BRAFV600E, but tumors treated with RAF inhibitors, even when they respond dramatically, rapidly adapt and develop resistance. Thus, there is a pressing need to identify the major mechanisms of intrinsic and adaptive resistance and develop drug combinations that target these resistance mechanisms. In a combinatorial drug screen on a panel of 12 treatment-naïve BRAFV600E mutant melanoma cell lines of varying levels of resistance to mitogen-activated protein kinase (MAPK) pathway inhibition, we identified the combination of PLX4720, a targeted inhibitor of mutated BRaf, and lapatinib, an inhibitor of the ErbB family of receptor tyrosine kinases, as synergistically cytotoxic in the subset of cell lines that displayed the most resistance to PLX4720. To identify potential mechanisms of resistance to PLX4720 treatment and synergy with lapatinib treatment, we performed a multi-platform functional genomics analysis to profile the genome as well as the transcriptional and proteomic responses of these cell lines to treatment with PLX4720. We found modest levels of resistance correlated with the zygosity of the BRAF V600E allele and receptor tyrosine kinase (RTK) mutational status. Layered over base-line resistance was substantial upregulation of many ErbB pathway genes in response to BRaf inhibition, thus generating the vulnerability to combination with lapatinib. The transcriptional responses of ErbB pathway genes are associated with a number of transcription factors, including ETS2 and its associated cofactors that represent a convergent regulatory mechanism conferring synergistic drug susceptibility in the context of diverse mutational landscapes.Fifty percent of cutaneous melanomas are driven by activated  BRAFV600E as an oncogenic driver is demonstrated by the rapid response of most BRAFV600E mutant melanomas to the BRaf inhibitors vemurafenib or dabrafenib [BRAFV600E mutant melanomas showed objective response after treatment [The BRaf kinase is activated by mutation from Valine to Glutamate at position 600 (V600E) in approximately 50% of cutaneous melanomas , resultibrafenib ,5: Over reatment ,5.MITF overexpression has been shown to repress the ability of BRaf inhibition to kill melanoma [MITF activity, class III and class IV melanosomes can sequester drugs [MITF activity has been implicated as an indication of resistance to BRaf inhibition [BRAF splice variants facilitate dimerization with RAF1 and result in MEK activation [MEK can be activated by mutation or by activation of COT [PI3K activation [Unfortunately, response to vemurafenib is not durable, with most patients relapsing within 2 to 18 months of initial treatment –8. Severmelanoma ,10 and ier drugs . In morehibition ,13. Finativation ; MEK cann of COT ; the cyttivation ; and thetivation –19. LessBRAFV600E mutant melanoma cell lines, with a wide range of intrinsic sensitivity to the BRaf inhibitor PLX4720, in combination with a library of 60 secondary drugs and tool compounds. Notably, Held et al. [BRAFV600E melanoma cell lines. We also found that the 6 cell lines that were most resistant to PLX4720 displayed synergistic cytotoxicity with lapatinib. In order to determine mechanisms of resistance to PLX4720 and synergy to lapatinib as well as help develop systematic approaches to better predict which combinations might be effective/synergize, we performed a functional genomics and genetics profiling of the12 melanoma cell lines. Novel results from our study include coupling the roles of mutant BRAF zygosity and mutations in RTKs in determining basal drug resistance to broad up-regulation of ErbB pathway genes including ErbB family RTKs in response to PLX4720 treatment. Further analysis revealed enrichment of transcription factors including ETS family members and their associated co-factors as likely regulatory drivers of adaptive PLX4720 resistance, providing a potential convergence point of adaptive resistance within the diversity of response mechanisms.Combination therapies are predicted to overcome intrinsic, adaptive and acquired resistance . For resd et al.  found PLMITF expression level that correlated weakly with drug sensitivity. Dividing the cell lines into groups based on unsupervised clustering of all the single drug and combination cytotoxic responses across a three by three dose response matrix yielded five phenotype groups. Strikingly, these cytotoxicity groupings closely mimicked the groupings observed in the basal transcriptome based on a principal component analysis (PCA). The transcriptional and proteomic responses to PLX4720 treatment were then analyzed to identify molecular responses that were common between the cell lines in each group. The lists of differentially expressed genes and phosphoproteins were subjected to the Mutational Signatures Database (MSigDB) [In order to gain insights into the mechanisms of synergy and sensitivity, and potentially to identify clinically relevant biomarkers, we broadly profiled our panel of lines with multiple functional genomic and genetic assays . Analysi(MSigDB)  enrichme(MSigDB)  to identTo determine whether the transcriptional profile of treatment-naïve cells could predict sensitivity to the drugs, singly or in combination, we classified the 12 cell lines based on unsupervised clustering of the basal (not drug-treated) transcriptome . ClusterTBL1X, IL6, CST7, NRG1, CPA4, SERPINB7, STC2, CALB2, RAB27B) that all contain the JUN motif in their promoter. Among these genes, TBL1X and CALB2 were the only two that were expressed at a relatively high level in both A375 and DM331 while the rest including NRG1 were highly expressed in DM331 only. We note that exposure to NRG1 has been shown to induce resistance to PLX4720 treatment in A375 cells [NRG1 in DM331 contributes substantially to DM331’s intrinsic resistance to PLX4720 treatment.Cluster III (49 genes) contained relatively highly expressed genes in DM331, which is of interest because it is the most resistant line to PLX4720 treatment. A subset of the genes in this cluster was also relatively high-expressed in A375, our second most sensitive line to PLX4720 treatment. To identify the potential functional significance of the genes within cluster III, we performed a MSigDB enrichment analysis using the transcription factor gene set . The top75 cells . DespiteMITF as a hub with 32 MITF target genes  of which 32 are shown in MITF transcriptional program driving this separation. To confirm whether MITF is a regulatory hub of cluster IV genes, we compared the genes in cluster IV with MITF target genes identified by Strub et. al. via a ChIP-Seq experiment [MITF and the expression of its associated targets as potentially driving lineage separation in our cell lines, but not contributing consistently to drug sensitivity.Clustering the expression profile of the 73 genes in Cluster IV separates our cell lines into two distinct groups. Ingenuity Pathway analysis of this list of 73 genes revealed that cluster IV contained periment  and obseMITF expression is associated with response to PLX4720 treatment in our panel, we correlated MITF expression in each of our lines with their respective IC50 values. We observed a modest anticorrelation of -0.40 between the MITF expression level of our lines, and their respective IC50 doses, consistent with Konieczkowski et al. [MITF gene expression and IC50 across the remaining eleven cell lines. Because our synergy studies were performed over a dose range that included concentrations below the IC50 (centered around IC20), we also examined the correlation of MITF expression and cytotoxicity at the low, medium and high doses. We found a relatively strong anticorrelation between MITF gene expression level and PLX4720 cytotoxicity (excluding DM331): -0.79, -0.68 and -0.67 for our low, medium and high doses, respectively. Notably, our PLX4720 doses, which were intended to assess drug synergy, are much lower, 125 nM, in our studies compared to other studies which tend to use ~1μM PLX4720 [MITF across our cell lines are associated with increased sensitivity to PLX4720 treatment while at IC50 doses, we find essentially no correlation.To assess the extent to which the i et al. . In our  PLX4720 –13,19. TMITF expression level in our lines  which reveals dominant sources of variability in the first principal component as well as sub-dominant sources of variability in the second and higher order principal components. As shown in the PCA plot , the sepThe strong correlation of the second principal component with the Bliss synergy score of the cell lines raised the possibility that the basal transcriptional profile could yield insights into mechanisms of synergy and resistance. To explore this possibility, we correlated each gene expression profile across cell lines with PC2  and idenNRG1, a known mechanism of PLX4720 resistance [Given the modest association between basal gene expression and synergy, we wanted to determine if there was a phenotypic classification that better correlated with the basal gene expression analyses. We re-examined the grouping of the cell lines according to cytotoxic drug response over the low dose ranges used to identify synergy. These analyses of cytotoxic responses to PLX4720, lapatinib and the combination of the two drugs revealed that the lines that did not demonstrate synergy had low synergy scores for fundamentally different reasons (detailed below). To organize the cell lines according to drug cytotoxicity profiles, we clustered the cell lines using their cytotoxicity values across drug treatments , again asistance . For thiBRAF locus and response to treatment, with homozygous BRAF mutant lines being more sensitive to BRaf inhibition, as has previously been reported in a panel of cell lines by Sondergaard et al [BRAF locus , which tends to yield repressed chromatin [EZH1 has been shown to maintain stem cell identity and pluripotency [EZH1 and EZH2 have been identified as candidate oncogenes in diffuse large B cell lymphoma [We then searched for genes that contained variants that were unique to individual cytotoxicity groups and could be dominant drivers of drug sensitivity within each group. We found that lines in both the CGE and CGD groups contained unique genetic variants. Variants previously identified by dbSNP were removed, and we further filtered the list by identifying novel nonsynonymous variants; nonsense and missense variants only. The list of 10,000 variants was narrowed to 143 variants across 83 genes that were identified as being known drivers of cancer . We impoer types ,29 and ihromatin . EZH1 haipotency . Importalymphoma .BRAF zygosity potentially explains the response of three of the four CGC and two of the three CGA cell lines to PLX4720 treatment respectively. Consequently, we searched for genes that contained variants specifically in the cell lines that were cytotoxicity group exceptions with regard to BRAF zygosity (SKMEL28 and SKMEL24) that were not shared by other group members. We also identified variants that were specific to SKMEL28 and SKMEL24, which could explain their being placed in CGC and CGA groups, respectively.While we found no cytotoxicity group specific variants in CGA and CGC cell lines, BRAF lines. SKMEL28, uniquely in our panel, contains a mutation in NOTCH1, a transmembrane protein that has been shown to interact with membrane bound ligands [ARID1A and PTEN are mutated in SKMEL28 but not in its cytotoxicity group members. However, ARID1A is also mutated in VMM15, and PTEN is not mutated in any other line. ARID1A is a SWI/SNF family member and potential tumor suppressor in melanoma [APC, a tumor suppressor that antagonizes WNT signaling [BRAF status.In SKMEL28, which was the second most PLX4720 resistant homozygous cell line among the six shown in  ligands  and it c ligands . SKMEL28melanoma . Lastly,ignaling . We hypoTNFRSF14, DNER, ERBB3 and EGFR  and applying a stringent 1% FDR cutoff . The CGAreatment . When weETS2, GATA1, and POU2F1. Additionally, we saw a number of well-established oncogenic transcription factor binding motifs, including JUN and MYB. Lastly, based on ChIP-seq data [MITF targets a number of ErbB pathway genes [Given the striking, simple result that the ErbB pathway is broadly upregulated in response to PLX4720 treatment only in the group of cell lines that require treatment with the PLX4720 and lapatinib combination in order to achieve an optimally cytotoxic response, we asked which transcription factors were likely activated by mutant BRaf inhibition and targeting ErbB pathway genes. Using MSigDB , we idenseq data , we founay genes .LEF1, SP1, AP4, ETS2, and PAX4 that putatively target multiple upregulated genes in the largest central cluster of the heat map. Additionally, ErbB family members contain binding motifs for LEF1, SP1, AP4, NFAT, MAZ, and FOXO4 at their promoters. Notably, many of these factors have been associated with melanoma. LEF1 has been shown to be highly expressed in proliferative cells in melanoma [HMGB1 knockdown in melanoma, a marked decrease in cell proliferation was observed mediated by p21 in a SP1 dependent manner [NFAT has been identified as a potential drug target in melanoma using aminobisphosphonate zoledronic acid [FOXO4 activation can induce senescence in BRAFV600E mutants [We further analyzed transcription factor motifs that appear across multiple cytotoxicity groups in order to identify those that were more strongly enriched in CGC. For motifs that appeared in more than one group, we added the motif’s rank in CGC to the motif’s rank in the other group subtracted from 100. For motifs that appeared in more than the CGC and one other group, we calculated the arithmetic mean of the 100 minus the other groups’ ranks, and added that to the CGC rank. Using these averaged ranks summed with the rank for CGC, we found that CGC is more enriched than any other group for an additional 28 transcription factor binding motifs. We then found which of these transcription factors were associated with the promoters of ErbB signaling pathway genes. To visualize the ErbB pathway gene responses to PLX4720 treatment together with the transcription factors that putatively regulate them in one view, we plotted the log2 fold change of PLX4720 treated versus control of genes in the ErbB signaling pathway for each cytotoxicity group , Left anmelanoma ,41. In at manner . NFAT hanic acid . Lastly, mutants . Putting mutants  are appaTo test our hypothesis that resistant lines upregulate ErbB signaling activity, we performed reverse phase protein array analysis. We assayed approximately 132 protein epitopes across 105 different proteins sampling various oncogenic pathways including MAPK, PI3K, ErbB, etc. eight hours after initial treatment with PLX4720. We log2 transformed the normalized values, performed cytotoxic group-wise paired t-tests as described above and applied a 5% FDR cutoff. We identified 36 differentially expressed phosphoproteins, one cleavage site, and two proteins, whose total amount changed in response to PLX4720 treatment. Using these epitopes, we plotted their fold changes for each group due to treatment with PLX4720 in a heat map . SurprisERBB4 protein levels in response to PLX4720 treatment is the most anticorrelated epitope with PLX4720 cytotoxicity  and cytotoxicity in response to PLX4720 treatment across cell lines (including DM331) for each of the three doses separately. We found the change in total toxicity . Other hBRAFV600E is confounded by a wide variety of mechanisms of resistance, both intrinsic and acquired. Thus, there is considerable interest in understanding the general principles of resistance to BRaf inhibition so that druggable targets could be identified that would guide the development of effective drug combinations. In a high throughput screen, we found that the combination of the mutant BRaf inhibitor PLX4720 with the ErbB family inhibitor lapatinib caused synergistic cytotoxicity in the six lines of our 12 cell-line panel that showed the greatest intrinsic resistance to BRaf inhibition. By performing hierarchical clustering of the cytotoxicity dose-response data, we identified five different cytotoxicity groupings, reflecting extraordinary heterogeneity in biological responses to drug treatment in this panel. Novel results from our systems-level molecular profiling analysis include linking lineage (reflected by MITF expression), BRAF zygosity, cell-line specific mutations in proto-oncogenes and tumor suppressors and widespread transcriptional upregulation of ErbB family members and other receptor tyrosine kinases in the response and resistance of melanoma cells to PLX4720. Examination of the transcriptional responses to PLX4720 treatment revealed the involvement of transcription factors with the potential to be responsive to the MAPK pathway, providing a possible convergent mechanism underlying the diversity of phenotypic responses.Inhibition of BRaf signaling as a therapy for the 50% of cutaneous melanomas that are mutant MITF expression, which has been implicated by several reports in drug resistance and oncogenesis in melanoma [MITF levels and MITF transcriptional target activity as associated with increased sensitivity to PLX4720 treatment [MITF and IC50 for PLX4720, an association that was driven almost entirely by our single most resistant cell line; when this line (DM331) was dropped from the analysis, no association with MITF appeared. However the study by Konieczkowski et al. [MITF expression levels than occurred in our panel; MITF gene expression levels would be classified as MITF high for all our cell lines according to the criteria established by Muller et al. [MITF expression and number of cell lines, was better powered to detect a modest association.Principal Component Analysis of the basal (untreated with drugs) transcriptomes of the five cytotoxicity groups revealed a correlation between drug synergy and the second Principal Component. The strongest transcriptional correlation and anticorrelation to synergy was among genes encoding cell surface receptors including GPCRs and neuroactive ligand signaling GPCRs. Many GPCRs are capable of activating the MAPK pathway, either directly or by transactivating RTKs utilizing neurotropin –48, thusmelanoma ,19,49. Freatment . Howeveri et al.  utilizedr et al. . Thus, tBRAFV600E zygosity status. The high proportion of homozygous BRAF mutants in our study (4 out of 12) helps to highlight this tendency, and was strengthened by comparison with data in the Cell Line Encyclopedia. As shown in BRAFV600E mutations. Regarding cytotoxic response to vemurafenib treatment, the cell lines tend to be more resistant on the left. Additionally, the cell lines on the top of the PCA plot respond synergistically to the combination of vemurafenib and lapatinib while those on the bottom do not. It has been shown that vemurafenib binding to BRAFV600E paradoxically activates downstream MAPK signaling via dimerization with non-mutant RAF family members and allosteric activation of the non-mutant partner [BRAFV600E cell line, opportunities for this paradoxical activation are limited to the heterodimers with RAF1, which could partially explain increased dependency on the output of the mutationally activated BRaf.The groupings of our cell lines according to cytotoxic response to drug treatment and basal expression profile can be rationalized in part by  partner . In a hoBRAFV600E suppresses MITF gene expression: in our cell lines, MITF levels tend to be lower in homozygous versus heterozygous lines. We hypothesize that BRAFV600E levels are inhibiting MITF levels, which in that sense may be acting more as a passenger. Nevertheless, MITF is a potent regulator of melanoma lineage and to a large extent determines the basal transcriptional profile, hence its association with the first Principal Component of our basal transcriptional analysis.Our results support the findings of Konieczkowski et al.  that oveBRAFV600E zygosity and MITF levels in affecting sensitivity to PLX4720, are mutations in additional oncogenes and tumor suppressor genes. Mutations in cell surface proteins and RTKs potentially explain (1) cytotoxicity group-BRAF zygosity exceptions including SKMEL28 (homozygous BRAF yet combination sensitive) and SKMEL24 (heterozygous BRAF yet PLX4720 sensitive), (2) lapatinib resistant cell lines, which are heterozygous BRAF and (3) DM331, which is an outlier from a cytotoxic and transcriptional profile grouping perspective. (1) In SKMEL28, the most striking variant was the EGFR exception mutation, which has previously been identified as a gain of function mutation [BRAFV600E. In SKMEL24 we found a number of potentially damaging mutations in cell surface proteins including TNFRSF14, ERBB3 and EGFR, which could limit SKMEL24’s ability to activate alternative growth pathways in the presence of BRaf inhibition, thereby, potentially explaining its sensitivity to PLX4720 treatment despite its heterozygous BRAF status. The ERBB3 mutation is particularly interesting given that ErbB family members are lapatinib targets; however, this mutation was not found to be damaging by polyphen2 analysis and would require more study to determine if it alters ERBB3 function. (2) Both lapatinib resistant cell lines contained a damaging mutation in ALK, which is a receptor tyrosine kinase that has been previously associated with multiple cancer types [ALK mutation could be stimulating growth, thereby explaining why the cells in the secondary resistant group are resistant to lapatinib. (3) DM331 is an exception in that it is homozygous for BRAFV600E yet it is by far the most resistant cell line to PLX4720 treatment. Additionally, its expression profile at the basal state most closely associates it with A375, one of the most sensitive lines. However, A375 resistant lines have been generated multiple times, indicating that its sensitivity is not immutable [NRG1, an ErbB family ligand is highly upregulated in DM331 only, and not in A375. On the other hand, addition of NRG1 to A375 causes resistance to PLX4720 [EGFR in DM331 this may explain the profound resistance of DM331 to BRaf inhibition.Layered over and interacting with mutation ,52 and cer types  and is ammutable ,18. We fERBB3 [V600E melanoma in response to PLX4720 treatment.We observed broad upregulation of ErbB pathway gene expression 8 hours after PLX4720 treatment, only in the group of cell lines that were sensitive to the combination of PLX4720 and lapatinib. This result is consistent with that of other groups that have found ERBB3 ,21,53 anERBB3 ,13 upregERBB4 total protein and VEGFR phosphorylation levels were anticorrelated with PLX4720 cytotoxic response across cell lines. This indicates that treatment with PLX4720 induces receptor tyrosine kinase expression, most likely as a compensatory survival mechanism in lines resistant to treatment as demonstrated in other studies [NRAS signaling and MAPK pathway reactivation.Surprisingly, we observed a relatively strong downregulation of oncogenic phosphoprotein levels after 8 hours of PLX4720 treatment in CGD and CGE cell lines with a similar but much weaker downregulation of the same proteins in CGA and CGC. We hypothesize that the downregulation observed at the protein level is the direct result of PLX4720 treatment followed by delayed, compensatory signaling in the combination and PLX4720 sensitive cell lines. As partial confirmation of this, we found that  studies ,53. ThisERBB3 activation due to vemurafenib treatment; however they saw activation via FOXD3. Chandarlapaty et al. [ERBB3 activation in response to AKT inhibition via phosphorylated FOXO3 in cancer cell lines from multiple tumor types, which were resistant to AKT inhibition. However, the correlation between changes in phosphorylated FOXO3 induced by PLX4720 and PLX4720 cytotoxicity in our melanoma lines indicate that FOXO3 phosphorylation is increased in the most sensitive lines suggesting alternative regulatory programs in the BRAFV600E melanoma cell lines that we studied. If FOXO3 phosphorylation activates ERBB3 as a compensatory mechanism to BRaf inhibition, we would expect its log2 fold change to be anticorrelated with cytotoxic response to PLX4720, similar to the response of ERBB4. We do see FOXO4 among the transcription factors whose motifs are enriched at the promoters of upregulated ERBB pathway genes. Importantly, while previous studies have observed ERBB3 upregulation in response to various inhibitors [Lastly, it appears that PLX4720 treatment potentially activates ETS family transcription factors and co-factors to upregulate ErbB pathway genes as well as those of other receptor tyrosine kinase signaling pathways. Abel et al. observed  ERBB3 acy et al.  identifihibitors ,24,54, wBRAFV600E melanoma cell lines. Additionally, we have observed that inhibition of other RTKs, also elicits a synergistic response when combined with BRaf inhibition. Taken together, our results suggest RTK mediated signaling is an adaptive response to mutant BRaf inhibition in melanomas and suggests combination therapy with specific RTK inhibitors to have potential clinical benefit.It has been noted that multiple receptor tyrosine kinases confer resistance to many forms of treatment . We findMelanoma cell lines used in this study were derived from tumors taken from tumor-involved lymph nodes from patients at the University of Virginia  or from patients at Duke University  as described previously –60. A375Four hours after being plated in 96-well plates, cells were treated with inhibitors or vehicle control in phenol red–free RPMI-1640 (Invitrogen) without FBS and incubated for 3 days at 37°C. alamarBlue (Invitrogen) was added to wells and incubated for 4 hours at 37°C. Fluorescence was measured at 560 nm excitation/590 nm emission on a Synergy 2 plate reader (BioTek Instruments). Mean results and SE were calculated for triplicate samples.Cell Culture and Generation of Transcriptome, Proteome and Exome Data.Cells were plated and incubated overnight before being treated, in duplicate, with inhibitors or vehicle control in phenol red–free RPMI-1640 without FBS for 8 hours at 37°C. Cells were placed on ice and rinsed with cold 1× PBS. Cells were collected and RNA was isolated using the Qiashredder (Qiagen) and RNeasy Mini Kit (Qiagen). RNA was quantified on the NanoDrop 2000 spectrophotometer (Thermo Scientific) and RNA quality was inspected on a 1% agarose gel. Biotin labeled RNA was hybridized to Illumina 3′IVT human HT-12 BeadChip arrays.Cells were plated in p60 dishes and incubated overnight to allow the cells to adhere before being treated with inhibitors or vehicle control in phenol red-free RPMI Medium 1640 without fetal bovine serum for 8 hours. Cells were washed and lysed in 1:1 2x Sample Buffer:Tissue Extraction Reagent (T-PER) (Life Technologies). Following lysis the samples were sonicated and centrifuged to clear. Samples were diluted to requested concentration and sent to the lab of Emanuel (Chip) Petricoin at George Mason University for Reverse Phase Array Analysis. Raw sequencing reads were generated using a combination of Nimblegen v2 and Agilent v1 capture probes.Cells were grown to 80% confluent, trypsinized, and collected in normal growth media. Cells were centrifuged to pellet, wash with PBS, and centrifuged a second time. DNA was extracted using the DNeasy Blood and Tissue Kit (Qiagen) and quantified on the Nanodrop 2000 spectrophotometer (Thermo Scientific). Exome sequencing was performed by Hudson Alpha Institute for Biotechnology .lumi Bioconductor package in R [limma to perform one way ANOVA and derive Benjamini-Hochberg False Discovery Rate (FDR) adjusted p-values [pvclust [pvclust with a correlation distance measure and average linkage  data was generated using methods described in [Illumina microarray data was variance stabilized transformed ,62 usingage in R . Genes tp-values ,65 and a[pvclust  with the linkage . We idenSeattleSeq. Genes with novel variants were identified by removing variants previously identified in dbSNP, or 1000Genomes. Novel variants were further filtered by selecting only nonsense and missense mutations as identified by SeattleSeq. The list of genes containing novel nonsynonymous variants were selected by retaining only those that were identified by [PolyPhen2 to determine the potential functional severity of these nonsynonymous variants. Genes were identified that uniquely contained variants in each cell line, and within each cytotoxicity group.Exome probes were genotyped using the Genome Analysis Toolkit and the HG19 build as a reference genome. Single nucleotide variants were annotated by ified by  as beingGene sets found to be significant underwent pathway enrichment analysis via Pathway Express and Gene Set Enrichment Analysis (GSEA) using MSigDB datasets. Significant gene sets and pathways were identified as having a FDR enrichment value of 0.05 for both tools. To ascertain significance of transcription factor binding motifs, we identified motifs unique to the CGC. For motifs not unique to the CGC, we summed the rank of CGC with 100 minus the rank of the other group. When multiple groups contained the motifs, we averaged the ranks, then summed the rank of CGC with 100 minus the averaged rank.S1 FigCombination − (1 – (1 – CSecondary Drug)*(1 – CPrimary Drug)) where CCombination is the observed cytotoxicity of the combination treatment, CSecondary Drug is the observed cytotoxicity of the secondary drug, and CPrimary Drug is the observed cytotoxicity of the primary drug.Synergy score (left y-axis) across all 12 cell lines (bottom x—axis) for escalating doses of lapatinib (top x-axis) and PLX4720 (right y-axis). Synergy Score was calculated using the Bliss model of independence. Synergy Score = C(EPS)Click here for additional data file.S2 FigMITF ChIP-Seq binding sites (blue) and Cluster IV gene list (pink).Number of overlapping genes (purple) between (EPS)Click here for additional data file.S3 FigBRAF mutants denoted with a “*” (A). Relative expression in Log2 of AXL (Red) and MITF (Blue) (B).Basal PCA plot with cytotoxicity groups encircled, and homozygous (EPS)Click here for additional data file.S4 FigBox plots of cytotoxic response to single drug treatment (top 2 rows) and escalating PLX4720 doses of the combination drug treatment (bottom 3 rows). Doses along the bottom refer to either the single drug treatment (top 2 rows), or the secondary drug treatment dose for the combination drug treatment (bottom 3 rows). Red dots denote negative cytotoxic responses, and blue dots denote positive cytotoxic responses.(EPS)Click here for additional data file.S5 FigThe responses for each group are CGD (top left), CGE (top right), CGA (bottom left), and CGC (bottom right). Nodes in red denote an upregulation, nodes in blue denote a downregulation. Refer to (EPS)Click here for additional data file.S6 FigThe responses for each group are CGD (top left), CGE (top right), CGA (bottom left), and CGC (bottom right). Nodes in red denote an upregulation, nodes in blue denote a downregulation. Refer to (EPS)Click here for additional data file.S7 FigThe responses for each group are CGD (top left), CGE (top right), CGA (bottom left), and CGC (bottom right). Nodes in red denote an upregulation, nodes in blue denote a downregulation. Refer to (EPS)Click here for additional data file.S8 FigThe responses for each group are CGD (top left), CGE (top right), CGA (bottom left), and CGC (bottom right). Nodes in red denote an upregulation, nodes in blue denote a downregulation. Refer to (EPS)Click here for additional data file.S9 FigUnsupervised clustering of gene expression response to PLX4720 treatment (y-axis) of the ErbB signaling pathway of each cell line (Left). Genes were selected for plotting based on passing the FDR corrected P Value threshold of 1% or better in at least one cytotoxicity group. Genes that demonstrated a FDR corrected P Value of 1% or better for the t-test for an individual cell line are denoted with an “*”. Transcription factors were identified that have binding motifs in the promoters of these genes (Right).(EPS)Click here for additional data file.S1 TableClusters III and IV were identified from the one way ANOVA analysis . Each cl(XLSX)Click here for additional data file.S2 TableList of probes from gene expression microarray that demonstrated a correlation coefficient greater than 0.8 or less than -0.8 with the loadings of the first principal component.(XLSX)Click here for additional data file.S3 TableList of probes from gene expression microarray that demonstrated a correlation coefficient greater than 0.8 or less than -0.8 with the loadings of the second principal component.(XLSX)Click here for additional data file.S4 TableProbes that demonstrated significant correlation  Tables u(XLSX)Click here for additional data file.S5 TableVariants that were present in individual cell lines of interest or present in all cell lines of a cytotoxicity group were submitted to Polyphen2 to ascertain potential functional significance.(XLSX)Click here for additional data file.S6 TableLog2 Fold changes, p values, and Bonferroni corrected p values (FDR) for differential expression analysis of each cytotoxicity group.(XLSX)Click here for additional data file.S7 TableDifferentially expressed genes for each cytotoxicity group that passed a FDR threshold of 1% or better were submitted to Pathway Express to identify pathways significantly affected by treatment.(XLSX)Click here for additional data file.S8 TableDifferentially expressed genes for each cytotoxicity group that passed a FDR threshold of 1% or better were submitted to MSigDB to identify pathways and transcription factors significantly affected by treatment.(XLSX)Click here for additional data file.S9 TableTable of correlations between log2 fold changes of cell lines’ protein responses to treatment of PLX4720 with cell lines’ cytotoxic responses to PLX4720.(XLSX)Click here for additional data file.


AXL is a well-characterized, protumorigenic receptor tyrosine kinase that is highly expressed and activated in numerous human carcinomas and sarcomas, including aggressive subtypes of liposarcoma. However, the role of AXL in the pathogenesis of well-differentiated (WDLPS), dedifferentiated (DDLPS), and pleomorphic liposarcoma (PLS) has not yet been determined.Immunohistochemical analysis of AXL expression was conducted on two tissue microarrays containing patient WDLPS, DDLPS, and PLS samples. A panel of DDLPS and PLS cell lines were interrogated via western blot for AXL expression and activity and by ELISA for growth arrest-specific 6 (GAS6) production. AXL knockdown was achieved by siRNA or shRNA. The effects of AXL knockdown on cell proliferation, migration, and invasion were measured in vitro. In addition, AXL shRNA-containing DDLPS cells were assessed for their tumor-forming capacity in vivo.In this study, we determined that AXL is expressed in a subset of WDLPS, DDLPS, and PLS patient tumor samples. In addition, AXL and its ligand GAS6 are expressed in a panel of DDLPS and PLS cell lines. We show that the in vitro activation of AXL via stimulation with exogenous GAS6 resulted in a significant increase in cell proliferation, migration, and invasion in DDLPS and PLS cell lines. Transient knockdown of AXL resulted in attenuation of these protumorigenic phenotypes in vitro. Stable AXL knockdown not only decreased migratory and invasive characteristics of DDLPS and PLS cells in vitro but also significantly diminished tumorigenicity of two dedifferentiated liposarcoma xenograft models in vivo.Our results suggest that AXL signaling contributes to the aggressiveness of DDLPS and PLS, and that AXL is therefore a potential therapeutic target for treatment of these rare, yet devastating tumors. Soft tissue sarcomas include over 50 histologically distinct subtypes of mesenchymal malignancies and account for approximately 1 % of all adult solid cancers in the United States .Approximately 40–45 % of LPS are ALT/WDLPS, which are locally aggressive and rarely metastatic. These tumors are characterized by amplification of the 12q13-15 chromosomal region, which ultimately results in the formation of supernumerary rings and/or giant rod chromosomes . CurrentWe recently showed that AXL was highly upregulated in LPS cells versus normal preadipocytes and adipocytes . AXL is It has also been reported that RNAi-mediated knockdown of AXL in pancreatic adenocarcinoma and breast and lung cancer cells decreases migration, invasion, tumor growth, and metastasis and also impairs angiogenesis , 28–31. n = 12) specimens were included as a control.This study was approved by The University of Texas MD Anderson Cancer Center (UTMDACC) institutional review board and informed consent was obtained from all patients. By using the UTMDACC prospective sarcoma database, institutional tumor registry, and pathology archives, 46 WDLPS and 19 DDLPS formalin-fixed paraffin-embedded samples from 45 patients who presented to UTMDACC from January 2000 to February 2009 and subsequently underwent surgical resection were selected for tissue microarray (TMA) inclusion. The TMA was constructed as previously described, with two 0.6-mm punches per sample embedded into a TMA block . HematoxHuman DDLPS cell lines Lipo-224A, Lipo-224B, Lipo-246, and Lipo-863, the DDLPS cell strain Lipo-573, and the PLS cell line PLS-1 were derived and cultured in our laboratory as previously described . The DDLCommercially available antibodies were purchased for immunoblotting or immunohistochemical detection of AXL. pAXL Y702, AKT, pAKT S473, ERK, and pERK T202/T204 ; MER ; TYRO3 ; Ki67 and cleaved caspase 3 ; and β-actin–horseradish peroxidase . Recombinant human GAS6 was purchased from R&D Systems .Standard protocols for western blot analyses of whole cell extracts were followed . For GASAntibody sandwich ELISAs  were used to measure secreted GAS6 in human primary preadipocytes, adipocytes, and DDLPS and PLS cell lines in low-serum conditions . Samples of conditioned DMEM (1 % FBS) were obtained from preadipocytes, adipocytes, and cell lines after 24 h, after which the total number of cells was calculated. Secreted GAS6 levels were normalized to cell number.3 cells/well in 100 μL of medium and assayed for proliferation after 48 h. For experiments with GAS6, 24 h after seeding, the medium was replaced with low-serum medium (1 % FBS) with or without 400 ng/mL recombinant GAS6. Cell proliferation was assayed 48 h later . For experiments with small interfering RNA (siRNA), cells were seeded 48 h post-transfection and proliferation was measured 48 h thereafter (96 h after transfection). Following the addition of MTS reagent, plates were incubated for 2 h at 37 °C, and absorbance was read at 490 nm. For each experiment, proliferation was assayed in triplicate, and the percent cell proliferation was normalized to the control.Cell proliferation was evaluated using a CellTiter 96 AQueous One Solution Cell Proliferation Assay -5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt; MTS)  according to the manufacturer’s instructions. Briefly, cells were seeded in 96-well plates at a density of 1.5 × 104 cells were seeded in the inserts of modified Boyden chambers in low-serum (1 % FBS) DMEM/F12 medium. DMEM/F12 medium containing 5 % FBS was used as a chemoattractant for these studies. Triplicate wells were used for each experiment. For studies evaluating the effect of GAS6-mediated AXL activation on migration and invasion, 100 ng/mL GAS6 was added within the inserts at the time of cell seeding. For experiments with siRNA, cells were seeded 48 h post-transfection. After 14 h incubation at 37 °C, cells were fixed with 10 % glutaraldehyde and stained with 0.2 % crystal violet in 20 % methanol . Migratory and invasive cells were quantified using ImageJ version 1.47 software (http://rsbweb.nih.gov/ij/index.html) [Migration and invasion assays were performed using cell culture inserts with 8 μm polyethylene terephthalate (PET) filters with or without Matrigel . Briefly, 2.5 × 10For transient knockdown of AXL, 20 nM pools of ON-TARGETplus human AXL-targeting siRNA or a 20 nM pool of control nontargeting (NT) constructs  were introduced into cells using X-tremeGene  per the manufacturer’s instructions. Cells were transfected at 45–50 % confluency in six-well plates and seeded for MTS, migration, and invasion assays 48 h after transfection. Knockdowns were evaluated via western blot analyses of whole cell extracts harvested 72 h after transfection.Stable knockdown of AXL was achieved by transfecting cell lines with the short hairpin RNA (shRNA) pGIPZ lentiviral constructs containing anti-AXL or NT sequences . Lentiviruses were generated at the MD Anderson shRNA and ORFeome Core. Target cells were infected with lentivirus, followed by selection with 1 μg/mL puromycin. AXL knockdown was confirmed via western blot analyses.6 or 3.5 × 106 cells/0.1 mL of phosphate buffered saline (PBS) per mouse, respectively) expressing either AXL-specific or NT shRNA lentiviral constructs were subcutaneously injected into the flanks of 6-week-old female hairless severe combined immunodeficiency (SCID) mice (n = 8 per construct) . Tumor size was measured biweekly. When control tumors reached a volume of 1500 mm3, all mice were sacrificed and their tumors resected, weighed, and frozen or fixed in formalin. Formalin-fixed tissues were subsequently embedded in paraffin for IHC analyses.All animal procedures were approved by the UTMDACC Institutional Animal Care and Use Committee. Animals received humane care in accordance with the Animal Welfare Act and the National Institutes of Health Guide for the Care and Use of Laboratory Animals. To evaluate the effect of AXL knockdown on tumorigenicity, suspensions of Lipo-246 or Lipo-863 cells . Tumor cores missing from the TMA sectioned slides were omitted from analyses. Staining was classified as either negative (intensity of stain scored as 0 or 1) or positive (intensity of stain scored as 2 or 3). Xenograft-derived specimens were further analyzed for Ki67 (cell proliferation marker) and cleaved caspase 3  expression.t-tests were used to assess significance. Means and SEMs were also calculated for xenograft studies. Differences in xenograft volumes and weights between AXL-specific and NT constructs were assessed using a two-tailed Student t-test (* = p < 0.05 or *** = p < 0.001).In vitro assays were repeated at least three times; the mean and standard error of the mean (SEM) were calculated for each assay. Two-tailed Student We first assessed AXL expression in human tumors by conducting IHC analyses on two TMAs containing samples from WDLPS, DDLPS, and PLS patients. AXL expression was observed in 27.7 %  of all evaluable LPS samples, with no staining in NF specimens Fig. . AXL wasFor the in vitro assessment of AXL expression and function, we utilized several previously established DDLPS and PLS cell strains and cell lines; however, WDLPS cell strains do not grow in culture and therefore could not be experimentally tested. Similar to our IHC analysis, immunoblotting of whole cell lysates from DDLPS and PLS cell lines demonstrated that four out of 6 DDLPS cell lines and cell strains and all three PLS cell lines expressed higher levels of both total AXL and its activated form (pAXL Y702) compared with preadipocyte and adipocyte controls Fig. . In compNext we assessed the levels of the AXL ligand GAS6 in tumor cell-conditioned media in order to determine the potential for autocrine activation of AXL in LPS oncogenicity. We found significantly elevated levels of secreted GAS6 in six out of the seven DDLPS and PLS cell lines when compared to adipocyte control cells, indicating that autocrine stimulation by GAS6 may play a role in AXL activation Fig. .In order to determine the enhancing effects of GAS6-mediated signaling on the activation of AXL and its downstream effectors in DDLPS and PLS, two DDLPS cell lines (Lipo-246 and Lipo-863) and two PLS cell lines (LiSa2 and PLS-1) were cultured in low-serum conditions overnight, followed by acute exposure to GAS6. Serum starvation did not completely abrogate AXL activation in Lipo-246, Lipo-863, and PLS-1, suggesting that cell line-secreted GAS6 may allow for a baseline level of AXL activation. Exogenous GAS6 stimulation increased AXL activation in all cell lines evaluated; furthermore, Lipo-246, PLS-1, and LiSa2 cells displayed increased phosphatidylinositol-3-kinase (PI3K) pathway activity, as measured by AKT phosphorylation at S473 Fig. . In mostAfter determining that AXL activation confers proliferative, migratory, and invasive properties to DDLPS and PLS cells in vitro, we wanted to assess whether transient knockdown of AXL could abrogate these processes. Using an anti-AXL siRNA pool, we knocked down AXL in DDLPS and PLS cells; a control pool of four NT constructs was used in parallel. As expected, AXL knockdown resulted in reduced total AXL protein expression and decreased AXL activation Fig. . We obseNext, we stably knocked down AXL to analyze the long-term effects of decreased AXL expression on tumorigenic phenotypes in vitro. DDLPS and PLS cells were transduced either with one of two different AXL-specific shRNA-containing lentiviruses (hereafter referred to as shRNA #1 or #2) or with the NT control construct. Following selection and expansion, western blots were used to examine the degree of stable AXL knockdown in all four cell lines Fig. . Interesp < 0.001) compared with that for NT control xenografts  was observed with AXL knockdown in Lipo-246 xenografts; the weight of Lipo-863 tumors with AXL knockdown were decreased compared to the NT control although statistical significance was not achieved   Figs.  and 6c. 2) Figs.  and 6d. In this study, we have confirmed that AXL is overexpressed and activated in DDLPS and PLS cell lines and corresponds to enhanced oncogenic signaling through downstream PI3K/AKT and MAPK pathways, which facilitate certain tumorigenic processes such as proliferation, migration, and invasion. While the mechanism of AXL overexpression is outside the scope of this study, we found that DDLPS and PLS cells are capable of producing and secreting GAS6 in an autocrine manner, and that exogenous administration of GAS6 enhances activation of AXL and downstream effectors in these cells. Taken together, these findings support an autocrine mechanism of AXL activation via GAS6 secretion in liposarcoma disease progression. Our observations support previous reports that the role of AXL in cancer is a consequence of receptor overexpression and/or its inappropriate activation –40.AXL signaling is involved in many cellular processes, including survival, migration, invasion, and metastasis in both normal and cancer cells –12. We oInitially, we observed that AXL signaling following stimulation with exogenous GAS6 is potentiated primarily through the PI3K/AKT pathway in all cell lines tested, as measured by an increase in pAKT, while a mild increase in signaling through the MAPK pathway was observed in 3 out of 4 cell lines. However, siRNA or shRNA-mediated AXL knockdown did not have a uniform effect on either downstream pathway evaluated. For instance, knockdown of AXL by siRNA resulted in a slight decrease in PI3K/AKT signaling in two cell lines (Lipo-246 and LiSa2), while both pAKT and pERK were elevated in PLS-1. We noted that the effect on downstream pathways were variable following stable AXL knockdown. It should also be noted that Lipo-863 had consistently low or absent basal levels of pAKT and therefore could not be included when assessing pathway preferences for AXL. These data indicate that AXL signaling can be propagated through both the PI3K/AKT and MAPK pathways without any apparent preference, and therefore merits further investigations into alternative downstream pathways.Our findings for DDLPS and PLS cell lines were similar to those of previous studies in which transient AXL knockdown with siRNA was associated with decreased cell proliferation and a concomitant reduction in migration and invasion , 29–31. The effect of AXL knockdown on in vivo tumorigenic potential is cell line-dependent and may vary with the degree of knockdown. Knockdown of AXL in Lipo-246 cells increased apoptosis and decreased cell proliferation, greatly reducing tumor volume and weight. Lipo-863 shRNA-containing xenografts resulted in decreased cell proliferation, but had minimal effect on apoptosis. These results likely reflect alternate growth factor signaling mediators that are active in different tumors. Knockdown via shRNA #2 did not completely eliminate AXL expression in Lipo-863; however, a significant reduction in tumor volume and weight was noted. These results indicate that moderate knockdown of AXL may be sufficient to reduce tumorigenicity. Additionally in vivo studies interrogating the use of a pharmacological inhibitor against AXL in vivo could further validate our findings and shed more light onto the role of the receptor in disease progression.Several small-molecule inhibitors of AXL are being investigated as both single agents and in combination for their anti-tumor effects in cancers with high AXL expression , 41–45. AXL has been identified as a predictor of poor prognosis for several types of malignancies, including breast cancer, pancreatic cancer, lung adenocarcinoma, osteosarcoma, oral squamous cell carcinoma, and acute myeloid leukemia , 46–48. Overall, our data suggests that AXL is a potential therapeutic target in DDLPS and PLS; further investigations into the effects of AXL-specific targeted therapies for the treatment of LPS are warranted.


Streptomyces pluripotens MUSC 137 was isolated from mangrove soil obtained from Tanjung Lumpur, Pahang, Malaysia. We investigated the phylogenetic, genomic, biochemical, and phenotypic characteristics of this strain. Uniquely adapted microorganisms from mangrove habitats have previously yielded compounds of biopharmaceutical interest. In order to examine the bioactivities possessed by the strain, fermentation extract was prepared through solvent extraction method prior to bioactivities screenings. Antioxidant activity was examined via DPPH assay while the cytotoxic effect was assessed by means of examining the activity of the extract against selected human cancer cell lines, namely colon cancer cells , breast cancer cell (MCF-7), lung cancer cell (A549), prostate cancer cell (DU145), and cervical cancer cell (Ca Ski). The results revealed MUSC 137 possesses significant antioxidant activity and demonstrates cytotoxic effect against several cancer cell lines tested. The results indicated MCF-7 cells were most susceptible to the extract with the lowest IC50 (61.33 ± 17.10 μg/mL), followed by HCT-116 and A549. Additionally, selective index (SI) showed that MUSC 137 extract was less toxic against normal cell lines when compared to MCF-7 and HCT-116 cells. The extract was further subjected to chemical analysis using GC–MS and revealed the presence of deferoxamine and pyrrolizidines related compounds which may account for the antioxidant and cytotoxic properties observed. Streptomyces from the different mangrove environments globally, such as the isolation of Streptomyces sanyensis  located in the Tanjung Lumpur mangrove forest in the state of Pahang, Peninsular Malaysia. Topsoil samples of the upper 20-cm layer (after removing the top 2–3 cm) were collected and sampled into sterile plastic bags using an aseptic metal trowel, and stored at –20°C. Air-dried soil samples were ground with a mortar and pestle. Selective pretreatment of soil samples was performed using wet heat in sterilized water  using MEGA version 6.0 . The designation of colony color was determined by using the ISCC-NBS color charts  as vehicle reagent prior to assay.Strain MUSC 137 was grown in seed medium (TSB) for 14 days prior to fermentation. The fermentation medium, FM3 was sterilized by autoclaving at 121°C for 15 min prior to experiment . FermentAntioxidant activity of MUSC 137 extract was examined by measuring radical scavenging ability using DPPH (Sigma–Aldrich). Scavenging activity on DPPH free radicals was determined using previous protocol with minor modifications . Reducti2 in air at 37°C). Cells were seeded into a sterile flat bottom 96-well plate at a density of 5 × 103 cells/well and allowed to adhere overnight. MUSC 137 extract was added to the cells (0–400 μg/mL) and further incubated for 72 h before performing MTT assay. Curcumin (1.56–25 μg/mL) was included as positive control. Twenty microliter of 5 mg/mL of MTT (Sigma) was then added to each well and the plates were incubated at 37°C in a humid atmosphere with 5% CO2, 95% air for 4 h. The medium was then gently aspirated, and 100 μL of DMSO was added to dissolve the formazan crystals. The amount of formazan product was determined spectrophotometrically at 570 nm (with 650 nm as reference wavelength) using a microplate reader. The percentage of cell viability was calculated with follows:For evaluation of cytotoxicity, a variety of human derived cancer cell lines were selected for testing: (a) colon cancer cell, HCT-116, HT-29, Caco-2 and SW480; (b) breast cancer cell, MCF-7; (c) cervical cancer cell, Ca Ski; (d) lung carcinoma cell line, A549; (e) prostate cancer cell, DU 145. Cells were grown in RPMi 1640 supplemented with 10% FBS in humidified incubator (5% CO50) was derived from the plotted graphs for each cell line. VERO cells were used as a normal cell model for determination of selective index (SI). SI value was calculated by dividing IC50 of cancer cell line with IC50 value of normal cell line.The 50% inhibitory concentration of extract . One-way analysis of variance (ANOVA) was used for comparison of more than two means. A difference was considered statistically significant when Streptomyces . The colors of the aerial and substrate mycelium were yellowish gray and brilliant yellow. Growth occurred at pH 5.0–9.0 (optimum pH 5.0–8.0), with 0–6% NaCl tolerance (optimum 0–4%) and at 24–40°C (optimum 28–32°C). MUSC 137 was positive for catalase but negative for hemolytic activity and melanoid pigment production. Hydrolysis of soluble starch and carboxymethylcellulose were positive; but negative for hydrolysis of tributyrin (lipase), casein, chitin, and xylan. Strain MUSC 137 was found to be able to utilize various compounds as carbon sources (Supplementary Table S1). The strain was sensitive to ampicillin, ampicillin sulbactam, cefotaxime, cefuroxime, cephalosporin, chloramphenicol, ciprofloxacin, erythromycin, gentamicin, nalidixic acid, Penicillin G, streptomycin, tetracycline, and vancomycin.Strain MUSC 137 was Gram-positive and aerobic. The strain grew well on ISP2 medium, SA and SCA after 1–2 weeks at 28°C, it grew moderately on Luria Bertani agar, ISP7 medium and nutrient agar, whereas it grew poorly on AIA. The morphological observation of the 15-day-old culture grown on ISP2 medium showed a smooth spore surface, with aerial and vegetative hyphae which was well developed and not fragmented. These morphological characteristics were consistent with its assignment to the genus ptomyces ,  formed a distinct clade with type strains S. pluripotens MUSC 135T, S. mexicanus NBRC 100915T, S. cinereospinus NBRC 15397T, S. coeruleofuscus NBRC 12757T, and S. chromofuscus NBRC 12851T. Strain MUSC 137 and S. pluripotens MUSC 135T formed a distinct subclade at 100% bootstrap value, indicating the high confidence level of the association . Strain MUSC 137 exhibited highest 16S rRNA gene sequence similarity to S. pluripotens MUSC 135T (100%), followed by S. cinereospinus NBRC 15397T (99.18%), which corresponds to 12 nucleotide differences at 1462 locations with gaps, and lower similarity values to S. mexicanus NBRC 100915T (99.17%) and S. coeruleofuscus NBRC 12757T (98.97%).The almost-complete 16S rRNA gene sequences were determined for strain MUSC 137 (1491 bp) and aligned manually with the corresponding partial 16S rRNA gene sequences of the type strains of representative members of the genus S. pluripotens MUSC 135T were 83 ± 3.2%, which were significantly higher that 70%, the threshold value for the delineation of bacterial species .The DNA–DNA relatedness values between strain MUSC 137 and  species . These rTable 1). In addition, the extract of MUSC 137 displayed variable levels of cytotoxic activity against all the tested human cancer cell lines, with our work recording viability of tested cells ranging from 18.5 to 76.2% . A dose response pattern was evaluated using different doses which indicated that the extract may be effective against some cancer cell lines even at the lowest tested concentration (25 μg/mL). The lowest IC50 value of MUSC 137 extract was recorded at 61.33 ± 17.10 μg/mL for MCF-7 cells, followed by HCT-116 (83.72 ± 7.17 μg/mL), A549 (147.20 ± 19.23 μg/mL) as shown in Table  Higher IC50 values of MUSC 137 extract were observed for Ca Ski and HT-29, at 300.50 ± 19.76 μg/mL and 300.98 ± 27.52 μg/mL, respectively.The results obtained from DPPH free radical scavenging assay showed significant antioxidant activity of MUSC 137 extract with 35.03 ± 3.74% scavenging activity at 2 mg/mL .Of particular note, the extract showed higher level of cytotoxicity against some cancer cell lines with low cytotoxic effects in normal cell lines. The calculation of selectivity index (SI) allows comparison of cytotoxicity by dividing ICTable 4) and the chemical structures  as 2,2-dimethoxybutane (1), Benzeneacetamide (2), Phenol, 2,5-bis- (3), -3-methyl-1,2,3,4,6,7,8,8a-octahydropyrrolopyrazine-1,4-dione (4), 2,5-cyclohexadiene-1,4-dione (5), Pyrrolopyrazine-1,4-dione, hexahydro- (6), 1,4-diaza-2,5-dioxo-3-isobutyl bicyclo[4.3.0]nonane (7), Deferoxamine (8), Pyrrolopyrazine-1,4-dione, hexahydro-3-(2-phenylmethyl)- (9).Gas chromatography–mass spectrometry analysis identified nine compounds present in the methanolic extract of MUSC 137  of interest. In this current study, DPPH assay revealed significant antioxidant activity of MUSC 137 extract (Table 1), which suggests that the strain may produce valuable compound(s) that could potentially reduce cancer occurrence and be further developed as chemopreventive drugs.Cancer initiation and progression is associated with accumulation of free radicals or oxidative stress . In genein vitro living cells and is often employed in cytotoxicity studies to evaluate drug efficiency (50 (61.33 ± 17.10 μg/mL), followed by HCT-116 (83.72 ± 7.17 μg/mL) and A549 (147.20 ± 19.23 μg/mL). Among the tested colon cancer cell lines, the treatment of MUSC 137 extract at the highest tested concentration (400 μg/mL) showed strongest growth inhibition against HCT-116 (18.5 ± 7.8%), while higher percentages of cell viability were still observed in HT-29 (44.1 ± 2.9%), Caco-2 (50.8 ± 2.1%), and SW480 (56.4 ± 1.3%). The tested colon cancer cell lines are known to possess dysfunctional p53 tumor protein, except HCT-116. Therefore, the varied levels of cytotoxic activity among these colon cancer cell lines exhibited by extract of MUSC 137 might be due to the p53 tumor suppressor protein status of these cancer cell lines -3-methyl-1,2,3,4,6,7,8,8a-octahydropyrrolopyrazine-1,4-dione were also found in other activity . As antiactivity , these pactivity . This tractivity . The detS. pluripotens strain MUSC 137 as this strain has demonstrated its ability to produce bioactive compounds which may account for its antioxidant and cytotoxic activities. Future studies should focus on the elucidation of mechanism action of cell death to provide more convincing evidence of selective cytotoxic activity observed and work investigating this phenomenon is in fact currently underway.This study has revealed the biopharmaceutical potential possessed by H-LS, B-HG, and L-HL contributed to the data analyses and writing of the manuscript. N-SA, W-FY, and K-GC contributed by providing vital technical support for the project. K-GC, B-HG, and L-HL contributed to the inception of the experimental design and funding of the project.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Soluble growth factors present in the microenvironment play a major role in tumor development, invasion, metastasis, and responsiveness to targeted therapies. While the biochemistry of growth factor-dependent signal transduction has been studied extensively in individual cell types, relatively little systematic data are available across genetically diverse cell lines.amp cancers (which overexpress ErbB2) the least. The ratio of ERK to AKT activation varies with ligand and subtype, with a systematic bias in favor of ERK in hormone receptor positive (HR+) cells. The factors that correlate with growth factor responsiveness depend on whether fold-change or absolute activity is considered the key biological variable, and they differ between ERK and AKT pathways.We describe a quantitative and comparative dataset focused on immediate-early signaling that regulates the AKT (AKT1/2/3) and ERK (MAPK1/3) pathways in a canonical panel of well-characterized breast cancer lines. We also provide interactive web-based tools to facilitate follow-on analysis of the data. Our findings show that breast cancers are diverse with respect to ligand sensitivity and signaling biochemistry. Surprisingly, triple negative breast cancers  are the most broadly responsive to growth factors and HER2http://lincs.hms.harvard.edu/niepel-bmcbiol-2014/.Responses to growth factors are highly diverse across breast cancer cell lines, even within the same subtype. A simple four-part heuristic suggests that diversity arises from variation in receptor abundance, an ERK/AKT bias that depends on ligand identity, a set of factors common to all receptors that varies in abundance or activity with cell line, and an “indirect negative regulation” by ErbB2. This analysis sets the stage for the development of a mechanistic and predictive model of growth factor signaling in diverse cancer lines. Interactive tools for looking up these results and downloading raw data are available at  Receptor tyrosine kinase (RTK) signaling is an important form of cell-cell communication involving 58 human receptors falling into 20 families . Each RTamp breast cancer subtype [Many RTKs are mutated, over-expressed, or dysregulated in cancer and a large number of anti-cancer drugs targeting RTKs are in use or in development . In some subtype , or ErbB subtype . In othe subtype ,6. Conve subtype -9 and HG subtype . The lat subtype .To date, systems-level studies of immediate-early signal transduction have focused primarily on increasing the number of proteins analyzed. For example, mass spectrometry has revealed the kinetics of phosphorylation of approximately 1,000 substrates in EGF-treated HeLa cells , and revamp lines the least. The magnitude of ligand responses appeared to be determined by four primary factors: the identity of the ligand, the abundance of the cognate RTK, regulators common to all receptors but different for AKT and ERK and differing in levels or activity with cell type, and indirect negative regulation mediated by the ErbB2 receptor. The significance of these factors varies depending on whether fold-change or absolute response is regarded as the biologically significant feature.In this paper we analyzed growth factor responsiveness in a canonical collection of 39 breast cancer cell lines of the NCI-ICBP43 set, whose genotypes span many of the mutations observed in primary disease . The datWe generated a multidimensional growth factor response dataset with axes corresponding to cell line, ligand identity, ligand dose, exposure time, and downstream target  and approximately 50% of combinations resulted in pAKT induction. Ligands that bind to ErbB receptors were the most broadly active (every tested ErbB ligand elicited a significant response in at least 35 lines), followed by FGF-1/2, HGF, and IGF-1/2, consistent with the known importance of these ligands in breast cancer biology. pERK induction ranged from statistically insignificant to more than 600-fold in CAMA-1 cells exposed to HRG. Moreover, induction of pERK was transient in some cell lines , or TNBC subtypes based on the expression levels of the ErbB2 tyrosine kinase and the estrogen and progesterone nuclear hormone receptors (ER/PR). We found that TNBC cell lines as a group . As expected, HER2amp cell lines exhibited the highest expression of ErbB2 and this correlated with high basal pErbB1-4, pERK and pAKT , strong responsiveness to FGF1/2 and HRG  of the time-series data identified trajectories that correspond to sustained, transient, late, or no response Figure . These cComparing response data between the two doses tested, we found that pERK was maximally responsive to 1 ng/ml EGF in some cell lines . We found that whereas EGF elicited symmetric pERK or pAKT induction, the FGF-1 response was heavily biased towards pERK, and the response of IGF-1 was biased to pAKT . Unexpectedly, basal pErbB2 and pERK levels were negatively correlated with pERK fold-change . This suggests that, for fold-change induction of pERK by EGF, EGFR is a positive regulator, pErbB2 a negative regulator and ERK level does not matter. The absolute level of induced pERK behaves differently, exhibiting little correlation with pERK or pErbB2 levels, but strong positive correlation with total EGFR level  to over 10her RTKs . Thus, eher RTKs .amp lines, which express the highest levels of ErbB2, are quite insensitive to exogenous ligand: high ErbB2 levels correlate negatively with fold-responsiveness of pERK to growth factors such as the ErbB2/3 ligand HRG. In addition, pAKT responsiveness to IGF ligands and insulin is strongly negatively correlated with ErbB2 levels. In marked contrast, TNBC cells, which are notionally “negative” for ErbB2, are highly responsive not only to HRG but to ErbB ligands in general.Sensitivity to ErbB1-4 ligands is a dominant feature of the breast cancer cell lines we examined: all 39 lines exhibited a statistically significant response to at least one ErbB ligand  and 31 of the 39 lines responded to all four ligands. ErbB receptors are ubiquitously expressed in epithelial cells and breast cancers originate primarily from epithelial ductal or lobular linings . Howeveramp cancers are relatively insensitive to their microenvironment and are among the most treatable breast cancers. Few antibody therapeutics other than those against ErbB receptors have proven effective in breast cancer and this might be a matter not only of the importance of ErbB2 in HER2amp cells, but also the down-regulation of other signaling pathways in these cells. It nonetheless seems plausible that identifying the subset of HR+ and TNBC cells responsive to IGFs, FGFs, and HGF might serve as means to identify those sensitive to therapeutic intervention with anti-receptor antibodies other than trastuzumab. For example, a subset of TNBC tumors might be sensitive to the inhibition of the c-Kit and PDGFR receptors.Other ligands to which breast cancer cells commonly respond include FGFs, IGF/INS, and HGF. Responsiveness to SCF and PDGF is observed more sporadically, but can be as strong as responses to ErbB ligands when the cognate receptor is present. In general, responsiveness within the same family of ligands is highly correlated, likely because such ligands can bind overlapping sets of receptors. TNBC cells exhibit the greatest fold-change responsiveness to ErbB ligands, FGFs, IGF/INS, and HGF, and they also account for the preponderance of responsiveness to SCF and PDGF. It is tempting to speculate that one of the reasons TNBC tumors respond poorly to targeted therapies  is that + cell line with a high level of cMet to have a dose-sensitive response to HGF that is strongly skewed toward pERK.The available data is as of yet insufficient for construction of a mechanistic model of the responsiveness of breast cancer cell lines to growth factors. We therefore propose a four-factor heuristic Figure  which se+ lines are biased toward high pERK response). The fourth and most unexpected factor is an “indirect negative regulation” by ErbB2: expression levels of pErbB2 negatively correlate with pERK fold-change for all ligands examined and are a dominant factor for IGF/INS response at the level of pAKT. Ligand-induced absolute pERK levels are not correlated with pErbB2, however, so the impact of the indirect negative regulation by pErbB2 signaling depends on whether fold-change or absolute level is the critical biological feature. Moreover, for all but IGF/INS, the phenomenon is reversed for pAKT: pErbB2 and basal pAKT levels  were non-adherent and, therefore, not suited to our measurement methodology. Cells were plated to achieve approximately 75% confluence following growth in complete media for 24 h and then starved in serum-free media for 18 h , confirmed to be 10 domain. Responses with values less than two standard deviations above the mean of the controls were considered to be non-significant (corresponding to approximately 1.2-fold change). For the calculation of pathway bias, the pAKT response was linearly scaled by a factor of 2.386 to minimize the Cramér-Von Mises distance between the distribution of pERK and pAKT fold-change responses. A detailed description of data collection and processing can be found in Niepel et al. [Basal expression and phosphorylation levels were normalized using recombinant standards and all measurements below the level of detection were set to the value of the detection threshold. The ligand responses were measured in duplicate and averaged in the logl et al.  and all l et al. .PKS is a founder of Merrimack Pharmaceuticals and chair of its scientific advisory board; this relationship is actively managed by Harvard Medical School (HMS) in accordance with NIH regulations. BS is a shareholder in Merrimack Pharmaceuticals and a board member of SilverCreek Pharmaceuticals.MN, EAP, MC, DHC, and LZ performed all experiments. MH performed all analysis. MN, MH, and JLM designed the website; PKS and BS supervised the work and preparation of the manuscript. All authors read and approved the final manuscript.Description of the dataset, cell lines, ligands and assays used in this study.Click here for fileConnections between ligands and receptors.Click here for fileNetwork maps of all cell lines used in this study.Click here for fileCorrelation between selected basal measurements.Click here for fileResults of kinetic clustering and dose-dependence classification.Click here for fileDistribution of responses and their fold-change according to the kinetic clusters and dose-dependence and distribution among sensitivity classes of response fold-change.Click here for fileEnrichment for basal RTK levels among cell lines with significant responses.Click here for fileRelationship between basal pErbB2, PI3K/PTEN mutational status and the basal levels of pAKT and pERK.Click here for file


TP53 wild type tumor at diagnosis and inhibition of p53 is required for tumorigenesis. Neuroblastomas in particular have previously been shown to have 17q amplification, harboring the WIP1 (PPM1D) gene and associated with poor clinical outcome. We therefore sought to determine whether inhibiting WIP1 with a selective antagonist, GSK2830371, can attenuate neuroblastoma cell growth through reactivation of p53 mediated tumor suppression. Neuroblastoma cell lines with wild-type TP53 alleles were highly sensitive to GSK2830371 treatment, while cell lines with mutant TP53 were resistant to GSK2830371. The majority of tested neuroblastoma cell lines with copy number gains of the PPM1D locus were also TP53 wild-type and sensitive to GSK2830371A; in contrast cell lines with no copy gain of PPM1D were mixed in their sensitivity to WIP1 inhibition, with the primary determinant being TP53 mutational status. Since WIP1 is involved in the cellular response to DNA damage and drugs used in neuroblastoma treatment induce apoptosis through DNA damage, we sought to determine whether GSK2830371 could act synergistically with standard of care chemotherapeutics. Treatment of wild-type TP53 neuroblastoma cell lines with both GSK2830371 and either doxorubicin or carboplatin resulted in enhanced cell death, mediated through caspase 3/7 induction, as compared to either agent alone. Our data suggests that WIP1 inhibition represents a novel therapeutic approach to neuroblastoma that could be integrated with current chemotherapeutic approaches.The wild-type p53-induced phosphatase 1 (WIP1) is a serine/threonine phosphatase that negatively regulates multiple proteins involved in DNA damage response including p53, CHK2, Histone H2AX, and ATM, and it has been shown to be overexpressed or amplified in human cancers including breast and ovarian cancers. We examined WIP1 mRNA levels across multiple tumor types and found the highest levels in breast cancer, leukemia, medulloblastoma and neuroblastoma. Neuroblastoma is an exclusively  The p53 pathway plays a critical role in maintaining the genomic fidelity of stem cell populations, including neural stem/ progenitor cells ,2. Wild-PPM1D) behaves as an oncogene, contributing to the transformation by other driver oncogenes in in vitro transformation assays and in genetically engineered mice . A. APPM1D) gliomas , medullo gliomas , and neu gliomas . Activatn cancer . These cn cancer ,28.Here, we focus on the role of PPM1D in neuroblastoma pathogenesis. This gene is amplified in a small subset of tumors and over expression of WIP1 has been shown to correlate with poor outcome . ImportaTP53 wild-type neuroblastoma cell lines. In addition, WIP1 inhibition markedly sensitizes TP53 wild-type neuroblastoma cell lines to genotoxic chemotherapy. These data suggest that WIP1 meditated repression of p53 is a novel therapeutic target for neuroblastoma.We have recently reported the identification and characterization of a novel series of allosteric, small molecule WIP1 antagonists including the orally bioavailable compound, GSK2830371 . Here, wPPM1D probeset (204566_at) were analyzed using Oncomine Powertools  across >25K samples. Overexpression was determined by setting the median to 1 and filtering values 4 fold above the median per tumor type.Quantile-normalized values from Human Genome U133 GeneChip arrays  corresponding to http://r2.amc.nl). The Neuroblastoma public-Versteeg-88 patient cohort [Analysis of PPMID was performed using the R2: microarray analysis and visualization platform  and treated with 10 Gy ionizing radiation followed by a 30 minute recovery. Flag-tagged p53 proteins were subsequently immunopurified using anti-flag antibody conjugated beads (Sigma Aldrich) and the protein bound to the beads was used as a substrate in the in vitro phosphatase assay. The phosphatase activity towards the intact proteins was determined by western blotting using antibodies against p53 phosphorylated at serine 15  and total p53 (Santa Cruz Biotechnology). GST-tagged PPM1A and PPM1B recombinant protein was provided by Dr. Wendell Yarbrough . Purified PP2A was purchased from Millipore . Purified PPM1G and PPM1H were purchased from OriGene Technologies .p53 pS15 (Ac-VEPPLpSQETFS-amide), p38 MAPK Thr180 (Ac-TDDEMpTGpYVAT-Amide), CHK1 pS345 (Ac-QGISFpSQPTCP-amide), CHK2 pT68 (Ac-LETVSpTQELYS-amide) and H2AX pS139 (Ac-GKKATQApSQEY-amide) phosphopeptides used were synthesized by New England Peptide . The RRA(pT)VA phosphopeptide is a component of the Serine/Threonine Phosphatase Assay System . One hundred ng of each phosphatase was diluted in PP2C buffer  and incubated with 1 mg/mL BSA, 30 mM MgCl2 and 100 μM of the specified phosphopeptide for 1 hour at room temperature. Free phosphate released from the reaction was determined using BIOMOL Green reagent  according to the manufacturer’s protocol. Optical density at 630 nm was detected on a Victor2 multilabel reader . Recombinant WIP1 was purified as previously described using the WIP1-ΔExon6-His-pET-23a+ construct kindly provided by Dr. Ettore Appella . To obta2, in a humidified incubator. GSK2830371 was synthesized at GlaxoSmithKline [Cell lines tested at GlaxoSmithKline were obtained from the American Type Culture Collection, Manassas, VA , and SK-N-FI), Deutsche Sammlung von Mikroorganismen und Zellkulturen, Braunschweig, Germany , and the European Collection of Cell Cultures (ECACC: KELLY and LA1-5S). Cell lines tested at Baylor College of Medicine  were supplied by Dr. Metelitsa  and validated previously . NeuroblithKline . AdditioPPM1D forward 5’-tagtggtgctcagcctgcaa-3’ and reverse 5’-tctgcgtcgcatggtgagt-3’, CDKN1A forward 5’-cctcatcccgtgttctccttt-3’ and reverse 5’-gtaccacccagcggacaagt-3’, and MDM2 forward 5’-tccccgtgaaggaaactgg-3’ and reverse 5’-tttcgcgcttggagtcg-3’. Target mRNA was normalized to GAPDH and relative expression compared to undamaged samples calculated by the standard ΔΔCT method.Cells were exposed to 10 Gy ionizing radiation and harvested at six hours post treatment. RNA was purified using an RNeasy kit . One μg of RNA was used for cDNA synthesis using qScript cDNA Supermix  according to the manufacturer’s protocols. One tenth of the cDNA reaction was used for quantitative RT-PCR using SYBR Green PCR Mastermix (Life Technologies) according to the manufacturer’s protocol in a Step One Plus PCR machine (Life Technologies). Sequences for primers used are: GAPDH forward 5’-ggagtccctgccacactcag-3’ and reverse 5’-ggcccctcccctcttca-3’, 50 of single agents and fixed ratio combinations using the equation described by Chou [Cells were seeded into 96 well plates at 500 cells per well and incubated overnight. Serial dilutions of GSK2830371 were added to cell plates. For combination studies, chemotherapeutic agents were added to cell plates one hour after GSK2830371. Cell viability was determined at days 0 and three (combination) or five and seven (single agent) post-drug addition using the CellTiter-Glo cell viability assay (Promega) or Cell Counting Kit-8  according to the manufacturer’s protocol. CellTiter-Glo luminescent signal was detected on an EnVision 2104 multilabel reader (Perkin Elmer) and CCK-8 optical density at 450 nm was detected on a Victor2 multilabel reader (Perkin Elmer). Mutually nonexclusive combination index was calculated at the ICIMR-32 and SK-N-AS cells were suspended at 1.5 million cells per mL, and dispensed into 6 well tissue culture plates in 2 mL volumes. After an overnight incubation, DMSO or GSK2830371 serial dilutions were transferred to duplicate wells of cell plates and incubated for 6 hours. Cells were then washed with ice-cold PBS without calcium chloride or magnesium chloride, and lysed in ice-cold RIPA lysis buffer  containing Complete Mini protease inhibitor and PhosSTOP phosphatase inhibitor . Lysates were briefly sonicated, clarified by centrifugation, and total protein was determined . SDS-PAGE was performed using pre-cast 4–12% Bis-Tris or 3–8% Tris-Acetate gels with the Novex NuPAGE system (Life Technologies). Western transfer to nitrocellulose membranes was carried out using the iBlot dry blotting system (Life Technologies) set to 9 minutes or wet transfer using the Novex NuPAGE system at 30 V (1 hour). Membranes were blocked with Odyssey Blocking Buffer  for one hour prior to being probed with the following antibodies from Cell Signaling Technology : p53 (S15), H2AX (S139), H2AX, p21, CHK1 (S345), CHK1, CHK2 (T68), CHK2, ATM (S1981), ATM, p38 (T180/Y182), p38, and PUMA. Membranes were also probed with antibodies to WIP1  and p53 (Millipore). A GAPDH antibody  was used to correct for protein loading. Secondary antibodies conjugated to IRDye 680 and 800 (Li-Cor Bioscience) were used for detection. Antibodies were diluted in 50% Odyssey Blocking Buffer, 50% PBS, 0.1% Tween-20. Membranes were probed overnight with primary antibodies and one hour with secondary antibodies. Odyssey molecular weight markers were used to determine protein size (Li-Cor).PPM1D  and MYCN  copy number status were determined by running the Qiagen qBiomarker Copy Number assays . Assays were performed in quadruplicates using 2 ng of genomic DNA per reaction following manufacturer’s protocol (Qiagen) on a QuantStudio real time PCR instrument . Data was normalized to a multi-copy reference assay (VPH000-0000000A) and analyzed by the calibrator genome method (Qiagen’s software).http://www.broadinstitute.org/ccle/home) and NCBI's Gene Expression Omnibus (GEO: http://www.ncbi.nlm.nih.gov/geo/)  using the tool developed by the Broad Institute (http://www.broadinstitute.org/gsea) [TP53 signatures extracted from MSigDB collection (http://www.broadinstitute.org/gsea/msigdb/) were interrogated by GSEA following geneset permutations in cell lines sensitive or resistant to GSK2830371 (see Publicly available Affymetrix microarray data for the cell lines used in this study were downloaded from Cancer Cell Line Encyclopedia (CCLE: eo/) see . CEL filrg/gsea) . Select 0371 see  using a TP53 but normal PPM1D copy number and expression, exemplified by AML, and (ii) those derived from solid tumors with both wild-type TP53 and aberrantly high WIP1 levels. To expand upon these observations and further define a potentially responsive clinical population, we used publicly available microarray data in Oncomine to determine the level of WIP1 mRNA expression across several different tumor types. Our results confirm that, in addition to breast cancer, nervous system malignancies have a high frequency of elevated WIP1 expression, relative to the cognate normal tissue (PPM1D gene expression in neuroblastoma using the R2 genomics platform of annotated databases (http://r2.amc.nl) demonstrates a correlation of PPM1D mRNA level with higher stage tumors  and -dependent (PPM) subclasses of the type 2 protein phosphatase family. Specifically, we measured the activity of each phosphatase on a generic substrate, RRA(pT)VA, in the presence or absence of GSK2830371  and SK-N-AS, are resistant to GSK2830371 while TP53 wild-type cell lines have varying degrees of sensitivity ranging from 0.2 μM for IMR-32 to >10 μM for NGP cell line (CDKN1A (p21) and PPM1D itself  did have a 5-fold increase in p21. These results suggest that sensitivity of neuroblastoma cell lines to growth inhibition by GSK2830371 is dictated not only by TP53 mutational status, but also by the magnitude of a functional p53 response.Due to the high frequency of WIP1 mRNA overexpression and amplification in neuroblastomas , we hypoell line . Our resell line . To analD itself . The celTP53 full length and determined a relationship, if any, with GSK2830371 antiproliferative response in a larger panel of neuroblastoma cell lines. In this panel of 13 cell lines, once again TP53 mutational status clearly determines sensitivity or resistance to GSK2830371. Seven of 11 TP53 wild-type cell lines tested had some degree of sensitivity to GSK2830371 while all 5 TP53 mutant cell lines were resistant  enrichment (NES >1.5) in the sensitive group (n = 6) relative to the resistant group (n = 6) of lines  is reported to be altered in neuroblastoma, we investigated if there was a relationship between the sensitivity of neuroblastoma cell lines and PPM1D copy number status. Among cell lines with a gain in PPM1D by copy number analysis  majority of cell lines with wild-type TP53 showed sensitivity to GSK2830371  cell line and the resistant SK-N-AS (mutant) cell line to increasing concentrations of GSK2830371 for six hours and examined known WIP1 substrates and p53 response proteins by western blot  mRNA expression from >25K microarray expression profiles across multiple human tissues. Overexpression is determined above the indicated threshold (see Survey of hold see .(TIF)Click here for additional data file.S2 FigFree phosphate is released from four WIP1 substrate peptides in the complete reaction . GSK2830371 inhibits this phosphate release to levels similar to that of the “no enzyme” and “no Mg” conditions.(TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.


CDH1 is critical for intercellular adhesion. In our previous work, we reported a nonfunctional CDH1 transcript that lacks the final 83 base pairs of exon 8 (1054del83). In this work, we probed the role of histone epigenetic modifications as well as DNA methylation in selection of this isoform.The tumor suppressor gene CDH1 RNA expression. Methylation of CDH1 was analyzed by bisulphite sequencing PCR. ChIP assay was performed to show histones level. Cell lines were treated with DNA methyltransferase inhibitor AZA, HDAC inhibitor TSA, or siRNA oligonucleotides to test regulation of CDH1 splicing.RT-qPCR was used to detect del83 transcripts were observed in gastric cancer (GC) cell lines than human gastric mucosal epithelial cell line GES-1. All the cell lines showed significant methylation pattern at the CpG sites of CDH1 exon 8. AZA treatment did not influence selection of 1054del83 transcripts. A significant decrease in acetylation for histones H3 and H4K16Ac in an internal region of the CDH1 gene surrounding the alternative exon 8 were detected in GC cell lines. Treatment with TSA preferentially expressed the correctly spliced transcript and not the exon 8 skipped aberrant transcripts, showing that histone acetylation was involved in the splicing regulation. SiRNA-mediated knockdown of SETD2 (The specific methyltransferase of H3K36) decreased exclusion of exon 8, suggesting that the presence of this mark correlates with increased skipping of the final 83 base pairs of CDH1 exon 8. However, CDH1 splicing was not affected by SRSF2 knockdown.Greater CDH1 1054H3K36me3 correlates with increased skipping of the final 83 base pairs of CDH1 exon 8. Histone acetylation was involved in the splicing regulation as well.The online version of this article (doi:10.1186/s12885-015-1983-5) contains supplementary material, which is available to authorized users. Gastric cancer (GC) is one of the most common malignancies worldwide, with the highest incidence rates in Eastern Asia . It is bCDH1 (E-cadherin) is critical for intercellular adhesion [CDH1 gene mutations occurred frequently in hereditary diffuse gastric cancer (HDGC) [CDH1 gene [The tumor suppressor gene adhesion , 3. CDH1r (HDGC) , 5. In oDH1 gene , 7.del83) of the gene. This non-functional transcript has a premature termination codon (PTC) with 358 aminos and is degraded by the nonsense mediated decay (NMD) pathway. We demonstrated this transcript is a frequent event in Chinese GC patients [Most human genes are alternatively spliced in a cell type–and tissue-specific manner, and abnormalities of pre-mRNA alternative splicing contribute to disease. In our previous work, we reported an alternatively spliced, nonfunctional CDH1 transcript that lacks the final 83 base pairs of exon 8  and human gastric mucosal epithelial cell line GES-1  were cultured in DMEM medium, supplemented with 10 % fetal calf serum, at 37 °C with 5 % COTotal RNA from GC cell lines or human gastric mucosal epithelial cell line was extracted using RNAiso Plus  Co., Ltd.). RT–qPCR was performed in two steps. First strand cDNA synthesis was performed using PrimerScript RT reagent Kit (TaKaRa) with random DNA hexamers and oligo-dT primer according to the manufacturer’s protocol. CDH1 expression is analyzed by a TaqMan qPCR experiment on ABI StepOne Plus Real-Time PCR System . The qPCR reaction condition and sequences of PCR primers and TaqMan probes are as previous reported .CDH1 exon 7–9 was analyzed by bisulphite sequencing PCR (BSP) on an ABI 3130-Avant automated sequencer (Applied Biosystems). Primer sequences for BSP are CDH1-E7-BSP-F:5′-TGAATTTTTTTAGGAATTTTTGTGAT-3′, CDH1-E7-BSP-R:5′-ATCCAACCCAATAATAACCACACTA-3′, CDH1-E8-BSP-F: 5′-GGGTTAGGTTAAAGGTGGTTAGTGT-3′, CDH1-E8-BSP-R: 5′-AAACCTTTCTTTAAAAACCCTCTAAAA-3′, CDH1-E9-BSP-F:5′-AGTATAAGGGTTAGGTGTTTGAGAA-3′ and CDH1-E9-BSP-R:5′-CTACATCTTACCAAATACCATACAAACC-3′.DNA was treated with bisulfite using the CpGenome™ DNA Modification Kit  according to manufacturer’s protocol. Methylation status of CpG sites of 6 cells were fixed with 1 % formaldehyde for 10 min at 37 °C. The cells were washed extensively with PBS, and the chromatin was sheared by sonication (Bioruptor sonicator) to 200–500 bp fragments. The cross-linked histone-DNA complex was immunoprecipitated with anti-H3K36me3, anti-H3K4me2, anti-H4K16ac, or anti-pan acetylated-H3 (anti-acH3) antibodies (Millipore). Normal rabbit IgG was used as negative controls. DNA was obtained from the crosslinked complex and equal amounts of input and immunoprecipitated DNA 1.0 ng were used to perform SYBR Green real-time PCR on ABI StepOne Plus Real-Time PCR System (Applied Biosystems). The qPCR reaction mixture contained DNA, the forward primer, reverse primer, ROX Reference Dye, SYBR and Premix Ex Taq™ (TaKaRa). Primers are described in Additional file ChIP assays were performed by using the EZ-Magna ChIP Kit  according to the manufacturer’s instructions. Briefly, 1 × 10To test whether CDH1 splicing is affected by DNA methylation or histone acetylation status, the GC cell lines SGC-7901, BGC-823 and MGC80-3 and human gastric mucosal epithelial cell line GES-1 were treated with DNA methyltransferase inhibitor AZA or HDAC inhibitor TSA. 5-aza-2′deoxycytidine (Sigma-Aldrich) was added to the medium to be 1 μM. Treatments were maintained for 72 h. TSA (Sigma-Aldrich) was added to culture to be 0.5 μM 12 h before the end of the experiment. CDH1 RNA expression was determined by RT-qPCR analysis.Downregulation of SETD2 or SRSF2 was performed using siRNA oligonucleotides . SiRNA oligos against human SETD2 or SRSF2 were delivered to cells at 50 nM following the manufacturer’s instructions. Normal negative control was used as control. Forty eight hours after transfections, cells were harvested and the knockdown efficiencies were analyzed by RT-qPCR and changes in CDH1 splicing were analyzed as mentioned above.http://www.fruitfly.org/seq_tools/splice.html) were used to predict splice acceptors and donors around CDH1 exon 8. We used the ESEfinder program (http://rulai.cshl.edu/tools/ESE) to identify exonic splicing enhancers (ESEs) [NNSPLICE (s (ESEs) .P values are two-sided; P < 0.05 was considered statistically significant.Differences in transcripts level between groups were analyzed by ANOVA  and SNK-q test. All CDH1 exon 8 and flanking sequences which will produce the 1054del83 transcript and normal transcript, respectively [We find two donor sites in ectively  Fig. 1)CDH1 exondel83 transcript in the GC cell lines SGC-7901, BGC-823 and MGC80-3 and human gastric mucosal epithelial cell line GES-1. A lower expression of the normal CDH1 and a higher level of CDH1 1054del83 transcript are detected in GC cell lines SGC-7901 and BGC-823 compared to GES-1  siRNA . To explore a putative regulation of the alternative splicing of CDH1 exon 8 by means of epigenetic, we carried out in vitro experiment.There might be a natural balance of the two alternative splice products  to be recognized by the splicing machinery, and thus the CDH1 1054del83 transcript. However, knockdown of SRSF2 did not influence ratios of CDH1 1054del83 transcript vs CDH1 normal transcript in the GC cell lines and GES-1 cell line (Fig. Analysis of histone methylation revealed an increase for H3K36 tri-methylation surrounding the CDH1 exon 8 regions in GC cell lines SGC-7901 and BGC-823 compared to human gastric mucosal epithelial cell line GES-1 Fig. . Down-redel83 isoform than GES-1 cell line, the difference did not exist between GC cell lines MGC80-3 and GES-1 (Fig. Though GC cell lines SGC-7901 and BGC-823 carry significant more CDH1 1054The chromosomal region encompassing the CDH1 exon 8 is highly enriched in H3K36me3 marks in GC cells compared to human gastric mucosal epithelial cells, and the presence of this mark correlates with increased skipping of the final 83 base pairs of CDH1 exon 8 in the mature CDH1 mRNA. We propose that the epigenetic modification patterns, such as histone acetylation might have a role in CDH1 exon 8 alternative splicing regulations as well. The linking between histone modifications and splicing regulation might be important in GC occurrence.


Scientific Reports6: Article number: 2239610.1038/srep22396; published online: 03032016; updated: 07202016This Article contains errors. In the last paragraph of the Results section,“Given the ability of atRA and compounds 3 and 4 to induce apoptosis in animal cancer cell line context, we surveyed several human cell lines and found that CRABP1 expression was lost in human ovarian cancer cell line A2780 and human pancreatic ductal carcinoma cell line KPC”.should read:“Given the ability of atRA and compounds 3 and 4 to induce apoptosis in animal cancer cell line context, we surveyed several additional cell lines and found that CRABP1 expression was lost in human ovarian cancer cell line A2780 and mouse pancreatic ductal carcinoma cell line KPC”.In addition, the legend of Figure 4D,D) Compounds lose ability to induce ERK activity in human CRABP1 null cancer cell lines A2780 (ovarian) and KPC  after 100 nM, 30 min treatment (upper). Compounds are able to induce apoptosis after 24 hr treatment (lower) in CRABP1 positive MCF7 breast cancer cell line (lower right). Quantification of cleaved caspase 3 is shown for MCF-7. Data (A–D) are representative of at least 3 independent experiments”.“(should read:D) Compounds lose ability to induce ERK activity in human CRABP1 null cancer cell line A2780 (ovarian) and mouse KPC  after 100 nM, 30 min treatment (upper). Compounds are able to induce apoptosis after 24 hr treatment (lower) in CRABP1 positive MCF7 breast cancer cell line (lower right). Quantification of cleaved caspase 3 is shown for MCF-7. Data (A–D) are representative of at least 3 independent experiments”.“(