Cdkn2a and of epigenetic regulators, in comparison with PN and normal human mesothelial cells, respectively. In particular, we identified the involvement of the relative expression of the Ten-Eleven Translocation (TET) family of dioxygenases and Dnmt3a in relation to the 5-hydroxymethylcytosine level in malignant transformation and the acquisition of metastatic potential.Malignant mesothelioma (MM) is one of the worst cancers in terms of clinical outcome, urging the need to establish and characterize new preclinical tools for investigation of the tumorigenic process, improvement of early diagnosis and evaluation of new therapeutic strategies. For these purposes, we characterized a collection of 27 cell lines established from F344 rats, after 136 to 415 days of induction with crocidolite asbestos administered intraperitoneally. Four mesotheliomas were distinguished from 23 preneoplastic mesothelial cell lines (PN) according to their propensity to generate tumors after orthotopic transplantation into syngeneic rats, their growth pattern, and the expression profile of three genes. PN cell lines were further discriminated into groups / subgroups according to morphology in culture and the expression profiles of 14 additional genes. This approach was completed by analysis of positive and negative immunohistochemical MM markers in the four tumors, of karyotype alterations in the most aggressive MM cell line in comparison with a PN epithelioid cell line, and of human normal mesothelial and mesothelioma cells and a tissue array. Our results showed that both the rat and human MM cell lines shared in common a dramatic decrease in the relative expression of  Malignant mesothelioma (MM) is a rare, aggressive cancer mainly related to asbestos exposure , the lonCell lines are universal model systems in cancer research and their characterization has provided crucial insights into the mechanisms leading to malignant transformation. Nevertheless, only a small number of cell lines meet the minimum standards of authentication and characterization , emphasiTET expression have systematically compared tumor tissues from various origins relative to their normal counterparts. In all cases, the reduced levels of hmC in tumor tissues were associated with a decrease in the relative expression of all three TET genes when compared with their matched normal tissues ) and “sarcomatoid” (named PNsarc) morphology in culture . This obc-Myc, a proto-oncogene gene the deregulation of which contributes to deregulated DNA synthesis and genomic instability, and the lowest expression of Igf1, a gene involved in the deregulation of cellular energetics  was identified by the highest expression of s Figure . A seconn Figure . Finally1 Figure .Ezr and Hmgb1, compared with the PN-[Epith] group of preneoplastic cell lines, however their expression profile was comparable to that of the PNsarc group  and malignant pleural mesothelioma cell lines (MPM) . Finally, among preneoplastic rat cell lines, a significant decrease in the expression of Dnmt3b and all TET genes was observed in the three invasive neoplastic cell lines, relative to the PNsarc group  Figure . Of the rc group .Representative images of the M5-T1, F5-T1, F4-T2 and M5-T2 tumors  increased Figure .During development, the mesothelium, which consists of a monolayer of specialized cells with a typical cobblestone-like morphology, retains its potential to differentiate into cells of different phenotypes . This mein vivo after orthotopic transplantation into the peritoneal cavity of syngeneic rats. Our finding that Lgals3 was up-regulated at the mRNA level in neoplastic relative to preneoplastic cell lines is in good agreement with accumulating evidence indicating that galectin-3 is closely involved in tumor cell transformation, migration, invasion and metastasis for a number of cancers  and PNsarc groups is in good agreement with the differences in the expression profiles previously observed at both the mRNA and protein levels  group, could also be due to instability of Msln mRNA, according to the presence of a polymorphic variant and regulation by micro-RNA  cell lines, the first significant  process . In para lesions , in rela lesions . Under c lesions , suggest decades . Finallyas Acta2 .Pdpn, Ezr, Msln, Hmgb1, Wt1 and Cdh1 shared by these three cell lines and the PNsarc group, compared with the PN-[Epith] group, tend to confirm that these three cell lines and the PNsarc group present in common the characteristics of a mesenchymal phenotype. In agreement with previous findings  group. The singular situation of the M5-T2 cell line was reinforced by the observation of a maximum expression of five other genes, Rassf1, Zeb1, Vim, Acta2 and Tgfb1, which was common to the PNsarc group, in contrast with the three invasive mesothelioma cell lines which presented significantly lower expression levels.In contrast, in the M5-T2 cell line, this common decrease was restricted to p16 deletion is a good marker of malignancy, both in pleural and peritoneal mesotheliomas with invasive components . To establish this biocollection, at 8 weeks of age, a group of 33 rats  received an intraperitoneal inoculation of 10 mg crocidolite fibers suspended in 0.5 ml 0.9% NaCl . Between 136 and 415 days after induction, after euthanasia, 2 ml sterile RPMI medium was injected intraperitoneally, the peritoneal lavage fluids were collected under sterile conditions and then placed into culture. This step led to the establishment of 23 transformed mesothelial cell lines. After 378 days of induction, one male rat was necropsied about one hour after death, presenting signs of hemorrhage, widespread neoplastic implants and nodules in the peritoneal cavity as previously described [6 cells.The cell lines used in this study belong to a biocollection established in 2011 [escribed , which, Mesothelioma cell lines were established from pleural effusions collected by thoracocentesis of patients with cancers, as previously described . All pat2 at 37°C.All cell lines were maintained in RPMI 1640 medium (Invitrogen) supplemented with 10% heat-inactivated fetal calf serum (Eurobio), 100 U/ml penicillin, 0.1 mg/ml streptomycin, 2 mM L-Glutamine  and cultured at low passage level (6 to 10) in a humidified atmosphere of 5% COin vivo after transplantation, cell lines were seeded at 3 × 105 cells/well at time 0 in 6-well plates  and counted in a Malassez hemocytometer, the entire growth curve being obtained from at least 12 time points. Cell cycle analysis was conducted on a NucleoCounter® NC-3000™  using NC-Slide A8™ after trypsinization, centrifugation, fixation of cells with ethanol, and incubation with DAPI at 37°C for 5 minutes. The cell number in each phase of the cell cycle was determined and calculated as a percentage of the total cell population.For determination of the population doubling time in the mid-log phase and the saturation density at plateau, in order to approach the conditions of proliferation 2 / well), one wound line in the form of a cross was made by scratching the cell monolayer with a plastic pipette tip. Floating cells were washed out and fresh medium was added. The narrowing width of the scratch was recorded by taking photographs under an inverted Zeiss Axio Observer.Z1 microscope from 17 h after the scratch.Cell migration was analyzed for all rat cell lines by the scratching test . When ce2 culture plate at preconfluence for each cell line using the NucleoSpin RNA II Kit according to the manufacturer's instructions . The total RNAs were next treated with an rDNase solution to remove contaminating genomic DNA, and subsequently purified. One microgram of total RNA was reverse-transcribed using Moloney-Murine Leukemia Virus Reverse-Transcriptase (Invitrogen). PCR reactions were performed using an Mx4000 thermocycler (Stratagene) with 10X QuantiTect Primer Assays (Qiagen), and the RT2 Real-Time SYBR-Green/ROX PCR Mastermix , according to the manufacturer's instructions. The thermal cycling protocol was followed by a melting curve analysis to check for the absence of nonspecific products. For each transcript, the efficiency of the PCR reaction was determined by the slope of the standard curve generated from serial dilutions of the same cDNA sample (pool of reverse-transcribed RNA samples). The relative amount of target RNA, called the Starting Quantity (SQ), was determined using the MxPro software, by comparison with the corresponding standard curve for each sample performed in duplicate. Each transcript level was normalized by division with the expression values of the acidic ribosomal phosphoprotein P0 housekeeping gene (RPLP0), used as an internal standard.Total RNA was extracted from one 75 cm2 were analyzed both by manual counting and by color deconvolution of TIF images using Fiji software and the Otsu method [For histological examination, the paraformaldehyde-fixed, paraffin-embedded sections of rat tumor samples , and surrounding normal/invaded tissues when present, were cut with a Bond Max automaton  and stained with hematoxylin-phloxine-saffron (HPS). A human mesothelioma array with normal mesothelium tissue (lung and cardiac pericardium) was obtained from US Biomax Inc. . Antibodies used for immunohistochemical analyses were anti-WT-1 ab15249, anti-calretinin ab16694 and anti-vimentin ab8978 from Abcam, anti ESA/EPCAM (MOC 31) LS-C331328 from LSBio, and anti-5-hmC 39769 (1/200) and anti-5-mC 61255 (1/60) from Active Motif Europe , with an anti-mouse secondary antibody and the N-Histofine Simple Stain Mouse MAX Peroxidase  as the detection reagent. Histopathology slides were scanned with a Nanozoomer 2.0 HT . To semiquantify histochemical staining on rat tumor slices, five randomly selected fields of 27,000 μmu method . HistochDNA samples were denatured by the addition of denaturation buffer (0.1 mM NaOH) and subsequently heated for 5 minutes at 95°C. The reaction was then stopped by addition of 6.6 mM ammonium acetate. Samples were rapidly chilled for 5 minutes on wet ice and then applied to a positively charged nylon membrane , previously charged and rehydrated by dipping for 15 seconds into ethanol and 5 minutes in tris-buffered saline with Tween 20 (TBST). The membrane was then air dried for 15 minutes. Denatured DNA was spotted onto the membrane and air dried for 45 to 60 minutes, UV-cross-linked to the membrane , and incubated for 1 hour with saturation buffer (TBST 1%BSA). Primary antibody  was incubated overnight at 4°C with agitation. The membrane was washed three times with TBST and incubated with the anti-rabbit secondary antibody . The membrane was washed again three times with TBST and the signal was revealed using enhanced chemiluminescence (ECL) substrate  and read with a Biorad Chemidoc MP Imaging System. To control the DNA load, DNA was quantified using gel red and the membrane was scanned with the Biorad imager. Dot blot signals were quantified and normalized to the intensity of the corresponding gel red signal. To ensure specificity of the antibody, naked DNA, methylated DNA, and 5-hmC DNA control (Active Motif) were used (data not shown).Metaphases were obtained using standardized harvesting protocols as described previously. Briefly, colcemid solution (0.03 μg/ml) was added to F3-1 and M5-T1 cells 90 minutes and 30 minutes before harvesting, respectively. Cells were treated with hypotonic solution (0.075M KCl), fixed three times with Carnoy's fixative (3:1 methanol/acetic acid) and spread on prechilled glass slides. M-FISH was performed with the aim of identifying numerical and structural alterations in both cell lines. The probe cocktail containing differentially labeled, chromosome-specific painting probes – except for chromosomes 13 and 14, which were distinguished by their DAPI banding  – was denatured and hybridized to denatured metaphase spreads according to the manufacturer's protocol for the Multicolor FISH probe kit for rat chromosomes (MetaSystems). After counterstaining with DAPI (1 μg/ml), the signal detection and analysis of subsequent metaphases used the Metafer system and Metasytems' ISIS software, respectively. F3-1 and M5-T1 karyotypic formulas were reported using an abbreviated format of the International System for Human Cytogenetic Nomenclature (ISCN), omitting breakpoint information either when the quality of counterstained chromosomes or the blending of colors through fluorescence flaring prevented the accurate definition of breakpoints. Chromosomal gains or structural aberrations had to be detected in at least two metaphases and chromosomal losses in three metaphases to be acknowledged as clonal. Chromosome identification and band nomenclature were indicated according to the standard rat karyotype .p value < 0.05 was considered to be statistically significant. All graphs present the mean and standard deviations of at least three independent experiments.Statistical analyses were performed using the Mann-Whitney nonparametric test for comparison of the means, using the Graph-Pad Prism software. A


Melanoma researchers utilize cell lines to model many tumour phenomena. It is thus important to understand similarities and differences between cell lines and the tumours that they represent, so that the optimal models can be chosen to answer specific research questions. Herein, we compared the transcriptomes of 42 melanoma cell lines to hundreds of tumours from The Cancer Genome Atlas and thousands of single melanoma cells. Tumour purity was accounted for using the ESTIMATE algorithm, so that differences likely resulting from non-tumour cells could be accounted for. In addition, UV mutational signatures and the expression of skin-associated genes were analyzed in order to identify the putative origin of various cell lines. We found the transcriptional and mutational characteristics of melanoma cell lines to mirror those of the tumours, with the exception of immune-associated transcripts, which were absent in cell culture. We also determined cell lines that highly or poorly recapitulate melanomas and have identified colon (COLO 741) and lung (COLO 699) cancer cell lines that may actually be melanoma. In summary, this study represents a comprehensive comparison of melanoma cell lines and tumours that can be used as a guide for researchers when selecting melanoma cell line models. BRAF and immune checkpoint inhibitors, have achieved success in extending patient survival, however, innate or acquired therapy resistance and tumour recurrence is almost unavoidable  but poorly with AXL enriched cell [r = 0.46]); or cell lines that were fair transcriptional representations of both states (eg. COLO 849 correlates with intermediate strength to MITF enriched cells [r = 0.53] and to AXL enriched cells [r = 0.51]). Interestingly, the three cell lines that had the lowest correlations to all malignant cells but that were characterized as AXL program enriched, still had low correlations to AXL-defined single cell entire transcriptomes, indicating that they are likely poor representations of both MITF- and AXL-enriched melanoma.To dissect out some of the subtleties likely due to transcriptional state, we classified the highest and lowest 400 rs Table . This anin vitro, we assessed the mutational spectrum of annotated melanoma versus non-melanoma cell lines and found that the average melanoma cell lines had over a two-fold increase in C>T substitutions at dipyrimidine sites  at such high levels that they are outliers when compared to all other annotated colorectal . Our analysis found that the correlation coefficient of individual cell lines to malignant cells varied from 0.22 to 0.54. This was somewhat similar to the ranges of 0.41-0.58 and 0.43-0.60 that has been previously observed in similar analyses conducted on breast and ovarian cell lines, respectively [MITF-defined malignant cells [r = 0.60] but not AXL-defined malignant cells [r = 0.46]). While several melanoma cell lines appear to be good tumour models, there are also three cell lines  that display exceptionally poor transcriptional resemblance to their tumour counterparts. As well, these three cell lines cluster with non-melanoma cell lines using tissue of origin analyses and unsupervised hierarchal clustering. While it’s difficult to conclude if these three cell lines have been misidentified as melanoma lines, it is convincing that they represent exceptionally poor models of melanoma.Despite the transcriptional differences that we observed between human melanoma cell lines and tumours, we did observe cell lines that clustered closer to their tumour counterparts than others in the principal component analysis. Accordingly, we predicted that some cell lines would more accurately represent the transcriptional profile of melanoma tumours. Recent single cell RNA-sequencing of over 4,000 melanoma cells provides an unprecedented opportunity to directly compare the trancriptomes of ectively , 11. It On the other hand, it may be more feasible to identify annotated non-melanoma cell lines as being of probable melanoma origin. The precedent for this was set in 2000, when the annotated breast cancer MDA-MB-435 cell line was shown to cluster with melanoma cell lines and express melanoma-specific genes . Follow-This study is the first of its kind to determine the extent to which melanoma cell lines represent tumours in terms of gene expression and mutational burden. While generally quite similar, we determined that cell lines and tumours differ at the transcriptional level, likely due to a loss of immune components in cell culture. Additionally, we were able to rank cell lines based on their transcriptional similarity to malignant cell tumour counterparts and identified three cell lines that were exceptionally poor models of melanoma. We have also identified two cell lines, COLO 741 and COLO 699, which are of probable melanoma origin. Knowing the strengths and weaknesses of our widely used tumour models can help direct tumour model choice and improve the clinical value of future research efforts.Level 3 TCGA RNAseqV2 gene expression and Level 2 TCGA somatic mutation data  was obtained from the TCGA Data Portal in January 2016. Patient survival information, pigment scores and lymphocyte density values for TCGA samples was obtained from the supplementary information of the SKCM TCGA landmark paper . RNA-se6, and for 42 melanoma cell lines by converting RPKM (reads per kilobase of exons per million mapped reads) to TPM. To avoid infinite values in log calculations, a value of 1 was added to TPM values before log2 transformation. Values for the genes that were available on both datasets were used in downstream analyses . For single cell analysis, TPM values were divided by 10 as in the original paper, given that the complexity of single cell libraries is estimated to be in the order of 100,000 transcripts.Relative abundance  was calculated for 471 melanoma patient samples (104 primary tumours and 367 metastases) by multiplying the scaled estimate data by 10The top 5,000 genes by variance across the combined cell line-tumour dataset were chosen for principal component analysis. Significant differences in relative transcript abundances between cell lines and tumours were calculated with Welch’s t test and p values were corrected for multiple testing using the Benjamini-Hochberg method. Unsupervised hierarchal clustering was done using Euclidean distance and Ward’s agglomeration method (ward.D2). Enrichment for functionally related genes between the two datasets was tested using Generally Applicable Gene-set Enrichment  with KEGG gene sets.Stromal and Immune scores were defined for tumours through the use of the ESTIMATE  algoritImmune Differentially Expressed Gene (DEG) Values were calculated for tumours by creating and summing Z-scores for genes that were (1) in the top 5% of differentially expressed between cell lines and tumours and (2) strongly correlated with immune score (r > 0.4). Immune DEG Values were dichotimized by median and ten year survival curves were constructed using the Kaplan-Meier method on metastatic samples that had survival information available (n=292). Significance was determined by log-rank test.MITF and AXL gene programs as defined by Tirosh et al. were used to characterize cell lines [MITF and AXL cell line enrichment scores and clusters were defined using Gaussian kernels.ll lines . EnrichmMITF and AXL gene set enriched single melanoma cells, MITF and AXL cell scores were calculated as in the original paper [MITF/AXL average relative expression cell scores and control gene set cell scores. Control gene set scores were subtracted from their respective MITF/AXL cell scores. All genes were used to compute Pearson’s correlation of all cell line-single malignant cell pairs and cell lines were ranked based on their average correlation with all malignant cells. In MITF/AXL specific analyses, Pearson’s correlation of the malignant single cells with the highest or lowest 400 MITF/AXL ratios were used instead of all malignant cells.To define the top al paper  and a raNon-synonymous coding mutations with neighbouring nucleotide information were extracted from all non-synonymous coding mutations and used in mutational spectrum analyses. Patterns of particular nucleotide substitutions were determined for melanoma (n=42) and non-melanoma (n=567) cell lines as a mean percentage of total somatic variants. Cell lines were classified as UV signature positive if the fraction of C>T transitions at dipyrimidines was >0.6 as defined in the landmark TCGA SKCM paper .In our genomics summary, selected mutational events and copy number alterations were considered if they were found to be significantly altered from normal tissue in the landmark TCGA SKCM study . Copy nubioinfo.wilmer.jhu.edu/tiger/). Gene symbols were filtered for presence in the SKCM RNA-sequencing dataset. TiGER Skin Scores were determined for each cell line as previously described [The skin tissue-specific panel of genes was retrieved from the TiGER portal (escribed . The TiGwww.pubmed.com) on February 29, 2016. Several punctuation alternatives were used for the cell line names.The number of PubMed abstracts that mentioned one of the 42 melanoma cell lines was determined as an estimator of frequency of use. Hits were determined using the PubMed search function . R/Bioconductor packages ggplot2, plyr, gplots, ggdendro, survival, pdfCluster, and GAGE were used where appropriate.


Mechanistically, ATM mediates crosstalk between the prosurvival MEK/ERK and AKT/mTOR pathways. ATM loss also enhances the sensitivity of KRAS- or BRAF-mutant lung cancer cells to MEK inhibition. Thus, ATM mutational status in lung cancer is a mechanistic biomarker for MEK inhibitor response, which may improve patient stratification and extend the applicability of these drugs beyond RAS and BRAF mutant tumours.Lung cancer is the leading cause of cancer deaths, and effective treatments are urgently needed. Loss-of-function mutations in the DNA damage response kinase ATM are common in lung adenocarcinoma but directly targeting these with drugs remains challenging. Here we report that ATM loss-of-function is synthetic lethal with drugs inhibiting the central growth factor kinases MEK1/2, including the FDA-approved drug trametinib. Lung cancer cells resistant to MEK inhibition become highly sensitive upon loss of ATM both  ATM is a tumor suppressor often mutated in lung adenocarcinoma. In this study, the authors starting from a synthetic lethal screen, demonstrate that tumor cells with mutations in ATM exhibit increased sensitivity to MEK1/2 inhibition through the modulation of the AKT/mTOR pathway. Lung cancer remains the leading cause of cancer-related deaths23467The majority of lung cancer mutations, such as ATM (ataxia–telangiectasia mutated) loss-of-function mutations, which are found in 5–10% of patients, are not actionable with small-molecule inhibitors. Some of these so-called ‘undruggable' mutations may result in vulnerabilities through synthetic lethal interactions that can be exploited with drugsATM gene. A–T patients are predisposed to cancer, particularly those of lymphoid origin, and ATM variants have also been associated with cancer predisposition10ATM somatic mutations in a variety of solid tumours, including lung (∼10%), pancreatic (∼12%) and bladder (∼4%) cancers5The serine/threonine kinase ATM is well known for its involvement in the DNA damage response (DDR) and maintaining genome stability but it has also been implicated in other cancer-relevant processes including cell growth, metabolism and mitochondrial homeostasisreatic ∼1% and blabladder ∼% cancerscreatic ∼% and blaRAS or its downstream signalling components , which occur in a large number of cancers. The first MEK inhibitor (trametinib) has recently been approved for treating BRAF mutant melanomas but in lung cancer results have not been as encouraging14ATM, remains of great interest and could have immediate clinical applications. MEK1/2 kinases have not been directly linked with DNA repair. Thus, our experiments reveal an unexpected link between growth factor signalling pathways and ATM loss, providing a strong rationale for testing MEK inhibitors in the context of ATM mutant tumours.MEK1/2 are kinases that regulate cell proliferation and survival. MEK inhibitors are currently in clinical development for a variety of cancers, including lung cancer, with mutations specifically in the oncogene in vitro screening strategy using isogenic cell lines16APC, ATM, CDKN2A, ERBB4, NF1, PRKDC, PTEN, SMAD4, SMARCA4 and STK11) that recur in human lung cancer based on publicly available tumour sequencing data and the Catalogue Of Somatic Mutations In Cancer (COSMIC) database245To search for synthetic lethal interactions between lung adenocarcinoma tumour suppressor genes and anti-cancer drugs, we employed an The top synthetic lethal interaction hit identified in the screen involved knockdown of ATM, a DNA damage signalling kinase, and the Chk1 kinase inhibitor AZD7762 ref. . This int-test, P<0.0001, The second strongest drug sensitivity hit in ATM knockdown cells was unexpectedly with a MEK1/2 inhibitor (PD0325901)KRAS and BRAF wild type) and H1755 (BRAF mutant). This suggests that ATM knockdown can affect the response to MEK inhibition in both the presence and absence of KRAS or BRAF mutations, both of which are common in lung cancer. Importantly, sensitization by ATM knockdown was not observed in the naturally ATM mutant H1666 cell line, supporting an on-target effect of the used shRNA sequences  targeting exon 6 of ATM was employed, and the genomic region flanking the Cas9 cleavage site in single cell clones was analysed by Sanger sequencing  and poor transfectability of the lung cancer cell lines, we next employed RNA-guided nucleases (RGNs)quencing . ATM fra editing . Complet editing . As expe editing . In agrecontrols . TogetheATM mutations, we assembled a set of nine ATM mutant and seven wild-type control lung cancer cell lines. To control for confounding mutations, we included equal numbers of KRAS/BRAF mutant cell lines in the ATM mutant and wild-type groups and the status of the ATM mutant cell lines was confirmed by Sanger sequencing. As the ATM mutations have not been functionally validated we employed a polymorphism phenotyping algorithm (PolyPhen v2) to predict which mutations were most likely to affect protein function, for example by causing a premature stop codon or a missense mutation affecting an evolutionary conserved amino acid, and further stratify the cell linesATM wild-type cells responded to ionizing radiation. In contrast, three out of the seven ATM mutant cell lines did not display an appreciable induction of SMC1 or KAP1 phosphorylation. This included the heterozygous ATM mutant cell line NCI-H1666. This experiment confirms that some of the cell lines with ATM mutations display aberrant DDR. However, it does not rule out that other aspects of ATM function are normal in the other cell lines that harbour ATM mutationsTo investigate whether the ATM-MEK gene–drug association also occurs in lung cancer cells harbouring P<0.0001) between the potency of the two MEK inhibitors across the cell lines, indicating a shared mechanism of action  mutations co-occur at a rate that is consistent with a lack of genetic interaction (Sensitivity to trametinib and TAK733 was determined in dose–response experiments and the area under curve (AUC) was calculated to compare the cell lines . There wf action . Out of nockdown . Analysieraction .ATM mutation in this collection  was observed  associated with ATM mutations ll lines . Remarkall lines . Similarll lines . This fuobserved . Furtherutations , ruling ATM as a predictive biomarker in the CCLE data set, we calculated sensitivity (fraction of responsive lines that is identified), false positive rates (fraction that is predicted to be responsive but is not) and true positive rates (fraction that is responsive and correctly predicted) and compared these with KRAS/BRAF  of the cell lines predicted to be sensitive based on ATM mutation indeed responded to MEK inhibition. Importantly, a genetically stratified clinical trial with a 57% response rate would be considered successful (compared with the 23% that would have been observed using KRAS/BRAF). Adding ATM status to KRAS/BRAF further increased sensitivity and true positive rate, while reducing false positive rate.To further assess the performance of RAS/BRAF . EncouraATM mutations is associated with high sensitivity to MEK inhibition.Together our results reveal that a heterogeneous panel of lung cancer cells carrying ATM are directly involved in MEK inhibitor sensitivity, we sought to restore resistance in ATM mutant cells. NCI-H23 cells have a homozygous ATM point mutation (c.5756A>C) resulting in an amino-acid substitution of glutamine with proline: ATM(Q1919P). Accordingly, these are highly sensitive to MEK inhibitors We next analysed whether an altered DDR was involved in the synthetic lethal interaction. MEK inhibition in ATM null cells did not alter levels of the DNA double-strand-break marker γ-H2AX . MoreoveATM knockout cells. Instead of upregulation, we observed a downregulation of p4EBP1 and pS6K upon MEK inhibition whereas phospho-mTOR was unchanged and pAKT was slightly lower.One candidate pro-survival pathway is the PI3K/AKT/mTOR pathway, the activity of which is reciprocally linked to MEK/ERK signalling27ATM mutations. We subjected 13 wild-type (n=6) and ATM mutant (n=7) cell lines to MEK inhibitor treatment for 6 h and determined the phosphorylation status of 4EBP1, AKT, mTOR and S6K. The individual cell lines did not respond identically, as expected from a set of (epi-) genetically heterogeneous cell lines. Strikingly, we observed a significant difference between the two groups for 4EBP1, mTOR and S6K, where the ATM mutant cell lines consistently displayed diminished or inverted feedback response  model carrying a heterozygous ATM mutation (F858L) on the background of an activating KRAS mutation. This ATM mutation has been associated with increased radiosensitivity and cancer susceptibility, indicating impaired signalling function31ATM in the sensitivity to selumetinib, lack of an isogenic control prevented us to rule out that other factors  are chiefly responsible for the response to MEK inhibition in this model.Next, we addressed whether the sensitization to MEK inhibition through ATM depletion in lung cancer cell lines was paralleled by a tumour response to MEK inhibitors in vivo  protein and it is likely that different domains are critical for its various functions and it is conceivable that some ATM mutations impact these non-canonical functions while maintaining largely normal DNA damage signalling. Indeed, some of the MEK sensitive, ATM mutant cell lines responded to IR by phosphorylating KAP1 and SMC1. Although this may suggest normal ATM function in the DDR, other (not tested) functions of ATM could be affected in these cell lines. Thus, a seemingly normal response to IR in ATM mutant cells should not be interpreted as compelling evidence for normal ATM function. Inversely, however, absence of ATM kinase activity likely indicates a broad functional defect in ATM.RAS or BRAF mutant lung cancer. However, these mutations alone do not adequately predict response to MEK inhibitionRAS and BRAF wild-type lung cancer cell lines display strong dependency on MEK. Indeed, the most sensitive cell line in our panel (EBC-1) is KRAS and BRAF wild type but carries an ATM mutation. These observations indicate that the determinants of sensitivity to MEK inhibitors in lung cancer are still largely unresolved. Yet, unraveling the precise molecular requirements for MEK inhibitor efficacy will likely be a key determinant for the clinical success of these drugs in this highly challenging and genetically heterogeneous tumour type. Until now, only experimental compounds  and gamma-H2AX (S319) antibodies were obtained from Millipore. SMAD4, ATM (2C1) and 53BP1 (H300) antibodies were purchased from Santa Cruz Biotechnology, β-actin from Sigma-Aldrich, phospho-KAP1 (S824) and KAP1 antibody from Bethyl Laboratories. All the other antibodies were from Cell Signaling Technology. All antibodies were used at a 1:1,000 dilution, except for α-tubulin that was used at a 1:5,000 dilution. Pemetrexed was obtained from Santa Cruz Biotechnology, TAK-733, trametinib, crizotinib, KU60019 and MK2206 from Selleck. Etoposide, paclitaxel, vinblastine, irinotecan, topotecan, gemcitabine, ifosfamide and neocarzinostatin (NCS) were purchased from Sigma. All other compounds were purchased from SynThesis Medchem (China).MEK constructs were obtained from Addgene. To generate mutants, a QuikChange Site-Directed Mutagenesis Kit (Agilent Technologies) was employed. Introduced point mutations were verified by Sanger sequencing and mutants were shuttled into a gateway compatible pBABE-puro vector. All shRNAs were cloned into lentiviral pLKO.1-puro vector .shRNAs for tumour suppressors were introduced into cells by lentiviral transduction followed by puromycin selection. All of them were validated using western blotting or qRT–PCR. For the STK11 and NF1 isogenic cell lines, untransformed AALE cells were used. For all other tumour suppressors, HRAS-V12G transformed cells were employed. Stable cell lines were then individually tagged with DNA barcoded lentivirus and pooledFor determining screening conditions, the drug concentration resulting in 50% AALE cell killing (IC50) was determined by performing 9-point dose–response experiments for all compounds. Based on these results, three concentrations were selected for the screen (IC50 and IC50 −/+ 4-fold). One day after seeding pooled cells, drugs (or DMSO) were added in quadruplicates. After 6 days, genomic DNA was extracted and the barcodes were amplified by PCR using a biotinylated primer, labelled with streptavidin–phycoerythrin, and hybridized to Luminex xMAP beads coupled to the antisense barcode sequence. Samples were measured on a Flexmap 3D plate reader (Luminex). Data were analysed1616Unless indicated otherwise, all cell viability assays were performed using the Cell Titer-Glo assay (Promega). Cells were counted and seeded in 96-well plates in triplicates. Next day, compounds were added and 3–5 days later, cell viability was assessed. AUC and EC50 determination was performed using GraphPad Prism. The percentage deviation from Bliss independency model was calculated by using the following formula:E is the effect on viability of drugs x and y expressed as a percentage of the maximum effect.Here, Chou–Talalay drug combination (CI) indices were calculated by using the formula:Edrug1 is the effect (in percent of maximum effect) of drug 1 alone, Edrug2 the effect of drug 2 alone and Eci is the effect of both drugs combined. A drug combination index <1 is considered synergistic.Here For colony formation assay, 10,000 cells were seeded on six-well plates and treated with drug or vehicle control for ∼3 weeks until clear colonies were formed. Colonies were fixed with 3.7% formaldehyde and stained with 0.1% crystal violet.For determination of apoptosis, cells were treated with MEK inhibitor or vehicle and analysed for annexin V positivity (Biolegend) and DNA content  by FACS.GAPDH as a control gene.RNA was extracted using an RNeasy MinElute Cleanup kit (Qiagen). Isolated RNA was then subjected to DNAse treatment . Reverse transcription was carried out using random hexamer primers and RevertAid reverse transcriptase (Fermentas). Quantitative real-time PCR was performed employing the KAPA SYBR FAST ABI Prism (Peqlab). Analysis was carried out in triplicates, using Cells were lysed in RIPA lysis buffer  supplemented with protease and phosphatase inhibitors. Lysates were sonicated, centrifuged and cooked with reducing sample buffer. Protein samples were separated by SDS–polyacrylamide gel electrophoresis on 4–12% gradient gels (Invitrogen) and transferred onto PVDF or nitrocellulose membranes. Quantification of band intensity on digital images was done in ImageJ and intensity of phosphorylation normalized to total protein staining. Uncropped images of all the blots in main figures are included as ATM target region located in exon 6 was amplified by PCR using flanking primers  and subsequently analysed by Sanger sequencing. Confirmation of monoallelic indels was determined by TA cloning of individual PCR products into a pCR 2.1 Vector  followed by Sanger sequencing of bacterial colonies.Cells were plated at high density and co-transfected with a gBlock (IDT) encompassing the guide RNA (5′-GGATGCTGTTCTCAGACTGACGG-3′) expression cassette and a plasmid encoding the Cas9 nucleaseTo restore the mutated ATM allele in NCI-H23 cell line, cells were plated at high density and co-transfected with a plasmid encoding the Cas9 nuclease, a PCR product encompassing the guide RNA (5′-ACTACATGAGAAGACCAAAGAGG-3′) and a double-stranded 120 bp oligonucleotide containing the wild-type sequence (5′-TGCTGTTTGGATAAAAAATCACAAAGAACAATGCTTGCTGTTGTGG ACTACATGAGAAGACAAAAGAGGTAATGTAATGAGTGTTGCTTCTTACGTTTAGGATCTAGAG TGTAACTTGTT-3′). An oligonucleotide containing a silent codon change resulting in the same amino-acid substitution present in NCI-H23 was used as a control. After selection with puromycin, cells were plated and treated with DMSO, trametinib or TAK-733 for 3 weeks. DNA was isolated from surviving colonies and the mutated locus was amplified by PCR using flanking primers  followed by Sanger sequencing.Potential off-target sites for the CRISPR sgRNA were predicted using the online tool crispr.mit.edu. Top five predicted exonic off-target sites were amplified from two independent NCI-H322 knockout clones and one control NCI-H322 wild-type clone by PCR using specific primers . PCR products were cleaned up by ExoSAP (Affymetrix) and labeled with BrightDye Terminator Kit (Nimagen). Samples were purified by gel filtration through Sephadex resin (Sigma) and sequenced on ABI 3500 Genetic Analyzer (Applied Biosystems).Data sets were downloaded from the respective data portal  and mutation and drug sensitivity data was compiled in a single file. Only drug sensitivities were considered that had been tested on >80% of the lung cancer cell lines. All data was analysed in PRISM.Polyphen analysis was performed using the online PolyPhen V2 tool. Mutations were considered damaging when the score was >0.9.ATM and other lung-cancer genes in the database and the simulated cohorts.For mutation co-occurrence analysis, we looked at mutation profiles defined in the COSMIC database for cancer census genes (v70). We simulated randomized cohorts while keeping constant the empirical gene-wise mutation rates and the patient-wise mutation burden. Then, we compared the co-occurrence of mutations in −1 NCS or 2 μM TAK-733 for 30 min and 24 h, respectively, and DMSO treatment used as a control. Cells were allowed to recover for 2 h, washed twice with ice-cold PBS and fixed with 4% PFA+0.1% Triton X-100 in PBS for 20 min on ice. Cells were permeabilized with 0.5% Triton X-100 in PBS for 20 min at room temperature and blocked with 10% FCS+0.1% Triton X-100 in PBS for 1 h with always three washes between individual steps. Primary and secondary  antibodies were diluted in blocking solution and incubated for 1 h at room temperature. Finally, cells were stained with DAPI  for 20 min at room temperature in the dark. Cell images were acquired on a deconvolution microscope (Leica).Cells were plated onto coverslips (VWR) in a 24-well plate. Next day, cells were treated with 50 ng mlV=1/2(L × W2), where L is the longest dimension (length) and W is width (shortest dimension). The experiment was not blinded. For the Kaplan–Meier survival curves, animals bearing tumours larger than 1,000 mm3 were considered as dead.Experimental procedures were approved by the Medical University of Vienna ethics committees and conform to Austrian regulations. NCI-H460  were injected subcutaneously in nude mice and allowed to form palpable tumours before randomization and starting treatment with pimasertib. Number of animals (four for each treatment arm) in the study was chosen based on a large expected effect size. Animals that did not form tumours were excluded from the experiment. Drug was administered daily per oral gavage and tumours were measured using calipers and tumour volume was estimated using Ki67 immunohistochemistry stainings were prepared using a Ventana Benchmark Ultra automated staining device, applying the CONFIRM anti-Ki67 rabbit monoclonal primary antibody  according to the manufacturer's protocol. Immunohistochemical stainings for cleaved Caspase 3 and phospho-4EBP1 (T37/46)  were performed according to the manufacturer's protocol.−1, daily, for 28 days. Vinorelbine was dosed i.v. at 5 mg kg−1 once a week, Carboplatin was dosed i.p. at 60 mg kg−1 once a week and Paclitaxel was dosed i.v. at 10 mg kg−1 once a week. All test agents were formulated according to the manufacturer's specifications. Beginning day 0, tumour dimensions were measured twice weekly by digital caliper and data, including individual and mean estimated tumour volumes (Mean TV±s.e.m.), are recorded for each group. Tumour volume was calculated using the formula: TV=width2 × length × π/6.For PDX models, a tumour sample was removed from the tibia of a NSCLC patient and a tumourgraft model was generated. Selumetinib was dosed orally at 100 mg kgThe authors declare that the data supporting the findings of this study are available within the paper and its How to cite this article: Smida, M. et al. MEK inhibitors block growth of lung tumours with mutations in ataxia–telangiectasia mutated. Nat. Commun.7, 13701 doi: 10.1038/ncomms13701 (2016).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Supplementary Figures 1-13 and Supplementary Tables 1-5Hit list from the small molecule screen for all ten tumor suppressors.Drug sensitivity data for lung cancer cell lines tested through the Cancer Cell Line Encyclopedia.


XL888, in combination with BRAF inhibitors for the treatment of therapy‐resistant melanomas. Unfortunately, our data show that this combination elicits a heterogeneous response in a panel of melanoma cell lines including PDX‐derived models. We sought to understand the mechanisms underlying the differential responses and suggest a patient stratification strategy. Thermal proteome profiling (TPP) identified the protein targets of XL888 in a pair of sensitive and unresponsive cell lines. Unbiased proteomics and phosphoproteomics analyses identified CDK2 as a driver of resistance to both BRAF and Hsp90 inhibitors and its expression is regulated by the transcription factor MITF upon XL888 treatment. The CDK2 inhibitor, dinaciclib, attenuated resistance to both classes of inhibitors and combinations thereof. Notably, we found that MITF expression correlates with CDK2 upregulation in patients; thus, dinaciclib would warrant consideration for treatment of patients unresponsive to BRAF‐MEK and/or Hsp90 inhibitors and/or harboring MITF amplification/overexpression.Novel therapies are undergoing clinical trials, for example, the Hsp90 inhibitor,  In the majority of the cases, it harbors BRAF (~60%) or NRAS (~20%) mutation. Other notable genomics alterations include the tumor suppressor CDNK2A deletions, KIT aberrations, MITF amplifications (10–20%), PIK3 mutations, loss of NF1 , such as vemurafenib and dabrafenib, as standard of care for the treatment of BRAF‐mutant melanoma has significantly improved the response rate among patients , although the clinical benefit of immunotherapy is often more stable (~2 years) and patients with tumors lacking BRAF mutations can also benefit from such treatment  targets of Hsp90i has not been thoroughly investigated. The off‐targets might cause a paradoxical activation of mechanisms of resistance to the drug therapy as was shown previously for the BRAFi PLX4032  approach to investigate the protein targets of the Hsp90i XL888 in sensitive and resistant cell lines and tease apart their eventual differences in terms of drug targets so as to provide insight into the mechanisms of resistance to XL888. In parallel, we employed orthogonal unbiased proteomics and phosphoproteomics approaches, which offer a systems‐level understanding of the cell signaling pathways that contribute to the inherent unresponsiveness to Hsp90i and BRAFi classes. The results provided by these complementary approaches enabled us to design drug strategies to overcome melanoma resistance to both BRAFi and Hsp90i monotherapies and their combination. Our et al, et al, in vitro (A375 DR1 and MNT‐1 DR100) or in vivo  collected before treatment with vemurafenib M026.X1.CL) and after the onset of resistance due to an acquired NRAS mutation . Interestingly, XL888 drastically reduced the viability in the previously unresponsive SK‐Mel 24 cells, but not in SK‐Mel 28, or in cells with acquired resistance to BRAFi  and ATP levels were measured as a surrogate for cell viability. Our results indicate that treatment with the Hsp90i panel, with the exception of novobiocin, yielded complete loss of viability in SK‐Mel 24 . Overall, the two cell lines show poor overlap in terms of drug–protein engagement. Regarding the (few) shared entries in the intact cell layout (drug/DMSO), we observed stabilization of the chaperone CDC37, a known Hsp90 interactor  in SK‐Mel 28 proteome thermal stability of resistant versus sensitive cells in different settings using TTP identified six potential targets, whose inhibition could sensitize the resistant cells to XL888 (henceforth referred as Hsp90i): CDK2, CDK4, CDK6, CRAF, PAK2, PAK4, and STAT1.2 phosphopeptide enrichment step. Overall, we identified ~7,000 proteins and ~15,500 phosphosites with a localization probability > 0.75  or unresponsive (SK‐Mel 28) to XL888 treatments were grown under conditions as detailed in Fig y > 0.75  and EV3.y > 0.75 C.et al, et al, et al, In both cell lines, we observed upregulation of Hsp70 upon treatment with XL888 or BRAFi‐XL888 . Both SKet al, To gain insight into the differential Hsp90i response, we compared unresponsive SK‐Mel 28) versus sensitive (SK‐Mel 24) cells. However, as the two cell lines are not isogenic, we compared each cell line (upon treatment with Hsp90i) to its untreated control (DMSO) and subtracted the upregulated entries of the sensitive cells from those of the resistant cells  to be the main kinases involved in the phosphorylation events  and the phosphopeptides that were uniquely upregulated in SK‐Mel 28 were analyzed by kinase‐substrate enrichment analysis  to analyze further with other cell lines. We exploited the same subtractive rationale shown previously in Fig We applied the same subtractive strategy for the analysis of SK‐Mel 28 undergoing the combined treatment BRAFi‐Hsp90i/DMSO and a shortlist of twelve kinases was generated , and the overlapping analyses retrieved two proteins involved in regulation of cell cycle progression, for example, ATM and CDKN2A Fig E. Our pret al, Based on these results, we proceeded with further investigations using dinaciclib. Importantly, this CDK2i is already used in clinical trials against leukemia  and melanoma ; hence, its toxicity in patients has been already assessed favorably  in a panel of 11 BRAF‐mutated cell lines, including two PDX‐derived cell pairs, obtained before BRAFi treatment, M026.X1.CL and M029.X1.CL, and after treatment, upon tumor relapse, M026R.X1.CL and M029R.X1.CL, respectively , a manually curated database from which the over‐representation of transcription factors in a dataset is predicted  between MITF and CDK2 mRNA expression levels  containing 935 cell lines. Among the different cancer types, melanoma‐derived cell lines exhibited the highest and most significant correlation  among the available cancers  transcriptomics data. We observed a similar positive correlation between MITF and CDK2 in melanoma patients Fig B, where  cancers . FurtherOverall, our orthogonal analyses point to CDK2 as a candidate whose inhibition overcomes inherent resistance to BRAFi and Hsp90i (driven by MITF) and their combination in melanoma.et al, et al, et al, Considering that the list of driver oncogenes is enriched in protein kinases  that would explain the different response to XL888 treatment.Considering that TPP is biased against membrane proteome, we pursued complementary unbiased genomewide proteomics and phosphoproteomics approaches to gain a systems‐level understanding of the molecular mechanisms underlying the resistance.et al  and resistant cell lines/tumors. This refinement will certainly benefit from the in vivo analyses of patient‐derived material generated by the ongoing clinical trial studies (NCT01657591 and NCT02721459).To date, there is no strategy that would be able to predict (i) which patients would benefit from BRAFi‐Hsp90i/BRAFi‐MEKi‐Hsp90i and (ii) monitor their response. The only attempt to address these issues was performed by Rebecca et al , where tet al H; thus, et al E. TherefWe show that the resistance to Hsp90i can be overcome by targeting different kinases  in our model system; however, in‐depth analyses reveal that CDK2 is the only shared upregulated druggable kinase that governs resistance to both the BRAF and Hsp90 classes of inhibitors and the combination thereof.et al, et al, et al, We investigated the mechanisms that govern the CDK2 expression and in agreement with previous studies ; XL888 ; fludarabine ; CHIR‐99021 HCl (CT99021) ; palbociclib ; LDC000067 ; Ro 3306 ; roscovitine ; K03861 ; CHIR‐99021 ; dinaciclib ; FRAX597 ; PF‐3758309 ; AUY922 ; BIIB021 ; novobiocin ; 17‐DMAG . All the drugs were dissolved in DMSO (Sigma D2650).et al, et al, SK‐Mel 24, SK‐Mel 28, A375, A375DR1 , MNT‐1  and MNT‐1‐DR100  cells were grown in Gibco Medium Essential medium  supplemented with 10% FBS (15% for SK‐Mel 24), 1% non‐essential amino acids, 1% sodium pyruvate, 1% penicillin and streptomycin. ESTDAB37 and ESTDAB102 [received from The European Searchable Tumour Line Database (ESTDAB)], SKMEL2, M026.X1.CL, M026R.X1.CL, M029.X1.CL, and M029R.X1.CL , and one tablet of PhosSTOP phosphatase inhibitor mixture per 10 ml of lysis buffer (Roche Applied Science). The cell suspension per cell line (SK‐Mel 24 and SK‐Mel 28) and condition (+/− drug) was divided into ten aliquots of 100 μl and transferred into 0.2‐ml PCR tubes. TPP was performed as previously described  or drug (100 μM) for 2 h. Cells were harvested, resuspended in 1.1 ml of HBBS (Gibco) supplemented with 20 mM MgCl2, 1 mM sodium orthovanadate, 1 tablet of Complete mini EDTA‐free mixture (Roche Applied Science), and one tablet of PhosSTOP phosphatase inhibitor mixture per 10 ml of lysis buffer (Roche Applied Science)) by freeze and thaw, and the lysates were centrifuged at 20,000 × g for 30 min at 4°C to separate the soluble fractions from precipitates. Each cell line's supernatant was incubated with either DMSO or 100 μM drug at room temperature for 30 min. The cell suspension per cell line (SK‐Mel 24 and SK‐Mel 28) and condition (+/− drug) was divided into ten aliquots of 100 μl and transferred into 0.2‐ml PCR tubes. TPP was performed as previously described  supplemented with 20 mM MgClEach supernatant for cell line (SK‐Mel 24 and SK‐Mel 28); condition (+/− drug); and experiment (intact cells/lysate) was reduced by 2 mM DTT at room temperature for 1 h; alkylated by 4 mM chloroacetamide for 30 min at room temperature at the dark. A first enzymatic digestion was performed using Lys‐C (1:75 w/w) at 37°C overnight; a second enzymatic digestion was performed using trypsin (1:75 w/w) at 37°C overnight. One hundred μg of each sample was labeled by TMT10plex according to the manufacturer's instructions. Samples of each set (10 different temperatures) were mixed 1:1 (v/v) and cleaned by Strata™‐X‐C 33 μm Polymeric Strong Cation (Phenomenex). The whole procedure was executed in two biological replicates.g 30 min at 4°C), the protein concentration was determined by Bradford assay (Pierce). Proteins were reduced with 2 mM DTT at room temperature for 1 h, alkylated with 4 mM chloroacetamide at room temperature for 30 min in the dark. A first enzymatic digestion step was performed using Lys‐C at 37°C for 4 h (enzyme/substrate ratio 1:50). Moreover, the sample was digested overnight at 37°C with trypsin (enzyme/substrate ratio 1:50). Peptides were desalted by reverse phase using Waters Sep‐Pak 1cc (50 mg) cartridges . The resin was rinsed with ACN and then equilibrated with 0.6% acetic acid. The samples were loaded and washed with 0.6% acetic acid and eluted with 80% ACN/0.6% acetic acid.SK‐Mel 24 and SK‐Mel 28 were grown in four different conditions: in the presence of DMSO (control); 1 μM dabrafenib; 200 nM XL888; and 1 μM dabrafenib plus XL888200 nM each for 48 h. Cell pellets were harvested and resuspended in lysis buffer , and one tablet of PhosSTOP phosphatase inhibitor mixture per 10 ml of lysis buffer (Roche Applied Science)). Cells were then lysed by 10 rapid passages through a 23‐gauge hypodermic syringe needle and by sonication on ice. After centrifugation  complemented with a list of common contaminants and concatenated with the reversed version of all sequences. See Label‐free quantification was performed by analyzing the raw data by MaxQuant  was purchased from Promega .4 cells per well were cultured and treated with XL888 or dinaciclib for 48–72 h. Then, the cells were collected and rinsed in PBS, pelleted, and resuspended in incubation buffer  containing 1% annexin V and 1% propidium iodide (PI) for 10 min.To evaluate the presence of apoptosis/necrosis, we used annexin V‐Fluos (cat. no. 11828681001 Roche) and propidium iodide and analyzed by NovoCyte flow cytometer . Between 4 and 10 × 10et al, et al, et al, 2‐mediated transfection of HEK293T cells, and the produced lentiviral particles were used to infect SK‐Mel 28 cells together with 0.4 μg/ml hexadimethrine bromide, which were then selected for successful integration with 1 μg/ml puromycin over 5 days.Inducible shRNA constructs were created by ligating annealed shRNA‐coding oligonucleotides see  into an http://proteomecentral.proteomexchange.org) via the PRIDE partner repository  via the PRIDE partner repository  via the PRIDE partner repository (Vizcaino et al, The mass spectrometry proteomics and phosphoproteomics data have been deposited to the ProteomeXchange Consortium (AA performed cell viability experiments, FACS analyses, validation of the MS data by Western blot analysis, and cell viability experiments using dox‐inducible cell lines, designed the experiments, wrote the manuscript, and approved the final draft; SC performed drug assay experiments on multiple cell lines, designed the experiments, analyzed data, wrote the manuscript, and approved the manuscript; BS‐L performed cell viability assay, isobolograms, analysis of phospho‐TPP data, designed the experiments, wrote the manuscript, and approved the final draft; JB generated the dox‐inducible cell lines, analyzed the data, and approved the manuscript; JLR performed analysis of CCLE and TCGA data, contributed to the manuscript preparation, and approved the final draft; FE supplied the IHC images, contributed to the manuscript preparation, and approved the final draft; RT performed cell viability experiments, contributed to the manuscript preparation, and approved the final draft; KK and OK obtained the PDX‐derived cell lines, contributed to the manuscript preparation, and approved the final draft; DSP, JN, JH, SEB, MA, MU contributed to the manuscript preparation and approved the final draft; GM performed the TPP, phospho‐TPP, proteomics, and phosphoproteomics experiments, performed the data analyses, designed the study, analyzed the data, wrote the manuscript, and approved the final draft.The authors declare that they have no conflict of interest.AppendixClick here for additional data file.Expanded View Figures PDFClick here for additional data file.Dataset EV1Click here for additional data file.Dataset EV2Click here for additional data file.Dataset EV3Click here for additional data file.Dataset EV4Click here for additional data file.Dataset EV5Click here for additional data file.Dataset EV6Click here for additional data file.Dataset EV7Click here for additional data file.Dataset EV8Click here for additional data file.Code EV1Click here for additional data file.Review Process FileClick here for additional data file.


In results from the NCI 60-cell line screen, the lead conjugate 28 displayed potent and highly selective antiproliferative activity towards the MCF-7 human cancer cell line (IC50 = 5 nM). In the ER-binding assays, the lead conjugate 28 demonstrated potent ER competitive binding in ERα  and ERβ . Preliminary biochemical results also demonstrate that the lead conjugate 28 may exhibit pure antagonism. This series makes an important addition to the class of ER antagonists and may have potential applications in anticancer therapy.Nuclear-receptors are often overexpressed in tumours and can thereby be used as targets when designing novel selective chemotherapeutic agents. To date, many conjugates incorporating an estrogen receptor (ER) ligand have been synthesised in order to direct chemical agents to tissue sites containing ERs. A series of ER ligand conjugates were synthesised incorporating an antagonistic ER ligand scaffold based on endoxifen, covalently-bound via an amide linkage to a variety of combretastatin-based analogues, which may act as antimitotic agents. These novel endoxifen-combretastatin hybrid scaffold analogues were biochemically evaluated in order to determine their antiproliferative and cytotoxicity effects in both the ER-positive MCF-7 and the ER-negative MDA-MB-231 human breast cancer cell lines. ER competitive binding assays were carried out to assess the binding affinity of the lead conjugate  Estrogen receptors (ER), principally present as two main isoforms; ERα and ERβ, are found in abundance in female reproductive tissues such as the breast, uterus and ovary, while also found in bone, liver and brain tissue ,2,3,4,5.Various strategies attempt to improve the selectivity of chemotherapeutic agents by specifically targeting cancer cells and tumour environments ,12. Conj1H, 13C and 19F nuclear magnetic resonance (NMR) spectra were recorded at 27 °C on a Brucker Avance DPX 400 spectrometer   at 20 °C in either CDCl3 ) or CD3OD by Dr. John O’Brien and Dr. Manuel Ruether in the School of Chemistry, Trinity College Dublin. For CDCl3, 1H-NMR spectra were assigned relative to the TMS peak at 0.00 δ and 13C-NMR spectra were assigned relative to the middle CDCl3 triplet at 77.00 ppm. For CD3OD, 1H and 13C-NMR spectra were assigned relative to the centre peaks of the CD3OD multiplets at 3.30 δ and 49.00 ppm respectively. 19F-NMR spectra were not calibrated. Electrospray ionisation mass spectrometry (ESI-MS) was performed in the positive ion mode on a liquid chromatography time-of-flight (TOF) mass spectrometer , equipped with electrospray ionization (ES) interface operated in the positive ion mode at the High Resolution Mass Spectrometry Laboratory by Dr. Martin Feeney in the School of Chemistry, Trinity College and a Micromass spectrometer (E.I. Mode) by Dr. Dilip Rai at the Centre for Synthesis and Chemical Biology, University College Dublin. Mass measurement accuracies of <±5 ppm were obtained. Low resolution mass spectra (LRMS) were acquired on a Hewlett-Packard 5973 MSD GC-MS system  in electron impact (EI) mode. Rf values are quoted for thin layer chromatography on silica gel Merck F-254 plates, unless otherwise stated. Compounds were visually detected with UV at 254 and 366 nm. Flash column chromatography was carried out on Merck Kieselgel 60 (particle size 0.040–0.063 mm), Aldrich aluminium oxide,  or Aldrich aluminium oxide, . All products isolated were homogenous on TLC. Analytical high-performance liquid chromatography (HPLC) to determine the purity of the final compounds was performed using a Waters 2487 Dual Wavelength Absorbance detector, a Waters 1525 binary HPLC pump, a Waters In-Line Degasser AF and a Waters 717 plus Autosampler . The column used was a Varian Pursuit XRs C18 reverse phase 150 × 4.6 mm chromatography column . Samples were detected using a wavelength of 254 nm. All samples were analyzed using acetonitrile (70%): water (30%) over 10 min and a flow rate of 1 mL/min. Combretastatin A-4 (CA4) 26 was prepared as previously reported 1H, 13C a 1H, 13C a  211H, 132 . To. To51]. 13–24 were prepared in yields of 36%–71%  isomeric mixtures in high yield (88%–94%) following chromatographic purification. In the 1H NMR spectrum the characteristic protons of the ethyl group are observed in the region 0.90–0.96 ppm (CH3) and 2.40–2.50 ppm (CH2), the amine methyl group is found between 3.04 and 3.25 ppm, while the methylene protons of the basic side chain are identified at 3.53–4.20 ppm. The amide compounds 47–53 were also prepared by reaction of the acrylic acids 13, 14, 21 and 23 with pyrrolidine and piperidine using the Mukaiyama reagent (2-chloro-1-methylpyridinium iodide) as the coupling reagent. The amines 54 and 55 were obtained by reduction of the corresponding nitro compounds 50 and 51 using iron in hydrochloric acid.From the panel of combretastatin type acrylic acids elds See . The sil27–46 synthesised to inhibit the proliferation of the human breast tumour MCF-7 cell line was investigated using a standard MTT cell viability assay while the compounds were concurrently tested to assess the extent of their cytotoxicity using a LDH assay [50 value obtained for the control 26 (CA4) in this assay is 0.008 μM for MCF-7 and is in good agreement with the reported values for CA4 using the MTT assay on human MCF-7 breast cancer cell lines [50 value obtained for the control endoxifen 11 in this assay is 0.029 nM while the reported IC50 value for endoxifen in the MCF-7 cell line is 50 nM [The ability of the compounds DH assay . The MCFDH assay . Selectell lines ,68,69; tis 50 nM .26 (IC50 = 8 nM), . The substitution pattern in the A and B rings of 13 are similar to that of the potent combretastatin CA4. It is interesting that the compound 23, in which the A ring having the 3,4,5-trimethoxyphenyl substituent is positioned on the carbon β to the acrylic acid is inactive with IC50 > 50 μM. The acrylic acids 14, 16, 21 and the ester 24 all demonstrated IC50 values less than 10 μM against MCF-7 cell line. The antiproliferative activity of acrylic acids has been previously reported [47–55 prepared from pyrrolidine and piperidine were also evaluated for antiproliferative activity in the MCF-7 cell line (49 (IC50 = >50 μM in comparison with the acrylic acid 13 (IC50 = 0.120 μM) which suggested that a more bulky amide such as endoxifen may be required for activity in MCF-7 cells.In general, combretastatin acrylic acid derivatives have a lower antiproliferative potency compared to the lead compound, combretastatin A-4 (CA4), nM), see . The acrell line . The com50 values in the range 0.005–6.15 µM , endoxifen 11 (29 nM) and the novel hydroxyendoxifen analogue 12 (IC50 = 28 nM). In many examples, coupling of the acrylic acid to the novel hydroxyendoxifen 12 resulted in a more potent conjugate product than the coupling of the same acid with endoxifen 11, . Since there is much interest in combretastatin analogues having the 3-amino-4-methoxy substituion patern in Ring B [50 = 0.959 and 6.15 for compounds 45 and 46 respectively, these compounds were not as potent as the corresponding Ring B 3-hydroxy-4-methoxy substitute compounds 27 and 28. The ring B 3-fluoro-4-methoxy substituted compounds 43 and 44 displayed impressive antiproliferative activity with IC50 values of 180 and 194 nM respectively.The conjugates 27–46 displayed a wide range of antiproliferative activity with IC5 µM see . In all n Ring B  it was o29 and 30 substituted with 4-methoxy group in Ring B of the acrylic acid component, also display potent activity with IC50 values of 87 nM and 43 nM respectively. Compound 31 which contains 3,4,5-trimethoxy substitution pattern in the Ring B acrylic acid component and the 4-methoxy substituent on Ring A demonstrate a reduced activity (IC50 = 2.08 µM), however in compound 32 which is the corresponding conjugate with hydroxyendoxifen, the antiprolifertive activity is determined at 41 nM, indicating the potent effect on activity of conjugation with hydroxyendoxifen. Inclusion of the 4-thiomethyl substituent or the 3,4-methylenedioxy substitution on Ring B of the acrylic acid component resulted in potent activity for the hydroxyendoxifen conjugated compounds 34 and 40 (IC50 = 49 nM and 64 nM respectively). Introduction of the 2-naphthyl Ring B as the acrylic acid component resulted in a reduced antiproliferative effect for compounds 36 and 37 . It would have been interesting to determine whether the addition of estradiol would alter the IC50 values in Conjugate compounds 27–36, 41–46) as expcted displayed reduced antiproliferative activity  compared with results obtained for the ER-positive MCF-7 cell line, indicating that the conjugates show a selectivity for the ER expressing cell line  and 4-hydroxytamoxifen (IC50 = 18 µM) in this ER negative cell line.The most active compounds from the MCF-7 assay were selected for evaluation in the ER-negative MDA-MB-231 human breast cancer cell line. The selected conjugates . These binding values are greater than the endogenous ligand estradiol, 4-hydroxytamoxifen, endoxifen 11 and significantly greater than that of the parent ER-ligand, hydroxyendoxifen 12   see .28 was evaluated in the National Cancer Institute  Division of Cancer Treatment and Diagnosis(DCTD)/Developmental Therapeutics Programme(DTP) in which the activity of the compound is determined using a 60-cell line screen facility of different cancer cell lines of diverse tumour origin [50) and cytotoxicity(LC50). These studies were performed at the NCI as part of their drug screening programme. Initially, the compound was evaluated against the 60 cell lines at a single dose of 10 μM; if significant growth inhibition was exhibited the compound was evaluated against the 60 cell panel at a further five concentration levels, 0.01–100 μM,  and NCI-H460 (98%); colon cancer HCT-116 (98%); breast cancer MCF-7 (94%) and MDA-MB-435 (91%); ovarian cancer OVCAR-8 (97%) and SK-OV-3 (95%); leukaemia RPMI-8226 (91%); renal cancers ACHN (96%), CAKI-1 (91%), RXF 393 (95%) and SN12C (90%); melanoma SK-MEL-2 (97%) and CNS cancer U251 (93%). The compound caused between 80%–89% growth inhibition in a further 14 cell lines and was very toxic to the non-small cell lung cancer NCI-H226  and melanoma SK-MEL-5  cell lines.On the basis of potency, compound r origin . Compoun μM, see . In the 28 displayed low micromolar GI50 (IC50) values for the majority of the 60 cancer cell lines. However, 28 demonstrated a high selectivity towards MCF-7 breast cancer with a GI50 (IC50) value of 9.5 nM and a LC50 value greater than 50 μM for this cell line. The mean GI50 value compound 28 across all 60 panel cell lines is 1.45 µM, (log GI50 = −5.79). As 28 has been shown to be a high-affinity ER-binding ligand, the high specificity towards MCF-7 cells is most probably due to its selective antagonistic effects on the over-expressed ER within this cell-line. This is an impressive and promising result as it confirms the suitability of the selected prototypes and project strategy for the possible therapeutic application of the ER-conjugates synthesised in the study. The LC50 value is a measure of the cytotoxicity of the compound in each cell line; the mean LC50 for compound 28 is 46.77 µM and was greater than 50 µM in all but 4 cell lines. . The mean LC50 for 28 across all 60 panel cell lines is 46.77 µM;  by the Ishikawa cell line of human endometrial adenocarcinoma cells . The Ish27, 31 and 32 was evaluated in phosphate buffer at pH values in the range 4–9 and the half-life was determined to be greater than 24 h for each compound at these pH values. The compounds were found to stable without any significant degradation of the conjugates suggesting that the combretastatin fragment remains intact at physiological pH; and indicating that the combretastatin component of the conjugate has a role in displacing helix-12 of the ER resulting in the potent (and possibly pure) antagonistic activity.The stability of the selected target compounds The intricate molecular basis of estrogen receptor agonism and antagonism has been well studied ,79,80,8128 was modelled in both ERα and ERβ isoforms in order to rationalise the biochemical data obtained for this compound. The modelling strategy protocol involved calculating the appropriate protonation state at physiological pH using MARVIN [The interactions of compound g MARVIN ; the detg MARVIN ; the geng MARVIN , followeg MARVIN . Graphicg MARVIN . High-ra28 is shown in 28 is found 3.9 Å away from the His524 residue and does not form a strong H-bond. The Asp351 residue is located 3.7 Å away from the amide nitrogen of 28, which would translate to a weak interaction. The Cys530 residue forms a H-bond with the C-4 methoxy group on the A-ring of the combretastatin fragment as illustrated in 28 may displace Helix-12 to a significant extent and could explain the antagonistic activity and impressive binding affinity of this lead compound 28 .The top-ranking docking solution in human ERα for compound 28 is shown in 28 forms an additional hydrogen bond interaction with the His430 residue. There is good agreement/overlap of the raloxifene core and the endoxifen moiety of 28. A methoxy group present on the combretastatin moiety of 28 forms another favourable hydrogen bond through a water molecule with the Asp258 residue within the LBD. This interaction could help “lock in” the ligand within the cavity site, resulting in the good binding affinity. Additional waters unaccounted for in this determined crystal structure may introduce further favourable hydrogen bond interactions with the other methoxy groups of the combretastatin moiety through a network of water molecules. The alkyl groups of Ile328 are in close proximity to a phenyl group present on the endoxifen moiety, which could produce a lipophilic-lipophilic interaction. The bulky side chain of 28 would have a greater degree of rigidity due to the presence of the amide linkage and may displace Helix-12 significantly in ERβ to produce antiproliferative effects observed.The top-ranking docking solutions in rat ERβ for compound 27–46 were successfully synthesised incorporating an endoxifen-combretastatin hybrid scaffold with potential SERM properties. A number of these novel compounds displayed potent antiproliferation activity in the MCF-t human breast cancer cell line with low cytotoxicity values. The conjugate 28 in the series was the most promising compound in the study, with potent antiproliferative activity in MCF-7 human breast cancer cell line (IC50: 5 nM), low cytotoxicity and impressive ER competitive binding IC50 values . A number of these conjugate compounds also display activity in the ER negative cell line MDA-MB-321 at low micromolar and sub-micromolar concentrations, e.g., compound 34 (IC50 = 0.68 µM). Preliminary stability studies show that these conjugates do not degrade significantly at physiological pH values. Interestingly, from a structural biology standpoint, this result indicates that the ER can tolerate a large substituent such as the combretastatin analogue, at the basic side chain of the triarylethylene ER ligand scaffold without detrimental effect on the overall antiproliferative and ER binding characteristics and biochemical activity. The lead conjugate compounds 27 and 28 are currently under further investigation by our research group to further explore its mechanism of action and potential applications in the development of useful ER ligands. These compounds demonstrate a novel and interesting class of ER ligand and may have potential future application as medicinal agents for anticancer therapy.A series of prototype ER-ligand conjugates


In metastatic cells, pAKTThr308 is highly expressed, contributing to anoikis resistance. We showed that PDK1Ser241 and PKCβIISer660 are activated by Timp1 in different stages of melanoma progression, contributing to colony formation and anoikis resistance. Moreover, simultaneous inhibition of Timp1 and AKT in metastatic cells resulted in more effective anoikis inhibition. Our findings demonstrate that Timp1 promotes cell survival with the participation of PDK1 and PKC in melanoma. In addition, Timp1 and AKT act synergistically to confer anoikis resistance in advanced tumor stages. This study brings new insights about the mechanisms by which Timp1 promotes cell survival in melanoma, and points to novel perspectives for therapeutic approaches.High TIMP1 expression is associated with poor prognosis in melanoma, where it can bind to CD63 and β1 integrin, inducing PI3-kinase pathway and cell survival. Phosphatidylinositol -trisphosphate (PIP3), generated under phosphatidylinositol-3-kinase (PI3K) activation, enables the recruitment and activation of protein kinase B (PKB/AKT) and phosphoinositide-dependent kinase 1 (PDK1) at the membrane, resulting in the phosphorylation of a host of other proteins. Using a melanoma progression model, we evaluated the impact of Timp1 and AKT silencing, as well as PI3K, PDK1, and protein kinase C (PKC) inhibitors on aggressiveness characteristics. Timp1 downregulation resulted in decreased  Cutaneous melanoma is a melanocytic tumor whose incidence and mortality are on the rise worldwide. Although melanoma accounts for only 4% of all skin cancers, its mortality rate corresponds to 74% of those related to skin cancer in general . The oveMAPK signaling pathway (mitogen activated protein kinase), including the cascade of proteins NRAS, BRAF, MEK1/2, and ERK1/2, is one of most-studied signaling pathways in melanoma. This path is considered the main route changed in melanoma and is involved in cell survival, proliferation, and migration, and is related to both development and melanoma progression .The phosphatidylinositol-3-kinase (PI3K) pathway is one of the most important pathways in cancer metabolism and growth. One of the key effector proteins in this pathway is known as PKB (protein kinase B) or AKT, which has three isoforms , with AKT3 being found frequently activated in melanomas . Howeveranoikis and form colonies in soft agar [anoikis-resistant phenotype through the PI3K signaling pathway [anoikis resistance phenotype. We also observed that the simultaneous deletion of Timp1 and AKT prevents more efficiently cell survival, providing a possible new therapeutic strategy for metastatic melanoma.In our laboratory, we developed a model to study different stages of melanoma progression. Murine melanocytes were subjected to sequential cycles of substrate adhesion impediment, thereby obtaining different cell lines representing pre-malignant melanocytes, non-metastatic melanoma, and metastatic melanoma . These coft agar . Moreoveoft agar . In addi pathway . Howeveranoikis-resistant phenotype to both the murine melan-a melanocyte lineage and melanoma cell lines [anoikis resistance in 4C, 4C11−, and 4C11+ cell lines silenced for Timp1. Only the melanoma cell lines 4C11− and 4C11+ were sensitized to cell death via anoikis in the absence of Timp1, as shown by MTT assay  by Western blot assay. We can see that only metastatic melanoma cell line 4C11+ has activated AKT  in the PI3K pathway is modulated by Timp1, we analyzed PDK1, a molecule activated after being recruited to PIP3, in the cell lines silenced for Timp1. Recently, several studies have investigated the role of PDK1 in tumors and there is evidence that PDK1 can activate other molecules involved in cell survival, senescence, and proliferation independent of its AKT phosphorylating activity [We found that Timp1 is highly expressed from the pre-malignant 4C cell line to metastatic cell line 4C11+ and that this high expression leads to increased activity ,14,15. Wanoikis resistance. The above data indicate that Timp1 is related to the PDK1 activation, which could be related to anoikis resistance. To confirm this hypothesis, we submitted cell lines to the treatment with a PDK1 inhibitor, GSK2334470 , 200 nM 12-phorbol-13-myristate acetate , 100 U/mL penicillin, and 100 U/mL streptomycin (Invitrogen) [Cell culture: Non-tumorigenic melan-a melanocyte lineage was cultured at 37 °C in humidified 95% air-5% COitrogen) . A murinitrogen) .Briefly, cell lines were established after melan-a melanocytes were submitted to 1–4 cycles of substrate impairment adhesion , giving rise to cell lines representing pre-malignant cells. Pre-malignant 4C melanocytes were subjected to a new cycle of anchorage blockade and the spheroids formed were submitted to a limiting dilution, giving rise different melanoma cells lines  . Pre-malReagents: All reagents were diluted in DMSO at the following concentrations: 1 µM LY294002 (PI3K inhibitor), 1 µM GSK2334470 (PDK1 inhibitor) and 2 µM Bisindolylmaleimide I (PKC inhibitor). All reagents were purchased from Sigma-Aldrich .® shRNA Plasmid DNA (pLKO.1-puro) from Sigma-Aldrich. Two plasmids carrying different shRNA sequences for Timp1  and a control plasmid containing a non-target sequence . These sequences were previously validated for the target. Lentiviral particles were produced by the calcium phosphate precipitation method in 293T cells, co-transfecting the lentiviral plasmids containing the shRNA sequences, with packaging plasmids. Viral supernatants were aliquoted and stored at −80 °C. Lentiviral titers were determined by limiting dilution and puromycin selection in HT1080 cells  and quantified as the number of transducing units (TU) per milliliter. These lentiviral particles were used to establish cell lines with knockdown of Timp1 or expressing the non-target shRNA sequence as a control. All transductions were performed using a MOI (multiplicity of infection) of 0.5, in the presence of 8 µg/mL polybrene. After transduction puromycin-resistant cells were selected. The target silencing was verified by RT-qPCR.Stable silencing of Timp1 by shRNA: Timp1 was silenced by shRNA—MISSION4 cells per well) and maintained in culture for 24 h. Nine microliters of Lipofectamine® RNAiMAX Reagent  were diluted in 150 μL of Optim-MEM® medium (Life Technologies). Thirty picomoles of siRNA  were diluted in 150 μL of Optim-MEM® medium (Life Technologies). Both solutions containing Lipofectamine® RNAiMAX Reagent (Life Technologies) and siRNA  were mixed and incubated for 5 min at room temperature. After 5 min, the solution containing the siRNA was dripped into wells. The cell lines were maintained in culture for 48 h and, after that time, cells were used for anoikis resistance assay. For the control, cells lines were transfected with a non-targeting negative control siRNA  as described above. The negative control has the same chemical modifications as Silencer® Select siRNA but without targeting any RNA.Transient silencing of AKT: Metastatic melanoma cell line 4C11+ was plated in six-well plates , kept for 15 min on ice, followed by centrifuging at 10,000 rpm for 15 min at 4 °C. The supernatant was collected and the protein concentration was measured by Bio-Rad protein assay dye reagent concentrate . Equivalent amounts of protein (50 μg) were denaturated in SDS-sample buffer  for 5 min, then separated by electrophoresis in SDS-polyacrylamide gels and transferred to a polyvinylidenedifluoride membrane . After protein transfer, the membranes were blocked with 5% non-fat dry milk in PBS , incubated with the indicated antibodies: anti-GAPDH , anti-actin , anti-phospho-AKTThr308  and anti-AKT , anti-phospho-PDK1Ser241 , anti-PDK1 , anti-phospho-PKC(pan)(βIISer660) , anti-PKC , caspase-3 , and Timp1  overnight at 4 °C and the signal was detected using horseradish peroxidase-conjugated anti-immunoglobulin G antibody  followed by development using chemiluminescence substrate .Western blotting: Subconfluent cell cultures were trypsinized, washed with PBS and membrane-enriched protein extracts were prepared using cold NP-40 lysis buffer   was added to the culture. The cells were kept in an incubator (37 °C and 5% CO2) for one hour. MTT was removed from the wells by inverting the plate and 100 µL of isopropanol were added to each well . After 15 min, the absorbance was measured with a spectrophotometer (OD = 620 nm) . The same procedure described above was carried out to determine viable cells both after treatment with the drug and anoikis resistance assay.MTT assay: To estimate cell proliferation, 2 × 10Anoikis resistance assay: Cell lines 4C wild-type, 4C non-target (TRC2), 4C shTimp1#2, 4C shTimp1#3, 4C11− wild-type, 4C11− non-target (TRC2), 4C11− shTimp1#2, 4C11− shTimp1#3, 4C11+ wild-type, 4C11+ non-target (TRC2), 4C11+ shTimp1#2, 4C11+ shTimp1#3, 4C11− non-target (AKTi CTL), 4C11− AKTi, 4C11+ non-target (AKTi CTL), and 4C11+ AKTi (5 × 104) were maintained for 96 h in 100 mm2 dishes coated with 1% agarose, with or without treatment with specific inhibitors. After 96 h, cell viability was assessed by MTT and/or analyzed for caspase-3 cleavage by Western blotting.6) were treated or not with the inhibitors. After 24 h of treatment, cells were trypsinized and 200 cells were plated to visualize the formation of colonies. After 10 days, the colonies were washed with PBS, fixed with 3.7% paraformaldehyde for 15 min, stained with 1% toluidine blue for 5 min and washed with water. For quantification of viable cells, the cells were lysed and the dye solubilized in 1% SDS for 4 h and the absorbance at 620 nm was measured using a plate reader.Colony formation assay (clonogenicity). Cell lines (103) were plated in 96-well plates. Twenty-four hours after cell adhesion, serial dilution of the drug was accomplished in the following concentrations: 0, 200, 400, 600, 800, and 1000 µM in order to determine the IC50. After 48 h, cell viability was assessed by MTT assay.Determination of IC50 dose: To determine the IC50 dose of the drug dacarbazine, melanoma cell lines (2.5 × 105) were inoculated in the subcutaneous tissue of the dorsal region of female C57BL/6 mice . After 12 days, tumor formation was observed and measured every two days. On day 18, mice were sacrificed and the tumor weight determined. All procedures involving animals were performed after approval from the Research Ethics Committee of the Universidade Federal de São Paulo, Brazil .Tumor growth assay: Cells (2 × 105) were inoculated on the lateral tail vein of female C57BL/6 mice. After 21 days, the animals were sacrificed and the presence of colonies in the lung was analyzed.Lung colonization assay: Cells . The level of significance utilized was anoikis resistance in metastatic melanoma cells.Our data provides evidence that Timp1 confers cell survival by activating PDK1 signaling pathway and that Timp1 and AKT have synergistic effects to confer