AbstractRattusnorvegicus   embryonic fibroblasts (rEFs) and in cultured in vitro rat pluripotent stem cells (rPSCs), embryonic stem cells (rESCs) and induced pluripotent stem cells (riPSCs), on early passages and after prolonged cultivation. Among studied cell lines, rESCs showed the lowest level of telomere dysfunction, while the riPSCs demonstrated an elevated level on early passages of cultivation. In cultivation, the frequency of dysfunctional telomeres has increased in all studied cell lines; this is particularly true for dysfunctional telomeres occurring in G1 stage in riPSCs. The obtained data are mainly discussed in the connection with the specific structure of the telomere regions and their influence on the differential DNA damage response in them.We studied the level of spontaneous telomere dysfunction in  Telomeres are specialized structures on animal chromosome ends; they preserve chromosomes from degradation and end-to-end fusions contributing to genome stability and play an important role in the maintenance of chromosome integrity. The lagging strand of telomeric DNA consists of repeat arrays of TTAGGG and are ended by G-rich overhang, which inserts into double strand telomere region forming t-loop. As a result, the chromosome ends are no longer recognized as DSBs (Double Strand Breaks). In addition, telomeres are protected from inappropriate recombination by a specialized protein complex shelterin consisting of six proteins , 2018. TSorexgranarius   fibroblasts without of hallmarks of growth crisis  signaling containing not phosphorylated CHK2 (Check point 2 protein) and partial telomere deprotection with sufficient TRF2   . Moreovecomplex) . In thisγ-H2AX is usually used for detection of both manifest and invisible non repaired DNA breaks and also for visualization of deprotected telomeres . γH2AX iRattusnorvegicus  embryonic fibroblasts (rEFs) and in cultured in vitro rat pluripotent stem cells (rPSCs), embryonic stem cells (rESCs) and induced pluripotent stem cells (riPSCs), on early passages and after prolonged cultivation. Most of the metaphases of these cell lines showed dysfunctional telomeres occurring before and after DNA replication, the last prevailed. The rESCs on the early passages of cultivation showed the lowest level of telomere dysfunction, while the riPSC lines demonstrated an elevated level. The level of telomere dysfunction increased in all studied cell lines after prolonged cultivation, especially noticeable increase in the level of telomere dysfunction arisen after G1. In addition, we have revealed a preferred accumulation of such dysfunction telomeres in riPSC lines than in rESCs. Note also the significant heterogeneity of the level of spontaneous telomere dysfunction between lines in the studied groups.Here we study the level of spontaneous telomere dysfunction in R.norvegicus 12-day embryos of laboratory strains Wag (RNFM1 and RNFF1 lines) and Brattleboro Wag (RWF1 and RWM1 lines). Three rESC lines  were isolated from blastocysts of different Wag rats. Three riPSC lines  were established as clonal lines from RNFF1 on 6 passage of cultivation by transduction. Briefly, rEFs were seeded at 104 cells/cm2 in a six-well plate. One hour before transduction, the growth medium was supplemented with 4 mg/ml hexadimethrine bromide  and the virus particles diluted in culture medium were added to wells for 18 hours .Four rEF lines were obtained from 18 hours . Then th18 hours . The rES18 hours . The rEFFISH (Fluorescent In Situ Hybridization) procedure with probe to telomeric DNA. Preparation of metaphase spreads for experiment was performed as described in FISH procedure. FISH with a high sensitive telomere PNA (peptide nucleic acid) probe (CCCATT)3 conjugated with FITC (Applied Biosystems) was performed as previously described , equipped with filter sets No. 49 (Zeiss),SP101 FITC and 103v1 (Chroma Technology), CCD-camera  and running ISIS5 software (METASystems GmbH). Microscopy was performed in the Microscopic Centre of The Federal Research Center Institute of Cytology and Genetics of SB RAS, Novosibirsk, Russia. Statistics were performed using the t-test in STATISTICA 10. The results are presented as mean ±SD.R.norvegicus telomere status was based on the accumulation of γ-H2AX histone in telomeres of metaphase chromosomes (Meta-TIF). A fluorescent signal located on one of the two sister chromatids demonstrates the chromatid type of dysfunction (Meta-TIF of chromatid type) that has arisen after DNA replication while signals located on both sister chromatids on the one chromosomal arm confirm the chromosome type dysfunction (Meta-TIF of chromosome type) arose in prereplication period R.norvegThe obtained data are presented in Table rEFs (97 percent) on 5–9th passage of cultivation contained Meta-TIFs  between lines on 5–9th and 23–29th passages, correspondingly. At the same, there observe wide disparities between studied lined in both groups on early passages and after prolonged cultivation. For instance, an average number of Met-TIFs on metaphase were 1.46 in RNFF1 on 9th passage of cultivation and 4.53 in RWM1, on 5th passage, and 2.66, in RWF1 on 29th passage.The on Table , Fig. 2.The standard method of embryonic fibroblasts generation suggests a presence of fibroblasts at different stages of differentiation, as well as the other types of cells in primary cultures; as a rule, further cultivation leads to changes in cell content. These factors appear can affect the level of telomere dysfunction in the lines.rPSCs)The level of spontaneous telomere dysfunction in rat pluripotent stem cells . In rESCs, the average number of Meta-TIFs of chromosome type per metaphase was 0.14±0.04 an average, and 1–5 percent of metaphases contained five and more of such Meta-TIFs. During cultivation, the number of Meta-TIFs on metaphase has increased. RES28 showed 9 percent metaphases containing five and more Meta-TIFs of chromosome type on 29th passage and 5 percent on 9th passage. RES27 on 42nd passage contained 52 percent of such metaphases, whereas on 10th passage only 2 percent. The maximum number of such Met-TIFs on metaphase was 18.Similar to the ed Table , Fig. 2.riPSCs were established from RNFF1 on 6th passage of cultivation. This line had the least number of dysfunctional telomeres among the rEFs  and 0, correspondingly; and in rECSs, 0.91±0.10 on average and 1–5, correspondingly.Fs Table . HoweverriPSCs has increased during cultivation. Some indicators characterized Meta-TIFs of both types differed significantly (P<0.05) in riPSCs on 4–6th and 15–22nd passages of cultivation  in the rESCs and the riPSCs; and one indicator, the number of metaphases with Meta-TIFs of chromosome type, was significantly higher (P<0.05) in the riPSCs, than in the rEFs. In general, the pattern of telomere dysfunction in the riPSCs is looking closer to those in the rEFs than in the rESCs.Similar to the other studied cell lines, the level of telomere dysfunction in the in vitro pluripotent stem cells showed low levels of spontaneous telomere dysfunction at the early stages of cultivation. The least level of telomere dysfunction characterized the rESCs whereas the riPSCs demonstrated elevated level. This applies to both types of dysfunction, chromatid and chromosomal, but increasingly to the chromosome type. For instance, the mean number of metaphases containing five and more Meta-TIFs of chromosome type in riPSCs exceeded that in normal human cells while the mean number of such Meta-TIFs on metaphase did not  telomeres, containing 213 kb of telomeric repeats on average . However, such telomeres activate DDR signaling and recruit ɤ-H2X (TRF2 deficit takes place, the activation of NHEJ, chromosome ends fusion with the following genome instability, a cell growth crisis, and cell death occur. Unlike whole genome, only five arisen in G1 partially deprotected telomeres with DDR signaling are able to induce replicative senescence in human cells and a more such telomeres can be accumulated without impact on growth cells in the absence of p53 signaling (TRF2. Since ALT cells have larger telomeres and, as a rule, inactive p53 the partially deprotected telomeres may be involved in the occurrence of telomere dysfunction in them (A special protein complex shelterin protects telomeres from inappropriate recombination . In thisit ɤ-H2X . If TRF2ignaling . The tel in them  Possibly in them . These fSpontaneous telomere dysfunction or telomere signaling are associated with the special mode of telomeric DNA replication and with a functioning of telomere safeguard complex shelterin, contributing to repression of NHEJ activation to avoid a numerous chromosomal rearrangements. They are involved in regulation of chromosome length and maintaining of genome stability.rEFs and riPSCs, the rESCs showed a reduced frequency of spontaneous telomere dysfunction on early passages of cultivation while the riPSC lines demonstrated an elevated level; moreover, the level of dysfunction was very different in studied riPSC lines. As far as cultivation, the number of dysfunctional telomeres has increased in cells of all lines; this is especially true for riPSCs. riPSCs are a model system, the study of which shows how important in personalized medicine the deeper monitoring of human iPSC attributes including the level of spontaneous telomere dysfunction before clinical implementation.Compared with


These Ds-containing DNA aptamers were generated by a method called cell-ExSELEX to target three breast cancer cell lines: MCF7, MDA-MB-231, and T-47D. Aptamer 14A-MCF7, which targets MCF7 cells, specifically binds to MCF7 cells, but not other cancer cell lines. Aptamer 07-MB231, which targets MDA-MB-231 cells, binds to a series of metastatic bone and lung cancer cell lines. Aptamer 05-MB231 targets MDA-MB-231 cells, but it also binds to all of the cancer and leukemia cell lines that we examined. None of these aptamers bind to normal cell lines, such as MCF10A and HUVEC. In addition, aptamers 14A-MCF7 and 05-MB231 are internalized within the cancer cells, and aptamer 05-MB231 possesses anti-proliferative properties against most cancer cell lines that we examined. These aptamers and the generation method are broadly applicable to cancer cell imaging, biomarker discovery, cancer cell profiling, anti-cancer therapies, and drug delivery systems.The potential of genetic alphabet expansion technologies using artificial extra base pairs  has been rapidly expanding and increasing. We present that the hydrophobic unnatural base, 7-(2-thienyl)imidazo[4,5- The creation of unnatural base pairs (UB pairs or UBPs) that exhibit high fidelity in replication as a third base pair has led to novel biotechnologies. The UBP technologies provide new material production platforms, such as semisynthetic organisms that generate novel proteins containing unnatural amino acids15 different sequence species by an evolutionary engineering method called SELEX .22DNA aptamers are single-stranded oligonucleotides that specifically bind to target molecules and materials of interest. They are isolated from a nucleic acid library containing ∼10b]pyridine) and Px (2-nitro-4-propynylpyrrole)  A.24, 25 Here, to explore the versatility and further availability of Ds-DNA aptamers, we applied ExSELEX to Ds-DNA aptamer generation by targeting three representative breast cancer cell lines: MCF7, MDA-MB-231, and T-47D. From each cell-ExSELEX used with the respective cell line, we obtained several Ds-DNA aptamers with unique specificities, from target cell specific to broadly cancer cell specific, and/or with some biological activities, such as internalization within the target cells and anti-proliferative activity. These results indicate the high potential of the Ds-DNA aptamers obtained by cell-ExSELEX for a wide range of applications toward cancer diagnostics and therapeutics and also suggest that genetic alphabet expansion increases the diversification of DNA aptamer generation and functionalization.In this cell-ExSELEX B, we use15 or 1.2 × 1015 different sequence combinations. After repetitive rounds of cell-ExSELEX, each DNA sequence in the enriched libraries was determined with an Ion PGM sequencing system (Life Technologies). In the sequencing system, the Ds bases in each DNA were replaced with the natural bases by replacement PCR, and the Ds positions in each DNA sequence were identified by the barcode embedded in each sub-library.We chose three breast cancer cell lines—MCF7, MDA-MB-231, and T-47D—as the targets and started cell-ExSELEX for each target using the Ds-predetermined DNA library with 0.6 × 10The cell-ExSELEX conditions are summarized in The enriched sequences of each library were determined by standard deep sequencing with the Ion PGM system, followed by the barcode identification of the Ds positions.To optimize each aptamer sequence and to predict its secondary structure,We chemically synthesized the selected aptamers—08B-MCF7DsDs51), 14A-MCF7(DsDs53), 07-MB231(DsDs43), 05-MB231(DsDs49), 23-MB231(DsDs49), and 03-T47DDsDs33)—and their variants to assess each aptamer’s binding affinity. First, we replaced one or two Ds bases in each aptamer with adenine to identify which Ds bases are essential  for bind1, 14A-MC—and theiWe assessed the binding affinities of each aptamer and its variants by flow cytometry, using its respective target breast cancer cell line . We usedAll of the aptamers originally contained two Ds bases, which existed within the initial Ds-predetermined sub-libraries. From the examination of Ds → A variants, one or two Ds bases strongly affected the target binding of each aptamer. For example, for 08B-MCF7  and S14,D = 1–3 nM) by flow cytometry , 14A-MCF7(DsDs42mh), 05-MB231(Ds45mh), 07-MB231(DsDs52mh), 23-MB231(DsDs58mh), and 03-T47D(DsDs42mh)—that target each breast cancer cell line are shown in We confirmed the high affinities of the aptamers 08B-MCF7, 14A-MCF7, 05-MB231, and 07-MB231 to their target cell lines  B and 3C We tested the binding specificity of each Alexa 488-labeled aptamer to the three breast cancer cell line targets and other cancer and leukemia cell lines , as well as normal cell lines (MCF10A and HUVEC), with fluorescent microscopic imaging . AptamerWe confirmed the binding of 14A-MCF7(DsDs38mh), 07-MB231(DsDs52mh), and 05-MB231(Ds45mh) to each target cell by flow cytometry at 4°C and 37°C . AlthougWe also examined the stabilities of 14A-MCF7(DsDs38mh), 07-MB231(DsDs52mh), and 05-MB231(Ds45mh) in human serum at 37°C, in comparison with AS1411-26 . AS1411 We performed competition experiments between high-affinity aptamer pairs to determine whether each aptamer binds to different targets from the others  and S23.For further exploration of the specificity of each Ds-DNA aptamer to each cancer cell line, the binding properties of each aptamer-cell combination were analyzed by flow cytometry assays using six Ds-DNA aptamers (14A-MCF7(DsDs42mh), 08B-MCF7(Ds54mh2), 07-MB231(DsDs52mh), 23-MB231(DsDs58mh), 05-MB231(Ds45mh), and 03-T47D(DsDs42mh) , AS1411-26, and Cont26, with 15 cancer cell lines and two normal cell lines. The binding pattern panels of each DNA sample to each cell line are summarized in These cancer cell panels can be classified into four categories, based on each aptamer’s binding tendency : categorWe found that categories 1–3 each have a common aspect. The category-1 cell lines are metastatic cancers of the bones (MDA-MB-231 and PC-3) and lungs (A549). The category-2 cell lines, except for T-47D, are low cell adhesive. The category-3 cell lines are related to gastrointestinal or gynecological cancers of endocrine organs. Thus, a new type of cancer profiling might be possible, with classifications using several Ds-DNA aptamers that have different binding specificities to cancer cell lines.Furthermore, the differences in the fluorescent intensities of each category might reflect the unique expression level of each aptamer target on the cell, when the aptamer’s affinity is sufficiently high. For example, the fluorescence intensity of the aptamer 05-MB231 binding to T-47D cells was much higher than that to MDA-MB-231 cells . Thus, hThe cytostatic activities of five Ds-DNA aptamers, 08B-MCF7, 05-MB231, 07-MB231, 23-MB231, and 03-T47D, were examined by a WST-8 assay using several cancer cell lines. The activities were compared to those of AS1411-26 and Cont26, as a positive and a negative control, respectively. In addition, other anti-cancer drugs, 5-fluorodeoxyuridine (FUdR) and Paclitaxel (PTX), were also used as positive controls , and S2750 = 120 ± 30 μM against CCRF-CEM, 56 ± 39 μM against HT-1080, and 190 ± 140 μM against WI-38).Salmonella typhimurium  and Escherichia coli (WP2uvrA) in an Ames test (data not shown).The anti-cancer activity of 05-MB231 does not result from the Ds nucleoside or nucleotide itself. The Ds nucleoside exhibits slight toxicity against both cancer and normal cell lines . An analysis of the mechanisms by which 05-MB231 exhibits the cytostatic activity might lead to the development of a better anti-cancer drug and synergistic combinations with other types of anti-cancer drugs.Based on the binding and profiling data and the competition experiments of each aptamer, the target antigen for 14A-MCF7 and 08B-MCF7 would be the same, while the antigens of 14A-MCF7, 05-MB231, and 07-MB231 would be different. It is notable that the expression level of each antigen could be predicted from the binding amount (fluorescent intensity) of each labeled aptamer when high-affinity aptamers are used, as in the case of 05-MB231 .D = 30 nM), generated by cell-SELEX using their UBP, Z-P.D = 2.6–108 nM) that target MDA-MB-231 cells by conventional cell-SELEX, and these aptamers specifically bound to MDA-MB-231 and T-47D cells.D = 5.9–138.2 nM) that targets the metastatic colorectal carcinoma LoVo bound to only the target cells.D = 1–5 nM measured by flow cytometry) will be required.As shown here, ExSELEX could provide a new cancer-profiling method, using a series of Ds-DNA aptamers for personalized medicine to select appropriate anti-cancer drugs.The cell-ExSELEX method could provide valuable information for cancer research and pharmaceutical applications toward individualized cancer medicine. In addition, cell-ExSELEX can be used to target other types of cells, including stem cells and induced pluripotent stem cells (iPSCs).The unnatural nucleoside triphosphates, dDsTP and dPxTP, and the Ds phosphoramidite were synthesized as described previously.The DNA library used for cell-ExSELEX was prepared by mixing 24 different single-stranded DNA sub-libraries 93-mer; . Each su2 atmosphere.The human breast cancer cell line MCF7 (RCB1904) was provided by the RIKEN BioResource Center (BRC), through the National Bio-Resource Project of the MEXT, Tsukuba, Japan. The MCF7 cell line was maintained in Minimum Essential Media (Life Technologies) supplemented with 10% fetal bovine serum , non-essential amino acids, and 1 mM sodium pyruvate (Life Technologies). The MCF10A cell line (CRL-10317), used in the counter-selection in cell-ExSELEX, was purchased from the American Tissue Culture Collection (ATCC). The base medium  for MCF10A was prepared with the additives provided in an MEGM kit (Lonza). Other human breast cancer cell lines, T-47D  and MDA-MB-231 , were maintained according to the supplier’s instructions. For the specificity profiling of the obtained aptamers, other types of cell lines—MDA-MB-453 , HCT-116 , A549 , MKN45 , NIH:OVCAR-3 , PC-3 , MIA PaCa-2 , Tohoku University, #TKG0227), Panc-1 , PLC/PRF/5 , HeLa , KG-1 , CCRF-CEM , and HUVEC —were also cultured according to the suppliers’ instructions and used for evaluations of the aptamers’ binding. These cell lines were cultured in a 37°C incubator under a 5% CO2, 0.1 mg/mL tRNA, and 1 mg/mL BSA, in D-PBS (−)) and incubated with approximately 5 × 106 target cells in a culture dish at 4°C for 60 min with gentle agitation. Unbound DNA species were removed by washing with ice-cold washing buffer (4.5 g/L glucose and 5 mM MgCl2 in D-PBS (−)) and then 0.5 mL water was added to the culture dish. To collect the cell-bound single-stranded DNA (ssDNA) species, the cells were scraped off from the culture dish with a cell scraper. The recovered cell-DNA solution was then heated at 95°C for 10 min, followed by centrifugation at 14,000 rpm for 20 min to pellet the cell debris. The supernatant, which contained the eluted DNA, was then subjected to PCR amplification, as described previously.The initial single-stranded DNA library (1 or 2 nmol), diluted in Dulbecco’s PBS (D-PBS (−)), was denatured by heating at 95°C for 5 min and then kept at room temperature for 20 min. The folded DNA library solution was diluted in binding buffer  values were calculated by curve fitting, using the OriginPro 9 software (OriginLab) according to the following formula: Y = BmaxX/(KD + X), where X is the DNA concentration, and Y is the geometric mean fluorescence intensity (MFI). The MFI without DNA was subtracted from that with each aptamer, and these values were plotted and used for curve fitting.The enrichment process of the DNA library during the cell-ExSELEX, as well as each aptamer’s binding capability, was analyzed by flow cytometry. The cultured cells were dissociated by an incubation with 0.2% EDTA, after washing with D-PBS (−). After washing with ice-cold washing buffer twice, the cells were incubated with Alexa 488-labeled DNA  in 100 μL binding buffer at 4°C for 30 min. After the incubation, the cells were washed three times with ice-cold washing buffer, re-suspended in 200 μL binding buffer, and then filtered with a 40-μm cell strainer  to remove clumped cells. The fluorescence intensity of the DNA bound to the cells was monitored with a cell sorter, S3e (Bio-Rad), by counting 10,000 events. The obtained data were analyzed with the FCS Express software (De Novo Software). Dissociation constant  and purified by denaturing gel electrophoresis. Each Alexa 488-labeled aptamer was subjected to a binding analysis with the S3e cell sorter, as described earlier.For further optimization and characterization of each aptamer candidate, partially randomized, doped libraries corresponding to 08B-MCF7, 14A-MCF7, 05-MB231, 07-MB231, 23-MB231, and 03-T47D were prepared, as described previously.To visualize and compare the binding specificity of each selected aptamer, fluorescence images of various cell lines bound with the aptamer were observed by fluorescence microscopy  or confocal laser microscopy . Each cell line was cultured on an 8-well chamber slide (Corning) and incubated with 250 nM Alexa 488-labeled aptamer, either on ice or at 37°C for 30 min. After the incubation, the supernatant was removed and the cells were washed with ice-cold washing buffer three times. The cells were fixed with 4% formaldehyde  for 10 min at room temperature, stained with DAPI , and mounted with VECTASHIELD mounting medium . In the confocal imaging, prior to the treatment with the aptamer, the cells were incubated at 37°C for 30 min in the presence of 75 nM LysoTracker Red DND-99 (Molecular Probes).4 cells per milliliter for cancer cell lines, except for KG-1, and 4 × 104 cells per milliliter for HUVEC and MCF10A cells) were plated into each well of a 96-well microplate, 1 day before the addition of DNA aptamers. In the case of KG-1 cells, 90 μL cell solution was plated into each well, 4 hr before the DNA addition. Each DNA aptamer was folded in D-PBS (−), similar to the DNA library used for cell-ExSELEX, at a 0.1mM concentration. The folded DNA aptamer, as well as FUdR , was used as a positive drug control and was diluted with D-PBS (−) and the medium used for each cell line, and added to the cells at final concentrations of 1, 2.5, 5, or 10 μM for DNA and 10 or 100 nM for FUdR in 10% D-PBS (−)/media (0.1 mL per well). As another positive control, PTX  in DMSO was also used for the analysis and added at the final concentration of 10 nM in 0.1% DMSO/media (0.1 mL per well). After incubation for 96 hr, 10 μL Cell Count Reagent SF  was added to each well, and the absorbance of the formazan product obtained by WST-8 reduction was measured at 450 nm with an ARVO multilabel counter (PerkinElmer).The cytostatic activity of representative aptamers against each cell line was examined through WST-8 assays, as follows. Aliquots (0.1 mL) of each cell solution


The use of EGFR inhibitors on oral squamous cell carcinoma (OSCC) as monotherapy yielded modest clinical outcomes and therefore would benefit from biomarkers that could predict which patient subsets are likely to respond. Here, we determined the efficacy of erlotinib in OSCC cell lines, and by comparing sensitive and resistant lines to identify potential biomarkers. We focused on the 4717C > G polymorphism in periplakin (PPL) where the CC genotype was associated with erlotinib resistance. To validate this, erlotinib-resistant cell lines harbouring CC genotype were engineered to overexpress the GG genotype and vice versa. Isogenic cell lines were then studied for their response to erlotinib treatment. We demonstrated that overexpression of the GG genotype in erlotinib-resistant lines sensitized them to erlotinib and inhibition of AKT phosphorylation. Similarly, the expression of the CC genotype conferred resistance to erlotinib with a concomitant increase in AKT phosphorylation. We also demonstrated that cell lines with the CC genotype generally are more resistant to other EGFR inhibitors than those with the GG genotype. Overall, we showed that a specific polymorphism in the PPL gene could confer resistance to erlotinib and other EGFR inhibitors and further work to evaluate these as biomarkers of response is warranted. It is most prevalent in India, Bangladesh and Pakistan due to the practice of known risk habits such as smoking, excessive alcohol consumption and betel quid chewing2. Patients diagnosed at early stage can be treated by surgery or radiotherapy alone while concurrent radio-chemotherapy is often used in patients with locally advanced disease3. About a third of the patients will progress into metastatic stage, with palliative chemotherapy as the only therapeutic option. Recently, pembrolizumab4 and nivolumab5 have been approved for patients with metastatic OSCC with disease progression during or after chemotherapy. Despite this advancement, therapies for advanced OSCC remains limited and targeted therapies are actively being explored to improve the survival of OSCC patients.Oral squamous cell carcinoma is one of the top ten cancers among men in the world6. Increased activity of EGFR results in activation of downstream signalling cascade such as PI3K/PTEN/AKT, ERK, and Jak/STAT pathways to promote cell proliferation, invasion and metastasis. Hence, increased protein expression of EGFR is a prognostic marker for poor survival in OSCC patients6. To target EGFR for therapeutic purposes, inhibitors have been developed and several of these have been tested in OSCC7. The success of cetuximab, a recombinant monoclonal antibody targeting EGFR in extending the progression-free survival (PFS) in patients with recurrent/metastatic OSCC, resulted in its approval by US Food and Drug Administration (FDA) in 20067. Whilst this is encouraging, this success has not been recapitulated with small molecule inhibitors targeting EGFR. One of these small molecule inhibitors is erlotinib or known as OSI-774 or Tarceva. Erlotinib is an orally active small molecule that blocks EGFR-mediated intracellular signalling by binding competitively to the ATP binding region8. It is approved for the treatment of patients with locally advanced or metastatic non-small cell lung cancer (NSCLC) with stable disease after standard platinum-based first-line chemotherapy9. A clinical study showed the efficacy of erlotinib by tumour shrinkage in 9 out of 35 locally advanced OSCC patients in a neoadjuvant setting before surgery10. However, a further phase II clinical trial on OSCC patients from 2006 to 2011 did not demonstrate a significant increase in PFS when erlotinib is combined with cisplatin and radiotherapy11. Similar results were also shown using another EGFR inhibitor, gefitinib12. Despite the conclusions, detail analysis showed that 52% of patients treated with erlotinib and cisplatin had a complete response as compared to 40% of patients who responded to cisplatin alone11; for gefitinib, 12.5% of patients who received docetaxel and gefitinib showed response as compared to 6.2% for patients treated with docetaxel alone12.OSCC has been characterized by high expression of epidermal growth factor receptor (EGFR)13 and olaparib for breast cancer patients who are HER-2 negative and carrying BRCA mutations14. Studies in NSCLC showed that 60–80% of the patients with EGFR mutations respond well to erlotinib, but it was evident that patients without these mutations also benefited from erlotinib15, suggesting that EGFR is not a reliable biomarker that could predict for drug response. Furthermore, EGFR mutations are not frequently observed in OSCC and hence may not be a useful biomarker in this context16. Further biomarker analysis examining the mutational status of EGFR and KRAS, copy number of EGFR and protein expression of EGFR, cMET, HER2, HER3 and PTEN from the TORCH trial in patients with advanced NSCLC was not able to identify other biomarkers for erlotinib17 that could be further tested in the OSCC setting. In OSCC, Martins and colleagues attempted to investigate the efficacy of erlotinib on human papillomavirus (HPV) positive tumour by examining p16 expression, but the test was performed in less than 50% of the patients, rendering it difficult for further analysis11. Taken together, there is currently no biomarker identified for EGFR inhibitors for OSCC, and this has in part stalled the advancement of these targeted therapies in OSCC7.Recent clinical trials on small molecule inhibitors were proven to be more effective when the patients were stratified based on biomarkers. For example, the approval of trametinib and dabrafenib for melanoma patients with BRAF V600 mutationsIn order to progress with further testing of EGFR inhibitors, it is pivotal to identify biomarkers that could predict the response to these drugs. In this study, we combined genomic information on cancer cell lines with drug response data to identify potential biomarkers that could predict response to EGFR inhibitors. Focusing primarily on the EGFR inhibitor erlotinib, we demonstrated that a single nucleotide polymorphism (SNP) in PPL could inhibit AKT phosphorylation and modulate response to erlotinib.50 at sub-micromolar concentrations (0.15 ± 0.02 µM to 0.62 ± 0.14 µM) and 6 cell lines were resistant with IC50 from 1.39 ± 0.05 µM to >4 µM. A431 with IC50 less than 1 µM was used as sensitive control18 whereas MCF7 was used as a resistant control with IC50 of more than 4 µM19  were sensitive to erlotinib with ICM19 Fig. .Figure 1p = 0.019) and 78.3% in ORL-214 (p = 0.011); Fig. Further, we determined the cytostatic effect of erlotinib on 2 representative cell lines from the erlotinib sensitive and resistant groups respectively by measuring the active DNA synthesis using the EdU assay. We showed that the proliferation rate of sensitive cell lines  reduced significantly by >75% (p < 0.001) when treated with 1 µM erlotinib for 24 hours compared to the control (0.01% DMSO). Meanwhile a large proportion of the cells in the resistant group were still proliferating at 1 µM  were significantly associated with increased sensitivity towards erlotinib treatment (p < 0.001 for both). Previous studies have suggested that the linker domain of PPL could bind to AKT21. Therefore, we decided to focus on rs2037912 harbouring homozygous GG at codon 4717, coding for glutamine, or homozygous CC, coding for glutamate located in this domain. The polymorphism was confirmed using Sanger sequencing on the cell lines ; ORL-214 (p = 0.043); Fig. , demonstrating that PPL is very likely to be important in modulating response to erlotinib.In order to validate the importance of the PPL gene in modulating the response of OSCCs lines to erlotinib, transient knockdown using siRNA was carried out in erlotinib-resistant lines (ORL-153 and ORL-214). The success of PPL knockdown was validated by western blotting as shown in Fig. Since cell lines harbouring the GG genotype in PPL are more sensitive to erlotinib, we went on to confirm the effects of this genotype in response to erlotinib. To do this, we overexpressed GG in ORL-153 and ORl-214 which originally carry the CC genotype. The same lines were also transfected with the vector control (VC) as a control and the plasmid expressing the CC genotype to ensure that our observations were not due to the transfection. The success in transfection was confirmed with Sanger sequencing using DNA from the transfected clones Fig. . With th50 of ORL-153 (VC) at 29.02 ± 1.7 µM, the IC50 of ORL-153 (GG) was reduced by ~3 fold to 11.67 ± 2.1 µM (p = 0.015). Similarly, IC50 of ORL-214 (GG) was ~2-fold lower than ORL-214 (VC), at 13.1 ± 1.4 µM and 27.6 ± 1.9 µM respectively . Overexpressing the CC genotype in ORL-153 and ORL-214 did not alter the IC50 significantly in comparison to VC as these lines originally harbour the CC genotype ; ORL214 (p = 0.040). Biochemical analyses were done to determine the changes of expression in PPL and the molecules within the EGFR signalling pathway in VC, GG, and CC clones after being treated with erlotinib . In Cal27, overexpressing the CC genotype increased the IC50 of VC by 4.4-fold, from 0.62 ± 0.1 µM to 2.7 ± 0.4 µM . Consistently, the CC genotype conferred resistance to erlotinib where EdU assays demonstrated that in ORL-115, EdU expressing cells were higher in CC than VC clones, albeit not statistically significant. For Cal27, the cells expressing CC were less responsive to erlotinib, as evidenced by higher proliferating cells as compared to VC . Western blot analyses were carried out to determine the biochemical changes in EGFR signalling in VC, GG, and CC after being treated with erlotinib. Protein expression of PPL was increased by 2.3-fold and 1.5-fold respectively in VC and GG clones of ORL-115 when treated with erlotinib. For Cal27, PPL expression increased by 4.3-fold and 2.6-fold respectively in VC and GG clones upon erlotinib treatment. Erlotinib treatment did not alter the level of PPL in CC clones. Phosphorylation of AKT levels were not changed in the CC clones of ORL-115 and Cal27 despite erlotinib treatment as compared to VC and GG clones where pAKTser473 were reduced as clearly demonstrated in the western blot analysis . Both pERK1/2 and pS6 expression levels were reduced in VC, GG and CC clones upon erlotinib treatment . To validate the hypothesis that genotype GG and CC would modulate the response to EGFR TKIs in other head and neck cancer (HNSC) cell lines, we looked into the Genomics of Drug Sensitivity in Cancer (GDSC)22. We were able to analyse data on HNSC cell lines treated with afatinib and gefitinib from the database. As for afatinib, the mean IC50 of cell lines with GG genotype was 12.36 µM ± 18.76, lower compared to cells harbouring CC genotype with IC50 at 17.37 µM ± 27.6 for afatinib treatment even though it was not statistically significant  and GDSC facilitates the identification of putative biomarkers as drug responses can be correlated to the well-characterized genomic features50 remained relatively high. Although these data suggest that PPL could modulate sensitivity to erlotinib, the modest difference could be due to the fact that the level of PPL knock-down was not sufficient to sensitize cells to erlotinib to the IC50 levels seen in inherently sensitive cells. Perhaps, the level of PPL may not be the only factor driving erlotinib sensitivity, and other factors such as its ability to interact with other proteins depending on its genotype could also be an important factor. Notwithstanding, our data is supported by Tonoike and his colleagues27 who showed that knocking down PPL reduced cell proliferation in pharyngeal cancer. This demonstration that the disruption of endogenous PPL levels could affect response to erlotinib suggests that PPL is involved in EGFR signalling. To examine this in more detail, we identified and validated the role of a polymorphism at rs2037912 on PPL in modulating sensitivity to erlotinib. We focused on rs2037912 where cell lines with the CC genotype (a glutamine at amino acid 1573) were significantly associated with resistant to erlotinib compared to cell lines with a glutamate (GG) at the same position. This polymorphism is particularly interesting in the context of EGFR signalling because previous immunoprecipitation studies have indicated that codon 4717 resides in a section of the PPL protein that binds to AKT21. To demonstrate whether the PPL polymorphism would directly affect erlotinib sensitivity, we engineered the GG genotype in erlotinib-resistant cells endogenously carrying CC genotype and showed that the change could sensitize cells that were originally resistant to erlotinib. This response was accompanied by a reduction in the expression of pAKTser473 rendering the GG clones more sensitive to erlotinib treatment. Meanwhile, overexpression of the CC genotype into erlotinib responsive cells endogenously harbouring GG genotype sustained the expression of pAKTser473 and cells were less responsive to erlotinib. Our data clearly demonstrated that this particular polymorphism in PPL directly affects response to erlotinib and this is likely due to modulating the activation of AKT, a key signalling molecule downstream of the EGFR. We noted that although overexpressing GG into erlotinib-resistant cells significantly reduced the IC50 when treated with erlotinib, however the IC50 was still relatively high and this could be possibly due to the effects of the second polymorphism (rs1049205), which changes the amino acid position at 589 from arginine (GG) to glutamine (AA) that was also significantly associated with erlotinib sensitivity. We did not test the effect of both polymorphisms simultaneously in this study so that the effect of each polymorphism can be delineated independently for their effects on drug sensitivity. Both of the loci have high LOD score and codon change from GG to AA at codon position 1766 occur together with CC change to GG at codon position 4717 in cell lines sensitive to erlotinib  response29. PPL which interacts with AKT, acts as a localization signal to AKT or a shuttle for delivery of AKT to the various cellular compartments21. For example, when PPL localizes to the cell membrane, AKT becomes activated and translocated to other compartments in the cell30, further underscoring that fact that PPL could play an important role in affecting the treatment response by EGFR TKIs.In addition to erlotinib, we also found the CC genotype at rs2037912 to be associated with resistance to other EGFR inhibitors including afatinib. Afatinib is approved for metastatic NSCLC with common EGFR mutations as well as L861Q, G719X, and/or S768I32. Whilst one of the factors is that Asian populations have a higher mutational rate in EGFR33, this could also be due to other genetic factors. Looking at the PPL polymorphism in the HapMap study of Asian and Caucasian populations (available from dbSNP website)34, ~47% of Asians including Japanese and Han Chinese in Beijing have the GG genotype as compared to only ~27% of Europeans. By contrast, ~23% of Caucasian have CC genotype in comparison to ~8.5% in Asians. The effect of this polymorphism in PPL between Asian and Caucasian populations could provide some additional insights into the differential response to EGFR inhibitors and should be investigated further.Clinical trials on NSCLC patients have demonstrated a better response to EGFR inhibitors amongst patients of Asian ethnicity compared to Caucasian patientsin vivo models to further investigate the role of 4717C > G polymorphism in PPL in modulating sensitivity to erlotinib. Cal27 overexpressing CC or GG in PPL were transplanted into NOD.CB17-Prkdcscid/NCrCrl mice but at the end of the study, genotyping showed that overexpression was not retained. Future investigations on establishing a suitable model to overexpress GG or CC stably and how the polymorphism alters the structure of PPL and affect binding to AKT is warranted. Nevertheless, our data add to the array of possible biomarkers for EGFR targeted therapy and suggest that other proteins that interact with molecules within the EGFR signalling pathway could confer resistance to treatment. In conclusion, our data provide evidence that a polymorphism in PPL could play a role in conferring resistance to EGFR inhibitors by affecting the activation of AKT. This finding is significant because many other tyrosine kinase receptors signal through AKT, and PPL could also play a role in conferring resistance in these pathways. We show that proteins such as PPL that interacts with signalling molecules could confer resistance to kinase inhibitors and affords an opportunity to investigate PPL as a biomarker for cancer therapeutics that directly or indirectly inhibits AKT phosphorylation.One of the limitations of the study is the lack of suitable Y1068 (#2234), AKT (#2938), pAKTser473 (#4060), ERK1/2 (#9102), pErk1/2 (#9101), S6 ribosomal protein (#2317), pS6 ribosomal protein , periplakin  and α-tubulin . The secondary antibodies goat anti-rabbit IgG-HRP (SB4010-05) and goat anti-mouse IgG-HRP (SB1010-05) were purchased from Southern Biotech. 3--2,5-diphenyltetrazolium bromide  solution was prepared at 5 mg/ml in phosphate-buffered saline (PBS).Erlotinib HCl  was dissolved in dimethyl sulfoxide  at a stock concentration of 10 mM and diluted in culture medium for use. Afatinib  was dissolved in DMSO at a stock concentration of 40 mM, and further diluted in culture medium to the required concentrations. Primary antibodies used in these studies were EGFR (#2232), pEGFR20. These cell lines were cultured in Dulbecco’s Modified Eagle’s Medium/Nutrient mixture F-12 HAM’s medium  supplemented with 500 ng/ml hydrocortisone (Sigma-Aldrich), and 10% heat-inactivated fetal bovine serum . The breast cancer cell line MCF7 was cultured in RPMI 1640 medium (Gibco) supplemented with 10% FBS and 100 IU Penicillin/Streptomycin. Cal27 and A431 were cultured in Dulbecco’s Modified Eagle’s Medium High Glucose  with 10% heated-inactivated FBS and 100 IU Penicillin/Streptomycin. Cell line authentication was carried out by comparing the STR profiles of cell lines to the matched blood or tumour from the patients for 16 ORL lines20 or to ATCC database for other lines. Cell lines were routinely checked for mycoplasma using MycoAlert™ Mycoplasma Detection Kit (Lonza). All cultures were incubated in a humidified atmosphere with 5% CO2 at 37 °C.The ORL cell lines used were established in our laboratory as described previously4 cells were seeded per well in 96-well plates and treated with erlotinib at ~1 nM to 4 µM. 72 hours after treatment, MTT solution was added to each well and incubated at 37 °C for 3 hours. Next, 100 µL of dimethyl sulfoxide (DMSO) was added to dissolve the purple formazan and the optical density was determined using Biotek microplate reader set at 570 nm. IC50 of the cell lines was presented as the mean ± SEM of three independent experiments with DMSO (0.01%) as the control. MCF7 which is resistant to erlotinib19 and A431 previously reported to be sensitive18 to erlotinib were used as resistant and sensitive controls.Cell viability of OSCC lines was determined using MTT assay as described previously. Briefly, 2–5 × 104, Alexa Fluor azide, reaction buffer additive) for 30 minutes in the dark. After washing (3% BSA), cells were incubated with 1X Hoechst 33342 solution (5 μg/mL) for 30 minutes. Coverslips were mounted with VECTASHIELD mounting medium (Vector Laboratories Inc) after washing with PBS. Olympus IX81 imaging system equipped with XcellencePro imaging software (Olympus) was used to capture EdU positive cells with Alexa Fluor 647: excitation 650 nm and emission 667 nm while live cells stained with Hoechst were captured using a filter with excitation at 360–370 nm and emission at 420 nm at 20x magnification. Images were merged using XcellencePro imaging software. Unmerged images were analyzed using Quick Count® to determine the number of Edu positive cells and live cells . Percentage of EdU positive cells was calculated using the following formula: (EdU positive cells/number of DAPI cells) × 100. The percentage of EdU-positive cells was normalized against vehicle control. The proliferation rate of cell lines was presented as the mean ± SEM of two independent experiments with DMSO (0.01%) as the control.Two OSCC cell lines shown to be resistant to erlotinib in this study (ORL-214 and ORL-153) and two other sensitive lines  were subjected to cell proliferation assay using Click-iT EdU Alexa Fluor 647 Imaging Kit (Invitrogen) according to manufacturer’s protocol. Briefly, cells were plated on coverslips in 12-well plates and treated with erlotinib at 1 µM for 24 hours. Following EdU incubation at 10 µM for 2 to 4 hours at 37 °C as optimized previously for each cell line, cells were fixed with 3.7% (v/v) formaldehyde in PBS for 15 minutes and permeabilized using 0.5% (v/v) Triton-X in PBS for 20 minutes at room temperature. Cells were washed with 3% (w/v) BSA in PBS. Next, the cells were stained with the Click-iT reaction cocktail  identified, the SNV was transformed into a two-way contingency table with two categories (i) sensitive and (ii) resistant. Cell lines ORL  were grouped as sensitive and ORL  were grouped as resistant. Fisher’s exact test was performed in R (version 3.20) using the function “fisher.test”. SNV with p value < 0.05 was considered significant.Identified mutations from the OSCC cell lines were tested for statistical significance between the erlotinib sensitive  or non-targeting control (Sigma-Aldrich) using HiPerfect transfection reagent (Qiagen) according to the manufacturer’s instructions. Cells were collected 48 hours post-transfection to be plated for MTT assay and western blotting.The pcDNA3.1 FLAG plasmids construct carrying the PPL CC or GG genotype respectively at position 4717 were purchased from GenScript. Plasmids were transfected into the ORL-153, ORL-214, ORL-115 and Cal27 using Turbofect transfection reagent (Fermentas) according to the manufacturer’s instructions. Control cells were transfected with the pCDNA3.1 FLAG vector. Transfected cells were selected using G418 (Sigma-Aldrich) at 400 µg/ml for 7 days.2), 100 µM of each dNTP, 50 µM of each primer and 1.25 U of Taq DNA polymerase (Promega) using 200 ng of gDNA template. PCR was performed using Veriti DNA thermocycler (Applied Biosystems) with an initial denaturation at 95 °C for 3 minutes, followed by 30 cycles of denaturation at 95 °C for 30 seconds, primer annealing at 55 °C for 30 seconds and DNA extension at 72 °C for 30 seconds. The PCR product was subjected to 1% agarose gel electrophoresis and viewed using FluorChemTM HD2 imaging systems (Alpha Innotech). The 462 bp fragment was purified using QIAquick Gel Extraction Kit (Qiagen) and sequenced using Sanger sequencing.Genomic DNA (gDNA) was extracted using the Blood and Cell Culture DNA Kit (Qiagen) from the following samples: blood genomic DNA from donors of the ORL cell lines, ORL cell lines, Cal27, ORL-214, ORL-153, ORL-204, ORL-115 transfected with PPL[G/G], PPL[C/C]. Polymerase chain reaction (PCR) was conducted to amplify exon 22 of the PPL gene using the primers: PPL-seq-F 5′ACCGAGCAAGAGATCCAGAG3′ and PPL-seq-R 5′TGTGTCAGGGTGGATGACTACG3′. PCR was set up in 50 µl containing 1X amplification buffer (containing 1.5 mM MgClTM HD2 imaging systems (Alpha Innotech). Blots were exposed for 1 minute to detect the bands except for PPL, pEGFRY1068, pS6 and S6, where blots were exposed for 3 minutes. To normalize the loading, tubulin was used as loading control. Densitometry analyses of protein bands were performed using Image J36 and normalized by α-tubulin. Fold-change of band intensity was normalized by comparing to DMSO-treated control.Cells were lysed on ice using RIPA lysis buffer , 1 mM HALT proteinase and phosphatase inhibitors (Thermo Scientific). Proteins were collected from untreated cells to establish basal levels of protein expression as well as treated cells. Treated cells were serum starved for 16 hours before treatment with 1 µM of erlotinib (or DMSO control) in serum-free medium for 1 hour before the cells were washed and stimulated with 10 ng/ml of recombinant human EGF (R&D Systems Inc) for 10 minutes before harvesting. Protein concentration was measured using BCA protein assay (Thermo Scientific). 20 µg of protein was resolved in 8% SDS-polyacrylamide gels and transferred to a polyvinylidene fluoride membrane  in a tank transfer apparatus for 2 hours at 100 V . The membrane was blocked with 5% (w/v) skim milk in Tris-buffered saline with 1% (w/v) tween 20 (TBST) for 1 hour and probed with primary antibodies at 1:1000 in 1% BSA in TBST overnight at 4°C. Blots were washed in TBST (x3 for 5 minutes). The blots were then incubated with secondary antibodies conjugated with horseradish peroxidase (dilution 1:10000). After washing using TBST, detection was performed by ECL™ detection reagents  and viewed using FluorChemp value was indicated as p < 0.05, p < 0.01 and p < 0.005.Statistical analyses were performed using Graph Pad Prism (GraphPad Software). Data are presented as mean ± SEM of at least 2 independent experiments conducted in triplicate. Unpaired t-test with two-tailed was used to calculate statistical significance between control and treatment groups. ANOVA Kruskal-Wallis test was carried out to determine the significance between control and more than one treatment groups. Significant Supplementary information


DEFA6, ABCB1 and GAL), whereas analysis of chromosomal copy number aberrations revealed no significant variation over time. These data indicate that although CTC lines derived from sequential blood samples during therapy have common traits, treatment-resistant CTC clones with distinct phenotypic characteristics are selected over time.Circulating tumor cells (CTCs) are important clinical indicators for prognosis and treatment efficacy. However, CTC investigation is hampered by their low number, making the establishment of permanent CTC lines very challenging. We derived and characterized nine CTC lines using blood samples from a patient with metastatic colorectal cancer collected before and after chemotherapy and targeted therapy, and during cancer progression. These cell lines displayed an intermediate epithelial/mesenchymal phenotype, stem-cell like characteristics, angiogenesis potential, an osteomimetic signature and the capacity to escape from the immune system. Moreover, they showed changes in mRNA and protein expression (e.g.,  Several large-scale clinical trials and meta-analyses have shown that CTC number is an important indicator of the risk of cancer progression or death in patients with metastatic solid tumors7. However, CTC investigation is hampered by their very low number, especially in blood of patients with colorectal cancer. Therefore, the establishment of in vitro cultures and permanent lines from CTCs has become the most challenging task over the past years. Indeed, CTC lines could be used to identify proteins and pathways involved in cancer cell stemness and dissemination, and also to test new drugs to inhibit metastasis-competent CTCs. Ex vivo CTC cultures have been established for breast9, prostate10, lung11, and head and neck cancer12. We previously described the first permanent cell line (CTC-MCC-41) from circulating colon cancer cells13. However, its establishment was very difficult (blood samples of 168 patients were tested). This could be partly explained by the much lower CTC number in the peripheral blood of such patients than in patients with breast or prostate cancer, making very difficult their enrichment and culture. In addition to its capacity to expand in vitro for more than 4 years, the CTC-MCC-41 cell line shows specific stem-cell like characteristics and shares some features of the original primary tumor and lymph node metastasis13.Over the past years, liquid biopsy of circulating tumor cells (CTC) has received major attention as a strategy to obtain real time diagnostic and prognostic informationWe then established another eight CTC lines from blood samples collected from the same patient at different time points during his follow-up. This unique biological material represents a chance to study clonal selection and resistance mechanisms during tumor progression and treatment. Here, we report the establishment of these new CTC lines from the same patient with metastatic colon cancer, and their characterization . Comparison of all nine autologous CTC lines (the previously described CTC-MCC-41 line and the eight new lines) highlighted their common features and main differences acquired over time.13 from the patient with the highest CTC number (302 CTCs/7.5 mL of blood) and with 38 CTC clusters (<2 to 5 CTCs/microemboli) (patient 044) . Before the first-line treatment, CTC number was evaluated in 7.5 mL of peripheral blood using the CellSearch system, and then another 10 mL of peripheral blood was used for CTC enrichment and culture. CTC number was ≥1 in 57.5% of patients and ≥3 in 39.4% . Only one colon CTC line (CTC-MCC-41) could be established44) Fig. .Figure 1in vitro. In addition, based on the CellSearch cell images, CTCs were present as single cells and clusters in the first samples, and as single cells in the last sample at the time of the third relapse  and colospheres with a size that could reach 100 µm (50%) Fig. , as obseIn the CTC-MCC-41.5 lines, CTCs started to proliferate in different wells at different time points. Therefore, they were kept separated, giving rise to the different lines . The first three CTC-MCC-41.5 lines  appeared after 18 days of culture. They showed different morphologies and behaved differently. CTC-MCC-41.5D cells grew as isolated cells (50%) and also as big colospheres (up to 100 µm). The CTC-MCC-41.5F and CTC-MCC-41.5G lines were composed essentially of isolated cells (70%) and small colospheres (up to 50 µm) for the remaining 30%. After 24 days of culture, three additional CTC lines appeared: the CTC-MCC-41.5A line, characterized by the presence of isolated tumor cells and colospheres (50%-50%); the CTC-MCC-41.5B line, with 70% of small isolated cells and few small colospheres (30%); and the CTC-MCC-41.5C line, made mostly of large colospheres  and few isolated cells (20%). Finally, after 34 days of culture, the last CTC line (CTC-MCC-41.5E) displayed isolated cells (50%) and big colospheres (50%)  were assessed genome-wide in two single cells and one colosphere for each CTC line by next-generation sequencing (NGS). This analysis revealed a wide spectrum of chromosomal aberrations Fig. . TypicalBRAFV600E mutation with a stable allele frequency of 50%, in agreement with a heterozygote mutation (Supplementary Table KRAS was wild type (wt) in all CTC lines. This mutation profile (BRAFmutKRASwt) mirrors the one detected in the primary tumor, lymph node metastasis, and also tumor xenografts from immunodeficient mice after injection of CTC-MCC-41 cells13.Concerning specific mutations, all samples carried the 14, and included genes encoding molecules involved in many tumor cell features .To characterize these CTC lines, the transcriptome profiles of the eight new colon CTC lines were compared with those of the first line (CTC-MCC-41) and of two commercial colon cancer cell lines . The panel of messenger RNAs (mRNAs) to be analyzed (Supplementary Table EPCAM), cytokeratin 19 (CK19), E-cadherin (ECAD), CMET, carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1), vascular endothelial growth factor (VEGF), snail family transcription repressor 1 (SNAIL) and aldehyde dehydrogenase 1 (ALDH1), but not CD45. This confirmed the epithelial origin of all these cell lines. A second group included transcripts that were differentially expressed in the colon CTC lines and in HT-29 and SW620 cells: (i) interleukin 33 (IL33), cyclin D2 (CCND2), semaphorin 6A (SEMA6A) and osteoprotegerin (OPG) that were expressed only in the colon CTC lines; and (ii) SWI/SNF related, matrix associated, actin dependent regulator of chromatin, subfamily A, member 1 (SMARCA1) and dickkopf WNT signaling pathway inhibitor 1 (DKK1) that were expressed only in HT-29 and SW-620 cells. The third group included genes that (i) were expressed in the colon CTC lines and in SW620 cells: B-cell lymphoma 11A (BCL11A), tumor necrosis factor receptor superfamilly 1B (TNFRS1B), bone morphogenetic protein 7 (BMP7) and fibronectin 1 (FN1); and (ii) were not expressed in the CTC lines and SW620 cells: adam metallopeptidase with thrombospondin type 1 motif 6 (ADAMTS6), Gata binding protein 2 (GATA2), gap junction protein beta 6 (GJB6), transforming growth factor beta 2 (TGFB2), prostaglandin endoperoxidase synthase 2 (PTGS2), bone marrow stromal cell antigen 2 (BST2), Wnt family member 11 (WNT11) and TWIST. The last group included genes that were downregulated in all colon CTC lines and in HT-29 cells, but expressed in SW620 cells: SRY-related HMG-box gene 2 (SOX2) and vimentin (VIM). Hierarchical clustering analysis based on the mRNA expression profile clearly segregated the CTC lines from the HT-29 and SW-620 cell lines , but none of the tested mesenchymal markers (N-cadherin and vimentin). In addition, the expression of CD133 and CD44v6 and the CD24(low)/CD44(high) ratio indicated stem cell-like characteristics15. All CTC lines weakly expressed VEGF and PanCD66 , also known as CEACAM . The absence of CD45 expression confirmed that these cells were not leucocytes. Moreover, the eight new colon CTC lines did not express endothelial proteins (CD31 and CD105), hematopoietic progenitor markers (CD34) and C-X-C chemokine receptor type 4 (CXCR4), a receptor expressed on breast tumor cells and involved in the chemoattraction to specific organs16  was assessed in the nine colon CTC lines by flow cytometry Fig. . As alrein vitro endothelial cell tube formation assay, and ALDH1 activity assay , and secrete VEGF (95% of cells)  could be established from CTCs isolated from the blood of a patient with metastatic colon cancer13 and present data on the establishment of colon CTC lines suggest that a CTC count of approximately 300 (using the CellSearch system) in blood samples from patients with colon cancer is a prerequisite for successful CTC expansion in culture. However, the number of CTCs is lower in the blood of patients with colorectal cancer that with other cancer types. In patients with colon cancer, viable CTCs can be captured in mesenteric blood samples, indicating that many of them are trapped in the liver22. Furthermore, this specific patient had the BRAF mutation, which is relatively rare (about 10%) and highly aggressive in CRC23. Indeed, only 8 out of 95 patients were positive for this rare colorectal mutation. Interestingly, no correlation between the BRAF mutation and a high number of CTCs has been observed in the COLOSPOT study. All these data clearly show how challenging it is to establish a CTC line from patients with colon cancer even with distant metastases, and may explain why no CTC culture or cell line has been reported in the literature, with the exception by our group.Our previousBRAF mutations that were detected with a stable frequency in all CTC lines.To verify that these cell lines are of cancer origin, we performed genotyping analyses. CNA analysis by NGS demonstrated that these CTC lines were cancer cell lines with cancer–specific genomic aberrations. This analysis highlighted CNA differences between samples (colosphere and single cells) from the same CTC line and also between lines, indicating that these lines have a polyclonal origin. However, we did not observe any CNA associated with disease progression. Indeed, the first CTC line (obtained at diagnosis) and the other lines (obtained at different time points after treatment) were all highly heterogeneous at the inter- and intra-line level. This suggests that therapy did not cause any significant change in the cancer CNA signature. However, we cannot exclude therapy-driven selection of resistance-conferring mutations, other than 24. Recent studies have emphasized that CTC clusters may have an increased malignant capacity25, and microemboli were observed in the blood samples of this patient with metastatic colon cancer during his follow-up.Visual monitoring of the cultures showed again a clear intra- and inter-line heterogeneity: colospheres as well as small and big single cancer cells. This suggests the absence of a preferred morphology for these tumor cells, consistent with the observation that both small and large CTCs are linked to unfavorable prognosis26. All nine CTC lines presented a stable epithelial phenotype, as indicated by the expression of epithelial markers, such as EPCAM and cytokeratins . Concerning markers of mesenchymal transition, all nine CTC lines expressed FN1 and SNAIL, but not vimentin or N-cadherin (mRNA and protein). Moreover, in all CTC cell lines, ECAD was weakly expressed and ADAMTS6, GATA2, GJB6, TGFB2, PTGS2 and BST2, genes encoding cell-cell junctions, focal adhesion or cytoskeletal proteins, were clearly downregulated14. This is in accordance with CTC loss of adherent cell properties to leave the tumor and reach the bloodstream. These data indicate that these colon CTC lines are tumor cells with an intermediate EMT phenotype. The acquisition of mesenchymal features by CTCs could reveal some plasticity required for dissemination to distant organs26. In addition, the expression of TNFRSF1B and BCL11A, linked to apoptosis suppression27, could indicate another CTC hallmark called «anoikis resistance», a mechanism to avoid cell death when tumor cells are not attached, but swim in the bloodstream18.Epithelial-to-mesenchymal transition (EMT) is a complex process that supports the migratory capacity of epithelial tumor cells. EMT also plays a crucial role in promoting cancer metastasis by various mechanisms, including CTC release from the primary tumor and distant metastases15, BMP7, GAL and SEMA6A, and the CD44high/CD24low ratio28, were detected in all nine CTC lines. Moreover, they all showed ALDH activity, a well-known stem cell marker17. The combination of EMT phenotype and stem-cell characteristics should favor their dissemination and ability to form distant metastases29.Stemness features, such as expression of CD133, CD44v6 OPG, BMP7 and TNFRSF1B (involved in osteogenic activity) suggests that these CTCs have the ability to lodge in the bone marrow. However, this patient developed liver metastases, without any evidence of bone metastases. Therefore, we hypothesize that these CTCs could have been released from the bone marrow where they were preserved in special niches as dormant tumor cells with the ability to mimic osteogenic cells.In addition, the expression of 30. However, PD-L1 expression was not detected in any of the nine CTC lines from this patient with colon cancer. Indeed, PD-L1 expression can vary, and it is still debated whether this protein is expressed more at early31 or late, metastatic stages33. Conversely, CEACAM1, DEFA6 and IL33, three genes encoding proteins also involved in immune escape, were expressed by all nine CTC lines. CEACAM1 upregulation at advanced stages of colorectal cancer is associated with invasiveness, metastasis, tumor progression and escape from the immune system34. Upregulation of DEFA614 and IL3335 also is involved in immune mechanisms. Therefore, these three factors could be attractive targets for immunotherapy.Concerning the immune system escape and survival, the PD-1/PD-L1 axis has been extensively studied in the last years after the introduction of cancer immunotherapy. Recently, we demonstrated that CTCs in breast cancer can express PD-L1 as a camouflage against T-lymphocytes13. Moreover, a functional test based on in vitro tube formation using endothelial cells showed that CTC-MCC-41, 41.4, 41.5B, 41.5E and 41.5F cell supernatants induced a denser network of endothelial tubes than those of CTC-MCC-41.5C, D and G cells. These data highlight again the heterogeneity of the nine CTC lines. As the fluoro-EPISPOT assay showed that VEGF secretion was comparable in all nine CTC lines, other factors must be involved in CTC-induced angiogenesis. For instance, semaphorin 6A, a member of the semaphorin family involved in cell motility and proliferation, also contributes to vascular development and particularly to angiogenesis by modulating VEGFR2 signaling in endothelial cells36. The stronger SEMA6A expression in the CTC-MCC-41, 41.4, 41.5B, 41.5E and 41.5F lines could explain the heterogeneous results of the functional assay. Moreover, Loria et al. showed that forced SEMA6A overexpression induces anchorage-independent growth and significantly increases invasiveness. Interestingly, SEMA6A is overexpressed in BRAFV600E melanoma cells36. This BRAF mutation was detected (i) in the primary tumor, (ii) in all the CTC lines, (iii) the lymph node metastasis, and also (iv) in tumor xenografts obtained by injection of CTC-MCC-41 cells13. Thus, SEMA6A might represent a new potential therapeutic target in BRAFV600E cancers.Another important feature of aggressive cancer cells is the capacity to induce neo-angiogenesis that is required to import nutrients and oxygen in the growing tumor. All the new CTC lines expressed and secreted VEGF, a key inducer of angiogenesis, as already shown for the CTC-MCC-41 lineIn conclusion, we established nine permanent colon CTC lines from the same patient with metastatic colon cancer at different time points during disease progression. We then thoroughly characterized these CTC lines, highlighting their heterogeneity, which suggests that they are derived from different clones, and also their common features. Indeed, they all show (i) an intermediate epithelial/mesenchymal phenotype, (ii) stem-cell like characteristics, (iii) the potential to induce angiogenesis, (iv) an osteomimetic signature, and (v) the capacity to escape from the immune system. These features suggest that they could initiate metastases at distant sites. Moreover, in these CTC lines, we observed changes in RNA and protein expression, while the analysis of chromosomal CNA revealed no significant pattern of selected genomic alterations. For instance, the stem-cell like characteristics progressively increased in the CTC lines isolated at later stages of cancer progression. These data indicate that CTCs cultured from sequential liquid biopsies during therapy have common traits, but that treatment-resistant clones with distinct phenotypic characteristics are selected.in vitro CTC culture.After signature of the informed consent, peripheral blood samples from patients with non-resectable metastatic colorectal adenocarcinoma were collected before the start and during first-line chemotherapy (FOLFIRI and bevacizumab) in the framework of the COLOSPOT study . The study was carried out in accordance with the World Medical Association Declaration of Helsinki and the guidelines for experimentation with humans by the Bioethical committee “Sud Méditerranée III”. The experimental protocol was approved  by the Bioethical committee “Sud Méditerranée III”. All included subjects provided written informed consent for participation in this study and the publication of results. The Blood (10 mL) was collected in CellSave tubes  for CTC enumeration and in EDTA tubes (10 mL) for Patient 044 was diagnosed at an advanced stage of colon cancer . He showed good tolerance to the first-line treatment (FOLFIRI + bevacizumab), but, after 5 cycles, had to stop it because of cancer progression (RECIST criteria). A second-line treatment was immediately initiated (FOLFOX + bevacizumab), but again, the treatment was stopped after three cycles due to clinical progression , and no other treatment was planned. The patient was hospitalized for abdominal pain and occlusive syndrome, and a new relapse was diagnosed. He died 6 months after the diagnosis of cancer, 2 months after the last treatment, and 1 week after the last blood sample collection. Additional clinical data about this special patient are available in the Supplementary Table (+) cells using the RosetteSep Human Circulating Epithelial Tumor Cell Enrichment Cocktail , according to the manufacturer’s protocol. Then, CD45(−) CTCs were incubated at 37 °C in 24-well non-adherent plates with CTC culture medium , as previously described13. Colospheres and tumor cells were observed during the first weeks of culture and were then transferred to new 24-well plates for further growth and to T25 flasks for cell expansion. In these conditions, CTCs could be quickly expanded and after a few months, billions of tumor cells were obtained.The blood collected in EDTA tubes was immediately used for CTC culture. First, viable CTCs were enriched through depletion of hematopoietic CD4537. HT-29 and SKBR3 cells were maintained in Dulbecco’s Modified Eagle’s medium (DMEM), 10% fetal bovine serum (FBS), and 1% penicillin/streptomycin. A549 cells were maintained in DMEM supplemented with 10 mM HEPES, 10% FBS and 1% penicillin/streptomycin. SW620 cells were maintained in RPMI 1640 with L-glutamine and 10% FBS, and the colon CTC lines and BC-M1 cells in CTC culture medium. Endothelial cells were cultured in M199  containing 0.4 ng/ml endothelial growth factors , 20 µg/ml heparin , 1% penicillin/streptomycin and 20% FBS .The colorectal cell lines HT-29 (ATCC HTB-38) and SW620 (ATCC CCL-227) were used as control for several tests, as well as the lung cancer cell line A549 (ATCC CRM-CCL-185), the mammary cancer cell lines SKBR3 (ATCC HTB-30) and BC-M1 (an established breast cancer cell line from a single disseminated tumor cell in the bone marrow of a patient with non-metastatic breast cancer patient)One colosphere and two single cells were transferred to individual PCR tubes (0.2 mL) for whole genome amplification using the PicoPlex WGA Kit, according to the manufacturer’s protocol . Next, libraries were prepared using the TruSeq DNA Sample Prep Kit according to the manufacturer’s protocol. NGS was performed on a HiSeq. 2500 apparatus  using single reads for 100 cycles. Sequence analyses and CNA identification were performed using Control-FREEC and a custom-made script in MatLab.DNA was extracted from the nine colon CTC lines using the QIAamp DNA Mini Kit , according to the manufacturer’s instructions. DNA yield and purity were assessed with a Qubit spectrophotometer . DNA was stored at −20 °C in 10 mmol/L Tris and 0.5 mmol/L EDTA (pH 7.6).38.Genomic DNA samples were genotyped using MassArray iPlex Pro (AGENA BIOSCIENCE), a Matrix Assisted Laser Desorption Ionization Time of Flight Mass Spectrometry assay. A specific colorectal cancer panel  was customized for the detection of mutations in six oncogenes  was used as reference housekeeping gene. One µL of each RT product was added to 9 µL of mix containing the two primers (6 µM each) and SYBR Green . PCR reactions were performed on a QuantStudio 5 real-time PCR instrument  as follows: 95 °C for 3 min, followed by 40 cycles of 95 °C for 5 s, 60 °C for 10 s, and then 1 cycle at 95 °C for 1 min, 55 °C for 5 s, 95 °C for 30 s, 37 °C for 30 s. Results were analyzed using the QuantStudio Design and Analysis Software. Hierarchical clustering was computed with the Cluster and Treeview software packages (PMID: 9843981).Messenger RNA (mRNA) was extracted from each CTC line using the RNeasy Mini Kit , and complementary DNA (cDNA) was obtained by reverse transcription (RT) using the SuperScript III First-Strand Synthesis Super Mix kit , according to the manufacturer’s instructions. Semi-quantitative PCR that targeted a wide range of sequences  and data interpreted with the Kaluza software (BECKMAN COULTER). In parallel, labeled tumor cells were also loaded on glass slides, using a cytospin, and observed under a fluorescent microscope .40.The fluoro-EPISPOT assays for CK19, VEGF and OPG were performed as previously described5 cells/well) were then seeded and cultured for 6 h with different media: RPMI 1640 alone , endothelial cell medium with 20% FBS (positive control), and supernatant from the CTC lines cultured with basal medium for 48 h.24-well plates were coated with 250 µL/well Matrigel  and left at 37 °C for 30 min. Endothelial cells (10ALDH1 activity was measured using the ALDEFLUOR kit (01700 – STEMCELL TECHNOLOGIES) following the manufacturer’s instructions. Briefly, 5 µL of ALDEFLUOR Reagent was added to the cell suspension and half of the cell mixture was immediately transferred to another tube with the ALDEFLUOR DEAB Reagent (an ALDH activity inhibitor) to serve as internal negative control to quantify ALDH activity in each cell line. A549 cells were used as positive control, as recommended by the manufacturer.Only metastasis-competent CTCs lead to cancer relapse, but their specific signature is not known. The nine sequential autologous CTC lines we derived from a patient with metastatic colorectal cancer will help to study CTC changes during cancer progression, a prerequisite to discover new therapeutic targets and to propose personalized treatments.Supplementary information


Ubiquinol cytochrome c reductase binding protein (UQCRB) is known to play crucial roles in the development of various types of diseases. However, the link between UQCRB and microRNAs remains unknown. In the present study, we performed microRNA sequencing of mutant UQCRB-expressing stable cell lines that exhibited pro-oncogenic activities caused by expression of the mutant UQCRB gene. Results showed that hsa-miR-10a-5p was significantly downregulated in the mutant UQCRB-expressing cell lines. Furthermore, mRNA sequencing and gene ontology analysis of differentially expressed genes (DEGs) revealed that the cholesterol biosynthesis pathway might be activation by mutant UQCRB expression. Moreover, inhibition of cholesterol synthesis in mutant UQCRB-expressing cells via treatment with the specific inhibitors suppressed the cell proliferation. Transfection with a hsa-miR-10a-5p mimic validated that lanosterol synthase (LSS) is a target of hsa-miR-10a-5p. In addition, hsa-miR-10a-5p was found to be downregulated in liver cancer cell lines overexpressing UQCRB. Taken together, our findings highlighted the potential use of hsa-miR-10a-5p as a biomarker for UQCRB related diseases. The electron transport complex (ETC) in the mitochondria consists of five complexes that involves in producing an electrochemical proton gradient for energy production by ATP synthesis1. The ubiquinol-cytochrome c reductase binding protein (UQCRB) is one of the subunits of mitochondrial complex III that plays a role in electron transport and maintenance of the mitochondrial complex III6. Identification of a target protein of terpestacin, an anti-angiogenic natural product, revealed a new role of UQCRB in regulation of mitochondrial ROS (mROS) generation and angiogenesis7. Moreover, many reports have implicated UQCRB variants in a number of diseases, including hepatocellular carcinoma8, ovarian cancer9, pancreatic ductal adenocarcinoma10, and colorectal cancer11.Mitochondrial dysfunction has been implicated to play a key role in various diseases, such as metabolic diseases and cancer12. Based on the above findings, our group generated mutant UQCRB-expressing stable cell lines, namely, MT1 and MT2, and investigated their angiogenic properties. The MT1 showed a higher expression level of mutant UQCRB protein than MT2 and both cell lines showed significantly faster cell growth and pro-angiogenic activities than those of control host human embryonic kidney cells 293 (HEK293). In addition, we demonstrated that treatment of these mutant UQCRB-expressing stable cell lines with UQCRB inhibitors significantly suppressed cell proliferation of the cells13.In a recent case study, a Turkish female harboring a deletion in the gene encoding the UQCRB and isolated complex III defect presented hypoglycemia and lactic acidosis during a metabolic crisis in her babyhood; however, these conditions did not continue to her childhood15. Multiple studies have reported the use of miRNAs as biomarkers for specific diseases17. Furthermore, miRNAs have been implicated in mitochondrial function, metabolism, and metabolic disorders, such as cholesterol metabolism20.MicroRNAs (miRNAs) are non-coding, single-stranded RNAs containing approximately 22 nucleotides. MicroRNAs are crucial regulators of numerous physiological and pathological processesHowever, the link between UQCRB and miRNAs remains to be mostly uncovered. In the present study, we performed microRNA and mRNA deep sequencing of mutant UQCRB-expressing stable cell lines with control host cell HEK293 to identify novel microRNA biomarkers for UQCRB related diseases.Mutant UQCRB-expressing stable cell lines were subjected to miRNA sequencing to identify differentially expressed miRNAs that are specific to mutant UQCRB. The mutant UQCRB-expressing cell lines (MT1 and MT2) were previously generated based on a human case report expressing the mutant UQCRB gene. miRNA sequencing was conducted to compare the expression patterns of the generated mutant UQCRB-expressing cell lines with those of normal HEK293.2FC| > 1, log2CPM > 2, and FDR < 0.15. Of these, nine miRNAs were downregulated , whereas three miRNAs were upregulated  compared to the control  and related pathways to identify novel mutant UQCRB-associated phenotypes. mRNA sequencing was performed to compare gene expression patterns between HEK293 wild-type cells and mutant UQCRB-expressing cell lines (MT1 and MT2). For DEG analysis, candidate differentially expressed mRNAs were selected based on the criteria |fold change| > 1.5 and p-value < 0.01.In addition, we performed gene set analysis to identify novel pathway. Results of gene set analysis showed significant upregulation (Benjamini <0.05) of eight different ontologies in both mutant UQCRB-expressing cell lines, which include sterol metabolic process, cholesterol metabolic process, steroid metabolic process, sterol biosynthetic process, steroid biosynthetic process (GO:0006694), terpenoid backbone biosynthesis, isoprenoid biosynthetic process, and cholesterol biosynthetic process. Additionally, the MT1 cell line showed upregulation in lipid biosynthetic process, steroid biosynthesis (hsa00100), MAPK signaling pathway, and biosynthesis of unsaturated fatty acids, whereas the MT2 cell line showed upregulation of mRNAs involved in isoprenoid metabolic process , Tarbase (ver. 5), and mirTarbase (ver. 4.5), which contained information on the validated mRNA targets of miRNAs. We also used miRNA target prediction databases, namely, TargetScanHuman , microRNA.org (August 2010), Microcosm (ver. 5), and miRDB (January 2012) Fig. .Figure 2Results showed that seven mRNA targets, namely, ANKRD1, SCD, CNST, SLC2A3, DUSP3, LSS, and HSPA1B had significantly high target prediction values for hsa-miR-10a-5p Fig. . The resNext, we performed mimic transfection to validate that LSS could be the putative target of hsa-miR-10a-5p. Treatment with 5 nM and 20 nM of hsa-miR-10a-5p mimic miRNA was found to upregulate miRNA levels in a dose-dependent manner Fig. . LSS mRNTo further investigate the relationship between hsa-miR-10a-5p and UQCRB, we analyzed hsa-miR-10a-5p levels in cell lines of UQCRB expressing cancer, namely, HepG2 (human hepatocellular carcinoma), PC3 (human prostate cancer) and HCT116 cells (human colon cancer cell). Results of qRT-PCR analysis showed that hsa-miR-10a-5p levels were downregulated in all three cell lines tested with a variable degree than HEK293. In addition, hsa-miR-10a-5p level was also down-regulated in HeLa , which is also cell line of UQCRB expressing cancer (data is not shown). Notably, hsa-miR-10a-5p level was significantly downregulated in HepG2 was even lower than that in MT1 cells Fig. . FurtherGiven that hsa-miR-10a-5p levels were significantly downregulated in HepG2 cells than HEK293, we evaluated expression levels in a different liver cancer cell line, Huh7. Similar to the results from HepG2 cells, hsa-miR-10a-5p expression levels were significantly downregulated in Huh7 cells relative to those in Chang, a normal liver cell line Fig. . The mRN21; Mevastatin, a HMG-CoA reductase (HMGCR) inhibitor; and YM-53601, a squalene synthase (FDFT1) inhibitor22. These inhibitors act on distinct points in the cholesterol synthesis pathway.To further validate the relationship between mutant UQCRB and cholesterol synthesis, cell proliferation was evaluated after treatment with the following three different cholesterol synthesis inhibitors: Fatostatin, a sterol regulatory element-binding protein (SREBP) inhibitor13. Notably, treatment of the mutant UQCRB-expressing cell lines with these inhibitors suppressed the cell growth in a dose-dependent manner as similar as HEK293. Similar results were obtained using liver cancer cell lines, as well. However, the efficacy of the cholesterol inhibitors varied depending on the liver cancer cell line used. Treatment with Fatostatin significantly inhibited cell proliferation in Huh7 cells. Mevastatin only slightly inhibited cell proliferation in both HepG2 and Huh7 cells. However, YM-53601 significantly inhibited cell proliferation in both HepG2 and Huh7 cells  as the metric to reliable factors which even considered the basal expression level not only the difference of expression level in cell type.de novo cholesterol biosynthesis and for the production of various intermediate metabolites that participate in other essential cellular processes23. For example, farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP) are critical for post-translational modifications of GTPase proteins, including Ras and RhoA24. The mevalonate pathway has been demonstrated to be activated or inhibited in a number of different tumors and has been implicated in tumorigenesis. Furthermore, inhibitor of HMG-CoA reductase (HMGCR), a rate-limiting enzyme of mevalonate pathway, has been demonstrated to exert inhibitory effects on various types of cancers, including breast cancer30. Based on the above-mentioned findings, the oncogenic phenotype in mutant UQCRB-expressing cell lines (MT1 and MT2) could be induced at least in part by promoting cholesterol biosynthesis.Results of mRNA sequencing showed that cholesterol biosynthesis was activated in mutant UQCRB-expressing cell lines. Cholesterol synthesis is initiated by the mevalonate pathway, in which acetyl-CoA is a starting precursor and isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP) are the end products. The mevalonate pathway is crucial for et al. reported that hsa-10a-5p was down-regulated in CaOV3 and RMUG-S cells under hypoxia as compared to normoxia31. Consistent with this, our study also suggested that mutant UQCRB stable cell lines down-regulated hsa-miR-10a-5p that might lead to higher cell proliferation via promotion of cholesterol biosynthesis. On the contrary, however, Wu C et al. suggested that miR-10a was up regulated in hypoxia condition32 and Zhi F et al., suggested that hypoxia caused up-regulation miR-10a in rat liver33. Accordingly, the cause of down regulation of miR-10a-5p in mutant UQCRB stable cells and exact mechanism underlying this regulation needs to be investigated for addressing the complicated responses of miR-10a-5p upon cell types and physiological conditions in coming studies.In the present study, we performed integrated analysis using established databases that provide validated microRNA-mRNA target pairs, as well as prediction databases that provide predicted microRNA-mRNA target pairs. Hsa-miR-10a-5p has been reported to regulate the metabolism in various cells including hypoxia related mechanism. Kinose Y In the present study, the pro-proliferative activity of mutant UQCRB-expressing cell lines was regulated by cholesterol synthesis inhibitors. Accordingly, we thought that cholesterol synthesis pathway could play a role in pro-proliferative activity of these cell lines. As up-regulation of specific genes could influence to increase sensitivity or resistance to drug, further studies are also needed to explore the exact cause of these effects of inhibitors in respect with their sensitivity or toxicity to the cells.Notably, the regulation of proliferative activity by cholesterol synthesis inhibitors in liver cancer cells was not so much effective compared to mutant UQCRB stable cell lines. This is probably because cholesterol synthesis may partially play a role in proliferative activity of mutant UQCRB cells. Because the other miRNAs also were down regulated in MT cells and these miRNAs could also play roles in proliferation through regulating other pathways of the cells, the effect of cholesterol synthesis inhibitors could be different in liver cancer cell lines.7 and PTD-UQCRB treatment in cells and tissues34 increased mROS, stabilized HIF-1 and increased expression of VEGF. Consistently, mutant UQCRB-expressing cell lines showed similar properties as like these activities of wild UQCRB overexpression13. However, we are not able to exclude the fact that mutant UQCRB-expressing cell lines are not same with wild UQCRB expressing cell lines, exactly. To address this issue, detail comparative studies using wild and mutant UQCRB expressing cells are under investigation.In this study, we used mutant UQCRB stable cell lines to identify miRNAs and novel pathways related UQCRB. We consider that mutant UQCRB stable cell lines are similar with wild UQCRB expressing cells as UQCRB related pathways are similarly activated in the mutant UQCRB-expressing cells. According to previous studies of our group, supplement of UQCRB protein by transfection of wild type UQCRB geneCollectively, our present study revealed that hsa-miR-10a-5p is associated with genes involved in the cholesterol synthesis pathway in mutant UQCRB-expressing cells. Moreover, LSS was validated as a hsa-miR-10a-5p target gene based on mimic transfection of mutant UQCRB-expressing cell lines. These results demonstrated that hsa-miR-10a-5p can be used as a biomarker for UQCRB related diseases.2 in air (pH 7.4). PC3 and HCT116 cells were grown in RPMI1640 (Invitrogen) under the same conditions described above. Mutant UQCRB stable cell lines were constantly provided with 1 mg/mL G418.Control (HEK293) cells, mutant UQCRB-expressing cell lines (MT1 and MT2), HeLa and HepG2 cells were grown in Dulbecco’s modified Eagle’s medium  supplemented with 10% fetal bovine serum  and 1% antibiotics (Invitrogen). All cells were incubated at 37 °C in a humidified incubator with 5% COTM RNA isolation kit (Ambion) according to the manufacturer’s instructions.Cells were collected using TRIzol reagent . Total RNA was extracted using PureLink35. Data were normalized via the quartile method using the edgeR package. Finally, candidate miRNAs were identified based on fold change (FC), count per million (CPM), and false discovery rate (FDR). The FDR was controlled by adjusting the p-values based on the Benjamini-Hochberg algorithm. The transcriptome analysis data of mutant UQCRB-expressing cells are available at Korean BioInformation Center (KBRS20171018–0000001~KBRS20171018–0000336).Isolated total RNAs were subjected to miRNA sequencing (Illumina HiSeq2000). Raw sequencing reads were provided by Macrogen . Briefly, for miRNA-sequencing, small RNA sample preparation was performed according to the standard the Illumina protocols. The 5′ and 3′ adapters were sequentially ligated to small RNAs (18–30 bp) that were gel-purified from 5–10 µg of total RNA. Adapter-ligated small RNAs were reverse transcribed, amplified, and sequenced on a HiSeq2000 instrument (Illumina) according to the manufacturer’s instructions. Next, adapters were trimmed and mapped to the reference sequence using bowtie, an ultrafast, memory-efficient short read aligner designed to align large sets of short DNA sequences (reads) to large genomeshttp://genome.ucsc.edu). Read counts were analyzed, and differentially expressed mRNAs were identified based on fold change and p-value cutoffs using Cuffdiff.Isolated total RNAs were subjected to mRNA-sequencing (Illummina HiSeq2000). Raw reads were obtained from Macrogen . cDNA library construction was performed using the TruSeq RNA library kit. Briefly, 1 µg of total RNA was subjected to polyA-selected RNA extraction, RNA fragmentation, random hexamer primed reverse transcription, and 100-nt paired-end sequencing on an Illumina HiSeq2000 instrument. The generated libraries were quantified via qPCR according to the qPCR Quantification Protocol Guide and validated using an Agilent Technologies 2100 Bioanalyzer. Next, the adapters were trimmed, and the resulting reads were mapped to the reference sequence using TopHat2. The reference genome sequence  and annotation data were downloaded from the UCSC website  and Taqman primers (Applied Biosystems). Real-time PCR was performed on a HT Fast Real Time PCR system (Applied Biosystems) using Taqman Fast Universal PCR master mix (Applied Biosystems) or on a LightCycler 96 system (Roche) with FastSTtart Essential DNA Probes Master (Roche) and TaqMan primers (Applied Biosystems) according to the manufacturer’s instructions. Data were analyzed based on the 2Isolated RNAs were reverse-transcribed using the following target-specific primers: wild-type UQCRB, forward: 5′-ATGTGAATTCATGGCTGGTAAGCAGGCC-3′ reverse: 5′ ATGCCTCGAGCTTCTTTGCCCATTCTTC-3′; mutant UQCRB, forward: 5′-ATGTGAATTCATGGCTGGTAAGCAGGCC-3′; reverse: 5′-CTC GAGGCCGTCCTCGTAGCAGCTGCAGCCGCACACCTCCACCACGTGGTTGCTGCGCTGGCCGTTCTTTTCTTTTCTTTCCCGAAT-3′; HMG-CoA reductase (HMGCR), forward: 5′-GTC ATT CCA GCC AAG GTT GT-3′; reverse: 5′-GGG ACC ACT TGC TTC CAT TA-3′; Mevalonate (diphospho) decarboxylase (MVD), forward: 5′-CTG CCT GAC TGC CTC AGC-3′; reverse: 5′-ACC TCT CCT GAC ACC TGG G-3′; lanosterol synthase (LSS), forward: 5′-GGC AGA CGT GGA CCT ACC T-3′; reverse: 5′-GAA AAG TGG GCC ACC ATA ATC-3′; GAPDH, forward: 5′-AACAGCGACACCCACTCCTC-3′; reverse: 5′-GGAGGGGAGATTCAGTGTGGT-3′). Expression levels were quantified using ImageJ 1.41o  and Image LabTM software (Bio-rad).TM software (Bio-rad).Cell lysates were analyzed via 10% and 12.5% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and subsequently transferred to polyvinylidenedifluoride membranes  following standard methods. Blots were incubated at 4 °C overnight with the following primary antibodies: anti-UQCRB , anti-HMGCR (Millipore), anti-MVD , anti-LSS , and β-actin . Immunolabeling was performed using Clarity Western ECL substrate . Images were quantified using Image Lab5 cells were seeded onto six-well plates at 24 h prior to transfection. Transfection was performed using Lipofectamine 2000 (Invitrogen) reagent according to the manufacturer’s instructions. Next, cells were transfected with 5 and 20 nmol/L miRNA mimics after 48 h of incubation. Cells were then lysed in TRIzol reagent (Invitrogen).Mutant UQCRB-expressing cells were transfected with a miR-10a-5p mimic purchased from Ambion . Afterwards, 1.5 × 1050 values in cells. Liver cancer cells  were seeded onto 6-well plates and incubated for 36 h with of A1938 (30 µM), a UQCRB inhibitor. A1938 was dissolved in DMSO.Cells were seeded onto 96-well plates and incubated for 24 to 72 h. Cell proliferation was measured using 3--2,5-diphenyltetrazolium bromide  colorimetric assay. Cells were treated with Fatostatin (Sigma), Mevastatin (Sigma) and YM-53601  at or above their reported IChttp://david.abcc.ncifcrf.gov/)36. Data were filtered based on the cutoff value of Benjamini <0.05.Forgene functional analysis of differentially expressed mRNAs (DEGs) was performed using the DAVID tool (www.graphpad.com). Student’s tests were performed to determine significant differences between the control and test groups. Statistically significant differences were considered at p-value < 0.05 .Results were expressed as mean ± standard error (±S.E.M.). All statistical analyses were performed using GraphPad Prism (ver. 5.00 for Windows, GraphPad Software, San Diego, CA, Supplementary Information


The Klotho protein is a recently discovered protein and its overexpression is associated with life extension. Klotho deficiency or silencing of the Klotho gene in mice leads to an accelerated aging and short life, whereas overexpression of Klotho in mice extends lifespan. Klotho participates in many metabolic pathways and is highly expressed in the kidneys, the choroid plexus and neurons. It plays a key role in the calcium-phosphate metabolism, remyelination, and cognitive processes. The present paper is a short review of the literature on the role of Klotho in neurodegenerative disorders, with special attention paid to multiple sclerosis. The neuroprotective function of Klotho is also reported. It is also important to consider potential clinical applications of Klotho that might be useful in the treatment of many diseases. The Klotho protein was discovered in 1997 . The KloBoth in mice and humans, the Klotho gene is located on chromosome 13q12; it encompasses 50 kb and consists of 5 exons. As in the mouse gene, in the human Klotho, two transcripts encoding membrane or secretory protein are formed from one gene by alternative RNA splicing . The Klo2+ absorption by stabilizing the Ca2+ transient receptor potential vanilloid 5 (TRPV5) channel in the plasma membrane. It inhibits renal 1-alpha 25 hydroxylase activity, thus decreasing the levels of circulating calcitriol. Therefore, hyperphosphatemia, hypercalcemia, elevated plasma calcitriol, vascular calcification, and premature aging can be observed in Klotho-deficient mice [Three types of the Klotho protein are distinguished, i.e., cell membrane-related, intracellular, and secretory forms. Klotho associated with the cell membrane (transmembrane Klotho) can be detected in the kidneys, pituitary gland, inner ear, brain, parathyroid glands, pancreas, large intestine, skeletal muscles, bladder, ovaries, testes, and epithelial breast cells . The larent mice . FGF23 lent mice .Klotho has been shown to inhibit 1-α hydroxylase, an enzyme responsible for the production of 1,25-dihydroxyvitamin D3, which is an active form of vitamin D . Lower lOn the molecular level, permanent chronic inflammation, cell proliferation disorders, or cellular aging leads to the formation of a number of age-related chronic diseases, such as obesity, diabetes, atherosclerosis, Alzheimer’s disease (AD), cancer, renal diseases, or degenerative diseases . The secKlotho is produced in large amounts in the brain by ependymal cells of the choroid plexus, Purkinje EC cells, and hippocampal neurons. It is also detected in the cerebral white matter . It was Aging of the brain is related to structural atrophy of the cortex and functional deterioration, especially in the frontal and temporal regions. The process depends on genetics. Inhibition of aging was detected in mice which were carriers of one copy of the KL-VS haplotype , which is associated with longevity . YokoyamM146V, APPswe, tauP301L) and healthy animals [The neural Klotho can protect against the development of age-related neurodegenerative diseases. Masso et al. compared the levels of secreted Klotho (s-KL) in different brain regions between young and older mice and between triple transgenic 3xTg-AD models of AD  . BindingMultiple sclerosis (MS) is a chronic inflammatory disease that is identified as one of the major neurological disorders in young adults. The disease is characterized by the infiltration of autoreactive immune cells into the central nervous system, resulting in degeneration of the myelin and axons . Costa eZeldich et al.  conducte3) for 6 months and 22 patients without MS (control group). In each group, the amount of the secreted form of Klotho and the “redox status” of CSF samples were investigated (using the ELISA kit and a ferric reducing antioxidant power assay—FRAP). The amount of Klotho in the CSF in RRMS was significantly lower (233.62 pg/ml) compared to that in the control group (541.62 pg/ml)—a nearly 2.3-fold decrease in comparison to the controls. The level of the FRAP in MS patients was 1.5-fold lower than in the controls—the mean for the control group was 314.93 μM, and 211.98 μM for MS patients. Additionally, a significant negative correlation was found between Klotho concentration and the Expanded Disability Status Scale (EDSS). However, a significantly positive correlation was observed between Klotho concentration and the FRAP in the CSF. Those results indicated that a decreased level of Klotho in the CSF was related to increased disability in patients with MS. Furthermore, Klotho regulates antioxidant properties of the CSF. The antioxidant power of the CSF was significantly lower in MS patients. Those results were consistent with the hypothesis according to which Klotho exerts neuroprotective effects by modulating oxidative stress.Emami Aleagha et al. conducted another interesting study on the role of Klotho in MS . CerebroAhmadi et al.  tried toCurrently, scientists try to find a potential strategy to increase the level of Klotho in the brain. The administration of exogenous Klotho is limited by the protein weight (130 kDa)—such a large protein cannot cross the blood-brain barrier. Another way is to find molecules that act as Klotho mimetics which could enhance DNA transcription and translation and reduce Klotho degradation. Abraham et al. initiated a program to identify small molecules that could increase Klotho at the transcriptional level . Two comLatanoprost (LA) is an ocular hypotensive agent used in the treatment of glaucoma targeting prostaglandin F2α (FP) receptor. The study of Yamamoto et al. demonstrated that a neuroprotective effect of LA is due to the Klotho-mediated suppression of calpain activation . It was Klotho is highly expressed in the brain. Many studies have demonstrated a neuroprotective role of Klotho. In the human brain, the secreted isoform of the Klotho protein plays a crucial role in neuronal activity, which is the key to aging and neurodegenerative processes. Due to activation of OPCs, soluble Klotho could be used as a therapeutic strategy for brain tissue regeneration. As a cofactor that modulates affinity for various receptors, Klotho regulates calcium homeostasis and oxidative stress. In the CSF, Klotho can participate in the suppression of cytokine/chemokine expression in the central nervous system, supporting the activation of neuronal cells. From a clinical point of view, the Klotho level in the CSF can be used as a biomarker of MS severity. Although it is still not known why Klotho suppresses aging, maintaining Klotho levels by stimulating endogenous Klotho production or administering exogenous Klotho protein may be a potential therapeutic target to slow down the aging process and reduce the incidence of age-related diseases. Finally, as a regulator of remyelination in the white matter, Klotho can become a new therapeutic substance for MS and other demyelinating and neurodegenerative diseases.