Few diagnostic and prognostic biomarkers are available for head-and-neck squamous cell carcinoma (HNSCC). Long non-coding RNAs (lncRNAs) have shown promise as biomarkers in other cancer types and in some cases functionally contribute to tumor development and progression. Here, we searched for lncRNAs useful as biomarkers in HNSCC.Public datasets were mined for lncRNA candidates. Two independent HNSCC tissue sets and a bladder cancer tissue set were analyzed by RT-qPCR. Effects of lncRNA overexpression or downregulation on cell proliferation, clonogenicity, migration and chemosensitivity were studied in HNSCC cell lines.Data mining revealed prominently CASC9, a lncRNA significantly overexpressed in HNSCC tumor tissues according to the TCGA RNAseq data. Overexpression was confirmed by RT-qPCR analyses of patient tissues from two independent cohorts. CASC9 expression discriminated tumors from normal tissues with even higher specificity than HOTAIR, a lncRNA previously suggested as an HNSCC biomarker. Specificity of HNSCC detection by CASC9 was further improved by combination with HOTAIR. Analysis of TCGA pan-cancer data revealed significant overexpression of CASC9 across different other entities including bladder, liver, lung and stomach cancers and especially in squamous cell carcinoma (SCC) of the lung. By RT-qPCR analysis we furthermore detected stronger CASC9 overexpression in pure SCC of the urinary bladder and mixed urothelial carcinoma with squamous differentiation than in pure urothelial carcinomas. Thus, CASC9 might represent a general diagnostic biomarker and particularly for SCCs. Unexpectedly, up- or downregulation of CASC9 expression in HNSCC cell lines with low or high CASC9 expression, respectively, did not result in significant changes of cell viability, clonogenicity, migration or chemosensitivity.CASC9 is a promising biomarker for HNSCC detection. While regularly overexpressed, however, this lncRNA does not seem to act as a major driver of development or progression in this tumor.The online version of this article (10.1186/s12885-019-6021-6) contains supplementary material, which is available to authorized users. Long non-coding RNAs (lncRNAs) have moved into the focus of cancer research as good candidates for tumor biomarkers and as regulators of various neoplastic cell properties. In general, lncRNAs are defined as being longer than 200 nucleotides and lacking a functional open reading frame. Apart from this general definition, they are highly diverse in structure and function. Many lncRNAs resemble mRNAs in being spliced, poly-adenylated and located in the cytoplasm. Some lncRNAs  are transcribed from separate loci, whereas others are transcribed divergently from promoters of protein-coding genes or in antisense direction to these. A number of lncRNAs have been shown to regulate cellular processes including proliferation, apoptosis and differentiation in diverse physiological and pathological contexts . ImportaHNSCC is a common malignancy caused mostly by exposure to carcinogens from cigarette smoking and alcohol consumption, or alternatively, by tumorigenic strains of human papillomavirus (HPV). Radiation therapy, surgery, chemotherapy, therapy with EGFR antibodies, immune checkpoint inhibitors or combined treatments are used for primary tumors and recurrent or metastatic disease. Patients with localized HNSCC and low tumor stage have a high chance of cure. Recurrent disease appears in up to 50% of the cases. High stage, metastatic and recurrent HNSCC have limited treatment options and therefore an unfavorable outcome . To dateA large number of studies have investigated the expression of various microRNAs in HNSCC as potential biomarkers . In contWe validated CASC9 overexpression in two independent HNSCC tissue sets by RT-qPCR and further investigated CASC9 expression in various other cancers. Finally, we performed in vitro experiments to explore the effect of CASC9 expression on cell proliferation, clonogenicity, migration or chemosensitivity. We found that CASC9 is upregulated in many HNSCC cases, particularly in late stages and tumors with extracapsular spread. A pan-cancer analysis revealed that CASC9 is also strongly overexpressed in different other entities including bladder, liver, lung and stomach cancers and especially in squamous cell carcinoma (SCC) of the lung. Analysis of a further tissue set comprising bladder cancers with different histologies by RT-qPCR demonstrated CASC9 overexpression predominantly in urothelial carcinomas with squamous differentiation or pure squamous bladder cancers. Collectively, these findings indicate CASC9 as a valuable diagnostic marker particularly for HNSCC and other squamous cell carcinomas. Discrimination between HNSCC tumor and non-cancerous tissues may be further improved by combination with HOTAIR detection, whose upregulation in HNSCC was confirmed in our study. However, since experimental modulation of CASC9 expression in HNSCC cell lines did not appear to exert a major influence on tumor cell properties in vitro, CASC9 may not be crucially involved in the establishment of the neoplastic phenotype in all HNSCC tumors, but may reflect the transformed state.INK4A. Information about p16INK4A state was missing for six patients. Median follow-up time for this cohort was 43.6 months. The Bonn HNSCC cohort (BN) consisted of 79 patients. Expression data was obtained from 66 tumor and 56 normal adjacent tissues. Median age was 62 years. Eleven tumors were categorized as pT1, 33 as pT2, 24 as pT3, 10 as pT4; pT category of one tumor was unknown. Median follow-up time for the complete cohort was 48.0 months. HPV status of the BN cohort was determined using the HPV 3.5 LCD-Array Kit .The Duesseldorf set of HNSCC tissue samples (DUS) used for quantitative real time RT-PCR analysis (RT-qPCR) comprised 32 tumor and 12 normal adjacent tissues, median patient age was 64.5 years. Six tumors were staged according to TNM version 7 as pT1, 13 as pT2, 6 as pT3, and 7 as pT4, six tumors were HPV positive determined by immunohistochemistry for p16Both tissue sets were collected according to the principles expressed in the Declaration of Helsinki and with written patient informed consent as approved by the ethics committees of the medical faculties of the Heinrich Heine University Duesseldorf (study number 4698) and Friedrich Wilhelms University Bonn (Nr. 187/16), Germany.A bladder cancer tissue set comprising 11 muscle-invasive pure urothelial carcinomas without any histological signs of squamous differentiation (UC), nine mixed tumors consisting of muscle-invasive urothelial carcinomas displaying histological areas with squamous differentiation patterns (MIX), 10 pure squamous carcinomas of the bladder (SCC) and 5 normal adjacent tissues were kindly provided by the RWTH centralized Biomaterial Bank Aachen  in accordance with the regulations of the biomaterial bank and the approval of the ethics committee of the medical faculty, RWTH Aachen .http://cancergenome.nih.gov/) accessed via the TANRIC database (http://ibl.mdanderson.org/tanric/_design/basic/index.html) . (DOC 255 kb)Additional file 2:Figure S1. Expression of CASC9 in cell lines of other tumor entities. (a) Relative expression of CASC9 was determined by RT-qPCR across 16 urothelial carcinoma cell lines compared to the benign urothelial control cell line HBLAK and a primary urothelial cell culture (UEC). (b) Relative expression of CASC9 in prostate cancer cell lines, benign control cells  as well as normal (N) and cancerous (T) tissue. (c) Relative expression of CASC9 in testicular cancer cell lines compared to normal (N) and cancerous (T) tissue. Figure S2. Expression of a putative downstream target gene PDCD4 in HNSCC and UCC cell lines. (a) Relative expression of PDCD4 mRNA was determined by RT-qPCR across 21 HNSCC cell lines compared to benign HaCat cells. (b) Relative PDCD4 expression across 16 urothelial carcinoma cell lines compared to the benign urothelial control cell line HBLAK and a primary urothelial cell culture (UEC). (c) Relative expression of PDCD4 in cells with CASC9 overexpression or downregulation (sh). No significant changes were observed. Figure S3. Expression of a putative downstream target genes CDK4, CCND1, CDH1 and BCL2 in HNSCC cell lines. (a) Relative expression of CDK4 mRNA was determined by RT-qPCR in cell lines following CASC9 modulation and across 21 HNSCC cell lines compared to benign HaCat cells. (b) Relative expression of CCND1 mRNA was determined by RT-qPCR in cell lines following CASC9 modulation and across 21 HNSCC cell lines compared to benign HaCat cells. (c) Relative expression of E-Cadherin mRNA was determined by RT-qPCR in cell lines following CASC9 modulation and across 21 HNSCC cell lines compared to benign HaCat cells. (d) Relative expression of BCL2 mRNA was determined by RT-qPCR in cell lines following CASC9 modulation and across 21 HNSCC cell lines compared to benign HaCat cells. (ZIP 1403 kb)


Sequential courses of anticancer target therapy lead to selection of drug-resistant cells, which results in continuous decrease of clinical response. Here we present a new approach for predicting effective combinations of target drugs, which act in a synergistic manner. Synergistic combinations of drugs may prevent or postpone acquired resistance, thus increasing treatment efficiency. We cultured human ovarian carcinoma SKOV-3 and neuroblastoma NGP-127 cancer cell lines in the presence of Tyrosine Kinase Inhibitors  and Rapalogues (Temsirolimus and Everolimus) for four months and obtained cell lines demonstrating increased drug resistance. We investigated gene expression profiles of intact and resistant cells by microarrays and analyzed alterations in 378 cancer-related signaling pathways using the bioinformatical platform Oncobox. This revealed numerous pathways linked with development of drug resistant phenotypes. Our approach is based on targeting proteins involved in as many as possible signaling pathways upregulated in resistant cells. We tested 13 combinations of drugs and/or selective inhibitors predicted by Oncobox and 10 random combinations. Synergy scores for Oncobox predictions were significantly higher than for randomly selected drug combinations. Thus, the proposed approach significantly outperforms random selection of drugs and can be adopted to enhance discovery of new synergistic combinations of anticancer target drugs. Emergence of target drugs significantly increased success rates for the therapy of many cancer types. For example, in renal cell carcinoma, which is not sensitive to radiation or generalized chemotherapy, the introduction into the clinical practice of a new class of target drugs—tyrosine kinase inhibitors (TKIs) increased the response rate of the therapy to the impressive value of up to 70% of patients . AnotherMultiple target drugs are currently under investigation in thousands of clinical trials. For example, novel Janus Kinase-2 inhibitor, Fedratinib, showed promising results in patients with myelofibrosis . Despite50) of the corresponding drugs according to MTT test . Thus, after four months of culturing, most of the cell populations developed drug-resistant phenotypes. Every four weeks, using microarrays  we profiled gene expression in aliquots of the cells cultured  compared to the NGP-127 cells A,B. In STo get a deeper insight into the mechanisms of acquiring drug resistance in these cell types, we focused on the signaling pathways that were altered after exposure to target drugs. For each investigated timepoint, we selected ten percent of the most strongly affected pathways and then intersected lists of gene products, which formed these pathways. We hypothesized that targeting proteins corresponding to the most frequently occurring intersected gene products may be beneficial to suppress acquisition of drug resistance. The targets predicted at 4-week time point were used to select combinations in naïve cells, and those predicted at a 16-week time point were used to select combinations in adapted cells. The predicted molecular targets varied significantly across drugs, cell lines, and timepoints investigated .50) on both cell lines  SKOV-3 and NGP-127 cells to combinations of the drugs/inhibitors. Each such combination contained one initial drug  and another drug or inhibitor, which was predicted to complement activity of the former.20, while concentrations of the initial drugs were variable in order to measure their IC50 parameters in the presence of an additional predicted drug/inhibitor. As before, the MTT test was used to investigate cell viability under all the conditions. Cytotoxicity curves on the viability graphs reflect the effects of titration of the initial target drug with the addition of a constant inhibitor concentration  for 800 mg bid. Median time to achieve peak concentration is 2 to 4 h after the dose (data from FDA website). Sapitinib is undergoing clinical trials for several cancer types. In this study we used an 11 µM Sapitinib concentration . At the same time, previous clinical trials have indicated that Cmax for this drug was 1.53 µg/mL for 300 mg bid and that peak concentration could be observed 1 to 2 h after administering the dose [max of at least 5 µg/mL, was not previously tested in clinical trials. However, lower concentrations of Sapitinib could possibly act in a synergistic manner with Pazopanib. The latter could be tested in further preclinical trials, which are needed to determine clinical feasibility of this combination.The Bliss synergy score for combination of U73122 and Sorafenib was the highest across this study. Unfortunately, U73122 was not yet tested in clinical trials, thus we are unable to estimate clinical feasibility of such combination. Another synergistic combination revealed was Pazopanib and Sapitinib. Pazopanib is already FDA approved for treatment of renal cancer, and concentration used in this study (0.8–50 µM) is lower than Cthe dose . SapitinBliss synergy scores for drug combinations predicted by Oncobox were significantly higher than for the randomly selected drug combinations. Thus, the proposed method significantly outperforms random selection. However, standard deviation of synergy scores was high for some combinations. Thus, additional studies using other cell lines and/or initial drugs might be required.Apparently, most of the predicted combinations failed to show synergism. This may be due to the fact that the differential genes previously identified from drug-resistant cell models may largely represent drug-induced transcriptional changes, rather than active resistant mechanisms. Nevertheless, this approach could potentially be used to find synergistic combinations in cell line experiments. These combinations should be further tested in clinical trials. In addition, such an approach can be used to identify personalized combinations of target drugs in cases, when both pre- and post-treatment material is available, e.g., after neoadjuvant therapy. However, drug combinations acting synergistically in cell lines may cause severe side effects when administered to patient. Thus, further trials are needed in order to investigate such critical issues as required dosage of drugs supplemented in combination and their possible side effects.L-glutamine  at 37 °C and 5% CO2. The cells were grown in 25 cm2 or 75 cm2 flask  and passaged for every 72 h. In order to obtain drug-resistant cell lines the cells were exposed to Sorafenib, Sunitinib, Pazopanib, Temsirolimus, or Everolimus for 16 weeks to obtain drug-resistant cell lines. The cells were washed three times a week and each time new media was added. The media was also supplemented with drug in initial concentration. Thus, concentration of the drug in the media was constant during 16 weeks of culturing. Drug combinations were further tested in MTT-test (described below). Together with culturing of cells with target drugs, we cultured intact SKOV-3 and NGP-127 cells in drug-free media for 4 months. We profiled gene expression in aliquots of control cells every four weeks and thus obtained five gene expression profiles for each cell line . We pooled the control samples for pathway activation quantification: each experimental gene expression profile was normalized by geometric mean of corresponding control samples.We used two human cell lines to profile responses to anticancer drugs. Cell lines were cultured as described previously . Brieflyg for 10 min, followed by the removal of supernatant. Thirty microliters of 0.5 mg/mL solution of MTT  was added to each well, and the plates were incubated for 2 to 4 h, then 100 µL of DMSO was added to each well for formazan crystals dissolving. The optical densities (OD) at 540 nm were measured using a plate reader Multiscan FC . Cell viability was calculated using the formula: (OD treated cells − OD blank)/(OD control cells − OD blank) × 100%, where OD blank means OD in control wells containing no cells. IC50 values were deduced from dose–response curves using SigmaPlot software . IC50 values are given in For cell viability assay, we used intact cell lines and cell populations resistant to anticancer drugs. Cell viability was determined as described previously . Brieflyt-test with default parameters.The effect was considered synergistic if the corresponding Bliss score was more than 5, antagonistic—if less than −5, and additive if otherwise. The difference between Bliss scores for Oncobox predicted combinations and Bliss scores for random combinations was estimated using R p is as follows.Gene expression profiles were deposited to GEO database with the accession numbers GSE97750 and GSE97751. Gene expression was quantile normalized using R preprocessCore package . ExtentsnCNR, is the ratio of expression levels for a gene n in the sample under investigation to the same average value for the control group of samples. ARR (activator/repressor role) equals to the following fixed values: −1, when the gene product n is a repressor of pathway excitation; 1, if the gene product n is an activator of pathway excitation; 0, when the gene product n can be both an activator and a repressor of the pathway; 0.5 and −0.5, respectively, if the gene product n is rather an activator or repressor of the signaling pathway p, respectively.Here the case-to-normal ratio, PAS can take both positive and negative values meaning over- or underactivation relative to control (untreated) samples. In total we calculated activation level of 378 intracellular signaling pathways. The signaling pathways knowledge base developed by SABiosciences  was usedHere we report a method for predicting effective combinations of target drugs, which outperforms random guessing in selecting synergistic or at least additive combinations. The method is based on gene expression profiling of drug-resistant cell lines and subsequent pathway analysis with Oncobox software. Our algorithm provides a list of potential targets, which are involved in most pathways activated during acquiring of drug resistance. Synergy scores for Oncobox predicted combinations were significantly higher than for random combinations.


Relapse after chemotherapy treatment depends on the cancer initiating cells (CICs). PEDF (Pigmented Epithelium Derived Factor) is an anti-angiogenic, neurotrophic and self-renewal regulator molecule, also involved in CICs biology. Acute and chronic exposition of colon cancer cell lines to CT/CTE PEDF-derived peptides decreased drug-resistance to conventional colorectal cancer treatments, such as oxaliplatin or irinotecan. We confirmed a reduction in the irinotecan and oxaliplatin IC50 doses for all tested tumour cell lines. After xenograft transplantation, CT/CTE treatments also produced a reduction in resistance to conventional chemotherapy treatments as in culture-assays. Metastatic capacity of these treated cell lines was also depleted. The PEDF signaling pathway could be a future therapeutic tool for use as an adjuvant therapy that decreases IC50 dosis, adverse effects and treatment costs. This pathway could also be involved in an increase of the time relapse in patients, decreased tumourigenicity, and decreased capacity to produce metastasis. There are different hypotheses about the origin of tumours, none of which has yet been able to explain all cases. One such hypothesis is based on the high susceptibility of stem cells to accumulate mutations, together with the existence of epigenetic changes that are essential for the initiation and development of tumourgenic behaviour. For this reason tumour stem cells display some same general characteristics as healthy stem cells (SCs) such as quiescence, self-renewal, asymmetric cell division, cell regulation via Wnt, Notch, Sonic Hedgehog signaling (Shh), and resistance to toxic substances and drugs. All of these characteristics were postulated more than fifteen years ago –4. ThereSelf-renewal can be defined as the capacity of the cell to, at a minimum, originate a daughter cell with identical characteristics to the previous one. Consequently, stem cells can undergo multiple cell divisions maintaining their cell de-differentiation. Self-renewal is also produced by asymmetrical cell divisions of stem cells. This ability is essential for maintaining stem cells throughout the organism′s lifetime. Every stem cell must maintain an equilibrium between self- renewal and cell differentiation .PEDF (Pigment Epithelium-Derived Factor) protein has been related to the modulation of self-renewal mechanisms in stem cells , 7. PEDFPEDF exhibits various collaborative effects against tumour progression. PEDF may induce cellular differentiation and induce apoptosis in tumour cells –13. Alsoin vivo and in vitro effects of the C-terminal part of PEDF on tumoural cell lines’ growth and tumourogenicity. We have measured the IC50 of different chemotherapeutic treatments in combination with two fragments of the PEDF protein: the CT and CTE peptides . Also, we have measured the resistant population at the end of the treatment, which is an important date for comparing the effectivity of those treatments. We have used three different colorectal cancer cell lines, with different genetic expression hallmarks . A fourth cell line, HT29 was also used for comparison in some of the experiments due to the high oncogenicity of those cells. PEDF derived peptides have been used in combined treatment with conventional chemotherapy, oxaliplatin and irinotecan, both in first and second line chemotherapy for colorectal cancer patients. These two PEDF derived peptides are designed from the carboxi-terminal part of the PEDF protein. CTE is the same molecule as CT, from the C-terminal part of PEDF protein, but with a glutamic acid instead of the phosphorylable serine of this small molecule. The treatments employed in this paper were acute treatments, lasting two hours, and chronic treatments, lasting 6 weeks, with the cell culture medium. In both cases the optimum concentration was 8 nM, optimized in a previous work-group in murine neural models [Here we present the We have selected three different colorectal cancer cell lines in order to study the generic effect in multiple source colorectal tumours, and the chemotherapy used will be oxaliplatin and irinotecan, which are the most common first line chemotherapy agents used for colorectal cancer patients.The resistance to chemotherapy decreased in the cell lines  treated with PEDF derived peptides (CT and CTE). All the cell lines showed statistically significant reduction of IC50, oscillating between 20 and 70% depending on every cell line in both acute and chronic treatments.SW-480 and SW-620 cell lines showed a significant reduction, between 30 to 50% in the variables studied: IC50 and resistant population. All these parameters and the survival curves with PEDF-derived peptides are always under control survival curves Figure .In the SW-480 cell line there is a sharp 50% decrease of oxaliplatin and irinotecan IC50 value when they are combined with CT and CTE chronic or acute treatments Table . A reducp < 0.5 and n ≥ 3 for every condition)  Table . This den) Table  (Supplemn) Table . Resistap < 0.5 and n ≥ 3 for every condition)  Table . Resistan) Table .HT-29 also showed a decrease in the IC50 of the chemotherapy combined with PEDF-derived peptides. A significant response was observed in acute CT/CTE combined with oxaliplatin treatment, obtaining a decrease of 40% in the oxaliplatin IC50. Reduction of IC0 was also visible  and the In vivo xenograft assays were started with the injection of 500 and 5,000 tumoral cells in nude mice to follow the tumour growth in the mice flank. Those cells were previously exposed to chronic treatment with CT and CTE peptides during six passages/2 weeks. Controls are xenografts with the same number of untreated injected cells. The assays were performed in parallel with the three colorectal cell lines. In this type of assays we compared the growth of the tumour in the flank between control untreated cells and CT or CTE previously treated cells.In vivo assays showed a significant decrease in the tumourigenicity of tumour cells after treatment with PEDF derived peptides in DLD-1, SW-480 and SW-620 cells lines. Consequently, the observed behaviour varied depending on the origin and the molecular characteristics of every cell line. CT treatment showed less effectivity than CTE-PEDF in vivo. CTE peptide is more stable due to the negative charge in its glutamic acid (instead of the serine found in the CT peptide).Notably, SW-480 cell line assays confirmed a 25% reduction in the number of tumours after CTE treatment, and similar results with CT treatment Figure  right.SW-620 and DLD-1 cell lines exposed to PEDF-derived peptides in chronic treatment, resulted in a reduction of the developed tumour number in the same way as SW-480. We observed 50% fewer tumours as compared to the untreated cells.When taken together, the colorectal cell lines results from injections within 35 mice showed that the number of tumours was significantly lower after treatment than of the control tumours. Comparing the data of all tumour lines used, 90% of the tumours of untreated cells are observed at four weeks and only 50% of the treated xenografts developed tumours.Concerning the volume of the tumours, untreated SW-480 cells reached four times larger tumour sizes after four weeks, than those treated did in eight weeks. After 6 weeks, treated tumours were 94% smaller in size than untreated control ones. In addition, the beginning of the tumours was drastically delayed in treated cells when compared to the control’s: In the untreated control group, 90% of tumours appeared in the fourth week, while in treated groups this percentage was not reached until the eighth week Figure . Similarp < 0.05 and n ≥ 3 every tumour groups  that corresponds temporally with chemotherapy doses application, even oxaliplatin or irinotecan. This way, we investigated the mechanisms involved in the interaction between the chemotherapeutic compounds and the PEDF derived peptides. The second one, a chronic 6 week treatment, with 8 nM CT and CTE peptides, was carried out to study the effect of TICs self-renewal inhibition in metastatic transformation.The peptide concentration (8 nM) was selected based on the team’s previous experience with the effect of this peptide on stem cell populations , 7. An aRegarding the acute treatment approach, we observed a decrease from 50% to 75% in the resistance to chemotherapy. This decrease was observed in the four studied cell lines and with both chemotherapeutic agents, irinotecan and oxaliplatin. Even though acute treatments demonstrated lower efficacy than the chronic ones, the improvement of the acute treatment effects remains an essential task concerning possible future applications. In this sense, we have obtained important IC50 reductions in some of the treatments and cell lines. This promising fact would present potential advantages such as easier application, a less tedious application procedure for patients, and would be economically advantageous for the health system , 24. In Additionally, we observed a great improvement in the chronic treatments: tumour cell lines that were previously treated for 6 weeks with a constant 8 nM amount of CT and CTE peptides. The treatment application time was coordinated with the day of the cells passing process to obtain a more homogeneous response. During this assay, the main objective was to identify the role that these PEDF-derived peptides might be playing as regards cell division or signaling pathway activation related to stress induced by toxic substances , 20, 36.Here, we report the decreased resistance to chemotherapeutic agents, oxaliplatin and irinotecan, in four colorectal cancer cell lines when such chemotherapy is combined with a dose of PEDF-derived peptides (CT or CTE). Interestingly, this resistance varied depending on the chemotherapeutic agent used. Thus, we observed a major synergy between irinotecan and PEDF-derived peptides. This fact is possibly due to differences in the action mechanism of both drugs. As chemotherapeutics, oxaliplatin and irinotecan are cytotoxic compounds that affect cell division. In this sense, both irinotecan and oxaliplatin target different cellular mechanisms . TherefoConsidering the effect between PEDF peptides and the chemotherapeutic molecules used in this study, it is not possible to speak about synergism. We have tried to calculate the Combination Index to show if it was CI = 1 (additive effect) CI > 1 (antagonism) or CI < 1 (synergy). We have also performed a bibliographical study and concluded that the best methodology for this is based on the Median-Effect Equation (Chou) and the Combination Index Theorem  ,  and the In vivo tumourigenicity of colorectal cell lines decreased with the application of a dose of PEDF-derived peptides. According to the previously presented results, PEDF-derived peptides, CT and CTE, showed to be notably effective against one of the most aggressive colorectal tumour cell lines, SW480, and also against their metastatic SW620 cell line, both derived from the same patient. In this sense, we observed the effect on the chemotherapy combined treatment, showing a decrease in the resistance of cells derived from xenografts previously treated with CT/CTE . The most significant effect was observed after re-injection of treated xenograft cells. 70% of xenograft cells treated with CTE were not able to produce a new tumour (metastasis-like model). Regarding CT treatment, the effects were not so drastic, and we observed a 40% reduction in tumour growth. This is the first time that a treatment is able to modify the behaviour of cancer; modified initiating cells hindered the tumour′s re-growth, and therefore that of the metastasis animal model.The difficulty of finding an experimental model of the metastasis inhibition complicates the study of the treatments’ effects. There are few models of e assays , 46. HerTo sum up, this data suggests that PEDF-derived peptides play a potentially therapeutic role in colorectal cancer. In light of the findings reported here, it would be necessary to confirm the low toxicity and high efficacy of these peptides with additional pre-clinical and clinical trials. Moreover, it would be essential that these trials be carried out with purified CT/CTE peptides instead of conditioned medium. Nonetheless, preliminary studies indicate a similar effect in all of the cell lines when conditioned medium is replaced with purified peptides (data not shown). Furthermore, it would be interesting to evaluate the effect of PEDF-derived peptides on other types of cancer and also perform combinatorial analyses for a larger ratio of doses. The group is currently focused on elucidating the molecular mechanisms that underlay these observed effects. Tumour initiating cells are long-retaining labeling cells with asymmetric divisions . We post2 at 37° C in a humidified incubator.The human colon cancer cells DLD-1, HT-29, SW-480 and SW-620 were obtained from Mario Fraga′s laboratory and maintained in DMEN F12  supplemented with 10% fetal bovine serum , 200 nM L-glutamine  and 1% penicillin/streptomycin (Biowhittaker) in an atmosphere of 5% COn1nu, at 1 month of age and of indistinct gender were obtained from Charles River International Laboratories. Animals were housed and bred at 20–25° C, humidity of 50–60% and a 12 hours light-dark cycle. All of the animals were treated in accordance with the approval of the local ethics committee (University of Castilla–La Mancha).FOXC-terminal (CT) fragment of PEDF protein was cloned in the mammalian expression vector pcDNA 3.1 (-)-myc-His as we describe previously . The recColon cancer cell lines were treated with PEDF derived peptides (CT or CTE) at a final concentration of 8 nM (200 ng/ml) in the medium. Cells were treated in two ways. On the one hand, acute treatments were performed, that is, a single treatment with the peptides. On the other hand, chronic treatments were carried out consisting of a total of 6 treatments with the peptides for a total of fourteen days. Peptide treatment was added with culture medium in the passage time. Timing for the treatments was: two hours with 8 nM peptide exposition before chemotherapy treatment for acute treatments and six weeks with 8 nM peptide added to the culture medium for chronic ones.5,000 cells were seeded per well in 24-well plates, in a volume of 250 uL. The next day the cells were treated with increasing doses of chemotherapeutic agents and left in a humidified incubator at 37° C and 5% CO2 for 4 days. Subsequently the cells were fixed with 0.5% glutaraldehyde (Sigma) for 10 minutes. Next, the cells were stained with 0.1% crystal violet for 20 minutes. After several washes, the staining was solubilized with 10% acetic acid. Finally, a spectrophotometric reading was performed at an onsa length of 590 nm with a plate spectrophotometer . The IC50 was determined (the dose of drug necessary to eliminate 50% of the cell population), while the IC0 (the dose of drug necessary to eliminate 100% of the population) was obtained through logarithmic regressions made with the program DE.0 plus v1.0.™ Basement Membrane Matrix, BD) with a 25 gauge-needle. The growth of the tumours was monitored weekly by means of a caliper. The final tumour volume was calculated using the formula V= 2 × L1 × L2 × π/6. Tumours were mechanically and chemically disintegrated with EDTA at 37° C. Later they were washed with PBS and seeded in DMEN F12 medium  supplemented with 200 nM L-glutamine , 1% penicillin/streptomycin (Biowhittaker), 0,5% EGF and 0,04% FGF in an atmosphere of 5% CO2 at 37° C in a humidified incubator.Untreated cells (control) and cells treated with peptides derived from PEDF suspended in PBS were injected subcutaneously on both flanks of immunocompromised mice. The injections were of 5000 or 500 cells, in a final volume of 200 ul of a 1: 1 dilution of matrigel (BD Matrigelhttp://www.combosyn.com/) from Combosyn Inc. company. Also we have analyzed the results with the Combenefit software, also indicated to analyze synergy between molecules (https://www.cruk.cam.ac.uk/research-groups/jodrell-group/combenefit).To analyze the interaction between molecules we have used the software Drug Combination Computer Simulation CompuSyn (U test or the Chi-square test when necessary. The data is expressed as the mean plus the standard error (+SE). The obtained results are considered statistically significant when p < 0.05(*), p < 0.01(**) and p < 0.005(***).The statistical analysis was carried out using a Mann–whitney


The title compounds were less toxic to PBMC, since IC50 was 1.5 to eighteen times higher (IC50 = 5.51 to 17.61 µg·mL−1) than values shown by tumour cell lines. The mechanism of cell growth inhibition and structure–activity relationships remains as a target for future investigations.The cytotoxicity of a series of aminonaphthoquinones resulting from the reaction of suitable aminoacids with 1,4-naphthoquinone was assayed against SF-295 (glioblastoma), MDAMB-435 (breast), HCT-8 (colon), HCT-116 (colon), HL-60 (leukemia), OVCAR-8 (ovarian), NCI-H358M  and PC3-M (prostate) cancer cells and also against PBMC . The results demonstrated that all the synthetic aminonaphthoquinones had relevant cytotoxic activity against all human cancer lines used in this experiment. Five of the compounds showed high cytotoxicity and selectivity against all cancer cell lines tested (IC Approximately 518,510 new cases of cancer were estimated for Brazil between 2012 and 2013, including cases of non-melanoma skin cancer, which is the most common type for both men and women , followed by prostate , female breast , colon and rectum , lung , stomach  and cervix   propanoate (9). Obtained as an orange solid in 64% yield from ethyl L-alanine ester after 2 h reaction, mp 65–68 °C  8.01 , 7.99 , 7.69 , 7.59 , 6.30 , 5.62 , 4.18 , 4.04 , 1.50 , 1.22 ; 13C-NMR  183.1, 181.3, 171.5, 146.5, 134.6, 133.1, 132.1, 130.3, 126.3, 126.0, 101.8, 61.9, 50.4, 17.4, 14.0. Anal. Calcd. for C15H15NO4: C, 65.92; H, 5.53; N, 5.13. Found: C, 65.32; H, 5.80; N, 4.89.ot found ); IR butanoate (10). Obtained as an orange solid in 64% yield from ethyl 4-aminobutanoate ester after 2 h reaction, mp 120–122 °C  8.05 , 8.01 , 7.69 , 7.59 , 6.08 , 5.73 , 4.12 , 3.24 , 2.41 , 1.99 , 1.23 ; 13C-NMR  182.8, 181.6, 172.8, 148.0, 134.7, 133.4, 132.0, 130.3, 126.3, 126.1, 100.7, 60.7, 42.0, 31.6, 17.4, 23.1, 14.1. Anal. Calcd. for C15H15NO4: C, 65.92; H, 5.53; N, 5.13. Found: C, 65.71; H, 6.24; N, 4.77.ot found ); IR butanoic acid (11). To a mixture of LiOH (30 mg), THF (2 mL) and water (0.5 mL) was added the ester 10a (100 mg). The mixture was stirred at room temperature and inspected by TLC until complete disappearance of the ester 10 (after 2 h reaction). The mixture was filtered under vacuum, acidified with 10% HCl and an amorphous orange solid filtered off in 80% yield, mp 210–212 ° C  12.17 , 7.95 , 7.93 , 7.80 , 7.67 , 5.7 , 3.20 , 2.30 , 1.78 ; 13C-NMR : 181.5, 181.3, 174.3, 148.6, 134.8, 133.2, 132.1, 130.4, 125.8, 125.3, 99.3, 41.2, 30.6, 22.6. Anal. Calcd. for C16H17NO4 × H2O: C, 62.94; H, 6.27; N, 4.59. Found: C, 61.14; H, 5.63; N, 4.73.0–211 °C ,26); IR in vitro. This analysis is part of an initial screening for determination of the potential antitumor of these samples. The human cancer cell strains used were SF-295 (glioblastoma-human), MDAMB-435 (melanoma), HCT-8 (colon), HCT-116 (colon), HL-60 (leukemia), OVCAR-8 (ovarian cancer), NCI-H358M  and PC3-M (highly metastatic prostate cancer cell line). All cancer cells were provided by the National Cancer Institute  and were cultured in RPMI 1640 supplemented with 10% fetal bovine serum and 1% antibiotics, maintained at 37 °C and atmosphere containing 5% CO2. The peripheral blood mononuclear cells (PBMC) were isolated by gradient of density (Ficoll-Hypaque) and cultivated in RPMI 1640 medium, supplemented with bovine foetal serum (20%), antibiotic (1%) and phytohemagglutinin (4%). The cells were maintained at 37 °C and atmosphere containing 5% CO2.This experiment evaluated the cytotoxicity of aminonaphthoquinones (ANPQs) against human cancer cells, −1. In order to determine the IC50 the substances were tested at increasing concentrations in serial dilutions (5–0.010 µg·mL−1). Doxorubicin (5–0.0010 µg·mL−1) was used as positive control only for the determination of IC50.The different samples of ANPQs were diluted in pure sterile DMSO. The substances were tested at a concentration of 5 µg·mLAnalysis of cytotoxicity by the MTT method has been used in the screening program at the National Cancer Institute of the United States (NCI), which tests over 10,000 samples per year . It is a6 cell mL−1 to OVCAR-8, NCI-H358M, PC3-M, SF-295 and MDA-MB435 strains, 0.7 × 105 cell mL−1 to HCT-8 and HCT-116 strains, 0.3 × 106 to HL-60 strain, 1.0 × 106 cell mL−1 to PBMC. The plates were incubated for 72 h at 5% CO2 at 37 °C. After, they were centrifuged and the supernatant removed. Then the solution of MTT  was added, and the plates were incubated for 3 h. The absorbance was read after dissolving the precipitate with 150 µL of pure DMSO in a spectrophotometer plate at 595 nm.The cells were plated at a concentration of 0.1 × 10The experiments were analyzed by mean ± standard deviation of the mean (SDM) of the percentage of cell growth inhibition using GraphPad Prism. 2, 9, 6, 5 and 7) showed high cytotoxic activity and selectivity against the eight human cancer cell lines tested. Furthermore, the aminonaphthoquinones in this study were less toxic to peripheral blood mononuclear cells, since the IC50s were 1.5 to eighteen times higher than values shown by tumor cell lines. The mechanism of cell growth inhibition and structure–activity relationship of the compounds remains the target of future investigations.Our findings have demonstrated the cytotoxic potential against human cancer lines of a series of synthetic aminoacids conjugated with a naphthoquinone nucleus. As shown by various tumor cell lines, only five samples (


Personalized cancer therapy relies on identifying patient subsets that benefit from a therapeutic intervention and suggest alternative regimens for those who don’t. A new data integrative approach, based on graphical models, was applied on our multi-modal –omics, and clinical data cohort of metastatic melanoma patients. We found that response to chemotherapy is directly linked to ten gene expression, four methylation variables and PARP1 SNP rs1805407. PARP1 is a DNA repair gene critical for chemotherapy response and for which FDA-approved inhibitors are clinically available (olaparib). We demonstrated that two PARP inhibitors (ABT-888 and olaparib) make SNP carrier cancer cells of various histologic subtypes more sensitive to alkylating agents, but they have no effect in wild-type cells. Furthermore, PARP1 inhibitors act synergistically with chemotherapy in SNP carrier cells , but they are additive at best in wild-type cancer cells. Taken together, our results suggest that the combination of chemotherapy and PARP1 inhibition may benefit the carriers of rs1805407 in the future and may be used in personalized therapy strategies to select patients that are more likely to respond to PARP inhibitors. Advances in cancer management have improved the overall outlook of patients with metastatic malignancies but chemotherapy remains a mainstay of treatment for most common cancers. Virtually all patients develop resistance to chemotherapy after prolonged exposure given the first order kinetics of cytotoxics that generally cannot eradicate cancer. Understanding the mechanisms of this resistance presents new opportunities to improve the therapeutic index of cytotoxic agents and identify novel drug targets.1. Given the critical role of PARP1 in base excision repair, PARP inhibition emerged as a therapeutic target and early studies demonstrated dramatic potentiation of chemotherapeutic agents in the presence of PARP inhibition3. Recent evidence indicates that, in addition to the catalytic inhibition of PARP activity, PARP inhibitors (PARPi) induce cytotoxic PARP-DNA complexes through PARP “trapping” that augment the cytotoxicity of alkylating agents. It is therefore of utmost importance to identify molecular features that act not only as biomarkers for patient stratification but also offer insights into the mechanisms of resistance to chemotherapy. Metastatic melanoma remains an excellent model for chemotherapy resistance given its refractory nature, despite the fact that current management of metastatic melanoma is mostly based on non-chemotherapy based strategies .A large proportion of cytotoxic agents exert their effect through DNA damage. Thus, DNA repair pathways constitute cells’ main resistance mechanisms and potential drug targets. Base excision repair, a predominant pathway for single strand break (SSB) damage repair, utilizes a family of related enzymes termed poly-(ADP-ribose) polymerases (PARP), which are activated by DNA damageCausalMGM6, in high-throughput data from a cohort of metastatic melanoma patients on chemotherapy. We identified a number of features directly linked to chemotherapy response, including a SNP in the PARP1 gene that is highly predictive of resistance to chemotherapy. Subsequently, in vitro studies investigated the impact of this PARP1 variant on PARPi sensitivity and demonstrated its utility as a predictive biomarker. Given the role of PARP1 in DNA repair, we propose this SNP as a characteristic biomarker for PARPi sensitivity to guide patient selection for chemotherapy treatment alone or in combination with PARPi.In this study, we used a probabilistic graphical method we have developed, Using a retrospective cohort study design Table , we eval7.Total RNA was isolated from melanoma tissues using the PerfectPure RNA Tissue kit . RNA was quantified using Ribogreen RNA quantitation Kit  and its quality was evaluated by RNA Integrity Number using the Agilent Bioanalyzer. Whole genome gene expression analysis was carried out using the Illumina HT-12 Expression BeadChip  which targets > 25,000 genes  derived from the RefSeq  and UniGene databasesDNA was isolated from melanoma tissues using ArchivePure DNA Cell/Tissue kit . DNA samples (0.5 μg) were treated with sodium bisulphite using the EZ DNA methylation Gold kit , and bisulphite-treated DNA was applied to an Illumina Infinium HumanMethylation27 BeadChip  for DNA methylation profiling. This microarray permits the quantitative measurement of DNA methylation for 27,578 CpG dinucleotides spanning 14,495 genes. Methylation status of the interrogated CpG sites was determined by comparing β-values, the ratio of the fluorescent signal from the methylated allele to the sum from the fluorescent signals of both methylated and unmethylated alleles.2 ≥ 0.8). We selected ~6 SNPs per gene (where possible) to complete the 384 SNP Illumina design  minor allele frequency (MAF) <10%; (2) screening using the Illumina Assay Design Tool  to ensure SNPs can be incorporated in an optimal custom array design. The remaining SNPs of each gene were in strong LD . Each reaction contained 12.5 µL TaqMan Universal PCR Master Mix, 0.625 µL TaqMan SNP Genotyping Assay, 10.875 µL distilled water and 1 µL gDNA (10 ng/µL), with a final reaction volume of 25 µL. Positive control samples for the three possible genotypes were included with each genotyping reaction. In addition, at least two no template (negative) controls were included.Genomic DNA was extracted from the melanoma tumor or the full panel of cancer cell lines using QIAamp DNA Mini kit  and subjected to SNP Genotyping using a Taqman SNP Genotyping assay  , colon , breast , ovarian  and lung . HT-29, HCT-116 and H-522 cell lines were generously provided by Dr. John Schmitz ; Dr. Jian Yu (LoVo and SW620); Dr. Vera Levina ; Nupur Gangopadhyay ; Dr. Lisa Butterfield ; Dr. John Kirkwood (FEMX); Dr. Anda Vlad (Magee Woman Cancer Institute) (A2780); Dr. Hassane Zarour (SK-Mel-2); WM-115, A375, WM-852 and SK-MEL-31 were purchased from American Type Culture Collection (ATCC). All cell lines were cultured according to their recommendations (in RPMI-1640 or DMEM medium supplemented with 10% FBS) without antibiotics and routinely tested for Mycoplasma. Cell lines were grown in monolayer at 37 °C in a humidified atmosphere containing 5% CO50 values were defined as the concentrations of drug(s) that inhibited growth by 50% relative to controls.Cytotoxicity was assessed using the MTT colorimetric dye reduction assay, according to the manufacturer’s instructions (TACS® MTT Cell Proliferation Assay (MTT-CPA) from Trevigen, Inc.). Briefly, cells were seeded in triplicates in 96 wells at appropriate cell density , in their respective regular culture medium. After 24h, medium was aspirated and cells were incubated with increasing concentrations of test agents for 72h at 37 °C. The MTT Reagent was subsequently added to each well and the plates were incubated for another 4h at 37 °C in the dark. In each well, 100 µl of Detergent Reagent was then added and incubated at 37 °C until complete solubilization of formazan crystals. Cell viability was measured by reading the absorbance at 570 nm using a Safire 2 microplate reader . All values were normalized to vehicle treated control wells. The values obtained for each of the triplicates were averaged, and ICRPMI 1640 and Dulbecco’s modified Eagle medium (DMEM) with L-Glutamine, cell culture media were purchased from Cellgro . Heat-inactivated Foundation Fetal bovine serum (FBS) was purchased from Gemini bio-products. The PARPi veliparib  used in the cell proliferation assays was generously provided by Dr. Jan Beumer . The ABT-888 used in the drug interaction assays was purchased from Selleck Chemicals LLC . ABT-888 and olaparib were dissolved in dimethyl sulfoxide (DMSO) to a stock solution of 100 mM. Working solutions were then prepared in PBS for subsequent serial dilutions. The final concentration of DMSO in drug-treated cultures was always less than 0.5% (v/v) and did not contribute to cytotoxicity (data not shown). MMS, an alkylating agent that results in DNA damage at the same bases as dacarbazine and temozolomide and that is used extensively as an alternative in the laboratory setting, was purchased from Sigma.50) values of MMS alone or in combination with PARPi were calculated by curve fitting in Graph Pad Prism . IC50 values from three independent experiments were averaged and expressed as mean ± standard deviation (S.D.). A chemopotentiation factor was defined as the ratio between IC50s of MMS in the presence or absence of PARPi. Cells were classified as “resistant” if their potentiation factor (ratio) was less than 1, and “sensitive” if the ratio was >2. For each cell line, p-values were calculated using the Student t-test  to compare the IC50 means of each treatment group .The cytotoxic effects of the alkylating agent MMS, alone or in combination with PARP inhibitors veliparib (ABT-888) or olaparib were assessed by MTT assay (see above). Cells were treated with increasing concentrations of MMS either alone (0 to 1 mM) or in the presence of a fixed concentration of PARPi  for 72h at 37 °C. Data were normalized to vehicle alone (0.5% DMSO) for MMS treatments, or 10 nM ABT-888 or 5 nM olaparib, for combination treatments. Half-maximal inhibitory concentration  and fixed ratio combinations thereof based on equipotency of the two test agents. Five two-fold dilutions ranging from one fourth of the IC50 to four times the IC50 of each drug in combination plus a control were tested in three independent experiments with duplicate samples. MTT data were then analyzed using the Compusyn Software , which provides a convenient tool to perform median effect analysis as described by Chou and Talalay8. The program calculates a combination index (CI) for each actual data point from the combination curves that is used to identify synergistic, additive, and antagonistic drug interactions. The program also calculates predicted CI values for every effect level (affected fraction or Fa), which when plotted as a function of Fa serve to visualize and quantify the effects of drug combinations over the entire dose range. CI values of 1, <1, and >1 represent additivity, synergism, or antagonism, respectively. Predicted CI values of three independent experiments were averaged and analyzed by one-sample Student’s t-test for significant deviation from additivity .Interactions between PARPi (ABT-888 or olaparib) and MMS were investigated by the Chou and Talalay median effect principleMixed Graphical Models (MGM) we refer to graphical models that are learned over variables of mixed type, i.e., continuous and discrete variables. We used CausalMGM, an algorithm we recently developed6 and used for biomarker discovery9, to learn a directed graph over the variables in our dataset, which consisted of continuous  and discrete (single nucleotide variants (SNPs), response to TMZ treatment) variables. The resulting directed graph selected those variables that are directly connected to response to TMZ treatment. We used subsampling to identify stable edges as we describe previously4. This is standard procedure, since the number of independence tests required is exponential to the number of variables.With the term response” clinical variable, a binary variable indicating response/no response to TMZ treatment dichotomized at presence of a response or stability of disease at 4 months of therapy. The 1,000 most correlated features in the input dataset included 557 mRNA expression probes, 425 methylation probes, 14 miRNA probes and 4 SNPs. BRAF mutation status was also included in the input variables to see if it had an effect on any of the features linked to response, although its direct correlation with it in this dataset was poor (R2 = 0.025). The largest interconnected output network included 20 features directly connected to the response variable in our initial (undirected) learned network  correlated features with response, as other (indirect) association yield higher pairwise correlation values. ID2 expression is also directly linked to response. ID2 is known to induce growth and proliferation in squamous cell carcinoma13. No miRNA was found to be directly linked to TMZ response in our dataset.Our dataset consisted of gene and microRNA expression, DNA methylation, SNP data from a selected panel and the clinical variable “response to TMZ”. We used CausalMGM to learn a network over the top 1000 features most correlated (pairwise) with “ork Fig. . We emph it Fig. , but alsresponse. We found this SNP to be an excellent predictor of worse outcome since all 21 patients that had the SNP (C/T or C/C) showed no response to TMZ treatment . Because of the strong direct association, the role that PARP1 plays in repairing TMZ mediated DNA damage, and the availability of PARP inhibitors, we decided to further investigate this finding. rs1805407 is located on the 2nd intron of PARP1, ~4 Kbp downstream of the PARP1 transcription start site and 35 bp downstream of the 3’ splice site of exon 2. Since this SNP does not appear to have any functional or regulatory role, we used SNAP14 to find other SNPs in strong linkage disequilibrium (LD) with it. The CEU population panel of 1000 Genomes pilot 1 contained 51 variants in perfect LD (R2 = 1)  and is annotated as a ‘Splice region variant’ because it is located 5 bp upstream from the splice site between intron 2 and exon 3. Finally, we found a strong dependence between rs1805407 and two other SNPs that have been previously associated with melanoma susceptibility: rs3219090 22. Due to high LD values with rs1805407 none of the above SNPs was included in our dataset.Another SNP (rs1805405) is also in perfect LD with rs1805407  vs WT (T/T). The Affymetrix 500k SNP arrays used by Cell Miner did not include rs1805407, so we used the k-nearest neighbors method with three of the 51 perfectly correlated variants with probes in the array  to infer the rs1805407 genotype in each cell line. Analysis of the IC50 values in cell lines predicted to have at least one allele of rs1805407  vs wild type  showed statistically significant resistance or sensitivity to four compounds, three of which are alkylating agents with action similar to TMZ (TMZ is not included in the NCI-60 dataset)  were resistant .Literature search identified 15 cell lines (Supplementary Table ). MMS was used as alkylating agent and ABT-888 was used to inhibit PARP activity. All four SNP cell lines were found to be significantly more sensitive to the combination treatment in agreement with our hypothesis; while in all five WT cell lines the combination treatment had no potentiation effect : five WT (T/T) and four C/T for SNP rs1805407  µM and for MMS + olaparib was 39.22 (±8.80) µM . By comparison, the corresponding values for SW620 (WT) cells were not significant: 342.71 (±68.67) μM with MMS only and 357 (±55.57) μM with MMS + olaparib (5 nM).  (Supplementary Table We extended those results to olaparib treatment of two cell lines: A2780 (SNP) and SW620 (WT); and we observed similarly significant potentiation of MMS cytotoxicity with the addition of olaparib in A2780 Fig. . Specifi50 value (drug combination treatment). Cell survival was assessed by MTT assay. We then determined the Chou-Talalay combination index (CI)8 of ABT-888 with MMS in four established cell lines: two with the variant C/T  and two WT T/T . Although CI is not a statistical measure, it nevertheless provides insight into whether the effect of both agents is additive (CI = 1), synergistic (CI < 1), or antagonistic (CI > 1). Both SNP carrier cell lines  exhibited strong and moderate effect, respectively across different concentrations  and PARPi (ABT-888) on cell lines with different rs1805407 genotypes. Exponentially dividing cells were exposed for 72h to increasing concentrations of ABT-888 (0–500 µM) or MMS (0–1 mM) (single drug treatment) or combined at a fixed ratio based on their corresponding ICns Table . In WT cns Table .Table 2AApplication of different therapies in well-defined subgroups of patients is an important step towards cancer precision medicine. A probabilistic graphical analytic framework identified ten gene expression and four DNA methylation variables that were directly linked to TMZ response that provided useful information about potential mechanisms. We also discovered a novel biomarker (PARP1 SNP rs1805407) of potentially high impact in stratifying patients in chemotherapy.50 values, two of which were DNA damage agents. We found the WT cell lines to be more sensitive to these drugs. Follow-up in vitro experiments in cells from different cancer histologies showed that the sensitivity of SNP carriers to alkylating agent is increased after PARP inhibition. Furthermore, we showed that in SNP cell lines the combination treatment was synergistic, while in WT cell lines was at best additive or even antagonistic. We also note that this effect was independent of BRCA1 mutation since none of the tested cell lines carried this mutation. Since this SNP has high prevalence  it has the potential of transforming the current chemotherapeutic strategies. SNP rs1805407 can be potentially used in the future as companion diagnostic marker for selecting the patients that are more likely to benefit from chemotherapy. Adequately powered future validation studies will test the clinical application of this SNP as response biomarker.All patients carrying this mutation responded poorly to chemotherapy in our cohort. Since chemotherapy induces DNA damage and PARP1 is a key factor in DNA repair, the previous results led to the hypothesis that rs1805407 may be associated with improved DNA repair, which results in poor efficiency of the drug. Comparison of drug sensitivity values in WT and SNP carrier cell lines (NCI-60) showed four drugs with statistically significant differences in IC35. Extensive autoPARylation of PARP1 results in dissociation from DNA, which is required for DNA repair completion36. It has been proposed that PARP1 inhibition acts on the autoPARylation stage, thus preventing PARP1 dissociation from the DNA (“PARP1-trapping”)38. PARP-DNA complexes are themselves cytotoxic and may underlie the differential efficacy of clinically relevant PARPi’s, which differ markedly in their PARP trapping potency24. Based on our findings we postulated that SNP rs1805407 affects PARP1 activity during treatment with alkylating agents. We found that SNP cell lines were sensitive to combination therapy with ABT-888, while WT in general were not. We subsequently conducted experiments with the FDA approved PARPi olaparib in A2780 (SNP carrier) and SW6220 (WT) cells and the results were more profound than with ABT-888. These are very important results as SNP rs1805407 can be potentially used in the future to decide whether a patient will receive a combination therapy or not.The mechanism of action of this SNP is currently unknown and is beyond the scope of this paper. However, one can postulate a possible scenario, based on publicly available data and what is known in the literature about PARP1 action and inhibition. PARP1 acts as a “molecular sensor” to identify DNA single strand breaks. Upon binding, PARP-1 undergoes autoPARylation and promotes PARylation of chromatin proteins, which recruit DNA repair factorsIn summary, we showed that (1) SNP carrying metastatic melanoma patients have poor response to chemotherapy; and (2) SNP carrying cells have increased sensitivity to combination treatment . Both these results are consistent with the theory that the identified SNP rs1805407 is associated with higher PARP1 expression. In this case, cancer cells in SNP carrying patients will repair the DNA damage, caused by chemotherapy, more efficiently. At the same time, inhibition of PARP-1 in SNP carrying cells can lead to higher number of PARP-DNA complexes, which will increase cytotoxicity. Supportive of the hypothesis of increased PARP1 expression in SNP carrying cells is the fact that rs1805407 is in perfect linkage disequilibrium with two PARP1 promoter SNPs (rs2077197 and rs6665208), which overlap ChIP peaks of cancer transcription factors. Based on GTEx data, rs1805407 is indeed an eQTL for PARP1.41. However, its overexpression can lead to inhibition of DNA repair42. Also, the short isoform uniquely maintains the auto-modification domain, which is most critical to PARP trapping. Experiments on measuring PARP1 trapping cannot be conducted in the context of this study, because cell lines have drastically different baseline response to alkylating agents  and -in addition- PARP1 is induced by alkylating agents (presumably at different levels in different cell lines). A future detailed analysis in cell lines or mouse models will shed more light in the mechanisms in which SNP rs1805407 may affect chemotherapy response.In addition, we note that SNP rs1805405, which is in perfect linkage disequilibrium with our rs1805407, causes a mis-splicing event leading to increased expression of the shorter PARP1 form, PARP1-003. PARP1-003 includes only exons 1 and 2, thus coding only for the first zinc finger (ZF1) of PARP1. In PARP1 protein, ZF1 is responsible for recognition of DNA lesions and for initiation of DNA repairin vitro and in a clinical setting.We also found that patients with one or two alleles of rs1805407 in TCGA have significantly increased relative abundance of isoform PARP1-003 vs PARP1-001 (the full length mRNA), but the difference was small in absolute numbers. However, TCGA data are generated from pre-treatment samples, generally from primary tumors, and in melanoma predominantly from metastatic sites . PARP1 expression is increased by DNA damage (treatment). The presence of SNP rs1805405 may affect the splicing rates and relative abundances of PARP1 isoforms, thus altering their relative protein levels. This is relevant in patients, especially knowing that TMZ is utilized in regimens that span either 5 days or 21 days out of 28-day cycles. Since the focus of this paper is in the discovery of an important biomarker for chemotherapy, the exact mechanism of action was not fully elucidated. The above stated hypotheses can guide future experiments The accurate prediction of therapy outcomes based on the molecular characteristics of tumors may alter the current landscape of cancer therapy given that immunotherapy results in substantial objective responses in some patients only. One can therefore expect that the accurate patient stratification into therapy-response categories may allow the overall percentage of disease control to increase through the current therapeutic armamentarium. In addition, insights into the molecular mechanism that characterizes a therapy-resistant phenotype may usher in new strategies to overcome therapy resistance.Supplementary Information


SIRT2 expression increases in acute myeloid leukemia blasts. Downregulation of SIRT2 using siRNA causes apoptosis of HeLa cells. Therefore, selective inhibitors of SIRT2 are candidate therapeutic agents for cancer. Adult T-cell leukemia/lymphoma (ATL) is a T-cell malignancy that has a poor prognosis and develops after long-term infection with human T-cell leukemia virus (HTLV)-1. Sirtuin 1 inhibition has been shown to induce apoptosis and autophagy in HTLV-1-infected cell lines, whereas the effects of SIRT2 inhibition alone have not been elucidated.Sirtuin 2 (SIRT2) is a member of the sirtuin family, nicotinamide adenine dinucleotideWe assessed the efficacy of our small molecule selective SIRT2 inhibitors NCO-90/141 to induce leukemic cell death. Cell viability was examined using the cell proliferation reagent Cell Count Reagent SF. Apoptotic cells were detected by annexin V-FITC and terminal deoxynucleotidyl transferase dUTP nick end labeling assays by flow cytometry. Caspase activity was detected using an APOPCYTO Intracellular Caspase Activity Detection Kit. The presence of autophagic vacuoles was assessed using a Cyto-ID Autophagy Detection Kit.Our novel small molecule SIRT2-specific inhibitors NCO-90/141 inhibited cell growth of leukemic cell lines including HTLV-1-transformed T-cells. NCO-90/141 induced apoptosis via caspase activation and mitochondrial superoxide generation in leukemic cell lines. However, a caspase inhibitor did not prevent this caspase-associated cell death. Interestingly, NCO-90/141 increased the LC3-II level together with autophagosome accumulation, indicating autophagic cell death. Thus, NCO-90/141 simultaneously caused apoptosis and autophagy.These results suggest that NCO-90/141 are highly effective against leukemic cells in caspase-dependent or -independent manners via autophagy, and they may have a novel therapeutic potential for treatment of leukemias including ATL.The online version of this article (10.1186/s12885-018-4710-1) contains supplementary material, which is available to authorized users. SIRT2 hunctions . Howeverunctions . SIRT2 munctions . High exunctions , 9.+ T-cells and has a poor prognosis, which develops after long-term infection with human T-cell leukemia virus (HTLV)-1 [ATL is a T-cell malignancy derived from mature CD4(HTLV)-1 –12. Alth(HTLV)-1 –16. Ther(HTLV)-1 . Despite(HTLV)-1 –21. TherWe previously reported that NCO-01 and NCO-04 inhibit both SIRT1 and SIRT2 activities in enzyme assays and induce apoptotic cell death , 22. SIR+ T-cell line derived from an ATL patient; kindly provided by Dr. Naomichi Arima, Kagoshima University), [Cell lines S1T (HTLV-1-infected CD4ersity),  MT-2 (HTersity),  Jurkat (ersity), .The novel SIRT2 inhibitors evaluated in this study, NCO-90 and NCO-141, have been described in a previous report . ChemicaWe purchased primary antibodies against β-actin, histone H3, p53, acetyl-p53K382 (ac-p53), and SIRT2 from Cell Signaling Technology , a primary antibody against acetyl-histone H4K16 (ac-H4) from Abcam , and a primary antibody against LC3 from MBL. Horseradish peroxidase-conjugated secondary antibodies were purchased from Vector Laboratories .Cell lysates were obtained using RIPA Lysis Buffer RIPA Lysis Buffer . Nuclear extracts were obtained using NE-PER Nuclear and Cytoplasmic Extraction Reagents , according to the manufacturer’s protocol. Western blotting was performed as described previously . Images 50) was calculated using Grafit .Cell viability was examined using the cell proliferation reagent Cell Count Reagent SF  as described previously . The halApoptotic cells were stained with annexin V-FITC (MBL) and 7-amino-actinomycin D , and analyzed by flow cytometry using a Cell Analyzer EC800  as described previously , 28. TheDNA fragmentation was detected by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assays using a MEBSTAIN Apoptosis Kit Direct (MBL) .Measurements of mitochondrial superoxide generation were performed using MitoSOX Red  as described previously .Caspase activity was detected using an APOPCYTO Intracellular Caspase-3 or − 8 Activity Detection Kit (MBL) and CaspGLOW Fluorescein Active Caspase-9 Staining Kit  as described previously [The presence of autophagic vacuoles was assessed using a Cyto-ID Autophagy Detection Kit , according to the manufacturer’s instructions , 30. Autt-tests and Wilcoxon matched-pairs tests were performed using Excel 2010  and Statcel2 software . In all tests, values of P < 0.05 were considered as statistically significant.Data are expressed as means ± SD. For data analyses, two-tailed Student’s 50 values of 1.0 and 0.5 μM for NCO-90 and NCO-141, respectively, whereas NCO-90/141 did not inhibit SIRT1 at up to 300 μM. NCO-90/141 inhibited the growth of all four cell lines in a dose-dependent manner. NCO-90 showed potent activities with GI50 values of 38.3, 48.5, 48.2, and 40.2 μM in S1T, MT-2, Jurkat, and HL60 cells, respectively. NCO-141 showed potent activities with GI50 values of 34.9, 26.4, 36.7, and 12.1 μM in S1T, MT-2, Jurkat, and HL60 cells, respectively. In contrast, commercially available SIRT2 inhibitor AKG2 had no GI50 values in leukemic cell lines


The cytotoxic impacts of the novel tubugi-1–NPY peptide–toxin conjugate, as well as of free tubugi-1, and tubugi-1 bearing the thiol spacer (liberated from tubugi-1–NPY conjugate), and native tubulysin A as reference were investigated by in vitro cell viability and proliferation screenings. The tumor cell lines HT-29, Colo320 (both colon cancer), PC-3 (prostate cancer), and in conjunction with RT-qPCR analyses of the hY1R expression, the cell lines SK-N-MC (Ewing`s sarcoma), MDA-MB-468, MDA-MB-231 (both breast cancer) and 184B5  were investigated. As hoped, the toxicity of tubugi-1 was masked, with IC50 values decreased by ca. 1,000-fold compared to the free toxin. Due to intracellular linker cleavage, the cytotoxic potency of the liberated tubugi-1 that, however, still bears the thiol spacer (tubugi-1-SH) was restored and up to 10-fold higher compared to the entire peptide–toxin conjugate. The conjugate shows toxic selectivity to tumor cell lines overexpressing the hY1R receptor subtype like, e.g., the hard to treat triple-negative breast cancer MDA-MB-468 cells.Tubugi-1 is a small cytotoxic peptide with picomolar cytotoxicity. To improve its cancer cell targeting, it was conjugated using a universal, modular disulfide derivative. This allowed conjugation to a neuropeptide-Y (NPY)-inspired peptide [K Until recently, the medication of tumor diseases was primarily based on more or less unspecific chemotherapeutics and the corresponding combination therapies –5. HowevMore recently, peptide–drug conjugates (PDCs) have been recommended as targeted therapeutics –15. WhilGenerally, a useful PDC must exhibit at least four major skills that are all required for the selective and potent treatment of, for instance, cancer cells: (1) a sufficient in vivo half-life, ideally hours to days, to reach the diseased cells with a high portion of intact PDC; (2) a selective conjugate binding to a specific target molecule, e.g., a cell-surface receptor, that is characteristic for the diseased cells; (3) a fast and efficient but target-dependent binding, or better internalization, of the PDC into targeted cells; and (4) the efficient cleavage of the linker structure and, thereby, efficient liberation of the drug molecule from the conjugate at or within the diseased cell, resulting in an efficient intracellular drug dose, ideally killing the tumor cells.2 receptor subtype (hY2R) -pNPY, which is comparable to the human NPY analogue -hNPY – has been identified and claimed to be especially selective for the NPY Y1 receptor subtype in comparison to the other, very closely related NPY receptor subtypes hY2R, hY4R and hY5R -pNPY as well as a peptide–tubulysin A conjugate -pNPY – representing a comparable PDC – compared to wildtype pNPY for their binding affinities at the NPY Y1 receptor subtype. While -pNPY (IC50 = 1.3 nM) showed a comparable binding affinity as pNPY (IC50 = 1.8 nM), the Y1 receptor binding of the peptide–tubulysin A conjugate -pNPY (IC50 = 47.6 nM) was detected to be slightly reduced. However, when testing the functional receptor activation – using an second messenger (IP) accumulation assay, Ahrens and co-workers found all three peptides and PDC, respectively, in the same EC50 range (1.7 to 2.6 nM) at the NPY Y1 receptor. Interestingly, at the NPY Y2 receptor subtype the EC50 value of the subtype-unspecific wildtype pNPY was found in the same range, but the EC50 values of -pNPY and -pNPY were detected with higher than 100 nM, i.e., around two magnitudes higher than at the Y1 receptor subtype. Furthermore the authors illustrated the Y1 receptor subtype-specific endocytotic internalization of the aforementioned peptides -pNPY, several approaches of peptide conjugates have been published with diagnostic indications -pNPY -pNPY -pNPY-based conjugates proved a convincing in vivo efficacy. To further improve the general setting of peptide–drug conjugates, major efforts have been made to enhance target affinity and specificity as well as metabolic stability of the peptide moiety, and to identify novel PDC payloads permitting superior PDC efficacies.Meanwhile, based on this hY1R-prefering peptide [Fications –34. In 234]-pNPY . This st34]-pNPY –37. Howe34]-pNPY . More re34]-pNPY ,39–40. H50 like in taxanes or epothilones) .–46.44–46464(C-βA-),F7,L17,P34]-hNPY was conjugated with tubugi-1 (2) as therapeutic payload, using a linker that promises a more general use than just for the present case. The study furthermore comprises the investigation of the PDCs’ in vitro efficacies on the viability and proliferation of colon and prostate as well as several breast cancer and Ewing`s sarcoma cell lines. Thereby the correlation with the hY1R expression levels of the latter three cell lines was determined as proof for targeted delivery.The aim of this work was to prepare a novel branched NPY Y1-receptor-selective peptide–toxin conjugate version with a tubugi toxin. Therefore, the NPY analogue -hNPY (8), was synthesized by reacting the tubugi-1-SSPy (3) with the free thiol function of a β-alanine–cysteine dipeptide (βAC) linked to the side chain of Lys4 of the targeting peptide. For this purpose, 1 mol equiv of the tubugi-1-SSPy building block 3 and one molar equivalent of the targeting peptide, -hNPY, were reacted for 60 min in an air- and moisture-free atmosphere  with DTT  healthy cells.2), tubugi-1–NPY-derived conjugate 8, as well as its reduced linker product 9 were initially tested against HT-29, PC-3 and Colo320 tumor cells. Contrarily to, for instance, SK-N-MC cells shown in 2 and 8, respectively, as shown in To assess the impact of the chemical modifications due to the linker-assisted peptide–toxin conjugation and toxin liberation on the tubugi-1 toxin’s, in vitro antitumor efficiency of free tubugi-1 (1), used for comparison, and the synthetic analogue tubugi-1 (2) expressed similar cytotoxic activities against the selected cancer cell lines in medium pM concentrations. Both compounds, 1 and 2, are able to penetrate the cells’ membrane by unspecific, receptor-independent pathways, not discriminating between normal and transformed cells. For that reason it is very difficult to adjust a practicable therapeutic window for these toxins. All the more, it is important to mask the high toxicity of tubulysin A and tubugi-1 until the toxins are delivered to the cells targeted. Indeed, in its conjugated form, represented by 8, attached to the peptide moiety designed to target the NPY Y1 receptor, the toxicity of tubugi-1 was found to be masked, with IC50 values increased ≈1,000-fold compared to the free toxin. The restoration of the tubugi-1 toxicity presupposes the intracellular cleavage of the disulfide linker within the reducing environment of the endo-lysosomal compartments of the addressed tumor cells, what should be simulated by testing tubugi-1-SH (9). As shown in 8. The only slight increase of cytotoxic activity of compound 9 compared to the complete conjugate 8 in Colo320 cells is most likely caused by a generally weak responsiveness of Colo320 cells towards tubugi-1-SH and the entire conjugate tubugi-1–SS–NPY. When compared with HT-29 and PC-3 cells, the IC50 value of tubugi-1-SH is by factor 10 higher in Colo320. Since the membrane passage of tubugi-1-SH is not depending on a NPY receptor, there have to be other explanations for the reduced cytotoxic impact of tubugi-1 and corresponding derivatives in Colo320, rather than the NPY Y1 receptor expression level.The antiproliferative activities of the investigated compounds and conjugates are summarized in 8 permits in the systemic situation before reaching the target cells much lower toxicity than the cytotoxic compound tubugi-1 alone, thus opening a feasible therapeutic window for the class of tubugi toxins. In that context, a loss of tubugi-1 activity is expectable due to its chemical modification caused by the linker-based conjugation, and after linker cleavage the intracellular activities of 8, i.e., the activities of the linker cleavage product 9, are within an acceptable range, and are comparable or higher than that of some commercially used anticancer compounds . Further in vitro cell proliferation and viability assays were conducted to investigate the impact of various durations of incubation of 8, and for the correlation of its potency with the hY1R expression levels of the cells.A significant aspect of the present concept of a hY1R-targeting peptide–toxin conjugate is the fact that intact tubugi-1–NPY conjugate 8 in two treatment regimens. One regime considered a pulsed setting, i.e., initial treatment with the drug for 6 h, washing and subsequently culturing without the PDC to reach 72 h , both tested at even higher concentration of the PDC than for the Y1 cell lines. This points out good selectivity not only between tumor cell lines with the different hY1R expression levels but also good discrimination against normal (non-cancerous) cells.For that reason, a collection of tumor cell lines was used that represents a wide range of cellular hY1R expression levels, i.e., highly hY1R-overexpressing Ewing`s sarcoma SK-N-MC cells, the triple-negative breast cancer cell lines MDA-MB-468 and MDA-MB-321 which are moderately and weakly expressing, respectively, as well as the 184B5 cell line, representing a normal, but chemically immortalized mammary gland epithelium with very weak hY1R expression. These cell lines were incubated with PDC ach 72 h . In the ach 72 h . As to b RT-qPCR . Both th8) with a reductively cleavable disulfide linker. The tubugi-1–NPY conjugate has a strongly masked antitumor activity against HT-29, PC-3 and Colo320 cells in comparison to the active compound alone, but the activity is restored to a sufficient extent upon linker cleavage (tubugi-1–SS–NPY → tubugi-1-SH). Most importantly, the cytotoxic potential of tubugi-1–SS–NPY correlates very well with the hY1R expression levels of a panel of tumor cell lines. For instance, the hY1R-overexpressing Ewing`s sarcoma cell line SK-N-MC was much more affected by the PDC than the normal  cell line 184B5 with weak hY1R expression. However, further efforts should be made to improve activity after internalization of the PDC.The highly active cytotoxin tubugi-1 was successfully conjugated to a truncated and modified neuropeptide-Y mimetic to form a new peptide–toxin conjugate (PDC Overall, the investigations carried out up to this point provide a biological validation of the developed conjugate. The principally modular conjugation protocol for tubugis bears promise for further cancer targeting conjugates.File 1Complete experimental procedures and characterization data.