Molecular chaperones are key components of the protein homeostasis system against protein misfolding and aggregation. It has been recently shown that these molecules can be rationally modified to have an enhanced activity against specific amyloidogenic substrates. The resulting molecular chaperone variants can be effective inhibitors of protein aggregation in vitro, thus suggesting that they may provide novel opportunities in biomedical and biotechnological applications. Before such opportunities can be exploited, however, their effects on cell viability should be better characterised. Here, we employ a rational design method to specifically enhance the activity of the 70-kDa heat shock protein (Hsp70) against the aggregation of the human islet amyloid polypeptide . We then show that the Hsp70 variant that we designed  is significantly more effective than the wild type in recovering the viability of cultured pancreatic islet β-cells RIN-m5F upon hIAPP aggregation. These results indicate that a full recovery of the toxic effects of hIAPP aggregates on cultured pancreatic cells can be achieved by increasing the specificity and activity of Hsp70 towards hIAPP, thus providing evidence that the strategy presented here provides a possible route for rationally tailoring molecular chaperones for enhancing their effects in a target-dependent manner. Molecular chaperones help maintain protein homeostasis by transiently binding their protein substrates to assist them in folding and trafficking ,2, and tAmong molecular chaperones, heat shock protein 70 kDa (Hsp70) has been the focus of much interest for protein misfolding diseases since it plays essential roles in protein homeostasis in physiological and stress conditions ,28,29, iOur strategy here is to modify rationally Hsp70 to increase its activity and specificity for a selected target substrate by grafting onto it a binding peptide designed to interact with a given epitope in the target substrate. By increasing the binding efficiency towards the target substrate, this strategy avoids the need for overexpression or modification of the ATPase domain by increasing the efficiency towards the target substrate. We have previously shown that this approach can be used in vitro to alter Hsp70 to target specifically the amyloidogenic regions of α-synuclein, a disordered protein associated with Parkinson’s disease, resulting in a reduction of aggregation even at low molar ratios . In this study, we targeted human islet amyloid polypeptide (hIAPP) since this peptide is the major component of the amyloid deposits found in patients with non-insulin dependent (type II) diabetes mellitus ,46. hIAPOur choice of targeting Hsp70 among other possible molecular chaperones was prompted by its known association with hIAPP-mediated pathology ,53. We pIn this work we rationally designed an Hsp70 variant with improved activity against hIAPP aggregation. The rational design procedure consists of two steps. First, we used a recently developed method  to constThe viability of RIN-m5F cells was used to compare the effects of Hsp70 WT and the designed variant GHsp70-hIAPP on the cytotoxicity of hIAPP. A tetrazolium salt reduction assay -2,5-diphenyltetrazolium bromide) (MTT) was chosen since this assay is widely used in the literature to assess hIAPP toxicity in RIN-m5F cells ,56.p < 0.01; at 0.4 μM 81 ± 1% and 103 ± 1%, respectively, p < 0.001; at 0.2 μM 79 ± 1% and 100 ± 1%, respectively, p < 0.001; We first checked that cell viability was significantly compromised in the presence of 10 μM hIAPP. We observed a reduction to a level (66 ± 1%), consistent with previous reports ,56 Figua. By conThese findings are significant since they indicate that the action of the GHsp70-hIAPP variant takes place via a combination of a generic activity of Hsp70 and a specific activity of this molecular chaperone through the additional engineered interaction with hIAPP that we designed ,57. We tWhite-light images of RIN-mF5 cells exposed to hIAPP for 24 h displayed a marked difference in cell morphology b. hIAPP-We next used the GHsp70-Aβ variant as a control since this variant contains a C-terminal grafted extension of equal length and similar hydrophobicity and charge to the variant we grafted onto GHsp70-hIAPP. This is, therefore, a rather stringent test, as GHsp70-Aβ could be expected to enhance non-specific binding towards hIAPP aggregates compared to the Hsp70 WT, since its grafted peptide endows it with an enhanced inhibitory activity on the in vitro aggregation of Aβ42 . In suppdK of 2.2 ± 0.3 μM. As expected, the designed variant GHsp70-hIAPP’s binding affinity was ten-fold higher (dK= 0.2 ± 0.06 μM), while the affinity of the control variant GHsp70-Aβ (dK = 2.8 ± 0.5 μM) was effectively unchanged with respect to that of Hsp70 WT.In order to understand whether the results from the cellular experiments could be explained by a stronger direct binding of GHsp70-hIAPP with hIAPP, we determined the affinity of this interaction and compared it to the corresponding affinities of Hsp70 WT and GHsp70-Aβ. To do so, we performed a fluorescence titration assay using a variant of hIAPP, which was N-terminally labelled with the fluorophore carboxyfluorescein, also known as FAM . In partAs a further validation, we performed an ELISA-based binding assay . This anGiven the enhanced binding affinity of GHsp70-hIAPP for hIAPP, we then assessed whether this Hsp70 variant was also more effective in directly inhibiting its aggregation in vitro. To do so, we monitored the formation of amyloid fibrils in 10 μM solutions of hIAPP alone or in the presence of the three Hsp70 variants by means of thioflavin-T (ThT) aggregation assays in vitro a. In ordThese aggregation experiments suggest that the reduction in hIAPP cell toxicity observed in the presence of the Hsp70 variants can be achieved via their anti-aggregation activity. As previous studies demonstrated that soluble oligomers formed during the aggregation of hIAPP are the most toxic species for RIN-m5F cells , we can To obtain more insights into the specific mechanism of the Hsp70-mediated inhibition of hIAPP aggregation, we performed transmission electron microscopy for the same mixtures  at the plateau of aggregation (15 h). We found that the morphology of the aggregates was significantly changed in the presence of all Hsp70 variants b, indicaAs a control, we performed the same analysis at the beginning of the aggregation reaction of hIAPP alone, and after 15 h of a reaction containing the Hsp70 variants alone , findingWe have described a rational design strategy to generate a molecular chaperone variant targeting an epitope within the amyloidogenic region of hIAPP. We have applied this strategy to Hsp70 and showed that it resulted in a variant with an enhanced ability to inhibit the hIAPP-induced toxicity in cultured RIN-m5F pancreatic islet β-cells. We have also shown that this beneficial effect results from an increased anti-aggregation activity of the Hsp70 variant, which diverts the aggregation pathway of hIAPP towards the formation of non-toxic aggregates. The therapeutic potential of molecular chaperones is a field in its infancy. As a consequence, there are still concerns regarding the use of these molecules as therapeutic agents for protein misfolding diseases. These concerns include whether therapeutic molecular chaperones would effectively engage their targets and whether their administration would result in pro-inflammatory responses ,11,12,13Although further methodological improvements are needed, our results provide support for the concept that engineered molecular chaperones could provide effective tools in biomedical and biotechnological applications related to protein misfolding and aggregation.E. coli BL21(DE3) Gold Strain  and purified as previously described [The different complementary peptides were grafted at the C-terminal end of human Hsp70  by means of mutagenic polymerase chain reaction (PCR) with phosphorylated oligonucleotides. Recombinant wild type and designed N-hexa-His-tagged Hsp70 variants were overexpressed from the pET-28b vector  in escribed .hIAPP  was dissolved at 1 mg/mL in hexafluoroisopropanol (HFIP) and incubated over night at room temperature in order to dissolve preformed aggregates. The following day, the peptide was lyophilised and resuspended in the cell medium just prior the toxicity experiments. v/v) fetal bovine serum (F9665), 1 mM sodium pyruvate , 4500 mg/L glucose (G8644), 2 mM Glutamax supplement , 20 mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) and maintained at 37 °C, 5% CO2, and 95% relative humidity in tissue culture flasks . Cultures were routinely split (1:2) at ~80% confluency by rinsing the cells with Dulbecco’s phosphate buffered saline  without calcium or magnesium and released for sub-cultivation using 0.25% (v/v) trypsin-EDTA.All cell culture reagents were purchased from Sigma-Aldrich  unless otherwise specified. Pancreatic insulinoma RINm5F β-cells  from Rattus norvegicus were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium  supplemented with 10%  for 24 h prior to the experiments with hIAPP or Hsp70 variants. Cell viability assays were conducted during passage number 15–26 . hIAPP wH-tetrazolium bromide  tetrazolium salt reduction assay, in 96-well plates. The intensity of the formazan produced from enzyme cleavage of the tetrazolium salt by metabolically active cells is proportional to the number of viable cells. After the exposure to treatments for 24 h at 37 °C, cells were incubated in a 0.5 mg/mL MTT solution per well for 2 h at 37 °C and then in a cell lysis buffer . The plate was gently mixed for 60 min at 400 rpm before the absorbance was measured at 570 ± 10 nm using a plate-reader . Untreated cells and cells lysed with 9% (v/v) Triton X-100 were used as controls for the expected maximum and minimum MTT reduction, respectively. Data are expressed as the percentage of MTT reduction compared to an untreated cell control corrected for the absorbance of cell media containing MTT without cells. RIN-m5F cell viability was assessed using the 3--2,5-diphenyl-2w/v) BSA in PBS (BSA/PBS). Samples of 60-µL of the different Hsp70 variants at 7 µM of protein concentration were then incubated in the coated wells. Primary mouse monoclonal antibody against human Hsp70  and secondary antibody conjugated with the fluorophore Alexa-488  were diluted 1:1000 in BSA/PBS and added to the wells. All incubations were performed at room temperature for 1 h under shaking and were followed by six consecutive washes with PBS/Tween 0.02%. Fluorescence measurements were performed using a CLARIOstar plate reader .The wells of the ELISA plates  were coated with 2.5, 5, and 10 µM of hIAPP and blocked with 5% (2 were incubated in darkness for 1 h at room temperature (RT). At the end of the incubation, samples were transferred into a low-binding, clear-bottomed half-area 96-well plate . Fluorescence intensities were then recorded at 520 nm by exciting the samples at 490 nm with a CLARIOstar plate reader . They were subtracted for the value at 0 µM of chaperone and fitted using a one-site binding model with the software Prims . Data are represented as fraction of bound hIAPP, by setting the fitted value of the fluorescence plateau to 1.Solutions containing 0.15 µM FAM-hIAPP  and different concentrations (from 0 to 36 µM) of either Hsp70 WT, GHsp70-Aβ, or GHsp70-hIAPP in 50 mM Tris pH 7.4, 150 mM KCl, and 5 mM MgClAggregation solutions containing 10 µM monomeric hIAPP alone or in the presence of 0.8 µM Hsp70 WT, GHsp70-Aβ, or GHsp70-hIAPP in cell medium and 20 µM ThT were incubated in quiescent conditions at 37 °C. ThT fluorescence of the samples was monitored at 480 nm in low-binding, clear-bottomed half-area 96-well plates  upon excitation at 440 nm by means of a CLARIOstar plate reader . Fluorescence data were then converted into relative fibril mass, assuming the fluorescence value of hIAPP at the plateau alone was equal to 1.2O, and negatively stained with 2% (w/v) uranyl acetate.TEM images of hIAPP aggregates obtained in absence or presence of the chaperone variants were acquired using a Tecnai G2 80–200 kv transmission electron microscope . Samples of 10 µL were applied to 400 mesh copper grids, washed twice with ddH


Integrating data from multiple regulatory layers across cancer types could elucidate additional mechanisms of oncogenesis. Using antibody-based protein profiling of 736 cancer cell lines, along with matching transcriptomic data, we show that pan-cancer bimodality in the amounts of mRNA, protein, and protein phosphorylation reveals mechanisms related to the epithelial-mesenchymal transition (EMT). Based on the bimodal expression of E-cadherin, we define an EMT signature consisting of 239 genes, many of which were not previously associated with EMT. By querying gene expression signatures collected from cancer cell lines after small-molecule perturbations, we identify enrichment for histone deacetylase (HDAC) inhibitors as inducers of EMT, and kinase inhibitors as mesenchymal-to-epithelial transition (MET) promoters. Causal modeling of protein-based signaling identifies putative drivers of EMT. In conclusion, integrative analysis of pan-cancer proteomic and transcriptomic data reveals key regulatory mechanisms of oncogenic transformation. Profiling molecular and phenotypic characteristics of large collections of cancer cell lines can be used to identify distinct and common oncogenic pathways across cancer types. So far, most large-scale data obtained from cancer cell lines have been at the genomic, transcriptomic, and phenotypic levels. Recently, high-quality data at the level of cell signaling through protein abundances and phosphorylation sites has become available. By integrating this newly generated protein data with prior transcriptomic data, and by visualizing all cancer cell lines using dimensionality reduction techniques, pan-cancer cell lines are strikingly shown to organize into a gradient of epithelial to mesenchymal types. Interestingly, many of the measured proteins and transcripts display bimodality; the expression of genes, proteins, and protein phosphorylations is either high or low, strongly suggesting that they act as molecular switches. Focusing on further characterizing molecular switches of epithelial-mesenchymal transitions, we identify candidate regulators and small molecules that can induce or reverse such transition, as well as potential causal relationships between proteins. Since the mesenchymal state of tumors is known to be associated with metastasis and later-stage cancer development, better understanding the regulatory mechanisms of epithelial-to-mesenchymal transition can lead to improved targeted therapeutics. CDH1 loci, Snail recruits protein complexes containing histone deacetylases (HDACs) that deacetylate H3 and H4 histones, silencing the transcription of E-cadherin [Central to the understanding of cancer cells are their epithelial or mesenchymal traits, which are governed by epithelial-mesenchymal transition (EMT). Cells that have undergone EMT display increased invasiveness and metastatic potential . The tracadherin . Other kcadherin . The regcadherin , predictde novo [The reverse phase protein array (RPPA) is a high-throughput proteomics method that utilizes antibody binding to quantify protein expression and post-translational modifications including phosphorylation, acetylation, and protein cleavage. Compared to mass spectrometry proteomics, RPPA has higher sensitivity for low-abundance proteins and is characterized by increased throughput; however, it relies on high-quality antibodies, so it cannot identify proteins or post-translational modifications de novo . Using Rde novo . This dade novo . In addide novo , which ade novo .Most genome-wide studies of EMT in cancer cell lines and tumors have focused on particular cancer types. Combining EMT signatures based on cell lines and tumors of multiple cancer types can identify general transcriptomic features of EMT in cancer cells, which are clinically relevant for multiple types of cancer. More recently, transcriptomic data from TCGA and Cancer Cell Line Encyclopedia (CCLE) have been used to define a pan-cancer EMT signature based on the expression of E-cadherin and Vimentin alone . In thisThe Cancer Cell Line Encyclopedia (CCLE) contains 1037 cancer cell lines with profiled transcriptomes , and theTranscriptional profiling of human tumor samples accurately predicts the tissue of origin for common cancer types . This suTo test the hypothesis that EMT governs the molecular states of cell lines across cancer types, we colored the z-scores of E-cadherin expression on the points on the t-SNE maps. For both transcripts and proteins, the cancer cell lines were globally organized by a gradient of E-cadherin expression , middle.Oncogenesis is a multi-step process by which cells acquire cancerous traits, which often mimic physiological cellular processes such as embryogenesis . Such prb > 0.5) bimodal expression of mRNA and protein. Slightly fewer proteins (10.8%) were uniquely bimodal only at the protein level, including important cancer-related proteins such as MEK1, mTOR, E2F1, TTF1, EIF4G, and JAB1. Hence, these proteins are bimodally expressed due to post-transcriptional regulatory mechanisms such as protein translation and degradation. In addition, we compared the bimodality of proteins and their phosphosites as measured by antibody binding in the RPPA data (b = 0.46), most likely due to autophosphorylation on increased dimerization at higher expression [CDH1 genotyped cell lines in COSMIC had either nonsense (MDA-MB-453 and HT115) or frameshift (MDA-MB-134-VI) mutations in CDH1, which validate our ability to identify effects on E-cadherin translation. Inactivating mutations in CDH1 are frequently observed in breast and gastric cancers with cancer type-specific mutational patterns and are associated with loss of cell-cell adhesion and increased cell motility [To identify whether the observed bimodal protein expression across cancer cell lines correlate with transcriptional regulation, we evaluated the bimodality of matching transcripts from CCLE . We thenPPA data . Here, wpression . The bimmotility . The natb| > 0.3. This yielded a network of 172 protein nodes connected by 507 edges, from which network communities were defined based on the leading non-negative eigenvector [b = 0.70), but found in a separate community (EMT3), along with Rab25 and N-cadherin. Looking closer at this correlation, cell lines had high E-cadherin and low Claudin7 expression but not conversely  with |renvector . In totaenvector . E-cadheenvector . Here wenversely . Two othnversely . In summb| > 0.5) between E-cadherin protein expression and transcriptomic measurements from CCLE, resulting in 239 transcripts—215 positively and 24 negatively coupled  algorithm implemented in the ‘mixtools’ R package with default parameters. To compare the fitted distribution to unimodal Gaussian distributions, we calculated the difference between the Bayesian Information Criterion (BIC). The data were determined to be bimodal if the BIC difference was larger than 2. Based on the fitted Gaussian mixture model, we calculated, using Bayes’ theorem, the posterior probabilities of measurements being generated from the high expression component. Note that the probability of belonging to the low component is 1-p. To estimate the tissue diversity of each bimodal fit, we first calculated the frequencies of tissues assigned to the low (p < 0.5) and the high (p > = 0.5) component. We then calculated the Shannon entropy of the tissue distributions associated with the low- and the high components. The bimodal RPPA measurements were classified into groups of low, medium, and high tissue diversity by the tertiles of the minimum tissue entropy associated with low- and high expression. The bimodal expression was considered common if the fitted mixture coefficients were above 1/4 and rare if below. Based on the posterior probabilities of the bimodal fits associated with high tissue diversity, we calculated a network of bimodal coupling coefficients defined as Spearman’s correlations between the posterior probabilities. To detect robust communities in this network, we set a cutoff of |rb > 0.5). Enrichment analysis was performed with Enrichr [The coupling coefficients between the E-cadherin RPPA measurements and matched CCLE transcript data were used to define an EMT signature Click here for additional data file.S2 Fig(EPS)Click here for additional data file.S3 FigArrows point to infection points where the cell lines are organized into clusters. Such inflection points are not expected for random distances between cell lines.(EPS)Click here for additional data file.S4 Fig(EPS)Click here for additional data file.S5 Fig(EPS)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(CSV)Click here for additional data file.


HIGHLIGHTSPreliminary results of this work were presented at the 2016 Academic Surgical Congress, Jacksonville (FL), February 2–4 2016 This study was funded by “Sapienza—University of Rome” (Funds for young researchers) and “AIRC” Hedgehog inhibitor was kindly provided by Genentech, Inc.®.GLI1 and GLI2, two Hh target genes, and CC survival and recurrence by gene expression microarray from a cohort of 382 CC patients. We found that patients with increased expression of GLI1 showed a statistically significant reduction in survival. In order to demonstrate a causal role of Hh pathway activation in the pathogenesis of CC, we treated HCT 116, SW480 and SW620 CC cells lines with GDC-0449, a pharmacological inhibitor of Smoothened (SMO). Treatment with GDC-0449 markedly reduced expression of Hh target genes GLI1, PTCH1, HIP1, MUC5AC, thus indicating that this pathway is constitutively active in CC cell lines. Moreover, GDC-0449 partially reduced cell proliferation, which was associated with upregulation of p21 and downregulation of CycD1. Finally, treatment with the same drug reduced migration and three-dimensional invasion, which were associated with downregulation of Snail1, the EMT master gene, and with induction of the epithelial markers Cytokeratin-18 and E-cadherin. These results were confirmed by SMO genetic silencing. Notably, treatment with 5E1, a Sonic Hedgehog-specific mAb, markedly reduced the expression of Hedgehog target genes, as well as inhibited cell proliferation and mediated reversion toward an epithelial phenotype. This suggests the existence of a Hedgehog autocrine signaling loop affecting cell plasticity and fostering cell proliferation and migration/invasion in CC cell lines. These discoveries encourage future investigations to better characterize the role of Hedgehog in cellular plasticity and invasion during the different steps of CC pathogenesis.Colon Cancer (CC) is the fourth most frequently diagnosed tumor and the second leading cause of death in the USA. Abnormalities of Hedgehog pathway have been demonstrated in several types of human cancers, however the role of Hedgehog (Hh) in CC remain controversial. In this study, we analyzed the association between increased mRNA expression of  In colon carcinoma (CC) as for other cancers, the understanding of cellular and molecular mechanisms involved in tumor progression is essential to provide the rationale for novel therapies. According to the 2016 NCCN Panel, CC is the fourth most frequently diagnosed tumor and the second leading cause of death in the USA. Incidence in Italy raised from 27.06% in 1970 to 52.65% in 2010 Ciatto, . ReactivHh pathway plays an important role in tissue development and organogenesis . Upon binding of Hh ligands to PTCH1, the G-protein–coupled receptor, Smoothened (SMO) is activated and promotes nuclear translocation of GLI family of zinc finger transcription factors. GLI activation induces the transcription of Hh target gene products, including ubiquitous genes such as GLI1, PTCH1, and Hh-interacting protein (HIP1) , lung, and brain tumors and rhabdomyosarcoma  with CC patients survival, we first treated CC cell lines with a pharmacological SMO inhibitor, GDC-0449, a small molecule already approved by the US Food and Drug Administration (FDA) for the treatment of advanced basal cell carcinoma (BCC)  and metastatic (SW620) human CC cell lines were grown in DMEM supplemented with 10% FBS  and antibiotics. These cellular lines were reported to express detectable levels of SMO and GLI1 . Differences were considered significant at P < 0.05 .RNA, extracted from cell cultures with ReliaPrep™ RNA Tissue Miniprep System , was reverse transcribed with iScriptTM c-DNA Synthesis Kit  according to the manufacturer's instructions. cDNAs were amplified by qPCR reaction using GoTaq® qPCR Master Mix . The specific primer pairs are listed in Table Monoclonal antibody against E-cadherin was from BD . Monoclonal antibodies against Snail1 and against GLI1 were from Cell signaling . Monoclonal antibody against pan-cytokeratin was from Sigma . Polyclonal antibodies against SMO (N-19) and tubulin were from Santa Cruz biotechnology . GDC-0449, a Hedgehog/Smoothened pharmacological inhibitor, was a kind gift from Genentech . 5E1, Shh specific mAb was purchased by Developmental Studies Hybridoma Bank   or the same amount of ON-TARGETplus NontargetingPool (Dharmacon Ref#D-001206-14-05) and 2 μl Lipofectamine® RNAiMAX Reagent from Thermo Fisher Scientific  in 200 μl Optimem from Gibco . 1 ml of supplemented medium per well was also added. Forty-eight hours after transfection, knockdown efficiency was determined by RT-PCR and western blot.200 × 10Monolayers of HCT 116 cells were lysed in modified RIPA buffer containing: 50 mM Tris-HCl, pH 7.4; 1% NP-40; 0.1% SDS; 0.25% Nadeoxycholate; 150 mM NaCl; 1 mM EDTA; 1 mM EGTA; 1 mM PMSF; 1 μg/ml each of aprotinin, leupeptin, and pepstatin; and 25 mM NaF . Equal amounts of proteins were resolved by SDS-PAGE. Proteins were transferred to nitrocellulose membranes, probed with antibodies and detected as in Battistelli et al. .CC cell lines were pretreated with DMSO or 1 μM GDC-0449 for 24 h in culture medium until reaching 100% confluency. A scratch wound was created on the cell surface using a micropipette tip in low serum (0.5% FBS) culture medium to inhibit cell proliferation. Micrographs were taken at time 0 and 24 h after the scratch. Cell devoid areas at time 0 and 24 h after the scratch were quantified through ImageJ software . Three independent experiments for each cell line were performed3-dimensional invasion assays were performed as in Strippoli et al. . HCT 116t-test using GraphPad Prism version 5.0 . Differences were considered significant at P < 0.05.Survival analysis significance was based on Logrank Test. Statistical significance of experiments performed with cell lines was determined with a GLI mRNA expression and CC survival by exploring gene expression data microarray from a cohort of 382 CC patients in the cancer genome atlas (TCGA) database   Figure . Patientl Figure . These fGLI1, PTCH1, HIP1, and MUC5AC, four of the known Hh target genes, thus demonstrating that Hh pathway is constitutively active in all three cell lines. GLI1 protein expression upon treatment with GDC-0449 was shown in Figures We analyzed whether treatment with GDC-0449 affects the expression of Hh pathway specific genes in CC lines. We exposed HCT 116, SW480, primary CC lines, and SW620, a metastatic CC cell line, to GDC-0449 for 24 h at a concentration (1 μM) that is compatible with cellular viability. As shown in Figures Similar results were obtained by genetically silencing SMO, and by using 5E1, a mAb specifically binding to Shh Figures . The extConversely in the MeT5A cell line, a non-tumorigenic mesothelium cell line, the same genes were found not responsive to the treatment with GDC-0449, therefore suggesting that these cells do not maintain a constitutively active SMO-GLI pathway Figure .Cyclin D1) while inducing the expression of cell cycle inhibitors (p21) in all of the three CC lines analyzed , this implying a constitutive activation of Hh pathway. The activity of Hh pathway in CC cell lines has been already analyzed in other studies, with controversial results  were decreased, whereas cell cycle blockers (p21) were increased. Therefore, the use of GDC-0449 directly impacts cellular proliferation and potentially tumor growth. These results were confirmed by performing SMO genetic silencing and by using a 5E1 Shh-specific inhibitory mAb. The effect observed using 5E1 presupposes the existence of an autocrine activatory loop involving Shh-SMO-dependent canonical pathway activation. A previous report analyzing loss-of-function mutation in PTCH1 suggested a role of autocrine Hh signaling in colorectal tumorigenesis  and with a significant downregulation of Snail1, a main effector of TGF-β pathway. With respect to cellular motility and Snail1 expression, our results appear equally provocative in the light of the fact that in several circumstances TGF-β facilitates tumor invasiveness through induction of epithelial-mesenchymal transition (EMT). EMT is a complex phenotypic conversion of epithelial cells leading to the acquisition of a highly motile phenotype and other mesenchymal traits, and is a key mechanism by which pre-malignant epithelial cells acquire a highly invasive phenotype that leads to metastatic spreading. The evidence of EMT in CC Cell migration and invasiveness are driven by actin cytoskeleton dynamic polarization, which is controlled by the members of the Rho GTPase superfamily Rho, Rac, and Cdc42 Ridley, . At the Hh pathway has been shown to intersect and cooperate with other pathways by regulating the differentiation/proliferation status of the tissue. In the intestinal mucosa, Bone Morphogenetic Protein (BMP) and Hh pathways are preferentially activated in differentiated layers, whereas Notch and WNT pathways are activated in the basal cells of the crypt . Besides the well-known effect on tumor invasion, Snail1 downregulation may be effective in limiting chemoresistance, as recently demonstrated in CC , inhibiting the immune response against the tumor (Otsuka et al., in vitro study established a causal link between constitutive Hh activity and the acquisition of pro-invasive, mesenchymal-like properties in CC cell lines.In conclusion, our When looking for a possible translational relevance of these discoveries, one may hypothesize that subsets of colorectal tumors endowed with abnormal Hh signaling due to mutations, such as a recently reported PTCH1 loss-of-function mutation (Chung and Bunz, PM, CB, and RS: equally contributed to the final manuscript and are responsible for study design, lab experiments, and manuscript drafting; GN, MT, and GR: supervised all the study process and revised the final manuscript; NP, LR, and FD: are responsible for literature review and data analysis; PA and LM: are responsible for lab experiments duplicates and statistical analysis. TP is responsible for the analysis of TCGA database.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.


Polo-like kinase 1 (Plk1) is a serine/threonine kinase that is a key regulator of multiple stages of mitotic progression. Plk1 is upregulated in many tumor types including colorectal cancer (CRC) and portends a poor prognosis. TAK-960 is an ATP-competitive Plk1 inhibitor that has demonstrated efficacy across a broad range of cancer cell lines, including CRC. In this study, we investigated the activity of TAK-960 against a large collection of CRC models including 55 cell lines and 18 patient-derived xenografts.50) and Tumor Growth Inhibition Index, respectively. Additionally, 2 KRAS wild type and 2 KRAS mutant PDX models were treated with TAK-960 as single agent or in combination with cetuximab or irinotecan. TAK-960 mechanism of action was elucidated through immunoblotting and cell cycle analysis.Fifty-five CRC cell lines and 18 PDX models were exposed to TAK-960 and evaluated for proliferation  and apoptotic markers was observed. Cell cycle analysis demonstrated that TAK-960 treatment induced G2/M arrest and polyploidy. Six out of the eighteen PDX models responded to single agent TAK-960 therapy (TGII< 20). The addition of TAK-960 to standard of care chemotherapy resulted in largely additive antitumor effects.CRC cell lines demonstrated a variable anti-proliferative response to TAK-960 with ICTAK-960 is an active anti-proliferative agent against CRC cell lines and PDX models. Collectively, these data suggest that TAK-960 may be of therapeutic benefit alone or in combination with other agents, although future work should focus on the development of predictive biomarkers and hypothesis-driven rational combinations.The online version of this article (10.1186/s12885-018-4036-z) contains supplementary material, which is available to authorized users. All antibodies were obtained from Cell Signaling Technologies .2.Human colorectal cancer cell lines were obtained from ATCC , DSMZ Cell Line Bank , ECACC  and the Korean Cell Line Bank (KCLB) . The GEO cell line was a generous gift from Dr. Fortunato Ciardiello . KM20 were a generous gift from Dr. Scott Kopetz from MD Anderson Cancer Center, Houston, TX, USA. The 55 human colorectal cancer cell lines used in this study were: CL-11(DSMZ ACC 467), CL-34(DSMZ ACC520), COLO201 (ATCC® CCL-224™), COLO205 (ATCC® CCL-222™), COLO678 (DSMZ ACC 194), DLD1 (ATCC® CCL-221™), GP2D (SIGMA 95090714), GP5D (SIGMA 95090715), HCA-24 (SIGMA 06061903), HCA-46 (SIGMA 07031601), HCA7 (SIGMA 06061902), HCT116 (ATCC® CCL-247™), HCT15 (ATCC® CCL-225™), HCT8 (ATCC® CCL-244™), HT15 (SIGMA 85061104), HT29 (ATCC® HTB-38™), HT55 (SIGMA 85061105), LOVO (ATCC® CCL-229™), LS1034 (ATCC® CRL-2158™), LS123 (ATCC® CCL-255™), LS174T (ATCC® CL-188™), LS180 (ATCC® CL-187™), LS513 (ATCC® CRL-2134™), MDST8 (SIGMA 99011801), Mip101 (ECACC CVCL-H689), NCI-H508 (ATCC® CCL-253™), NCI-H716 (ATCC® CCL-251™), NCI-H747 (ATCC® CCL-252™), RKO (ATCC® CRL-2577™), SKCO1 (ATCC® HTB-39™), SNU-1235 (KCLB 01235.1), SNU-1411 (KCLB 01411.1), SNU-1544 (KCLB 01544.1), SNU-1684 (KCLB 01684), SNU-1746 (KCLB 01746), SNU-254 (KCLB 00254), SNU-70 (KCLB 00070), SNU-796 (KCLB 00796.1), SNU-977 (KCLB 00977.1), SNU-C1 (KCLB 0000C1), SNU-C2B (KCLB 0000C2B), SNU1460 (KCLB 01460.1), SW1116 (ATCC® CCL-233™), SW1417 (ATCC® CCL-238™), SW1463 (ATCC® CCL-234™), SW403 (ATCC® CCL-230™), SW48 (ATCC® CCL-231™), SW480 (ATCC® CCL-228™), SW620 (ATCC® CCL-227™), SW837 (ATCC® CCL-235™), SW948 (ATCC® CCL-237™), T84 (ATCC® CCL-248™), WiDr (ATCC® CCL-218™). All cell lines were cultured in RPMI media supplemented with 10% fetal bovine serum, 1% penicillin-streptomycin and 1% MEM nonessential amino acids and routinely screened for the presence of mycoplasma . Cell lines were maintained at 37 °C with 5% CO50 was calculated from at least 3 independent experiments for each cell line. Each bar represents this mean IC50 +/- SEM and corresponds to the matrix of mutational status of KRAS, BRAF and PIK3CA, p53, and APC.Cell proliferation was assessed using CyQuant assay . Variable numbers of cells, relative to their logarithmic growth phase, were suspended in 200uL of media and plated in sterile 96 well black wall plates. Plates were incubated for 24 h to allow cells to attach. Cell lines were exposed to TAK-960 at increasing concentrations (0-.75 μmol/L) for 72 h. After 72 h dye/lysis buffer was added to the plate and fluorescence was measured on a Synergy 2 microplate reader . ICCRC cell lines were seeded in 6-well plates (density determined for each cell line based on growth rate) as previously described . The folCRC cell lines were seeded in 6-well plates . The following day cell lines were exposed to TAK-960  or mock treated control for 72 h. After 72 h of drug exposure, drug containing media was removed, each well was washed with 1 mL PBS and media containing no drug was added for an additional 72 h for regrowth. After the 72 h regrowth phase, cells were fixed with 100% methanol and stained with 1X crystal violet for 30 min. Methanol and crystal violet was removed and each well was washed three times with water and allowed to air dry. Colony area was quantified using Image J (Colony Area Plugin) .CRC cell lines were seeded in 6-well plates (density determined for each cell line based on growth rate). The following day, cell lines were treated with one of two concentrations of TAK-960 (0.1 and 1 μM) or mock treated control for 24 and 48 h. Cells were trypsinized, washed in PBS + 2% FBS and re-suspended in Krishan’s stain, incubated overnight at 4 °C and analyzed for cell cycle and ploidy using flow cytometry by the University of Colorado Cancer Center Flow Cytometry Core Facility.3 tumor sections were injected subcutaneous into both flanks of the mice. Tumors were injected into 5–6 mice  per group. When tumor volumes reached ~ 150–200 mm3 the mice were randomized into either vehicle or TAK-960 groups. Mice were treated daily with TAK-960 (10 mg/kg) or vehicle control daily by oral gavage for at least 28 days. Mice were monitored daily for signs of toxicity. Tumor size was evaluated twice per week by caliper measurements using the following equation: tumor volume = (length × width2) × 0.52 and recorded in the Study Director Program . Tumor growth inhibition index was calculated from average volume of the treated (Vt) and vehicle control (Vvc) groups, with the equation: TGII (%) = / × 100. Therefore, if TAK-960 treatment resulted in no change in growth vs vehicle treated controls, TGII (%) =100. If TAK-960 treatment results in 80% tumor growth compared to vehicle treated control tumors, TGII (%) < 20. For combination studies, mice were randomized as above into 4 groups and were treated with either vehicle, TAK-960 (5 mg/kg) daily, cetuximab  twice weekly, irinotecan (KRASMT 15 mg/kg) once weekly, or the combination for at least 20 days. Tumor measurements were obtained as above.Female athymic nude (nu/nu) mice were purchased from Harlan Laboratories . Approximately 3 mmp < 0.05 and ** indicates p < 0.01. In the cell cycle analysis assay ## is used to indicate a significance of p < 0.01 for the aneuploid (N > 4) cell population.Results from clonogenic colony formation and cell cycle analysis assays were analyzed for statistical significance with GraphPad Prism V5.04 software using paired and unpaired T-Tests, respectively. * indicates 50 values ranging from less than 0.001 μM to greater than 0.75 μM. Mutations in KRAS, BRAF, PIK3CA, p53, or APC did not correlate with response to TAK-960  and HCT116 (KRASG13D/PIK3CAH1047R). Non-responsive cell lines chosen were DLD1 (KRASG13D/ PIK3CAD549N E545K) and COLO678 (KRASG12D/PIK3CAWT).The anti-proliferative effects of TAK-960 were assessed across a panel of 55 colorectal cell lines  with little to no visible colonies in doses of TAK-960 greater than 10 nM  for 8, 24, 48, and 72 h Fig. . Recent Histone H3 becomes phosphorylated during chromosome condensation in mitosis and meiosis. Following a complete cellular division, histone H3 is dephosphorylated . AccumulSimilar to pHH3, cyclin B1 accumulates during mitosis, peaking at the G2-M interface. During the anaphase step of mitosis, cyclin B1 is rapidly degraded by APC , 37. AccPresence of caspase 3 and PARP cleavage products are well-established markers of apoptosis –41. CaspAs mentioned above, tumor suppressor p53 and Plk1 function to counterbalance each other in the cell, contributing in opposite action to arrest and progress the cell cycle, respectively. There were no appreciable changes in p53 levels in all CRC cell lines with length or concentration of TAK-960 exposure (data not shown).Levels of β-actin (loading control) were consistent across TAK-960 exposure duration and dose.p < 0.01) under all treated conditions. Interestingly, in HCT116 cells there was a dramatic increase in the percentage of aneuploid (> 4 N) cells at the 0.1 μM concentration (p < 0.01) of TAK-960 at 48 h that was not observed in the WIDR cells  for 24 and 48 h Fig. . This rells Fig. . DLD1  were treated with TAK-960 for at least 28 days. PDX models were treated daily (QD) with 10 mg/kg of TAK-960. Response to TAK-960 varied among PDX models with tumor growth inhibition indices (TGII) ranging from − 4.17 to 111.48 Fig. . Six PDXWT PDX models were treated with TAK-960, cetuximab (EGFRi) or the combination of the two. All PDX KRASWT models were treated with 10 mg/kg of TAK-960 orally once daily and 400 μg/mouse of cetuximab dosed twice per week by intraperitoneal injection. TAK-960 did not sensitize either of the 2 PDX models to cetuximab. Antitumor effects were largely driven by single-agent treatment with either cetuximab or TAK-960  , cytotoxicity without regrowth in colony formation assays and the induction of polyploidy. In vivo, TAK-960 therapy resulted in moderate antitumor activity (33% response rate) in our panel of 18 patient-derived colorectal tumor models, however, this response was not enhanced with the addition of standard of care agents.Anti-mitotic therapies are a growing field in oncologic research. Plk1 is an essential gene that regulates the progression of the cell cycle through mitosis. The current study was designed to evaluate the efficacy of the investigational Plk1 inhibitor TAK-960 in colorectal cancer (CRC) models as a single agent and to determine the anti-cancer effects of TAK-960 in combination with standard agents in patient-derived CRC xenografts. The results reported here demonstrate that exposure of CRC cell lines to TAK-960 in vitro resulted in a robust anti-cancer response . However, Plk1 inhibition has been shown to sensitize cancer cells to gemcitabine and vincristine in vitro [Often, targeted therapeutics are not used clinically as a single agent. In a phase 1 trial, TAK-960 was shown to be an ineffective therapy for solid tumors when administered as a single agent  was completed on sensitive and resistant cell lines to gain insights to possible molecular vulnerabilities and combination partners to anti-Plk1 therapy. GSEA pathway analysis revealed that TAK-960 resistant cell lines exhibited increase expression of multiple cell cycle signaling nodes in series and parallel to Plk1 signaling (data not shown). While it is unsurprising that resistant cell lines would exhibit an increase in multiple alternative cell cycle signaling pathways, the extent to which global dysregulation was observed was unanticipated. These data suggest molecular inhibition of multiple targets in cell cycle pathways may be required for effective anti-cancer therapy.Perhaps Plk1 inhibition should be rationally combined with MAP kinase pathway inhibition in TAK-960 resistant CRC. This novel combination was recently found to be robustly effective in NRAS mutated melanoma tumor models, whereas synergistic antitumor activity was observed both in vitro and in vivo . These dTAK-960 has been shown to be an efficacious inhibitor of proliferation in a large collection of CRC models, however mechanism of action in colorectal cancer has yet to be fully elucidated. These data suggest a potential for TAK-960 to be of therapeutic value as a single agent or in combination therapy under the right conditions. Future development of Plk1 inhibition as a therapeutic strategy for CRC will require more study into the selection of patients based upon molecular vulnerabilities, but also the development of mechanism-based rational combinations.We have demonstrated that the Plk1 inhibitor TAK-960 is a potent anti-colorectal cancer therapy through in vitro cell line assays and patient-derived tumor xenograft models. TAK-960 was shown to reduce proliferation and induce cell cycle arrest. These data form the basis of future work to elucidate rational combination partners for TAK-960, as well as strategies to select patients responsive to Plk1 inhibition.


SMAD4 gene, while P-STS had a loss on 11q. BON-1 had a homozygous loss of CDKN2A and CDKN2B, and QGP-1 harboured amplifications of MDM2 and HMGA2. Whole-exome sequencing revealed both disease-characteristic mutations (e.g. ATRX mutation in QGP-1) and, for patient tumours, rare genetic events . A large-scale inhibitor screening showed that cell lines from pancreatic NETs to a greater extent, when compared to small intestinal NETs, were sensitive to inhibitors of MEK. Similarly, neuroendocrine NET cells originating from the small intestine were considerably more sensitive to a group of HDAC inhibitors. Taken together, our results provide a comprehensive characterisation of GEPNET cell lines, demonstrate their relevance as neuroendocrine tumour models and explore their therapeutic sensitivity to a broad range of inhibitors.Experimental models of neuroendocrine tumour disease are scarce, and no comprehensive characterisation of existing gastroenteropancreatic neuroendocrine tumour (GEPNET) cell lines has been reported. In this study, we aimed to define the molecular characteristics and therapeutic sensitivity of these cell lines. We therefore performed immunophenotyping, copy number profiling, whole-exome sequencing and a large-scale inhibitor screening of seven GEPNET cell lines. Four cell lines, GOT1, P-STS, BON-1 and QGP-1, displayed a neuroendocrine phenotype while three others, KRJ-I, L-STS and H-STS, did not. Instead, these three cell lines were identified as lymphoblastoid. Characterisation of remaining authentic GEPNET cell lines by copy number profiling showed that GOT1, among other chromosomal alterations, harboured losses on chromosome 18 encompassing the  Patient tumour-derived cell lines, as models of tumour disease, have been widely used for studying the molecular mechanisms of tumours and their response to therapy. However, cell lines do not perfectly reflect their tumour of origin and in terms of genomic alterations, protein expression and therapeutic sensitivity, they can differ substantially (The establishment of cell lines from human neuroendocrine tumours (NETs) of the gastrointestinal tract and pancreas (GEPNETs) has proved to be difficult. This has been attributed, in part, to the low proliferation rate of GEPNETs . The GOTThe generation of cell lines from human pancreatic NETs (PanNETs) has also been proven difficult, and only few cell lines are available. Among the most frequently used PanNET cell lines are QGP-1 and BON-1. QGP-1 was established in 1980 from a somatostatin-producing islet cell carcinoma  were trypsinised and counted using Countess cell counter (Invitrogen).The GOT1 cell line was established from a liver metastasis of a midgut carcinoid from a female patient . The PCR was performed using a 7500 Fast-Real-time PCR system (Applied Biosystems).DNA was isolated from cell lines at the same passage as used for the inhibitor screening using the DNeasy Blood & Tissue Kit (Qiagen). DNA from cell lines, and from GOT1 patient tumour tissue, was subjected to STR analysis at a DANAK/ILAC DS/EN ISO 15189:2008 accredited laboratory  at Aarhus University Hospital, Denmark) . Sectioning and staining were carried out as previously described  were grown on µ-slide 8-well chamber slides  for 3 days and fixed in 4% buffered formaldehyde in PBS. Cells growing in suspensions  were spun onto microscope slides with a Cytospin 2 cytocentrifuge  before fixation. The slides were incubated with primary antibodies for 1 h at room temperature in 1% BSA and 0.2% Triton X-100 in PBS and for 1 h with secondary antibodies conjugated to Alexa Fluor 594  or to FITC . All experiments included negative controls wherein the primary antibody was omitted. The fluorescent cells were analysed using a Zeiss LSM 700 confocal microscope and Zen black software was supplied by the manufacturer. Images were captured using a Zeiss LSM 700 inverted microscope with a 63× oil immersion objective.P = 10−8 to P = 10−18, where the higher stringency threshold was used for arrays with QC >0.2. The log2 ratio thresholds for aberration calls of gain and loss were set to 0.2 and −0.2 for arrays with a significance threshold P≥10−8, and to 0.3 and −0.3 for arrays with a significance threshold P<10−8. Thresholds for amplification and homozygous loss were set to 1.5 and −1.5 in all cases. Sex chromosomes and regions partially or completely covered by a previously reported copy number variation  were excluded from the analysis  as recommended by the manufacturer and as previously described . FastQC (version 0.11.2) was used to assess the quality of the data. Paired end reads were aligned to the human reference genome (hg19) using a Burrows-Wheeler Aligner (mem version BWA_0.7.13) . Samtooln = 8), and each experiment included an additional plate with DMSO control wells (n = 96), as well as one cell-free control plate. The screening library was diluted 1:50 in 100 µL medium and added to the treatment plates . Cell viability was estimated using a fluorescence-based assay to measure the reducing capacity of metabolically active cells . All assay plates were incubated for 72 h at 37°C, followed by addition of 1:100 alamarBlue reagent. The plates were further incubated for 6 h at 37°C and were then analysed using a 96-well fluorescence plate reader . For vorinostat and trametinib dose–response curves, first-passage primary cultures were thawed and seeded onto 96-well plates, allowed to settle for 48 h before treatment with vorinostat or trametinib for 72 h. Each concentration was added in triplicate and plates were analysed as described earlier. Fitting of the curves was done in GraphPad Prism software, v7.02 using inhibitor vs. response nonlinear fit with variable slope.The screening library consisted of 1224 compounds . The library was prepared by two-step dilution of the compounds from 10 mM to 100 µM in DMSO and batched in 96-well plates. Inhibitors were subjected to a maximum of five freeze-thaw cycles. From frozen stocks, cells were expanded 2–5 passages before being used in screening experiments. Seeding density was adjusted for each cell line so that control cells were approximately 70‒80% confluent at treatment endpoint in 100 µL cell medium/well in black solid-bottom 96-well plates. The plates were incubated for 72 h at 37°C to allow for cell attachment. Each treatment plate included DMSO control wells  were also investigated. VMAT1 was strongly stained in GOT1, weakly stained in BON-1 and QGP-1, and not stained in P-STS, while 5-HT was strong in GOT1, moderate in BON-1 and weak in P-STS and QGP-1. The localisation of synaptophysin, chromogranin A, VMAT1 and 5-HT to secretory granules was confirmed by confocal laser scanning microscopy .Figure GEPNETs are derived from progenitor cells in the intestinal crypts, or pancreatic ducts, which express epithelial markers such as cytokeratins. To confirm the epithelial phenotype of NET cell lines, we stained for high-molecular-weight cytokeratins (AE1/AE3) and cytokeratin 8/18. The staining for high-molecular-weight cytokeratins was strong in all cell lines, while staining for cytokeratin 8/18 was weak to moderate in GOT1, P-STS, BON-1 and QGP-1 .Neuroendocrine marker, linage-specific marker and epithelial marker expression for GOT1, P-STS, BON-1 and QGP-1 overall well reflected that of clinical samples of GEPNETs. However, the Ki67 index of the cell lines was not representative of typical GEPNETs. GOT1 originated from a SINET liver metastasis with positivity for SYP, CgA, 5-HT, CK8/18 and with a Ki67 index of 1.3% . Furthermore, the Ki67 index of the cell lines correlated poorly to their doubling time .Supplementary Fig. 2). We therefore conclude that these cell lines are lymphoblastoid, and do not represent true GEPNET cell lines.For the three putative SINET cell lines KRJ-I, L-STS and H-STS, we however, with the exception of NCAM, found only weak or no staining of neuroendocrine markers and could not detect any linage-specific markers or cytokeratins. These observations, in addition to the fact that these three cell lines grow non-adherently and form aggregates in cell culture (in contrast to the other GEPNET cell lines), made us question their authenticity. We hypothesised that KRJ-I, L-STS and H-STS were EBV-driven lymphoblastoid cell lines and thus performed immunohistochemical staining for lymphoid (CD45) and B cell markers (CD20). Both markers were highly expressed in KRJ-I, L-STS and H-STS, and the cell lines also contained high levels of EBV DNA, while the GOT1, P-STS, BON-1 and QGP-1 were negative (Somatostatin receptors are frequently over-expressed in GEPNETs and are used as a target for imaging and therapy. We performed staining for the somatostatin receptor subtype 2 (SSTR2) and found strong staining in GOT1 cells, weak staining in BON-1 and no staining in P-STS and QGP-1 . ConfocaSupplementary Table 1).Chromosomal changes have been linked to tumourigenesis and disease progression in patients with GEPNETs. Recurrent copy number alterations (CNAs) differ between SINETs and PanNETs, suggesting different mechanisms for tumour initiation and progression. To study CNAs in GEPNET cell lines, we performed whole-genome copy number profiling and found CNAs in all GEPNET cell lines (range 7‒44) . The GOT1 cell line showed loss of a 1.8 Mb segment of 18q, including the tumour suppressor SMAD4 . The GOTnd HMGA2 . The lymCDKN1B, which occurs in less than a tenth of all tumours . Using the same filtering process, the number of indels was narrowed down from an average of 31,729 per cell line  to an average of 275 (range 134‒653) protein-modifying events (Supplementary Table 3). Among the four GEPNET cell lines, P-STS had the highest number of protein-altering SNPs and indels and also displayed a higher degree of transitions compared to the other cell lines .We detected an average of 196,067 single-nucleotide polymorphisms (SNPs) per cell line . After quality filtering according to GATK best practices with the tool HaplotypeCaller, the number of reads was narrowed down to 188,133 on average . In order to remove frequently occurring SNPs, all variants were further filtered against the 1000 Genomes Project database  Pan-Cancer effort. (Supplementary Table 4). Among affected genes, we found the key tumour suppressor TP53 mutated in three out of four cell lines. TP53 is seldom inactivated in GEPNETs, but here found mutated in P-STS, BON-1 and QGP-1. GOT1 was the only GEPNET cell line with wild-type TP53. The cell-cycle regulators CDKN2A and CDKN2B were inactivated by homozygous loss in BON-1. The SMAD4 gene, involved in cell growth inhibition signalling, was lost in GOT1, had a heterozygous mutation in P-STS and a homozygous mutation in BON-1.We first investigated the cell lines for mutations in genes linked to hereditary endocrine tumour syndromes, i.e. re found . None ofGEPNET cell lines have been used to study selected inhibitors for their efficacy and mechanism of action (Supplementary Table 5). In the data, we observed an overall high degree of similarity in inhibitor sensitivity between cell lines derived from SINETs and PanNETs. We could however identify several groups of inhibitors that were significantly more selective for PanNET cells or SINET cells . FDA-approved MEK inhibitor trametinib was used to treat these cells  and had a heterozygous mutation in GOT1 , possibly leading to a bi-allelic inactivation of the gene. No mutations in DAXX or MEN1 were found in any of the cell lines. While expression studies have indicated that mTOR signalling is upregulated in most PanNET tumours  further strengthens this concern. The original publications on GEPNET cell lines do not contain sufficient data to determine the grade of the tumours from which cell lines were derived. We have therefore reinvestigated the origin of the GOT1 cell line and found it to be a liver metastasis from a well-differentiated, serotonin-producing (enterochromaffin cell type) ileal NET. The liver metastasis was of grade 1 (WHO 2010) and harboured a loss of chromosome 18. We conclude that it remains to be proven to what extent GEPNET cell lines are representative for the tumour disease from which they were derived. Experimental data obtained from GEPNET cell lines should therefore be carefully validated using patient tumours.From these findings combined, we conclude that the four cell lines GOT1, P-STS, BON-1 and QGP-1 are indeed authentic neuroendocrine tumour cell lines, harbouring genomic events characteristic of GEPNETs, but also cancer-associated mutations that most likely have occurred in cell culture. The identification of lymphoblastoid cell lines among putative GEPNETs emphasises the need to thoroughly characterise GEPNET cell lines used as models for neuroendocrine tumour disease. Our observations also emphasises the need to be cautious when drawing conclusions from studies performed on GEPNET cell lines. One major concern is the origin of GEPNET cell lines and whether they were derived from well-differentiated neuroendocrine tumours or poorly differentiated neuroendocrine carcinomas. The difficulties in establishing GEPNET cell lines are usually attributed to the low proliferation rate of these tumours. One may argue that available GEPNET cell lines were established from more aggressive tumours that should be classified as neuroendocrine carcinomas. The occurrence of The authors declare that there are no conflicts of interests that could be perceived as prejudicing the impartiality of the research reported.This study was supported by grants from the Swedish Cancer Society, the Assar Gabrielsson Research Foundation, the Wilhelm and Martina Lundgren Foundation for Scientific Research, the Sahlgrenska University Hospital Funds, the BioCARE National Strategic Research Program and by ALF grants from the Sahlgrenska University Hospital.


Klotho mRNA with unknown function. The purpose of this review is to highlight the recent advances in our understanding of Klotho's function in mineral homeostasis.α-Klotho (Klotho) occurs in three isoforms, a membrane-bound form acting as a coreceptor for fibroblast growth factor-23 (FGF23) signalling, a shed soluble form consisting of Klotho's large ectodomain thought to act as an enzyme or a hormone, and a secreted truncated form generated by alternative splicing of the Klotho and Fgf23-deficient mouse models demonstrated that all isoforms of Klotho lack any physiologically relevant, FGF23-independent functions in mineral homeostasis or ageing. Finally, it was demonstrated that the alternatively spliced Klotho mRNA is degraded and is not translated into a secreted Klotho protein isoform in humans.A number of seminal discoveries have recently been made in this area, shifting existing paradigms. The crystal structure of the ternary FGF receptor (FGFR)-1c/Klotho/FGF23 complex has been uncovered, revealing how the ligand FGF23 interacts with FGFR1c and the coreceptor Klotho at atomic resolution. Furthermore, it was shown that soluble Klotho lacks any glycosidase activity and serves as a bona fide coreceptor for FGF23 signalling. Experiments with a combination of Taken together, there is now overwhelming evidence that the main physiological function of transmembrane and soluble Klotho for mineral homeostasis is their role as coreceptors mediating FGF23 actions. In light of these findings, the main pathophysiological consequence of the downregulation of Klotho observed in acute and chronic renal failure may be the induction of renal FGF23 resistance. In this complex, FGF23 binds in a groove formed between the D2 and D3 domains of FGFR1c, and between the KL1 and KL2 domains of Klotho, respectively. The ternary complex is stabilized by the interaction of Klotho's receptor binding arm with FGFR1c  and FGF23 (32 kDa) in intact tissues. Therefore, it is unclear whether the latter model is predictive of the in-vivo situation.Current open questions in this context are: First, what is the relative contribution of transmembrane Klotho vs. sKlotho–mediated FGF23 signalling in target tissues under normal circumstances? Second, is shedding of sKlotho in the kidney regulated? Third, as FGFR1c is expressed almost ubiquitously, does sKlotho enhance FGF23 signalling in a body-wide fashion? In principle, biologically relevant levels of circulating sKlotho should render all target cells expressing FGFR1c FGF23-sensitive. In an attempt to answer the question regarding the relative contribution of transmembrane vs. soluble Klotho for renal FGF23 signalling, we recently cotreated 200 μm thick live kidney slices isolated from wild-type and nd FGF23 ▪▪. We fomstances ▪▪. Howevet al. [▪▪] conclude that ‘In light of these data, we propose that the pleiotropic antiaging effects of α–Klotho are all dependent on FGF23’. A recent study performed in our laboratory fully corroborates this conclusion. To examine the existence of physiologically relevant, FGF23-independent effects of Klotho on mineral homeostasis in vivo, we generated triple knockout mice with simultaneous deficiency in Fgf23 and Klotho and a nonfunctioning vitamin D receptor (VDR) . The rationale behind crossing Fgf23 and Klotho-deficient mice with VDR mutant mice is that Fgf23 and Klotho null mice can be studied at older ages only when vitamin D signalling is ablated . These results strongly argue against any physiologically relevant, FGF23-independent effects of Klotho, regardless of its isoform, on mineral homeostasis or ageing. Taken together, there is now overwhelming evidence that the main physiological function of transmembrane and soluble Klotho for mineral homeostasis in vivo is their role as coreceptors mediating FGF23 actions  to R.G.E.The author declares no conflicts of interest.▪ of special interest▪▪ of outstanding interestPapers of particular interest, published within the annual period of review, have been highlighted as:


Expression levels of 90 TKs in the cell lines were examined by western blotting. Among these, the levels of PDGFRB, DDR1, AXL, MET, and PYK2 were higher, and those of FGFR1 and VEGFR3 were lower in the 1273/99 cell line than the other cell lines. Gene silencing analysis of the TKs upregulated in 1273/99 cells showed differing effects on cell growth: PDGFRB, MET, and PYK2 knockdown induced cell growth inhibition, whereas DDR1 and AXL knockdown did not influence cell growth. Using the PamChip peptide microarray, we found that 18 peptide substrates were highly phosphorylated in the 1273/99 cell line compared with other cell lines. Using the PhosphoNet database, we found that kinases FGFR3, RET, VEGFR1, EPHA2, EPHA4, TRKA, and SRC phosphorylated these 18 peptide substrates. Moreover, the results for overexpressed and aberrantly activated TKs in pazopanib-resistant cells showed no overlap. Taken together, our study indicates that identification of comprehensive TK profiles represents an essential approach to determining the molecular background of pazopanib resistance in synovial sarcoma.Pazopanib, a multitarget tyrosine kinase (TK) inhibitor, has been approved for treatment of soft tissue sarcoma. Elucidation of the molecular background of pazopanib resistance should lead to improved clinical outcomes in sarcomas; accordingly, we investigated this in synovial sarcoma using a proteomic approach. Pazopanib sensitivity was examined in four synovial sarcoma cell lines: SYO-1, HS-SYII, 1273/99, and YaFuSS. The 1273/99 cell line showed significantly higher IC Pazopanib is a molecular targeted drug approved by the US Food and Drug Administration for soft-tissue sarcoma . It is aProteomic analysis is an essential approach for the identification of proteins associated with drug resistance , 7. In pTo elucidate the molecular background of pazopanib resistance in sarcoma, we examined the protein expression levels of 90 TKs by western blotting. Further, we performed TK activity profiling in synovial sarcoma cell lines using the PamChip peptide microarray. Via this approach, we identified TKs whose expression or activity was considerably different.50) of pazopanib for the SYO-1, HS-SYII, 1273/99, and YaFuSS cells were 1.41 ± 0.19, 1.92 ± 0.22, 10.3 ± 1.45, and 2.59 ± 0.05 μM, respectively  to examine the kinase activity among the four cell lines, and found that the kinases in pazopanib-resistant cells were not consistent with the overexpressed TKs identified by western blotting. We considered that the kinase activity of some overexpressed TKs was not regulated by protein expression in synovial sarcoma cells. Previous studies have shown that kinase activity is not regulated only by protein expression: Lutz et al. found that Src kinases participate in growth regulation of pancreatic cancer cells, and that the kinase activity level of Src is not regulated by protein expression levels . In lighWe examined the kinase activity profiling of synovial sarcoma cell lines using a commercially available array (PamChip) consisting of 144 protein TK substrates. Several studies have discussed the potential of this array for target identification in clinical samples , and othPrevious studies have reported that kinome profiling is a highly useful approach for elucidating the molecular mechanisms underlying drug resistance in cancer. Cox et al. reported that alterations in the kinome affected drug resistance in leukemia . KurimchThe current study has some limitations. First, an important consideration in the selection of cell lines for drug response is the ethnic background of the individuals from whom the cell lines have been derived. Numerous potential issues result from population heterogeneity. In this study, SYO-1, HS-SY II, and YaFuSS cell lines were derived from Japanese patients, while the 1273/99 cell line was derived from a European patient. We did not examine the effect of cell origin as sufficient synovial sarcoma cell lines are not available. Second, the results for kinase activity were not validated by other proteomic approaches, such as mass spectrometry-based phosphoproteomics. Further research is required to validate these results.In conclusion, our study demonstrated that identification of comprehensive TK profiles is an essential approach for determining the molecular background of pazopanib resistance in synovial sarcoma.2. Cells from exponentially growing cultures were used in all experiments.Four synovial sarcoma cell lines were used in this study: SYO-1 was a gift from Akira Kawai  , HS-SY-IThe tumor cells were plated on 96-well plates  and incubated in the presence of pazopanib  for 72 h. Then, cell growth was measured using the Cell Counting Kit-8  according to the manufacturer’s recommendations.Each protein sample (5 μg) was subjected to separation using sodium dodecyl sulfate (SDS)–polyacrylamide gel electrophoresis (PAGE) on a 12.5% polyacrylamide gradient gel; this was followed by blotting onto a nitrocellulose membrane. Immunoblot analysis was performed using the antibodies listed in http://bioconductor.org/packages/agilplimma/). Differences between two sample groups were established using an unpaired t-test. A p-value < 0.05 was considered to represent statistical significance.Total RNA was extracted from synovial sarcoma cell lines using the RNeasy kit . In brief, mRNA expression profiles of the samples were obtained by hybridizing the RNA to the SurePrint G3 Human GE DNA microarray , following the manufacturer’s instructions. Hybridized microarrays were scanned with a microarray scanner (Agilent G2565BA) with default protocols and settings. The microarray data were normalized and standardized using the Bioconductor agilp limma package  in accordance with the manufacturer’s protocol. Twenty-four hours later, the culture medium was replaced with DMEM-low glucose (Sigma). The cells were harvested for western blotting or treated with pazopanib for 72 h after transfection.siRNAs were purchased from Sigma, and non-specific control siRNA duplexes (AllStar Negative Control siRNA) were purchased from Life Technologies. The target sequences have been provided in −1 monoclonal anti-phosphotyrosine FITC conjugate (clone PY20); the total volume was adjusted to 40 μl with distilled H2O. All chemicals were provided by PamGene International BV. Each array was blocked with 0.2% bovine serum albumin and washed with PK solution. A kinase reaction was then performed at 30 °C. The reaction mix was pulsed back and forth through the porous material of the PamChip for 60 cycles. An image was obtained with a built-in CCD camera every fifth cycle. The study was performed in triplicate. Kinases that phosphorylate substrate peptides were predicted by referring to Phospho.elm (http://phospho.elm.eu.org) and Uniprot (http://www.uniprot.org) databases.TK activity profiles were determined using the PamChip TK peptide microarray system , as described previously . Briefly


Advanced biliary tract cancer (BTC) has a poor prognosis and limited treatment options. The PI3K/Akt/mTOR signalling pathway is hyperactivated in a subset of BTCs, and clinical activity of the mTOR inhibitor everolimus has been observed in some patients with BTC. The goal of this study was to identify biomarkers predictive of everolimus response. Twenty BTC cell lines were assessed for everolimus sensitivity with a spectrum of growth inhibitory responses observed. Molecular biomarkers of sensitivity and resistance were identified by interrogation of the activation status of the Ras/MAPK and PI3K/Akt/mTOR pathways. K‐Ras mutations and/or amplifications were identified in 45% of cell lines and were associated with resistance to everolimus. Activating mutations in PIK3CA or loss of PTEN was not predictive of everolimus response; however, high basal levels of pAKT were associated with sensitivity, independent of Ras/MAPK pathway activation status. Notably, everolimus inhibited mTOR signalling to a similar extent in sensitive and resistant cell lines, suggesting that relative dependence on the mTOR pathway rather than the magnitude of pathway inhibition determines everolimus response. Consistent with the known limitations of rapalogs, everolimus induced feedback‐mediated activation of AKT in BTC cell lines, which could be overcome by cotreatment with an AKT inhibitor or ATP‐competitive mTORC1/mTORC2 inhibitors. However, both approaches failed to induce greater apoptosis compared to everolimus, and mTORC1/mTORC2 kinase inhibitors induced compensatory activation of pERK, identifying an inherent limitation of these agents in BTC cell lines. These findings suggest that future trials of everolimus in BTC would benefit from preselecting patients based on their K‐Ras and PI3K/mTOR pathway activation status. The study also identifies strategies for enhancing inhibition of the PI3K/mTOR pathway in BTC cell lines. BTCbiliary tract cancerDCRdisease control rateMAPKmitogen‐activated protein kinaseORRoverall response rateRTKreceptor tyrosine kinaseS6KS6 kinase1in situ, and finally invasive carcinoma. The incidence of the disease varies around the world, with highest rates in north‐eastern Thailand and neighbouring Laos and Cambodia where liver fluke infestations (Opisthorchis viverrini) are endemic  are a heterogeneous group of anatomically distinct adenocarcinomas, which include intra‐ and extrahepatic cholangiocarcinoma and gallbladder cancer that are extremely challenging to diagnose and treat. BTCs arise as a result of epithelial cells lining the biliary tract (cholangiocytes) undergoing dysplasia, hyperplasia, carcinoma et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., One potential treatment target is the PI3K/AKT/mTOR pathway, which is activated in a subset of BTCs . Activated RTKs recruit and phosphorylate IRS‐1 causing activation of PI3K and subsequent activation of AKT. Activated AKT phosphorylates a number of substrates including TSC2, which prevents TSC1/TSC2 complex formation  and its derivatives everolimus and temsirolimus . These are allosteric inhibitors that bind and form a complex with the intracellular protein FBP12, which interacts with mTORC1 and inhibits the activity of the complex. Of note, the FBP12/rapalog complex does not inhibit the mTORC2 complex. Consequently, due to relief of the feedback loops described above, rapalogs induce paradoxical activation of AKT  of 5%, a disease control rate (DCR) of 45% at 8 weeks and a median overall survival time of 7.7 months , at 37 °C with 5% CO2. The anatomical location from which each cell line is derived is listed in Table A panel of 20 BTC cell lines derived from distinct anatomical locations within the biliary tree were obtained from the following sources: HuH‐28, OZ, OCUG‐1, NOZ , SNU‐1079, SNU‐1196, SNU‐245, SNU‐478, SNU‐869, SNU‐308 (KCLB – Korean Cell Line Bank), TKKK, TGBC2TKB, TGBC18TKB, TGBC14TKB, G‐415, HuCCT1 , SK‐ChA‐1, Mz‐ChA‐2 , TFK‐1, EGI‐1 (DSMZ). STR profiling was performed on all lines using the Promega StemElite ID System at the Queensland Institute of Medical Research DNA Sequencing and Fragment Analysis Facility , and all lines were found to be unique. All cell lines were maintained in Dulbecco's minimal essential media/F12 (DMEM/F12) supplemented with 10% FBS, 1% HEPES buffer and penicillin/streptomycin .2.3® AQueous Non‐Radioactive Cell Proliferation Assay as per manufacturer's instructions . Cell cycle distribution was assessed by propidium iodide (PI) staining and FACS analysis as previously described ‐5‐(3‐carboxymethoxyphenyl)‐2‐(4‐sulfophenyl)‐2H‐tetrazolium, inner salt) assay using the CellTiter 962.4Genomic DNA was extracted from cell pellets using DNeasy Blood and Tissue Kit . The mutation status of K‐Ras , BRAF (V600) and PIK3CA (exons 9 and 20) was validated in the 20 BTC cell lines by conventional Sanger sequencing. Primers used for amplification of exon 2 of K‐Ras were: F: AGGCCTGCTGAAAATGACTGAATA and R: CTGTATCAAAGAATGGTCCTGCAC, for exon 3, F: TAAAA GGTGCACTGTAATAATCC and R: TAAAAACTATAATTACTCCTTAATG. Primers used for the amplification of exon 15 of BRAF were F: AACACATTTCAAGCCCCAAA and R: GAAACTGGTTTCAAAATATTCGTT, for exon 9 of PIK3CA, F: GCTTTTTCTGTAAATCATCTGTG, and R: CTGAGATCAGCCAAATTCAGT and for exon 20 of PIK3CA, F: CATTTGCTCCAAACTGACCA and R: TACTCCAAAGCCTCTTGCTC (for codon 1023 mutation) and F: ACATTCGAAAGACCCTAGCC and R: CAATTCCTATGCAATCGGTCT (for codon 1047 mutation).2.5et al., in situ hybridization (FISH) was performed using the ZytoLight SPEC KRAS/CEN 12 Dual Color Probe  per the manufacturer's instructions.Quantitative PCR was performed on 10–30 ng of genomic DNA per reaction with Power SYBR Green on a ViiA7 Real‐Time PCR System  using the following primers as described previously  per the manufacturer's instructions.2.7m Tris/HCl pH 7.5, 150 mm NaCl, 0.5% sodium deoxycholate, 1 mm EDTA, cOmplete EDTA‐free Protease Inhibitor Cocktail , PhosSTOP phosphatase Inhibitor cocktail (Roche/Sigma‐Aldrich) and 3 m urea. 15–30 μg of total protein per sample was electrophoresed on NuPAGE™ 4–12% Bis/Tris gels and transferred onto PVDF membranes using iBlot transfer stacks . Antibodies used in western blot analysis were phospho‐Akt Ser473 (cat # 4051), AKT1 (2938), PTEN (9559), phospho‐S6 Ser240/244 (5364), S6 (2317), phospho‐4EBP1 T37/46 (2855), 4EBP1 (9644), beclin (3495), phospho‐ERK Thr202/Tyr204 (4370), ERK1/2 (9107) and phospho‐mTOR Ser2448 (2976) from Cell Signalling Technologies , β‐tubulin  and anti‐β‐actin . Secondary antibodies used were fluorescent‐labelled goat anti‐mouse (IRDye(R) 680 CW, LI‐COR, 1 : 15 000) and goat anti‐rabbit (IRDye(R) 800 CW, LI‐COR, 1 : 15 000). Signal was detected using an Odyssey (R) Infrared Imaging System , and fluorescent intensities were quantified using odyssey software, version 3.0.29 (LI‐COR Biotechnology).Cells were lysed in 1% NP‐40, 50 m2.8A tissue microarray was constructed from formalin‐fixed, paraffin‐embedded cell blocks. Slides underwent microwave heat‐induced epitope retrieval in Tris/EDTA pH 9.0 followed by incubation with primary antibodies overnight at 4 °C. Sections were incubated with REAL EnVision‐HRP secondary antibody, diaminobenzidine substrate , and counterstained with haematoxylin.2.9t‐tests. Ras activity was log‐transformed and groups were compared using one‐way anova and post hoc Tukey–Kramer tests. Continuous variables were compared using Pearson's correlation coefficients.Groups were compared using unpaired 33.1m), and the effect on cell proliferation was assessed after 72 h using the MTS assay. Shown in Fig. m, with G‐415, TGBC2TKB and SK‐ChA‐1 the three most sensitive and TGBC18TKB, HuCCT1 and SNU‐1196 the three most resistant cell lines in the panel  and nonamplified line (TFK‐1) are shown in Fig. post hoc Tukey–Kramer, P > 0.05), which were significantly greater that K‐Ras WT/nonamplified lines    Fig. D.3.4We next determined whether the activation status of the PI3K/PTEN/AKT pathway was predictive of everolimus response. Activating mutations in exons 9 and 20 of PIK3CA occur in a subset of BTCs and direct sequencing identified activating mutations in exon 9 in two cell lines, HuH‐28 and SNU‐869 Table . In addiHowever, separation of all lines as PIK3CA mutant/PTEN null or PIK3CA/PTEN WT revealed no difference in everolimus response Fig. , suggestr = −0.604, P = 0.007, Fig. r = −0.691, P = 0.026) . Everolimus had no effect on pERK in any of the cell lines, confirming its specificity for targeting the PI3K/AKT/mTOR pathway, and lack of compensatory activation of this alternate signalling pathway.To determine whether the differential sensitivity to everolimus was also linked to the magnitude of inhibition of the mTOR pathway, we compared changes in p‐mTOR and downstream signalling components in the three most sensitive and resistant cell lines. Everolimus decreased p‐mTOR, pS6 and p4EBP1 in all cell lines after 48‐h treatment  and resistant (HuCCT1) cell lines with everolimus for 96 h resulted in strong suppression of pS6 in both cell lines, which remained suppressed over the duration of the time course, indicating that the differential sensitivity is not due to differences in the duration of pathway inhibition Fig. B. Collecet al., Finally, a known limitation of rapalog treatment is feedback‐mediated activation of AKT . While K‐Ras mutations and amplifications do occur in primary BTC, the collective frequency of these events in the cell line panel (45%) is significantly greater than that observed in primary cancers (18%)  (Cho et al., In addition to establishing K‐Ras mutation and amplification status as a marker of resistance, our findings also established high basal expression of pAKT as a positive predictor of everolimus sensitivity. This finding is consistent with two prior We also investigated whether everolimus response was dependent on the magnitude or duration of mTOR pathway inhibition. These analyses demonstrated that everolimus inhibited downstream signalling to a similar extent in sensitive and resistant cells, indicating that differential pathway blockade was not the primary determinant of sensitivity. Instead, they support the conclusion that cell lines inherently dependent on mTOR signalling, reflected by high basal pAKT levels, are preferentially sensitive to pathway inhibition, while cell lines dependent on the Ras/MAPK pathway are less responsive.et al., et al., et al., et al., et al., Consistent with the known limitation of rapalogs, which has been observed in several tumour types (Yeung et al., A further observation made during these studies was the feedback‐mediated activation of pERK in BTC cells following treatment with ATP‐competitive TORC1/TORC2. Similar findings have previously been reported in pancreatic cancer cells (Soares In summary, our findings establish a significant association between high basal pAKT expression and sensitivity to everolimus and K‐Ras mutations/amplifications with resistance. An implication of these findings is that subsequent trials of everolimus for this indication may benefit from enrichment of patients according to K‐Ras mutation/amplification status and pAKT expression. Our findings also indicate that while therapeutic targeting of the PI3K/AKT/mTOR pathway using combinations of everolimus with AKT inhibitors or ATP‐competitive mTOR inhibitors that inhibit both mTORC1 and mTORC2 represents strategies for more robust pathway inhibition, they do not induce greater levels of cell death and present their own inherent limitations.The study was conceived by AJW, MN, AMS, NCT and JMM. Experiments and data analysis were performed by YY, DL, FC, HT, JT and JMM. All authors were involved in writing and approval of the manuscript.


Hepatocellular carcinoma is highly refractory cancer which is resistant to conventional chemotherapy and radiotherapy, carrying a dismal prognosis. Although many anticancer drugs have been developed for treating HCC, sorafenib is the only effective treatment, but it only prolongs survival duration for about 3 months. Recently, oncolytic virotherapy has shown promising results in treating HCCs and the effects can be more enhanced by adopting immune modulatory molecules. This review discusses the current status of treating HCC and the effective strategy of oncolytic virus-based immunotherapy for the treatment of HCCs. Hepatocellular carcinoma (HCC) is the third most common cancer with a leading cause of cancer-related death worldwide and is the only carcinoma with increasing mortality , 2. The Therefore, HCC is considered a highly refractory cancer and is resistant to conventional chemotherapy. To overcome these issues, the focus is shifting from antiangiogenic therapy  to novelThis review discusses the current status and the barriers of immunotherapy against HCC and effective immunotherapy strategies using oncolytic virus for HCC.Treatment options for HCC are divided into three categories as follows: (1) surgical therapies , (2) liver-directed nonsurgical therapies , radio frequency ablation (RFA), TACE, radiation, and radioembolization), or (3) systemic nonsurgical therapies . Since surgical therapies and liver-directed surgical therapies are generally used in the early stages of HCCs and HCC is typically diagnosed late in the course of patient with chronic liver diseases , here weDoxorubicin is the most studied chemotherapy agent for advanced HCC. Among the agents tried, doxorubicin-based regimens appear to have the greatest efficacy with response rates of 20–30% and a minimal impact on survival . Mitoxan5-Fluorouracil (5-FU) has broad antitumor efficacy with acceptably low toxicity. Although the response rate with only 5-FU has been low, combination with leucovorin can bring response rates to as high as 28% .Gemcitabine has modest activity at best. It is reported that 5 of 28 patients that received gemcitabine had a partial response of short duration (~13 weeks) . IrinoteCisplatin-based combination therapy appears to show higher objective response rates than that in noncisplatin-based therapy, although it is not clear whether it confers a survival benefit. Cisplatin plus doxorubicin had 18 and 49% objective responses, respectively , 36. TheTreatment approaches are now directed against a specific molecular defect, which is termed “molecular-targeted therapies.” Although the molecular pathogenesis of HCC remains poorly understood, the following signaling pathways are notably involved: (1) the epidermal growth factor receptor (EGFR)/EGF (HER1) in the carcinogenesis and proliferative behavior of HCC –49. (2) Oncolytic viruses (OVs) have been recently recognized as an effective treatment for cancer in preclinical models and promising clinical responses in human cancer patients . OVs havMost of the preclinical studies use adenoviruses and vesicular stomatitis virus (VSV) as the vector, due to their natural ability to kill or target HCC cells. For instance, adenoviruses have excellent tropism towards hepatocytes , in caseE1A and E1B genes of adenovirus have made it as oncolytic vector against the cancer cells [E1A gene is responsible for inactivation of several proteins, including retinoblastoma, allowing entry into S-phase [Adenoviruses are nonenveloped viruses, consist of double-stranded DNA (dsDNA) about 36 kb in size. There are seven subgroups reported, based on cross-reactivity patterns of neutralizing antibodies, from A to G. These are further classified as serotypes. As of now, 57 serotypes have been reported. The pathogenicity and tissue tropism are similar within the subgroups . Adenovier cells . The E1A S-phase . The E1B S-phase  whereas E1b gene, was engineered. The efficacy of this virus was checked in HCC cell lines and animal models in terms of tumor targeting, which showed selective gene expression, tumor regression, and prolonged survival period. The results compared with those of CNHK300 confirmed antitumor efficacy and selective replication of CNHK500 in HCC models [In order to overcome to this hurdle, adenovirus vector, ZD55, was constructed with -Smac (second mitochondria-derived activator of caspases)/TRAIL as ZD55-Smac/ZD55-TRAIL. The combined antitumor effect of these vectors was evaluated in mice xenograft models and cell lines. A significant regression of tumor size was reported in contrast to partial effect of single treatment of ZD55-Smac or ZD55-TRAIL. Moreover, in vitro-based analysis showed activation of caspases and significant reduction of X-linked inhibitor of apoptosis protein (XIAP) expression . VariousC models .β was generated by homologous recombination of ZD55 vector with interferon-β (IFN-β) containing vector. The antitumor efficacy and IFN-β expression of ZD55-IFN-β were evaluated in HCC cell lines and mice xenograft model. In contrast to Ad5-IFN-β-treated model, 100% cytopathic effect was examined in cell lines and more IFN-β expression were observed in both ZD55-IFN-β received mice and cell lines. This study confirmed the ability of adenovirus as a carrier of potential anticancer genes [Another viral strain, a monoregulated adenovirus CNHK300, was constructed with hTERT, and its antitumor efficacy was evaluated in different cell lines. The detection of hTERT expression and cytotoxicity, especially at low multiplicity of infection (MOI), confirmed the oncolytic activity of CNHK300 . ZD55-IFer genes .let7T was constructed as adenovirus with microRNA (miRNA), let-7. It was generated as introducing eight copies of let-7 target sites (let7T) into the 39 untranslated regions of E1A. A higher selective replication and cytotoxicity were reported in HCC cell lines when compared to normal liver cell lines. These were also verified in animal models [SG7011l models . Telomell models .γ (IFN-γ) production were also reported in this study [α-fetoprotein- (AFP-) E1A-IRES-E1B bicistronic cassette derived from Ad5, CV890 showed a synergic antitumor effect in vitro as well as in vivo [Adenoviruses armed with apoptosis-induced and immune-stimulatory molecules, such as human TRAIL and IL-12, were constructed as Ad-ΔB/TRAIL and Ad-ΔB/IL-12, respectively. The combined antitumor effects of these vectors were evaluated in Hep3B and HuH7 HCC cell lines. In addition, in vivo efficacy was examined in transplanted Hep3B-orthotopic model. A significant reduction in tumor size of orthotopic model and increased HCC cell death in cell culture were reported. Interestingly, this study documented a remarkable suppression of VEGF with infiltration of natural killer cells (NK cells) and antigen-presenting cells (APCs) at tumor microenvironment (TME). Moreover, enhanced apoptosis and upregulation of interferon-is study . In addi in vivo .Herpes simplex viruses (HSV) consist of dsDNA (154 kb) as genetic material. Cell receptors, such as herpesvirus entry mediator (HVEM), nectin 1, and nectin 2, are used for cell entry. Using HSV as an oncolytic agent, only very few studies were conducted in HCC model. In order to enhance HCC cell-specific tropism, liver-cancer-specific oncolytic virus (LCSOV) was created. It was constructed by linking the essential viral glycoprotein H gene with the liver specific apolipoprotein E (apoE)-AAT promoter. Then, miR-122a complimentary sequence was further inserted to the 3′ untranslated region (3′UTR). In addition, let-7 was also engineered into the same 3′UTR to increase the safety of this virus. The highly selective replication and tumor cell lysis were reported in xenograft as well as in cell culture models . HSV, G4TK)  . Intratuft model .β-gal into Buffalo rats bearing orthotopically implanted multifocal HCC showed efficient viral transduction, tumor-selective viral replication, and extensive oncolysis. There were no significant vector-associated toxicities and damage to the hepatic parenchyma reported, whereas prolonged survival was reported in vector-treated group [α/β) was checked in rVSV-NDV/F- (L289A-) treated HCC cell lines and rat models. Potential replication efficacy and no toxicity were documented as major outcomes. This study reported an increased index of oncolytic VSV virotherapy in interferon-treated advanced HCC models [VSV consists of single strand RNA (ssRNA) as a genetic material. Due to its intrinsic infectivity against cancer cells, most of the earlier preclinical studies used VSV for oncolytic virotherapy. VSV was recombined with green fluorescence protein (GFP) to examine antitumor efficacy and toxicity in HCC cell panels. The efficient selective replication and cytotoxicity were documented. IT injection of the virus in rat liver showed tumor destruction and tumor growth inhibition, which leads to prolonged survival of animals . Hepaticed group . Recombied group . AntivirC models . In ordeC models .The effect of the combination of an oncolytic MeV with the novel oral HDACi resminostat (Res) was checked in HCC cell panels. The combination effect showed a boosted cytotoxic effect as an enhanced induction of apoptosis with improved rate of primary infections . In ordedI1520 vector [9 pfu [Despite a number of OVs were tried in the preclinical studies, only a few among them have entered into the clinical studies . The rea0 vector . Most of0 vector , 81, 92.r [9 pfu . In order [9 pfu .A sequential therapy of JX-594, followed by sorafenib, a multikinase inhibitor and antagonist to vascular endothelial growth factor receptor (VEGFR) in three HCC patients, showed well-tolerance, associated with significantly decreased tumor perfusion and tumor responses  . In addiDue to limitations in conventional therapies for HCC, new treatment approaches have been established using modern molecular techniques. Oncolytic virotherapy is using oncolytic viruses which selectively infect and kill cancer cells. In order to enhance immunity against cancer cells, genes elevating onco-immunity have been engineered within oncolytic virus. Past and ongoing clinical trials have investigated anticancer efficacy and toxicities of oncolytic viruses, particularly JX-594 (GM-CSF encoding VV) for HCC treatment. GM-CSF as an immune-stimulatory cytokine boosts host immune activity through the infiltration of dendritic cells (DCs) and CD4+ and CD8+ T cells at tumor sites. Despite these outcomes have shown favorable results for prognosis of HCC treatment, as an immunotherapeutic approach, more advanced methods, that is, combination therapy with immune checkpoint inhibitors (ICIs) such as monoclonal antibodies (mAbs) against cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4), programmed death-ligand 1 (PD-L1), and/or programmed cell death protein 1(PD-1), would be taken into the account . This is


Proper expression of key reproductive hormones from gonadotrope cells of the pituitary is required for pubertal onset and reproduction. To further our understanding of the molecular events taking place during embryonic development, leading to expression of the glycoproteins luteinizing hormone (LH) and follicle-stimulating hormone (FSH), we characterized chromatin structure changes, imparted mainly by histone modifications, in model gonadotrope cell lines.Cga), αT3–1 , and LβT2 , to assess changes in chromatin status and transcription factor access of gonadotrope-specific genes.We evaluated chromatin status and gene expression profiles by chromatin immunoprecipitation assays, DNase sensitivity assay, and RNA sequencing in three developmentally staged gonadotrope cell lines, αT1–1 (progenitor, expressing Cga, to have an open chromatin conformation in all three cell lines. In contrast, chromatin status of Gnrhr is open only in αT3–1 and LβT2 cells. Lhb begins to open in LβT2 cells and was further opened by activin treatment. Histone H3 modifications associated with active chromatin were high on Gnrhr in αT3–1 and LβT2, and Lhb in LβT2 cells, while H3 modifications associated with repressed chromatin were low on Gnrhr, Lhb, and Fshb in LβT2 cells. Finally, chromatin status correlates with the progressive access of LHX3 to Cga and Gnrhr, followed by PITX1 binding to the Lhb promoter.We found the common mRNA α-subunit of LH and FSH, called Cga, Gnrhr, Lhb, and Fshb are not only controlled by developmental transcription factors, but also by epigenetic mechanisms that include the modulation of chromatin structure, and histone modifications.Our data show the gonadotrope-specific genes  Cga, glycoprotein hormones also known as αGSU) is detectable at E11.5 in the mouse, followed by the nuclear receptor Steroidogenic Factor 1 (Sf1 or Nr5a1), and the gonadotropin-releasing hormone receptor (Gnrhr). Finally, the gonadotropin hormone beta subunit mRNAs emerge at E16.5 for Lhb and E17.5 for Fshb . In. InCga. Cga, Gnrhr, Lhb, and Fshb in these model cell lines and demonstrate that these promoters are not only controlled by developmental transcription factors, but also by epigenetic mechanisms that include the modulation of chromatin structure, and histone modifications.Our data show exciting new evidence of gonadotrope-specific chromatin changes taking place during development using immortalized model gonadotrope cell lines. Although the cell lines utilized remain an in vitro system, representative of developing gonadotrope cell lines, we show a progressive opening of the gonadotrope-specific gene promoters,