck squamous cell carcinoma (HNSCC) cell line panel. A panel of NSCLC lines consisting of both adenocarcinomas and squamous cell carcinomas was also studied to determine the variation between tumor types and histologies within one tumor type. Moreover, the effect of AKT inhibition on hypoxic cell survival was tested to increase the insight in the dependence of hypoxic cell survival on AKT activity. Lastly, the expression pattern of pAKT was studied in patient biopsies from HNSCC and NSCLC to assess whether the observed 2). Cells were maintained in DMEM (HNSCC lines) or RPMI1640 (NSCLC lines) containing 2 mM l-glutamine, 1% nonessential amino acids, 20 mM Hepes, 10 units/ml penicillin, 10 units/ml streptomycin and 10% fetal bovine serum.Four human HNSCC cell lines  and six human NSCLC cell lines (obtained from the ATCC) were used in this study. The characteristics of the cell lines are shown in 2, H35 hypoxystation, Don Whitley Scientific Ltd., West Yorkshire, UK) for 1 h, 4 h, 8 h, 16 h or 24 h. To determine protein expression after AKT inhibition, cells were treated overnight (16 h) with 0 or 2 μM MK-2206  under standard normoxic conditions and thereafter incubated under normoxic conditions or under hypoxic conditions  for 4 h, 8 h or 16 h.To determine protein expression after hypoxia, cells were incubated under hypoxic conditions (0.5% O2) for 72 h. To prevent interference of hypoxia in the determination of cell survival with the cell-counting Kit-8 assay , cells were placed back into normoxic conditions before measurement of cell survival. Cell survival was measured 72 h after hypoxic incubation in HNSCC lines and 24 h after hypoxic incubation in NSCLC lines.To assess cell survival after hypoxia and/or AKT-inhibition, cells were seeded in 96-well plates. After the cells were allowed to attach overnight under standard normoxic conditions, 0, 0.5, 1, 2, 4 or 8 μM, MK-2206 was added and cells were incubated under normoxic conditions or under hypoxic conditions (0.5% OCells were lysed in radioimmunoprecipitation assay (RIPA) buffer and protein was quantitated using a standard Bradford absorbance assay. Proteins were separated by SDS-PAGE and blotted onto polyvinylidene fluoride (PVDF) membrane. Membranes were incubated with the appropriate primary antibodies followed by incubation with horseradish peroxidase (HRP)-conjugated antibodies. Finally, proteins were detected using chemiluminescence. Antibodies against the following antigens were used: HIF1-alpha , GLUT-1 , CAIX , pAKT, AKT 1/2/3 , HRP-conjugated goat-antirabbit IgG , HRP-conjugated goat-antimouse IgG  and α-tubulin . Given the many intricacies in quantifying Western Blots, we chose only to make qualitative statements on the intensities of the stainings.Biopsies were obtained from 16 patients with stage I–IV squamous cell carcinoma of the oral cavity or oropharynx planned for curative treatment with fractionated radiotherapy with or without concurrent chemotherapy. Before onset of treatment, biopsies were taken for routine diagnostic purposes and additional biopsies were taken for multiple marker analyses. The latter were snap-frozen in liquid nitrogen and used in this study.et al. [Fresh frozen biopsies of 75 NSCLC tumors of a total of 74 patients were included from a study described in detail by Meijer et al. . Only tuAll patient studies were performed according to institutional ethical rules and regulations.in vivo, frozen tumor sections (5 µm) were thawed, fixed in acetone (4ºC) and rehydrated in phosphate-buffered saline (PBS). Tumor sections were stained for pAKT in combination with vessels. The antibody against pAKT(S473) was obtained from Santa Cruz Biotechnology and the antihuman endothelium antibody PAL-E was obtained from Euro Diagnostica AB . pAKT was detected by incubation with Cy3-conjugated goat-antirabbit F(ab’)2 fragments , and PAL-E by incubation with Alexa647-conjugated chicken-antimouse IgG . Stained sections were mounted in Fluorostab .To determine localization of protein expression Stained tumor sections were scanned on a digital image processing system on a fluorescence microscope  and a computer-controlled motorized stepping stage, using IPLab software. Each section was sequentially scanned at 100× magnification, yielding an image of pAKT-expression and an image of the vasculature (PAL-E).One pseudocolored composite image was reconstructed from the individual microscope images. Using this composite image and a hematoxylin-eosin stained tumor section, total tumor area was delineated and nontumor tissue, necrotic area and staining artifacts were excluded from the analysis. Thereafter, thresholds for the fluorescence signals were interactively set at intensities where the steepest gradient occurred between background and foreground intensity levels, and gray value images were converted to binary images. To quantify the distribution of pAKT in relation to the vasculature, zones were chosen at increasing distances from the nearest vessel . Using Image J software , the fraction positive for pAKT in each zone was calculated by dividing the zone area positive for pAKT by the total zone area. Zones with areas <200 pixels were excluded from the analysis.Statistical analyses were performed using Prism  or SPSS . p-values < 0.05 were considered significant. Synergism between hypoxia and MK-2206 was determined by correcting the cell survival after hypoxia and MK-2206 by the effect of hypoxia alone. Differences between the effect of MK-2206 under normoxia and the corrected effect of MK-2206 under hypoxia (difference = supra-additive effect) were tested for significance using Mann–Whitney tests. Bonferroni correction was applied to correct for multiple testing. Correlations between parameters were assessed using the Spearman correlation test.2) on pAKT levels in a time-dependent manner  squamous cell lines and one out of three (H522) adenocarcinoma lines of the lung showed upregulation of pAKT under hypoxia. Interestingly, both the HNSCC and the NSCLC cell lines that did not upregulate pAKT had already high levels of pAKT under normoxic conditions. In almost all cell lines a decrease of pAKT at later incubation times was seen irrespective of the induction of AKT by hypoxia. Although clear differences between cell lines of the same tumor type and histology were observed, these data suggest that the AKT pathway is activated by hypoxia in HNSCC and to a lesser extent in NSCLC.As observed before, pAKT expression increased by incubation with 0.5% OSupplementary Figure 1).To determine whether HIF-1 signaling under hypoxic conditions is regulated by the AKT pathway in the tested cell lines, the expression levels of HIF-1α and downstream proteins glucose transporter-1 (GLUT-1) and carbonic anhydrase IX (CAIX) were investigated under hypoxia with or without addition of the allosteric AKT inhibitor MK-2206 . IncreasAKT inhibition decreased the hypoxic induction of GLUT-1 and/or CAIX in HNSCC lines UT-SCC15, -24A and -38. Whereas in UT-SCC24A this was associated with a concomitant decrease in HIF-1α, in UT-SCC15 and -38 HIF-1α levels increased after AKT inhibition. In UT-SCC5 reduced hypoxic HIF-1α levels were observed after AKT inhibition, but this did not result in reduced GLUT-1 or CAIX levels. Overall, no unequivocal effects of AKT inhibition on HIF-1 signaling were found in these lines. Interaction of the AKT and HIF-1 pathway under hypoxia was not correlated with hypoxic induction of pAKT in these HNSCC cell lines A. In genOverall, these data show that the AKT pathway regulates hypoxia-induced HIF-1 signaling in part of the HNSCC lines and that this regulation is independent of hypoxic-induction of pAKT. No interaction between the AKT pathway and hypoxia-induced HIF-1 signaling was observed in the NSCLC lines.We have shown before that cell survival of UT-SCC5 and UT-SCC15 was decreased by AKT inhibition under hypoxic, but not normoxic conditions . HypoxicAs shown before, hypoxic cells of UT-SCC5 and UT-SCC15 were more sensitive to MK-2206 compared with normoxic cells A. In conAn overview of the data from s = 0.58; p < 0.0001). In contrast, in NSCLC biopsies, either squamous cell or adenocarcinoma, pAKT expression did not increase with increasing distance from the vessels; that is, pAKT was expressed throughout the tumor or not  is induced by hypoxia in head and neck squamous cell carcinoma (HNSCC) cells and to a lesser extent in NSCLC cells.Interaction between AKT activity and hypoxic hypoxia inducible factor 1 (HIF-1) signaling is not correlated with induction of pAKT by hypoxia.Hypoxic sensitization to AKT inhibition is observed in HNSCC and NSCLC lines and is independent of effects of AKT inhibition on HIF-1 signaling.pAKT is expressed in a hypoxia-induced pattern in HNSCC, but not in NSCLC patient biopsies.The interaction between hypoxia, AKT activation and HIF-1 signaling varies widely between tumor types and cell lines of the same tumor type, which could have important consequences for response to treatments targeting hypoxia or growth factor receptor signaling.Click here for additional data file.


All the synthesized derivatives were selected by National Cancer Institute (NCI), Bethesda, and evaluated for their 4a, b and c were found to have good growth inhibition % against numerous cell lines that belong mainly to leukemia, non-small cell lung, CNS and breast Cancer subpanels.Schiffs like compounds  Normal cells are constantly subjected to signals that dictate whether the cell should divide, differentiate into another cell or die. Cancer cells develop a degree of autonomy from these signals, resulting in uncontrolled growth and proliferation. If this proliferation is allowed to continue and spread, it can be fatal. In fact, almost 90% of cancer- related deaths are due to tumor spreading or dissemination . PhenomeAlthough advances in the field of chemo-preventive and therapeutic medicine have been made regularly over the last ten years, the search for novel anticancer treatments continues as it became an urgency to develop new anticancer agents with fewer side effects.1A) that acts as Sulphatase inhibitors , the percentage growth was calculated at each of the drug concentrations levels. Percentage growth inhibition was calculated as:Using the seven absorbance measurements  x 100 for concentrations for which Ti ≥ Tz[(Ti − Tz)/Tz] x 100 for concentrations for which Ti < Tz.[-11 were synthesized as depicted in (Scheme 1 and 2).The intermediates 1 was prepared as reported in literature [4a-d. The structures of 4a-d were confirmed with elemental analyses and spectral data. IR spectra elicited a band at 3460-3188 cm-1 corresponding to NH group. 1H NMR of 4a-c displayed a singlet at 7.89-8.22 ppm corresponding to azo methane proton and another singlet at 8.50-11.57 ppm assigned to NH. While compound 4d revealed a singlet signal at 2.53 ppm corresponding to the N=CCH3 protons and two singlet signals at 3.81 and 3.84 ppm corresponding to the added two OCH3 protons. Finally, MS spectra revealed their molecular ion peaks. Compound 5 was obtained by stirring a mixture of acid hydrazide 3 with glacial acetic acid at room temperature for 24 h. The structure of 5 was elucidated by the elemental analysis and spectral data. IR spectrum showed bands at 3446, 3238 cm-1 corresponding to 2 NH groups. 1H NMR spectrum revealed a singlet signal at δ = 1.88 ppm corresponding to COCH3 proton and 2 singlet signals at δ = 9.89 and 10.04 ppm corresponding to 2 NH groups. The MS spectrum revealed its molecular ion peak at 394. Synthesis of substituted oxadiazoles can be achieved through one pot reaction of three components, phosphorus oxychloride, acid hydrazide and acid derivatives [6 in good yield. Compound 6 was confirmed through the elemental analysis and spectral data. IR spectrum showed disappearance of bands corresponding to 2 NH groups and only one band at 1691 cm-1 corresponding to benzopyrone C=O group. 1H NMR spectrum revealed disappearance of any signals corresponding to the 2 NH protons. MS spectrum showed its molecular ion peak at m/z 367. Reaction of acid hydrazide derivatives with acid chloride as tosyl chloride led to the formation of benzene sulfone hydrazide derivatives, this reaction was reported to be performed in glacial acetic acid at room temperature for 24 h [7 was deduced as disubstituted derivative by the elemental analysis and spectral data. IR spectrum showed a band at 3446 cm-1 corresponding to NH group and bands at 1354, 1188 cm-1 corresponding to SO2 groups. 1H NMR spectrum showed a singlet signal at δ = 2.29 ppm corresponding to two p-CH3 protons, a double of doublet at δ = 7.12 and 7.50 ppm corresponding to 3ʺ, 5ʺ and 2ʺ, 6ʺ protons of the two p-methylbenzenesulphone moieties, respectively. The MS spectrum of 7 revealed the molecular ion peak at 660.The starting compound terature . Benzyliivatives . Anotherivatives . In the 3 with many substituted carbonitrile derivatives as malononitrile, ethyl cyanoacetate and ethoxy methylene malononitrile was reported to yield the corresponding amino pyrazoles 8, pyrazolone 9 and pyrazole carbonitriles 10, respectively. The target compounds 8, 9, and 10 were synthesized through refluxing acid hydrazide derivative 3 with appropriate carbonitrile compound in ethanol (Scheme 2).Refluxing acid hydrazide 8 showed bands at 3504, 3448 and 3404 cm-1 assigned to NH2 and NH groups. 1H NMR spectra of 8 displayed three singlet signals at 1.88 and 4.65 and 9.93 ppm corresponding to CH2 pyrazoline, NH and NH2, respectively. 1H NMR spectra of 9 revealed two singlet signals at δ = 1.87 and 9.67 ppm corresponding to CH2 pyrazolone and two NH protons, respectively. IR spectrum of 10 revealed a characteristic band at 2196 cm-1 corresponding to the added cyano group. Finally, MS spectra revealed the molecular ion peaks of the titled compounds. Condensation of acid hydrazide with acetyl acetone in ethanol containing triethylamine afforded the corresponding dimethyl pyrazoles 11 (Scheme 2). The structure of 11 was deduced from microanalytical and spectral data. 1H NMR spectra showed new two singlet signals at 2.40 ppm assigned to six protons of the two CH3 substituting the pyrazole ring and at 5.66 ppm corresponding to the CH of pyrazole, finally 11 revealed the molecular ion peak at 416.The IR spectrum of 3.23.2.14a-d, 5, 6, 7, 8, 9, 10 and 11) were selected by National Cancer Institute (NCI) Developmental Therapeutic Program (www.dtp.nci.nih.gov), Bethesda, MD, U.S.A. The synthesized compounds were subjected to the NCI’s disease-oriented human cell lines screening assay to be evaluated for their in vitro antitumor activity. The anticancer assays were performed in accordance with the protocol of the Drug Evaluation Branch, NCI, Bethesda [1).Newly synthesized compounds  with GI value of 30.93% and a weak activity over the leukemia subpanels CCRF-CEM and SR with GI values of 19.49 and 25.42%, respectively. A noticeable effect was achieved upon many non-small cell lung cancer subpanels with cell line NCI-H226 having the greatest effect of GI value of 46.05% while NCI-H23, NCI-H332M and NCI-H522 was inhibited by values of 15.35, 22.25 and 19.71%, respectively. An overall weak activity was revealed over the other subpanels tested with GI values of 15.28, 11.02, 10.69, 21.11, 12.95, 15.27, 10.23, 28.13, 11.26, 10.65 and 13.56 for CNS cancer subpanels SF-268, SNB-19, U251, melanoma subpanels LOXIMVI-MALME-3M, UACC-62, ovarian cancer OVCAR-8, renal cancer A498, SN12C, UO-31, prostate cancer PC-3 and finally breast cancer MCF-7 and MDA-MB-231/ATCC, respectively. The 4-methoxybenzylidene derivative 4b revealed similar activity to that of the N,N-dimethylaminobenzylidene derivative 4a with the highest effect achieved over the CNS cancer subpanel SNB-75 with GI value of 44.61%. The other cell lines tested were inhibited with weak to moderate effect with GI values of 15.78, 23.39, 13.84 and 20.78% for leukemia cell lines CCRF-CEM, HL-60 (TB), MOLT-4 and RPMI-8226, respectively. While the non-small cell lung cancer subpanels A549/ATCC, HOP-92, NCI-H226, NCI-H23, NCI-H322M and NCI-H522 with GI values of 17.44, 14.88, 12.03, 13.99 and 22.18%, respectively. Colon cancer subpanels HCT-116 and KM12 were inhibited by GI values of 13.60 and 10.00%, respectively. A moderate effect was achieved upon melanoma subpanel UACC-257 with GI value of 21.41%, the renal cancer RXF393 with GI value of 18.22% and prostate cancer cell line PC-3 was inhibited by 21.06%. Finally, GI values of 21.06 and 19.15% were achieved over breast cancer subpanels MDA-MB-231/ATCC and MDA-MB-468, respectively. The trimethoxybenzylidene 4c inhibited leukemia subpanels RPMI-8226 and SR with GI values of 24.64 and 29.69%. A good effect was shown upon the non-small cell lung cancer subpanel NCI-H522 with GI value of 31.83%. GI values of 34.48 and 30.04% were achieved over renal cancer UO-31 and breast subpanel MDA-MB-468, respectively. The dimethoxyphenylethylidene derivative 4d showed a weak activity over a single cell line, the renal cancer UO-31 with GI value of 15.52%.Regarding the activity towards individual cell lines, Schiffs like compounds 5, oxadiazole 6 and bis-methylphenylsulphonyl derivative 7 showed a similar inhibitory pattern. The three compounds revealed only a weak effect upon leukemia subpanel RPMI-8226 with GI values of 15.42, 12.64 and 11.32% for 5, 6 and 7, respectively. Also the non-small cell lung cancer cell line NCI-H522 was inhibited by 13.08, 10.75 and 15.72% for 5, 6 and 7, respectively. Finally, 5, 6 and 7 exhibited a weak activity over renal cancer UO-31 with GI values of 21.08, 11.03 and 21.24%, respectively.The acetohydrazide derivative 8 derivative, 5-iminopyrazolidin-3-one derivative 9 and 5-imino-2,5-dihydropyrazole-4-carbonitrile 10 revealed no noticable effect, all three derivatives shared activity towards non-small cell lung cancer subpanel NCI-H522 with GI values 22.46, 21.43 and 20.63%, respectively, they also shared activity towards renal cancer UO-31 with GI values of 29.22, 35.81 and 24.45%, respectively. 8 also possessed weak activity towards breast cancer T-47D with GI value of 29.36% where it showed the highest mean GI among the three derivatives of value 3.66% compared to mean GI value of 2.08 and 0.95% corresponding to 9 and 10, respectively. Compound 11 did not exhibit considerable activity as GI values were 12.22 and 15.14% against non-small cell lung cancer NCI-H522 and renal cancer UO-31, respectively.The 3-amino-5-imino-4,5-dihydropyrazol in vitro anticancer activity in the full NCI 60 cell lines panel assay by a single dose test (10 µM). Substituted benzylidene derivatives 4a, b and c had the best activity with mean GI values of 6.17, 10.84 and 7.99%, respectively. Results revealed that, Schiff’s like compounds of benzopyrone scaffold with substituted benzylidene derivatives 4a-c had overall good effect. However, Schiff’s like compounds comprised of disubstituted phenylethylidene derivative 4d had no significant effect. In addition, aetohydrazide derivatives 5 and 7 or hybrids with oxadiazole 6 or substituted pyrazoles 8-11 had a weak or no significant effect.New benzopyrone derivatives were prepared in this study. All compounds were selected by National Cancer Institute (NCI), Bethesda, and evaluated for their


Got1), a key enzyme in the MA shuttle, which generates cytosolic glutamate, and the vesicular glutamate transporters , which participate in glutamate transport into secretory vesicles. Got1 knockout (KO) β-cell lines were defective in cytosolic glutamate production from glucose and showed impaired IIIS. Unexpectedly, different from the previous finding that global Slc17a7 KO mice exhibited impaired IIIS from pancreatic islets, β-cell specific Slc17a7 KO mice showed no significant impairment in IIIS, as assessed by pancreas perfusion experiment. Single Slc17a7 KO β-cell lines also retained IIIS, probably due to compensatory upregulation of Slc17a6. Interestingly, triple KO of Slc17a7, Slc17a6, and Slc17a8 diminished IIIS, which was rescued by exogenously introduced wild-type Slc17a7 or Slc17a6 genes. The present study provides direct evidence for the essential roles of AST1 and VGLUTs in β-cell glutamate signaling for IIIS and also shows the usefulness of the CRISPR/Cas9 system for studying β-cells by simultaneous disruption of multiple genes.Incretins (GLP-1 and GIP) potentiate insulin secretion through cAMP signaling in pancreatic β-cells in a glucose-dependent manner. We recently proposed a mechanistic model of incretin-induced insulin secretion (IIIS) that requires two critical processes: 1) generation of cytosolic glutamate through the malate-aspartate (MA) shuttle in glucose metabolism and 2) glutamate transport into insulin granules by cAMP signaling to promote insulin granule exocytosis. To directly prove the model, we have established and characterized CRISPR/Cas9-engineered clonal mouse β-cell lines deficient for the genes critical in these two processes: aspartate aminotransferase 1 (AST1, gene symbol  The latter involves amplification of the effects of the triggering pathway by metabolic signals other than ATP evoked by glucose metabolism, but the mechanism remains poorly understood. In addition to glucose, neuro-hormonal amplification is critical in normal regulation of insulin secretion, as reviewed in detail by Yokoi et al. . Proteins were separated by SDS-PAGE and electrophoretically transferred onto a PVDF membrane . The membranes were blocked in blocking solution , incubated with anti-GOT1 antibody  for 1h, washed with tris-buffered saline  containing 0.1% Tween 20 (TBS-T), incubated with HRP-conjugated secondary antibodies for 1h, and washed with TBS-T. Immunoreactivity was visualized with an enhanced chemiluminescence system, ECL Prime detecting reagents  and detected by ImageQuant LAS 4000mini .Got1 or INS1, pcDNA3.1(+) vectors containing cDNA of Slc17a7 or Slc17a6 were purchased from Genscript . Got1 KO β-cell lines were reverse-transfected with a combination of INS1 (0.4 μg/mL) and either Got1 (0.46 μg/mL) or empty pcDNA3.1(+) N-DYK construct . VGLUTs  triple KO β-cell lines were reverse-transfected with either INS1  or a combination of INS1 (0.4 μg/mL) and Slc17a7 (0.0025 μg/mL) or Slc17a6 (0.0025 μg/mL). For transfection, Lipofectamine 2000 transfection reagent (Life Technologies) was used according to the manufacturer’s instruction. After 48 hours of incubation, human C-peptide secretion experiment was performed in the same manner as in the insulin secretion experiment. Human C-peptide released in the incubation buffer and cellular C-peptide content were measured by Ultrasensitive C-peptide ELISA .The pcDNA3.1(+) N-DYK empty vector, pcDNA3.1(+) N-DYK vectors containing cDNA of Got KO β-cell lines were preincubated for 60 min in H-KRB with 2.8 mM glucose and then incubated for another 30 min in H-KRB with [U-13C]-glucose (2.8 mM or 16.7 mM). Extraction and quantification of cytosolic glutamate contents was performed as previously described -glucose. M and M+1 glutamate isotopomers are both naturally existing glutamate in cells, while M+2, M+3, M+4, and M+5 glutamate isotopomers are synthesized de novo from [U-13C]-glucose. We found that M+2 to M+5 glutamate isotopomers were significantly increased by glucose stimulation in WT cell line, whereas they were not increased in Got1 KO cell line  cell lines established  cell line with WT Got1 significantly restored incretin-induced C-peptide secretion (Got1 KO (A60) cell line, dmGlu amplified insulin secretion in a dose-dependent manner  or pharmacological inhibition of glutamate transport reduced insulin secretion in response to incretins while knockdown of VGLUT2 (Slc17a6) did not [Slc17a7 KO mice at pre-weaning two weeks of age [Slc17a7 would be enough to cause a severe defect in IIIS.Our previous study showed that knockdown of VGLUT1 ( did not . In addis of age . Based oSlc17a7 knockout (βSlc17a7−/−) mice by using the Cre-loxP system (Slc17a7−/− mice (Slc17a7−/− mice and Slc17a7flox/flox mice (control)  between βSlc17a7−/− mice and Slc17a7flox/flox mice  . Unexpecperiment . RT-PCR lox mice . VGLUT3 ereafter , while tSlc17a7−/− mice, we also established Slc17a7 single KO (SKO) β-cell lines. In WT MIN6-K8 cell line, Slc17a7 and Slc17a6 are expressed at similar levels and Slc17a8 is not expressed  triple knockout (TKO) β-cell lines. The above-mentioned Slc17a7 single KO cell line was further transfected with plasmids expressing Cas9 nickase and two pairs of sgRNA each targeting Slc17a6 and Slc17a8, followed by clonal isolation. After confirmation of deficiency of Slc17a6 in these colonies by sequencing, the Cas9 target region of Slc17a8 of these colonies were examined. Finally, three TKO cell lines, V22, V39, and V61, were established. As for Slc17a6, all of these cell lines were compound heterozygous mutants harboring frameshifts on both alleles  was administered to mice after 6 hours of fasting. For meal tolerance test, 10 μL/g (1.5 g carbohydrate/kg body weight) Twinline  was administered orally to mice after 6 hours of fasting. For intraperitoneal insulin tolerance test, insulin  was administered to mice after 6 hours of fasting. Blood glucose levels were measured by a portable glucose meter .(TIFF)Click here for additional data file.S1 FigBoth alleles of the KO cell line A60 and allele 1 of the KO cell line A64 were distinct from the WT allele. Allele 2 of the KO cell line A64 was not detected probably due to low expression.(TIFF)Click here for additional data file.S2 FigGot1 KO cell lines. ***p < 0.001.mRNA expression levels of KO cell lines are presented as fold-change relative to those of WT (n = 4). The data are expressed as means ± SEM. Representative results are shown. Similar results were found in 3 independent experiments. Dunnett's method was used for statistical comparisons between WT and (TIFF)Click here for additional data file.S3 Fig Cells were stimulated with glucose and GLP-1 (A) or GIP (B) (n = 4 for each). Insulin secretion was normalized by cellular insulin content. The data are expressed as means ±SEM. Representative results are shown. Similar results are found in 3 independent experiments.(TIFF)Click here for additional data file.S4 FigloxP sites. The FRT-flanked neo gene was excised via Flp-FRT recombination. Floxed exon 2 was deleted via Cre-loxP recombination.A targeting vector was constructed so that exon 2 coding the first transmembrane domain was flanked by (TIFF)Click here for additional data file.S5 FigSlc17a7−/− mice during (A) oral glucose tolerance test, (B) oral meal tolerance test, and (C) intraperitoneal insulin tolerance test. n = 6 each. Differences between the groups were analyzed with the Welch’s t-test. n.s., not significant.Changes in blood glucose levels of control and β(TIFF)Click here for additional data file.S6 FigSlc17a6 and Slc17a8 are presented as fold-change relative to those of Slc17a7 (n = 3). The data are expressed as means ± SEM. Representative results are shown. Similar results were found in 3 independent experiments. n.d., not detected.mRNA expression levels of (TIFF)Click here for additional data file.S7 FigSlc17a7 KO cell lines 34 and 39 were distinct from the WT allele. Detection of allele 1 and 2 required specific primer sets, respectively.Both alleles of (TIFF)Click here for additional data file.S8 FigSlc17a7 KO cell lines. *p < 0.05; ***p < 0.001.mRNA expression levels of KO cell lines are presented as fold-change relative to those of WT (n = 4). The data are expressed as means ± SEM. Representative results are shown. Similar results were found in 3 independent experiments. Dunnett's method was used for statistical comparisons between WT and (TIFF)Click here for additional data file.S9 FigSlc17a7 single KO (34 and 39) cell lines were stimulated with glucose and GLP-1 (n = 4). Insulin secretion was normalized by cellular insulin content. The data are expressed as means ±SEM. Representative results are shown. Similar results were found in 3 independent experiments.WT MIN6-K8 and (TIFF)Click here for additional data file.S10 FigSlc17a6 exon 2 in triple KO cell lines induced by the CRISPR/Cas9 nickase system. (B) Mutations in Slc17a8 exon 2 in triple KO cell lines induced by the CRISPR/Cas9 nickase system. Slc17a8 allele 2 in cell lines V22 and V61 were not detected by PCR probably due to large deletions. WT sequence is shown with target sites of sgRNAs. PAM and mutations are shown in red.(A) Mutations in (TIFF)Click here for additional data file.S11 Fig(A) Both alleles of TKO cell line V22 and allele 1 of TKO cell line V39 were distinct from the WT allele. Allele 2 of TKO cell line V39 was not detected probably due to low expression. Both alleles of TKO cell line V61 were indistinguishable from the WT allele. (B) Specific primer sets for allele 1 or 2 of TKO cell line V61 proved the mutation.(TIFF)Click here for additional data file.S12 Fig Cells were stimulated with glucose and GLP-1 (A) or GIP (B) (n = 4 for each). Insulin secretion was normalized by cellular insulin content. The data are expressed as means ±SEM. Representative results are shown. Similar results are found in 3 independent experiments.(TIFF)Click here for additional data file.


Background: Breast cancer (BC) represents the most common cancer in women worldwide. Due to its heterogeneous nature, breast cancer management might benefit from differential treatments toward personalized medicine. Additionally, drug resistance is a common phenomenon. We systematically investigated the effect of 14 different drugs administered on BC cell lines in combination with microRNAs .Methods: Thirty-eight miRNAs, all associated with BC by clinical and molecular parameters including progression, prognosis and subtypes, were tested for their effects on the viability of 12 different BC cell lines. Four miRNAs with the strongest impact on viability were further assayed in combination with 14 BC drugs. Mann–Whitney U-test with Bonferroni correction was used for statistical analysis.Results: In a miRNA only pre-screen we observed effects on BC cell lines' viability for 34 out of 38 candidate miRNAs. We then identified 14 miRNA/drug combinations for which the combination IC50 was lower than that of both miRNA and drug as single agents. miR-181a, paired with GSK1070916, Doxorubicin, XL765 and AMG511, was the only miRNA active on the triple negative (TNBC) MDA-MB-468 cell line. miR-126 was the only miRNA (in combination with CDK4/6 or PIK3CA inhibitors) with significant effects on cell lines from different subtypes: MCF7  and MDA-MB-453 (HER2+). Because of its activity on different BC subtypes, we investigated the genome wide effects of miR-126 using transcriptomics and confirmed that expression of miR-126 in BC cell lines affected cell cycle and mitosis.Conclusion: Our results show that a combination treatment with miRNAs, in particular miR-181a, miR-326, miR-9 and miR-126, enhance the activity of specific BC drugs in vitro, even on the most aggressive BC subtypes, HER2+ and TNBC. Finally, as expected from its drug interactions, based on a whole transcriptome study we could confirm a role for miR-126 in cell cycle regulation. Breast cancer (BC) is the most common cancer in women worldwide representing about 25% of all cancers, with nearly 1.7 million new cases diagnosed in 2012 . This corresponds to about 12% of all new cancer cases and 25% of all cancers in women as reported by the World Cancer Research Fund International website+-enriched (HER2 positive), and Basal-like or Triple-Negative  , progesterone receptor (PR), and human epidermal growth factor receptor-2 (HER2) status, in association with gene expression profiling revealed a subtype classification of BC. The classification includes luminal A , luminal B , HER2Nowadays there is an acceleration toward implementation of individualized treatment and thus an ever larger need for the widest possible range of therapeutic tools. Notwithstanding more sensitive diagnostics and advances in early detection, drug resistance is still one of the major problems that remain to be solved. Too often, in fact, patients go on to develop aggressive malignancies, which exhibit resistance to one or more drugs. The underlying mechanisms of acquired resistance to chemotherapeutic agents being still poorly understood.MicroRNAs, among the most investigated actors in the non-coding RNA (ncRNA) panorama, can potentially be used to increase the response of cancer to therapeutic interventions . MDA-MB 468, MCF7, ZR75.1, MDA-MB-361, and MDA-MB-231 were cultured in Dulbecco's modified Eagle's medium , while SKBr3 in McCoy's (Lonza). All media were supplemented with 10% fetal bovine serum (FBS), 1% penicillin and streptomycin antibiotics (PS) and 1% L-glutamine. T47D, MDA-MB-453, BT474 and HBL100 were maintained in RPMI-1640 medium with L-glutamine (Sigma-Aldrich) supplemented with 10% FBS and 1% PS. Immortalized MCF-10A and 184A1 cells were cultured in mammary epithelial cell growth medium F12 (Gibco) supplemented by 10% FBS, 1% PS, 10 μg/ml bovine insulin, 100 ng/ml cholera toxin, 20 ng/ml recombinant human epidermal growth factor, 0.5 μg/ml hydrocortisone . All cell lines were grown in a 37°C humidified incubator with 5% COThe cells were transiently transfected with miRNA mimics  using siPORT™ NeoFX™ Transfection Agent  as recommended in the instruction protocol. Briefly, we operated a dilution of miRNA and siPORT™ NeoFX™ in separate tubes containing medium without FBS. Upon mixing, the transfection complex was incubated at RT (room temperature) for 10 min, and then transferred to a 96-well plate containing cells in appropriate medium with 0.1% FBS. Final miRNA concentration for transfection was 100 nM.3 cells, for each BC cell lines, or 12 × 103 cells, for breast epithelium cell lines (MCF10A and 184A1) were seeded into 96-well flat bottom microplates in a final volume of 100 μl, with 0.1% FBS, per well. The MTS/PMS mix was added to each well and the microplates further incubated for 1–4 h at 37°C and 5% CO2. The absorbance (OD) of the samples was read with a SUNRISETM ELISA plate reader operating at 492 nm . All experiments were repeated at least three times in duplicates.The MTS assay was performed to assess cell viability after miRNA transfection in 0.1% FBS. MTS -5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) CellTiter 96 Aqueous Reagent Powder  and PMS (phenazine methosulfate) (SIGMA), were diluted in phosphate buffered saline (PBS) in a 20:1 ratio and used to assess cell proliferation according to the manufacturer's protocol. Briefly, 6 × 103 cells were seeded into 96 wells plates. The reagent was added in 1:10 proportions to the cells in culture. After 4–6 h of incubation at 37°C, absorbance was monitored at 570 and 620 nm wavelengths. Again, as for MTS assays, all experiments were repeated at least three times in duplicates.AlamarBlue cell viability assays were performed as recommended in the instruction protocol. Briefly, for all BC cell lines, 6 × 103 cells/well in 96-well plates and treated with increasing concentration of drug in a range starting from 1.28 nM up to 100 μM, or from 0.64 nM up to 1 μM for Docetaxel, in 10% FBS medium, for 72 h. We determined the inhibition curves using Graph Prism. To evaluate possible synergisms or interactions between miRNAs and drugs, cells were also transfected using siPORT™ NeoFX™ with miRNAs final concentration of 50 nM in 10% FBS medium. After 16 h, the medium was changed and the drug added. Passed 72 h, the alamarBlue cell viability assay was performed. Experiments were performed at least in duplicate for three times.Drug sensitivity was specifically investigated to determine the inhibition dose for each cell line. Cell viability assays were performed in triplicates with alamarBlue. MDA-MB-468, T47D, MCF7, and MDA-MB-453 were plated at 6 × 10U-test was used for the alamarBlue assay (cell lines treated with miRNA and/or drug in combination). The Bonferroni correction was applied, dividing the critical P-value (α = 0.05) by the number of comparisons for each experiment. Normalized miRNA profiles (n = 796) for the METABRIC study .For the analysis of MTS experiments we calculated the ratio between the mean (quadruplicate samples) of each miRNA and the global median from all transfections. We then calculated, for each miRNA, the absolute deviation from the median, i.e., MAD. We deemed significant the effect of a miRNA for which the median MTS-value was larger  than the global median +/- 2 times its MAD. The Mann–Whitney https://gdac.broadinstitute.org/). Lentiviral assays for miR-126 were obtained from GEO (GSE40458). Filtering was enabled to filter out genes that had <20% of expression data with at least a 1.5-fold change in either direction from gene's median value. Correlation between miRNAs and mRNAs was performed using Spearman correlation. The genes for the regulated mRNAs were studied for functional enrichment on PantherDB (http://pantherdb.org/) and Gene Set Enrichment Analysis  to Invasive Ductal Carcinoma (IDC) (Supplementary Table The key criteria for the inclusion of a miRNA in our study were based on microRNA profiles in BC cohorts: (i) differentially expressed miRNAs in solid tumors vs. normal breast samples (TCGA), and (ii) miRNAs related to the transition from Ductal Carcinoma Prior to cell assays we tested the transfection efficiency of MDA-MB-453, MDA-MB-468, MCF7, and T47D cell lines using siPORT and a plasmid containing the green fluorescence protein EGFP. After 48 and 72 h the mean efficiency of transfection for all cell lines explored was acceptable and comparable, 70 ± 10% (data not shown) and prompted us to carry on with the experiment. We then investigated the miRNA effects on the proliferation of 10 breast cancer cell lines and of 2 cell lines derived from normal breast epithelium. This analysis was performed to gain experimental evidences on the miRNA functional involvement in cancer mechanisms, where often the growth signals are constitutively activated by a host of mutations . We thus assayed cell proliferation upon miRNA transfection in condition of serum deprivation (0.1% FBS). Figure + and MDA-MB-468 as Triple-Negative.Thus, we selected 4 of the miRNAs with higher anti-growth effect as candidate enhancer of anticancer drugs . Figure ,We used 14 cancer drugs with different targets of action to be combined with the 4 active miRNAs in subsequent studies for miRNA-drug interactions. Most of these drugs have been assessed in clinical trials50), cell viability assays were performed using increasing drug concentrations. We treated all four BC cell lines with scalar concentration of each drug, in a range from 1.28 nM to 100 μM, or from 0.64 nM to 1 μM for Docetaxel; alamarBlue was used to evaluate cell growth inhibition , and α = 0.007 for MDA-MB-468 and T47D (each n = 7). To generate robust results, we deemed the effect of combined miRNA/drug as a true interaction only if all p-values for the 3 comparisons were significant. For a stringent statistics the Bonferroni correction was applied also for the single miRNA effects: α = 0.017 for MDA-MB-453 and MCF7 (each n = 3), and α = 0.025 for MDA-MB-468 and T47D (each n = 2). Figure In most assays, the miRNA acted decreasing cell viability also in complete medium, as expected from our initial results with miRNA transfection in 0.1% FBS. For what concerns the miR-drug combinations, some pairs had remarkable effects. To identify them statistically, we operated 3-way comparisons: single miRNA vs. combined, single drug vs. combined and untreated vs. combined. The Bonferroni correction was applied, to yield stringent p < 0.05. For example, miR-126 and XL765 were significantly more active in combination than alone both in MCF7 e MDA-MB-453.Among the many combinations with positive effects against cell proliferation, the miR-126/LEE011 and miR-126/BYL719 pairs were significant (p < 0.005) in both cell lines we evaluated, namely MCF7 and MDA-MB-453. Therefore, we went on to treat with these combinations the remaining two cell lines, T47D and MDA-MB-468. But for these two additional cell lines miR-126, alone or in combination with LEE011 or BYL719, did not show significant effects . We performed a Spearman correlation test of miR-126 with all coding genes and applied Bonferroni correction for multiple testing. In Supplementary File p < 0.05). We then determined the cellular pathways and processes inversely correlated to miR-126 in BC, analyzing the 661 down-regulated genes for enrichment of GO, REACTOME terms and pathways . Other studies supported the anti-tumor role of miR-126 , AZD5363 (p = 0.007), Trametinib, and SCH772984 (p = 0.006) drugs, reach a p-value very close to the significance. Somehow revealing, still in MCF7, for the ambiguous role of miR-9 in cancer, its positive combination with the ATP-competitive mTORC1/C2 inhibitor drug AZD8055. Although miR-9 alone doesn't show particular effects on MCF7 proliferation, the association with AZD8055 clearly affects the cell growth. The recent founding that miR-9 suppress the expression of the oncogene androgen receptor in MCF7, could suggests a possible explanation of the successful combination with AZD8055 , whereas miR-9 and miR-181a reach a + subclass. It appeared to be the most sensitive cell line to miR/drug combination treatments. The effects of single miRNAs were confirmed for miR-126 and miR-9 . miR-126 was active in combination with LEE011, BYL719, AZD8055, AZD5363, Trametinib, and SCH772984. AZD5363, a PKB/AKT isoforms inhibitor, acts like BYL719 and AZD8055 on the PI3K/AKT/mTOR pathway whereas Trametinib and SCH772984, as MEK 1/2 inhibitor and ATP competitive inhibitor of ERK1 and ERK2, respectively, acts on the MEK/ERK pathway. The wide involvement of miR-126 in viability of MDA-MB-453 is entirely coherent with its in vivo expression, inversely correlated with HER2 overexpression . There are six combinations between miR-326 and drugs with significant interactions on BC viability: LEE011, BYL719, AZD5363, SCH772984, all already seen for miR-126, Gefitinib and Erlotinib, two epidermal growth factor receptor (EGFR) tyrosine kinases inhibitors. Curiously was the behavior of these last two drugs, that alone seemed to have a “proliferative action,” in particular Erlotinib, but they revert their trend in combination with miR-326. Considering HER3 can act as signaling platform when it forms heterodimers with a signaling-competent HER family member (as HER2), this finding can be explained by the observed role of miR-326 in reducing HER3 mRNA and protein levels , more focused investigation are needed . These are very interesting results indicating a novel, albeit still ambiguous and undefined role, for miR181a in BC. Indeed, for example it has been demonstrated how an up-regulation of miR-181a expression due to TGF-β promotes breast cancer metastasis , known to have generally the worst outcome. As TNBC lacks molecular targets, chemotherapy is the only currently available systemic treatment for TNBC, and prognosis remains poor  and the related protein p107 and p130, tumor suppressor proteins that allow the cell cycle to proceed from G1 to S phase (Nielsen et al., In conclusion, our results confirm that a combination treatment involving miRNAs and drugs can enhance drug activity for specific and important cancer targets. Our experiments further unravel the effects of non-coding RNAs on the potency of BC drugs and open up questions about the cellular mechanisms of interaction that might themselves address novel drug targets.FB, SV, MP study concept and design; All authors acquisition, analysis, or interpretation of data; FB, SV, MG drafting of the manuscript; FC, LM, CA, MP, CC critical revision of the manuscript for important intellectual content; FB, LM, CZ, SV, MG statistical analysis; SV, CC obtained funding.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.