The pentose phosphate pathway (PPP) is critical for anabolism and biomass production. Here we show that the essential function of PPP in yeast is the synthesis of phosphoribosyl pyrophosphate (PRPP) catalyzed by PRPP-synthetase. Using combinations of yeast mutants, we found that a mildly decreased synthesis of PRPP affects biomass production, resulting in reduced cell size, while a more severe decrease ends up affecting yeast doubling time. We establish that it is PRPP itself that is limiting in invalid PRPP-synthetase mutants and that the resulting metabolic and growth defect can be bypassed by proper supplementation of the medium with ribose-containing precursors or by the expression of bacterial or human PRPP-synthetase. In addition, using documented pathologic human hyperactive forms of PRPP-synthetase, we show that intracellular PRPP as well as its derived products can be increased in both human and yeast cells, and we describe the ensuing metabolic and physiological consequences. Finally, we found that PRPP consumption appears to take place “on demand” by the various PRPP-utilizing pathways, as shown by blocking or increasing the flux in specific PRPP-consuming metabolic routes. Overall, our work reveals important similarities between human and yeast for both synthesis and consumption of PRPP. A) has two key functions .Synthesis of NADPH by the PPP is particularly important for human erythrocytes and a defect in glucose-6-phosphate dehydrogenase (G6PDH), the first step of the oxidative branch of PPP from glucose-6-phosphate, results in hemolytic anemia . In yeasthionine . Importathionine , 6, 7, 8survival , presumasurvival A.PRS1-5, encoding PRPP synthetase subunits. Based on specific activity measurements from single and combined knockout (KO)   , 11, as : p: pPRS1-5PRPS1 expression was found to result in an anti-apoptotic effect in B-ALL cell lines and was associated with a poor prognosis for disease progression . The PRPS1 and PRPS2 enzymes are 95% identical and are expressed in all tissues examined, while PRPS3 is specifically expressed in the testis , 17. PRPgression . Finallygression . PRPS2, gression  leading gression . The reqIn this work, we first established that the synthesis of ribose-5-phosphate and PRPP is the essential function of PPP in yeast. We then aimed at deciphering how PRPP synthesis and consumption affect metabolism and proliferation in yeast cells. We report that PRPP synthetase, but not ribose-5-phosphate, is limiting PRPP synthesis. We also show that PRPP-utilizing enzymes compete for PRPP and that, under physiological conditions, PRPP is limiting for nucleotide synthesis but not for proliferation and that it is competitively consumed by the downstream pathways. Finally, we investigated PRPP metabolism in human cells and found that the main conclusions drawn from yeast apply quite similarly to human cells.RKI1 is the only essential gene in the PPP. This is most likely due to the fact that the PPP can be fed by the glycolysis pathway via several intermediates, namely, glucose-6-phosphate, fructose-6-phosphate, and glyceraldehyde-3-phosphate (A). It ensues that there is a redundancy in the synthesis of PPP intermediates with a possible exception for ribose-5-phosphate synthesized by Rki1 and for its derived product, PRPP, synthesized by Prs1-5 which are also essential for yeast cells in combinations . The supplementation could not simply be done by feeding the cells with preformed nucleotides since nucleotides are not taken up by yeast cells. We hence fed the cells with nucleosides, which are nucleotide precursors carrying the ribose moiety but no phosphate. The nucleosides provided to cells were adenosine, uridine, and nicotinamide riboside (Nr) for purines, pyrimidines, and pyridines, respectively. Of note, since Nr is chemically unstable, cells were fed with nicotinamide mononucleotide that is metabolized to Nr by the Pho5 phosphatase enzymatic activity and immediately taken up by the Nrt1 permease, as shown previously  to facilitate uptake. In addition, tryptophan and histidine were supplied to bypass the requirement of PRPP for the synthesis of these amino acids. This combination of nucleoside and amino-acid supplements clearly bypassed the inviable phenotype of the rki1 mutant (B) lacking ribose-5-phosphate ketol-isomerase and hence unable to synthesize ribose-5-phosphate from ribulose-5-phosphate (A). We conclude that the main function of the non-oxidative branch of the PPP, in yeast, is to provide ribose-5-phosphate that is used for nucleotide and amino acid synthesis via the synthesis of PRPP by Prs1-5. PRPP synthesis from ribose-5-phosphate hence appears to be the essential role of the PPP in yeast.As mentioned above, hosphate A. It ensinations . We thusno acids B. The sueviously . Since a1 mutant B lackinghosphate A. We conprs1 prs3 double knock-out mutant) or the minor forms (prs2 prs4 prs5 triple knock-out mutant) were compared with an isogenic wild-type strain. Intracellular PRPP was in the 0.1 mM range for the wild-type strain, lowered by 25% in the prs2 prs4 prs5 triple mutant, and was decreased almost 10-fold in the prs1 prs3 mutant (A). This result is in good agreement with previous results showing a much more severe defect in PRPP synthetase activity in the prs1 prs3 mutant than in the prs2 prs4 prs5 triple mutant (B). This suggests that PRPP is not stored but rather utilized on demand.We then evaluated the metabolic and phenotypic contributions of the various yeast PRPP-synthetase isoforms in an isogenic prototrophic background. Prototrophic strains were used to ensure that the observed effects did not result from metabolic interferences with the auxotrophic markers commonly used to facilitate genetic studies in yeast and which often affect PRPP-requiring pathways. Two mutant strains lacking either the major enzyme (3 mutant A. This re mutant B. This sA) were associated with a significant decrease in the ability of the mutant cells to produce biomass, although the effect was much stronger in the case of the prs1 prs3 mutant (C). Quantifying biomass production rate does not allow, in a single measurement, to differentiate the effects on cell division from those on cell growth. We hence appraised these two parameters separately, by measuring cell volume (D) and population doubling time (E). Clearly, in the triple prs2 prs4 prs5 mutant, the lower biomass production was associated with a lower cell volume, while doubling time was not increased (D and E). By contrast, in the prs1 prs3 mutant, both factors were severely affected (D and E). Finally, metabolic profiling of the three strains revealed that the prs1 prs3 mutant was the most affected strain for all four nucleotides as well as the sum of NAD+ and NADH (hereafter referred as NAD(H)), while the prs2 prs4 prs5 mutant was less severely, although significantly, affected . This discrepancy could be due to the fact that the strains used in our work are prototrophic, while in the previous work, the used strains were mutated in four PRPP-utilizing pathways . Interestingly, uracil, adenine and hypoxanthine, three pyrimidine and purine precursors, were higher in the prs1 prs3 mutant than in the control strain (E–G), suggesting that the low PRPP level in the mutant limited their consumption by the cognate phosphoribosyl transferases (A).The effects of the mutations on intracellular PRPP A were as3 mutant C. Quantil volume D and poping time E. Clearlncreased , D and Eaffected , D and El strain , E–G, susferases A.Figure A), and subsequently intracellular nucleotides , severely affected biomass production rate in both prs mutants (C), while division, as revealed by population doubling time, was only affected in the prs1prs3 mutant but not significantly in the prs2 prs4 prs5 mutant (E). Hence, PRPP shortage, apparently, first affected cell growth  before slowing down cell division .We conclude that decreasing intracellular PRPP A, and su mutants C, while 5 mutant E. Hence,PRS1 and PRS3) concomitantly on multicopy yeast plasmids and evaluated the metabolic and phenotypic effects , as well as nucleotide and NAD(H) concentrations (B–F). This result shows that increasing PRPP synthesis in yeast can significantly increase intracellular nucleotides. Accordingly, the purine intermediate metabolites IMP and inosine were also very significantly increased (G and H), as previously reported for purine overproducing ADE4 dominant mutants  and diminished the biomass production rate (J), while it had no significant effect on cell size (K). This establishes that there is no strict proportional relationship between PRPP concentration and generation time or cell size and that the connection between metabolism and these traits is probably more complex. The metabolic burden imposed by increased synthesis of nucleotides and accumulation of intermediates such as IMP and inosine in the mM range (G and H) could be responsible for the higher doubling time by diverting a significant part of glucose from glycolysis to nucleotide synthesis via the PPP.We then overexpressed the two major isoform genes  under the control of a yeast promoter. Expression of the human or bacterial enzymes restored colony growth on plates (A) and doubling time in liquid cultures (B). Importantly, expression of the exogenous PRPS in yeast restored intracellular PRPP, although only partially (C). Similarly, expression of exogenous PRPS in yeast only partially, but significantly, restored nucleotide levels, although to various extents, higher for purines than for pyrimidines for unknown reasons (D–H). We conclude that synthesis of PRPP derivatives is apparently limiting for proliferation in the prs1 prs3 mutant and that partial restoration of the intracellular pools, by expression of exogenous PRPS, is likely sufficient to recover doubling times not very different from that of the wild-type control. Of note, this result is highly consistent with the one observed previously for the prs2 prs4 prs5 mutant (E). It should be noted that the phenotypic and metabolic rescue by expression of bacterial PrsA or human PRPS in the prs1 prs3 mutant is probably contributed, in part, by the Prs2 Prs4 and Prs5 isoforms that are still present in this mutant . Furthermore, the rescue of doubling time by the combination of supplements was total in liquid cultures (B), suggesting that the partial rescue effect observed on plates (A) could be due to poor diffusion of the substrates in the agar plate, as previously reported (prs1 prs3 mutant did not increase intracellular PRPP concentration (C) but rather lowered it for a yet ununderstood reason, while, as expected, it significantly increased all intracellular NTPs (D–G) to levels comparable to those of the wild-type isogenic strain. Meanwhile, NAD(H) was only partially restored (H), the metabolization of nicotinamide mononucleotide to Nr, and/or the uptake of Nr by Nrt1 being possibly limiting. We conclude that most of the growth defect of the prs1 prs3 mutant is due to its inability to synthesize a sufficient amount of PRPP-derived metabolites.We then asked whether it is PRPP itself or its derived products that is limiting the growth of the 3 mutant ; however3 mutant A. Furthecultures B, suggesn plates A could breported . Importantration C but ratlar NTPs , D–G to restored H, the meprs1 prs3 mutant.Altogether, our results show that low synthesis of PRPP and more specifically of its derived products, such as nucleotides, is the principal cause of the slow growth phenotype of the prs1prs3 mutant slow growth, we then asked whether this proliferation phenotype could be suppressed genetically. Starting from a prs1 prs3 double KO mutant, we searched for suppressors that would alleviate the severe colony growth defect of the mutant and thereby possibly reveal metabolic processes limiting proliferation. Because PRPP is a central molecule in metabolism, the experiment was done on prototrophic yeast to avoid interference with auxotrophy markers. After UV mutagenesis, rapid growers were isolated from the prs1 prs3 slow growers and further characterized. The experiment was run in parallel on “rich”  and “poor”  media to establish whether different types of suppressors would be found when the salvage or de novo pathways are mostly challenged  were backcrossed to a prs1 prs3 parental strain and in both cases, the suppressor mutation was found to segregate as a single locus in the meiotic progeny. Sets of pooled segregants (PRS5 locus was found in the suppressed segregant pools but not in the non-suppressed segregant pools. The PRS5-C328T (Pro110Ser) and PRS5-G932T (Arg311Ile) mutations were confirmed by resequencing the PRS5 locus in the original supp3 and supp48 mutants, respectively, while none of these changes were found in the parental strains. Sequencing of the PRS5 locus from 15 suppressor mutants obtained from SD medium and 15 from SDcasaWAU medium revealed that they all carry a mutation in PRS5 , further confirming that they are necessary and sufficient suppressor mutations and revealing their dominance over the wild-type PRS5 allele. We conclude that several mutations in PRS5 can efficiently suppress the growth defect of the prs1 prs3 double mutant. All the tested PRS5 suppressor mutations were found dominant. A subset of three suppressors  was further analyzed revealing that the PRS5 suppressors decreased the population doubling time (C) and had a significant effect on intracellular PRPP (E–I). Finally, we observed that overexpression of the mutated forms of PRS5 on multicopy plasmids only slightly improved suppression compared to that obtained with centromeric plasmids (B). We hypothesized that this could be due to the limitation of one or both of Prs5 partners, namely, Prs2 and/or Prs4. We hence overexpressed PRS2 or PRS4 together with PRS5P110S and found that indeed the co-overexpression of either PRS2 or PRS4 strongly stimulated growth (J), indicating that the PRS5 suppressor acts through its interaction with its partners Prs2 or Prs4. Hence, suppressors of prs1 prs3 most likely act by increasing PRPP synthesis by the Prs2/4/5 enzyme rather than modulating downstream consumption.Having shown that supplementation or exogenous PRPS activity could rescue the allenged , respectgregants  carrying in PRS5 . Finally did not B, furtheing time C and hadlar PRPP  as well lar PRPP , E–I. Fiplasmids B. We hypd growth J, indicaprs1 prs3 mutant, PRPP is limiting for proliferation and that these effects can be alleviated by increasing PRPP  or by bypassing its requirement (via supplementation with nucleosides and amino acids).The results presented in this section and the previous one allow us to conclude that in the PRPS1 human enzyme that was able to suppress the growth defect of the prs1 prs3 mutant (A). Expression of the human enzyme did not increase intracellular PRPP nor NTPs or NAD(H) in the wild-type cells (A–F). This result suggested that ribose-5-phosphate, the substrate of PRPP-synthetase, could be limiting for the reaction to proceed or, alternatively, that the human enzyme is feedback inhibited by some end-product(s) (A) as well as of all NTPs and NAD(H) when expressed in wild-type yeast (B–F). We conclude that PRPP-synthetase activity, rather than the ribose-5-phosphate substrate, is limiting PRPP synthesis and downstream reactions in a wild-type prototroph yeast strain. Furthermore, the expression of these hyperactive forms of PRPP-synthetase significantly affected the doubling time (G). Of note, the accumulation of purine intermediates, such as SZMP, IMP and inosine (H–J) when the PRPS1 mutants are expressed in yeast, indicates that the flux in the purine de novo pathway is overloaded. This metabolic burden could be responsible for the higher doubling time by diverting a significant part of glucose from glycolysis to the PPP and nucleotide synthesis in the form of an unused excess of purines.We then asked whether PRPP is present in excess in yeast cells or whether it could be limiting for nucleotide synthesis even in wild-type cells. To address this question, we first expressed in wild-type yeast cells the 3 mutant A. Exprespe cells , A–F. Thoduct(s) . To settoduct(s) . Clearlyoduct(s) A as wellpe yeast , B–F. Weing time G. Of not inosine , H–J whePRPS1 mutants (B–F) suggested that, in wild-type yeast cells, PRPP-utilizing enzymes compete for PRPP as a limiting substrate. To address this question more directly, we asked whether affecting one PRPP-consuming pathway results in an effect on the products of other PRPP-consuming pathways. This was first done by blocking de novo synthesis of purine nucleotides, using a ade4 knockout mutation that blocks the first step of the pathway (A and B). This mutant strain can grow and proliferate as long as it is fed with a purine precursor such as adenine, utilizable through the salvage pathway. When shifted for 3 h in a medium lacking adenine, the ade4 mutant showed a strong decrease of adenylic and guanylic nucleotides (D and E) as expected but also CTP and NAD(H) (F and G), most probably because their synthesis is affected at low ATP concentration (ade4 mutant (H), suggesting that the PRPP that was not used for purine, CTP, and pyridine synthesis due to the ade4 block in the purine pathway could be used to make more UTP. In addition, the expression of an ADE4 hyperactive dominant allele (A and C) (I and J). Of note, here UTP and CTP both decreased reflecting the fact that under these experimental conditions, CTP is synthesized de novo from UTP. In the same experiment, ATP and GTP were not significantly affected (K and L), while the flux in the de novo purine pathway was high as attested by the accumulation of the metabolic intermediate ZMP and of the degradation product inosine (M and N). These results indicate that the flux in the purine pathway has a strong impact on intracellular UTP. We conclude that, in wild-type yeast, PRPP-utilizing enzymes are competing for PRPP as a substrate which is limiting for the synthesis of downstream products.The increase in all NTPs and NAD(H) in yeast strains expressing the two  mutants , B–F sug pathway , A and Bleotides , D and Ed NAD(H) , F and Gntration , 30. By 4 mutant H, suggest allele , A and CA and C) , 32 resuA and C) , I and Jaffected , K and L inosine , M and Nura2 block in the pyrimidine pathway (A). When these cells were grown in the absence of uracil, we observed a severe decrease of UTP and CTP and a small but significant increase of intracellular ATP (B–F)). Due to the different intracellular abundance of ATP and UTP  it could be expected that a variation of intracellular ATP would much more severely affect UTP levels than the reverse. Finally, intracellular NAD(H) was tightly correlated to intracellular ATP as reported before (M for ATP of this enzyme might be high).This competition between PRPP-utilizing enzymes was further confirmed using a  pathway A. When tular ATP , B–F). Dd before , and intAltogether, our results allow two conclusions. First, decreasing intracellular PRPP leads to a reduction of all downstream products, while increasing intracellular PRPP had the opposite effect, establishing that in wild-type yeast cells PRPP is limiting nucleotide synthesis. Second, when PRPP is over- or under-used in one pathway, it is compensated by its usage in other PRPP-utilizing pathways, indicating that phosphoribosyl transferases are competing for PRPP. We conclude that, in yeast, PRPP synthesis limits nucleotide synthesis but not cell proliferation. In addition, PRPP utilization appears to directly result from competition between the enzymes of the downstream pathways.PRPS1  or PRPS2 were expressed in U-87 MG cells using lentiviruses, and metabolic profiles were established. As in yeast cells, expression of the V142L and A190V PRPS1 mutants in U-87 MG resulted in a significant increase of PRPP (A) as well as of all NTPs, from 1.6-fold for ATP to more than 2-fold for the other NTPs (B–E) and of 1.2-fold for NAD(H) (F). A significant increase, although slightly lower, was also observed when these mutant PRPS1 alleles were expressed in the colon tumor cell line HCT116 (A), while such an effect was not observed in the HCT116 cell line (A) as well as in yeast cells (A), the reasons for this discrepancy are not known. However, while PRPS1 expression had no significant effect on NTPs (B–E) or NAD(H) (F), expression of PRPS2 resulted in a significant increase of all four NTPs and NAD(H) (B–F).Based on the conclusions obtained with yeast cells, we then questioned several aspects of the PRPP metabolism in human cells. We first aimed at evaluating the metabolic consequences of PRPS hyperactivity in the glioblastoma U-87 MG human cell line.  of PRPP A as wellher NTPs , B–E andr NAD(H) F. A signe HCT116 , indicate HCT116 A, while ell line A as wellst cells A, the re on NTPs , B–E or r NAD(H) F, expresd NAD(H) , B–F.FigG), indicating that, as observed in yeast, PRPP is limiting for nucleotide synthesis but not for cell proliferation. Actually, in all cases where intracellular NTPs were high, cell proliferation was severely slowed down (G), indicating that more NTPs is not “better” for human cells, although the precise reasons for this inhibitory effect are not known.In all experiments, cell proliferation was not accelerated by conditions resulting in higher intracellular NTP content G, indicawed down G, indicaA and E) and conjointly decreased all other triphosphate nucleotides (B–D and F–H), suggesting that the PRPP utilized to synthesize ATP from adenine in excess, is limiting the synthesis of other PRPP derived metabolites. We conclude that PRPP utilization in human cells . Importantly, the overproduction of PRPP decreased the proliferation of both the human cells (G and S4G) and the yeast cells (G). The reason for this defect is not established, but it could reflect the metabolic burden associated with the production of PRPP, and most importantly nucleotides, that are not further used for growth but degraded and excreted in the form of inosine in yeast and uric acid in humans.Clearly,  the PPP , is not  the PPP , and no an cells G and S4Gst cells G. The reprs2 prs4 prs5 triple mutant, Fig. 2A) results in a lower biomass production rate , while doubling time is not affected , indicating that in this condition, maintaining the doubling time is preferred over maintaining cell size. However, further decreasing intracellular PRPP (prs1 prs3) double mutant  affected both biomass production rate  and population doubling time . Strikingly, overexpression of PRS1/PRS3 resulting in increased PRPP concentration also diminished the biomass production rate and increased doubling time (F and H). These results parallel the fact that both gain and loss of function, in PRPS1, are associated with human diseases , 2% glucose. SDcasaW is an SD medium supplemented with 0.2% casamino acids (Difco) and tryptophan (0.2 mM). When indicated, adenine (0.3 mM) and/or uracil (0.3 mM) were added to SDcasaW medium, resulting in a medium named SDcasaWA (+adenine), SDcasaWU (+uracil) and SDcasaWAU (+ adenine + uracil). SC -Ura -Leu medium is SC medium supplemented with adenine (0.3 mM), histidine (0.06 mM), lysine (0.06 mM), and tryptophan (0.2 mM). The YPD medium contained 1% yeast extract, 2% peptone, and 2% glucose. Yeast cells were grown in liquid or solid media at 30 °C. Yeast strains , L-glutamine, penicillin, and streptomycin. Dialyzed serum (same FBS as above) was obtained after dialysis in PBS (3 kDa-molecular weight cutoff) and was filtered at 0.1 μm. Lentiviruses expressing PRPS1, PRPS2, and mutants were obtained at the Vect’UB platform  and U-87 MG (HTB-14) were obtained from the ATCC. Cells were grown at 37 °C, 5% CO2 and grown for 4 days at 37 °C with medium changes every 2 days. Triplicate or tetraplicate conditions were used. Cells were harvested by trypsinization and were counted using a Multisizer four Coulter counter (Beckman).For metabolites quantifications, cells were seeded in 6-well plates and grown up to half confluency. Cells were then washed and incubated for 24 h in the complete medium before metabolic extractions were performed as described in . Tri-, tPRPS1 and PRPS2 in yeast were obtained by PCR amplification of the open reading frames (ORF) with the following pairs of oligonucleotides (5562 + 5563 for PRPS1 and 5763 + 5764 for PRPS2) using, respectively, cDNA IRAUp969H0616D and IRCMp5012H0142D from Source Biosciences as templates. PRPS1 Hyperactive mutants were obtained by site-directed mutagenesis. PCR products were cloned in the pCM189 or its derivative p2714 plasmids in which gene expression is under the control of tetracycline-repressible promoter. For expression of the same genes in mammalian cells, ORF were amplified with the 5678 + 5563 (PRPS1) and 5763 + 5764 (PRPS2) and cloned in the pLenti-MND-IRES-PuroR-WPRE. The PrsA expression plasmid was obtained by PCR amplification of PrsA ORF with oligonucleotides 2278 + 2279 on E. coli genomic DNA as a template and cloning in pCM189. The hENT1 encoding plasmid was a generous gift from Drs. Cass, Damaraju, and Sawyer. The plasmid expressing the hyperactive form of ADE4 gene (ADE4-1 mutant R328K) was obtained by cloning in pCM189 the PCR fragment amplified with oligonucleotides 48 + 429 on genomic DNA of the m131 ADE4 mutant described in  genes were obtained by PCR amplification of ORFs using yeast genomic DNA as template  or multicopy (Yeplac181 or Yeplac195) plasmids as indicated in All plasmids and oligonucleotides are listed in 7 cells/ml) for at least 24 h hours by successive dilutions before any measurement. Population doubling time was then determined by following cell number during 8 h on at least four independent exponential growth culture using the Multisizer four Coulter counter (Beckman). Population doubling time was then determined by exponential growth regression of the proliferation curves using GraphPad Prism 5.Cells were kept in exponential growing phase (<1.5 107 cells/ml) grown in indicated medium for at least 24 h hours before measurement. Biomass was determined by filtration of aliquots of the cell culture (starting at comparable biomass for each strain around 10 mg corresponding to 50–100 ml at 1–2 × 106 cells/ml) on 0.45 μm polyamid pre-weighted filters  followed by two 10 ml washes with room temperature milliQ water and drying of the filter with successive 30 s cycles (until the mass of filter + cells is constant) in a microwave oven at 800 W. Dried biomass was measured in exponential growth conditions during 4 h and exponential biomass production rate was determined by linear regression of the experimental points  rates close to nominal for unequal variances.For yeast strains and/or human cell lines, growth and metabolites extraction were performed on three to eight biologically independent samples, as detailed in each figure legend. Statistics, given as All data are contained within the manuscript.This article contains The authors declare that they have no conflicts of interest with the content of this article.


PHYTOCHROME KINASE SUBSTRATE (PKS) proteins are involved in light-modulated changes in growth orientation. They act downstream of phytochromes to control hypocotyl gravitropism in the light and act early in phototropin signaling. Despite their importance for plant development, little is known about their molecular mode of action, except that they belong to a protein complex comprising phototropins at the plasma membrane (PM). Identifying evolutionary conservation is one approach to revealing biologically important protein motifs. Here, we show that PKS sequences are restricted to seed plants and that these proteins share 6 motifs (A to F from the N to the C terminus). Motifs A and D are also present in BIG GRAIN, while the remaining 4 are specific to PKSs. We provide evidence that motif C is S-acylated on highly conserved cysteines, which mediates the association of PKS proteins with the PM. Motif C is also required for PKS4-mediated phototropism and light-regulated hypocotyl gravitropism. Finally, our data suggest that the mode of PKS4 association with the PM is important for its biological activity. Our work, therefore, identifies conserved cysteines contributing to PM association of PKS proteins and strongly suggests that this is their site of action to modulate environmentally regulated organ positioning. Members of the PHYTOCHROME KINASE SUBSTRATE (PKS) family are characterized by six conserved sequence motifs, one of which is required for plasma-membrane association and biological activity of PKS4. Background: Plants can orient their leaves towards the light. This is known as phototropism and fascinated scientists since Charles Darwin. Phototropism is initiated by a blue light photoreceptor called phototropin. Light activates phototropin's protein kinase activity (an enzyme that phosphorylates proteins). This is followed by a series of poorly understood events, which lead to the redistribution of the growth hormone auxin in the stem resulting in growth towards the light. PHYTOCHROME KINASE SUBSTRATE 4 (PKS4) is phosphorylated by phototropin and is believed to act between phototropin light activation and auxin redistribution. The mechanism of PKS4 action is unknown because the protein does not contain any domains of known biochemical activity. PKS4 associates with the plasma membrane (PM), where it was postulated to act.Question: We wanted to understand how PKS4 associates with the PM and determine whether this subcellular localization is important for its biological activity.Findings: Functionally important parts of proteins are typically conserved over evolutionary timescales. We, therefore, identified genes coding for PKS proteins and found that they are present in seed plants and comprise 6 conserved sequence motifs. We showed that one of these motifs is important for efficient PKS association with the PM in Arabidopsis. Cysteine residues of this motif are modified with lipids, which presumably contribute to PM association. Moreover, in PKS4, these residues are important for its function in phototropin signaling in Arabidopsis.Next steps: Our study identified a PKS4 sequence motif that is important for biological activity and subcellular localization. However, we still don’t know what the rest of the protein does and how it may be important in linking phototropin activation and auxin redistribution. We hope that studying other conserved motives identified here will allow us to answer this question.Arabidopsis thaliana), etiolated (dark-grown) seedlings grow following the direction of the constantly present gravity stimulus: roots grow downwards while shoots grow upwards. However, in response to light, hypocotyls orient their growth following the integration of 2 main signaling pathways: gravitropism (repressed) and phototropism (activated)  . Growth tivated) . Additiotivated) . Althougtivated) .Gravitropism signaling comprises the perception of the gravity vector, which requires sedimentation of starch-filled amyloplasts for signal generation and transduction, resulting in hypocotyls growing upwards . In ArabArabidopsis has 2 phototropins, which exhibit specific and partially redundant functions: phot1 and phot2. Hypocotyl phototropism is mainly mediated by phot1 . PhototrPKSs are expressed in the hypocotyl elongation zone, consistent with their importance during hypocotyl growth regulation (PKSs are a family of basic hydrophilic proteins that do not contain domain(s) of known function. They were initially identified as phytochrome-binding proteins that regulate phytochrome signaling . Surprisgulation . PKS1, Pgulation . However2P2 sequence predictor (PKS homologs using the Orthologous matrix (OMA) browser , we identified 2 PKS genes each, which group into the PKS4 subfamily and a subfamily formed by the ancestors of PKS1, PKS2, and PKS3 . Based on the limited data available from gymnosperms and the limited bootstrap support throughout the tree, the most parsimonious interpretation of our data is that PKS was present as a single copy in the ancestral spermatophyte genome . We hypothesize that there was a duplication in the ancestral angiosperm to form 2 copies: PKS4 and PKS1/2/3. Another duplication occurred after the divergence of Amborella, P. americana, and N. colorata, giving rise to PKS1/2 and PKS3 in the ancestral Mesangiospermae. An additional Brassicaceae-specific duplication gave rise to PKS1 and PKS2. PKS4 genes are present in all analyzed monocot and dicot genomes .Using the Dredictor , we deteredictor . To defi browser  and manu browser . This annosperms . Howeverng MAFFT , removinng MAFFT , and theng MAFFT . The resng MAFFT . Howeverng MAFFT . In the  genomes . EudicotP<0.05) between motifs C and F, suggesting that parts of these motifs are related to each other  35 promoter. We first confirmed that the full-length (ABCDEF) PKS1-GFP signal was consistent with the previously reported PM localization  residues  and selected 3 independent single insertion lines accumulating PKS4 to levels comparable to a wild-type PKS4-HA control line (WT-3) (PKS4 WT-3 line showed complementation of the phototropic phenotype of the pks4 mutant as previously shown (PKS4 C* lines complemented the mutant (PKS4 C*-2) exhibits an even stronger phototropic defect than the pks4 mutant, suggesting that the expression of the PKS4 C* variant might interfere with the molecular mechanism underlying phototropism. To look at the rapid phototropin-mediated response, we analyzed the light-induced reduction of PKS4 mobility on SDS-PAGE gels triggered by phototropin-mediated PKS4 phosphorylation   (Schumace (WT-3) . The PKSly shown    with similar protein abundance as in our PKS4 WT control line  was fused to an N-terminal myristoylation (myri) sequence. We selected single insertion lines (myriPKS4 WT 1-4 and myriPKS4 C* 1-3) with comparable protein levels as in our PKS4 WT and PKS4 C* lines  to have a stronger phototropic response than PKS4 C* or Cys-12-Ser alone  displaying a slightly more robust phototropic response than the PKS4 C* construct . PKS1 and PKS2 were inferred by OMA to be in the same gene family (rooted at the Magnoliopsida level), and PKS3 and PKS4 were in 2 other families, rooted at the Pentapetalae and Mesangiospermae levels, respectively. A final, smaller gene family was found in OMA by searching for the A. trichopoda PKS4 gene, which was rooted at the Magnoliopsida level. Seventeen additional PKS sequences not present in the OMA database, selected to increase phylogenetic diversity, were added to cover the following: the basal angiosperms A. trichopoda and N. colorata; the monocots purple false brome (Brachypodium distachyon) and foxtail millet ; the magnoliid avocado (P. americana); the asterids potato (Solanum tuberosum) and tea plant (Camellia sinensis); the rosid wild strawberry (Fragaria vesca); and the Caryophyllales sugar beet (Beta vulgaris). The aforementioned sequences were obtained by reciprocal BLAST searches using Arabidopsis PKS1 and PKS4 protein sequences as query  from the January 2020 release of the OMA browser . The firas query . Two gymerms 1.0 . Thus, aerms 1.0 .The 172 sequences were aligned using MAFFT v7.313 , with thThe alignment was used to make a gene tree of all PKS sequences using IQ-TREE web server version 1.6.12  with theTo identify motifs in the highly gapped alignment, GLAM2 was utilized . Since Ghttps://meme-suite.org/meme/tools/tomtom)  . The Sanhttps://meme-suite.org/meme/tools/meme)   The BIG ls/meme)  using ArA. thaliana) Columbia-0 accession. The pks4-2 allele (GABI_312E01) was utilized in this study (35S:PKS1-GFP (pCF202), 35S:PKS4-GFP (pIS03), and PKS4pro:PKS4-3XHA (pPS09) in pks4-2 were previously described . The PKS1 cDNA from plasmid pCF173 was amplified with the following primer combinations (CF129/CF470) corresponding to amino acids (aa) 1–160 of PKS1 (AB); (CF129/CF471) corresponding to aa 1-273 (ABC); (CF472/CF473) corresponding to aa 274-439 (DEF); (CF507/CF471) corresponding to aa 161-273 (C), and (CF507/CF473) corresponding to aa 161-439 (CDEF). The PCR products were digested with Kpn1 and BamH1 and ligated into digested pCF203 to obtain pCF524 (AB), pCF525 (ABC), pCF526 (DEF), pCF534 (C), and pCF535 (CDEF). Mutations of the 3 conserved Cys residues in motif C were obtained by site-directed mutagenesis using pCF173 as a template to generate pCF546 (ABC*), pCF547 (ABC*DEF), and pCF550 (C*DEF). Mutations of the conserved Cys residues in motif F (Cys-378) were also obtained by site-directed mutagenesis using pCF173 as a template to obtain pCF393. For PKS4 C* lines, a fragment encoding PKS4 ABC (aa 1 to 186 of PKS4 with Cys-143 and Cys-145 mutated to Ser) was ordered at Eurofins and after digestion with XmaI/Bpu10I was ligated into a 35S:PKS4-GFP construct to replace the wild type with the mutant sequence to obtain pCF561. For the generation of the PKS4pro:PKS4 C*-3xHA lines (pAL10), a 517-bp DNA fragment containing PKS4 encoding the C143S and C145S variants was digested from pCF561 with the restriction enzymes EcoRV and NruI and replaced in the pPS9 construct , a synthetic 517-bp DNA fragment of the PKS4 coding sequence harboring the C143S or C145S mutation and flanked by the EcoRV and NruI sites was ordered from Eurofins and replaced in the pPS9 construct previously digested with the same restriction enzymes to replace the wild type with the mutant sequence. Similarly, for the generation of the PKS4pro:PKS4 F*-3xHA lines (pAL40), a synthetic 682-bp DNA fragment of the PKS4 coding sequence carrying the C358S mutation and flanked by the EcoRV and BamHI sites was ordered from Eurofins and replaced in the pPS9 construct previously digested with the same restriction enzymes to replace the wild type with the mutant sequence. For the generation of the PKS4pro:myriPKS4-3xHA (pAL63) and PKS4pro:myriPKS4 C*-3xHA (pAL64) and PKS4pro:myriPKS4 Cys12*-3xHA (pAL86) lines, a synthetic 171-bp DNA fragment of the PKS4 coding sequence containing the last part of the PKS4 promoter and the myristoylation signal sequence ATGGGAATTTGTATGTCTAGA followed by the beginning of the PKS4 coding sequence was ordered and digested with the restriction enzymes XhoI and NruI and replaced in the pPS9, pAL10, and pAL80 constructs previously digested with the same restriction enzymes. For the generation of the PKS4pro:PKS4-3xHAfarn (pAL70) and PKS4pro:PKS4 C*-3xHAfarn (pAL71) lines, a synthetic 225-bp DNA fragment including the last part of the PKS4 coding sequence, the 3xHA tag sequence followed by the farnesylation sequence TCT AAG GAT GGA AAG AAG AAG AAG AAG AAG TCT AAG ACT AAG TGT GTT ATT ATG, and a very short fragment of the backbone vector flanked by the unique restriction enzymes sites BamHI and PstI was replaced in the pPS9 and pAL10 previously digested with the same restriction enzymes. For the generation of PKS4pro:PKS4-GFP (pAL43) lines, a DNA fragment containing the PKS4 pro:PKS4-GFP:term in pED10 (OLE1pro:OLE1-FastRed for selection of transgenic plants) previously digested with the same enzyme. For the generation of the PKS4pro:PKS4 C*-GFP (pAL45) and PKS4pro:PKS4 F*-GFP (pAL65), PKS4pro:PKS4 Cys10*-GFP (pAL81), and PKS4pro:PKS4 Cys12*-GFP (pAL82) lines, a DNA fragment encoding PKS4 with C143S and C145S, or C358S, or C143S, or C145S mutations, respectively, was digested from the pAL10, pAL40, pAL79, and pAL80 vectors with the restriction enzymes NruI and ZraI and replaced in pAL43 previously digested with the same enzymes. For the generation of the PKS4pro:myriPKS4-GFP (pAL61), PKS4pro:myriPKS4 C*-GFP (pAL62), and PKS4pro:myriPKS4 Cys12*-GFP (pAL87) lines, the same synthetic 171-bp DNA fragment used for the generation of pAL63 and pAL64 was digested with the restriction enzymes XhoI and NruI and replaced in the pAL43, pAL45, and pAL82 previously digested with the same restriction enzymes. For the generation of the PKS4pro:PKS4-GFPfarn (pAL67) and PKS4pro:PKS4 C*-GFPfarn (pAL68) lines, the same synthetic 1,557-bp DNA fragment used for the generation of pAL70 and pAL71 was replaced in pAL43 and pAL45 previously digested with the same restriction enzymes. All constructs were verified by Sanger sequencing. Primers are provided in Agrobacterium tumefaciens)-mediated transformation with Agrobacterium GV3101  at 22 °C, and plates were placed back in the dark at 19 °C or 22 °C for 3 d before light exposure. For inhibition of gravitropism experiments, germination was induced by 1 h of RL (50 μmol m−2 s−1) at 22 °C and plates were placed back in the dark at 22 °C for 1 d before light exposure.For seed production, plants were grown on soil at 22 °C with 16 h of white light (WL) per day. For physiological experiments, seeds were surface-sterilized in 70% (v/v) ethanol and 0.05% (v/v) Triton-X for 5 min and in 100% ethanol for 5 min. Seeds were then sown on Petri plates containing half-strength MS medium, pH 5.7, 0.8% (w/v) agar. Plates were stored in the dark for 3 d at 4 °C for stratification. For experiments with dark-grown seedlings, germination was induced by 4 to 6 h of WL  of each hypocotyl with respect to the vertical after light treatment, as determined by a customized MATLAB script developed in the Fankhauser Lab. One-way ANOVA (aov) and Compute Tukey's Honest Significance Differences (HSD.test) [agricolae package] were performed in R. We considered 35S:PKS4-GFP and 35S:PKS4_C*- GFP. Excitation was accomplished using an Argon laser at 488 nm and detection used a bandpass emission between 505 and 530 nm. For some images, the PM was stained with the dye FM4-64  at a concentration of 50 μM, by soaking the seedlings for 1 min and then washing 3 times in liquid half-strength MS medium. In that case, the signal was detected with a long pass emission from 650 nm. Model LSM 880 was used for imaging PKS4pro:PKS4 F*-GFP and PKS4pro:PKS4 C*-GFPfarn. Samples were excited with an argon laser (488 nm) and detection was done between 495 and 518 nm for GFP and between 607 and 691 nm for chlorophyll and red fluorescent protein (RFP). Leica Stellaris 5 was used to image PKS4pro:PKS4-GFP, PKS4pro:PKS4 C*-GFP, PKS4pro:PKS4 Cys10*-GFP, PKS4pro:PKS4 Cys12*-GFP, PKS4pro:myriPKS4-GFP, PKS4pro:myriPKS4 C*-GFP, PKS4pro:myriPKS4 Cys12*-GFP. The objective used was HC PL APO 63×/1.40 OIL. Samples were excited with a 488-nm laser and fluorescence was detected between 496 and 520 nm for GFP, 580 to 585 nm for RFP, and 670 to 690 nm for protochlorophyllide. For each plant, between 30 and 40 images were taken at different z positions, every 1 µm starting in the epidermis and finishing in the second cortex layer. Images were taken below the apical hook, where PKS4 expression is the highest. Images were edited using ImageJ. In Confocal microscopy images were taken with a confocal microscope model LSM 510 (Zeiss), LSM 880 (Zeiss), or Stellaris 5 (Leica). Model LSM 510 was used for all PKS1 imaging and for µL of high capacity neutravidin-agarose beads (Thermo Fisher) instead of 15 µL.Palmitoylation of the PKS1 fragments was assayed as described in µL in an Eppendorf tube) were sown onto plates containing half-strength MS medium solidified with 08% (w/v) agar. After stratification for 3 to 5 d, germination was induced with 6 h of WL (80 μmol m−2 s−1). Individual plates were incubated at 22 °C for 3 d in darkness. Seedlings were collected, weighted, and ground with a mortar and pestle on ice. Two volumes of extraction buffer  were added. Samples were centrifuged for 5 min at 1600 rpm (200 g) in a benchtop centrifuge at 4 °C. The supernatant corresponds to the total fraction, S1. The supernatant was centrifuged again for 75 min at 100,000 × g at 4 °C. The supernatant corresponds to the soluble fraction, S2. The pellet was resuspended in 1 volume of extraction buffer with 0.5% (v/v) Triton X-100 and centrifuged again for 5 min at 14,000 rpm  in a benchtop centrifuge at 4 °C. The supernatant corresponds to fraction S3.One thousand seeds per replicate (100 µL of protein extract was incubated with 50 µL of magnetic beads for 90 min at 4 °C. The first 4 washes were done with extraction buffer (as defined above) + Triton X-100. Elution was done with 2× Laemmli buffer.Co-IP was done using a Miltenyi µMACS HA isolation kit (130-091-122), μ Columns (130-042-701), and a μMACS Separator (130-042-602) following the manufacturer's guidelines with a few modifications. The microsomal fraction S3 was used for the Co-IP. Briefly, 340 µL 2× Laemmli buffer for 20 mg fresh weight) and 10 µL was loaded per lane. Proteins were separated on 4% to 15% precast polyacrylamide gels except for data shown in For the biotin switch assay, proteins extracted in 2× Laemmli buffer were separated on 12% SDS/PAGE gels and transferred onto nitrocellulose membrane in CAPS buffer. Cell fractionation samples and Co-IP samples were mixed with 1 volume of 2× Laemmli buffer. For the cell fractionation assay, twice as much was loaded of S1 and S2 than S3. For all other immunoblots, total proteins were extracted in Laemmli buffer , PKS2 (At1g14280), PKS3 (At1g18810), PKS4 (At5g04190), NPH3 (At5g64330), BG1 (At5g12050), BG2 (At3g42800), BG3 (At1g54200), BG4 (At3g13980), BGL1 (At1g69160), BGL2 (At1g13670).The Arabidopsis Genome Initiative numbers for the genes mentioned in this article are as follows: koad096_Supplementary_DataClick here for additional data file.


Calotropis procera, Calotropis gigantea, and Asclepias currasavica that belong to the Asclepiadaceae family. All of these plants are recognised as medical traditional plants used in Asian countries. Calotropin is identified as a highly potent cardenolide that has a similar chemical structure to cardiac glycosides (such as digoxin and digitoxin). During the last few years, cytotoxic and antitumor effects of cardenolides glycosides have been reported more frequently. Among cardenolides, calotropin is identified as the most promising agent. In this updated and comprehensive review, we aimed to analyze and discuss the specific mechanisms and molecular targets of calotropin in cancer treatment to open new perspectives for the adjuvant treatment of different types of cancer. The effects of calotropin on cancer have been extensively studied in preclinical pharmacological studies in vitro using cancer cell lines and in vivo in experimental animal models that have targeted antitumor mechanisms and anticancer signaling pathways. The analyzed information from the specialized literature was obtained from scientific databases until December 2022, mainly from PubMed/MedLine, Google Scholar, Scopus, Web of Science, and Science Direct databases using specific MeSH search terms. The results of our analysis demonstrate that calotropin can be a potential chemotherapeutic/chemopreventive adjunctive agent in cancer pharmacotherapeutic management.Calotropin is a pharmacologically active compound isolated from milkweed plants like  This enzyme allows the active transport of Na+ and K+ ions through the cell membrane. It was observed that calotropin has a more perceptible impact on the myocardium compared to it on skeletal muscles, as myocytes have more active Na+/K+-ATPase  R. Br., Calotropis gigantea (L.) R. Br (Cg) and Asclepias subulata, the plants belonging to the family Asclepiadaceae . The leaf extract of Cp possessed a powerful antiulcer activity by to the reduction gastric acid secretion, as well as augmenting the mucosal defensive factors and inhibiting lipid peroxide levels  and melanoma (B-16) cells. The same authors also reported that those extracts induced a significant reduction in tumor weight as well as the growth of the sarcoma tumor in mice. Several previous studies of the biological activities of Cp have shown anti-inflammatory  primarily was isolated from iadaceae . Plants Pakistan . Other s America . The mend cancer . Differed cancer . Accidenarasites . Phytochycosides . A nativysentery . Despitee levels . In a pre levels . The pha strains . The eth in mice . The stuammatory , and antammatory  were shoCalotropis gigantea and showed that calotropin had the most pronounced cytotoxicity on A172 and U251 glioblastoma cell lines, and confirmed a possible anticancer mechanism that includes G2/M phase cell arrest . The IC50 value was by a previous study where was observed that calotropin has the same IC50 value on porcine cerebral cortex Na+/K+-ATPase activity  and Hs578T cells (IC50 = 0.06 ± 0.01 µM) compared to MDA-MB-231 . The results suggested that BT-459 cells are more sensitive to calotropin due to increases in intracellular Ca2+ levels, leading to cell death at lower concentrations than is required in other human TNBC cell lines. Another evidence that calotropin may have cytotoxic effects on cancer cell lines derives from its capability to inhibit the enzyme Na+/K + -ATPase viz altering through key transmembrane cellular protein homeostasis. The possible explanation of the different effects of calotropin in the different human triple-negative breast cancer cell lines may derive from the expression of different Na+/K+-ATPase isoforms and ↑Na+/Ca2+ exchanger (NCX) isoforms. It has been shown that α1 Na+/K+-ATPase isoform is more resistant to inhibition by some cardiac glycosides, and at the same time, its expression was lowest in BT-459 cells. However, in another cell line Hs578T the expression of α1 Na+/K+-ATPase isoform was intermediate. Related to another hypothesis with Na+/Ca2+ exchanger isoforms, it was observed that the mRNA of NCX1 isoform was significantly higher in BT-459 and Hs578T compared to the other human triple-negative breast cancer cell lines. Thus, the high mRNA expression of NCX1 may predict sensitivity to calotropin. The breast cancer BT-549 cells were treated with different doses of calotropin, 150 nM and 500 nM. The results showed that both doses of calotropin inhibit the efflux of Ca2+ via the Na+/Ca2+ exchanger. A major consequence of this inhibition is increased intracellular Na+, leading to increased Ca2+ entry and elevated concentrations of intracellular Ca2+. Cancer cells with defects in this signalling pathway might be particularly vulnerable, as an increase in intracellular Ca2+ triggers apoptosis  and downregulated expression of anti-apoptotic proteins: p53, B-cell lymphoma (Bcl 2) and Bclw  which is characterized by invasion, migration, rapid growth, and reoccurrence . In vitrand Bclw . On the and Bclw . In vivoese mice   involves changes in the intracellular cytoskeleton and extracellular matrix. After EMT, cells lost polarization, acquire an elongated morphology, and possess strong intercellular adhesion and polarity, which can be attributed to the reorganization of the actin cytoskeleton as well as increased expression of vimentin or keratin . EMT is 50 0.33 ± 0.06 μM at 48 h), the same inhibitory effect on A549 was weak (IC50 38.46 ± 1.16 μM at 48 h). Furthermore, while cell cycle arrest in the G2/M phase and an increased number of cells in the apoptotic phase were observed in A549/CDDP after treatment with calotropin, the same effects lack in A549. After the treatment with calotropin the expression of CDK1, CDK2, cyclin A, and cyclin B was significantly downregulated in A549/CDDP cell line, while p21 and p53 protein levels were increased. The same as in the previously mentioned research, the expression levels of the precursor forms of Cap- 3, Cap-8, and Cap-9 were downregulated and their active forms were upregulated. Also, the increased intracellular levels of ROS were observed in the same cell lines, as well as changes in mitochondrial membrane potential and poly (ADP-ribose) polymerase (PARP) after treatment with calotropin (in a dose of 0.5 and 1.0 μM) compared to a control group. The levels of anti-apoptotic protein Bcl-2 were decreased and pro-apoptotic Bax protein levels were increased in a concentration-dependent manner in the treatment group. All of the previously mentioned results were not observed after the treatment of calotropin on the A549 cell line. In A549/CDDP cell line treatment with calotropin was correlated with increased phosphorylation of JNK , while this was not observed in the A549 cell line. There is no provided explanation of the differences in the effects of calotropin on these 2 cell lines  and its parent’s cells lung adenocarcinoma A549 . Interesll lines .50 values of calotropin on the same cell line are 0.0013 µM, while antiproliferative activity was confirmed on another two cancer cell lines LS 180  and PC-3 (human prostate cancer cell line) with IC50 values 0.06 and 0.41 µM, respectively. Also, the cytotoxic effects of calotropin on lung adenocarcinoma A549 cells were confirmed in the research done by in vitro growth inhibitory activity of calotropin was tested on A549 cells, PC-3, and U373 (glioblastoma cell line) by Ibrahim and colleagues, and the same value of IC50 (0.005 µM) was reported for all 3 cell lines  mice. All of them were subcutaneously injected with H358 cells and after that divided into 2 groups. In the treatment group, mice were intravenously injected with calotropin (5.0 mg/kg) every day for 7 days, while PBS injections were used as the control. Related to in vitro experiments on H358 cells it was observed that calotropin suppressed cell growth in a time-dependent manner (after 72 h treatment). The cell proliferation was arrested at the G2/M phase. Also, calotropin inhibited protein level expression of cyclins (cyclin-dependent kinases: CDK1 and CDK2), fibronectin, vimentin and E-cadherin. The migration and invasion were inhibited after treatment with calotropin. Analysis of calotropin effects on H358 cells apoptosis showed that apoptosis was promoted. There was increased pro-apoptosis gene expression of caspase-3 (Cap-3), caspase-8 (Cap-8), and Apaf-1, while anti-apoptosis protein expression levels of p53, Bcl-2, and Bsl-w were decreased. Also, calotropin promoted Cyt-c and JNK protein expression levels. Additional effects of calotropin indicated that its treatment of H358 cells decreased the CTLA-4 (T-lymphocyte-associated-protein 4) protein expression (CTLA-4 protein is a checkpoint receptor that downregulates T cell activation), and inhibited the TGF-β protein expression levels and phosphorylation and protein expression levels of ERK1/2, indicated that probably TGF-β/ERK signalling pathway is included in calotropin-mediated apoptosis.Comprehensive research on the anticancer effects of calotropin was done by 50 value of calotropin was reported on the human hepatocarcinoma cell line (HepG2) and Raji cells (human B lymphoblastoid cell line), and values were 0.04 and 0.02 µM, respectively, which confirmed its cytotoxic effect on tumor cell lines . The anticancer activity of calotropin in HCC was linked to the suppression of fatty acid levels by a decrease in inflammatory cytokines and adipocyte shrinkage, as can be seen in a different model of liver carcinogenesis . During ll lines .Recently, the pro-apoptotic effects of calotropin were confirmed on HepG2 and human colorectal adenocarcinoma cells (Caco-2) .in vivo experiments of a 25-day short-term observation period were analysed it was suggested that injection of calotropin inhibited tumor growth. In the treatment group, the number of apoptotic cells and lymphocyte infiltration was increased. In excised tumors from the treatment group expression levels of Casp-3 and Casp-8 were increased. Besides the fact that the administration of calotropin has significantly inhibited tumor growth, after the 120-day observation period the prolonged mice survival was noted as well.Colorectal cancer (CRC) is a malignant neoplasm that develops in the colon and rectum, and its symptoms can be extremely varied; in its early stages, CRC is asymptomatic and is detected through screening examinations (2022). Calotropin markedly suppresses the proliferation of colorectal cancer cells by the Hippo pathway through the activity of YAP . Since tin vitro study was done on two types of human colorectal cancer cell lines HT-29 and HCT116, treated with calotropin for 24 h. Increased concentrations of calotropin (0.2–10 µM) inhibit proliferation in a dose-dependent manner in both cancer cell lines. Also, calotropin promoted dephosphorylation of YAP (Yes-associated protein) and induced its nuclear localization in colorectal cancer cells, therefore supporting the hypothesis that the Hippo signalling pathway is included in calotropin-induced inhibition of cell proliferation. Additionally, calotropin treatment shortened the half-life of LATS1 (large tumor suppressor 1) protein, the main kinase in the Hippo pathway that phosphorylates YAP. Calotropin probably promotes LATS1 degradation through the ubiquitination/proteasome pathway. Another part of the study was in vivo performed with BALB/c immunodeficient nude mice injected with HT-29 cells (6–8 weeks old). Mice were divided into two groups, the treatment group where calotropin was intravenously administered once every 2 days for 3 weeks, and the control group was treated in the same way during the same period with DMSO as a vehicle. It has been shown that the tumor obtained from the calotropin-treatment group was reduced in volume and weight. Another research on colon cancer cells (SW480 colon adenocarcinoma cell line) marked calotropin as an anticancer agent which inhibits the Wnt signalling pathway by decreasing nuclear and cytosolic located β-catenin in a dose-dependent manner and leading to degradation of β-catenin by increasing the phosphorylation of β-catenin through casein kinase 1α (CK1α) (In another research done by α (CK1α) . AdditioThe observation that the anticancer effects of calotropin are probably mediated in a caspase-dependent manner was for the first time reported on human chronic myeloid leukemia K562 cells in research done by https://www.wcrf.org/cancer-trends/colorectal-cancer-statistics/accessed September 2022), so only during 2020, there were more than 1.9 million of new cases  (2022). The combination of low concentrations of 5-FU and calotropin in comparison to a high dose of 5-FU reduced cell viability and abolish resistance to this conventional chemotherapeutic in HCT116 colorectal cancer cells. The same treatment significantly reduced the cellular ATP levels and increase ROS levels in HCT116 cells (Standard treatments currently involve chemotherapy with or without surgical resection but there are still certain limitations. Using traditional plant extracts as an alternative therapeutic strategy is subject to much research. Standard chemotherapy is associated with drug resistance and the occurrence of systemic adverse effects on different organs that limit its utility Therefore, to overcome these barriers and achieve better therapeutic success, there are recommendations in some studies of a combination regimen of traditional chemotherapeutic and medicinal plant extracts . In rece16 cells . Additio16 cells . The lev16 cells .in vivo studies of calotropin in experimental animal models for all cancers. As a result, these studies are needed in the future to confirm its anti-cancer therapeutic potential. Translational pharmacological studies are also needed to establish effective therapeutic doses in humans.The therapeutic limitations and clinical pitfalls of calotropin as an anticancer agent are represented by insufficient data on adverse and toxic effects in humans. Very few cases have reported some digestive, hepatotoxic adverse effects and only one case with severe cardiotoxicity . Other iin vitro, while there is a lack of in vivo evidence, which is especially noted regarding human clinical studies; there are no available clinical trials where calotropin was investigated. Increasing interest in the anticancer effects of calotropin and numerous evidences of its activity on cancer cell lines puts calotropin on the pedestal of cancer research. Additionally, the encouraging evidence that the cytotoxic effects of calotropin can be avoided on normal cell lines makes it a promising agent for further research. There is not a lot of evidence of anticancer activity for chemicals obtained from plant-like there are for calotropin. However, it is evident that in vivo experiments on animals are still deficient, and that should be the next step in calotropin research.Traditional medicine from the Middle East, India, and China has a long period of usage, development and impact on the later evolvement of a great number of approved drugs and supplements. Until today this traditional medicine has been recognized as one of the most diverse with daily consumption by a huge number of their citizens. Despite their long-term usage, for most of the traditionally used plants, the full mechanism of action has not been discovered yet. However, modern medicine in the last few decades turns to reveal its true impact on health improvement and to confirm its mechanism of action. Finding the critical molecules in these traditionally used plants has a further influence on the development of modern drugs approved by different regulatory bodies across the world. Studies have shown that calotropin has cytotoxic and antitumor effects, the most representative effects being on breast, colon, lung and leukemia cancers. The main limitation of calotropin is represented by the fact its anticancer activity has been confirmed


Increasing evidence points to a role of the circadian clock in the regulation of cancer hallmarks with a strong impact on the understanding and treatment of this disease. Anti-cancer treatment can be personalized considering treatment timing. Here we present a new mathematical model based on data from three colorectal cancer cell lines and core-clock knock-outs, which couples the circadian and drug metabolism network, and that allows to determine toxicity profiles for a given drug and cell type. Moreover, this model integrates external Zeitgebers and thus may be used to fine-tune toxicity by using external factors, such as light, and therefore, to a certain extent, help fitting the endogenous rhythms of the patients to a defined clinic routine facilitating the implementation of time-dependent treatment in clinical practice. To outgrowth in the body, cancer cells need to escape a series of safe keeping mechanisms known as the hallmarks of cancer3. Accumulating evidence points to a role for the circadian clock in regulating most, if not all, of these hallmarks4. Understanding the mechanisms that relate the circadian clock to cancer will help to develop new therapies or optimize existing ones.A wide range of diseases are reportedly associated with the disruption of circadian rhythms, such as obesity, sleeping disorders, neurodegenerative diseases and cancer development5. Colorectal cancer (CRC) is the third most common cancer worldwide. Estimates from 2020 point to 1.9 million incidence cases and 0.9 million deaths making CRC the second most deadly cancer type worldwide6. CRC occurs in the large intestine (colon) and the rectum of the gastrointestinal tract. Depending on the exact location, it is also referred to as large bowel cancer, colon cancer or rectal cancer. CRC affects mostly older patients, with a median age at diagnosis of 72 years in males and 75 years in females7. Many patients are treated with chemotherapy, during which they receive a drug that interferes with the mechanism of cell division, and thus induces cell death preferentially in fast-dividing cells. Fast dividing cells encompass not only cancer cells, but also, for example, cells in the gastrointestinal tract, which leads to side effects such as strong diarrhoea, particularly devastating for patients of old age. Chronotherapy aims to alleviate the side effects of anti-cancer treatment by aligning the drug exposure to the circadian time of the patient8, and several clinical studies reported an overall treatment improvement in cancer when considering drug administration time9. The desired timing where drug effectiveness is maximised and side effects minimized results from the regulation via the circadian clock over the expression of numerous genes including those responsible for drug metabolism10. In a clinical trial, in which patients received the drug irinotecan at different times of the day, an ideal time of treatment, for which patients showed the least severe side effects, could be identified for sex-specific patient groups10, this is also the case for other anti-cancer drugs11. For personalized treatment with irinotecan, we established previously a mathematical model, which relates circadian gene expression with the circadian profile of irinotecan toxicity12. The model combined a transcription-translation network of the core clock and of clock-regulated genes relevant for irinotecan with a model of irinotecan pharmacokinetics and -dynamics (PK-PD). Our previous work on SW480 and SW620 CRC cells showed different toxicity profiles, in particular a flatter circadian toxicity profile for the SW620 cell line with reduced circadian oscillations12.Cancer is a major health problem, it caused death in about one out of six persons, and was the second most common cause of death worldwide in 2020. This number is expected to increase with the continuous aging of the population12, we could improve the fit of the data by network refinements including new network connections. The fit quality improved the coefficient of determination from a R2 value of 0.23  to a R2 value of 0.29 with the here proposed refinements , also three different core-clock knock-outs (KOs), In patients, an analogue altered circadian rhythm amplitude, and the expected reduced circadian oscillation of the toxicity, would make chronotherapy less effective. To alleviate this problem, the usage of external Zeitgebers  is an interesting option, which may enhance circadian amplitudes of the core clock and thus impact toxicity profiles. In addition, external Zeitgebers may enable to manipulate the patients’ cytotoxicity curves, which would allow, for example, to fit a patient internal rhythm to a certain clinic schedule. We thus incorporated in our model the influence of external Zeitgebers, such as light, to the predicted toxicity profile. Our results show that our model, which can be fitted to different human CRC cell lines, also allows us to generate personalized cytotoxicity curves, which can be further fine-tuned via external Zeitgebers. Such an integrative approach using the individual biological data (here cell lines) allows for a personalization of treatment schedules, which may strongly benefit treatment outcome and that can be further adjusted, if need be, to implement an enhancement of the patients’ endogenous rhythms, as well as an integration in a realistic clinic treatment schedule, by using external cues to fine-tune the clock.Scheduling anti-cancer drug administration over 24 h may critically impact treatment success in a patient-specific manner. We address personalization of anti-cancer treatment toxicity time for irinotecan, an anti-cancer drug widely used against digestive malignancies. For this we developed a mathematical model that links cellular pharmacokinetics and -dynamics (PK-PD) of irinotecan to a representation of the core clock, which together predict treatment toxicity based on circadian gene expression profiles, and further allow for a fine-tuning of cytotoxicity profiles using external Zeitgebers Fig. .Fig. 1Bavs. a metastasis site, respectively), show strong differences in gene expression. We found an appropriate level of heterogeneity in a set of three core-clock KOs and the WT of the same CRC cell line (HCT116), as described below.We are particularly interested in heterogeneous circadian gene expression profiles to simulate the variety of expected circadian profiles within a patient cohort. The heterogeneity of different CRC cell lines is due to their pathological state rather large; even cell lines such as SW480 and SW620, which are derived from the same patient, but different tumour sites  required in Hesse et al. 202112 to fit the mRNA expression of CES2 and ABCC  gene), a negative regulator of mTOR that influences autophagy, shows a pronounced peak following irinotecan treatment start21. The full mathematical description of the model including the model equations with modifications marked in blue font colour is provided in the For the transcription-translation network, the model presented here refines the connections between network elements as compared to the previous model from Hesse et al. 2021BCC Fig. . For the12, see Supplementary Fig. For the SW480 cell line, we compared fits to the RNA-seq data (sampled over 30 h) with the rescaled microarray data (sampled over 24 h) used in Hesse et al.PER2KO, NR1D1KO, and ARNTLKO, as well as SW480 lied within the same order of magnitude . The noteworthy exception is UGT1A1, responsible for drug removal, which was in the HCT116 cell lines very low expressed, in agreement with the literature22. The RNA-seq datasets of HCT116 WT, PER2KO, NR1D1KO, and ARNTLKO, as well as SW480 showed consistent drifts, or linear trends, in addition to the actual oscillation, that were not observed in the SW480 microarray data . Genes with a linear trend in the RNA-seq data were still fitted in reasonable agreement with the microarray data. This highlights the potential of the model to uncover hidden oscillations in the data. As the microarray data was measured at an earlier time point and for a shorter time interval as compared to the SW480 RNA-seq data, these linear trends may hint at an underlying adaptation to the fresh cell culture media used for synchronization only visible in the longer time series of the RNA-seq data.For most genes, the Counts Per Million (CPM) values for the CRC cell lines HCT116 WT, 23. In the PER2KO cells, PER1 is upregulated, likewise in the NR1D1KO cells NR1D2 is upregulated , see Fig. 23.Our data shows paralog compensation for HCT116 cells, i.e. downregulation of one paralog leads to the upregulation of another, thereby maintaining the overall sum of expression profiles almost unchanged, in agreement with previous reports on other cell typesAs KO cell lines were derived with defined changes from the WT (single gene KOs), they should resemble the wild-type cell line, with strong differences in only few aspects of the gene regulatory network, which try to compensate for the gene loss. Regularization of the model fit resulted in a small subset of strongly deviating parameters that may give a hint as to which biological processes were altered in the KO as compared to the WT cell lines.Our transcription-translation network models the interaction between genes, and could be fitted to all CRC cell lines here tested, including the three HCT116 core-clock KO cell lines Fig. . The larλ, thus making the KO models more similar to the WT . On the other hand, weaker interactions result from an upregulation of nuclear protein degradation . Thereby, faster degradation implies faster removal and thus a reduced amount of nuclear proteins in the KO cell lines compared to the wild type, and as the amount of nuclear proteins regulate the translation of other genes, the reduced amount of nuclear proteins can be interpreted as a reduced interaction between core-clock genes. These changes seem to hint at an overall reduced interaction between the core-clock elements of the KO cell lines compared to the WT. These results are in line with previous observations that under certain conditions, such as a perturbation of the clock due to external or internal factors, the interaction strength of the core-clock network is affected25.Our data showed that about half of the parameters is altered by more than 5% in at least one KO, see Fig. 2 values at penalty 0, i.e. no regularization, with R2 values at penalty 5), and the fits do not resemble the WT data . To evaluate whether the transcription-translation network of the HCT116 KO cell lines is more similar to their WT as compared to other cell lines, we fit two different CRC cell lines, SW480 and SW620, using the same regularization of the parameters based on the HCT116 WT as for the KOs. In agreement with our assumption that the HCT116 KO cell lines show a larger similarity to the HCT116 WT as compared to other cell lines, we find stronger parameter deviations for the SW480 and SW620 cell lines compared to the HCT116 KO cell lines  of irinotecan via the expression of UGT1A1, CES2, ABCB and ABCC1, as well as the free parameters of the PK-PD model part are fitted to cytotoxicity from Hesse et al. 202112, see Methods for details. Protein translation of UGT1A1, CES2, ABCB and ABCC1 is implemented with circadian protein degradation, as common for many proteins19. As in Hesse et al. 202112, maximal protein expression is rescaled to the maximal concentrations used by Dulong et al. 201514. To account for the reduced expression of UGT1A1 in HCT116 cell lines, 10-fold reduction in the UGT protein expression is assumed for HCT116 cell lines. After this rescaling, UGT is increased in a sigmoidal way following treatment.The protein translation of the mRNAs et al. 202112 shows an oscillation whose phase is reset by treatment time. The death rate in the model presented here shows a circadian oscillation independent of treatment, plus a transient increase in death rate induced by treatment.Besides the UGT increase, we free the parameters of the PK-PD model part in the equations for the number of living and dead cells, and the cell death modulation. The death rate in Hesse UGT1A1 expression in HCT116 cell lines, as well as the here observed changes in other genes relevant for irinotecan metabolism 33, differ in the timing of the maximal phase shifts, making this an attractive alternative if the desired phase shift is not permissive with bright light therapy due to time restrictions. Pulses in PER (light) and NR1D  led to differential responses of the core clock, suggesting that the reaction of the core clock is specific to the affected gene(s). In the future, we plan to extend our model to explicitly include metabolic dynamics, such as those of the model by Woller et al. 201646. These specific interactions will affect a larger number of core clock genes in response to metabolic signals, and will modulate the altered temporal profile resulting from perturbation induced in core-clock genes. Approaches such as bright light therapy may potentially be used to entrain the peripheral clock of patients to a timepoint where the patient-specific treatment regime overlaps with the ideal treatment timepoint, in particular given that specific treatment hours might be unrealistic due to clinic opening hours.We complemented our model with a 24 h day-night rhythm and tested the effect of pulses in 47. Promising for irinotecan treatment is also the experimentally observed correlation between CES2 expression and the activation of irinotecan in tumour tissue48.Our approach to personalization of treatment timing uses a model that relates gene expression with irinotecan toxicity. The model may aid to bridge the observed gap between gene expression and toxicity; for example, mouse data shows only partly a relation between gene expression and toxicityOptimal treatment timing can be a combination of multiple factors, and dependent on the patient and cancer type, as well as development stage, the best timepoint of treatment could be where the least side effects occur or where the highest cytotoxicity lays, and mathematical models like the one here presented may support clinical decisions in such choices and enable a more personalized treatment planning, for the benefit of the patients.PER2KO, NR1D1KO, ARNTLKO) published in Yalçin et al. 202124 (accession number: E-MTAB-9701), and from SW480 and SW620 , as well as for comparison microarray data from SW480 .This study considered data from six different CRC cell lines, RNA-seq data from the wild-type HCT116 cell line and three core-clock knock-outs of the HCT116 cell line  and for comparison with the previous model the SW480 cell line and the SW620 cell line. For the SW480 cell line, the model is for comparison also fitted to microarray data, which was rescaled to the same mean as the RNA-seq data.Circadian dynamics in gene expression was modelled by a transcription-translation network including core-clock genes and core-clock regulated genes relevant for irinotecan metabolism. This model was fitted to mRNA expression derived from RNA-seq data for six different cell lines, the wild-type HCT116 cell line, three core-clock knock-outs of the HCT116 cell line  with free magnitude MUGT and free slope kUGT,t as time after treatment.The PK-PD model from Hesse et al. 2021The death rate in the model presented here shows a circadian oscillation, plus a transient increase in death rate, which is modelled by an alpha function, see 51 via the Python implementation pycma on a compute cluster. The cost function is the squared error between data and model fit, evaluating for each mRNA j the simulation sj(t) at the same time points ti as the data xji was sampled, normalized by the maximum gene expression of the mRNA expression data, summed for all genes in the network:Parameter optimization used the evolutionary algorithm CMA-ESHere the maximum is taken for each mRNA over the experimental time series. The division ensures equal weight to all mRNAs independent of their concentration.−4, absolute tolerance = 10−12). Model fits of mRNA were forced to oscillate with a minimum relative amplitude of 5%, i.e. for the maximum and minimum of the simulated time series we demanded (max-min)/max > 0.05, with the exception of UGT1A1 for the HCT116 cell lines, which was not expressed in these cell lines.For the numerical integration we used Python’s scipy.integrate.odeint .As for the transcription-translation network, parameter optimization used the evolutionary algorithm CMA-ES via the Python implementation pycma on a compute cluster. The cost function is the squared error between cytotoxicity data and model fit. For the numerical integration we used Python’s scipy.integrate.solve_ivp  can shift the toxicity profile of the model simulation. Light was implemented as a change in the maximal transcription rate of PER, i.e. the light-sensitive transcription rate V1maxlight is given asV1max is the maximal transcription rate of Per without light, and f > 1 is a constant factor accounting for the increase in transcription rate with light on, see Zeitgebers, such as bright light pulses, can shift circadian rhythms. We asked whether a simple implementation of light as a transient increase in the dynamical variable The strength of the rhythmic light entrainment was determined by the period of a harmonic oscillation of 24 h and a relative amplitude over 64 h that is at least similarly strong as without light.durpulse = 1 h and a strength of flight = 3 unless stated otherwise, given at a certain perturbation time Tpulse. If the transcription-translation network was not entrained, light pulse stimulation induced persistent shifts in the phase of the simulated mRNA expression. For a more realistic setting, we entrained the circadian oscillations of the model with a zeitgeber light that follows a 12 h dark-12h light cycle, with a strength of flight = 1.07. Giving a light pulse stimulus in the entrained system led to a transient shift in the phase of the mRNA oscillations.We investigated the response of the system in response to a short and strong light pulse with a duration of ARNTL. The relative phase shift was measured as the time difference of the maximum of ARNTL in the second maximum after the pulse comparing the condition with light pulse with the condition without light pulse.We evaluated the phase shift induced by light pulses with different parameters on the gene expression of NR1D, implemented in analogue to the pulses on PER.We also tested the effect of pulses that increase the expression of Further information on research design is available in the Supplemental materialReporting Summary


Helichrysum italicum (Roth) G. Don., immortelle, is a plant species used in ethnomedicine and the food industry as a spice added to food, beverages, and bakery products. It has been shown to possess various biological activities, such as antioxidant and antibacterial activity, making it useful as a natural preservative. We investigated the phytochemical profile and biological activity of H. italicum essential oils from wild-grown plant material collected from natural habitats in the Republic of Croatia and Bosnia and Herzegovina. Using high-resolution scanning electron microscopy (SEM), a visual investigation of plant organs  was performed, confirming the presence of essential oil reservoirs on the surface of all examined plant organs. Essential oils were isolated by hydrodistillation in the Clevenger apparatus. The chemical composition of the essential oils was determined using the GC-MS analytical technique. Cytotoxic activity tests were performed in vitro on three cell lines: skin (fibroblast), lung, and breast cancer. Using statistical tools, the synergistic and selective effects of H. italicum essential oil on healthy and tumor cells were correlated to chemical composition and cytotoxic activity. The synergistic and antagonistic effects of H. italicum essential oil’s individual components were simulated by testing pure compounds and their mixture of cytotoxic activity on fibroblasts and breast cancer cells. The results confirm that essential oil’s biological activity is much greater than the sum of the effects of its components. The present data are novel contributions to the body of knowledge on the biological activity of this species used in the food industry. Helichrysum italicum (Roth) G. Don., also known as curry plant or immortelle, is a perennial herb from the Asteraceae family. It is native to the Mediterranean region but can also be found in other parts of the world, such as Africa  vs. normalized response—Variable slope, was used to fit normalized cell viability curves. The mean and standard deviation are used to express the calculated ICficients . R valueH. italicum. Using SEM, we have determined that stems, flowers, and leaves contain essential oil reservoirs on their surfaces, proving that all areal plant organs are viable sources of EO. Further, we have investigated the chemical composition using GC/MS of nine H. italicum essential oils collected along the Croatian coast. The main compound in EOs of H. italicum is neryl acetate, whose share in the analyzed samples was 4.48–21.36%. Other terpene compounds found in all samples are limonene, β-selinene, α-copaene, italicene, α-terpineol, Ar-curcumene, and β-selinene. Eight out of nine obtained EOs could be classified as high quality considering the proportions of neryl acetate and α-pinene.We investigated the aerial parts of H. italicum EO using five pure compounds: linalool, α-terpineol, terpinene-4-ol, nerol, and nerolidol. The results showed a conclusive antagonistic effect on their individual cytotoxic potential, more evident in the MDA-MB-231 cell line, in which individual compounds were more toxic than in the HDF cell line. These results explain why this cell line was the most resistant to H. italicum EO. Furthermore, this study supports the idea of using H. italicum in the human diet, indicating its safety for dietary consumption.Obtained EOs showed moderate or low cytotoxic potential in three cell lines: skin (fibroblast), lung, and breast cancer. The lung cell line was the least resistant to EOs, while the breast cancer cell line was the most resistant of the three cell lines used. To gain insight into which compounds or compound groups impact EO’s cytotoxic potential, we performed a statistical analysis, which led to the identification of several compounds or compound groups with significant activity for the individual cell line. As EOs are complex mixtures, we simulated


VEGFR2 and PTEN mRNAs and impacts VEGFR2 downstream signaling, in part via PTEN regulation.Lung cancer is the most common and lethal malignancy worldwide. Musashi-2 (MSI2) is an RNA-binding protein that is overexpressed in advanced NSCLC. VEGFR2 protein expression contributes to NSCLC progression and several FDA-approved drugs are used to target it in the clinic. Here, we show that MSI2 is a strong positive regulator of VEGFR2 protein levels in murine and human NSCLC cell lines. Furthermore, we found that MSI2 protein directly binds to PTEN mRNA translation regulation. In silico prediction analysis suggested that both VEGFR2 and PTEN mRNAs have predicted binding sites for MSI2. We next performed RNA immunoprecipitation coupled with quantitative PCR, which confirmed that MSI2 directly binds to VEGFR2 and PTEN mRNAs, suggesting a direct regulation mechanism. Finally, MSI2 expression positively correlated with VEGFR2 and VEGF-A protein levels in human lung adenocarcinoma samples. We conclude that the MSI2/VEGFR2 axis contributes to lung adenocarcinoma progression and is worth further investigations and therapeutic targeting.Lung cancer is the most frequently diagnosed cancer type and the leading cause of cancer-related deaths worldwide. Non-small cell lung cancer (NSCLC) represents most of the diagnoses of lung cancer. Vascular endothelial growth factor receptor-2 (VEGFR2) is a member of the VEGF family of receptor tyrosine kinase proteins, which are expressed on both endothelial and tumor cells, are one of the key proteins contributing to cancer development, and are involved in drug resistance. We previously showed that Musashi-2 (MSI2) RNA-binding protein is associated with NSCLC progression by regulating several signaling pathways relevant to NSCLC. In this study, we performed Reverse Protein Phase Array (RPPA) analysis of murine lung cancer, which suggests that VEGFR2 protein is strongly positively regulated by MSI2. Next, we validated VEGFR2 protein regulation by MSI2 in several human lung adenocarcinoma cell line models. Additionally, we found that MSI2 affected AKT signaling via negative  KRAS and TP53, NSCLC is associated with numerous genetic alterations such as EGFR mutations, ALK translocation, VEGFR2 and c-MET amplification, and others [Lung cancer is second in incidence and first in mortality among all cancer types in recent years based on World Health Organization and American Cancer Society reports ,2. Most d others ,8. At thd others .VEGFR2 gain-of-function is associated with chemoresistance and poor survival in patients with lung cancer [p = 0.0019) [p = 0.023) [Vascular endothelial growth factor (VEGF-A) and its receptors  are the main pro-angiogenic drivers of solid tumors . In the g cancer . Currentg cancer . Adding  0.0019) . AdditioFurthermore, Musashi-2 (MSI2) protein expression is associated with advanced NSCLC ,25. MSI2VEGFR2 and PTEN mRNAs and impacts VEGFR2 downstream signaling via PTEN regulation. Finally, MSI2 protein expression correlated with VEGFR2 and VEGF-A protein levels in NSCLC patient samples.In the light of published papers, we have validated proteomic analysis data that suggested VEGFR2 regulation upon MSI2 depletion in murine lung cancer. We found that MSI2 depletion leads to a strong decrease in VEGFR2 protein levels in murine and human lung adenocarcinoma cell lines. Furthermore, we found that MSI2 protein directly binds to 34G>AKRAS, 1_471del471CDKNA), Calu-1 , H23 , and H441  were obtained from the American Type Culture Collection (ATCC). Human lung cancer cell lines Hcc1171  and Hcc461  were obtained from UT Southwestern Medical Center. The murine NSCLC cell line (344SQ) from Trp53R172HΔG/+ KrasLA1/+ mice was previously described [2, 94% N2, and 5% CO2.Human lung adenocarcinoma cell lines A549 (Anti-MSI2 (#ab76148) and anti-VEGF-A (#ab46154) were obtained from Abcam . Anti-VEGFR2 (#2479), anti-phVEGFR2 (#3817), anti-β-actin (#3700), anti-phAKT (T308) (#13038), anti-phAKT (S473) (#4060), anti-AKT total (#2920), anti-phGSKαβ (#9331), anti-GSKαβ total (#5676), anti-PTEN (#14642), anti-phERK (#4370), anti-ERK total (4695), anti-ph4EBP(#2855), anti-phP70SK (#9206), anti-P70S6K total (#2708), anti-rabbit HRP-linked (#7074), and anti-mouse HRP-linked (#7076) were obtained from Cell Signaling . Doxycycline (#HY-N0565), hygromycin (HY-B0490), and puromycin (HY-B1743A) were obtained from MedChemExpress .To generate cell lines with inducible knockdown of MSI2, we used self-complementary single-stranded DNA oligos from SiRNA transfection was performed using nonspecific control pool siRNAs and anti-human MSI2 SiRNAs . Detailst-test or ANOVA to determine statistical significance.Cell lysate preparation and Western blot analysis were performed using standard methods as previously described . Band si® assay  based on the manufacturer’s protocol to measure OD 562 nm.To evaluate the effects of MSI2 protein level on cell proliferation, we plated cells at a concentration of 500 cells/well in 96-well cell culture plates in triplicate. After 72 h of incubation to allow cells to grow, we added reagents for the CellTiter-Blue−ΔΔCt method using the primers indicated in RNA was extracted using a phenol–chloroform-based method. RNA concentration and quantity were measured using NanoDrop Lite . First-strand cDNA synthesis was performed using the iScript cDNA synthesis kit  according to the manufacturer’s instructions. The generated cDNA was diluted tenfold and used as a template for qPCR, which was performed with an Applied Biosystems QuantStudio 3 system using PowerTrack™ SYBR Green Master Mix . Relative quantification of gene expression was performed using the 2The concentration of VEGF-A in human lung cancer cell lines was determined by a VEGF Human ELISA kit . ELISA assays were performed according to the manufacturer’s instructions. Briefly, the collected condition media from cells were added to a well coated with primary antibody and then immunosorbented using a biotinylated primary antibody at room temperature for 2.5 h. The color development catalyzed by horseradish peroxidase was terminated with 2.5 mol/L sulfuric acid and the absorption was measured at 450 nm. The protein concentration was determined by comparing the relative absorbance of the samples with the standards.RNA-IP was performed using the Magna RIP RNA-binding Protein Immunoprecipitation kit  according to the manufacturer’s protocol. Immunoprecipitated RNAs were quantified by quantitative PCR (qPCR) using the primers indicated in The RPPA analysis procedure of 344SQ-SCR, 344SQ-m1, and 344SQ-m2 mouse cells was previously described .Human non-squamous NSCLC patient samples from the Rоstov Research Institute’s Human Tissue Repository Facility (HTRF) and from the Republican Clinical Oncology Dispensary named after Prof. M. Z. Sigal (RCOD at Kazan) were used. At the time of tissue acquisition, patients provided Institutional Review Board (IRB)–approved informed consent for the storage of tissues and the review of deidentified clinical data. Clinical information  from thep values) and Wang et al. [p values; Human and murine genome sequences for EGFR were obtained from the UCSC Human Gene Sorter December 2013 (GRCh38/hg38) assembly and scanned for the Musashi binding motifs previously defined by Bennett et al.  .All statistical analyses, including unpaired two-tailed VEGFR2 mRNA levels  [ACTB and GAPDH as negative controls. Antibodies to MSI2 specifically immunoprecipitated the VEGFR2 mRNA as efficiently as they did for the positive controls   as positcontrols D. Taken VEGFR2 mRNA in human lung adenocarcinoma cell lines, we established cell lines with MSI2 depletion and overexpression to evaluate the effect of MSI2 expression on VEGFR2 and VEGF-A protein levels. Depletion of MSI2 in human NSCLC cell lines led to a significant decrease in VEGFR2 protein levels and had a mixed effect on intracellular VEGF-A protein levels   . In para Calu-1) . TherefoSince MSI2 depletion leads to a reduction in VEGFR2 protein levels, we evaluated VEGFR2 downstream signaling in human NSCLC cell lines. Western blot analysis showed that MSI2 depletion reduces VEGFR2 and phAKT protein levels . To assePTEN mRNA levels and affect AKT signaling in lung adenocarcinoma. To test this, we performed in silico analysis and found that the 3′ UTR of PTEN mRNA has predicted binding sites for MSI2  and A549 as a control with a low level of VEGFR2 expression (VEGFR2 but not VEGF-A mRNA. These results expand our knowledge about the role of Musashi-2 in NSCLC progression.First, we selected human lung adenocarcinoma cell lines with high levels of pression A,B. MSI2pression C,D. In sVEGFR2 overexpression. This effect may be interpreted as the indirect positive regulation of VEGF-A by MSI2 [We then established human NSCLC cell lines with MSI2 KD and MSI2 OE to see how MSI2 levels affect VEGFR2 and VEGF-A protein and mRNA levels . We foun by MSI2 ,40.KRASmut, and published papers show that KRASmut results in constitutive activation of ERK [As MSI2 regulates VEGFR2 protein levels, we evaluated cell proliferation along with VEGFR2 downstream signaling in human lung adenocarcinoma cell lines upon MSI2 depletion. The viability assay showed that MSI2 does not affect cell growth in these KRAS-driven cells . Signalin of ERK ,42. NextPTEN mRNA binding, independent of VEGFR2 mRNA regulation.Wang et al.  previousIn addition, TMA analysis of human lung adenocarcinoma samples showed a positive correlation between MSI2 and VEGFR2 and VEGF-A protein levels . Taken tVEGFR2 and PTEN mRNA binding in lung adenocarcinoma. MSI2 protein expression was correlated with VEGFR2 and VEGF-A protein levels in non-squamous NSCLC patient samples. MSI2 is a promising therapeutic target for better control of NSCLC.MSI2 protein directly regulates VEGFR2 and PTEN protein levels via