It was demonstrated that SPARC expression was down-regulated to background levels in Cd+2-and As+3-transformed UROtsa cells and tumor transplants compared to parental cells. In the present study, the transformed cell lines were stably transfected with a SPARC expression vector to determine the effect of SPARC expression on the ability of the cells to form tumors in immune-compromised mice.This laboratory previously analyzed the expression of SPARC in the parental UROtsa cells, their arsenite . Transplantation of the cultured cells into immune-compromised mice by subcutaneous injection was used to assess the effect of SPARC expression on tumors generated from the above cell lines and urospheres.Real time PCR, western blotting, immunohistochemistry, and immunofluorescence were used to define the expression of SPARC in the As+3-and Cd+2-transformed UROtsa cells could undergo stable transfection with a SPARC expression vector and that the transfected cells expressed both SPARC mRNA and secreted protein. Tumors formed from these SPARC-transfected cells were shown to have no expression of SPARC. Urospheres isolated from cultures of the SPARC-transfected As+3-and Cd+2-transformed cell lines were shown to have only background expression of SPARC. Urospheres from both the non-transfected and SPARC-transfected cell lines were tumorigenic and thus fit the definition for a population of tumor initiating cells.It was shown that the As+3-and Cd+2-transformed cell lines have an inherent mechanism to suppress the expression of SPARC mRNA.Tumor initiating cells isolated from SPARC-transfected As SPARC iin situ . Low expin situ .+3 and Cd+2 exposure and the development of urothelial cancer. The UROtsa cell line is an immortalized, non-tumorigenic model that retains features of transitional urothelium when propagated using a serum-free growth medium [+2 and As+3 can cause the malignant transformation of human urothelial cells [+3- and Cd+2-transformed cell lines were all shown to retain a morphology consistent with human urothelial cancer and to display phenotypic differences characteristic of tumor heterogeneity. The histology of subcutaneous tumor heterotransplants produced by these transformed isolates displayed histologic features of human urothelial carcinoma with areas of squamous differentiation. This observation is important since urothelial carcinoma is the most prominent type of bladder cancer in western countries and accounts for over 95% of all cases and is 5th in overall occurrence [This laboratory’s interest in SPARC expression is the role it might have in the development and progression of urothelial cancer in general and in environmental-induced urothelial cancer in particular. SPARC has been shown to be expressed at the luminal surface of normal human urothelium  and primh medium , 26. Thial cells –30. Thescurrence .+3- and Cd+2-transformed cell lines, and the resulting tumors when the transformed cells were transplanted subcutaneously in immune compromised mice [+2- and As+3-transformed UROtsa cells compared to parental cells. In addition, it was shown by tumor transplantation into immune-compromised mice that the malignant epithelial component of tumors derived from these cell lines was also down-regulated to background levels for SPARC expression, but that the stromal cells recruited to these tumors was highly reactive for SPARC. This finding was shown to translate to specimens of human urotheial cancer where tumor cells were SPARC negative, but stromal cells were positive. It was also shown that acute exposure of the parental UROtsa cells to either As+3 or Cd+2 resulted in a marked reduction in SPARC expression. The finding that the As+3-and Cd+2-transformed cell lines had only background expression of SPARC offered the opportunity to re-introduce SPARC expression back into these cell lines. In the present study, the As+3-and Cd+2-transformed cell lines were stably transfected with a SPARC expression vector, and the effect of SPARC expression on the ability of the cells to form tumors in immune compromised mice and on in vitro cell growth, migration, and invasion was determined.A previous study from this laboratory analyzed the expression of SPARC in the parental UROtsa cells, their Assed mice . It was ICLAC Database of Cross-contaminated or Misidentified Cell Lines) that the UROtsa cell line from some sources have been contaminated by the human bladder cancer cell line, T24, this laboratory has a history of never having cultured T24. Thus the contamination event would have occurred through secondary sources after the UROtsa cell line was given to other investigators. Short tandom repeat analysis (STR) of the UROtsa cell line shown in the table below does not match any cell line in the ATCC STR database indicating that it is a unique cell line and certainly does not contain any T24 cells (2 tissue culture flasks using Dulbecco’s modified Eagle’s medium (DMEM) containing 5% v/v fetal calf serum in a 37°C, 5% CO2: 95% air atmosphere [+2-transformed isolates and the 6 individual isolates of the As+3-transformed UROtsa cells has been described previously [+3 and 2 Cd+2-transformed cell lines were used in the present study, As#3, As#6, Cd#1 and Cd#4, respectively. The transformed cell lines were all grown and maintained using identical conditions. Briefly, confluent flasks were sub-cultured at a 1:4 ratio using trypsin-EDTA  and the cells were fed fresh growth medium every 3 days. The MDA-MB-231 and Hs578T cell lines were obtained from the American Type Culture Collection (ATCC) and grown following the ATCC-supplied protocols. Cell viability and doubling times were determined by measuring the capacity of the cells to reduce MTT -2,5-diphenyltetrazolium bromide) to formazan. Triplicate cultures were analyzed for each time point and concentration.The UROtsa cell line was initially developed and characterized by Petzoldt , and fur24 cells . Mycoplamosphere . The isoeviously –30. Two +3-and Cd+2-transformed UROtsa cell lines were transfected with the SPARC ORF (2 culture well. Clones were selected using cloning rings and propagated in growth media containing 4 μg/ml Blasticidin (Invitrogen). Multiple clones from each cell line were analyzed to confirm SPARC expression and one clone from each line selected for further study. A blank vector control for each cell line was also selected for study.The SPARC ORF was cloned into the pENTR221 vector and transferred into the destination vector, pcDNA6.2/V5-DEST by the LR recombination reaction (Invitrogen). The purified plasmid constructs were verified by sequencing. The DNA constructs were linearized prior to transfection. The selected AsPARC ORF  in pcDNA6 cells were pelleted, and resuspended in PBS prior to the addition of proteinase K. To prevent carryover of RNA, the suspension was incubated with 1.8 μg/μL RNase A for 2 minutes at room temperature. An equal volume of lysis buffer was added to the cell suspension, mixed, and incubated at 56°C for 10 minutes. Ethanol was added to the suspension and mixed thoroughly before loading onto a DNeasy mini spin column. The bound DNA was washed 3 times prior to elution with 10 mM Tris-HCl, pH 7.5. For qPCR of the genomic DNA, the same conditions were used as that for qRT-PCR, except 100 ng of input DNA was used rather than 10 ng. Cycling parameters were 15 sec denaturation at 95°C, 30 sec annealing at 58°C, 30 sec extension at 72°C. The SPARC expression plasmid, SPARC-pcDNA6.2/V5-DEST, was used as a quantitative standard for the PCR reaction.Genomic DNA was isolated and purified from the transfected cell lines using DNeasy Blood and Tissue Kit  according to the manufacturer’s instructions. Approximately 5 x 105 cells in T-25 cm2 Ultra-low attachment flasks . The growth medium consisted of 1:1 mixture of Dulbecco’s modified Eagles’ medium and Hams’s F-12 growth medium supplemented with selenium (5 ng/ml), insulin (5 μg/ml), transferrin (5 μg/ml), hydrocortisone (36 ng/ml), triiodothyronine (4 pg/ml), and epidermal growth factor (10 ng/ml). The cells were allowed to form spheres for 8 days after which the urospheres were harvested by centrifugation and were used for RNA isolation or tumor transplantation into immune compromised mice.For the formation of urospheres, the SPARC transformed cell lines and the blank vector controls were seeded at a density of 106 cells. All mice were sacrificed by 10 weeks post-injection or when clinical conditions dictated euthanasia. The maximum tumor size was 2.5 cm. Tumor samples were either paraffin-embedded, sectioned, stained with hemotoxylin and eosin (H&E) and analyzed by light microscopy or snap frozen and processed for RNA and protein isolation. An identical procedure was used for the subcutaneous transplantation of the SPARC-transfected cell lines, blank vector (DEST) cell lines, and urospheres.All four transformed cell lines, corresponding SPARC-transfected (and blank vector-transfected) cell lines, and derived urospheres were capable of producing tumors when inoculated subcutaneously into immune compromised mice. The As#3 and Cd#1 lines also formed tumors when injected into the peritoneal cavity of immune compromised mice. The protocol for the transplantation of the transformed UROtsa cell lines into immune compromised mice has been described previously . Briefly2 inhalation and euthanasia conformed to AVMA Guidelines on Euthanasia.This study adhered to all recommendations dictated in the Guide for the Care and Use of Laboratory Animals of the NIH. The specific protocol was approved by the University of North Dakota Animal Care Committee (IACUC #1110-2C). All efforts were taken in order to minimize animal suffering, and mice were euthanized when clinical conditions dictated. Animals were sacrificed by 10 weeks post tumor transplantation by CO+2-and As+3-transformed cell lines and their subcutaneous transplants have been described previously [The preparation of total RNA and protein from the parental UROtsa cell line and from the Cdeviously –30. The eviously . The expeviously . See S2 The expression of secreted SPARC protein was determined by western analysis using slight modification in the protocol as previously described by Sage . Briefly+3-and Cd+2-transformed cells, the SPARC and DEST-transfected UROtsa cells and the breast cancer cell line MDA-MB-231 (positive control) were assessed. Analysis of migration was conducted using two assays, a wound/scratch assay and a trans-well chemotaxis migration assay. For the wound/scratch assay, cells were grown to confluence and treated with Mitomycin C (MMC) (Sigma Aldrich) for 2 h, to inhibit cellular division. Appropriate concentrations of MMC were specifically determined for each cell line by MTT analysis to insure inhibition of cellular proliferation while also insuring that levels were not toxic. A scratch was made within the cell monolayer using a sterile 200 μL pipette tip. The monolayer was then washed with phosphate-buffered saline, fresh growth medium was added, and cells were allowed to migrate for 24 or 48 h. Cells were photographed by light microscopy (using a 10X magnification lens) at 0, 24, and 48 h to analyze the migration of the cells towards the “wounded” area.The migration potential of parental UROtsa cells, the As4 cells in 300 μL of serum free media was added to the upper chamber and 500 μL of media containing 1.5% fetal calf serum was added to the bottom chamber of each well. The cells were allowed to migrate for 8 h at 37°C, 5% CO2: 95% air atmosphere following which they were fixed and stained with the supplied solutions and total cells were counted using light microscopy. After counting the total number of cells, the non-migratory cells were gently swabbed off the top insert membrane and the remaining migratory cells on the bottom side of the membrane were counted. Twenty fields were per insert was counted for each side of the membrane and the percentage of migrated cells was determined.Analysis of migration by chemotaxis was conducted using 24-well trans-well inserts with an 8 μm pore size polycarbonate membrane . 7.2 x10+3-and Cd+2-transformed cells, the SPARC and DEST-transfected UROtsa cells and the breast cancer cell line Hs578t (positive control) was assessed using basement membrane coated 24-well trans-well inserts. Cells were pretreated with MMC for 2 h before they were trypsinized and added to the upper chamber of the basement membrane coated 8 μm pore size polycarbonate membrane (Cell Biolabs) insert. The same concentrations of MMC were used as in the wound assay. 7.2 x104 cells in 300 μL serum free media was added to the upper chamber and 500 μL of media containing 10% fetal calf serum was added to the bottom chamber of each well. The cells were allowed to invade for 24 h at 37°C, 5% CO2: 95% air atmosphere. Cells were stained and counted in the same manner as the chemotaxis migration assay. The assay was performed in duplicate and the percentage of cells that invaded was determined.The invasion potential of the UROtsa parent cells, the As™ Real-Time Detection System (Bio-Rad Laboratories). Cycling parameters consisted of denaturation at 95°C for 15 seconds, annealing at 62°C for 30 seconds, and extension at 72°C for 30 seconds which gave optimal amplification efficiency. The expression level of ALDH1a1 was normalized to levels of 18S expression and is expressed in (mean ± SEM) transcripts ALDH1a1/106 transcripts 18S.The expression of ALDH1a1 mRNA was assessed by qRT-PCR. Purified RNA (100 ng) was subjected to cDNA synthesis using the iScript cDNA synthesis kit  in a total volume of 20 μl. Real-time PCR was performed utilizing the SYBR Green kit (Bio-Rad Laboratories) with 2 μl of cDNA, 0.2 μM commercially available primers (Qiagen) in a total volume of 20 μl. Amplification was monitored by SYBR Green fluorescence in a CFX96 Touch® software using separate variance t-tests or ANOVA with Tukey post-hoc testing. Unless otherwise stated, the level of significance was 0.05.All experiments were performed in triplicate unless otherwise stated and the results are expressed as the mean ± SEM. Statistical analyses were performed using GraphPad Prism+3 (#3 and #6) and 2 Cd+2 (#1 and #4) transformed UROtsa cell lines were stably transfected with the SPARC ORF under the control of the CMV promoter. An analysis of SPARC mRNA expression confirmed that the As+3-and Cd+2-transformed cells had significantly reduced levels of SPARC mRNA compared to the parental UROtsa cells  and to that previously published for the original non-transfected transformed cell lines [+3 and Cd+2 transformed cell lines  was significantly reduced when compared to both the SPARC-transfected cell line and to levels present in the parental UROtsa cells , were isolated from the Asboratory –30. The boratory –30. The +3-and Cd+2-transformed UROtsa cell lines. The results of this determination showed that ALDH1A1 mRNA was significantly enriched in TIC from both the SPARC-transfected and non-transfected As+3-and Cd+2-transformed UROtsa cell lines  isolated from all four SPARC-transfected As+3-and Cd+2-transformed UROtsa cell lines (+3-and Cd+2-transformed cell lines (TIC tumor transplants). In contrast to the consistently observed low levels of SPARC found in the SPARC-transfected urosphere (TIC) cultures, the level of SPARC mRNA in the SPARC-transfected urosphere (TIC) generated tumor transplants was variable  derived from the SPARC-transfected Asll lines . This isll lines . The levvariable . In partvariable  and apprvariable . Additiovariable . In ordevariable . The resWestern blotting of protein lysates prepared from tumors produced by the urospheres (TIC) isolated from all 4 SPARC-transfected and non-SPARC transfected cell lines all showed no expression of the SPARC protein (data not shown). Immunohistochemistry was used to determine if the SPARC protein might have focal expression in the tumors generated from the urospheres (TIC) generated from the 4 SPARC-transfected and non-SPARC transfected cell lines. Immunolocalization using a SPARC antibody demonstrated that 3 of the 4 TIC tumor transplants  generated from the non-SPARC transfected urospheres (TIC) had focal expression of the SPARC protein representing between 10 to 20% of the total tumor mass (+3-and Cd+2-transformed cell lines was determined and compared to that of the corresponding non-transfected As+3 and Cd+2 cell lines stably transfected with the blank vector (+3-and Cd+2-transformed cell lines containing no expression vector and there were no differences in cell morphology (data not shown). Overall, there were no major changes in the light level morphology of the As+3-and Cd+2-transformed cell lines when they were stably transfected with the SPARC expression vector.The light level morphology of the SPARC transfected Ask vector . All the+3-and Cd+2-transformed cell lines (+3-and Cd+2-transformed cell lines transfected with the blank vector showed no immunoreactivity for the SPARC protein (+3-and Cd+2-transformed cell lines had no immunoreactivity for the SPARC protein (data not shown). The majority of the cells in all four of the SPARC-transfected cell lines were immunoreactive for the SPARC protein, with only infrequent cell profiles negative for SPARC  and 2 of the Cd+2-transformed cell lines (#1 and #2) showed significant increases in chemotaxis compared to the parental UROtsa cells  and 2 of the 7 Cd+2-transformed cell lines  had rates of invasion greater than that of the parental UROtsa cells  in the tumor transplant generated from one SPARC-transfected cell line (As#3). Tumor generated from the other 3  SPARC-transfected cell lines showed no focal expression of SPARC. The combination of real time PCR, western blotting and immunohistochemistry convincingly demonstrated a complete suppression of SPARC expression in tumors generated from 3 of the 4 SPARC-transfected As+3-and Cd+2-transformed cell lines and only a low amount of focal expression of SPARC in the fourth cell line.This is an important observation since the malignant epithelial component of the tumor transplants generated from the SPARC-transfected As+3-and Cd+2-transformed cell lines is that the TICs responsible for tumor formation did not express SPARC. This is a possible explanation since it is now generally accepted that only a sub-population of cells within a transformed cell line are capable of initiating a tumor transplant. A method has been developed to isolate a putative TIC population from such cultures by subculture of the cell line on non-adherent plastic and isolation of a population of cell spheroids. These spheroids are putative TICs and are associated with enhanced expression of aldehyde dehydrogenase 1A1 (ALDH1A1). The method has become standard for many cancers and has been employed to isolate TICs from the urothelial cancer cell lines, HTB-2, 4 and 9 [+3-and Cd+2-transformed cell lines and that urospheres could initiate a tumor in immune-compromised mice. It was demonstrated that the urospheres isolated from the 2 As+3 (As#3 and As#6) and 2 Cd+2(Cd#1 and Cd#4)—transformed cell lines could initiate tumors in immune-compromise mice and that the tumors recapitulated the histology of the tumors produced when the parent cultures were inoculated into immune-compromised mice [+3 and Cd+2, two common environmental pollutants.One possible explanation for the finding that SPARC expression was suppressed in the tumor transplants from the SPARC-transfected As 4 and 9 , 33. A nsed mice –30. It w+3-and Cd+2-transformed cell lines. Total RNA was isolated from the urospheres representing the 2 As+3 and 2 Cd+2 SPARC-transfected cell lines and used to determine the level of ALDH1A1 and SPARC mRNA expression. The level of ALDH1A1 mRNA was elevated compared to the parent cultures and similar to the levels found in urospheres from the non-SPARC transfected As+3-and Cd+2-transformed cell lines. In marked contrast, the level of SPARC mRNA in the urospheres was reduced to near background levels of expression compared to the level present in the SPARC-transfected As+3-and Cd+2-transformed cell lines. The finding that the urospheres from the SPARC transfected cells had a very low expression of SPARC mRNA could explain the lack of SPARC expression in the tumor transplants generated from the SPARC-transfected As+3-and Cd+2-transformed cell lines. To confirm this notion, the urospheres from the non-SPARC transfected and SPARC-transfected As+3-and Cd+2-transformed cell lines were inoculated into immune compromised mice, with the assumpion being that there would be no expression of SPARC. However, this was found not to be the case. An immunohistochemical analysis for SPARC expression demonstrated focal staining for SPARC in 3 of the 4 tumors derived from urospheres isolated from the non-SPARC transfected As+3-and Cd+2-transformed cell lines. Similarly, immunostaining for SPARC demonstrated focal staining for SPARC in all 4 tumors derived from urospheres isolated from the SPARC transfected As+3-and Cd+2-transformed cell lines. The focal expression of SPARC in the tumor transplants was in agreement with the finding that SPARC mRNA was expressed in these tumors; however, western blotting did not detect the SPARC protein. The lack of SPARC protein on western analysis can be explained by the fact that only approximately 10% of the transplanted tumors expressed SPARC and that real time PCR is a far more sensitive method to detect mRNA expression. In addition, focal expression of a protein within a tumor is subject to sampling errors depending on the area of tumor dissected for lysate preparation. These results show that TICs isolated from both SPARC transfected and non-SPARC transfected As+3-and Cd+2-transformed cell lines can produce tumors with a low incidence of focal expression of the SPARC protein.An identical procedure was employed to isolate urospheres from the SPARC-transfected As+3-and Cd+2-transformed cell lines are suppressed for their expression of SPARC mRNA, while the majority of cells in the overall culture from which the TICs arise are permissive for the expression of SPARC mRNA. That such a population exists in the overall culture of the As+3-and Cd+2-transformed cell lines is supported by the examination of the cells for the intracellular localization of SPARC. In this examination, it was noted that there were rare profiles of cells in each SPARC-transfected cell line that did not express SPARC and these were estimated to be less than 1 in every 10 cells. These rare cells that do not express SPARC, or a subset thereof, are presumably the cells comprising the population of TICs that initiated the tumor transplants. The laboratory has not yet determined the mechanism underlying the ability of the TICs from the SPARC-transfected As+3-and Cd+2-transformed cell lines to silence the expression of SPARC mRNA under cell culture conditions. One explanation that can be removed from consideration is that there can be no interaction of the TICs regulatory components with the regulatory regions of the SPARC gene, since these sequences were not present in the expression vector used for the stable transfection of the SPARC gene. This includes the promoter region, the 3' untranslated region and intron sequences. Less likely explanations, but not inconceivable, are that some property of the TICs does not allow the uptake and stable transfection of the expression vector harboring the SPARC gene or that some undefined elements present in the TICs silences the CMV promoter of the expression vector. One can speculate that the most likely explanation will involve the expression of one or more microRNAs, or other regulatory RNAs, in the TIC population that are able to prevent the expression and translation of SPARC mRNA. This mechanism would be consistent with the finding that the TICs had a near background expression of SPARC mRNA. In addition, no literature could be found which indicates how common or rare it is to find a gene, such as SPARC in the present case that is silenced in a TIC population when the majority of other cells in the culture can be stably transfected with an expression vector.The results of this study show that TICs from the SPARC transfected As+3-and Cd+2-transformed cell lines were able to initiate tumors with the focal expression of SPARC. With the exception of the TIC-initiated tumor derived from one non-SPARC transfected cell line, the other 3 TIC-derived tumors all had a similar level of focal expression of the SPARC protein, showing moderate immunoreactivity in approximately 10% of the tumor. It is possible that the one tumor without SPARC is simply a sampling error. The finding that SPARC expression was similar in both the TIC-derived tumors from both the SPARC transfected and non-SPARC transfected suggests that the SPARC expression vector had no influence on the ability of the TIC-derived tumors to express the SPARC protein. This would suggest that the ability to initiate the focal expression of SPARC in the TIC-derived tumors is due to some unknown feature of the tumor environment. A change in tumor environment could be expected to alter the regulatory RNA profile of the TICs. What is unknown in the present study is if the focal expression of SPARC is a favorable sign of tumor differentiation or one of tumor progression. This is especially true for the SPARC protein since as detailed in the introduction it has been found to have contrasting properties and has been proposed to be a tumor suppressor or an oncogene depending on the tumor. Serial passage of the tumor transplants would be necessary to determine if the focal expression of SPARC is an overall feature of these tumors or an element lost upon serial passage.The involvement of a mechanism such as a regulatory RNA would also be consistent with the finding that TICs from the non-SPARC transfected and SPARC transfected As+3-and Cd+2-cells may only be indicative of one set of TICs that could exist in the cell culture and that can also form subcutaneous tumors in immune-compromised mice. The isolation of the TICs is dependent on a cell culture method and it is possible that another type of isolation could also identify a population of TIC that might have different properties. However, the correlation in the present study between SPARC expression in the TICs isolated from the cultured cells and that of the transplant tumors is noteworthy for one population.An additional caveat in the present study is that the urospheres isolated from the non-transfected and SPARC-transfected As+3-and 2 Cd+2- transformed cell lines. No effect of SPARC expression was noted for cell growth or the morphology of the cells. Three of the 4 SPARC transfected cell lines did show a reduction in chemotaxis when compared to their non-transfected counterparts. However, there was no effect of SPARC transfection on the ability of the transformed cells to close a wound that was generated using the “scratch assay”. The expression of SPARC also had no effect on the invasiveness of the transformed cell lines.The effect of SPARC transfection on cell growth, light level morphology, chemotaxis and invasion was determined on the overall cell population representing the 2 As+3 or Cd+2. The study also shows that a mechanism exists for these transformed cells to suppress the expression of SPARC mRNA in TICs isolated from both, SPARC transfected and non-SPARC transfected As+3-and Cd+2-transformed cell lines. However, tumor formation in immune compromised mice demonstrated that the focal expression of SPARC can be regained following inoculation of TICs from both SPARC transfected and non- SPARC transfected As+3-and Cd+2-transformed cell lines.In conclusion, this study demonstrates that TICs can be isolated from cultures of UROtsa cells malignantly transformed by either AsS1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.


In vitro models are essential to understanding the molecular characteristics of colorectal cancer (CRC) and the testing of therapies for CRC. Many efforts to establish and characterize primary CRC cell lines have been published, most describing a small number of novel cell lines. However, there remains a lack of a large panel of uniformly established and characterized cell lines. To this end we established 20 novel CRC cell lines, of which six were derived from liver metastases. Genetic, genomic and transcriptomic profiling was performed in order to characterize these new cell lines. All data are made publically available upon publication.in vitro models for CRC. All cell lines are available for further research.By combining mutation profiles with CNA and gene expression profiles, we generated an overall profile of the alterations in the major CRC-related signaling pathways. The combination of mutation profiles with genome, transcriptome and methylome data means that these low passage cell lines are among the best characterized of all CRC cell lines. This will allow researchers to select model cell lines appropriate to specific experiments, facilitating the optimal use of these cell lines as  With over a million cases diagnosed every year, colorectal cancer (CRC) is the third most commonly occurring cancer in the world (Globocan 2012). The occurrence of metastatic disease has a major impact on patient survival: CRC patients presenting with distant metastases have a 5-year survival rate of only 12%, while patients with local disease or regional spreading of disease show 5-year survival rates of 90% and 70%, respectively [RAS mutations are treated with EGFR inhibition therapies such as cetuximab and panitumumab. Patients without RAS mutations show a median overall survival of 23.8 months on this treatment [RAS mutant CRC are ineligible for EGFR inhibition therapy and show a median overall survival of 19.2 months.One third of CRC cases already show distant metastases at diagnosis and around 50-60% of these metastases are found in the liver –4. Metasin vitro testing of candidate compounds. The availability of a cell line model that closely resembles the tumor subtype under investigation is therefore pivotal. Many well characterized cell lines exist which represent most of the CRC subtypes [Development of new drugs for the treatment of cancer starts with subtypes , 7. Thessubtypes –11. For subtypes , 13. Lansubtypes .Several studies were performed to establish and characterize low passage CRC cell lines. For example Maletzki et al. established CRC cell lines from 5 tumors, and extensively characterized their morphology, growth kinetics, and molecular profile , 15. Sevin vitro drug research and may help further understanding of the molecular mechanisms underlying CRC.For this reason we sought to generate novel CRC cell models and have now established a panel of 20 new CRC cell lines. Six of these originated from CRC liver metastases, while the remaining cell lines all derived from primary CRC tumors. We performed characterization of these novel CRC cell lines, including somatic mutation profiling genomic and transcriptomic analyses. Additionally, sensitivity to oxaliplatin was tested as a measure of sensitivity to current CRC treatment regimens. The combined dataset are publically available. These novel CRC cell lines will serve as a valuable research tool in addition to currently available cell lines to be used for Here we report the establishment of 20 novel CRC cell lines, 14 of which were derived from primary colorectal cancers, while the remaining 6 were established from liver metastases. To assure these cell lines are permanent and stable, the cell lines were cultured for at least 30 passages. For the analyses described here cultures of approximately 13 passages were used. All cell lines have successfully been cultured multiple times from frozen vials to ensure they are able to survive the freezing process. The recovery rate was between 60-90%, except for JVE774 (10%).We performed comprehensive genomic profiling of the cell lines, including genome-wide gene expression, copy number and somatic mutation analyses.Of the cell lines derived from primary tumors, five originated from distal CRC, including rectal and sigmoid tumors, while 9 originated from proximal CRC tumors, amongst others from the cecum and the ascending colon. The various tumor locations are listed in Table Line JVE367 was derived from a large-cell neuroendocrine carcinoma (LNEC). LNEC account for only 0.2% of all CRC, and generally are associated with a very poor prognosis. , 20 To oConsiderable differences in cell morphology could be observed between the cell lines. The various morphologies are illustrated in Figure 50 > 25 μM, N = 4), the high sensitivity group with IC50's below 10 μM (N = 7) and an intermediate group with IC50's between 10 μM and 25 μM (N = 9). Dose-response curves for JVE059 and KP283T are shown in Figure t-test, p < 0.02). IC50 values for oxaliplatin, including 95% confidence intervals, are included in As a measure of resistance to chemotherapeutic regimens used in the treatment of CRC, sensitivity to oxaliplatin was assessed. Cell lines fell into one of three sensitivity groups; a resistant group  73 out of 147 CRC cell lines are MSI-H and recently Medico shows 41,7% of cell lines are MSI-H [MLH1 promoter, resulting in loss of the MutL complex. Therefore, MLH1 DNA promoter hypermethylation was assessed. JVE109 was found to be hypermethylated at the MLH1 promoter, while hypermethylation was absent in all other cell lines.While only 15% of primary CRC tumors show MSI-H  these ceTGFBR2 and ACVR2A. The microsatellites in exon 3 of TGFBR2 and the 3rd and 10th exons ACVR2A were screened in all cell lines . Hypermethylation of MGMT is observed in 38% [CDKN2A methylation is found in 25% of CRCs [CDKN2A is methylated in up to 75% of cases [CDKN2A hypermethylation has been proposed to be part of a cell line-specific DNA methylation pattern [MGMT: of these only JVE774 showed partial methylation. MSP of CDKN2A identified 12 cell lines with hypermethylation of CDKN2A, of which 6 cell lines were partially methylated. Partial hypermethylation can be explained by either methylation of one of the alleles or by a mixed population of cells with and without hypermethylation at these loci. A more accurate quantification of the percentage of methylated alleles could shed more light on this.In addition to d in 38%  and CDKN of CRCs . In CRC of cases , 25, and pattern . MSP ideKRAS, NRAS, PIK3CA and BRAF hotspots and part of the mutation cluster regions of APC and TP53. The most frequent mutated gene is TP53, with damaging mutations found in 13 cell lines.To further characterize the cell lines, somatic mutations were studied in mutation hotspot regions of 50 known oncogenes and tumor suppressor genes, including KRAS were found in 11 cell lines, including the less common KRAS activating mutations c.64C>A (p.Q22K) and c.351A>T (p.K117N). APC mutations were found in 9 cell lines.Activating mutations in BRAF c.1799T>A (p.V600E). Notably, all BRAF-mutated cell lines derived from primary CRC, while KRAS mutations were found in both primary and metastasis-derived cell lines. We identified PIK3CA mutations in 6 cell lines and all these cell lines also carried either a KRAS or a BRAF mutation. Co-occurrence of PIK3CA mutations with activating mutations in the mitogen activated protein kinase (MAPK) pathway was also observed in 74% of the TCGA colon adenocarcinoma samples. Damaging SMAD4 mutations were found in 2 cell lines. Additionally, next generation sequencing (NGS) data revealed homozygous deletions of SMAD4 in two additional cell lines. Similarly, PTEN homozygous deletions were observed in KP283T and JVE127, although no PTEN mutations were found. A possible activating EGFR c.2164G>A mutation as found in JVE059, however, the effect of this mutation is currently unknown. In the 4 MSI-H cell lines without MLH1 methylation, NGS of the DNA mismatch repair genes MLH1, MSH2, MSH6 and PMS2 was performed. MLH1 mutations were identified in JVE192 and JVE059. JVE192 carried the c.112A>C, p.N38H mutation, while JVE059 carried the compound heterozygous nonsense mutations c.551C>A and c.1975C>T. A homozygous MSH6 mutation, c.2718_2719del, p.V907Rfs*10, was found in JVE222. Analysis of KP7038T failed to show a variant, but did reveal lack of coverage of exon 16 of MLH1, suggesting the presence of a homozygous deletion. We carried out PCR analysis spanning exons 15-17 of MLH1 in KP7038T. The expected PCR product of 6497 bp was absent and was replaced by a PCR product of approximately 1350 bp. Sequencing analysis confirmed a deletion of 5146 bp at g.chr3:37084101-37089246, covering MLH1 exon 16 and resulting in an in-frame deletion of 55 amino acids. This deletion disrupts the PMS2 interaction domain, explaining the MSI-H profile of this cell line. An overview of all mutations, deletions and the promoter hypermethylation identified is provided in Table We identified 5 cell lines with a Complementary to the mutation profiles we generated genomic and transcriptomic profiles of all cell lines by hybridizing DNA and RNA respectively to Infinium HumanExome-12v1 BeadChips. Log2 relative gene expression values are included in SMAD4 and PTEN, and for promoter DNA hypermethylation of MLH1. MLH1 promoter hypermethylation was detected in JVE109. Expression of MLH1 was found to be 15-fold decreased in this sample compared to all other cell lines  pathways is given in Figure KRAS, BRAF or NRAS. Alternatively, by upregulating EGF gene expression, cancer cells establish an autocrine feedback loop, abolishing their dependency on systemic EGF. Upregulation of EGF gene expression in CRC occurs in 9.7% of tumors  [EGF was detected in 11 cases, 9 of which also showed a KRAS or BRAF mutation, and could therefore be considered to be no longer dependent on systemic EGF. JVE059 and JVE114 are the only cell lines that show neither a mutation in BRAF or KRAS, nor EGF upregulation. JVE059 harbors an EGFR mutation  within the tyrosine kinase domain, although it is not known whether this mutation leads to constitutive activation.Upregulation of the MAPK signaling pathway is a common alteration in CRC and is generally mediated by activating mutations in samples) . UpregulKRAS c.351A>T, p.K117N mutation was identified in JVE187. This mutation is known to result in constitutive activation of MAPK signaling, albeit to a lower extent than mutations in codons 12, 13 and 22 [ERBB2 mutation, c.2524G>A, which is also known to result in constitutive activation of the MAPK pathway [A 3 and 22 . Interes pathway .PIK3CA and inactivation of PTEN, PI3K signaling was activated in half of the cell lines. Alternative routes of activation for this pathway include transcriptional upregulation of IGF2 and IRS2. JVE103 and JVE371 both showed a 4-fold increase in IGF2 expression levels compared to the other samples. IRS2 is known to be upregulated in a small proportion of CRC tumors [IRS2 expression levels in almost all cell lines, although it is difficult to draw any conclusions from this without reference values. JVE371 showed substantially lower IRS2 expression levels, while it was amongs the highest IGF2 expressing cell lines. As JVE371 also carries an activating PIK3CA mutation, it is probably independent of upstream signaling. JVE103, on the other hand, is wildtype for both PIK3CA and PTEN, which suggests that this cell line may have upregulated the PI3K pathway through an autocrine upregulation of IGF2.As a result of activating mutations in C tumors . Our datDNA copy number profiles of each of the cell lines were generated using the Infinium HumanExome-12v1 data. In addition to copy number profiles, LOH and genomic imbalances were visualized by lesser allele intensity ratio (LAIR) analysis using the HumanExome-12v1 data . Cell liDifferences in copy number profiles between CRC metastases and primary CRC tumors have been reported previously . Liver mTo visualize the differences in copy number alterations (CNA) between primary tumor cell lines and metastasis-derived cell lines, the overall differences in CNA between the groups were plotted Figure . In addiWe established 20 novel cell lines from primary and metastasized CRC tumors. Using NGS and microarray-based approaches, we generated genetic, genomic and transcriptomic profiles and evaluated cell line sensitivity to oxaliplatin as a measure for chemoresistance. Thus we established a set of low passage CRC cell lines, which have all been thoroughly characterized in a uniform manner.in vitro experiments to further untangle the complexity of CRC tumorigenesis, and to the development of new therapies for both primary and metastatic disease.The characterization of these low passage cell lines identified a large diversity in mutation spectra and gene expression profiles. This extensive panel of new CRC cell lines represents a valuable research tool that can now be applied to in vitro models for CRC. The cell lines characterized in this manuscript are deposited at the Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures (www.dsmz.de).The combined dataset presented here elevates these cell lines to rank among the best characterized CRC cell lines. The SNP array data will also be made available through GEO (accession numbers GSE67773 and GSE67774), allowing researchers to select appropriate cell line models for their particular experiment, thus allowing optimal use of these novel cell lines as 3 fragments which were enzymatically dissociated using a 1% collagenase I-A (Sigma), 1% dispase (Gibco Life Technologies) solution. The dissociated cells were washed with RPMI-1640 and culturing was commenced in DMEM/F12 supplemented with 10% fetal bovine serum (FBS), 100 U penicillin and 100 μg streptomycin per mL. Once the cell line was established, further culturing was performed in either RPMI-1640 or DMEM/F12 medium (lines JVE222 and JVE241) supplemented with 10% FBS, 2 mM Glutamax-I, 50 U penicillin and 50 μg streptomycin per mL . Cells were cultured at 37°C with 9% CO2 in a humidified stove. Anonymized samples were handled according to the medical ethical guidelines described in the Code Proper Secondary Use of Human Tissue established by the Dutch Federation of Medical Sciences. All cell lines were checked for mycoplasma using a mycoplasma-specific PCR [Anonymized tumor material was thoroughly rinsed using RPMI-1640 medium under vigorous tapping. The tissue was cut into ± 1 mmific PCR .DNA isolation was performed using the Wizard Genomic DNA Purification Kit  according to the manufacturer's instructions.RNA isolation was performed on cells in exponential growth phase, using TRIzol® Reagent (Life Technologies). DNAse treatment was performed in suspension using rDNAse .Short tandem repeat (STR) profiles of the cell lines were established using the CellID system (Promega) according to the manufacturer's instructions. Fragment analysis was performed on an Applied Biosystems 3130 Genetic Analyser (Life Technologies). STR profiles for all cell lines are included in 50 concentration was determined using Graphpad Prism® software (version 5.01).Cells were seeded in 96-well plates at 10000 cells per well. Twenty-four hours after seeding, the medium was removed and fresh medium containing oxaliplatin  was added. After 72 hours of incubation with the compound, viability was assessed using the PrestoBlue® assay (Life Technologies) according to the manufacturer's instructions. Toxicity profiling was performed in triplicate and reproduced in 2 independent experiments. ICTGFBR2 and ACVR2 microsatellite analysis was performed as described previously [The microsatellite instability (MSI) status of each of the cell lines was determined using the MSI analysis system  according to the manufacturer's instructions. Fragment analysis was performed on an Applied Biosystems 3130 Genetic Analyser (Life Technologies). Samples with at least two out of five unstable mononucleotide markers were classified as MSI-H. eviously .Bisulfite conversion was performed using the EZ DNA methylation gold kit  according to the manufacturer's instructions, using an input of 200 ng of DNA. Bisulfite-converted DNA was eluted in 15 μL MQ water.MLH1 and MGMT MSPs were performed according to protocols developed at the Pathology Department molecular diagnostics lab at the LUMC [MGMT methylation-specific PCR (MSP) was performed using the same protocol, using the following primers: MGMT_Um_Fw: TTTGTGTTTTGATGTTTGTAGGTTTTTGT, MGMT_Um_Rev: AACTCCACACTCTTCCAAAAACAAAACA, MGMT_M_Fw: TTTCGACGTTCGTAGGTTTTCGC, MGMT_M_Rev: GCACTCTTCCGAAAACGAAACG.the LUMC . MGMT meCDKN2A, 1 μL of bisulfite converted DNA was used in combination with 1 pmol primers in a total volume of 10 μl, containing 1x IQ SYBR Green supermix . The primers used were as follows: CDKN2A-M-Fw: TGTAAAACGACGGCCAGTTTATTAGAGGGTGGGG CGGATCGC, CDKN2A-M-Rev: CAGGAAACAGC TATGACCGACCCCGAACCGCGACCGTAA, CDKN2A-Um-Fw: TGTAAAACGACGGCCAGTTTATTAGAG GGTGGGGTGGATTGT, CDKN2A-Um-Rev: CAGGA AACAGCTATGACCCAACCCCAAACCACAACCA TAA. PCR protocol: 5′ at 95°C, 40 cycles of 15″ at 95°C, 30″ at 69°C and 30″ at 72°C. CDKN2A methylation status was determined by gel analysis of the PCR product and by melting curve analysis.For the Genomic DNA (10 ng) from each sample was used to prepare barcoded libraries using IonXpress barcoded adapters (Life Technologies). Libraries were pooled to a final concentration of 15 ng/mL after quantification with a fluorometer , and emulsion PCR was performed using the Ion PGM Template OT2 200 kit on a OneTouch-2 instrument. Sequencing was performed on an Ion Torrent Personal Genome Machine, using 316v2 chips.Somatic mutations were analyzed using the Ion AmpliSeq™ Cancer Hotspot Panel v2. A list of the target genes included in this panel, along with mutation frequencies in CRC reported by the TCGA, is included in BRAF, codons 12 and 13 of KRAS and codons 545 and 1047 of PIK3CA were validated using Taqman genotyping assays [Mutations in codon 600 of g assays .Infinium HumanExome-12 v1 BeadChips were used with an input of 200 ng DNA. Raw data and preprocessed intensities per probe are available via the Gene Expression Omnibus (GEO) under accession numbers GSE67773 and GSE67774. For gene expression analysis using the same platform, 500 ng of RNA was converted to cDNA using the DyNAmo™ cDNA Synthesis Kit . cDNA was then purified using the QIAquick PCR purification kit  and eluted in 15 μL MQ water. Five μl of purified cDNA was used as input for the Infinium protocol.Lesser allele intensity ratio (LAIR) analysis was performed as described previously . Copy nuGene expression data was generated using the intensity data of the cDNA hybridization on the Infinium HumanExome-12 v1 BeadChips. Intronic probes and probes which were heterozygous in any sample were removed. Subsequently, intensity data related to the color of the genotyped allele was extracted for each probe. After quantile normalization using the Limma package , the ave


The mutant enzyme causes the production of D-2-hydroxyglutarate (D-2-HG), affecting CpG island and histone methylation. While mutations in IDH1/2 are early events in benign cartilage tumors, we evaluated whether these mutations play a role in malignant chondrosarcomas. Compared to IDH1/2 wildtype cell lines, chondrosarcoma cell lines harboring an endogenous IDH1 (n=3) or IDH2 mutation (n=2) showed up to a 100-fold increase in intracellular and extracellular D-2-HG levels. Specific inhibition of mutant IDH1 using AGI-5198 decreased levels of D-2-HG in a dose dependent manner. After 72 hours of treatment one out of three mutant IDH1 cell lines showed a moderate decrease in viability, while D-2-HG levels decreased >90%. Likewise, prolonged treatment (up to 20 passages) did not affect proliferation and migration. Furthermore, global gene expression, CpG island methylation as well as histone H3K4, -9, and -27 trimethylation levels remained unchanged. Thus, while IDH1/2 mutations cause enchondroma, malignant progression towards central chondrosarcoma renders chondrosarcoma growth independent of these mutations. Thus, monotherapy based on inhibition of mutant IDH1 appears insufficient for treatment of inoperable or metastasized chondrosarcoma patients.Mutations in  In 87% of these syndromic enchondromas and in 52% of sporadic enchondromas mutations are found, indicating that these are an early, driving and crucial event for the development of enchondroma [IDH1/2 in 38-70% of primary central chondrosarcomas (arising without a preexisting benign enchondroma) and in 86% of the secondary central chondrosarcomas [Mutations in a (~20%) , cholanga (~20%) -6 and ina (~20%) -10. Encha (~20%) , 12. Thehondroma . Malignahondroma . Centralsarcomas .IDH1 or IDH2 [Chondrosarcoma is the second most common primary malignant bone tumor and represents a heterogeneous group of tumors. So-call or IDH2 , 10.IDH1/2 mutations are associated with active Hh signaling [IDH1 and R140 and R172 in IDH2. These mutations in IDH-1 or -2 lead to gain-of-function, by which the mutant enzyme acquires the activity to convert α-ketoglutarate into D-2-hydroxyglutarate (D-2-HG), but not to its enantiomer L-2-hydroxyglutarate (L-2-HG). The newly formed oncometabolite D-2-HG shows structural similarities with α-ketoglutarate, and as a result D-2-HG is able to competitively inhibit α-ketoglutarate dependent enzymes such as the ten-eleven translocation (TET) enzymes [IDH1 or IDH2 mutation [IDH1/2 mutation [The signaling pathways that regulate endochondral ossification are thought to also play a role in the development of enchondromas and chondrosarcomas . Of thesignaling . Isocitr enzymes . TET enz enzymes -25. Indemutation , and DNAmutation , 9.IDH1/2 mutations are associated with DNA hypermethylation at CpG islands but not at other genomic regions [IDH1 R132H mutation [IDH1/2 are associated with stabilization of hypoxia inducible factor-1α (HIF1α) through an effect on the prolyl hydroxylases (PHD). Gliomas with an IDH1/2 mutation show upregulation of HIF1α, whereas PHD activity is inhibited in artificial mutant IDH1 cell lines [Genome-wide CpG methylation sequencing of chondrosarcoma biopsies revealed that  regions . In addi regions  and an imutation . Trimethmutation , 29. Furll lines .IDH1/2 mutations in enchondroma and chondrosarcoma suggest a causal rather than a bystander role. This led us to investigate the function of the IDH1/2 mutation in chondrosarcoma. Chondrosarcoma patients show a poor response to conventional chemotherapy and radiotherapy, and surgery is the mainstay of treatment. Alternative treatment strategies are urgently needed as no treatment options are currently available for patients with inoperable or metastatic disease. To evaluate the functional role of IDH1/2 mutations we used a chondrosarcoma cell line panel including five IDH1/2 wildtype, three endogenous IDH1 mutant and two endogenous IDH2 mutant cell lines, originating from conventional central as well as dedifferentiated chondrosarcomas. Remarkably glioma and leukemia cells harboring mutations in IDH1 or -2 cannot be maintained in culture, however we previously reported that chondrosarcoma cell lines retain these mutations [IDH1 or -2 mutations in their naturally occurring context and to evaluate whether inhibition of mutant IDH1/2 could be a potential treatment strategy.The high prevalence of utations  providinIDH1 and -2 in the cell lines confirmed the previously reported heterozygous mutations in JJ012 (IDH1 R132G), L835 (IDH1 R132C), SW1353 (IDH2 R172S), HT1080 (IDH1 R132C) and the homozygous R172W IDH2 mutation in L2975. Moreover, in all cell lines the mutant allele was expressed, as shown by sequencing of the corresponding cDNA samples , HT1080 (R132C) and L835 (R132C) with IC50s of 0.7 μM, 0.5 μM and 0.35 μM, respectively   Figure . We confs Figure . Howevers Figure . MoreoveAGI-5198 . Indeed,IDH1 are associated with inhibition of demethylases [IDH1 chondrosarcoma cell line L835, treated with 1.5 μM AGI-5198 for 10 passages, and the mutant IDH1 cell lines HT1080 and JJ012, treated for 20 passages, did not show any difference in lysine trimethylation of these specific histone marks  were only very lowly expressed in the cell lines before and after treatment, in comparison to the normal cartilage, growth plate and osteoblastoma controls  showed comparable expression levels between cell lines and controls. Treatment with D-2-HG did not influence the gene expression of these HIF1α target genes (data not shown), whereas treatment with AGI-5198 led to a decrease in the expression of VEGF and GLUT1 in L835 only  were variable and no consistent change in expression was seen upon inhibition of mutant IDH1 (data not shown).Analysis of gene expression using qPCR revealed that osteogenic and chondrogenic differentiation related genes  that, contradictory to what has been shown in the anaplastic astrocytoma WHO grade III cell line IMA [IDH1/2 cell lines. Thus, our panel provides us with an excellent model to functionally study the consequences of IDH1 or -2 mutations and to evaluate whether inhibition of mutant IDH1 could be a potential treatment strategy.The high prevalence of ulturing , 31. We line IMA , revealeIDH1 R132H and R132C mutations [IDH1 R132G mutant cells can also be decreased using AGI-5198. The selectivity of AGI-5198 was confirmed in the mutant IDH2 cell line SW1353, where treatment did not affect D-2-HG levels. Although inhibition of mutant IDH1 decreased levels of D-2-HG up to 90%, only one out of three IDH1 mutant chondrosarcoma cell lines (JJ012) showed a minor effect on viability. In addition, prolonged treatment during 10 or 20 passages also did not affect proliferation or migration in monolayer cultures nor in a 3D collagen scaffold model. Moreover, the colony forming capacity of the mutant IDH1 cell lines after 10 days was not affected by treatment with 10 μM AGI-5198. These results are in contrast to results in glioma since Rohle et. al. showed that AGI-5198 impaired soft-agar colony formation in the transformed glioma cell line TS603. Moreover, an effect on colony formation after knockdown of IDH1 with shRNAs was shown [IDH1/2, while being essential as an early event in the development of benign enchondroma, are not essential anymore after progression towards high grade chondrosarcoma. It is likely that other, additional genetic events occurred during tumor progression that have taken over driving chondrosarcoma cell proliferation.To inhibit the mutant IDH1 protein we used the commercially available small molecule inhibitor AGI-5198, which is a potent and selective inhibitor of IDH1 with mutations on position R132 . Inhibitutations , we hereas shown . A possiIDH1/2 mutant and IDH1/2 wildtype chondrosarcoma cell lines. Interestingly, only CpG islands probes show hypermethylation in the chondrosarcoma cell lines with an IDH1/2 mutation, whereas the shores and shelves show hypermethylation in the IDH1/2 wildtype cell lines. When all 480 000 probes were included, comparison of the mean β-values between mutant IDH1/2 and IDH1/2 wildtype cell lines showed general hypermethylation of the IDH1/2 wildtype cell lines. Thus, even though cell lines have been in culture for a long time, the mutation in IDH1/2 is associated with increased levels of methylation at the CpG islands, and decreased levels of methylation at other areas in the genome.Since the elevated levels of the oncometabolite D-2-HG caused by the mutation are reported to affect CpG island hypermethylation and histone methylation thereby affecting gene expression, as well as to interfere with HIF1α signaling, we next investigated the effect of inhibition of mutant IDH1 on these cellular processes in our cell lines. Mutant IDH1/2 causes hypermethylation of the genome via inhibition of α-ketoglutarate dependent enzymes in AML , gliomasGLUT1 and VEGF, upon inhibition of mutant IDH1. On the other hand, HT1080, formally derived from a “fibrosarcoma of bone” but in retrospect presumably representing dedifferentiated chondrosarcoma based on its IDH1 mutation, showed no alterations in gene expression, nor in CpG island methylation levels. Not surprisingly, this cell line does not express any of the differentiation markers either. It is tempting to speculate that HT1080 obtained many additional molecular alterations upon dedifferentiation  and prolongation in culture, thereby rendering the IDH1 mutation non-essential.In TS603 glioma xenografts treated with AGI-5198 for two weeks Rohle et. al. showed no statistically significant change in the genome wide distribution of DNA methylation . Likewisdeiodinase iodothyronine type 2 (D2) gene DIO2, which was confirmed using qPCR. The gene product of DIO2 catalyzes the conversion of intracellular inactive thyroid hormone (T4) to active hormone (T3) [IDH1/2 would upregulate DIO2, which would increase the levels of T3 thereby promoting chondrocyte maturation.In addition to downregulation of HIF1α downstream targets in L835 upon inhibition of mutant IDH1, RNAseq analysis revealed downregulation of the one (T3) . T3 is ione (T3) , 42. ThiDIO2 in L835. The latter finding is novel and suggests a role of DIO2 and thyroid signaling in the development of enchondroma and chondrosarcoma, which would warrant further study. Our results suggest that while mutations in IDH1 or-2 are an early event in tumorigenesis, chondrosarcoma is not dependent on mutant IDH1 anymore. Thus, other events are likely to be involved in chondrosarcoma progression, in which the mutation in IDH1 or -2 no longer seems to be a driver mutation. Even though promising results are being reported for IDH1 inhibitors in early clinical trials in leukemia [In summary, both short term as well as long term continuous inhibition of endogenous mutant IDH1 in three different chondrosarcoma cell lines show that although D-2-HG levels are decreased by the treatment, there is no significant effect on proliferation, migration (both in monolayer as well as in 3D), CpG island methylation or histone trimethylation of H3K4, H3K9 and H3K27. At the gene expression level we show inhibition of HIF1α target genes and of leukemia , 44, ourThe specific IDH1 mutant inhibitor AGI-5198  was dissolved in DMSO. D-2-hydroxyglutarate  and L-2-hydroxyglutarate  were dissolved in PBS.IDH1. Since fibrosarcoma is a diagnosis of exclusion [The chondrosarcoma cell lines CH2879 , NDCS-1 xclusion , in retrxclusion . STR typIDH1 and -2 mutation status was confirmed on DNA, as described previously [Both genomic DNA and cDNA were used for mutation analysis. DNA isolation was performed using the wizard genomic DNA purification kit  according to the manufacturer's instructions. RNA was isolated using TRIzol , and purified with the RNeasy mini kit . cDNA synthesis was performed with AMV-RT enzyme , oligodTs and random hexamer primers were used. Extracellular D/L-2-hydroxyglutarate was measured using an aliquot (1 mL) of culture medium conditioned for 72 hours. Intracellular D/L-2-hydroxyglutarate was measured in cell lysates. The cells were washed with PBS and taken up in Hanks balanced salt solution (HBSS). The cells were lysed by three cycles of freeze-thawing in dry ice. To distinguish between D-2-HG and L-2-HG, samples were measured via LC-MS/MS as described elsewhere . IntraceWhole cell lysates were made using the HOT-SDS protocol, lysis buffer contained complete inhibitors and phosphoStop. Proteins were separated by SDS-PAGE and transferred to a polyvinylidene difluoride membrane by a wet blotting system according to manufacturer's manual . After blocking with 5% non-fat dry-milk, the primary antibody, H3K4me3 ; H3K9me3 ; H3K27me3  was incubated overnight at 4°C, at dilutions of 1:1000, 1:2000 and 1:2000 respectively. Afterwards the membrane was washed with PBS/0,05% Tween-20, and the appropriate secondary antibody was incubated for 30 minutes at room temperature. After a final washing step, the membrane was developed using the ECL system  according to the manufacturer's protocol.Cells were seeded in 96 well plates and allowed to adhere overnight. For SW1353 and JJ012 5000 cells per well were seeded, for HT1080 and L835 10 000 cells and 15 000 cells, respectively. After 72 hours incubation with AGI-5198, a WST-1 assay  was performed according to the manufacturer's instruction and analyzed with the Victor3V, 1420 Multilabel plate reader . Assays were performed in triplicate for three independent experiments. For long term treatment with AGI-5198 and D-2-HG, cells were treated for 10 or 20 passages with 1.5 μM AGI-5198. Medium with compounds or solvent controls were refreshed twice a week.http://www.aceabio.com) was used to study either proliferation or migration in real time. Proliferation was monitored for 72 hours in E-plates. For the migration assay, SIM plates were used with the lower compartment filled with RPMI1640 with 20% FCS as a chemoattractant, cells were plated in the upper compartment in RPMI1640 only.The RTCA xCelligence system , HIF1α signaling , and differentiation . In addition, we evaluated the expression of COL1A2 and DIO2. PCR-primers are shown in http://www.broadinstitute.org/cancer/software/GENE-E/index.html).PCR reactions were carried out and relative gene expression levels were normalized for DNA input using the housekeeping genes eviously . We usedGenomic DNA was bisulfite-converted using the EZ DNA Methylation Gold Kit (Zymo Research) and used for microarray-based DNA methylation analysis, performed at ServiceXS  using the HumanMethylation450 BeadChip array . This array interrogates over 450,000 CpG sites representing about 99% of the RefSeq genes. The bisulfite-converted DNA was processed and hybridized to the HumanMethylation450 BeadChip , according to the manufacturer's instructions. Analysis was performed using ‘R project’. Samples were normalized using BMIQ .RNA was isolated using TRIzol . High througput sequencing of mRNAs was performed as previously described in detail . This pr


Glioblastoma (GBM) is considered to be one of the most aggressive tumors of the central nervous system (CNS). Even with the use of modern treatment protocols, the prognosis remains reserved, with children with GBM having a mean survival of 12–15 months.In the present study we investigated the potential radiosensitizing effect of PCI-24781, a potent pan-histone deacetylase inhibitor (HDACi), on the SF188 and KNS42 cell lines of pediatric GBM. Cell proliferation rates, clonogenicity and apoptosis were compared in the presence and absence of treatment with PCI-24781. We also compared the clonogenicity rates of the irradiated SF188 and KNS42 cell lines with or without previous treatment with PCI-24781 at the doses of 0.25–16 μM. In addition, we investigated the effects of PCI-24781 on the expression of some of the main proteins responsible for the repair of double-strand DNA breaks caused by irradiation.The inhibitor blocked cell proliferation, induced death by apoptosis and reduced the colony forming capacity of the cell lines, both of them showing a significant decrease of colony formation at all irradiation doses. The expression of the Rad51 protein, important for the homologous recombination (HR) repair pathway, and of the DNA-PKcs, Ku70 and Ku86 proteins, important for the non-homologous end joining (NHEJ) repair pathway, was more reduced when the irradiated cell line was previously treated with PCI-24781 than when it was treated exclusively with radiotherapy.These findings demonstrate that HDACi PCI-24781 has a radiosensitizing profile that compromises the repair of double-strand DNA breaks in cells of pediatric GBM treated with radiotherapy. Glioblastoma (GBM) is considered to be the most aggressive and most frequent central nervous system (CNS) tumor among adults, representing about 65 % of cases  in this The treatment strategy for GBM has not changed substantially over the last few years and essentially consists of surgical resection, radiotherapy and chemotherapy. Chemotherapy is usually employed to inhibit the replication of tumor clones or to provoke DNA damage in order to induce cell apoptosis , 4. ThisTemozolomide is the chemotherapeutic agent most frequently used for the treatment of adults and children with GBM but has been found to be poorly effective for the treatment of pediatric patients –9. In adRadiotherapy induces various types of DNA damage, among them double-strand breaks (DSB). DSB are defined as two simultaneous breaks in the opposite strands of the DNA helix. Although the cells are able to adapt to low levels of irreparable DNA damage, only one DSB is already potentially cytotoxic and can induce apoptosis in certain cell types –14. The New treatment strategies have been recently explored for gliomas, such as drug and therapy combinations directed at the molecular characteristics and genomic profile of the patients . These sAmong the various types of histone modifications, acetylation is the one that has been best characterized and that plays an important role in the modulation of the expression of genes that act on cell cycle control and contribute to the development and progression of neoplasia , 23–26. The objective of the present study was to assess the therapeutic potential of the HDAC inhibitor PCI-24781 against the pediatric GBM cell lines SF188 and KNS42, by analyzing the rates of cell proliferation, the clonogenic capacity and the apoptosis rates. We also investigated the effects of PCI-24781 as a possible radiosensitizing agent on both GBM lines by analyzing the clonogenic capacity and potential of the drug in changing the expression of proteins involved in the repair of double-strand DNA breaks caused by irradiation, i.e., Rad51, the heterodimer Ku70/Ku86 and DNA-PKcs.2 bottles using HAM F10 culture medium supplemented with 60 mg/L penicillin, 100 mg/mL streptomycin and 10 % (v/v) fetal calf serum , pH 7.2–7.4, in a moist atmosphere containing 5 % CO2 at 37 °C. The KNS42 cell line was cultured in MEM medium supplemented with 60 mg/L penicillin, 100 mg/mL streptomycin and 5 % (v/v) FCS , pH 7.2–7.4, in a moist atmosphere containing 5 % CO2 at 37 °C. The SF188 line originated from an 8-year-old male patient, has a TP53 mutation in codon 266 [GGA(Gly)/GAA(Glu)], and is not tumorigenic. The KNS42 line originated from a 16-year-old male patient with primary GBM located in the right frontoparietal lobe and has a TP53 mutation in codon 342[GGA(Arg)/TGA(Stop)] [The pediatric GBM lines SF188 (kindly provided by Dr. Nada Jabado and Dr. Damien Faury—McGill University—Canada) and KNS42 (obtained from the Japanese Collection of Research Bioresources Cell Bank through the Cell Bank of Rio de Janeiro) were used in the present study. The SF188 cell line was cultured in 75 cmA(Stop)] .According to the literature and to pilot experiments performed, a stock solution of the histone deacetylase inhibitor PCI-24781  was prepared at a final concentration of 50 mM in dimethylsulfoxide  and stored in aliquots at −80 °C. Working solutions of 10 mM were prepared also in DMSO and stored in aliquots at −80 °C.The drug was added to the culture medium and homogenized before being added to the cell culture. The DMSO concentration was 0.1 %. All controls were normalized by adding the same amount of DMSO as used for the treated cells.3 cells were seeded in 96-well plates and kept under culture conditions for 24 h. Next, the cells were treated with HDACi at the concentrations of 0.5, 1, 2, 4, 8 and 16 µM and incubated for 24, 48, 72 and 96 h. A resazurin solution was added to the plate  at each treatment interval. The plates were incubated for 4 h under standard culture conditions. The non-fluorescent blue reagent is reduced to highly fluorescent resorufin by the dehydrogenase enzymes present in metabolically active cells with an absorbance peak at 570 nm. Absorbance readings were taken with the iMax Microplate Reader , with the value detected being proportional to the quantity of cells in proliferation. These data were used to obtain the IC50 and IC30 values, which are defined as the concentrations necessary for a 50 and 30 % reduction of proliferation, respectively, using the Calcusyn software . Three independent experiments were performed in triplicate.Cell proliferation was determined by the assay using the Resazurin Cell Viability kit according to manufacturer instructions. A total number of 2 × 10The assay for the detection of cell death was carried out by labeling apoptotic cells with annexin V fluorescein isothiocyanate  and necrotic cells with propidium iodide (PI). Annexin V is a molecule with high affinity for phosphatidyl serine, to which it binds specifically. Phosphatidyl serine is a phospholipid present on the inner surface of the cell membrane which is externalized during the process of apoptosis and acts as a signal for the cells to be removed. Labeling with PI indicates that cells have lost their membrane integrity.After a 48 h treatment at concentrations of 2, 4, 8 and 16 µM of the PCI-24781 inhibitor, the cells were trypsinized, centrifuged at 1000 r.p.m. for 5 min, washed with ice-cold 1 X PBS and resuspended in 300 μL 1 X annexin V binding buffer . The cells were then labeled with 5 μL annexin V and 50 μL of a 50 μM PI solution, and analyzed with a BD FACSCalibur™ flow cytometer (BD Biosciences Pharmigen) for a total of 10,000 events per treatment. The values represent the mean and standard deviation of three independent experiments performed in triplicate.The effect of the PCI-24781 inhibitor on clonogenic capacity was assessed according to the protocol of Franken et al. . Cell suIC30 values, defined as the concentration necessary for a 30 % reduction of cell proliferation calculated with the Calcusyn software , were used for the assay combining the inhibitor with radiotherapy. The cells were then seeded as described earlier and treated with PCI-24781 for 48 h. Next, the cells were washed with 1X PBS and drug-free medium was added before irradiation with an RS-2000 X-Ray Irradiator Biological System . The irradiation rate was 1115 Gy/min and the doses were 0, 0.5, 1, 2 and 4 Gy. The irradiated cells were then incubated at 37 °C for 7–10 days, fixed in absolute methanol, stained with Giemsa, and counted. The values represent the mean and standard deviation of three independent experiments carried out in triplicate.® lysis buffer  together with protease and phosphatase inhibitors according to manufacturer instructions. Protein concentration was then determined by the method of Bradford, 1976 [Total protein extraction was performed using RIPArd, 1976  using boThe methodology proposed by Sambrook et al. 1989  was modiAll assays were carried out in triplicate and in three independent experiments. The mean and standard deviation of the experiments was considered for analysis by one-way ANOVA and two-way ANOVA followed by the nonparametric Bonferroni test.The effect of the HDACi PCI-2478 on the combination and the effect of the irradiation dose on percent colony numbers was tested by logistic regression.All analyses were performed with the aid of the SPSS 20.0 software , with the level of significance set at p < 0.05.Percent cell proliferation, clonogenic survival, radiosensitization and apoptosis are presented graphically in the form of histograms using the GraphPad Prism software, version 5.0. Protein quantitation was performed using the Image J software, version1.49t .We first investigated the effect of PCI-24781 on cell proliferation at doses of 0.5, 1, 2, 4, 8 and 16 µM for periods of 24, 48, 72 and 96 h on the cell lines SF188 and KNS42. A significant growth inhibition (p < 0.05) was detected compared to control at 48 and 96 h of treatment starting with the 2 µM dose. For the 72 h time point, this inhibition occurred starting with the 4 µM dose. After 24 h of exposure, the only dose that significantly reduced proliferation compared to control was 8 µM . Apoptosis was induced at 48 h at the doses of 2, 4, 8 and 16 µM in the pediatric GBM lines SF188 and KNS42. All doses tested showed a significant effect of increased apoptosis compared to control (p < 0.05). Starting at the 2 µM dose, the effect on the cell death rate of both lines was already quite significant compared to control, demonstrating that only a small dose of the PCI-24781 inhibitor was able to induce apoptosis. The cell death rate by apoptosis was almost 40 % for the SF188 line and 55 % for the KNS42 line at the maximum dose tested Fig. .Fig. 3PeIn order to determine whether the reduction of proliferation affected cell growth in the long term and to test the capacity of tumor renewal, the test of clonogenic capacity was performed at the 48 h time point at the doses of 0.25, 0.5, 1 and 2 µM on the pediatric GBM lines SF188 and KNS42. The analysis revealed that all doses induced a significant fall in colony formation compared to control (p < 0.001). The SF188 line exhibited a reduction of colony formation of almost 99 % and the KNS42 line was unable to form colonies at the maximum dose used  of PCI-24782 for the SF188 line for 48 h and irradiation doses of 4 and 6 Gy. The proteins were collected after 4 h of exposure to irradiation.The results showed that PCI-24781 was competent in reducing the efficiency of the SF188 line in diminishing the DSB induced by irradiation, with a reduction of the expression of the proteins tested. For the Ku 70, Ku 86 and DNA-PKcs proteins there was only a small difference between treatment with PCI-24781 alone or in combination Figs. .Fig. 7PCOver the last few years, the association between HDACs and carcinogenesis has increased the interest in the use of HDACi as antitumor agents. HDACi are new anticancer agents that induce tumor cell death and differentiation, chromatin decondensation and arrest of the cell cycle .Uncontrolled cell proliferation is a strong characteristic of aggressive tumors, including GBM . In the TP53 gene, but several studies have demonstrated no significant difference in the response to treatment with PCI-24781 between TP53-proficient and TP53-deficient cell lines [Both lines studied herein here have a mutation of the ll lines –39. Dalvll lines . Severalll lines , 39, 44.Regarding the clonogenic capacity, we observed that after treatment there was a significant reduction of colony formation in both lines. Several studies have detected results similar to those obtained here with PCI-24781 and various other HDACi in separate or combined treatments of neoplastic cell lines , 39, 42.Clinically, two HDACi inhibitors approved by the FDA, suberoylanilide hydroxamic acid (SAHA) and valproic acid (VPA), were used in phase I studies conducted on children with primary or relapsing CNS tumors. The treatments were carried out using the inhibitor alone or in combination with chemotherapy and were well tolerated in all studies –49.Radiotherapy is an important part of the treatment of pediatric GBM. Strategies increasing the efficiency of radiotherapy and permitting a reduction of toxic effects on normal tissue are important. In this respect, in the current study we also investigated the radiosensitizing potential of PCI-24781 using clonogenic assays, which showed that treatment with the inhibitor significantly increased the sensitivity to radiation of both lines.TP53 mutation showed a greater reduction of colony forming capacity, suggesting that TP53 plays an important role in the modulation of the effect of HADCi [Other studies have also assessed the radiosensitizing effect of PCI-24781 and of other HDACi based on clonogenic capacity in tumors other than GBMs. Chen et al. (2009) assessed the radiosensitizing capacity of the HDACi VPA on two colon tumor cell lines, one of them with a p53 mutation and the other without a mutation. The line with no of HADCi . Munshi of HADCi .Radiation remains an essential modality in the treatment of various brain tumors. Radiotherapy can be delivered alone or in combination with a radiosensitizer agent, in order to enhance the efficacy of irradiation. As revised by Harasaki and Waziri (2012) , the conIn the present study, in order to validate the radiosensitizing effects observed, we investigated the effect of PCI-24781 on the response of the SF188 line in terms of the repair of irradiation damage, affecting proteins of two important repair pathways, i.e., homologous recombination (HR) and non-homologous end joining (NHEJ). The results showed that PCI-24781 affected the expression of important proteins of both the HR pathway (the RAD51 protein) and of the NHEJ pathway , leading us to believe that the inhibitor proved to be competent in reducing the efficiency of the SF188 cell line for the repair of irradiation-induced DSB. RAD51 is an important repair protein responsible for mediating the search of a homologous DNA sequence to be used as a model and for the formation of molecular joining between the damaged and the undamaged strands . It has The present study was the first to assess the effect of the HDACi PCI-24781 on the expression levels of the proteins involved in double-strand DNA repair in pediatric GBM lines.Previous studies of colon and gastric tumor lines and of xenographic models have also observed that the level of expression of the RAD51 protein is drastically reduced after treatment with PCI-24781, leading to inhibition of DSB repair by homologous recombination , 53, 54.Despite the great interest in combining HDAC inhibitors with radiation as a form of clinical strategy for tumor treatment, the exact molecular mechanism of the radiosensitizing effect of these inhibitors is still not well known. HDAC inhibition facilitates the relaxation and opening of chromatin, rendering it more sensitive to radiation damage and also modifying the transcription of various genes, with these two processes playing an important role in the regulation of radiosensitivity. In addition, transcriptionally active genes have proved to be more sensitive to DNA damage produced by ionizing radiation, thus forming a favorable antitumor interaction between HDAC inhibitors and radiation. An explanation for this increased response to radiation following HDACi treatment may be the effect of these inhibitors on the DNA repair processes , 63–65.The present study demonstrates that the HDACi PCI-24781 can reduce the ability of the pediatric GBM line to repair radiation-induced DNA damage, increasing the radio sensitization of the line.In conclusion we demonstrated that the HDAC inhibitor PCI-24781 reduced proliferation and clonogenic capacity and increased apoptosis of pediatric GBM cell lines. In addition the HDACi PCI-24781 demonstrates high radiosensitizing activity by reducing the capacity of the cells to repair DNA damage induced by radiation by altering the expression of nonhomologous proteins (NHEJ). Thus, PCI-24781 can be considered to be an agent with a good potential for studies of in vivo models of pediatric GBM treatment.


MDP can be queried for drugs active in cancer cell lines carrying mutations in specific cancer genes or for genetic markers associated to sensitivity or resistance to a given compound. As proof of performance, we interrogated MDP to identify both known and novel pharmacogenomics associations and unveiled an unpredicted combination of two FDA-approved compounds, namely statins and Dasatinib, as an effective strategy to potently inhibit YAP/TAZ in cancer cells.Targeted anticancer therapies represent the most effective pharmacological strategies in terms of clinical responses. In this context, genetic alteration of several oncogenes represents an optimal predictor of response to targeted therapy. Integration of large-scale molecular and pharmacological data from cancer cell lines promises to be effective in the discovery of new genetic markers of drug sensitivity and of clinically relevant anticancer compounds. To define novel pharmacogenomic dependencies in cancer, we created the Mutations and Drugs Portal (MDP,  Genetic alterations in cancer cells often generate cancer-specific dependencies that are exploited to develop targeted therapies. Mounting evidence shows that the number of cancer patients responding to targeted therapies has dramatically increased. For example, about half of melanomas show mutations in BRAF, making these cells sensitive to specific BRAF (Vemurafenib) and/or MEK inhibitors. Although the beneficial responses are not always durable , the likin vitro, growth of human cancer cell lines from tissues as breast, prostate, lung, colon, ovary, kidney, central nervous system and from melanomas and leukemias. In the NCI60 screen, drug activity is measured in terms of GI50, the log of compound concentration required to cause 50% of growth inhibition whereas molecular data of cell lines includes DNA copy number, single nucleotide polymorphisms (SNPs) [from gene to drug) or gene mutations associated to drug response (from drug to gene).The MDP database builds on exome-sequencing and drug response data from the NCI60 and the CCLE datasets. The National Cancer Institute (NCI) anticancer drug screen was originally developed to identify compounds with growth-inhibitory or toxic effects on a panel of 60 human tumour cell lines (NCI60), representing different tumour types , 9. The s (SNPs) , 11, whos (SNPs) , and RNAs (SNPs) –15. In pAnalysis page, selecting the from gene to drug section, it is possible to start from a gene to retrieve compounds or drug families with growth-inhibitory effects on cancer cell lines carrying mutations on that gene. Instead, using the from drug to gene analysis, the user can input a drug to identify which genomic mutations affect the sensitivity of the cancer cell lines. Both types of queries can be performed selecting the genomic information of the 1,651 oncogenes from CCLE  or the whole set of 15,000 human genes from NCI60 repository .The web interface allows querying associations between genomic and pharmacological data in either direction depending on the particular question of interest. In the from gene to drug section, both run analysis on CCLE and run analysis on NCI60 buttons re-direct to a web page where the user can query for the gene names or the SNP Ids of interest [from gene to drug analysis is a list of molecules that show a statistically significant activity on cell lines bearing specific mutations on the selected gene/s  or resistance (red bubbles) to the selected molecule . The size of the bubbles is proportional to the total number of mutations found in cancer cell lines , in each tissue , and for each variant classification .When using the s Figure . The detTo determine whether MDP could be used to find out real pharmacogenomic associations between somatic gene mutations and sensitivity to specific drugs, we decided to interrogate MDP for genes on which targeted therapies have been already established and clinically proved. In particular, the Mitogen-activated protein kinase (MAPK) pathway is involved in the control of proliferation of both normal and transformed cells . MAPK cap = 6.48 × 10−7), Vemurafenib [p = 1.2 × 10−5) and SB-590885-AAD [p = 7.96 × 10−5); three MEK inhibitors: Hypothemycin [p = 9.49 × 10−5), Selumetinib [p = 0.0008) and protein LF  [p = 7.96 × 10−5)  to query CCLE genomic data. When considering all tissues, the analysis identified, out of 5,793 drugs, 197 drugs with significant selectivity for the BRAF-mutated cell lines. Interestingly, among the top 20 score-ranked molecules we identified seven compounds that are already associated with BRAF mutations. In particular, we identified three BRAF inhibitors: Dabrafenib [0885-AAD  ( p = 7. to queryumetinib  ( p = 0.= 0.001) ; and a t) Figure . Similarp-value = 0.03) [p-value = 0.03) with somatic missense EGFR mutations in CCLE cancer cell lines . Mutations that lead to EGFR overexpression or hyperactivity have been associated with a number of cancers, including lung cancer . To furt = 0.03)  and the s Figure .We also successfully identified a number of other known pharmacogenomic associations such as: cytidine derivatives with KRAS mutations , gemcitain vitro experiments. For example MDP suggests that BRCA1 and BRCA2 missense mutations are associated with sensitivity to Mitoxantrone; PTEN mutations with sensitivity to Brequinar; MET mutations with sensitivity to Dabrafenib; ATM mutations with sensitivity to Lapatinib; FBXW7 mutations with sensitivity to Corticosteroids (Prednisolone and Fluorometholone) and ERBB4 mutations with sensitivity to Acivicin  and are highly expressed in cells with cancer stem cells properties and metastatic potential . Increasfrom gene to drug) on all CCLE cell lines and identified the most enriched family drugs. Interestingly, the Enriched Drug Families table of this query highlighted Statins  and Imatinib analogues  as the only two classes of drugs significantly associated to drug sensitivity in cancer cell lines harbouring NF2 mutations  depending on the particular question of interest.Next generation sequencing of cancer cell lines, coupled with high throughput cell viability-based screening of small molecules, offers the possibility to rapidly identify many cancer-specific dependencies, potentially targetable with numerous chemical compounds. Nevertheless, the full exploitation of these “omics” databases is still hampered by gaps in the integration of genomics and pharmacological data. Here we developed MDP, an open access resource, which helps the researchers to unveil oncogene-induced dependencies by systematically coupling specific gene mutations with the pharmacological response to tens of thousands of compounds. MDP is available on-line and links genomic information from CCLE and NCI60 databases to pharmacological data from the NCI60 DTP screening allowing the identification of synthetic or natural compounds active in cancer cells with specific genetic features. The main goal of this web resource is to offer a very user-friendly platform for the execution of custom Since oncogenes may give rise to pharmacological dependencies only in particular tissues, MDP easily allows users to run the analysis selecting only cancer cells from a specific tissue thus leading to the identification of tissue-specific therapeutic biomarkers. Moreover the resource permits multiple mutation queries, enabling the identification of drugs active against cells with more than one mutated gene at the same time.from drug to gene analysis, is capable of retrieving the most enriched mutations associated with sensitivity to the specific drug selected by the users and, in this way, might help researchers to identify potential drug-mutation dependencies in cancer cell lines, or unveiling new correlations between these mutated cancer cell lines and their sensitivity to a particular drug.MDP, through the The reliability of this approach is guaranteed by the identification of already established and clinically proved gene-drug pairs, among them BRAF V600E mutation with clinically used BRAF/MEK inhibitors or EGFR mutations with EGFR inhibitors. Moreover, MDP suggests several novel dependencies, making it a powerful tool to guide the early phases of drug discovery or drug repositioning. As an example, querying MDP for drugs selectively active on cells bearing mutation in NF2 gene, an event that leads to the aberrant activation of the transcription cofactors YAP and TAZ, we found that the combinatorial use of two FDA-approved drugs, namely statins and Dasatinib, acts synergistically to strongly inhibit the activity of YAP and TAZ in different biological contexts.YAP and TAZ are critical downstream effectors of actomyosin cytoskeleton acting as nuclear transducers of mechanical stimuli  and we aThe evidence that statin and Dasatinib, as demonstrated from our work, act synergistically to inhibit YAP/TAZ suggests that they could be used in combination and thus at lower concentrations, to inhibit YAP/TAZ activity and tumour growth in cancers characterised by hyperactivation of YAP/TAZ and, among them, in triple negative breast cancer and metastatic mesothelioma .While the huge amount of compounds screened represents the strength of MDP, the limited number of cancer cells and genotypes used can depict a weakness. However, the NCI60 cell lines have been extensively used to identify novel predictive cancer biomarkers proving that the number of different genotypes is not necessarily a limitation –45. ReceIn conclusion, here we present MDP, an on-line tool able to perform prediction about cancer cell lines mutations and drugs sensitivity dependencies. MDP is, as far as we know, the largest database of pharmacogenomics structured to be a user-friendly tool for the investigation of drugs-mutations correlations, and can be queried either from the gene or drug perspective. Data obtained via MDP will help researchers in the challenge of patients' stratification, thus refining the parameters for personalized targeted therapies on the basis of their specific genetic abnormalities. We strongly believe that MDP provides a novel and comprehensive tool for the systematic identification of new biomarkers of drug sensitivity, setting the rationale for the design of new clinical trials and the discovery of novel anticancer drugs.50 Data , retrieved from the NCI60 DTP portal, and sequencing data and variant classifications retrieved from the CCLE (http://www.broadinstitute.org/ccle/data/browseData?conversationPropagation=begin) and NCI60 (http://discover.nci.nih.gov/cellminer/loadDownload.do) public repositories.Statistical analyses are based on the drug response data file GI50 Data file contains a matrix of GI50 values for 50,816 molecules tested on 115 cancer cell lines. GI50 values are computed, for any compound, as minus the log10 of IC50, i.e., the drug concentration necessary to inhibit 50% growth of treated cells relative to untreated controls. Prior to analysis, for any single compound, first the GI50 is transformed back to IC50 and then the IC50 value is normalized dividing the IC50 of any cell line by the average of the IC50 across all 115 cell lines.GI50  of a compound is used to define the response for any combination of drug and cell line in terms of i) good response if the normalized log2 IC50 is lower than two standard deviations of the distribution of all log2 IC50 in a given cell line, and ii) bad response otherwise.The normalized ICfrom gene to drug analysis, given a specific set of mutation/s, compounds with increased activity in cases (cancer cell lines treated with the given compound and bearing the specific set of mutation/s selected by the user) as compared to controls , are identified ranking all compounds based on a score given by the fraction of good responses in cases multiplied by the fraction of bad responses in controls. This score ranks each drug based on the enrichment of good responses in the case group. The statistical significance  of this ranking is computed, for each drug, using a one-tailed Fisher's exact test for the enrichment of good responses in cases as compared to bad responses in controls, given the number of bad responses in cases and good responses in controls.In the from drug to gene analysis. In this case, the normalized log2IC50 of the selected compound is used to first retrieve the two groups of cell lines with good and bad responses for that drug. Then, for each mutation, we calculate the number of cases, i.e., the fraction of cell lines in the good response group bearing the considered mutation, and the number of controls, as the fraction of cell lines in the bad response group bearing the considered mutation. The score given by the fraction of good responses in cases multiplied by the fraction of bad responses in controls is again used to rank mutations and a Fisher's test for the enrichment of good responses in cases as compared to bad responses in controls, given bad responses in cases and good responses in controls, to assess significance.The same ranking function and statistical test is also used to identify the most enriched mutations starting from a drug when performing a from gene to drug analysis, whereas Python 2.7 and Pandas were used to perform the from drug to gene statistical process. MDP is hosted on a Linux server with 512 GB RAM and 64 processors.MDP web interface has been written using PHP version 5.0, HTML 5.0 and CSS. Connections between pages and programming languages have been performed using Twig, a template engine that allows fast connections between PHP and HTML. Graphical representations in the results pages  have been coded using JavaScript and jQuery, basing on the structure of the latest Google APIs release (2015). Statistical methods of analyses were performed using Python 2.7, through Pandas 0.16 module, and R 3.0. R software was used in the MDA-MB-231, HT29, PC-3, SKBR-3 and PANC-1 were cultured in DMEM supplemented with 10% FBS  and antibiotics. H1299 cells were cultured in RPMI 1640 with 10% FBS and antibiotics. Cells have been authenticated by STR profiling and are free from mycoplasma contamination. The following compounds were purchased from Sigma Aldrich: statin , Zoledronic Acid (SML0223), Paclitaxel (T7191). Dasatinib was purchased from Selleck (S1021). Cell lines used to build MDP have been already authenticated .For the quantification of the number of cells with nuclear YAP/TAZ at least 300 cells from different fields were counted. Only cells with complete nuclear YAP/TAZ exclusion were scored as positive.For cell growth assay cells  were plated in 96-well plates and treated as indicated. Cells were trypsinized, collected and counted at day 1, 2 and 3. Cell viability was assayed with ATPlite (Perkin Elmer) according to the manufacturer's instructions using the EnSpireMultilabel Reader (Perkin Elmer).Cells were collected in Qiazol lysis reagent (Qiagen) for total RNA extraction, and contaminant DNA was removed by DNase treatment. Retrotranscription was performed with the Quantitect reverse transcription kit (Qiagen). The obtained cDNA was properly diluted and used in qPCR reactions performed with SsoAdvanced SYBR Green Supermix (Biorad) using the CFX96 Touch Real-Time PCR Detection System and analyzed with Biorad CFX Manager software. Each experiment was performed at least three times Expression levels are always given relative to histone H3. The primers used were previously described .siRNA transfections were performed with Lipofectamine RNAi-MAX (Life Technologies) in antibiotic-free medium according to the manufacturer's instructions. Negative control siRNA was: AllStars negative control siRNA Qiagen 1027281. siRNA sequences are: YAP (GACAUCUUCUGGUCAGAGA) and TAZ (ACGUUGACUUAGGAACUUU).DNA transfections were done with Lipofectamine LTX and Plus Reagent (Invitrogen) or Lipofectamine 2000  according to the manufacturer's instructions.Luciferase assays were performed in MDA-MB-231 cells with the established YAP/TAZ-responsive reporter 8xGTII–lux. Cell lysates were analyzed using the Dual-Luciferase Reporter Assay System . Luciferase reporter (300 ng cm−2) were transfected together with CMV–Renilla (30 ng cm−2) to normalize for transfection efficiency. Cells were collected 24 h after DNA transfection. For luciferase assays in siRNA-transfected cells, siRNA transfection was achieved first and, after 48 h, transfection of plasmid DNA was performed. Cells were collected 24 h after DNA transfection.The antibodies used for western blot and immunofluorescence were: anti-YAP/TAZ , anti-actin , anti-Cleaved Caspase 3 .Immunofluorescence staining was performed as previously described . Briefly


Skin cancers account for less than 1 % of all malignancies in India. Squamous cell carcinomas occurring over the waistline due to tying of cotton cloth called dhoti in males and sarees in females are predominantly seen in traditional Indian population. On wearing of these clothes for years, there is a constant irritation which produces depigmentation, glazing of the skin, acanthosis, scar formation, and later on malignant transformation. Presenting a case of a 65-year-old male with 7 × 5 cm ulceroproliferative growth over the right waistline with a history of prolonged use of dhoti. Wide local excision of the growth with 2-cm margin and primary closure of wound by mobilizing the skin was carried out. Histopathology showed well-differentiated squamous cell carcinoma. The patient is clinically disease free after postoperative follow-up of 1 year. Waistline cancer is a rare type of squamous cell cancer occurring over the waistline predominantly in Indian males and females wearing a piece of cotton cloth used to cover the lower part of the body. This cloth is called dhoti in men and saree in women. The dhoti is worn tightly around the waist with one of the shorter ends carried under the groin and tucked at the back, and the saree is adorned with a petticoat worn underneath by women, which is secured tightly to the waist by a cord. The tight knot around the waist at a similar position causes long-term friction and along with perspiration leads to pigmentation and scale-like changes in the skin which eventually undergo malignant transformation. The knowledge and existence of such malignancy is sparse in Indian medical fraternity  where itA 65-year-old male patient presented with a progressively increasing painless ulcer on the right side of the waist with a 6-month duration. Patient gave history of tightly tying a cotton dhoti over the waistline for the last 40 years. On local examination, there was a hypopigmented skin patch with scaling and ulceroproliferative growth of 7 × 5 cm with everted edges Fig. . There wHistopathology reports confirmed the presence of squamous cell carcinoma with tumor-free margin Fig. . PostopeThe term dhoti cancer was coined in 1945 by Khanolkar and Suryabai  which isThe exact causation of dhoti cancer is ill understood, and there are many hypothesis put forward to explain its probable mode of causation. Constant friction at the site of tying of dhoti or saree causes friction of the waistline which is associated with dermatoses . Other hAnother hypothesis put forward is depressed immunological state produced by the surrounding scar tissue  and absedhoti users for any malignant transformation.The age of presentation in the present case that was 65 years with more than 40 years of wearing dhoti is quite consistent with its occurrence in the age group after 50 years as reported in literature. This suggests a prolonged exposure of more than 40 years before malignant transformation . The feadhoti cancer will become further rare, but clinicians in India need to remember this condition as dhoti will continue to be a dress of traditional Indian males. The presence of acanthotic lesion in the waistline should be viewed with suspicion, and a constant monitoring to the patient is recommended.A rare case of squamous cell carcinoma due to chronic friction by wearing dhoti is presented with a treatment plan. It is expected that with change of lifestyle and lesser use of dhoti in new-generation Indians, the occurrence of The patient has given a written consent. Written and informed consent was obtained from the patient for the publication of case report and accompanying images. A copy of written consent obtained in vernacular language (Marathi) is available with us and is being held back to protect patient’s identity. It could be submitted with name blocked if the journal requires it.