The majority of cancer cells have a unique metabolic requirement for methionine that is not observed in normal, non-tumorigenic cells. This phenotype is described as “methionine dependence” or “methionine stress sensitivity” in which cancer cells are unable to proliferate when methionine has been replaced with its metabolic precursor, homocysteine, in cell culture growth media. We focus on the metabolic response to methionine stress in the triple negative breast cancer cell line MDA-MB-468 and its methionine insensitive derivative cell line MDA-MB-468res-R8.Using a variety of techniques including fluorescence lifetime imaging microscopy (FLIM) and extracellular flux assays, we identified a metabolic down-regulation of oxidative phosphorylation in both MDA-MB-468 and MDA-MB-468res-R8 cell types when cultured in homocysteine media. Untargeted metabolomics was performed by way of gas chromatography/time-of-flight mass spectrometry on both cell types cultured in homocysteine media over a period of 2 to 24 h. We determined unique metabolic responses between the two cell lines in specific pathways including methionine salvage, purine/pyrimidine synthesis, and the tricarboxylic acid cycle. Stable isotope tracer studies using deuterium-labeled homocysteine indicated a redirection of homocysteine metabolism toward the transsulfuration pathway and glutathione synthesis. This data corroborates with increased glutathione levels concomitant with increased levels of oxidized glutathione. Redirection of homocysteine flux resulted in reduced generation of methionine from homocysteine particularly in MDA-MB-468 cells. Consequently, synthesis of the important one-carbon donor S-adenosylmethionine (SAM) was decreased, perturbing the SAM to S-adenosylhomocysteine ratio in MDA-MB-468 cells, which is an indicator of the cellular methylation potential.This study indicates a differential metabolic response between the methionine sensitive MDA-MB-468 cells and the methionine insensitive derivative cell line MDA-MB-468res-R8. Both cell lines appear to experience oxidative stress when methionine was replaced with its metabolic precursor homocysteine, forcing cells to redirect homocysteine metabolism toward the transsulfuration pathway to increase glutathione synthesis. The methionine stress resistant MDA-MB-468res-R8 cells responded to this cellular stress earlier than the methionine stress sensitive MDA-MB468 cells and coped better with metabolic demands. Additionally, it is evident that S-adenosylmethionine metabolism is dependent on methionine availability in cancer cells, which cannot be sufficiently supplied by homocysteine metabolism under these conditions. Reprogrammed metabolism in cancer has gained greater attention in recent years, beyond the well-known Warburg effect . While m5-methyltetrahydrofolic acid or toward cysteine and glutathione synthesis in the transsulfuration pathway . To. To53]. In conclusion, the present study reveals dynamic changes in the metabolic phenotype associated with methionine dependency of breast cancer and further highlights the importance of SAM as a major regulator of tumorigenesis. Importantly, using a labeled precursor, we discovered that in both MB468 and MB468res-R8 cell types, homocysteine does not significantly contribute to formation of methionine and ultimately S-adenosylmethionine. Our study indicates that SAM metabolism, which is required for all methylation reactions, depends on availability of methionine in the cancer cell lines investigated and cannot be substituted by homocysteine-forming pathways. Moreover, these findings shed light on the metabolic reprogramming, which accompanies methionine stress beyond methylation potential. Such knowledge is instrumental for elucidating novel targets of therapeutic intervention, which can be exploited in future studies. Given the importance of SAM in lipid biogenesis and our observed changes in LLS, assessing the impact of methionine stress on lipid composition and synthesis is of considerable interest and warrants further investigation.


Chondroitin sulfate proteoglycan 4 (CSPG4) is a membrane bound proteoglycan, composed of an N-linked 280kDa glycoprotein, and a single 450kDa chondroitin sulfate linked proteoglycan . CSPG4 e16 pediatric tumor lines were assessed for CSPG4 expression. CSPG4 was detected by flow cytometry in 69% of the cell lines assessed: osteosarcomas: 143b,HOS-MNNG,MG63; synovial sarcomas: SYO-I,HSSY-II, alveolar/embryonal rhabdomyosarcomas: Rh18c,Rh30/Rh36, medulloblastoma: DAOY, melanoma: Mel624,Mel1300. CSPG4 expression site density was estimated with BD PE Quantibrite beads by flow cytometry.Using two different single chain variable fragments (scFv) known to target CSPG4 (225.28 and TP41.2), two anti-CSPG4 second generation CAR constructs were synthesized by transient transfection of retroviral packaging lines. Both constructs have identical intracellular signaling components . Healthy donor T cells were transduced with viral supernatants, and assessed for CAR expression. Anti-tumor activity was measured by cytokine release and tumor specific lysis. Cell surface expression of the CSPG4-based CARs was detected by flow cytometry using conjugated Protein L to target the expressed scFv light chain. 225.28 and TP41.2 based CAR T cells induced 25% and 31% lysis of target pediatric tumor lines, respectively (E:T ratio of 20:1). On further investigation, TP41.2 based CAR T cells were able to specifically lyse CSPG+ cell lines 143b, Rh30, and Rh18c at 40%, 37%, and 16%, respectively (E:T ratio of 30:1). Additionally, the TP41.2 and 225.28 CAR T cells were assessed for Interferon gamma (IFNy) production by flow cytometry. Compared to mock T cells coincubated with varying tumor lines , the TP41.2 and 225.28 CAR+ T cells had significantly positive populations for IFNy production (p < 0.0001), predominantly in the CD8+ population.in vitro at significant levels (p = 0.01). Targeting CSPG4 with CAR redirected T cells represents a new strategy for eliminating these pediatric malignancies in an antigen specific, cell-mediated manner.High levels of CSPG4 are present on a variety of pediatric solid tumors. TP41.2 CSPG4-based CAR T cells target, generate significant levels of IFNy, and lyse osteosarcoma


CDKN2A and NF2, were examined in primary cell lines and in two widely used commercial lines. Expression of MM-associated markers, as well as the status of CDKN2A, NF2, the ‘gatekeeper' in MM development, and their products demonstrated that primary cell lines are more representative of the tumor close to its native state and show a degree of molecular diversity, thus capturing the disease heterogeneity in a patient cohort. Molecular profiling revealed a significantly different transcriptome and marked metabolic shift towards a greater glycolytic phenotype in commercial compared with primary cell lines. Our results highlight that multiple, appropriately characterised, patient-derived tumor cell lines are required to enable concurrent evaluation of molecular profiles versus drug response. Furthermore, application of this approach to other difficult-to-treat tumors would generate improved cellular models for pre-clinical evaluation of novel targeted therapies.Malignant mesothelioma (MM) is an aggressive, fatal tumor strongly associated with asbestos exposure. There is an urgent need to improve MM patient outcomes and this requires functionally validated pre-clinical models. Mesothelioma-derived cell lines provide an essential and relatively robust tool and remain among the most widely used systems for candidate drug evaluation. Although a number of cell lines are commercially available, a detailed comparison of these commercial lines with freshly derived primary tumor cells to validate their suitability as pre-clinical models is lacking. To address this, patient-derived primary mesothelioma cell lines were established and characterized using complementary multidisciplinary approaches and bioinformatic analysis. Clinical markers of mesothelioma, transcriptional and metabolic profiles, as well as the status of p53 and the tumor suppressor genes  In recent years the demography of MM has changed; the age of MM patients has decreased and there is an increased incidence in females, likely reflecting exposure from non-occupational sources.3 The major histologic subtypes of MM, epithelioid, sarcomatoid and biphasic are all associated with poor patient survival, with sarcomatoid MM exhibiting the worst prognosis.4 The median overall survival for MM following frontline chemotherapy with pemetrexed and cisplatin is ~12 months.5 The disease occurs after a long (up to 40 years) latency period and the delay between asbestos exposure and MM onset suggests that multiple factors are involved in asbestos-induced tumorigenesis. Moreover, the non-specific early symptoms combined with the older age of MM patients and the absence of reliable biomarkers hinders early diagnosis.Malignant mesothelioma (MM) is an aggressive, fatal tumor strongly associated with asbestos exposure. MM is responsible for ~3000 deaths per year in the United States and 5000 deaths in Western Europe.in vitro model systems, thereby enabling mechanistic studies of tumor pathogenesis, as well as the identification of new biomarkers and novel therapeutic targets. A number of commercially available cell lines have been widely used for translational in vitro studies.6 In addition, to date, several primary MM cell lines have been described, although their degree of characterization varies.12 It is generally accepted that these primary cell lines are more physiologically relevant as in vitro models, although the generation of such lines is both challenging and labor intensive.There is an urgent need to improve MM patient outcomes and this requires both appropriate pre-clinical models and new therapeutic strategies. Mesothelioma-derived cell lines are essential for the development of in vitro models, a detailed comparison of long-established commercially available MM cell lines with freshly derived primary cell lines is essential. This is particularly important as MM is associated with chromosomal loss, deletions in CDKN2A, CDKN2B, and NF215 genes and mutations in BAP1 and CUL1.16 Thus, genomic instability limits the long-term usefulness of commercially available MM cell lines. To address this, and to develop more relevant pre-clinical models of MM, we established and characterised eight primary mesothelioma cell lines and employed a number of complementary multidisciplinary approaches to examine differences between these cell lines and two widely used commercial cell lines. Our data demonstrate that there is a significant difference between commercial and primary cell lines at the molecular level, including the transcriptome, expression of mesothelial markers and proteins associated with MM pathogenesis including p53, metabolic profile, status of the tumor suppressor genes CDKN2A and NF2 and their products. Importantly, the commercial cell lines lack many key molecular features known to be associated with MM, whereas the eight primary cell lines more accurately recapitulate human disease, thus providing a superior model for pre-clinical evaluation of novel targeted therapies.To evaluate MM is one of the most difficult cancers in terms of early diagnosis; as a result, tissue specimens representing early stages of MM are not generally available. Surgically resected tumor tissues were obtained from patients with advanced epithelioid (six cases) or biphasic (two cases) MM , as surg17 Primary MM cell lines MESO-3T, MESO-7T, MESO-8T, MESO-12T, MESO-14T and MESO-27T exhibited the ability to grow to high passage (>30), whereas MESO-9T and MESO-17T could only be cultured for up to 15 passages before displaying signs of senescence.Primary MM cell lines, including MESO-3T, MESO-7T, MESO-8T, MESO-9T, MESO-12T, MESO-14T, MESO-17T and MESO-27T, established in culture as an attached monolayer . Adheren20), western blot analysis showed that five out of eight primary cell lines were strongly positive for pan-Cytokeratin  have been implicated in mesothelioma development27 and are of great importance in determining appropriate therapeutic strategies.16 We examined the status of p16INK4A/p14ARF genetic loci in both primary and commercial mesothelioma cell lines (p16INK4A and p14ARF in seven out of eight patient-derived cell lines (with the exception of MESO-27T). The commercial cell line MSTO-211H retained the CDKN2A gene; however, the copy number was reduced by ~60%, corresponding to the loss of one allele (http://cancer.sanger.ac.uk/cosmic). Despite the presence of one p16INK4A allele, p16 protein was not expressed in this cell line . For example, type I interferon signaling pathway-related genes were enriched in most of the primary but not in the commercial cell lines . For each data set, the top genes associated with survival were selected . The gene groups were then used as reference knowledge, similar to standard GO data, to perform Enrichment Analysis. Enrichment Analysis demonstrated that the list of genes aberrantly expressed in MM primary cell lines was enriched by genes significantly associated with survival in breast and lung cancer. For example, out of the top 100 genes associated with survival in the METABRIC data set,34 32 were present in the list of aberrantly expressed genes in MM primary cell lines (P<<0.001). The similarity of the aberrantly expressed genes in primary MM cell lines with the gene sets associated with survival in other cancers is summarized in There are no large-scale clinically annotated gene expression data sets available for MM. Therefore, to gain new insight into the clinical relevance of the transcriptional profile of primary MM cell lines, we compared their transcriptome with 14 large-scale data sets of gene expression and clinical data for other cancers, including breast, lung, colon, prostate, ovarian cancer, glioma, glioblastoma and lymphomas  DNA profiling, their profiles were also matched to the original patient tumor genomic DNA (gDNA). The results showed some differences between STR profiles of gDNA from a primary cell line and the original tumor . MM is aCytogenetic analysis of primary MM cell lines identified both chromosomal structural abnormalities and changes in aneuploidy status, with structural abnormalities commonly seen in chromosomes 1, 2, 3, 6 and 7 . WhereasIn vitro cellular models have significantly contributed to our understanding of tumor biology and response to therapeutic agents.12 However, successful translation of promising pre-clinical data to the clinical setting is limited; for example, out of a number of novel therapies recently examined in clinical trials,40 to date none has been approved for treatment of mesothelioma.41 The development of new therapeutic strategies requires improved pre-clinical models that are more physiologically relevant than long-established, widely used commercial cell lines. Although 2D cell culture models lack the architectural and cellular complexity of real tumors, primary cells, nevertheless, are representative of the patient tumor close to its native state and provide a relatively simple model defined by the ‘robustness' of the system for evaluating new agents in early pre-clinical studies. Generation of primary human mesothelioma cell lines is therefore an important tool for studying the response of this highly chemoresistant tumor to new therapies. Eight new mesothelioma cell lines were established in vitro and characterized using complementary multidisciplinary approaches. Clinically relevant markers of mesothelioma, transcriptional and metabolic profiles, as well as status of the tumor suppressor genes CDKN2A and NF2, the ‘gatekeeper' in MM development,32 were examined in the primary cell lines and compared with those of two widely used commercial cell lines. FACS analysis and confocal microscopy of non-permeabilised cells immunostained for Podoplanin19 demonstrated that all primary cell lines displayed cell surface expression. In contrast, the commercial cell lines tested Podoplanin-negative or were similar to normal primary mesothelial cells. Another important feature of the primary cell lines was the loss of CDKN2A in seven out of eight lines. The CDKN2A genetic locus encodes the proteins p14 and p16; p14 activates p53 by rescuing it from proteasome-mediated proteolysis, whereas p16 antagonizes the cyclin-dependent kinases 4 and 6, consequently blocking cell cycle progression.43 Although the TP53 gene is rarely inactivated in MM,16 our observation of multiple p53 isoforms with variable expression levels in primary cell lines emphasizes an inter-patient variability characteristic of MM. A predominance of primary cell lines negative for p16 protein suggests that this may have arisen from clonal selection with p16-negative cells growing more readily ex vivo. The presence of p16INK4A and NF2 genes and their encoded proteins p16 and Merlin in MESO-27T indicates, however, that loss of these proteins is not a prerequisite for successful culturing of MM tumor cells in vitro. Alternatively, there is also the possibility that CDKN2A/p16 was lost as cells adapted to the culture environment. Either way, the status of key tumor suppressor genes15 in cellular models should be taken into consideration in experiments designed to target these pathways.44 Interestingly, although the commercial cell line MSTO-211H displayed only heterozygous loss of the p16INK4A and p14ARF loci, p16 protein was undetectable in these cells. This suggests an additional mechanism, transcriptional silencing, and exemplifies the possibility of two independent mechanisms simultaneously inactivating key tumor suppressor genes in MM. The NF2 gene is mutated or homozygously deleted in 40–50% of MM,45 although established primary and commercial cell lines show more frequent rates of mutation or undetectable protein.46 Comparison of NF2 gene copy number and its product Merlin, highlighted the importance of transcriptional silencing and posttranslational modification in MM,46; in seven out of eight primary cell lines it was observed that, despite at least one allele being retained, Merlin was undetectable. Furthermore, although Merlin was expressed in cell line MESO-27T, the protein was inactivated by phosphorylation.3347 Metabolic profiling of cultured tumor cells is therefore essential for validation of cell-based in vitro models.A striking difference in the transcriptional profile between primary and commercial cell lines was reflected by hierarchical clustering analysis, displaying that commercial cell lines clustered together and completely separately from primary cell lines. MESO-27T was positioned close to the other primary cell lines, but was not included in the same cluster; this is perhaps not surprising, considering that MESO-27T, unlike the other primary cell lines, expresses p16 and Merlin. Another very important difference between the primary and commercial cell lines was unveiled by examining their metabolic profiles. Commercial cell lines exhibited increased metabolism compared with both low- and high-passage primary MM cells, with a shift towards a more glycolytic phenotype. The commercial cell lines would therefore be an inappropriate model for studies exploring the cellular response to metabolic perturbation agents, including, for example, strategies based on synthetic lethality.48) and three other loci had altered STR values, with a further two loci altered at high passage, implying genomic instability of these cells. Although we cannot fully rule out that these changes existed before cell isolation, no peak imbalance  was observed on the electropherogram  and 10% FBS. Samples were transported to the laboratory for primary cell culturing within 1 h of collection. Human investigations were performed after Research Ethics Committee approval (LREC 08/H0406/226).All patients underwent surgery for radical decortication without prior chemotherapy or radiotherapy. The resected mesothelioma specimens were collected with patient consent. Solid tumors were immediately immersed in ice-cold RPMI-1640 supplemented with penicillin (100 U/ml), streptomycin (100 g followed by red blood cell lysis. Cells were seeded in gelatin-coated flasks at 5 × 104/cm2 and maintained in RPMI-1640 growth media supplemented with l-glutamine (2 mM), penicillin (100 U/ml), streptomycin (100 μg/ml), hEGF (20 ng/ml), hydrocortisone (1 μg/ml), heparin (2 μg/ml) and 2% FBS at 37 °C and 5% CO2. Differential trypsinisation with 0.05 and 0.1% trypsin was used to remove fibroblasts and select mesothelial cells, respectively. Culture purity varied from 97%  to 100% of malignant mesothelial cells. After the cell lines were established they were cultured in RPMI-1640 growth media supplemented with l-glutamine (2 mM), penicillin (100 U/ml), streptomycin (100 μg/ml), hEGF (20 ng/ml), hydrocortisone (1 μg/ml), heparin (2 μg/ml) and 10% FBS at 37 °C and 5% CO2. Cells were monitored regularly for their morphology. All cell lines were authenticated by STR DNA profiling  and matched to the original tumor tissue. All cell culture reagents were from Life Technologies , except for hydrocortisone and heparin .Tumor tissue was mechanically dissociated under sterile conditions and transferred into serum-rich dissection media (RPMI-1640 media supplemented with 10% FBS); cell release was encouraged by mixing of the tissue suspension. Cells were collected by centrifugation at 300 × l-glutamine (2 mM), penicillin (100 U/ml), streptomycin (100 μg/ml) and 10% FBS in a 5% CO2 humidified incubator at 37 °C. Adult human primary omental mesothelial cells (single or multi-donor) purchased from Cambridge Bioscience  served as normal controls  and were maintained in culture in growth medium  for up to three passages.Two mesothelioma cell lines, MSTO-211H and NCI-H2052, were obtained from the American Type Culture Collection  and cultured in RPMI-1640 supplemented with in situ before the plastic was removed, with propylene oxide, and the blocks re-embedded for orthogonal sectioning. Ultra-thin sections were stained with lead citrate and recorded using a Megaview 3 digital camera and iTEM software  in a Jeol 100-CXII electron microscope .For transmission electron microscopy (TEM), cell pellets were fixed in 2% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.4) at 4 °C overnight and postfixed with 1% osmium tetroxide/1% potassium ferrocyanide for 1 h at room temperature. After fixation, cells were stained en bloc with 5% aqueous uranyl acetate overnight at room temperature, dehydrated in a series of alcohols and embedded in Taab epoxy resin . Confluent cultures, in plastic dishes, were also processed For scanning electron microscopy, cells on glass coverslips were processed to absolute alcohol, as for TEM, transferred to hexamethyldisilazane and air-dried before being sputter-coated with a 15 nm layer of gold and examined in a FEI Quanta FEG 250 electron microscope .6/ml) was incubated at 4 °C for 1 h with anti-Podoplanin antibody  or isotype control antibody (Mouse anti-BCL-2). Cells were pelleted, washed and re-suspended in normal medium containing goat anti-mouse IgG conjugated with FITC  and incubated for 1 h at 4 °C. Cells were washed and re-suspended in PBS before analysis by BD FACS Calibur; data were acquired and analyzed with CellQuest software.MM cell suspension . Proteins were blotted onto polyvinylidene difluoride membranes . Immunoblotting was done after blocking with 5% non-fat milk with the following primary antibodies: anti-Merlin mAb D1D8 and anti-p53 (7F5) , anti-p53 (DO-1), and anti-pan-Cytokeratin (H-240) (Santa Cruz), anti-Vinculin mouse, anti-Merlin (phospho-S518) rabbit , anti-p16 (G175–405) and anti-Calretinin mouse . Bands were detected using fluorescently-labeled secondary antibodies  according to the manufacturer's recommendations. The membranes were subsequently scanned with an Odyssey Infrared Imager (LiCor Biosciences) and converted to greyscale.Cells were lysed for 20 min on ice in lysis buffer ), briefly sonicated and the resultant lysate clarified by centrifugation at 1000 × Immunohistochemistry of formalin-fixed, paraffin-embedded tissues from human explants was performed with the Histostain-Plus detection System according to the manufacturer's protocol (Life Technologies). Sections were dewaxed and rehydrated, and antigen retrieval was performed by heating the slides in 10 mM citric acid buffer (pH 6.0) for 15 min. Sections were incubated overnight at 4 °C in a humidified chamber with a 1 : 10 dilution of antibody to p16 (BD) and 1 : 50 NF2 (Santa Cruz). Control IHC experiments (data not shown) were performed without primary antibody. All sections were counterstained with Gill's haematoxylin and mounted for digital slide scanning.For immunofluorescence, isolated cells were first fixed in 4% paraformaldehyde and where indicated permeabilized with 0.1% Triton in PBS-Tween (0.1% vol/vol), prior to blocking in 5% goat serum in PBS-Tween. Cells were then incubated with primary antibody 1 : 50 anti-Podoplanin (Santa Cruz) without prior permeabilization; 1 : 50 anti-Calretinin  overnight at 4 °C. Cells were washed three times for 10 min each with 1 × PBS, incubated for 1 h with secondary antibody (in blocking solution), washed three times with 1 × PBS, and counterstained with DAPI and mounted for confocal microscopy.Total RNA was extracted by TRIzol (Life Technologies). Hybridization to 60 K whole human genome microarray gene expression chips was conducted following manufacturer's protocol . In brief, total RNA samples were Cy3-labeled using Agilent Low Input Quick Amp 1-colour Labelling Kit. The level of dye incorporation was evaluated by spectrophotometry . Labeled RNA was then fragmented and diluted (v/v) in hybridization buffer. Hybridization to 60 K high-density oligonucleotide microarray slides was performed in a microarray hybridization oven overnight at 65 °C. Following hybridization, the slides were rinsed and immediately scanned using a DNA Microarray Scanner .P<0.05) in mRNA expression compared to normal mesothelial cells were included in subsequent analyses. These were subjected to Anova unequal variations test with Benjamin–Hochberg corrections. Significant (P<0.05) changes were subjected to hierarchical clustering with average linkage. The clustered heat-map was visualized using GeneSpring. Significantly highly represented networks were identified using Ingenuity Pathway Analysis software . The PCA  was performed for the three most changing components to identify the principal directions in which the mRNA changes varied. The data were projected on three axes (components), ordered by decreasing significativity; the first principal component explains 20.2% of the variance, the second explains 14.99% and the third explains 14.45% of the variations in expression. GO term Enrichment Analysis was conducted as described previously.50The raw data were uploaded into Agilent's GeneSpring Software, normalized and fold changes calculated. For each cell line the probes with an absolute twofold-change  according to the manufacturer's instructions. An assay based on the paralogue ratio test and real-time PCR was used to determine the DNA copy number as described previously.μl reaction contained 3.4 μl of gDNA (10 ng), 0.3 μl of each of the two primers required for each loci (20 pM), 0.2 μl of each of the probes required (50–200 nM), and 5 μl of Genotyping MasterMix (Applied Biosystems). The PCRs began with 2 min at 50 °C, 10 min at 95 °C, followed by a cycle consisting of 15 s at 95 °C and 60 s at 60 °C. Fifty cycles were performed. CT values were determined using the automatic threshold settings. gDNAs from diploid control  and normal mesothelial cells were used as controls. The ΔCT was calculated for each of the reactions using the following equation: CT gene 1 (gene of common DNA loss)−CT gene 2 (reference)=ΔCT. The mean ΔCT from at least three reactions was used for subsequent analysis. The relative quantification of copy number was calculated using the 2−ΔΔCT value. Primers and probes are listed below:Real-time PCRs were performed on the ABI 7500 Fast system . Each 10-Rates of oxidative phosphorylation and glycolysis were determined by measuring the rate of OCR and extracellular acidification rate (ECAR), respectively, using a Seahorse XF24 extracellular flux (XF) analyser .4 cells/well 24 h before the assay and incubated at 37 °C under 5% CO2 in a humidified atmosphere. Primary lines were seeded on gelatin coated microplates. 24 h prior to assay, 1 ml of Seahorse calibrant was added to each well of a Seahorse XF24 utility plate and the probes on the sensor cartridge were allowed to soak at 37 °C under CO2-free conditions. 1 h prior to assay, cells were washed 3 × in unbuffered bicarbonate and serum-free DMEM (pH 7.4) containing 1 mM sodium pyruvate, 11 mM glucose and 2 mM glutamax; then covered in 675 μl of the same media, and incubated for 1 h at 37 °C under CO2-free conditions. The cell microplate was then loaded into the Seahorse XF24 and assayed to measure basal OCR and ECAR.Commercially available and primary MM cell lines were seeded in a XF24 cell culture microplate at 5 × 10μg/ml in PBS)  was added to semi-confluent actively dividing cultures 4 h before harvesting. Metaphase cells were harvested by trypsinizing, treated with a hypotonic solution (deionised water) for 15 min at 37 °C and fixed twice in acetic acid (one part)/methanol (three parts). Cells were dropped onto clean slides, heat-aged, treated with trypsin and stained with Leishman stain to produce G-banded chromosomes. In total, 4–10 metaphase spreads were analyzed from each culture, depending on availability, for numerical and structural rearrangements.Chromosome preparations of the cells were made as follows: Colcemid (Gibco Invitrogen, Paisley, UK; 10


This study is to investigate which Toll Like Receptors (TLRs) are expressed on human ovarian cancer cell lines and to evaluate the effect of TLR expression on the growth of ovarian cancer cells.Four human ovarian epithelial carcinoma cell lines SKOV3 (clear), OVCAR3 (serous papillary), SNU8 (serous papillary) and SNU251 (endometrioid) were used for the experiments. To find whether the expression of TLRs is functional on human ovarian cancer cell lines, cytokines  were investigated. For statistical analysis, one-way ANOVA with Tukey's multiple comparison post-test was carried out.In SKOV3 cell line, TLR 1-7 were expressed and among them, TLR-2, -3 and -5 have strong intensity; however, TLR 9 was absent and TLR 8 was weak. In OVCAR3 cell line, TLR 3 and TLR 5 were much more intense, and TLR-1, -2, -4, -7 and -8 were weak, while TLR 6 and TLR 9 were absent. Unlike other cell lines, SNU8 has strong intensity through TLR 1-9 although TLR9 was a little weaker than the others. In SNU251 cell line, TLR 1-5 and TLR 7-8 were expressed and TLR 6 and TLR 9 were absent or weak. The exposure of cells to ligands for TLR 1-9 showed significant upregulation of IL-6, especially in TLR 5. IL-10 after stimulating TLR 5 was significantly increased in SKOV3 and OVCAR3. Another proinflammatory cytokine TNF-α was assessed, and the result that only TLR 5 in OVCAR3 has a significant induction. IL-12p70 on TLR 9 was induced in the four cell lines but the result didn't show any significance and IFN-γ was induced insignificantly in SKOV3 and OVCAR3 for TLR 9 only.The expression and biologic function of TLRs are different in each ovarian cancer cell line. In order to target TLRs for the management of ovarian cancer, further studies are needed on the effects of TLRs in tumor development.


MAPK7/ERK5  functions within a canonical three-tiered MAPK (mitogen activated protein kinase) signaling cascade comprising MEK (MAPK/ERK kinase) 5, MEKK(MEK kinase) 2/3 and ERK5 itself. Despite being the least well studied of the MAPK-modules, evidence supports a role for MAPK7-signaling in the pathology of several cancer types.in situ hybridization (FISH) analysis identified MAPK7 gene amplification in 4 % (3/74) of non-small cell lung cancers (NSCLC) (enriched to 6 % (3/49) in squamous cell carcinoma) and 2 % (2/95) of squamous esophageal cancers (sqEC). Immunohistochemical (IHC) analysis revealed a good correlation between MAPK7 gene amplification and protein expression. MAPK7 was validated as a proliferative oncogenic driver by performing in vitro siRNA knockdown of MAPK7 in tumor cell lines. Finally, a novel MEK5/MAPK7 co-transfected HEK293 cell line was developed and used for routine cell-based pharmacodynamic screening. Phosphorylation antibody microarray analysis also identified novel downstream pharmacodynamic (PD) biomarkers of MAPK7 kinase inhibition in tumor cells (pMEF2A and pMEF2D).Fluorescence Together, these data highlight a broader role for dysregulated MAPK7 in driving tumorigenesis within niche populations of highly prevalent tumor types, and describe current efforts in establishing a robust drug discovery screening cascade.The online version of this article (doi:10.1186/s12885-015-1455-y) contains supplementary material, which is available to authorized users. MAPK7 gene [Mitogen activated protein (MAP) kinase signaling pathways are highly evolutionarily conserved throughout eukaryotes and represent a key mechanism in the transduction of intracellular signals. Of the four MAP kinases which exist in mammalian cells  3, commonly detected in advanced breast cancer , 16. TheC tumors . In the MAPK7 and protein overexpression in clinical samples of non-small cell lung cancer (NSCLC) and esophageal cancer (EC) of Asian origin, using array comparative genomic hybridization (aCGH) and FISH (fluorescent in-situ hybridization) technologies. Importantly, by suppressing expression within MAPK7 amplified cell lines, we were able to validate MAPK7 as a driver of tumor cell proliferation and engineer a stable cell line assay for screening of candidate MAPK7 small molecule kinase inhibitors. Lastly, using reverse-phase protein chip arrays, our work identified potential pharmacodynamic biomarkers of MAPK7 kinase inhibition within MAPK7-amplified tumor cell lines. In summary, the work here identifies and validates a novel role for dysregulated MAPK7 as a tumor driver in clinical samples of NSCLC and EC, and outlines aspects of preliminary work in developing a drug discovery programme to identify novel small molecule inhibitors of MAPK7 kinase activity.In the work reported here, we identified genetic dysregulation of in situ hybridization (FISH) analysis identified high level MAPK7 gene amplification in 4 % (3/74) of NSCLC (enriched to 6 % (3/49) in squamous cell carcinoma) and 2 % (2/95) of sqEC  analysis of the same NSCLC tissue samples was performed. Analysis revealed that all 3 MAPK7 amplified cases had corresponding high level MAPK7 protein expression (defined as IHC3+), suggesting a good correlation of MAPK7 gene amplification with high level protein expression  and a MAPK7 ‘normal’ cell line (NCI-H1793), were selected for study. Detailed MAPK7 gene copy number and MAPK7/CEP17 ratios for these cell lines are tabulated in Additional File MAPK7 gene amplification in cell lines KYSE30 and SNU449, but not in NCI-H1793  to silence MAPK7 gene expression. Cell lines were transfected over a 4-day period and cell lysates were taken for Western blot analysis of MAPK7 protein expression  with MAPK7 in CHO-K1 cells and demonstrated the kinase specificity of MEK5 via direct phosphorylation and activation of MAPK7 [50) were determined for all four agents  to induce direct cellular activation of MAPK7, this effect is only reliably detectable using a non-quantitative gel-shift assay . Using aof MAPK7 . Taking of MAPK7 . This conts Fig. .Fig. 4DeTo enable more comprehensive characterisation of MAPK7-pathway signaling, we sought to identify cellular pharmacodynamic biomarkers using a reverse-phase protein array (RPPA) platform. Protein extracts were prepared from KYSE30 cells, treated (and untreated) for 2 h with 10 μM XMD8-92, and then applied to the antibody microarray. Quality control and data normalization was performed using a standardized and validated antibody set and actual array chip images are shown in Fig. MAPK7 amplification and high level protein expression (IHC3+), we also identified a further 12 cases with IHC3+ protein staining in the absence of MAPK7 amplification, raising the question of whether MAPK7 gene amplification, protein overexpression, or both, are required for driving tumor proliferation. Despite our cell lines showing a good correlation between MAPK7 gene copy number/ratio and protein expression . Although our data demonstrate coordinate MAPK7 gene amplification and protein overexpression, it should be noted that previous studies have identified additional mechanisms through which MAPK7 activity can be regulated [Dysregulated MAPK7 signaling has been demonstrated to play key roles in uncontrolled cell proliferation across several tumor types , 17, 19.egulated , 24. Detegulated .MAPK7 in two dysregulated cell lines confirmed a driving role for MAPK7 in tumor cell proliferation in vitro. Interestingly, although a good correlation was observed between MAPK7 gene amplification and protein expression in both cell lines, targeted knockdown had slightly different functional effects. Partial knockdown of MAPK7 expression in the SNU449 cell line resulted in anti-proliferative activity but no appreciable increase in cell death above control levels. In contrast, near-complete suppression of MAPK7 expression in the KYSE30 cell line resulted in anti-proliferative activity and significant increases in cell death above background levels. It is currently unclear whether this difference was attributable to variation in the degree of siRNA MAPK7 suppression, or perhaps due to the cellular background and pathway ‘wiring’. Importantly however, significant suppression of MAPK7 expression and a lack of any effect on cell proliferation or cell death in the non-dysregulated MAPK7 cell line, NCI-H1793, confirmed a role for dysregulated MAPK7 in driving uncontrolled cell proliferation. The relatively benign effect of MAPK7 suppression in the MAPK7 ‘normal’ NCI-H1793 cell line is consistent with the results of previous inducible genetic MAPK7 knockout studies in adult mice, which despite perturbing vascular integrity, had no wider effects on the whole animal [Targeted siRNA knockdown of e animal .D = 80nM), followed by DCAMKL2 (KD = 190nM), TNK1 (KD = 890nM) and PLK4 (KD = 600nM) [MAPK7 gene knockout work described above, in vivo studies using XMD8-92 did not lead to any vascular abnormalities, supporting the concept that targeting MAPK7 kinase activity (as opposed to whole gene knockout) may be a viable therapeutic strategy. Our identification of MEF2A and MEF2D (‘myocyte enhancer factors’) as novel pharmacodynamic biomarkers of MAPK7 inhibition is notable. These transcription factors bind and activate the transcription of numerous muscle-specific, growth-factor and stress controlled genes involved primarily in (but not limited to) skeletal and cardiac muscle development. Interestingly however, oncogenic activity has been attributed to MEF2D fusion proteins in acute lymphoblastic leukaemia [To facilitate validation of our cell-based MAPK7 pharmacodynamic ELISA assay and provide a positive control in our biomarker screen, XMD8-92 served as an excellent tool compound due to its highly selective nature. Profiling of this agent against a panel of 402 diverse kinases identified MAPK7 as the most potently inhibited kinase  . Notablyeukaemia , underlieukaemia . AlthougMAPK7 expression in clinical samples of squamous cell lung and esophageal carcinoma. Using MAPK7 siRNA, we validate a role for dysregulated MAPK7 in driving cell proliferation in established tumor cell lines and describe the development of a cell based ELISA assay to support screening of novel MAPK7 kinase inhibitors. Finally, we highlight the identification of several putative novel pharmacodynamic biomarkers of cellular MAPK7 signaling. This data affirms MAPK7 as an attractive therapeutic oncology target and outlines aspects of preliminary work in developing a drug discovery program to identify novel small molecule inhibitors of MAPK7 kinase activity.Taken together, our data provide the first reported incidences of dysregulated 74 NSCLC and 95 sqEC cancer clinical samples were collected from Beijing tumor hospital  with signed patient informed consent. This study was approved by the ethics committee of the Beijing tumor hospital. Cell lines were obtained from commercial sources  or from AstraZeneca internal cell banks.The MAPK7 FISH probe was generated internally by directly labeling BAC (CTD-2387H16) DNA with Spectrum Red . The CEP17-Spectrum Green probe  for the centromeric region of chromosome 17 was used as internal control.FISH assays were performed on 4 μm dewaxed and dehydrated FFPE sections. The SpotLight Tissue pretreatment Kit  was used for pretreatment . Sections and probes were codenaturated at 80 °C for 5 min and then hybridized at 37 °C for 48 h. After a quick post wash off process , sections were finally mounted with 0.3 μg/ml DAPI , and stored at 4 °C avoiding light for at least 30 min prior to scoring.4/well in 96-well plate or 2 × 105/well in 6-well plate) were transfected with 40 nM of MAPK7 siRNAs (Sigma) or non-silencing siRNAs (Dharmacon) as control. The confluence of cells reflecting cell growth was monitored by Incucyte (Essen) over time after siRNA treatment. At 96 h post transfection, the cells were staining with final concentration of 1.5 μM of propidium iodide  and 10 μM of Hoechst , and the survival rate of cells was detected by Acumen X3 (TTP LabTech).In vitro siRNA transfection was performed using HiPerFect transfection reagent (Qiagen) following the manufacturer’s protocol. Briefly, cells -conjugated secondary antibody for 1 h at room temperature. Signals were visualized using Amersham ECL detection reagents . The primary antibodies included: MAPK7 antibody , pMAPK7 (T218/Y220) , pMEF2A (T312) , MEF2A , and HA .The constitutively active MEK5 (MEK5CA) and MAPK7 plasmids were constructed as published . HEK293 Co-transfected HEK293 cells (described above) were aliquotted and seeded at a density of 10,000 cells/100 μl/96-well for 4 h, and then treated with various concentrations of XMD8-92 for 2 h at 37 °C in a cell incubator (Thermo). After washing out cell culture medium, 110 μl/well of RIPA-cocktail buffer was added to lyse the cells overnight. On the same day, 96-well NUNC plates  were coated with 100 μl/well of MAPK7 capture antibody  at 1 μg/ml at 4 °C overnight. Next day, plates were then washed with buffer (PBS containing 0.05 % Tween20) 3 times, and blocked with 2 % BSA-wash buffer for 1 h. Cell lysates were then added (100 μl/well) for 2 h, followed by washing 3 times. pMAPK7 (T218/Y220) detection antibody  was added at 5 μg/ml for 2 h, and plates then washed before incubating with secondary antibody  for 1 h. Plates were given a final wash, enhancement solution added  for 30 min, before reading on the Envision reader(PerkinElmer).http://www.fullmoonbio.com/product/phospho-explorer-antibody-array/). KYSE30 cells (2 × 107 cells per 10-cm plate) were treated with or without 10 μM XMD8-92 for 2 h. Cells were washed with cold 1X PBS (4 °C) and collected by scraping from the plate. Cells were spun briefly in a microcentrifuge to remove culture media from the cells. Three additional washes were performed using cold 1X PBS (4 °C). Cells were centrifuged again at 4 °C and supernatant discarded. Cell pellets were shipped to the vendor and antibody microarray’s run according to the manufacturer’s instruction. Duplicate chips were run for both control and treated lysates groups and ‘% of control’ values calculated according to normalised average plate readings.The phosphorylation explorer antibody microarray chip PEX100, containing 1,318 well-characterized site-specific antibodies, was purchased from Full Moon Biosystems . Details of these antibodies can be accessed via the vendors website (


There is an urgent need for better therapeutics in head and neck squamous cell cancer (HNSCC) to improve survival and decrease treatment morbidity. Recent advances in high-throughput drug screening techniques and next-generation sequencing have identified new therapeutic targets in other cancer types, but an HNSCC-specific study has not yet been carried out. We have exploited data from two large-scale cell line projects to clearly describe the mutational and copy number status of HNSCC cell lines and identify candidate drugs with elevated efficacy in HNSCC.et al., and Barretina et al., were analyzed. Data from Garnett et al. was interrogated for relationships between HNSCC cells versus the entire cell line pool using one- and two-way analyses of variance (ANOVAs). As only seven HNSCC cell lines were tested with drugs by Barretina et al., a similar analysis was not carried out.The genetic landscape of 42 HNSCC cell lines including mutational and copy number data from studies by Garnett 50 values, suggesting higher drug sensitivity, in HNSCC lines versus non-HNSCC lines. Furthermore, the PI3K inhibitor AZD6482 demonstrated significantly higher activity (as measured by the IC50) in HNSCC cell lines harbouring PIK3CA mutations versus those that did not.Recurrent mutations in human papillomavirus (HPV)-negative patient tumors were confirmed in HNSCC cell lines, however additional, recurrent, cell line-specific mutations were identified. Four drugs, Bosutinib, Docetaxel, BIBW2992, and Gefitinib, were found via multiple-test corrected ANOVA to have lower ICHNSCC-specific reanalysis of large-scale drug screening studies has identified candidate drugs that may be of therapeutic benefit and provided insights into strategies to target PIK3CA mutant tumors. PIK3CA mutations may represent a predictive biomarker for response to PI3K inhibitors. A large-scale study focused on HNSCC cell lines and including HPV-positive lines is necessary and has the potential to accelerate the development of improved therapeutics for patients suffering with head and neck cancer. This strategy can potentially be used as a template for drug discovery in any cancer type.The online version of this article (doi:10.1186/2050-6511-15-66) contains supplementary material, which is available to authorized users. Despite advances in multi-modal treatment of head and neck squamous cell carcinoma (HNSCC), mortality rates for advance disease remain high . Thus thTargeted therapy has become an important method in personalizing treatment for cancer patients based on the genetic mutations present in their tumor(s). Such therapies enable the use of drugs to specifically target molecules within the tumor that are responsible for the malignancy. A search of the literature, as well as clinical trials that are currently underway in HNSCC, revealed a variety of agents being investigated that target various cellular molecules  . However, despite the multiple trials, only EGFR tyrosine kinase inhibitors and EGFR monoclonal antibodies (e.g. cetuximab) have been approved for clinical use and demonstrate only modest activity in a subset of patients [Cell lines are imperfect models of cancer: they tend to be generated from more aggressive, often metastatic tumors, can demonstrate genetic and epigenetic changes relative to the parent tumors, and lack interactions with the surrounding stroma and immune system –7. HowevBRAF inhibitors in BRAF mutant cell lines) and identified novel associations such as the marked sensitivity of Ewing’s sarcoma cells harboring the EWS-FLI1 gene translocation to poly(ADP-ribose) polymerase (PARP) inhibitors [The advent of next generation sequencing has allowed complete, affordable and rapid genomic characterization of both patient samples and of cell lines. In parallel, the development of high-throughput robotic drug screening platforms has facilitated the rapid testing of a large number of drugs. Together these techniques provide the ability to correlate mutation status, copy number variation and expression levels with drug response. Two recent, large-scale studies, involving hundreds of cell lines of different tissue types , 9 have hibitors . Howeveret al.) included 31 HNSCC cell lines , seven of which were screened with 24 anticancer agents [et al. [et al., were simply reported as 0 (deletion), between 0 and 8 , and greater than 8 (amplification). Barretina et al. reported CNVs as continuous variables, relative to control genes with 0 considered “non-amplified”. We considered values greater than 2 (reflecting at least 2 extra gene copies) as amplifications and less than -2 (representing homozygous deletion) as this appeared to agree with the TCGA data from http://cbioportal.org and correspond best with the amplifications and deletions noted in the study by Garnett et al. , we restricted drug sensitivity analysis to the data from Garnett et al. [50 in μM) for each drug between HNSCC cell lines and non-HNSCC cell lines via one-way analysis of variance (ANOVA) as computed through t-tests. Specifically, for each drug i, cell lines were partitioned into two groups j = {HNSCC, non-HNSCC} as per their cell-line type. Letting k denote replicate number, the linear model for each t-test was the standard yijk = μj + εijk, where yijk represents the observed log2(IC50), μj represents the mean response of group j, and each εijk represents a realization of p-values using Strimmer’s fdrtool [p-value” [yijk = μ + αi + βj + γij + εijk where group i = {copy-number unchanged, copy-number changed}, group j = {wild-type, mutant}, and k again denotes replicate number. As per standard ANOVA, αi and βj represent the mean additive responses of their respective groups, γij represents any non-additive interaction effect, εijk represents a realization of ε ~ N, and μ represents the grand-mean effect. The standard constraints ∑iαi = 0, ∑jβj = 0, and ∑ijγij = 0 were used to ensure that all parameters of each model were identifiable.Due to the small number of HNSCC cell lines that were treated with drugs in Barretina t et al. . All stat et al.  version  fdrtool , 12. Thep-value” , 14. A LTP53 , MYC and PIK3CA. A complete listing of mutations identified in HNSCC cell lines in Barretina et al. is provided in Additional file TP53, which was the most commonly mutated gene found in tumor samples  (insertion of adenine at position 112832307 (dbSNP ID: rs117108942) and deletion of cytosine at position 112832340) in virtually every HNSCC cell line in Barretina et al. Of note, 11 cell lines with the same name were characterized in both studies. However, two of these lines had significant discrepancies in terms of mutations between the studies  bringing the true identities of the lines into question  and CDKFour chemicals, Docetaxel (anti-mitotic chemotherapy), Bosutinib (combined SRC/ABL inhibitor), Afatinib (an EGFR and HER2 inhibitor), and Gefitinib (an EGFR inhibitor) were found to have significantly increased activity in HNSCC cell lines compared with the remainder of the cell line pool Table . Two druet al. and Garnett et al.  in the cell lines to allow analysis. It should be noted that not all cell lines were treated with every drug and some genetic changes occurred in a very small number of cell lines, which resulted in exclusion of analysis of certain drugs with a particular genetic change. Of the 131 drugs tested, three were PI3K inhibitors including AZD6482, GDC0941, and the combined mTOR and PI3K inhibitor NVP-BEZ235. We calculated a robust increase in sensitivity to AZD6482, explainable by the interaction of PIK3CA mutation status and HNSCC cell-line type . There were too few PIK3CA amplified cell lines to examine the effect of amplification alone on drug response, however when these were pooled with the PIK3CA mutant lines, no drugs were found to be preferentially active when compared to PIK3CA wild-type cell lines  demonstrated a trend towards selective inhibition of PIK3CA HNSCC mutant cell lines  inhibitors, have been approved for use in head and neck cancers . Despiteet al. and Barretina et al. [FGFR1, CCND1, MYC, EGFR), deletions (e.g. PTEN), activating mutations (e.g. PIK3CA) and fusions (e.g. FGFR3/TACC3) were observed. Despite a relatively limited number of cell lines, our HNSCC-specific reanalysis of these studies shows that the spectrum of mutations observed in HNSCC cell lines is similar to that of primary HNSCC patient samples [TP53 are frequent, while activating mutations in oncogenes  occur at lower frequencies.By pairing next-generation sequencing of cell lines with high-throughput drug screening techniques, the impressive studies by Garnett a et al. , 9, conf samples , 16. OveTP53, which is the most commonly mutated gene found in patient tumor samples  highlights the importance of comparing tumor sample or cell line DNA, to matched-normal tissue. Moving forward, it is crucial that matched-normal tissue and blood samples, representing the germ-line genetic profile are obtained when cell lines are established for improved sequencing analyses.As mentioned, our analysis revealed 22 genes that are more frequently mutated than ile NCAM1. Intereset al. [PIK3CA mutations appeared to be more frequent in three HPV-positive tumors sequenced by Stransky et al., and we confirmed this finding by sequencing the PIK3CA hotspots in a larger number of HPV-positive (46) and negative (43) oropharyngeal HNSCC samples [PIK3CA mutations occurred in 28% of the HPV-positive oropharyngeal tumors versus 10% of the HPV-negative samples, confirming that this is an important therapeutic target in HNSCC. There is great interest in targeting PI3K as it is frequently either amplified or mutated in a large variety of human cancers [The most obvious directly druggable target in HNSCC appears to be the PI3K pathway, which was mutated in 8% of the patient samples examined by Stransky et al. . We note samples . We foun cancers , howeverPIK3CA mutant lines. However, it is somewhat surprising that AZD6482 was the most tightly correlated with PIK3CA mutation status as it has approximately an 87-fold higher affinity for PI3Kβ than PI3Kα [in vitro activity, much less in vivo efficacy. As well, compounds such as NVP-BEZ235, a PI3K/mTOR inhibitor have been previously found to have selective activity in PI3KCA-mutant HNSCC cell lines and patient-derived xenografts (PDX) [PI3KCA mutant cell lines in these studies, reinforcing the importance of identifying the most promising preclinical candidates that have the strongest correlation with genomic changes to take forward into clinical trials.Cell lines are not perfect models of cancer due to their lack of three-dimensional stromal environment, lack of interactions with an immune system, and an inability to test drug delivery issues. Many of these concerns can be overcome in xenografts and human testing. The utility of cell line drug screening, given that poor correlation of drug testing in matched cell lines and patients have variably been observed , 20 whilan PI3Kα . It is nan PI3Kα , 25 indits (PDX) . HoweverPIK3CA mutant and amplified cell lines as well as tumor xenografts [et al. [PIK3CA amplifications and/or mutations including 8 HNSCC patients, six of which had stable disease and two had partial responses (25%) [http://clinicaltrials.gov/ct2/show/NCT01602315). BYL719 is an excellent example of a molecule initially identified in studies using cell lines, its efficacy confirmed in PDX models and results of the first Phase I clinical trials are now in the literature [There are two highly specific PI3Kα inhibitors, GDC-0032 (Genetech) and BYL719 (Novartis) that have demonstrated superior inhibition of nografts , 28. Ind [et al. , 28. Theterature , 30. ThiEGFR inhibitors have been integrated into routine clinical care for HNSCC patients based on the landmark Bonner study . EGFR isEGFR mutations, while resistance in colon cancer is mediated by downstream KRAS mutations [The mechanisms of sensitivity and resistance to EGFR inhibitors in HNSCC are poorly understood . In lungutations . These gutations , and EGFutations , 31. IndWhile EGFR amplification was not correlated with EGFR inhibitor efficacy, it was associated with increased response to the PI3K inhibitor AZD6482 and JNK inhibitor VIII. EGFR signalling is mediated through several pathways including PI3K and JNK , 33. As et al. only screened drugs to a maximum of 8 μM, so many resistant cell lines are merely listed as having an IC50 of 8 μM. In contrast, Garnett et al. screened cell lines up to 3 orders of magnitude higher (10 mM). However, if we compare cell lines based on their characterization of resistance (IC50 above 8 μM) in their respective study, we find that the majority of cell line IC50 values were concordant between the two studies  that could be tested. Another limitation is that not all drugs were tested in all cell lines. However, despite these limitations as well as discrepancies in drug responses described by Papillon-Cavanagh and colleagues , when coAn additional limitation of our analysis is that all of the cell lines utilized were HPV-negative to the best of our knowledge . Given tThe disagreements noted in the cell line sensitivities and mutations between the two studies have significant implications for future work of this type. They are many factors that can explain these discrepancies, however the most likely is that the identically named lines are in fact different, despite the fact that genotyping was completed in both studies. Other possible sources of disagreements in the data are differences in screening techniques, different drug concentrations, and different statistical models to calculate IC50 values from the dose–response curves. Ideally, the genotyping data can be compared to determine the discrepancies and provide the definitive genotype for cell lines. We also suggest that a standard methodology of cell line drug screening needs to be developed to allow external validation of future findings.High throughput drug screening of molecularly characterized HNSCC cell lines has the potential to rapidly identify promising agents to improve therapies for patients suffering with head and neck cancer. An expanded HNSCC specific study including HPV-positive cell lines has the potential to identify effective agents, as well as mechanisms of resistance and sensitivity to molecular agents.No ethics approval was required as this present work represents an investigation and analysis of publicly accessible data.et al. and Barretina et al. Legend: CNV - relative copy number variation, Disagreements highlighted in bold. (XLSX 46 KB)Additional file 1: Table S1: Comparison of mutations noted in the studies by Garnett et al., Nature 2012. Legend: Del - deletion, Ins - insertion, 5′ UTR - 5′ untranslated region, 3′ UTR - 3′ untranslated region, SNP - single nucleotide polymorphism. (XLSX 231 KB)Additional file 2: Table S2: Complete list of mutations found in the HNSCC cell lines in Barretina et al., Science 2011, **Most frequently mutated genes identified in cell lines in Barretina et al., Nature 2012. (XLSX 68 KB)Additional file 3: Table S3: Comparison of mutations noted in head and neck squamous cell tumors and cell lines. Legend: *Significantly mutated genes found in patient tumor samples from Stransky Additional file 4: Table S4: Associations of drug response and HNSCC cell line type versus non-HNSCC cell lines. Legend: t - t statistic calculated by ANOVA, p - p value calculated by ANOVA, LFDR - local falsed discovery rate, Effect - differences in the Log2(IC50(μM)) for HNSCC versus non-HNSCC cell lines, 95% lower limit and 95% upper limit delineate confidence intervals for the effect size. (XLSX 63 KB)Additional file 5: Table S5: Comparison of drug sensitivities in cell lines common to the studies by Garnett et al. and Barretina et al. (DOCX 16 KB)50(μM)) for wild-type cell lines, Other: Log2(IC50(μM)) for mutated and/or amplified cell lines, Effect: differences in the Log2(IC50(μM)) for WT/nor and mut/amplified cell lines, 95% lower limit and 95% upper limit delineate confidence intervals for the effect size, LFDR: Local false discovery Rate. (XLSX 120 KB)Additional file 6: Table S6: Associations of mutations and copy number alterations with drug response in HNSCC cell lines. Legend: wt - wild type, nor - normal (non-amplified), mt - mutation, amp - amplified, del - deletion, t - t statistic calculated by ANOVA, p - p value calculated by ANOVA, Wild-type: Log2(IC


Moringa oleifera leaves, bark and seed extracts. When tested against MDA-MB-231 and HCT-8 cancer cell lines, the extracts of leaves and bark showed remarkable anti-cancer properties while surprisingly, seed extracts exhibited hardly any such properties. Cell survival was significantly low in both cells lines when treated with leaves and bark extracts. Furthermore, a striking reduction (about 70–90%) in colony formation as well as cell motility was observed upon treatment with leaves and bark. Additionally, apoptosis assay performed on these treated breast and colorectal cancer lines showed a remarkable increase in the number of apoptotic cells; with a 7 fold increase in MD-MB-231 to an increase of several fold in colorectal cancer cell lines. However, no significant apoptotic cells were detected upon seeds extract treatment. Moreover, the cell cycle distribution showed a G2/M enrichment (about 2–3 fold) indicating that these extracts effectively arrest the cell progression at the G2/M phase. The GC-MS analyses of these extracts revealed numerous known anti-cancer compounds, namely eugenol, isopropyl isothiocynate, D-allose, and hexadeconoic acid ethyl ester, all of which possess long chain hydrocarbons, sugar moiety and an aromatic ring. This suggests that the anti-cancer properties of Moringa oleifera could be attributed to the bioactive compounds present in the extracts from this plant. This is a novel study because no report has yet been cited on the effectiveness of Moringa extracts obtained in the locally grown environment as an anti-cancer agent against breast and colorectal cancers. Our study is the first of its kind to evaluate the anti-malignant properties of Moringa not only in leaves but also in bark. These findings suggest that both the leaf and bark extracts of Moringa collected from the Saudi Arabian region possess anti-cancer activity that can be used to develop new drugs for treatment of breast and colorectal cancers.In this study we investigated the anti-cancer effect of  Moringa oleifera L (MO) (Family: Moringaceae) is a perennial angiosperm plants, which includes several other species [et al (2015) [ species , 2. It i species , 4. The  species –9. Additl (2015) .Moringa oleifera extracts from leaves (MOL), bark (MOB), and seeds (MOS) to observe its efficacy as an anti-cancer agent on breast and colorectal cancers. To elucidate the effectiveness of these extracts we analyzed cell motility and clonogenic survival assays to assess the phenotypic changes in MDA-MB-231(breast) and HCT-8  cancer cell lines. To elaborate our study further, we also analyzed the cell survival, apoptosis, and cell cycle progression of these two lines after challenging them with the extracts of MO as stated above. The rationale behind selecting these lines are; easy access to availability and more importantly, prevalence of these cancers in a major population of the Kingdom of Saudi Arabia.In this study we focused upon the effect of Colorectal cancer is the third most lethal cancer worldwide. Both genders, male and female are equally affected by this deadly disease. In the past year about 140,000 people were diagnosed with colon cancer in the United States and the estimated survival is 50% or less. FurtherThe important characteristic features of cancer cells include the ability to proliferate, invade through the extra cellular matrix and migrate to other body parts to form secondary tumors. The migration of cancerous cells is dependent on the tumor micro environment from where they get nourishment and support by forming new-vasculature  and allowing them to spread . It is aAs reported earlier, about 74% of the known anti-cancer medicines are derived from various plant species , 17. IndLycopene is a carotenoid compound abundant in tomatoes and also present in tomato products . As repoMoringa oleifera grown in the kingdom of Saudi Arabia, against breast and colorectal cancers. The importance of this study is that this locally grown plant has not been previously tested as an anti-cancer agent. Hence, the novelty of our research is that we have tested not only leaf extracts, but also the bark and the seed extracts against two cancer cell lines, which has not been studied in the past.In this study we have shown the remarkable effects of Moringa leaves and bark on MDA-MB-231 and HCT-8 cancer cell lines. Moringa is a common vegetable used by inhabitants of tropical and sub-tropical countries. The extracts of leaves and bark tested in our current study induced a significant level of apoptosis as well as G2/M enrichment in breast and colorectal cancer cell lines. In addition, a remarkable change in the normal phenotypic properties of the cells was also observed. However, surprisingly, we did not observe any significant changes in cell lines exposed to Moringa seed’s extract. Furthermore, when GC-MS analyses of these extracts were performed, we observed considerable amounts of bioactive components present in the extracts of leaves and bark, which we have describe elaborately in the results section. The effective role of these compounds present in the extracts as an anti-cancer agent is well documented –32. TherMoringa oleifera were collected from the city of Tabuk, Kingdom of Saudi Arabia. Geographically the latitude of the city is 28° 22`60N and longitude is 36° 34`60E on degree minutes second (DMS) unit. Due to the ease of cultivation and abundance, it does not fall under the endangered plant species and hence safe to use for research purpose. (http://faculty.ksu.edu.sa/74413/Pages/Endangeredplants.aspx). Additionally, the parts of the plant used in this study are the gift of one of the co-authors (SMA) of this manuscript obtained from his garden plants. The study was performed in the research center, Prince Sultan Military Medical City, Riyadh.Leaves (L), bark (B), and seeds (S) of The extractions were performed using Soxhlet apparatus. About 60g of dried leaves, bark, and seeds were grinded separately into coarse powder. Each of the coarsely powdered specimens was taken in round bottom flask and 600 ml of ethanol was added. The extraction was continued for 6–8 h until all the soluble constituents dissolved in the solvent. The soluble extracts were filtered and evaporated in rotary evaporator  to yield semi solid masses. Extracts thus obtained, were collected and stored at 4°C until further use. For this study 250mg of extracts were dissolved in 1.0 ml of ethanol and filtered through a 0.22μM filter. The sterile extracts were always used in a cell culture hood under aseptic conditions.−1 in split less mode. The injector temperature was adjusted at 250°C, while the detector temperature was fixed to 280°C. The column temperature was kept at 70°C for 1 min followed by linear programming to raise the temperature from 70° to 200°C (at 8°C min−1 with 2 min hold time), and 200°C to 250°C (at 10°C min−1 with 2 min hold time). The transfer line was heated at 280°C. Total run time was 27.2 min. Mass spectra were acquired in scan mode (70 eV); in the range of 50 to 550 m/z. Twenty microliter each of the extracts (250 mg/ml stock) were further diluted in 2 ml of methanol. One micro liter of this diluted sample was injected for GC-MS analyses.GC-MS analysis of the extracts were carried out in a GC system  equipped with split/splitless injector and auto-sampler attached to an apolar 5-MS (5% phenyl polymethyl siloxane) capillary column  and fitted to Mass Detector . The flow rate of the carrier gas, helium (He) was set to be at 1 ml.minInterpretation of mass spectra was conducted using the database of the National Institute of Standards and Technology . The database caters for more than 62,000 patterns of known compounds. The spectrum of the extracts was matched with the spectrum of the known components stored in the NIST library.Cancer cell lines namely HCT-8 .Cell viability assay: the cell viability assay was performed in 24-h pre-treated MDA-MB-231 and HCT-8 cell lines by different concentrations of plant extracts. The assay was performed as per the manufacturer’s protocol. Briefly, 12x104 cells from control and extract treated cells were taken in 200 μl of PBS and mix with 380μl of cell counting solution . Contents were gently mixed for a few seconds and immediately read on the machine using specific programming. A histogram and numeric values displayed the number of dead and live cells after treatment.Apoptosis assay: in order to evaluate the extent of early and late apoptosis induced by the plant extracts, 12x104 cells from MDAMB-231 and HCT-8 cell lines were treated for 24 h with different concentrations of leaves, bark and seeds extracts. Thereafter the extent of apoptosis was examined on the Muse cell analyzer. The assay was performed by utilizing the Annexin V and Dead Cell Kit . Using designated programming on the cell analyzer, the numbers of live, dead, early and late apoptotic cells were determined. Total apoptosis was calculated by combining the number of cells from late and early apoptosis quadrants of the histograms and is presented in the form of bar graphs.Cell cycle assay: to determine the effect of plant extracts on cell cycle arrest the assay was performed using the cell cycle kit . At the completion of the plant extracts treatment which was done in the same way as described above, cells were trypsinized, and counted. About 12x 104 cells from control and treated groups were fixed in chilled 75% ethyl alcohol for 3h at -20°C. Next cells were washed once with PBS and incubated with 200μl of assay solution for 30 min in the dark at room temperature. After completion of the incubation period the cells were vortexed gently and read on the cell analyzer. The number of cells at each event namely, Go/G1, S and G2/M phases were determined in control and extracts treated cells.p values were ≤ 0.05.Statistical calculations (Student’s t-test) were performed using Graph Pad Prism 4.0 software. The mean was reported with standard deviation (± SD). Differences were considered to be statistically significant when GC method was optimized by varying the oven temperature. In current gradient oven temperature programming, a good resolution of all the extracts has been seen in a relatively short duration of time. The fragmented ions were separated by the analyzer, according to their mass-to-charge ratio.The ethanol extracts of the leaves and bark of Moringa showed twelve and seventeen peaks on chromatogram respectively Figs  and 2A. The effect of MOL, MOB, and MOS were tested on MDA-MB-231 and HCT-8 cell survival. The analysis was done on the Muse Cell Analyzer as described in the Materials and Methods section. A significant decrease in cell survival was observed in both MDA-MB-231 and HCT-8 cancer cell lines when treated with the extracts of leaves and bark. Interestingly, we did not observe any remarkable decrease in cell population when these lines were exposed to the seed extract of MO. A bar graph was constructed summarizing the effect of MO extracts on growth of these two cancer cell lines . The selAs discussed above, the extracts of MO (leaves and bark) showed a promising effect on the inhibition of cell survival. Next, we performed a cell motility assay using all three extracts of MO. As expected, our cell motility assay shows a significant decrease in wound closing after 24-h of treatment of cell lines with the extracts when compared with corresponding controls. A noticeable decrease of about 90% in leaves extract and about 50% in bark extract was observed when cells were treated at 500μg/ml concentration in both MDA-MB-231 and HCT-8 cell lines . HoweverOur consistent findings on cell survival and also on phenotypic assays gave an indication that the low rate of cell survival after extract treatments could be due to the induction of apoptosis. In order to assess the degree of apoptosis caused by the MO extracts on breast and colorectal cancer cell lines, an apoptosis assay was performed on the Muse cell analyzer using the Annexin V staining procedure. The extent of apoptosis was increased as a function of extract concentrations. In this study we observed a significantly high population of late apoptotic cells when treated with plant extracts as compared to control. The number of apoptotic cells increase from 27% to 46% in leaves treatment and from 27% to 29% in case of bark treated MDA-MB-231 cell lines, while control shows only 5% late apoptotic cells . A similTo elucidate the mechanism of reduced cell survival and cell movement, the effect of MO extracts was investigated on cell cycle progression. MDA-MB-231 and HCT-8 cells were treated with 500μg/ml concentration of leaves, bark and seed extracts for 24-h. At the termination of the treatment time (24-h), the cell cycle distribution analysis was performed on the Muse cell analyzer. As shown in Moringa oleifera, a common vegetable plant in many Asian and South East Asian countries possesses numerous compounds with excellent health benefits including anti-oxidant and anti-cancer properties [in vivo models. There is evidence that D-allose (present in leaves of Moringa) inhibits the growth of cancer cells at G1 phase (G1- cell cycle arrest) through specific thioredoxin interacting protein (TXNIP) induction and subsequent p27kip1 protein stabilization without exerting appreciable effects on normal cells [in vivo model [in vivo anti-tumor activity in mice [operties . The plaoperties . The inhoperties , D-allosoperties , isopropoperties  etc. Keeal cells . The orgvo model . The pre in mice . Previou in mice –45, oste in mice , leukemi in mice , gastric in mice , 49, ski in mice , 51, mas in mice  and pros in mice . In summ in mice . Next, w in mice . In thiset al (2006) reported the role of eugenol  in MDA-MB-231 cell apoptosis by increasing Bax protein expression [et al (2013) reported the possible mechanism of action of eugenol by down regulating the E2F1 protein which shows promising outcomes in breast cancer [Moringa oleifera. The rate of increased apoptosis is gradual and also dose dependent. In conclusion, the increased apoptotic events in the presence of MO extracts could be the manifestation of down regulation of E2F1 and up-regulation of Bax proteins. A study by Berkovich et al (2013) showed similar findings in which cell cycle arrest was observed in a pancreatic carcinoma cell line when treated with Moringa leaves extract [Lacroix pression . Followit cancer . Our res extract . In this extract .The most widely used and accepted method for obtaining plant extracts is the Soxhlet technique and this operates at considerable amount of heat. Consequently the chances of thermal inactivation of the bio-active compounds cannot be ruled out. Therefore, we should still consider this step of extraction as the limitation of this technique.The implication of this study opens a new and fertile area of future research to elucidate the molecular mechanism by which signaling events take place after treating cancer cells with MO extracts. There are two probable possibilities which need future investigation. First, a decrease in the expression of PI3K/AKT pathway targeting proteins or second, inhibition of the phosphorylation of focal adhesion kinase (p-FAK), which plays a significant role in cell migration and in cell invasion.In summary, we show that the MO extracts act as an anti-cancer agent by decreasing cell motility and colony formation in colorectal and breast cancer cell lines. Additionally, low cell survival, high apoptosis and G2/M enrichment was detected upon treatment with the extracts of MO leaves and bark. In conclusion, our findings add to the growing evidence supporting the promising role of MO as an anti-cancer agent and open a new vista for molecular analysis of the action of MO on the predominant signaling mechanisms responsible for cancer development. Therefore, MO extracts may represent a valuable therapeutic tool for use as part of a therapy for the treatment of aggressive breast and colorectal carcinoma.S1 Fig(A) Typical TIC-GC/MS chromatogram of Moringa seed analyzed on GC system equipped with an apolar 5-MS capillary column attached with Mass Detector. It showed the presence of fourteen compounds, these are: 1-butanamine, 1-dodecene, 2-decenal, 3-tetradecene, 2-tetradecene, 1-octadecene, hexadecanoic acid, 10-octadecenoic acid, and heptadecanoic acid.(TIF)Click here for additional data file.


In this study we investigated whether co-targeting the MAPK and the PI3K-AKT pathway can prevent emergence of resistance or provide additional growth inhibitory effects Anti-tumor effects of the combination of the BRAF inhibitor (BRAFi) dabrafenib and GSK2141795B (AKTi) in a panel of 23 BRAF mutated melanoma cell lines were evaluated on growth inhibition by an ATP-based luminescent assay, on cell cycle and apoptosis by flow cytometry and on cell signaling by western blot. Moreover, we investigated the possibilities of delaying or reversing resistance or achieving further growth inhibition by combining AKTi with dabrafenib and/or the MEK inhibitor (MEKi) trametinib by using long term cultures.50 < 1.5 μM). The combination of dabrafenib and AKTi synergistically potentiated growth inhibition in the majority of cell lines with IC50 > 5 nM dabrafenib. Combinatorial treatment induced apoptosis only in cell lines sensitive to AKTi. In long term cultures of a PTEN-/- cell line, combinatorial treatment with the MAPK inhibitors, dabrafenib and trametinib, and AKTi markedly delayed the emergence of drug resistance. Moreover, combining AKTi with the MAPK inhibitors from the beginning provided superior growth inhibitory effects compared to addition of AKTi upon development of resistance to MAPK inhibitors in this particular cell line.More than 40% of the cell lines, including PTEN-/- and AKT mutants showed sensitivity to AKTi (ICAKTi combined with BRAFi-based therapy may benefit patients with tumors harboring BRAF mutations and particularly PTEN deletions or AKT mutations. V600 protein, which initially results in profound growth inhibition of the melanoma cells [NRAS[BRAFV600 gene or truncations in the BRAFV600 protein through alternative splicing resulting in lack of inhibition by the drug due to increased dimerization [MEK and overexpression of the Ser/Thr MAP kinase kinase kinases  has also been described in the context of BRAF inhibitor resistance [BRAF inhibitors such as vemurafenib or dabrafenib efficiently block signaling downstream of the mutated BRAFma cells ,2 and hima cells ,4. Howevma cells . Accumulrization ,8. Activsistance -11. A cosistance . It is hsistance ,14. In tsistance , the inssistance  or the esistance  .Given that the MAPK and the PI3K-AKT pathways are the predominant signaling pathways in melanoma and that MAPK-independent resistance to BRAF inhibitors can be mediated through enhancement of signaling through the PI3K-AKT pathway, it would be reasonable to combine a BRAF inhibitor with an inhibitor of the PI3K-AKT pathway to achieve synergistic antitumor activity -22. ThisBRAFV600 mutant melanoma cell lines compared to single agent dabrafenib. Furthermore, we investigated whether addition of the AKTi upon resistance to MAPK inhibitors could provide secondary responses, and whether upfront combination of dabrafenib, trametinib and AKTi could delay the emergence of drug resistance. Here we provide evidence that the combination of dabrafenib and AKTi synergistically inhibits proliferation in the majority of cell lines tested. Furthermore, we show that AKTi can delay the emergence of resistance to MAPK inhibitors and also provide further growth inhibition upon resistance to a combination of MAPK inhibitors in the only AKTi sensitive cell line tested in this study.In this study, we tested the hypothesis that combining the BRAF inhibitor dabrafenib, which recently has been approved for clinical use by the US Food and Drug Administration, with a novel AKT inhibitor tool compound GSK2141795B (AKTi), which is an analogue of the clinically tested AKT inhibitor GSK2141795, would have superior anti-tumor effects in BRAFV600 mutations , was also found in the sensitive group (blue bar).In this study, a panel of 23 previously described ,6 melanons Table  was usedhis drug . The MAPThe efficacy of the drugs in inhibiting the signaling pathways was verified by western blot analysis of phosphorylated proteins Figure C and 1D.After evaluating the growth inhibition resulting from treatment with each drug alone, we explored whether blocking both pathways by the combination of dabrafenib and AKTi would enhance the growth inhibitory effects. The IC50 values for the combination of the two drugs  were significantly lower compared to the IC50 values for each drug alone in the majority of cell lines sensitive and resistant to dabrafenib Figure A and 2B.Next we evaluated the responses seen in growth assays by quantitating basal levels of p-AKT in a representative panel of cell lines with differential sensitivity to single agent dabrafenib or AKTi Figure . The firTo further explore the effects of the two drugs, we selected 6 cell lines with differential sensitivity to single agent dabrafenib or AKTi Table  and analTo investigate whether dabrafenib or AKTi or the combination affect cell cycle, four representative cell lines  with different dabrafenib and AKTi sensitivities Table A. Howevein vitro culture as a model, we explored whether addition of AKTi upon emergence of resistance to dabrafenib in combination with the MEKi, trametinib, could provide further growth inhibition .Co-targeting the MAPK and the PI3K-AKT pathway is a compelling approach given the frequent cross-talk and regulating feedback loops between these two pathways ,27. MorePTEN null and other cell lines expressing high levels of p-AKT are among the dabrafenib sensitive cell lines indicates that activation of the PI3K-AKT pathway is probably not a reason for the innate resistance to BRAF inhibition. Another explanation for this finding could be that, although these cell lines are primarily dependent on MAPK for their proliferation, they also to some extend are dependent on PI3K-AKT pathway for their proliferation and survival. This idea can be supported by the fact that in growth assays, these cell lines exhibit sensitivity to both dabrafenib and AKTi as single agents, and the combination treatment induced apoptosis in one tested PTEN null cell line (M411). Other studies exploring dual inhibition of the MAPK and the PI3K-AKT pathway using a different panel of inhibitors also found that combinations of MAPK and PI3-AKT pathway inhibitors augment induction of apoptosis in melanoma cells compared to single drug treatments [The finding that eatments ,33,34. MIn our studies, reduction in p-S6 seemed to be a good predictor of sensitivity to either of the single drugs or their combination. Reduction in p-S6 as a predictor of response to RAF and MEK inhibitors has also been described by others ,36 and oIn the study of AKTi’s effects on the PI3K-AKT pathway, we observed a considerable increase in p-AKT at both phosphorylation sites namely T308 and S473. This induction suggests that the inhibition of AKT either abrogates a negative feedback loop or induces a positive regulation mechanism. Two different proteins have been reported to be responsible for phosphorylation at site T308 and S473, PDK1 acting from upstream and mTORC2 acting from downstream of AKT, respectively. A well-established feedback loop mediated by S6K inhibits the PI3K-AKT pathway through phosphorylation and inactivation of insulin receptor substrate-1 (IRS1), which activates PI3K ,37. HencMost patients with metastatic melanoma have early response with BRAF inhibitors as monotherapy, but acquired resistance frequently develops and the majority of patients experience relapse with a median of 6-7 months . SupportOvercoming resistance to BRAFi is a major problem in the treatment of metastatic melanoma. Multiple strategies including combinatorial therapies are evaluated in the attempt to solve this problem. Herein, we showed that combining the BRAF inhibitor dabrafenib with an AKTi potently inhibits growth in the majority of melanoma cell lines tested and induces cell death in a subset of cell lines. Moreover, AKT inhibition demonstrated ability to reverse acquired drug resistance to combination therapy with dabrafenib and trametinib in the single AKTi sensitive cell line that was tested (M397). Finally, triple drug administration delayed the emergence of drug resistance in that particular cell line. Thus, combining dabrafenib with an AKTi appears to be a promising strategy for more effective treatment of melanoma. This is the basis of a US cooperative group clinical trial , which has the goal of determining the safety of the combination of dabrafenib and the clinical grade AKTi GSK2141795, and early evidence of the antitumor activity of this combination in patients progressing on prior BRAFi-based therapy.Dabrafenib (GSK2118436A), trametinib (GSK1120212B) and GSK2141795B  powder were obtained under a materials transfer agreement with GlaxoSmithKline . The compounds were dissolved in dimethyl sulfioxide  to a stock concentration of 10 mM.in vitro acquired resistance to vemurafenib were generated as previously described [2 and passaged into new flasks as needed. Information of the mutational status of the cell lines used in the study has been previously described [Human melanoma cell lines from the M series were established from patient’s biopsies under the University of California Los Angeles (UCLA) Institutional Review Board approval IRB#02-08-067. Cell lines with escribed  and labeescribed .For short term growth assays, melanoma cell lines were seeded in 96-well plates (3000 cells/well). The following day, the cells were treated in duplicate with dabrafenib, AKTi or the combination in 10-fold serial dilutions starting from 10 μM for 72-120 hours depending on each cell line’s specific growth rate. Cell viability was measured by using the CellTiter-GLO® Luminescent Cell Viability assay . The IC50 values were determined by interpolation from the dose-response curve. Each experiment was repeated at least three times and the average of minimum two is presented. In long term assays, cells were seeded in 96-well plates . To study delays in emergence of resistance, the cells were treated with 200 nM dabrafenib (2 × IC80 for M397) alone or in combination with 2 nM trametinib (2 × IC80 for M397) or the combination of all three drugs including 2.5 μM AKTi. In another setup, cells were treated with dabrafenib and trametinib at the above mentioned concentrations and upon development of resistance to these two drugs, trametinib was replaced with 2.5 μM AKTi. Culture media containing the drugs was changed once a week. Growth of the cells was monitored and upon confluence of some wells, a gradient of the cells were plated to be used as a reference for the cell number. One hour before cell viability was determined using a tetrazolium compound [3--5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS)-based colorimetric cell proliferation assay (Promega) the drug media was replaced with fresh culture media without drugs to eliminate measurement of absorbance of the drugs and to reduce the drugs impact on mitochondrial activity.Cells were seeded in 12-well plates at a density of 200,000 cells/well. The next day, the culture medium was replaced with media containing DMSO, 50 nM dabrafenib, 2.5 μM AKTi or the combination. After 24 hours, the media was removed and the cells were lysed directly in the wells for 15-20 min with lysis buffer  containing phosphatase- and protease inhibitors (Sigma) and protein was extracted for western blot analysis as previously described . Blots w6 cells, BD biosciences) for 30 minutes. Next, for cell cycle analysis, the cells were washed with Perm/Wash (BD Bioscience) before resuspended in 3 μM DAPI (Sigma-Aldrich) solution diluted in PBS containing 1% bovine serum albumin at a concentration of 1 × 106 cells/mL. Flow cytometry was performed on a LSR-II (BD Bioscience) and data was analyzed using FlowJo .Cells were seeded at a density of 200,000 cells/well in 6-well plates. The following day, the culture medium was replaced by medium containing DMSO, 1 μM staurosporine (positive control for apoptosis), 50 nM dabrafenib, 2.5 μM AKTi or the combination. After 48 hours of exposure to the drugs, both adherent and floating cells were harvested by trypsinization and fixed for 20 minutes with Cytofix/Cytoperm solution . For apoptosis, cells were stained with Alexa Flour700 linked anti-cleaved-PARP antibody  were treated in triplicate with DMSO, 50 nM dabrafenib, 2.5 μM AKTi or the combination for 48 hours. Apoptosis was quantified by anti-body-mediated capture and detection of cytoplasmic mononucleosome- and oligonucleosome-associated histone-DNA complexes  according to the manufacturer’s instructions. Results were expressed as the average absorbance value of triplicates.IC50 values were calculated on the basis of the growth inhibition curves and define the concentration of drugs that resulted in 50% growth inhibition. Synergistic, additive or antagonistic effects of the drug combinations were determined by the use of the combination index (CI) method of Chou and Talalay using the Calcusyn software . Any CI values less than 1 indicates synergism, CI = 1 additive effect and CI > 1 antagonism. Error bars represent the standard error of the mean (SEM). A two-tailed unpaired t-test was used when applicable. P-values < 0.05 were considered to be statistically significant.AR has participated in scientific advisory boards from GSK. The honoraria from these boards are paid to Institutional accounts at UCLA.AL, MA, DW, LR performed the experiments and AL co-wrote the manuscript. MA participated in the design of the study and in the interpretation of data and revised the manuscript critically. BCA designed the experiments performed by flow cytometry and MC participated in collecting the data. AR conceived the study and participated in its design and coordination and co-wrote the manuscript. All authors read and approved the final manuscript.(A). Cells were treated with increasing concentrations  of dabrafenib, AKTi or the combination for 72-120 hours to assess cell viability. The graphs represent the average growth inhibition in percent of minimum two independent experiments in duplicates and the error bars represent SEM. Dose-effect parameters for the cell lines with synergy data (B). The r value is the linear correlation coefficient of the median-effect plot while m denotes the shape of the dose-effect curves.Effects of single agent dabrafenib, AKTi or the combination on cell proliferation and viability. Growth inhibition curves of melanoma cell lines with differential sensitivity to single agent dabrafenib and AKTi Click here for fileQuantitative analysis of p-S6 from western blots. Data corresponds to the p-S6 bands in the western blots presented in figure Click here for fileAnti-cleaved-PARP gating strategy. The fraction of anti-cleaved-PARP positive cells was determined by using an unstained control. Apoptosis induced by staurosporine was used as a positive control. The analysis was done by using FlowJo software (PC ver. 7).Click here for fileDetermining apoptosis using a cell death detection ELISA. Cells were treated with DMSO, 50 nM dabrafenib, 2.5 μM AKTi or the combination for 48 hours. The extent of apoptosis is reflected by the enrichment of nucleosomes in the cytoplasm, which was quantitated as the relative increase in absorbance (y-axis). The panel included one cell line sensitive to both dabrafenib and AKTi (M411), one cell line sensitive to AKTi but intermediate resistant to dabrafenib (M414) and one cell line demonstrating resistance to both drugs (M299). The bars represent the average absorbance of triplicates.Click here for fileExample of a standard curve used for the calculations of cell number in long term culture. Cells were plated in 1:2 serial dilutions starting from 50,000 cells. The measured absorbance (using a MTS-based assay) from these wells were plotted against the known cell numbers and by use of the equation for the trend line through (0.0) the unknown cell numbers could be determined.Click here for file