Eleven CRC cell lines were classified into molecular subtypes and tested for their response to pan-ERBB, MEK, and ERK inhibitors as single agents and in combination. All six inflammatory or TA cell lines were exquisitely sensitive to the combination of MEK and neratinib whereas all five stem-like cell lines were resistant. Growth inhibition in sensitive cell lines was greater with the combination than with either drug alone even in cell lines with KRAS mutations. The combination inhibited pERK in inflammatory cell lines but not in four out of five stem-like cell lines. MEK162 plus neratinib were synergistic in cell culture and xenograft models in inflammatory cell lines. The ERK inhibitor, SCH772984, down-regulated pERK, decreased cell viability, and was synergistic with neratinib in both inflammatory and stem-like subtypes. These results suggest that inhibition of pERK is a critical node in decreasing cell viability of stem-like CRC tumors. Our results also suggest that CRC molecular subtypes may yield predictive information and may help to identify patients who may respond to targeted inhibitors.Molecular subtypes of colorectal tumors are associated with prognosis and prediction for treatment benefit from chemotherapy. The purpose of this study was two-fold: 1) to determine the association of colorectal (CRC) molecular subtypes with response to  The current standard of care for stage II/III colon cancer is adjuvant chemotherapy with 5-fluorouracil + leucovorin (FULV) or FULV plus oxaliplatin. The addition of targeted therapies in adjuvant setting has not been shown to reduce recurrences. We examined the association of subtypes with prognosis and for prediction of oxaliplatin benefit, when added to FU/LV by subtyping tumors from patients enrolled into NSABP C-07 N = 1729), a clinical trial in which patients were randomly assigned to FU/LV with or without oxaliplatin. In agreement with other investigators 729, a cl, we showKRAS mutations. Anti-epidermal growth factor receptor (EGFR) monoclonal antibodies, panitumumab and cetuximab, have been shown to improve overall survival, progression-free survival, and overall response rates in patients with metastatic, KRAS WT tumors [KRAS WT tumors respond, and even for those who do, the response is limited [Unlike stage II/III disease, the standard of care for colorectal cancer patients with metastatic disease is driven by the presence or absence of T tumors , 6. Howe limited , 8 by re limited –11.KRAS mutants with intrinsic resistance to anti-EGFR antibodies should be sensitive to inhibition of downstream signaling elements. Preclinical models tested this hypothesis with agents targeting pathways downstream of KRAS, however, single-agent inhibitors were disappointing in both PDX models and patients [in vivo were successfully inhibited.Preclinical studies showed that resistance to an EGFR blockade consistently displayed persistent activation of mitogen-activated protein kinase (MEK) and extracellular signal-regulated kinase (ERK) irrespective of the upstream genetic alterations . Theoretpatients –14. Intepatients . Recentlpatients  have shoDifferences among CRC subtypes with respect to general biology, association with prognosis, and the important clinical observation that oxaliplatin benefit was limited to the enterocyte CRC subtype, led us to hypothesize that subtypes may show a differential response to targeted therapies . To expl2. Cells were cultured in DMEM medium with 1X penicillin/streptomycin, and 10% fetal bovine serum. Selumetinib, gefitinib, afatinib, neratinib, MEK162, and SCH772984, were purchased from Selleckchembio .All cell lines used in this study were purchased from American Type Culture Collection (ATCC) . The CRC cell lines NCI-H747, SW837, SW1116, SW1463, SNU-C1, NCIH508, SW480, SW620, C2BBE1, HS675.T and HCT116 were cultured at 37°C in a humidified atmosphere in the presence of 5% CO3/well) in 100 μl of growth medium were plated into each 96 microtiter well  plates. Cells were allowed to attach for 14 to 18 hours, then were exposed to different doses of inhibitors for 72 hours. The number of viable cells was determined by using the CellTiter-Blue cell viability assay . The CellTiter-Blue reagent, an indicator dye, was added to cells, incubated for 1 hour, then fluorescence was measured (560(20) Ex/ 590(10) Em). The release of fluorescence as a result of cellular metabolism indicates living cells. The percent of living cells was calculated by normalizing the absorbance of treated cells to untreated cells. Cells were treated with drug over a range of concentrations. Dimethyl sulfoxide (DMSO) (0.01%) was used as the control treatment. Results are shown as triplicates of mean ± SD.CRC cells . Intensities of spots were quantified using myImage analysis software .CRC cells were harvested after 48 hours of culture in the presence of drugs or control vehicle, lysed by using 1X cell lysis buffer containing 1mM PMSF. Cell lysates were subjected to PathScan RTK Signaling Antibody Array Kit  (Chemiluminescent Readout) as per manufacturer's protocol. Briefly, the PathScan RTK Signaling Antibody Array Kit is a slide-based antibody array founded upon the sandwich immunoassay principle. The array kit allows for the simultaneous detection of 28 receptor tyrosine kinases and 11 important signaling nodes when phosphorylated at tyrosine or other residues. Target-specific capture antibodies using biotinylated protein as positive control, and nonspecific IgG as negative control, were spotted in duplicate onto nitrocellulose-coated glass slides. Cell lysates were then incubated on the slide followed by a biotinylated detection antibody cocktail. Streptavidin-conjugated HRP and LumiGLO reagent were then used to visualize the bound detection antibody by chemiluminescence. An image of the slide was captured with myECLProtein was extracted from cells using 1X RIPA buffer containing a Halt Protease and Phosphatase Inhibitor Cocktail (ThermoFisher Scientific). Cell lysate proteins (30 μg) were separated on 4–12% NuPAGE Bis-Tris precast gel electrophoresis and transferred to iBlot 2 polyvinyl difluoride membranes . The blots were incubated with the appropriate antibodies to detect the protein level of interest, and the immune complexes were visualized by GE Healthcare Amersham ECL WB detection system (ThermoFisher Scientific). Western blots were probed with antibodies against phosphor-ERBB2/HER-2 (Tyr1248) , ERBB2 , phosphor-ERBB3 (Tyr1197) , ERBB3 , phosphor-ERK1/2 , phosphor-p44/p42 MAPK (diluted 1:1000), total p44/42 MAPK, (diluted 1:1000), total ERK1/2 , phosphor-AKT S473 , AKT , phosphor-EGFR , phosphor-p90RSK , and Actin .3 and were randomly assigned to different treatment groups and a vehicle control (0.5% carboxymethylcellulose) (seven mice/group). An initial study was performed for dose tolerability. Efficacy studies were subsequently undertaken with 10 mice in each of 4 cohorts: vehicle, MEK162 alone (3 mg/kg); neratinib alone (10 mg/kg); and MEK162 (3mg/kg) plus neratinib (10mg/kg). Each drug was suspended in 0.5% carboxymethylcellulose and orally administered q.d. Mice were treated daily for 4 weeks. Tumor volume  where L is the longest and W is the shortest axis) and body weight were measured twice weekly. Mice were euthanized by brief isofluorane anesthesia followed by cervical dislocation, two hours following the last dose of drug. Tumors were harvested and snap frozen in a dry ice/ethanol bath for immunoblot analysis. Mice exhibiting any signs of distress including weight loss >20%, tumor size >10% body weight, or tumor growth hindering mouse mobility and ability to eat drink/eat normally, were euthanized.The animal study protocol was approved by the Institutional Animal Care and Use Committee of the University of Pittsburgh (IACUC protocol #18022278) and in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Five million cells per mouse were inoculated subcutaneously in female athymic nude mice  at the axillary region using human CRC cell lines NCI-H747 (inflammatory subtype) or SW480 (stem-like subtype). Tumor xenografts were allowed to grow to an average size of 100–200 mmt test. Results were considered statistically significant with p values <0.05. The RTK data was quantitated with a digital imaging system using the myImage Analysis software (ThermoFisher Scientific). Raw data quantification of all figures is shown in Five replicate measurements were made for all cellular proliferation and apoptotic assays and these data are presented as a mean plus or minus on standard deviation (SD). Statistical differences were determined by Student KRAS mutation status and their molecular subtype. Three of the five CRCA subtypes  were included in this study and represent approximately 80% of stage II and III colon cancer based on our analysis of C-07 [Cell lines were selected for the testing of targeted agents based on  of C-07 .One of the goals of this study was to identify novel treatments for patients resistant to chemotherapy. As noted previously, the stem-like subtype has de novo resistance to chemotherapy and bodes for a poor outcome. Although TA and inflammatory subtypes (approximately 50% of CRC) have a more favorable prognosis when treated in early-stage disease (stage II/III), if disease recurs, these patients have limited options.Table 1shows the mutation status of BRAF, KRAS, PIK3CA, and NRAS, as well as the subtype designations of these cell lines based on the results of two other studies [ studies . Fig, pan-ERBB inhibitors (afatinib and neratinib)  Fig, and the EGFR inhibitor, gefitinib,  as single agents in five stem-like cell lines , two inflammatory subtypes , and four TA subtypes . The MEK inhibitors (MEK162 and selumetinib) were the most effective agents in decreasing cell viability of inflammatory and TA cell lines  Figs. The inflammatory cell lines had IC50 values below 1μM, and one TA cell line had an IC50 value at 1μM (MEK162), whereas the other four TA cell lines had IC50 values above 2μM (MEK162), however, IC50 values in stem-like cell lines were all above 2μM, and for a majority of stem-like cell lines, IC50 values were not achieved at concentrations of 5μM . The two inflammatory cell lines, although KRAS mutant, were sensitive to the MEK inhibitors. The pan-ERBB inhibitor, neratinib, had an IC50 above 4μM in both inflammatory, stem-like , and TA cell lines except NCI-H508 TA cell line, which had an IC50 at 1μM (neratinib) . One TA cell line, NCI-H508, had an IC50 value at 1μM (MEK162); all other TA cell lines had IC50 values from 2μM to 5μM (MEK162) . Afatinib and gefitinib had little effect on cell viability at concentrations between 125nM to 4μM  Figs.Recently published data has shown combining pan-ERBB and MEK inhibitors demonstrated strong synergistic inhibition of CRC cell proliferation . Therefo. For all inflammatory and TA cell lines, the combination of neratinib (0.5 μM) with varying doses of MEK162 (0.062 μM -1μM) treatment was significantly more effective in reducing the cell viability . The combination of MEK162 at 0.062μM and neratinib at 0.5μM inhibited approximately 70% of inflammatory cell lines and 60% of TA cell lines, but more than 80% of the stem-like cell lines remained viable even at 1μM MEK162 and neratinib at 0.5μM. The resistance of the stem-like cell lines was independent of KRAS mutation status. We observed the same pattern of sensitivity and resistance with a constant dose of MEK162 (0.5 μM) with variable doses of neratinib (0.062μM-1 μM) .To improve the efficacy of these agents, we combined two different classes of drugs: the MEK inhibitor, MEK162, and the pan-ERBB inhibitor, neratinib. This combination dramatically reduced the cell viability of inflammatory and TA cell lines compared to single-agent treatment V600E-mutated metastatic melanoma. The same pattern of sensitivity was seen when the MEK inhibitor, trametinib, was combined with neratinib . Stem-like cell lines were resistant to this combination. More than 80% of the stem-like cells remained viable after treatment with 6 nM trametinib and 62 nM neratinib. In contrast, at these same drug concentrations, the viability of inflammatory and TA cell lines ranged between 20–60% . Our data suggest that although intrinsic resistance to EGFR-targeted therapy could be overcome by combination targeted therapies in inflammatory or TA subtypes, this approach would not work for stem-like subtypes.We used the combination of neratinib and another MEK inhibitor, trametinib, which has US FDA approval as a single agent for the treatment of patients with BRAFKRAS mutations (n = 4) or KRAS wild-type (n = 2) was demonstrated by performing a CompuSyn analysis, which showed a combination index (CI) of ≤1  ) or unchanged  . More specifically, inhibition of pERK after treatment with the combination of MEK162 plus neratinib was demonstrated in inflammatory cells lines NCI-H747 (0.50-fold vs. control)  and SW837 (0.20-fold vs. control) . Conversely, pERK levels were either increased or unchanged after exposure to the MEK162 plus neratinib combination compared to the control in the stem-like cell lines SW480 (4.2-fold vs. control), C2BBE1 (no change vs. control), and HCT-116 .To determine the targets that might be responsible for the resistance in cell lines to the combination of MEK162 and neratinib, we used the PathScan RTK Signaling Array Kit. Both inflammatory (n = 2) and stem-like (n = 5) cell lines were incubated with either no drug (control) or with the combination of MEK162 plus neratinib for 48 hours. Thirty-nine different anti-phosph-tyrosine antibodies were interrogated using the PathScan RTK Signaling Antibody arrays. These receptor tyrosine kinases signal primarily through tyrosine phosphorylation events involving a wide range of downstream signaling cascades including the PI3K/Akt, MAPK, and JAK/Stat pathways, which are important for cell division, growth, metabolism, differentiation, migration, and survival. The most dramatic single difference in the phosphorylation levels between the stem-like cell lines (C2BBE1 and SW480) and the inflammatory cell lines (NCI-H747 and SW837), after treatment with the combination of MEK162 plus neratinib, was the phosphorylated protein p44/42 MAPK . In inflammatory cell lines the levels of pERK1/2 were reduced after treatment with MEK162 plus neratinib . Conversely, the MEK162 plus neratinib combination failed to inhibit pERK levels in four out of five stem-like cells lines . Only one stem-like cell line, SW620, showed down-regulation of pERK after exposure to the MEK162 plus neratinib combination . These results suggest that continued activation of pERK might result in resistance in most stem-like cell lines to the combination of MEK162 plus neratinib.To validate the PathScan RTK array results, we examined the RAF-MEK-ERK downstream targets using western blots. Cells were incubated with neratinib or MEK162, singly or in combination for 48 hours. Results were similar to those of the PathScan RTK array. The combination of MEK162 plus neratinib resulted in substantial reduction of pERK levels after 48 hours of treatment in inflammatory cell lines (NCI-H747 and SW837) Fig 4D). This suggests that the decrease of cell viability by the MEK162 plus neratinib combination in inflammatory cell lines may be due to the induction of caspase 3/7 activity, an essential event during apoptosis.To determine whether apoptosis was responsible for the decreasing cell viability in cell lines when treated with the combination of MEK162 plus neratinib, we assessed apoptosis activity in resistant and sensitive cell lines. Both inflammatory  and stem-like  cell lines were treated with the varying doses of neratinib in combination with a constant dose of MEK162 (0.5 μM) for 72 hours followed by evaluation of caspase 3/7 activity. Only the inflammatory cell lines, NCI-H747 and SW837, showed induction of apoptosis after treatment with the combination of MEK162 (0.5 μM) plus neratinib (0.062 μM- 1 μM), whereas stem-like cell lines, SW480,C2BBE1, HCT-116, and Hs.675.T, showed no sign of apoptosis after treatment (p≤0.002) and single-agent neratinib (p≤0.0001) inhibited growth in the inflammatory cell line xenograft. However, the combination resulted in greater tumor inhibition  in the inflammatory cell line than either agent alone . In tumor xenografts, with a stem-like subtype (SW480), MEK162 alone significantly inhibited tumor growth compared to the vehicle control (p≤0.006), however, neratinib alone did not (p = 0.145) . Furthermore, the combination of MEK162 plus neratinib had no additive effects on the inhibition of tumor growth compared to the MEK162 alone (p = 0.57) in the stem-like xenograft models . No effects on the body weight of the treated xenograft mice were seen , indicating that there were no toxic effects of treatments. Table 2shows the percent of tumor growth inhibition (TGI) in both inflammatory (NCI-H747) and stem-like (SW480) cell line xenograft models in three cohorts: neratinib alone, MEK162 alone, and the combination of MEK162 plus neratinib compared to control.We examined the effect of neratinib and MEK162 as single agents and in combination on an inflammatory (NCI-H747) and a stem-like (SW480) cell line in xenograft models. Both single-agent MEK162 (p≤0.0001) in inflammatory subtype tumors Fig . There was no inhibition of pERK levels by single-agent neratinib. p-ERK levels were significantly downregulated with the combination of neratinib and MEK162 compared to either drug alone (p≤0.001 vs. MEK162 only) (p≤2.8511E-10 vs. neratinib only) or control vehicle only (p≤1.060E-05) . In stem-like xenograft models, MEK162 significantly inhibited pERK as a single agent (p≤1.696E-06 vs. control), whereas neratinib showed no effect on pERK as a single agent Fig . pERK levels were not statistically different between MEK162 alone and the combination (p = 0.09 vs. combination) in the stem-like xenograft tissue . The inability to inhibit pERK and tumor growth with neratinib alone or in combination with MEK162 was correlated.Examination of the phosphorylation status from inflammatory and stem-like tumors was carried out on all seven mice from each of four cohorts . Single-agent MEK162 significantly reduced the pERK protein levels compared to the control (. The combination of neratinib (0.125 μM) and SCH772984 (1 μM) was more effective at decreasing cell viability in both inflammatory and stem-like cell lines than either drug alone . Moreover, inhibition of pERK was seen with single-agent SCH772984 in both inflammatory and stem-like cell lines and correlated with loss of cell viability . CompuSyn analysis showed that the combination of SCH772984 plus neratinib were highly synergistic CI ~0.19) in both inflammatory and stem-like subtypes .Most of our cell culture and xenograft data show that inhibition of pERK is associated with drug sensitivity. This observation suggests that pERK is a critical downstream target for signaling of the RAS/RAF pathway and led us to test the ERK inhibitor, SCH772984. The SCH772984 is a potent, ATP-competitive and non-competitive inhibitor of ERK1/2 with additional allosteric properties, which inhibits ERK activation/phosphorylation , 18. SCHIn this study, we demonstrated that the combination of MEK162 plus neratinib effectively inhibited cell viability in inflammatory or TA subtypes but not in the stem-like subtype. Tumor growth of an inflammatory cell line in mouse-xenograft models was inhibited by MEK162 and neratinib as single agents, however, the combination of MEK162 plus neratinib was more efficacious, synergistic, and independent of KRAS status. The finding that sensitivity appeared independent of KRAS status is a particularly important observation as currently there are no specific therapies for KRAS MT tumors . In contS2 File). Moreover, in resistant stem-like cell lines, we did not observe any change in ERBB3 expression levels with MEK, neratinib, or the combination . One plausible explanation for the difference in our findings and those of Sun et al., is that we used different pan-ERBB and MEK inhibitors, which may affect the amount of ERBB-RAS-RAF-ERK targets downregulation and assessment.Sun et al. , who tesOther investigators , 20 havePatients with stem-like tumors represent approximately 25% to 30% of the CRC population and have a very poor prognosis regardless of treatment  and thusMolecular subtyping of colorectal cancer with gene expression represents a relatively new approach to predicting treatment response. The development of a validated assay may be a valuable tool not only in determining prognosis but also by guiding more precise therapies.S1 Fig(TIF)Click here for additional data file.S2 Fig(TIF)Click here for additional data file.S3 Fig(TIF)Click here for additional data file.S4 Fig(TIF)Click here for additional data file.S5 Fig(TIF)Click here for additional data file.S6 Fig(TIF)Click here for additional data file.S7 Fig(TIF)Click here for additional data file.S8 Fig(TIF)Click here for additional data file.S9 Fig(TIF)Click here for additional data file.S10 Fig(TIF)Click here for additional data file.S11 Fig(TIF)Click here for additional data file.S12 Fig(TIF)Click here for additional data file.S1 File(XLSX)Click here for additional data file.S2 File(PDF)Click here for additional data file.


Klotho is a membrane-bound or soluble antiaging protein, whose protective activity is essential for a proper function of many organs. In 1997, an accidental insertion of a transgene led to creation of transgenic mice with several age-related disorders. In Klotho-deficient mice, the inherited phenotypes closely resemble human aging, while in an animal model of Klotho overexpression, the lifespan is extended. Klotho protein is detected mainly in the kidneys and brain. It is a coreceptor for fibroblast growth factor and hence is involved in maintaining endocrine system homeostasis. Furthermore, an inhibition of insulin/insulin-like growth factor-1 signaling pathway by Klotho regulates oxidative stress and reduces cell death. The association between serum Klotho and the classic risk factors, as well as the clinical history of cardiovascular disease, was also shown. There are a lot of evidences that Klotho deficiency correlates with the occurrence and development of coronary artery disease, atherosclerosis, myocardial infarction, and left ventricular hypertrophy. Therefore, an involvement of Klotho in the signaling pathways and in regulation of a proper cell metabolism could be a crucial factor in the cardiac and vascular protection. It is also well established that Klotho protein enhances the antioxidative response via augmented production of superoxide dismutase and reduced generation of reactive oxygen species. Recent studies have proven an expression of Klotho in cardiomyocytes and its increased expression in stress-related heart injury. Thus, the antioxidative and antiapoptotic activity of Klotho could be considered as the novel protective factor in cardiovascular disease and heart injury. The leading cause of death among Europeans and around the world is cardiovascular disease (CVD) . There aKlotho protein is associated with aging and the name of gene comes from Greek mythology . Clotho,Klotho (kl) gene spans approximately 50 kbp and is composed of 5 exons. Its two transcripts encode membrane and secreted proteins [ Kl gene highly correlates with the suppression of several ageing phenotypes [kl-/kl-) [ kl gene was not disrupted, but the expression was reduced and the loss of Klotho mRNA was observed [ kl gene expression in mice were accompanied by a short lifespan, infertility, and several age-related disorders such as atherosclerosis, osteoporosis, age-related skin changes, and ectopic calcifications [ kl gene is observed mainly in the kidneys and brain, as well as in the pituitary gland, placenta, skeletal muscle, urinary bladder, aorta, pancreas, testis, ovary, colon, and thyroid gland [proteins . Kl geneenotypes , 5. It hkl-/kl-) . The insobserved , 4. The ications , 4. At tications . The expid gland , 4, 6.Kl gene encodes a single-pass transmembrane glycoprotein type 1 (1014 and 1012 amino acids in both mouse and human), which is expressed in the cell membranes and Golgi apparatus [β-glucosidases. Those glucosidases hydrolyze a β-glycosidic linkage in the saccharides, glycoproteins, and glycolipids [pparatus . The molpparatus –11. Two colipids , 7, 12.αKlotho (Klotho)  activity. They cleave Klotho fragments from the extracellular domain of membrane-bound Klotho into the blood, urine, and cerebrospinal fluid [ kl gene leads to the generation of secreted Klotho (about 65 kDa). The transcript contains 3 exons in mice (lacking exons 4 and 5) and 5 exons in humans [ KL gene transcript of secreted form of Klotho protein predominates over the membrane form in human [The main type of Klotho protein is called (Klotho) . There aal fluid –14. ADAMal fluid , 15. Firal fluid , 12, 13.in human .β-Klotho and γ-Klotho proteins have been described. β-Klotho and γ-Klotho are the single-pass transmembrane glycoproteins type 1 likewise [β-Klotho is composed of KL1 and KL2 domains, shares 41% amino acid identity with α-Klotho, and is primarily expressed in the liver, gastrointestinal tract, spleen, kidney, and adipose tissue. γ-Klotho is composed of KL1 domain only and can be found mainly in the adipose tissue, eyes, and kidneys [Based on the sequence similarity, two other Klotho-related genes which encode likewise , 14, 17.αKlotho. As a result, they established ELISA test for detection of circulating soluble αKlotho protein for the first time [α-Klotho from other short forms (KL1 or KL2 domains) and secreted Klotho, present in the body fluids. Likewise, in 2000, Kato et al. established the anti-Klotho monoclonal antibodies, KM2119, with recognition site for KL2 domain, and KM2076 and KM2365 with recognition site for KL1 domain [αKlotho in the body fluids, it is possible to measure only total soluble αKlotho. It is difficult to characterize detected forms of αKlotho . Moreover, it is demanding to make a distinction between shed or secreted Klotho. A specific antibody against the variant peptide fragment may help resolve this problem, for example, polyclonal antibody against an additional C-terminal sequence in measurement of secreted Klotho. We expect technical advances will allow these key details to be filled in the future.It should be noted that there are some difficulties in the measurement of soluble Klotho in serum or urine. In 2010, Yamazaki et al. tested antibodies specifically recognizing an extracellular domain of human serum rst time . HoweverThe membrane-bound Klotho protein is needed to increase an affinity of endocrine fibroblast growth factor (FGF) to FGF receptors (FGFR) at their target organs. Twenty-two members of FGFs have been identified, a large superfamily of peptides which impact a wide range of biological processes in human. FGFs bind to FGFRs to form a complex which functions as a paracrine/autocrine factor . FGF19, i-IIa and downregulates the reabsorption of inorganic phosphate (Pi) in the renal proximal tubules [2+) via transient receptor potential vallinoid-5 channel (TRPV5) and regulates Ca2+ homeostasis [α-hydroxylase (CYP27B1) in the renal proximal tubules and consequently suppresses the synthesis of biologically active 1,25-dihydroxyvitamin D3 . Calcitriol is responsible for absorption of intestinal and renal phosphate and calcium. An elevated level of calcitriol in kl-/kl- mice led to increased plasma Ca2+ and Pi levels, as well as to vascular calcification [The main function of membrane-bound Klotho is to form a complex with FGFR1c, FGFR3c, and FGFR4 and to convert their canonical forms into the specific receptors for FGF23, a bone-derived phosphaturic hormone  7, 14, , 14, 17. tubules . In the eostasis . Moreovefication , 22. Henfication , 22. It β-glucuronidase and sialidase activity and modifies the function of renal and intestinal transporters [2+ and secretion of potassium (K+) are regulated by TRPV5 and renal outer medullary potassium channel 1 (ROMK1), respectively. TRVP5 is found predominantly along the distal tubules and ROMK1 is expressed in the distal and collecting tubules [α2,3, α2,6, or α2,8 linkage [α2,6-linked sialic acids from TRPV5 and ROMK1 N-glycan chains and uncovers the underlying disaccharide, galactose-N-acetyloglucosamine (LacNAc). LacNAc is a ligand for galactose-binding lectin, galectin-1. It is crucial that galectin-1 binds LacNAc and α2,3-sialylated LacNAc, but not α2,6-sialylated LacNAc. Therefore, removal of terminal α2,6-linked sialic acid  from N-glycans of TRPV5 and ROMK1 by Klotho exposes underlying LacNAc for binding to galectin-1 on the extracellular side of cell membrane. Then, the channels are accumulated on the plasma membrane because of binding to galectin-1 lattice at the extracellular surface. It prevents the TRPV5 and ROMK1 endocytosis. Consequently, the abundance of cell-surface TRPV5 and ROMK1 is increasing, and the renal reabsorption of calcium and the secretion of potassium into urine are enhanced [+ intake increases the activity of ROMK1 in the apical membrane of cortical collecting duct cells, to secrete more K+ into the urine and maintain K+ balance. Experimental data showed a defect in secretion of K+ and hyperkalemia in Romk1−/− mice under high K+ diet [+ homeostasis by Klotho was examined by Cha et al. (2009). The intravenous administration of soluble Klotho in rats increased urinary K+ excretion. It was due to enhanced net tubular secretion of K+. They found that soluble Klotho appeared in urine, which supports the thesis that Klotho regulates ion channels from the luminal side [+ level [+ [+ level needs to be further investigated. It also remains unclear how injected Klotho reaches urinary space. Hu et al. (2016) proposed that Klotho is expressed in the kidney, released into the systemic circulation by secretases-mediated shedding, and cleared from blood through the kidney into the urine. They showed that Klotho traffics across renal proximal tubules by transcytosis and is secreted into the urinary lumen [i-IIa in the renal proximal tubules. Through its β-glucuronidase activity, Klotho modifies NaPi-IIa glycans and leads to their internalization from the apical membrane leading to decreased absorption of phosphates, also independently of FGF23 [It needs to be pointed that soluble form of Klotho can maintain the ion homeostasis independently of FGF23. Klotho shows the sporters , 24, 25. tubules –29. It i linkage , 31. It enhanced –37. It wenhanced . Moreove K+ diet . The regnal side . Surpris[+ level . It may level [+ , 38–40. ry lumen . Other pry lumen . It shouof FGF23 .βKlotho, the another Klotho family member, is a coreceptor for FGF15/19 and FGF21 and influences energy metabolism [βKlotho and FGFR4 in the liver. In consequence, the gluconeogenesis and synthesis of bile acids are suppressed (negative feedback loop), while the production of hepatic glycogen and proteins is increased [βKlotho and FGFR1c in the white adipose tissue and consequently promotes the lipolysis. FGF21 also stimulates a glucose uptake in the adipocytes and induces an insulin synthesis in the pancreatic β cells [β-Klotho is necessary for FGF19 and FGF21 to regulate weight loss and glycemia [βKlotho, FGF19, and FGF21 activate the sympathetic nervous system and then metabolism of the brown adipose tissue. Therefore, the enhanced insulin sensitivity and thermogenesis are observed [tabolism , 43. Posncreased , 14. By  β cells . The recglycemia . Based oobserved .β1 (TGF-β1) signaling pathways [Soluble form of Klotho is recognized as having the activity of endocrine, autocrine, and paracrine hormone. It has been shown that Klotho is involved in the regulation of oxidative stress, inflammation, and fibrosis by the inhibition of insulin/insulin-like growth factor-1 (IGF-1) and transforming growth factor-pathways , 47–49.It is well documented that the forkhead box protein O transcription factors (FoxOs) are suppressed through phosphorylation by the phosphatidylinositol 3-kinase (PI3K)/serine-threonine kinase (Akt) signaling pathway. FoxOs control genes which are involved in the cellular differentiation, growth and survival, cell cycle, glucose and lipid metabolism, and detoxification of reactive oxygen species (ROS) , 50. The in vivo oxidative DNA damage) of long-lived Klotho-overexpressing transgenic mice was reduced. It suggests the Klotho-induced prevention of oxidative DNA damage [In 2017 Lim et al. analyzed the tacrolimus-induced oxidative stress in mice . They deA damage . klotho gene was disclosed in the study of mouse inner medulla collecting duct 3 (mIMCD3) cells that have undergone an oxidative stress. An oxidation was caused by an exposure to hydrogen peroxide (H2O2). Surprisingly, an insertion of exogenous kl gene into mIMCD3 cells resulted in the significantly diminished H2O2-induced apoptosis. This confirms that Klotho may protect cells from death caused by oxidative stress [ kl gene expression protected tissue from I/R injury [There are speculations that expression of Klotho is decreased under the sustained stress conditions , 53. TheR injury . SimilarR injury . TherefoR injury . KLOTHO (KL) gene were recently shown. They are characterized by their large degree of pleiotropic associations, especially with the kidney diseases, coronary artery disease, stroke, and bone mineral density [More than 10 mutations or single nucleotide polymorphisms (SNPs) in human density , 57.KL-VS, an allele of KLOTHO gene, is a common haplotype in general population. It was defined by the presence of 6 SNPs in exon 2 and flanking sequence. Three mutations of KL gene in exon 2 include two amino acid substitutions  and one silent mutation (K385K). KL-VS allele affects the metabolism and activity of Klotho, contributes to the human age-related phenotypes, and is connected with a reduced lifespan in homozygosity [ KL-VS could be a marker of enhanced risk for the development of cardiovascular diseases [ KL-VS is related to an enhanced level of serum high-density lipoprotein, systolic blood pressure, and an augmented risk of stroke. This supports the hypothesis of Klotho association with atherosclerosis [ KL gene, and carotid atherosclerosis in hypertensive patients [zygosity –62. It azygosity , 60. It diseases . The invclerosis . In addipatients . KL gene. They are recognized as being associated with hypercalcemia, hypophosphatemia, phosphaturia, and low bone mineral density [ KL gene and cardiovascular risk factors in an examination of healthy Korean females [The single nucleotide polymorphisms G395A located in the promotor and C1818T in exon 4 are other, well-studied, variants of human density , 65, 66. density . Interes density . Similar females . The ana females . Moreove females . It is k females . The out females . Substan females . KLOTHO gene polymorphisms and occurrence of atherosclerosis, coronary artery disease, or left ventricular hypertrophy. Thus, the allele A of KL gene could be considered as the cardiovascular risk factors.To conclude, the presented data showed the importance ofTaking into account the fact that Klotho is involved in a resistance to oxidative stress, some scientists have focused on the role of Klotho in the development of cardiovascular diseases .The first investigation of plasma Klotho in the aspect of CVD was performed in 2011 by Semba et al. . The anaβ-Klotho, FGF21, and FGF23 proteins are expressed in cardiomyocytes and subjects at higher cardiovascular risk had a reduced expression of cardiac Klotho and an elevated expression of cardiac FGFs [κB), which is accountable for the inflammatory responses to a variety of signals [κB and overexpression of iNOS in cardiomyocytes lead to the cellular dysfunction and damage and, hence, result in the cardiomyopathy, bradyarrhythmia, and sudden cardiac death [β1 intensified fibrosis [Based on the previous studies proclaiming a potentially positive effect of Klotho in CVD, in 2016 Corsetti et al. assessed that Klotho is expressed in myocardial tissue and proved its association with the prevalence of CVD [iac FGFs . An expr signals –75. Therac death –74. Impoac death , 73. An There is a link between Klotho deficiency and CKD, renal disorders, and kidney damage. Several studies mentioned that Klotho can protect the kidneys against decline of their function , 77. In  in vivo research of Klotho-deficient mice (kl-/kl-) subjected to restraint stress showed that kl-/kl- mice died more frequently than wild-type animals. The sinoatrial conduction block or sinus arrest in kl-/kl- mice (shown as bradyarrhythmias) was observed [Interestingly, Klotho protein was also found in the sinoatrial node pacemaker cells in mice . An in vobserved . Therefoobserved .The above data showed that Klotho deficiency is independently related to the development of cardiovascular disease and its proper expression could provide the cardiovascular protection. An intracellular distribution of Klotho in myocardium is necessary for appropriate metabolism of cardiomyocytes and heart physiology.Experimental studies of Klotho protein in ischemic tissues have apparently affirmed its protective role in several animal models. Zhou et al. (2018) investigation of ischemic injury in murine brain proclaimed the neuroprotection and inhibition of neuropathological changes after an induced overexpression of Klotho . SimilarκB and block its activation and can modulate the response to inflammatory cytokines [κB and led to decreased production of proinflammatory mediators. These novel findings suggest that the inflammatory response and disrupted cardiac function are associated with Klotho deficiency. Nevertheless, Klotho may serve as a therapeutic factor in the amelioration of endotoxemic cardiac disturbances related to aging [Klotho has been widely studied due to its protective role in toxemic heart damage . HSP70 iytokines , 84. In κB, confirming suppressed inflammatory responses. The antioxidative effect of Klotho was also constituted through the augmented expression of nuclear factor erythroid 2-related factor 2 (Nrf2), which plays a pivotal role in the regulation of oxidative stress [In 2018, Guo et al. presented a study of cardioprotection by Klotho in a hyperglycemia-induced injury of hearts in diabetic mice, as well as in cells subjected to high glucose . Regardie stress . Activate stress . Considee stress . For thie stress .The above findings constitute the importance of Klotho in reduction of cardiac dysfunction and morphological changes. Klotho is highly involved in cell defense against the oxidative stress and may induce the restoration of cardiac function in response to myocardial injury. Klotho could be tested as a potential therapeutic factor in myocardial tissue impairment.βKlotho in cardiac tissue is based on its interaction with FGF21 as a coreceptor , a major controller of cardiac lipid metabolism [Insight into the regulation of FGF21 gene expression in the heart showed that Sirt1-PPARechanism , 90. It tabolism . Interestabolism . MoreoveβKlotho [βKlotho network induced the phosphorylation of cell survival PI3K/Akt1 pathway. Then, Akt1 (Akt serine/threonine kinase 1) phosphorylated the Bcl-XL/Bcl-2-associated death promoter (BAD), and finally it exerted the separation of antiapoptotic proteins Bcl-XL and Bcl-2, which inhibited the activity of caspase-3 and subsequently apoptosis [βKlotho is associated with FGFR1/FGF21 signaling in cardiac tissue. Moreover, FGFR1/FGF21 interaction was declined due to suppressed expression of βKlotho, and deficiency of FGF21 led to high abundance of apoptotic cells in I/R model [Some scientists induced the myocardial I/R injury in mice by the occlusion of left anterior descending coronary artery. Interestingly, they tested the signaling mechanism which mediates the FGF21-based myocardial protection. An enhanced production and release of FGF21 from the hepatic cells and adipocytes during cardiac I/R were observed . Data alβKlotho . Activatpoptosis , 84, 94.poptosis . This co/R model .βKlotho complex was also proved in the study of Liang et al. (2017) [βKlotho in H9c2 rat myoblasts. Moreover, the induction of cell survival ERK1/2  signaling pathway by FGF21 abolished ER stress-induced injury of cardiomyocytes, increased cell viability, and reduced apoptosis [The stimulation of cell survival pathways by FGFR1/FGF21/. (2017) . Tunicampoptosis . The actpoptosis . Similarpoptosis .βKlotho functions as a coreceptor for FGF21. βKlotho and FGFR1 are present in the plasma membrane of cardiomyocytes. The reaction of FGFR1 and β-Klotho is necessary for FGF21 sensitivity and ability to activate intracellular signaling pathways [βKlotho signaling induces cell survival and antioxidative mechanisms and recovery of energy supply in cardiac cells [β-Klotho in cardiac cells improve the function of left ventricular cells. It suggests the beneficial impact of FGF21 and βKlotho [It was reported that the administration of recombinant FGF21 led to inhibition of inflammation and prooxidative signaling , 97. As pathways , 98. FGFac cells , 97. Wha βKlotho .βKlotho has been confirmed in the hearts of lean and obese rats that were perfused with Langendorff method [βKlotho [β-Klotho in adipose tissue and liver [α inhibits the expression of β-Klotho in adipocytes and impairs FGF21 action. As a result, cells lacking β-Klotho were unable to respond to FGF21 [βKlotho in obese animals, which may account for “FGF21 resistance” and disruption in the FGFR1/FGF21/βKlotho signaling [βKlotho is crucial in cardioprotective effect of FGF21 administration. What is more, FGF21 resistance could also be due to decreased expression of both βKlotho and FGF receptors [βKlotho remained unaltered [βKlotho background.The cardioprotective role of FGF21 and f method . After tf method . It is p[βKlotho , 95. Hownd liver , 100. Itto FGF21 . The benignaling . Thus, teceptors . It decrnaltered . These fβKlotho could be involved in the protection of cardiac cells from I/R. The cardioprotective effect of FGF21 was shown in the mouse model of myocardial infarction caused by the permanent ligation of the left anterior descending coronary artery (LAD). The treatment with intramuscularly injected adenoviral vectors expressing FGF21 led to mitigation of left ventricular systolic dysfunction and dilatation after LAD ligation [γ co-activator-1 α (PGC1α). PGC1α is a transcriptional coactivator, involved in the control of energy metabolism and oxidative stress in several tissues [γ represses expression of proinflammatory cytokines by targeting NF-κB signaling [α axis by FGF21 could be envisioned as a new therapeutic target in metabolic disorders and heart disfunction. Interestingly, in 2015 Planavila et al. revealed new action of FGF21 in prevention of ROS production in cardiac cells [FGF21 and ligation . The mailigation . The inh tissues , 104–106ignaling . So, theac cells . They shac cells .βKlotho. Thus, the modulation of FGFR1/FGF21/βKlotho signaling pathway could be a novel strategy in cardioprevention.The potential impact of these investigations is that FGF21 can improve the function of left ventricular cells and may be considered as a therapeutic agent. It should be emphasized that the cardioprotective action of FGF21 is possible only in the proper expression of FGFR1 and 2+-permeable cation channels, are expressed in heart and mediate in the Ca2+-dependent signal transduction [2+-signaling. It is elucidated that TRPC channels, including TRPC1, 3, 4, 5, and 6, promote the cardiac hypertrophy by an activation of calcineurin, nuclear factor of activated T cells (NFAT), the Ca2+-dependent hypertrophy-inducing pathway [Cardiac hypertrophy and remodeling occur in response to various pathogenic stimuli, such as chronic pressure overload, chronic volume overload, or myocardial infarction. Consequently, the pathological alterations of heart structure and function are observed , 109. Itsduction –113. The pathway –112. It  pathway . Additio pathway .The mechanism of Klotho cardioprotection in myocardial impairment is also based on its antioxidative activity. In 2015 Yang et al. tested an influence of indoxyl sulfate (IS), a uremic toxin, and Klotho protein on the heart function and their association with left ventricular hypertrophy . It is kIn favor of a rationale that supports the compensatory action of Klotho, an association between serum Klotho level and classic risk factors, as well as a clinical history of cardiovascular diseases, has also been investigated. The examination group included 168 older adults with the prior coronary artery disease, myocardial infarction, and stroke . Surpris in vivo study of mice with hypertension. There was more intensive cardiac interstitial fibrosis in the 130 kDa Klotho differentiation of myofibroblasts, whereas 65 kDa isoform has the antifibrotic effect [β-cell in β-islets was assessed. As a result, only short-form Klotho promoted insulin secretion in β-islets. The full-length 130 kDa Klotho did not have obvious effect [α-Klotho from short fragments, as well as shed from secreted Klotho. This could be the reason why investigators do not make a distinction regarding the treatment with Klotho isoforms and do not account for their suspected differential effect. They mostly describe the administration just of recombinant Klotho. The work of Liu et. al. (2016) highlighted the advantageous usage of soluble isoforms of Klotho as a potential strategy for the development of novel therapeutic interventions in cardiology.It has become clear that hypertensive heart disease is associated with the cardiac fibrosis. Myofibroblasts, the phenotypically transformed fibroblasts, are present in heart due to injury and tissue repair. However, myofibroblasts are also involved in cardiac remodeling and thus contribute to heart disorders –126. In c effect . This noc effect . So, thec effect . The stic effect , 129. Inc effect . Therefoc effect . It shous effect . The pros effect . HoweverSumming up, there is a rational basis for Klotho administration in the event of heart injury to restrain the further pathological modifications in cardiac tissue. The myocardial protection of Klotho is based on its contribution in the multifarious mechanism. However, it should be considered that the isoforms of Klotho may act as a counter-regulatory factor in the cardiac fibrogenic responses.3, it was proved that vitamin D receptor (VDR) ligands render the cardiovascular survival benefits [It might be important to consider that significant reduction of Klotho expression in vascular wall is associated with the development of artery dysfunction. An expression of endogenous Klotho in the human aortic smooth muscle cells (HA-SMCs) and in the medial layer of renal and epigastric arteries from healthy and CKD patients was described . It was benefits , 22, 133 via ADAM17 protease [A large number of studies over the last decade have greatly enriched our knowledge of the antioxidative activity of Klotho and its correlation with an endothelium function. In 2016, Richter et al. analyzed an influence of synthesis of NO mediated by FGF23 and oxidative stress on human coronary artery endothelial cells (HCAEC) . As mentprotease . Afterwaprotease .It is also suggested that an activity of NADPH oxidase 2 (Nox2) leads to the generation of ROS, while MnSOD and catalase detoxify ROS. The investigation showed that FGF23 increases the expression of Nox2 and antioxidant proteins . Essenti via an activation of PI3K/Akt/eNOS pathway. Consequently, the enhancement of eNOS activity and production of NO were observed [Reduction of oxidative stress may contribute to the development of effective treatment strategies for atherosclerosis. In 2017 Yao et al. analyzed an association between Klotho and atherogenesis in an ox-LDL-induced endothelial cell injury model with human umbilical vein endothelial cells (HUVECs) . HUVECs observed .Klotho treatment also reduced the expression of lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1). LOX-1 is a major ox-LDL receptor in endothelial cells, which is a crucial factor in the pathogenesis of atherosclerosis . Thus, tTo conclude, the presence of Klotho protein in vascular wall is necessary for the functional amelioration of vasculature and protects against the procalcific processes. KLOTHO gene polymorphism can be the risk factors for the most prevalent cardiovascular diseases. Thus, the regulation of serum Klotho and FGFs level and their expression in cardiomyocytes could be essential for the cell metabolism, proper heart function, and protection in some disorders. Given the apparent importance of Klotho, it may be considered as a novel vital factor in the ischemic heart injury, such as myocardial infarction. Conclusively, it appears plausible that an activity of Klotho could be protective in a damaged myocardial tissue and open new path for the treatment of cardiovascular diseases.There are many well documented evidences indicating the antioxidative and antiapoptotic activity of Klotho protein. It is also well established that Klotho plays an important role in the prevention of cardiovascular diseases, particularly in the maintaining of appropriate cardiac and vascular function. Klotho is involved in mechanisms of defense against the development of heart hypertrophy and remodeling, as well as the vascular calcification and atherogenesis. Many studies showed that Klotho deficiency or


Transcriptome profiling of 3D models compared to 2D models in various cancer cell lines shows differential expression of TGF-β-mediated and cell adhesion pathways. Presence of TGF-β in these cell lines shows an increased invasion potential which is specific to cell type. In the present study, we identified exogenous addition of TGF-β can induce Epithelial to Mesenchymal Transition (EMT) in a few cancer cell lines. RNA sequencing and real time PCR were carried out in different ovarian cancer cell lines to identify molecular profiling and metabolic profiling. Since EMT induction by TGF-β is cell-type specific, we decided to select two promising ovarian cancer cell lines as model systems to study EMT. TGF-β modulation in EMT and cancer invasion were successfully depicted in both 2D and 3D models of SKOV3 and CAOV3 cell lines. Functional evaluation in 3D and 2D models demonstrates that the addition of the exogenous TGF-β can induce EMT and invasion in cancer cells by turning them into aggressive phenotypes. TGF-β receptor kinase I inhibitor (LY364947) can revert the TGF-β effect in these cells. In a nutshell, TGF-β can induce EMT and migration, increase aggressiveness, increase cell survival, alter cell characteristics, remodel the Extracellular Matrix (ECM) and increase cell metabolism favorable for tumor invasion and metastasis. We concluded that transcriptomic and phenotypic effect of TGF-β and its inhibitor is cell-type specific and not cancer specific. Ovarian cancer is one of the most lethal gynecological malignancies, which accounts for 5% cancer deaths among women . The oveThe in vitro cancer cell line models of 2D cultures are not an ideal system to explain the molecular mechanisms controlling EMT, metastasis and chemoresistance. Moreover, 2D models usually lack biological requirements which explain hypoxia and angiogenesis. When cancer cells grown in 2D, they have a uniform and rapid proliferation across the plastic surface unlike the 3D models that show many zones of differential proliferation and cell division, mostly confined to outer zones ,7. The gIt is a well-known phenomenon that TGF-β plays an important role in the tumor progression and actin cytoskeleton reorganization . The rolGlobal gene expression studies by RNA-Seq analysis in anchorage-independent 3D ovarian cancer cells compared to 2D models have shown that TGF-β signaling is one of the most altered pathways in 3D spheroid models A Supple. MoreoveInvasion of 3D spheroids was measured by matrigel coated Boyden Chamber Assay C. These Seven cell lines  were used to analyze expression pattern of the EMT signature genes in the presence of TGF-β1. The 3D spheroids treated with TGF-β for 14 days partially activate EMT by inducing some of the EMT signature genes D. From the We established anchorage-independent models for five cancer cell lines by culturing them in an ultra-low attachment plate for 14 days. Cell lines displayed different sizes of 3D structures in anchorage-independent cultures A. PA-1 aA2780, APOCC, CAOV3, A2780 CIS cell lines showed significant increase in the spheroid size of the TGF-β-treated multiple cell spheroids. In contrast, SKOV3 cell line displayed a significant reduction in the size of the spheroids when treated with TGF-β. Among the 3D model systems, CAOV3 and A2780 cell lines increased the spheroid size significantly compared to the other cell types B. The spThe size of the TGF-β-treated and control spheroids was also analyzed. The average size of the spheroids of the ovarian cancer cell lines is as following: PA-1 [433 um], SKOV3 [339 um], OVCAR3 [55 um], SW626 [149um], APOCC [50 um], A2780 [53 um], A2780 CIS [44 um], CAOV3 [26 um] B. Most oOur next aim was to study whether TGF-β treatment could alter affinity towards various essential ECM components. Not all ovarian cancer cells exhibited a similar pattern of adhesion affinity towards ECM components alone or in combination. SKOV3 cells showed the highest affinity towards ECM components as shown in the intensity plot A. SKOV3 TGF-β increased cell growth percentage in CAOV3, APOCC, SKOV3, OVCAR3 and A2780 cis cell lines in 2D models compared to the other cell lines A. Only CCAOV3 and SKOV3 are the promising cell lines to study invasion and migration among the selected cancer cells both in 2D and 3D in vitro models. To validate the effect of TGF-β in these cancer cells, we used conventional 2D cultures for TGF-β inhibitor-based study. Both cell lines responded well with the TGF-β treatment and increased the invasive and migratory potential. TGF-β treatment fastened the wound healing process and facilitated invasion C,D,E. InLY364947 was able to induce cell proliferation reduction in a dose-dependent manner with an IC50 value of 52.73nM in CAOV3 and 118.5nM in SKOV3 in 2D models C. The TGIn conclusion, we demonstrated that both 2D and 3D phenotypes are efficient models in cancer invasion and migration studies. TGF-β—a well-known cytokine for its protumorigenic properties in cancer—can induce partial EMT in SKOV3 and CAOV3 cells both in 2D and 3D models. Our results suggest that TGF-β can induce partial EMT, cell survival, increased metabolism, ECM remodeling and sensitivity to LY364947 . FurtherResults of the current study revealed that 3D anchorage-independent spheroids turned out to be an aggressive phenotype as it activates most of the EMT inducing genes and downregulates E-cadherin in the cell lines that were studied. CAOV3 and SKOV3 3D models retain a better phenotype by upregulating most of the cell adhesion molecules like collagen 1, 3 and 5 . As repoThe 3D models show abnormal ECM that may promote invasion. An increase in collagen deposition or ECM stiffness alone or in combination upregulates integrin signaling and promotes survival and proliferation ,24. StudRNA-Seq analysis, showing differential expression in 3D and 2D phenotypes, revealed that 3D spheroids show better cell-to-cell interaction and cell adhesion . SurprisOur results suggest that 3D spheroids have more heterogeneity and activate tumor-promoting pathways compared to 2D models. The most noted one among those pathways is TGF-β pathway for EMT. EMT is thought to be activated in cancer cells, linked to their dissociation from the primary tumor and intravasation into blood vessels . HoweverTGF-β is a pleiotropic cytokine that controls proliferation, differentiation, embryonic development, angiogenesis, wound healing and other functions in many cell types. TGF-β acts as a tumor suppressor in normal epithelial cells and in early stage of tumor progression. In advanced cancers, the corrupted TGF-β pathway induces many activities that lead to growth, invasion and metastasis of cancer cells ,42,43. TIn our study, TGF-β seems to induce a partial EMT—like most tumor cells—in the cell lines studied in 3D spheroids. Our results suggest that TGF-β can induce partial EMT and enhance invasion potential in ovarian cancer cells. As per earlier studies depending on signaling context, epithelial cells that lose some epithelial characteristics or attain both epithelial and mesenchymal characteristics are called partial EMT . The proWe used ECM array to investigate the interaction between TGF-β induction in cancer cells and ECM stimuli. ECM array has 36 combinations of ECM protein conditions that are deposited onto the hydrogel surface as printed array spots. Among the cell lines studied, SKOV3 shows a better adhesion capacity and responds well with TGF-β treatment. SKOV3 cells show a better and stronger attachment to many of the ECM combinations compared to the other cell lines. The intensity plot shows that SKOV3 has a better attachment compared to the other cell lines. EMT in the SKOV3 cell line is ECM component-dependent. fibronectin and laminin combination show more attachment in SKOV3 cells after TGF-β induction. Combinations of ECM show a better attachment ability compared to individual ECM components. SKOV3 cells in 3D phenotypes show EMT induction and high level of attachment towards collagens and other ECM components, which may increase the complexity in EMT and matrix stiffness. A higher level of matrix stiffness condition has been implicated in TGF-β induced EMT and stronger ECM adhesion capability of spheroids . The revAmong the cell lines studied, CAOV3 and SKOV3 are the two promising cell lines to be used as a model system for studying EMT and invasiveness in ovarian cancer cells. Both 3D and 2D models of SKOV3 and CAOV3 cells responding to TGF-β and its receptor specific inhibitor treatments signify that both cell lines are promising to study EMT or invasion in cancer. Prolonged TGF-β induction in 3D models is more promising, as it can mimic the whole tumor microenvironment and explain many cell adhesion properties. As TGF-β inhibitors are mainly targeting the tumor microenvironments with a minimal cytotoxicity, they should be used in combination with cytotoxic agents to kill the cancer cells and revert tumor favorable microenvironments. LY364947 has a potential to be a future TGF-β-targeted anti-cancer therapy, since it efficiently reverses the effect of TGF-β in cancer cells.Eight ovarian cancer cell lines—PA-1, SKOV3, SW626, CAOV3, OVCAR3 , APOCC (cell line derived from ascites fluid), A2780 and A2780 CIS (A2780 cisplatin-resistant cell line) —have been used for the current study. Cells were cultured in DMEM  supplemented with 10% fetal bovine serum (FBS) . Cell cultures were incubated at 37 °C in a humidified atmosphere, 5% CO2-injected incubator. Cell confluency and morphology were routinely checked. Cells were washed with phosphate-buffered saline (PBS)  then fresh growth media 10% FBS DMEM- was added every two to three days. Cellular viability was determined by Trypan blue exclusion in TC20 Automated Cell Counter .2 incubator. Cells were grown in ultra-low attachment dishes for 2 weeks to make 3D anchorage-independent models. The 3D anchorage-independent models were imaged by Axio Imager 2 Research Microscope  and the average size of the spheroids was measured using the Zeiss ZEN Microscope Software for Microscopic Components.Multicellular cancer spheroids (MCS) were generated from a heterogeneous parental population by seeding cells growing during log-phase in 100mm ultra-low attachment culture dishes , culturing them in DMEM /F12 supplemented with 5% FBS then incubated at 37 °C 5% COSYBR Green Master (Rox)  was used to amplify the specific gene using cDNA primers obtained from PrimerBank (http://pga.mgh.harvard.edu/primerbank/) . cDNA synthesis was done using first strand cDNA synthesis kit for RT-PCR (AMV) (Roche Applied Science). FastStart Universal erbank/) . Each re®-3 Stain (life technologies). Cell adhesion patterns of TGF-β-treated and untreated samples were also analyzed by extracting the intensity of cells attached to the ECM using ImageQuant TL 8.1 Software.ECM-cell adhesion pattern of different ovarian cancer cells was analyzed by using MicroMatrix ECM Array . The array consisted of 36 different combinations of selected cell adhesion molecules. Cells were attached to the ECM combinations according to its specificity towards different ECM components. Images were extracted using a GE Typhoon Array Scanner after fixing and staining the array with TO-PROAnchorage-independent spheroids and 2D cells were treated with 10ng/mL TGF-β1  for 2 weeks. Spheroid size was measured by Axio Imager 2 Research Microscope  and the average size of the spheroids was measured using the Zeiss ZEN Microscope Software. Two weeks old spheroids (both TGF-β-treated and control) were seeded on the top of collagen gel  with a concentration of 2mg/mL in chamber slides . ® AQueous One Solution Cell Proliferation Assay (MTS) . Cells were grown in 2D (96 well plate BD) and 3D anchorage-independent condition using ultra-low attachment 96 well plate  with seeding density of 5000 cells per well for 3 days. The effect of cell proliferation in the presence of TGF-β and LY364947  in 2D and 3D models was measured.The effect of TGF-β on cell proliferation in different cell lines was measured by CellTiter 966 cells/well. Confluent cell monolayers were disrupted by standardized wound scratching using a sterile 200-μL pipette tip and incubated in serum-free culture medium with 10ng/mL TGF-β1 in both treated and LY364947 treated cells. Migration of cells into the bare area and recovering of monolayer was evaluated 18 h, 24 h, 48 h and 72 h using Axio Imager 2 Research Microscope  and the average distance of the scratch at 18h was measured using Zeiss ZEN Microscope Software.The CAOV3 cells were cultured in 6-well plates with seeding density of 1 × 105 cells/well) onto the top chamber of ECM-coated with 8µm pore membrane filter. ECM consisted of a combination of matrigel, collagen I and fibronectin (2:1:1) or matrigel alone (10 mg/mL). A total of 2 ml of DMEM supplemented with 10% FBS was placed in the bottom chamber as a chemoattractant. After 24 h, invasion was evaluated by counting the number of cells penetrating the membrane, counting the cells in the top chamber (noninvasive) and the bottom of the membrane (invasive). Those cells—which had undergone EMT induction either with or without the addition of TGF-β—were used for the in vitro invasion assay. Cells that migrated through the matrigel and 8-micron pore size membrane towards the medium with 5% serum were then counted [The transwell chamber  was used to measure the invasive abilities of cells according to the modified protocol. Briefly, the pre-treated with or without TGF-β1 (10 ng/mL) cells were seeded  with sequential injection of 1mM oligomycin A, 1mM FCCP and 0.5 mM rotenone/antimycin A.The Seahorse XFe96 Analyzer  was used to continuously monitor OCR and ECAR. A time of 72 h prior to the experiment, 10000 cells/well were seeded in a XF96 Cell Culture Microplate in DMEM media supplemented with 2.5% FBS, 25 mM glucose, 2mM glutamine, 1 mM sodium pyruvate and cultivated at 37 °C in humidified atmosphere and 5% COt-test. p < 0.05 was considered as significant difference between groups. The half maximal inhibitory concentration (IC50) of LY364947 was calculated using non-linear regression method. The analysis was done using Prism Software Version 7.Data represents means ± S.E.M. with at least two to three biological replicas in triplicate. Statistical analyses were determined by using Student’s


Signatures of normal and defective DNA repair and replication continued to be generated at roughly stable mutation rates. Signatures of APOBEC cytidine deaminase DNA-editing exhibited substantial fluctuations in mutation rate over time with episodic bursts of mutations. The initiating factors for the bursts are unclear, although retrotransposon mobilization may contribute. The examined cell lines constitute a resource of live experimental models of mutational processes, which potentially retain patterns of activity and regulation operative in primary human cancers.Multiple signatures of somatic mutations have been identified in cancer genomes. Exome sequences of 1,001 human cancer cell lines and 577 xenografts revealed most common mutational signatures, indicating past activity of the underlying processes, usually in appropriate cancer types. To investigate ongoing patterns of mutational-signature generation, cell lines were cultured for extended periods and subsequently DNA sequenced. Signatures of discontinued exposures, including tobacco smoke and ultraviolet light, were not generated   •Annotation of mutational signatures across 1,001 cancer cell lines and 577 PDXs•Activities of mutational processes determined over time in cancer cell lines•APOBEC-associated mutagenesis is often ongoing and can be episodic•Detection of mutational signatures by single-cell sequencing An analysis of 1,001 human cancer cell lines and 577 xenografts shows that mutagenesis associated with the cytodine deaminase APOBEC occurs in episodic bursts in contrast to mutation signatures associated with DNA replication and repair. Each mutational process operative in a cell leaves a mutational signature imprinted on its genome . Using min vivo suggest that some mutational processes show varying degrees of activity over time  and 577 patient-derived xenografts  derived from a broad spectrum of cancer types. The panel includes most widely used models in cancer research and therapeutics testing and is being extensively characterized genomically, transcriptomally, epigenomically, and for biological responses to therapeutics .in vitro culture and to characterize their temporal patterns of activity. Cell lines continuing to acquire mutational signatures represent informative models for future investigation of their underlying mechanisms.We subsequently used a subset of the cancer cell lines to experimentally assess whether mutational processes underlying mutational signatures continue to be active during The presence and relative contributions of single base substitution signatures (SBSs) were determined in each of 1,001 cancer cell lines  and 577 The majority of the base substitution signatures observed in primary cancers  were fouPOLE, in colorectal, endometrial, and stomach models; SBS36, associated with defective base excision repair and MUTYH mutations  cytidine deaminase DNA-editing activity , in cellutations , in pancutations , in breaFinally, signatures of uncertain and speculative origins  were alsA small subset of signatures was absent from the examined datasets  or found less often than expected  . These mPOLE). SBS1 and SBS5 were detected in most cell lines.To investigate the patterns of activity of mutational processes underlying a wide range of signatures, we selected 28 cell lines derived from cancers of the breast, colorectum, uterus, lung, stomach, cervix, ovary, head and neck, skin (melanoma and squamous), white blood cells (B cell lymphoma and leukemia), and neuroblastoma A. One orin vitro propagation between the two subcloning events  cell line overwhelmed with these signatures. All other signatures present in stock cell lines continued to be generated during culture of descendant clones from at least some cell lines.Certain mutational signatures present in stock cell lines were not generated during t clones . These iin vitro culture  or remained roughly stable   . In someer CW-2) . In otheer CW-2)  continueer CW-2) .Figure SPOLE  and endometrial (ESS-1) cancer cell lines with mutations in POLE  continuein vitro . Furtherro  and breast cancer (AU565) cell lines, which did not show obvious deficiencies in BRCA1 or BRCA2 function  was clearly visible following normalization of mutation frequencies to account for depletion of NCG trinucleotides in the human genome   .in vitro culture were highly variable between cancer cell lines, between different daughter lineages of the same cancer cell line, and over time in the same daughter lineages  and two B cell lymphoma  cell lines to further rounds of subcloning and the squamous carcinoma of the tongue cell line (CAL-27) to seven serial rounds of subcloning over short periods of time . These e100-fold .Figure 4in vitro in the same set of clones  (in vitro were largely enriched in the YTCN/YTCA sequence context (where Y is a pyrimidine), consistent with episodic mutagenesis mainly being mediated by APOBEC3A (The particular APOBEC family member(s) that causes the SBS2 and SBS13 mutations observed in cancer is currently a matter of discussion. utations . Howeverponsible . Indeed, purine) . The SBSAPOBEC3A .in vitro-acquired SBS2 and SBS13 mutations and expression levels of APOBEC genes were not detected  and two B cell lymphoma cell lines  . The cledetected B. It rem in vivo .in vivo and during in vitro culture represent collateral mutational damage on the host cell genomes from APOBEC-mediated responses directed against viruses and/or retrotransposons. The BC-1 and JSC-1 B cell lymphoma lines, which continue to generate SBS2 and SBS13 in culture, both carry Epstein-Barr virus (EBV) and human herpesvirus-8 DNA sequences  retrotransposons using the whole-genome sequence data from available clones and examined the relationship between retrotransposition mobilization and APOBEC-associated signatures , most pronounced in breast and lung adenocarcinoma cell lines , and can be deconvoluted from signatures of genome amplification-induced mutations.Single cells, equivalent to parent clones, after subtraction of mutations present in the stock cell line, provide information on mutations acquired since its most recent clonal expansion . HoweverHence, to determine the proportion of cell lines that experienced APOBEC mutagenesis at some point following the establishment of the most recent common ancestor of the stock cell line, we whole-genome sequenced 32 single cells from 16 cancer cell lines exposed to it in the past, alongside single cells from endometrial cell line with POLE-associated SBS10a-b and SBS28 (ESS-1) and an osteosarcoma cell line (G-292-clone-A141B1) with SBS40, which is characterized by roughly uniform representation of all 96 mutation types .Figure 6POLE (scA) and APOBEC activity (scB and scC) and a signature (scD) characterized in part by C>A mutations similar to SBS18, which probably results from in vitro culture (see above). Two novel signatures (scE and scF) were present exclusively in single cells and absent from the corresponding stock cell lines  substrate, changing access of APOBEC(s) to nuclear DNA and retrotransposon mobilization.The variability in acquisition of APOBEC-associated mutational signatures occurs in cell culture despite the absence of many proposed initiators and regulators of APOBEC activity, including the immune system and detectable exogenous virus infections , the clomrs@sanger.ac.uk).Further information and requests for resources and reagents should be directed and will be fulfilled by the Lead Contact, Michael R. Stratton  and the National Cancer Institute’s Patient-Derived Models Repository  and used for annotation of mutational signatures  listed in gnatures .https://www.cancerrxgene.org/gdsc1000/ and were anayzed here as per section ‘1,001 cell line panel expression and methylation datasets’ to find putative aberrations in genes associated with mutational signatures in examined cell lines .Cancer cell lines used in this study originate from the cryopreserved aliquots of 1,001 cell lines, sourced previously from collaborators or public repositories and extensively characterized as part of the Genomics of Drug Sensitivity in Cancer (GDSC)  and COSMA subset of 28 of these cell lines was used to establish 58 parent and 141 daughter clones, which were subject to whole-exome and/or whole-genome sequencing , dependi2 in RPMI or DMEM/F12 media . Single cell DNA was amplified by multiple displacement amplification (MDA) as per the manufacturer’s protocol , using a two-hour amplification time. A no-cell negative control wells were processed with the same reagents to control for background contamination. Amplified DNA was purified using a 1:1 volume ratio of AMPure XP beads (Beckman Coulter) and DNA, following the manufacturer’s protocol.Genomic libraries were prepared with the Custom SureSelect Library Prep Kit (Agilent Technologies), aiming for an average insert size of approximately 500bp and 150bp for, respectively, whole-genome and whole-exome sequencing. The Sure Select Targeted Enrichment kit (Agilent Technologies) was used for exome enrichment. Following cluster generation, 150 base (whole-genome) and 75 base (whole-exome) paired-end sequence data was generated on Illumina HiSeq 2000/2500 (whole-exome) and HiSeq X Ten (whole-genome) platforms. Sequencing reads were aligned to the reference human genome (GRCh37) using the Burrows-Wheeler Alignment (BWA) tool , with ‘bhttps://cancer.sanger.ac.uk/cell_lines). Individual clones obtained here were genotyped by comparing alleles presenting at the same set of SNP loci (not shown). Reads reporting alleles presenting at the enquired loci were quantified and pairwise comparisons generated among individual samples to confirm that all single-cell derived originate from their respective cell lines.All cancer cell lines were genotyped previously by SNP and STR profiling, as part of the COSMIC Cell Line Project (https://github.com/cancerit), by performing comparisons outlined in Base substitutions, indels and rearrangements were discovered using, respectively, the CaVEMan , cgpPindNumbers of mutant and wild-type reads presenting at the loci of the remaining base substitutions and indels were assessed across the related cell line samples using vafCorrect  and filtin vitro parts of the cellular lineages that are of known durations and span two single cell isolation steps used in establishment of the parent and daughter clones . For some cell lines, further series of clonal generations were established to study mutations acquired during multiple consecutive in vitro time frames  the mutation locus was covered in the reference clone by ≥ 15 reads, (2) mutation-reporting reads were not detected in the reference clone at variant allele fraction (VAF) ≥ 5%, (3) mutation-reporting reads were not detected in other available clones from the preceding part of the lineage at average VAF ≥ 5%, (4) mutation-reporting reads did not present at average VAF ≥5% across clones from the other available parent lineage(s) where mutation-reporting reads were detected, (5) mutation-reporting reads were not detected in sister clones derived from the same parent at average VAF ≥ 5% if the reference locus was covered by < 30 reads . Rearrangements were considered only if not detected in other related clones within 50bp of identified breakpoints.To retain mutations acquired predominantly https://cancer.sanger.ac.uk/cell_lines). These were generated previously using an unrelated normal human genome as a reference sequence and were filtered extensively against the common germline variation . To remove possibly pre-existing mutations acquired prior to this time frame, base substitutions and indels were only considered if (1) the mutated locus was covered in the corresponding stock cell line by ≥ 15 reads, (2) mutation-reporting reads were not detected in clones from the other available parent lineage(s) at average VAF ≥ 5%, (3) mutation-reporting reads were not detected in the corresponding stock cell line at VAF ≥ 5%.Mutations in whole-exome sequenced parent clones  were dishttp://www.completegenomics.com/public-data/69-Genomes/) (A subset of stock cell lines and subset of parent clones (from different sets of cell lines) were subject to whole-genome sequencing to be used as, respectively, references for their related single cells  and daugenomes/) , as descenomes/) , as wellTo derive proxies for mutational catalogs of the stock cell lines from which whole-genome sequenced parent clones were derived  and 4, wfref = allele frequency of 1000 genomes a0 allele, falt = allele frequency of 1000 genomes a1 allele, logRunadj = unadjusted logR, mean(logRunadj) = the mean value of logRunadj across all 1000 genomes loci. To identify regions with variable copy number, logR and BAF of related samples were jointly segmented using the multipcf function in the R package copynumber ((i)th and 95th centiles of the difference in logR between related clones in regions of ‘constant’ copy number regions were reclassified as ‘variable’.Regions with differences in BAF of > 0.03 and < 0.01 between related clones were classified as, respectively, ‘variable’ and ‘constant’ in copy number. Regions with differences in logR outside the range between the 5(ii)Regions with variable copy number states and variable mutation rates were identified as regions variable in copy number states with significant differences in the ratios of mutation rates compared to the ratio of rates calculated for regions constant in copy numbers (Poisson test p < 0.01).Using this approach, variants that were present in the most recent common ancestor cell of the related parent clones, but reside on an allele that was subsequently lost in only one of the clones would be misclassified as ‘private’. Loss of an allele in the setting of a polyploid cell line setting may not always manifest as B allele frequency ∼1 , expectpynumber . Joint aClonality of the established cell line clones was examined using the proportion of the mutation-reporting reads  at the mutation loci remaining after the described filtering steps. Most of the VAF distributions deviated from average 50%  expectedSome clones exhibited a shift toward lower distribution of VAF compared to other related clones, pointing to putative establishments of such clones from multiple cells or from single cells that underwent genomewide increases in the ploidy. Such instances may result in an increased mutational burden due to availability of more DNA bases and hence more opportunity for a mutation to occur  of single cell DNA leads to (1) loci dropouts, (2) allele dropouts where only some alleles at the loci are amplified or (3) imbalanced amplification of alleles presenting at given loci. To estimate the proportion of somatic mutations that were likely missed due to WGA-associated losses, we annotated a set of high confidence heterozygous germline SNPs  on the sMutations in single cells were discovered by using related stock cell lines as the reference sequences (comparisons in CN contexts) were enriched among mutations presenting at < 50% of the sequencing reads in single cells, both genomewide and when only haploid regions were considered . Germline mutations were filtered out from the lists of reported mutations using the complete list of germline mutations from dbSNP (http://www.completegenomics.com/public-data/69-Genomes/)  , as wellBase substitutions were classified into 96-channel catalogs based on their sequence context, by considering the bases immediately 5′ and 3′ to each mutated base using the ENSEMBL Core APIs for human genome build GRCh37. Because there are six classes of base substitutions  and 16 possible sequence contexts for each mutated base, there are 96 possible mutated trinucleotides.Indels were classified as described previously , by inteKataegis, or foci of localized hypermutation , exhibiting strand-coordination and have an average intermutation distance of ≤1,500 bp. This approach may miss some foci, sacrificing sensitivity of detection in obtaining higher positive predictive value of kataegis foci.mutation , were qumutation . A kataeCN) frequently arise during cell cultivation. Briefly, enrichment analysis quantifies how frequently C>T and C>G mutations occur in a sequence context of interest, compared to the total number of C>T and C>G mutations. For example, to calculate enrichment for mutations at TCN sites the following was used:Sequence contexts of interest were quantified in the reference human genome (GRCh37) across autosomal chromosomes and within regions considered by the CaVEMan algorithm in detection of base substitutions B and S4CMutTCN represents the number of TCN > TGN or TCN > TTN mutations; MutC the number of C>G and C>T mutations; ConTCN and ConC represent the number of occurrences of TCN contexts  and cytosines (or guanines), respectively.Doubling times were measured for a total of 26 available daughter and granddaughter clones from the BC-1, JSC-1, MDA-MB-453 and BT-474 cell lines. Clones were thawed and recovered in culture for 14-21 days prior to seeding. 100,000 cells were seeded from each clone and counted every 24 to 72 hours over the course of 8 to 15 days in triplicates. Alive cells, stained with Trypan Blue (Sigma-Aldrich), were counted with a hemocytometer. Doubling times were derived from the cell counts assuming the logarithmic growth,0 and Nt are the cell counts at the seeding time and at the examined time, respectively. T is the doubling time and t are days past seeding at the examination. A linear regression was performed in R using the ‘lm’ function. The 90% confidence interval was calculated using the ‘prd’ function. Numbers of divisions which took place during the examined in vitro periods were estimated for each clone from the measured doubling times and durations of examined time frames , read-pairs which had one or both reads unmapped were identified and bases with Phred quality score < 10 were removed. The remaining sequence was split into non-overlapping 30bp fragments. Terminal fragments were processed without further splitting (30-59bp). The obtained fragments were aligned to the viral GOTTCHA database , at the https://gitlab.com/mobilegenomes/TraFiC), an improved version of the TraFiC (Transposon Finder in Cancer) algorithm  clones using the corresponding parent (and daughter) clones as reference sequences to identify L1 insertions acquired during the examined in vitro time frames . Gene expression levels were estimated with the default settings of the Cufflinks tool (‘OK’) were considered in derivation of FPKM values and further analyses. A positive correlation between expected and observed burden of control Spike-In RNA implied the minimal technical variation affecting the data interpretation (not shown).RNA was extracted using RNeasy Plus kits (QIAGEN) from 26 daughter clones . To contnks tool  .The set of mutational signatures annotated across cell line and PDX datasets was extracted across 2,709 primary human cancers, as part of the Platinum version of ICGC PCAWG release . The 96-https://www.mathworks.com/matlabcentral/fileexchange/38724-sigprofiler. The first step (SigProfiler) encompasses a hierarchical de novo extraction of mutational signatures based on somatic mutations and their immediate sequence context, while the second step (SigProfilerSingleSample) estimates the numbers of somatic mutations in an individual sample associated with a given set of mutational signatures. Numerical and graphical patterns of the 48 Platinum set of PCAWG signatures, including 9 signatures associated with technology-associated artifacts (termed ‘R1-9’ signatures), are provided in The computational framework for identification of mutational signatures across the Platinum PCAWG dataset incorporated two independent and distinct steps, termed SigProfiler (v. 2.1) and SigProfilerSingleSample (v. 1.2) , based oMutational signatures were annotated on cell line and PDX datasets using SigProfiler (v.2.1) and the SigProfilerSingleSample (v.1.2), modified as described below.de novo discovery of mutational signatures across five separate datasets, including 96-channel mutational catalogs K × N, where K reflects the number of mutation types and G corresponds to the number of samples. The algorithm first deciphers the minimal set of mutational signatures that optimally explains the proportion of each mutation type and then estimates the contribution of each signature across the samples. More specifically, the algorithm makes use of a well-known blind source separation technique, termed nonnegative matrix factorization (NMF). NMF identifies the matrix of mutational signatures, N, is based on the robustness of the overall solution; the methodology has been previously described  could be explained by a combination of signatures from the global set (cosine similarity > 0.75) and hence none were considered as novel. Signatures extracted across complete mutational catalogs from single cells (dataset 5) revealed two novel mutational signatures, termed SBS scE and scF, likely associated with the process of single cell lysis and/or WGA of single DNA molecule .The extracted signatures were compared to the set of mutational signatures deciphered from the PCAWG Platinum release . Given tSigProfilerSingleSample. The core set of mutational signatures , using the Poisson family with a log link function. In the case of the null model, for each cell line experiment (i), the fitted model has a mean mutation count mi given by c is a constant, ti is the time spent in culture by the cells in experiment i, Ti is the tumor type from which the cell line was derived, a(Ti) denotes the random effect of this tumor type factor, and ei denotes a random effect which is specific to the experiment. In the case of the alternative model we introduce a predictor variable u which depends on the L1 insertion rate x is the L1 insertion count, and t is the time spent in culture). The predictor variable u is defined as ti. Furthermore, the log(t) term is an “offset” which ensures that the estimated effect sizes (including the slopes) do not depend on the time in culture. The fitted model has a mean mutation count mi given by bTi denotes the random effect of this tumor type factor on the slope of the predictor variable ui, and as before, aTi denotes the random (intercept) effect of this tumor type factor. The Wald test was used to test the null model against the alternative model. A separate test was performed for each signature (n = 5) and the Bonferroni procedure was applied to adjust the significance thresholds for multiple testing.The relationship between mutational signatures and somatic retrotransposition was first examined across 100 cell line daughter (and granddaughter) cell line clones. We applied generalized linear mixed model (GLMM) Poisson regression analysis to burdens of i referring to an individual primary cancer, and ti representing the age of the patient at diagnosis. The L1 insertion rate was defined as x is the L1 insertion count, and t is the patient age at diagnosis. A separate test was performed for each signature and the Bonferroni procedure was applied to adjust the significance thresholds for numbers of successful tests performed .The relationship between the individual mutational signatures and the rate of somatic L1 insertions was investigated further on 2,353 primary human cancers from patients with a known age at the diagnosis project  and somahttps://www.ebi.ac.uk/ega/) with accession numbers:Whole-exome sequencing: EGA: EGAD00001004201Whole-genome sequencing: EGA: EGAD00001004203RNA sequencing: EGA: EGAD00001004202Sequence data generated in this study has been deposited at the European Genome-Phenome Archive (https://dockstore.org/containers/quay.io/wtsicgp/dockstore-cgpmaphttps://dockstore.org/containers/quay.io/wtsicgp/dockstore-cgpwxshttps://dockstore.org/containers/quay.io/wtsicgp/dockstore-cgpwgsThe most recent versions of our mapping and mutation calling pipelines, and supporting documentation, can be accessed from:


Accelerated vascular aging is a condition that occurs as a complication of several highly prevalent inflammatory conditions such as chronic kidney disease, cancer, HIV infection and diabetes. Age-associated vascular alterations underlie a continuum of expression toward clinically overt cardiovascular disease. This has contributed to the striking epidemiologic transition whereby such noncommunicable diseases have taken center stage as modern-day global epidemics and public health problems. The identification of α-Klotho, a remarkable protein that confers powerful anti-aging properties has stimulated significant interest. In fact, emerging data have provided fundamental rationale for Klotho-based therapeutic intervention for vascular diseases and multiple other potential indications. However, the application of such discoveries in Klotho research remains fragmented due to significant gaps in our molecular understanding of Klotho biology, as well as hurdles in clinical research and experimental barriers that must first be overcome. These advances will be critical to establish the scientific platform from which future Klotho-based interventional trials and therapeutic enterprises can be successfully launched. Advancing age is a major risk factor for both subclinical and clinically overt cardiovascular disease (CVD) . Age-assThe kidneys are among the organs that are functionally most sensitive to the aging process, and the link between aging and kidney function is well-recognized and appears bidirectional ,6. PremaAging and age-associated changes of the vascular system involve fundamentally different alterations from atherosclerosis. Vascular alterations attributable to aging include arterial dilatation, vascular calcification, increased collagen-to-elastin ratio with fragmentation, endothelial dysfunction and hypertrophy of vascular smooth muscles cells (VSMCs) . These cMedial vascular calcification occurs at an accelerated rate in age-associated conditions such as CKD, cancer and diabetes with the consequence of loss of arterial distensibility and increasing arterial stiffening. As arteries stiffen, this leads to a lack of buffering capabilities during oscillatory changes in blood pressure caused by intermittent ventricular ejection and results in a more rigid aorta that can accommodate less stroke volume; these changes can be clinically detected by greater pressure augmentation in systole and higher pulse pressures . End orgα-Klotho, hereby referred to as Klotho, was first discovered by Kuro-o et al. in 1997 and has made a triumphant entry onto the center stage in the field of aging . The ideTaken together, emerging data suggest that arterial aging and age-associated arterial diseases are active, tightly regulated cell-mediated processes that may be potentially modifiable. Given the implications of these discoveries for human health, Klotho as a potential longevity-modulating therapeutic strategy has attracted widespread attention and as a result, the field of aging research has flourished over the past decade. This review will consider the biology of Klotho and contemporary evidence for its therapeutic applications in the treatment of age-associated vascular diseases ,29.Klotho .,99.98,99Several critical considerations, gaps and challenges must first be overcome before Klotho-based interventions can be successfully translated into potential therapies for vascular diseases and beyond: Firstly, the precise functional role of the various Klotho isoforms in health and disease, their molecular mechanisms and the identity of the Klotho receptor for FGF23 independent effects remains to be elucidated. Therapeutic strategies will need to carefully target the isoform with most clinical benefit and carefully consider their application from a standpoint of prophylactic treatment  or reversal of existing vascular diseases. Given the pleiotropic effects of Klotho and its inherent large globular form, significant molecular analysis is still needed to identify the mechanisms and active sites of Klotho responsible for its various activities. This knowledge would form a critical platform on which successful pharmaceutical engineering can then be built.From a clinical viewpoint, the precise concentrations of circulating Klotho and their specific isoforms that would be considered sufficient is still unknown and this is likely influenced by genetic variability between populations. Additionally, it is unknown whether supplementation above levels considered sufficient would be of therapeutic benefit or result in toxicity. In fact, high levels of Klotho may result in hypophosphatemic rickets and hyperparathyroidism . These lThese experimental and clinical studies remain hampered by significant challenges with current antibody-based techniques for assessing Klotho: While commercially available antibodies against Klotho are available, most of them appear to be unspecific and cross-react with other proteins . A novelA related technical challenge is that soluble Klotho appears to be highly unstable in blood and urine ,107. PreThe central thesis of this review was that accumulating evidence has stimulated significant interest and provided fundamental rationale for the therapeutic role of Klotho for age-related vascular diseases. As such, there is much reason for optimism toward the development of Klotho-based therapies. However, there are several significant gaps in our molecular and clinical understanding, as well as experimental challenges as discussed earlier. These gaps or hurdles must first be overcome before we can harness the clinical benefits of Klotho-based therapies as an elixir for vascular disease treatment or prevention. However, it seems likely that the pleiotropic nature of Klotho has brought together investigators from multiple different basic science and clinical disciplines that would have otherwise had traditionally disparate research emphases to work together toward a concerted strategy. What is interesting about a potential Klotho-based therapy is the possibility for a single drug to have multiple different disease indications. Transcending traditional barriers between disciplines offers immense opportunities for speeding innovative research that can address the growing burden of non-communicable diseases, in this case age-associated vascular diseases that remain a significant public health burden today.


Comparing the viability of normal fibroblast cells treated with compound 1a and TZQ, the LC50 value of the latter was 2.52 mM, indicating more toxicity than compound 1a. This significantly decreased cytotoxicity of compound 1a towards normal SF cells, while still maintaining the anticancer activity towards Hep2 cells is an interesting feature. Among the seven compounds synthesized, compound 1c has similar toxicity effects on the three cancer cell lines and SF normal cells as the TZQ monomer.Aziridine-containing compounds have been of interest as anticancer agents since late 1970s. The design, synthesis and study of triaziquone (TZQ) analogues with the aim of obtaining compounds with enhanced efficacy and reduced toxicity are an ongoing research effort in our group. A series of bis-type TZQ derivatives has been prepared and their cytotoxic activities were investigated. The cytotoxicity of these bis-type TZQ derivatives were tested on three cancer lines, including breast cancer (BC-M1), oral cancer (OEC-M1), larynx epidermal cancer (Hep2) and one normal skin fibroblast (SF). Most of these synthetic derivatives displayed significant cytotoxic activities against human carcinoma cell lines, but weak activities against SF. Among tested analogues the bis-type TZQ derivative  Mp＞300°C (DMF); IR (neat) νmax: 1643 cm-1; 1H-NMR (CDCl3): δ 2.28 , 2.30 , 2.42 , 2.98 ; 13C-NMR (CDCl3): δ 30.12, 30.31, 31.04, 33.15, 120.9, 140.0, 143.1, 153.7, 171.5, 179.8; Anal. calcd. for C26H28N6O4S2: C, 56.50; H, 5.11; N, 15.21. Found: C, 56.44; H, 4.98; N, 15.02.2,2’-bisbisbisbisbisbis[3,5,6-tris(aziridin-1-yl)benzo-1,4-quinone(1g): The crude product was purified by flash chromatography  to yield an opaque oil (26% yield). IR (neat) νmax: 1643 cm-1; 1H-NMR (CDCl3): δ 2.26 , 2.31 , 2.42 , 3.06 , 3.59 ; 13C-NMR (CDCl3): 29.36, 30.06, 31.84, 32.54, 70.18, 70.62, 70.94, 121.60, 139.85, 143.12, 153.66, 177.62, 179.76; Anal. calcd. for C32H40N6O7S2: C, 56.12; H, 5.89; N, 12.27. Found: C, 56.32; H,5.71; N, 12.10.2 at 37°C. Numbers of cells were counted after trypsinization by a Neubauer hemocytometer .The cell lines OEC-M1 , Hep-2 , BC-M1 (human breast adenocarcinoma) were cultured in the RPMI 1640 medium with 10 % fetal bovine serum (FBS), 2 mM L-glutamine, 25 mM Hepes. Skin fibroblasts (SF) were cultured in DMEM medium with 10 % fetal bovine serum (FBS), 2 mM L-glutamine, MEM non-essential amino acid. The cell culture medium for the four cell lines all contained pencillin-streptomycin and fungizone. All the medium and supplements were purchased from Gibco Laboratories . All cells were incubated in a humidified atmosphere of 5 % COet al. [50 values were calculated from the 50 % formazan formation compared with control without drugs addition. The MTT assay was according to the method of Skehan et al. . One day1a produced the most potent cytotoxicity to Hep2 cells and least toxicity to SF. This compound is a possible future anticancer drug.The synthetic bis-triaziquone derivatives herein displayed various cytotoxic activities against three tumor cell lines and normal skin fibroblasts (SF). Among all of these derivatives compound