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


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


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


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


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


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


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


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