N,N-dimethyl-N-β-lauryl/myristyl 7/3 polyethyleneoxy n = 3–18 ethylamines, LM(EO)nAT, are niche intermediates in the synthesis of homogeneous N-alkyl (C1–C18)-N,N-dimethyl-N-β-lauryl/myristyl 7/3 polyethyleneoxy n = 3–18 ethylammonium chlorides (unitary degree of oligomerization of ethylene oxide in the polyoxyethylene chain). This paper synthetically presents the dependence of the reductive methylation yields of homogeneous primary β-lauryl/myristyl 7/3 polyethyleneoxy n = 3–18 ethylamines, LM(EO)nAP, on the reaction time (10–90 min), the temperature (70 °C), the molar ratio formic aldehyde /LM(EO)nAP (1.1/1–2.5/1), the molar ratio HCOOH/LM(EO)nAP (5/1), the degree of oligomerization of ethylene oxide in the homogeneous polyoxyethylene chain in the 3,6,9,12,18 series, and the structure of the phase-transfer catalysts. The steric effects of hydrophobic groups CH3 and C18H37 grafted onto the ammonium function, and the micellar phenomena in the vicinity of their critical micellar concentration, directly proportional to the homogeneous degree of oligomerization, were highlighted. In all cases, a steady increase in reductive methylation yields was observed, with even quantitative values obtained. The high purity of the homologous series LM(EO)nAT will allow their personalization as reference structures for the study of the evolution of basic colloidal characteristics useful in forecasting technological applications. LM(EO)nAP were obtained either by direct amidoethylation nOH, to acrylamide monomer) or by cyanoethylation of LM(EO)nOH under basic catalysis at 25–50 °C, in the presence of Fe2+ cations as oligomerization/polymerization inhibitor, followed by partial acid hydrolysis of homogeneous β-alkyl (C12H25/C14H29) 7/3 polyethyleneoxy n = 3, 6, 9, 12, 18 propionitriles, LM(EO)nPN, to β-alkyl (C12H25/C14H29) 7/3 polyethyleneoxy n = 3, 6, 9, 12, 18 propionamides, LM(EO)nPD, which led to LM(EO)nAP by Hoffmann degradation. Homogeneous higher tertiary polyetheramines LM(EO)nAT were structurally characterized.Homogeneous tertiary  The recent literature in the field mainly considers two ways of carrying out the process: (a) indirect reductive alkylation (amination) or (b) direct reductive alkylation (amination) [4BF4), ammonium perchlorate (NH4ClO4), ammonium hexafluorophosphate (NH4PF6), or formamide, we distinguish the Leuckart reaction [N-formyl derivatives above 180 °C), which limited the generalization of these processes. The reductive methylation that employed formic aldehyde and formic acid has become widespread, the Eschweiler–Clarke reaction. The steadily growing interest in reductive methylation studies has been driven, with all the limitations mentioned, by the fact that tertiary amino groups are generally frequently present in biologically active compounds  in modern medicine, pharmaceutical synthesis, compounds specialized in the elimination of CO2, of sulfur impurities, monitored polymerization, stabilization of polymers against degradation induced by light radiation, etc. [N-formyl imines, N-formyl iminium cations, carbenium-onium ions, and amphions appear  . If formreaction . Both pron, etc. ,10. The on, etc. ,15,16,17s appear . The rea effects ,12.2 and the formation of the secondary amine, then, after resuming the cycle, of the tertiary amine [The literature  reports ry amine .N-alkylation (including methylation) with alkyl halides, O-alkylation and/or S-alkylation have also benefited from the competencies of phase-transfer catalysis [N,N,N-tricapryl-N-methylammonium chloride, N,N,N,N-tetrabutylammonium chloride, and N,N,N-triethyl-N-benzylammonium chloride. Soluble in both phases, they transfer the low energy carbenium-onium cation with positive charge delocalized in both directions. Among the most publicized examples, we mention aliphatic amines as such, aromatic, or mixed: hydrazobenzene, phenylhydrazone, diazoamine derivatives, and nitrogen heterocycles  [nAP, obtained by cyanoethylation or amidoethylation, i.e., nucleophilic addition under basic catalysis of homogeneous lauryl/myristyl 7/3 polyethyleneoxy n = 3, 6, 9, 12, 18 alcohols, LM(EO)nOH, [nPN, or the Hofmann degradation of homogeneous β-lauryl/myristyl 7/3 polyethyleneoxy n = 3, 6, 9, 12, 18 propionamides, LM(EO)nPD, obtained, respectively, on the main operating parameters .Conventional atalysis ,14,15, ae, etc.) ,16. In e(EO)nOH, ,18 to ac(EO)nOH, ,21,22,23nAP (not reported in the literature) is also an oxidation-reduction process with a pronounced heterogeneous ionic character, in which the direct contact of the reactants depends decisively on the homogeneity of the system. As a result, operating yields rarely exceed 80%. Preliminary experimental tests performed on the entire chain of transformations  led even from the cyanoethylation stages to mixtures of complex compounds. This confirms the aspects reported previously and for the cyanoethylation of the same hydroxyl substrates without polyoxyethylene chains: the existence of an induction period (approx. 3–5 min), the subsequent appearance of yellow-orange suspensions, of reddish-brown resin, the violent and even explosive evolution of the process, the simultaneous unfolding of two parallel, concurrent reactions (nucleophilic addition/anionic polymerization of the acrylic monomer), and the reversible character at long processing times and/or high alkalinity. The subsequent strategy followed after cyanoethylation attempted to monitor the other steps  transferred through batteries of processing columns filled with aqueous pyrogalol solution 40%, granular decarbonated KOH, and granular silica gel indicator impregnated with 2% cobalt chloride. Finely ground FeSO4 was suspended in the reaction mixture as polymerization inhibitor. The exhaustive preliminary purification of LM(EO)nOH from LM(EO)0OH, EOn, traces of water, secondary products consuming excess acrylic monomer, and subsequently other reagents was performed by repeated solid/liquid and liquid/liquid extractions in binary/ternary solvent systems (The reductive methylation of LM(EO)rmations  with heter steps  after th systems .nOH  in LM(EO)nOH or the technical product. The origin of the constantly growing interest that has fascinated for more than a century the scientific efforts of researchers towards the special characteristics of polyoxyethylene chains in different situations was due to the specific structure and reactivity of the oxirane ring of ethylene oxide, the promoter of peaked ethoxylated alcohol [Subsequent experimental tests performed on the entire chain of transformations  with purnOH  confirme alcohol  with a sFrom the simple working hypothesis  able to explain phenomena or processes, research based on X-ray investigations, microscopy, and electron diffraction has confirmed the ability of intra- and intercatenary contraction, dependent on structure, environment, with the formation of cavities , depending on the degree of oligomerization of ethylene oxide in polyoxyethylene chains with more than 8–9 oxyethylene units, and “sandwich” for chain sizes between 3–6 structural units, respectively ,27,28,29nAP.The synergistic cumulation in a unitary structure of the conformational and colloidal competencies of these chains, with the possibility of directed modification of the hydrophilic/hydrophobic balance, remains in perspective of wide technological interest. Although pros and cons persist in the profile literature, experimental data associated with cross-interpretations provide sufficient constructive elements that support the free catenary rotation, the release of conformational tensions, and explain ways of packing  in the macromolecular matrix of polyoxyethylene chains. For simplicity, the kinetic and energy efficiency of the phase-transfer catalysis was also becoming a major challenge for the study of the reductive methylation of homogeneous primary LM(EO)nAP addressed in the study, preliminary tests confirmed the role of direct contact of the reactants (water solubility). This becomes a major working premise that also affects the subsequent processing strategies. The homologous series of homogeneous primary LM(EO)nAP occupies from this point of view the entire range of values of the hydrophilic/hydrophobic balance, W/O emulsions, wetting agents (wetting/spreading), O/W emulsions, washing agents, micellar stabilizers, as shown by their functional structural analysis (In the casuistics of reductive methylation of homogeneous primary LM(EO)analysis A,B [23,2nAP and LM(EO)nAT , and N-methyl-N-benzyl-N-octadecyl-N-β-octylphenyl polyethyleneoxy n = 18 ethyl ammonium chloride [2), respectively, these operating premises can be argued in the proposed reductive methylation reaction. Preliminary experimental probes have confirmed the dependence of the processing yields of homogeneous LM(EO)nAP on the structure and nature of the reaction medium. Subsequently sequential evaluations also confirmed the following aspects.The rediscovery of crown polyethers by Pedersen, A.  and of tM(EO)nAT A,B, homochloride  0AP. The possible explanation may be the direct conformational participation of homogeneous polyoxyethylene chains in the processing, directly proportional to the size of the degree of oligomerization of ethylene oxide in the homogeneous polyoxyethylene chain. To confirm the hypothesis, the reductive methylation of homogeneous LM(EO)nAP was performedunder similar conditions . The same experimental test was compared with the reductive methylation yield of homogeneous LM(EO)18AP, noticing an even more obvious difference (over 99%). These results support the hypothesis of conformational participation proportional to the magnitude of the degree of oligomerization of ethylene oxide in the nonionic structure. Following the same reasoning, the reductive methylation of the homogeneous LM(EO)0AP was performed under the same parameters, with the addition of 0.0001 moles phase-transfer catalyst (CTF2) . The participation of CTF1 and/or CTF2 in the reductive methylation reaction of homogeneous LM(EO)0AP can also be explained by the encapsulation of the onium intermediates nditions . The obtt (CTF2) D, relatiles CTF1 C, the yimediates , either 18AP dissolved in the non-polar solvent suitable for the size of the homogeneous polyoxyethylene chain, and the ionic intermediates or polar reactants  in the aqueous phase, separated by an interface in the system. In the studied casuistics, the transfer from the aqueous phase to the organic one was performed by CTF1 or CTF2, charged catalysts , and simultaneously uncharged catalysts through the homogeneous polyoxyethylene chains n = 3–18 included in the same molecular assembly assembly .nAP lay below the value of their critical micellar concentration, being quasimonomolecularly ordered at the interface separating the two phases of the heterogeneous system with the primary amino group in the aqueous (polar) phase, and by effectively stirring, the direct contact of the reactants was considerably favored by the increase of the system interface; (b) micellar phenomena catalyzed by the quaternary ammonium functional group in the CTF1 or CTF2 surface-active structures in micelles with hydrophobic (non-polar) cavities in the aqueous phase and hydrophilic (polar) cavities in the organic phase, respectively. The interphase displacement of CTF1 or CTF2 was also accompanied by micellar inversion. In the development of micellar processes in the organic phase, both areas (with and without charge) in the CTF1 structure participated simultaneously through specific competencies, providing the direct contact between homogeneous primary polyetheramine/formic aldehyde and formic acid to initiate the mechanism of reductive methylation (The process was successively accompanied by (a) interface phenomena dependent on the direct contact of the reactant structures  by effective mechanical stirring of the heterogeneous system. The mentioned processes became possible when the concentration of homogeneous LM(EO)hylation . The kinhylation .2), triethylene glycol (EO3), acrylonitrile, acrylamide, acetic anhydride, p-toluene sulfochloride (TsCl), thionyl chloride (SO2Cl2), and chromatographic purity solvents were purchased from Sigma-Aldrich Chemie GmbH . LM(EO)0OH was purchased from Condea Chemie GmbH  (Diethylene glycol (EOGermany) . All subnOH n-2Na /myristyl 7/3 polyethyleneoxy n = 3–18 ethylamines  ethylammonium chloride (CTF1) (N-methyl-N-benzyl-N-octadecyl-N-β-octylphenyl polyethyleneoxy n = 18 ethylammonium chloride (CTF2) nOH ; homogennOH n-2Na ; tosylatylamines   C, and hoe (CTF2) D, were sThe chemical and physico-chemical characterization was performed by using previously reported experimental conditions ,41 Tabl.nAT, the reaction scheme in For the synthesis of homogeneous LM(EO)nAP, in the non-polar organic solvent selected depending on the degree of oligomerization of ethylene oxide in the homogeneous polyoxyethylene chain (n = 3–18), then under continuous stirring at 5–15 °C (controlled temperature) 0.5 moles formic acid 85% aqueous solution, followed by the solution of 0.12 moles formic aldehyde relative to each methyl group subsequently grafted on the homogeneous LM(EO)nAP and 0.0001 moles homogeneous N,N-dimethyl-N-benzyl-N-β-octylphenyl polyethyleneoxy (n = 18) ethylammonium chloride (CTF1) or homogeneous N-methyl-N-benzyl-N-octadecyl-N-β-octylphenyl polyethyleneoxy (n = 18) ethylammonium chloride (CTF2), respectively. The system was heated approx. 2–4 h (compared to 10–12 h in the classic procedure). As a guide, the reaction was considered complete after the cessation of CO2 emissions. The processing mixture was acidified with 35% HCl in the presence of Congo red indicator after which it was evaporated to dryness under vacuum (10−2–10−4 mm Hg). The obtained residue was dissolved/suspended in a minimum amount of cold water. Free homogeneous tertiary N,N-dimethyl-N-β-lauryl/myristyl 7/3 polyethyleneoxy n = 3–18 ethylamines were subsequently isolated by controlled addition of aqueous NaOH (KOH) 25% and purified by repeated extractions (2–3 times) with anhydrous ethyl ether. The ether phases, combined and thoroughly dehydrated by transfer to columns filled with anhydrous Na2SO4, were evaporated. Free homogeneous LM(EO)nAT were purified subsequently by molecular distillation (10−4–10−7 mm col Hg) and structurally characterized cterized .nAP under conditions of phase-transfer catalysis can be considered an achieved goal, a sustainable synthesis variant, accessible for derivatization to homogeneous LM(EO)nAT. The high yields and their purity do not suggest limits and restrictions and offer the real possibility of further diversification for new nonionic-ionic (cationic) surface-active structures, broadening the niche spectrum of these hybrid molecular architectures. The encouraging results also allow the process to be generalized to other higher primary amines, with the contribution of the phase-transfer catalysis conferred by homogeneous N-methyl-N-benzyl-N-alkyl(C1-C18)-N-β-octylphenyl polyethyleneoxy n = 3–18 ethylammonium chlorides CTF2 and others.The reductive methylation of homogeneous LM(EO)


Advanced age is the greatest risk factor for aging-related brain disorders including Alzheimer’s disease (AD). However, the detailed mechanisms that mechanistically link aging and AD remain elusive. In recent years, a mitochondrial hypothesis of brain aging and AD has been accentuated. Mitochondrial permeability transition pore (mPTP) is a mitochondrial response to intramitochondrial and intracellular stresses. mPTP overactivation has been implicated in mitochondrial dysfunction in aging and AD brains. This review summarizes the up-to-date progress in the study of mPTP in aging and AD and attempts to establish a link between brain aging and AD from a perspective of mPTP-mediated mitochondrial dysfunction. Recl status ,17,18,19l status ,21,22. I2O. Irreversible mPTP activation results in destructive consequences leading to severe mitochondrial dysfunction [Mitochondrial permeability transition (mPT) through the opening of the mitochondrial permeability transition pore (mPTP) is a mitochondrial response to calcium overloading ,24 and ofunction ,30,31,32function , mPTP infunction ,37,38,39function ,42,43,44function ,45,46,47Despite the consensus that mPTP is a hetero-multimer channel contacting the inner mitochondrial membrane (IMM) and outer mitochondrial membrane (OMM), the molecular identity of this mitochondrial pore remains as a conundrum to date. A prevailing opinion was that mPTP is composed of voltage-dependent anion channel (VDAC) and adenine nucleotide translocator (ANT) as the OMM and IMM components, respectively . However2+ triggers the dimerization of F1Fo ATP synthase to form a high-conductance channel displaying mPTP properties [2+-potentiated F1Fo ATP synthase dimerization is likely in a CypD-dependent manner, probably via the interaction of CypD with oligomycin sensitivity-conferring protein (OSCP), an integrative subunit of F1Fo ATP synthase [In recent years, the role of mitochondrial F1Fo ATP synthase in the formation of mPTP has begun to emerge ,59,60,61operties ,64. Moresynthase . The twosynthase ,67. As tAs a nonselective mitochondrial pore, mPTP has been repeatedly linked to mitochondrial dysfunction. Prolonged opening of mPTP results in mitochondrial depolarization, oxidative phosphorylation (OXPHOS) uncoupling, and reactive oxygen species (ROS) overproduction. In addition, uncontrolled entry of small molecular osmolytes and water into mitochondria through mPTP causes mitochondrial swelling, leading to the disruption of OMM ,30,31,322+ and ROS from mitochondria [2+ and ROS accumulation and further contributes to intracellular Ca2+ and ROS signaling [Despite the aforementioned deleterious effects of mPTP on mitochondrial function and structural integrity, the physiological function of mPTP is increasingly recognized. Contrary to persistent mPTP-mediated nonselective transportation of small and large molecules, the low-conductance state of mPTP allows a transient diffusion of Cachondria ,73. It iignaling ,75,76. Mignaling . In addiignaling . The phy2+, inorganic phosphates (Pi), magnesium ion (Mg2+) and proton (H+); (2) reactive oxygen species (ROS) such as superoxide, hydrogen peroxide (H2O2), and hydroxyl radicals; and (3) nucleotides and derivatives of nucleosides including ADP, ATP and NADH. These regulators have distinct impacts on mPTP activation. To be more specific, Ca2+, ROS and Pi are activators of mPTP opening, while matrix Mg2+, H+ and nucleotide derivatives desensitize mPTP [2+ was historically the first identified mPTP regulator [2+ and mPTP in regulating mitochondrial homeostasis in health and disease is still under intensive investigation [2+-mediated mPTP opening have not been fully elucidated, it is generally accepted that the influence of Ca2+ on mPTP opening is context dependent. Tenuous matrix Ca2+ augmentation triggers transient mPTP activation to alleviate mitochondrial Ca2+ overloading and mitochondrial stress; while excess and long-term matrix Ca2+ accumulation promotes persistent mPTP formation, resulting in destructive mitochondrial changes [2+ entry [2+, Sr2+, and Mn2+ that inhibit Ca2+-mediated mPTP opening through competitive inhibition [2+ dynamics and the associated mPTP activation exemplifies, to a certain extent, the interaction between MCU and mPTP.So far, studies have identified a series of modulators of mPTP opening. These regulatory agents could be further categorized into three groups based on their molecular nature including: (1) ionic regulators such as Caize mPTP ,80,81,82egulator , and thetigation . Althoug changes ,75,85,862+ entry . In addihibition . To this2+, ROS is another critical regulator of mPTP. A possible mechanism of ROS-mediated mPTP formation is that oxidative modified transmembrane proteins undergo structural misfolding with the assistance of CypD [2+ deregulation and the leakage of Ca2+ from affected mitochondria further strengthens the effect of ROS in mediating mPTP activation in neighboring mitochondria, resulting in severe mitochondrial stress and, in a worse scenario, the induction of a mitochondrial pathway for apoptosis [In addition to Ca of CypD ,89,90 an of CypD . A criti of CypD ,93. In tpoptosis ,93.Mitochondrial dysfunction develops with the aging process and constitutes a feature of aging brains ,95. Prev2+ and ROS are strong inducers of mPTP [2+ deregulation in neurons develops with age [2+ buffering capacity of endoplasmic reticulum (ER) [2+ influx [2+ homeostasis increases the propensity of mPTP opening [2+ crisis, culminating in a feeding-forward vicious cycle of Ca2+-induced mPTP activation during aging. Moreover, increased ROS generation and reduced GSH/GSSG are prominent with age [2+ deregulation may synergistically promote mPTP activation. It is proposed that oxidative modification of calcium transporters potentiates excess calcium influx to mitochondria, which subsequently triggers mPTP activation, promoting mitochondrial ROS generation by interfering with mitochondrial electron transfer [2+ pathways of mPTP activation, aging cells exhibit changes in mPTP formation-associated proteins. CypD is a crucial regulator of mPTP and increased CypD expression favors mPTP activation [mPTP activation leads to mitochondrial ROS production and release, as well as ATP deficiency and loss of metabolites including nicotinamide (NAD) ,30,31,32 of mPTP . Ca2+ dewith age  probablywith age , Ca2+ bulum (ER)  and glut+ influx . Such ag opening ,111, whiwith age ,112. ROSwith age . Moreovetransfer . In additivation ,113. Ourtivation ,36,45. Ftivation . Such antivation ,115 and tivation ,116. Pretivation  and an itivation . However2+ deregulation and mitochondrial dysfunction [2+-mediated mPT [2+ deregulation and oxidative stress [In line with the close relationship of aging and AD, the two neurological conditions demonstrate a variety of neuropathological overlaps including oxidative stress, Cafunction ,119. Of function . Moreovefunction . Althougfunction . In viewfunction ,40,121, function ,122,123.function . Aβs arefunction . Monomerfunction ,128,129.function . Previoufunction ,132,133.function . Our prefunction . The intfunction ,40. Morefunction  and ANT1ated mPT . Togethee stress , the inte stress . Modifice stress . The inte stress ,141. A pe stress . Moreovee stress . Tau proe stress ,144. Alte stress . Importae stress . These o2+ overloading. However, such a strategy unlatches persistent mPTP opening with the exacerbation of neuronal stress during aging. In addition, relatively low levels of Aβ are produced in normal aging [The sameness pertaining to mPTP activation in aging brains and AD implicates a potential link between the two conditions. First, mitochondrial stress demonstrated by elevated mitochondrial ROS production and calcium deregulation is a common change in aging and AD brains . These mal aging . TherefoGiven the critical role of pathological mPTP opening in AD pathogenesis, inhibiting mPT could be a promising target for the treatment of AD. Since CypD is the only determined mPTP component, inhibiting CypD could be a practical therapeutic strategy. Indeed, our previous studies and others reported the neuronal protective effecting of inhibiting CypD by either genetic abrogation of CypD or the direct inhibition of CypD by CsA. CypD deficient AD mice and CsA treated mitochondria showed preserved mitochondria membrane potential, enhanced resistance to Aβ-mediated mitochondria swelling, and synaptic function and learning memory ,160. HowWith the increase in the aging population, the incidence of AD is escalating. Despite the familial form of AD, the etiology of sporadic AD remains largely elusive. In recent years, although a mitochondrial cascade theory of AD has been accentuated , the det


Fascin, an actin-binding protein, is upregulated in different types of human cancers. It is reportedly responsible for increasing the invasive and metastatic ability of cancer cells by reducing cell–cell adhesions. This review provides a brief overview of fascin and its interactions with other genes and oncoviruses to induce the onset and progression of cancer.FSCN1 gene (located on chromosome 7). It triggers membrane projections and stimulates cell motility in cancer cells. Fascin overexpression has been described in different types of human cancers in which its expression correlated with tumor growth, migration, invasion, and metastasis. Moreover, overexpression of fascin was found in oncovirus-infected cells, such as human papillomaviruses (HPVs) and Epstein-Barr virus (EBV), disrupting the cell–cell adhesion and enhancing cancer progression. Based on these findings, several studies reported fascin as a potential biomarker and a therapeutic target in various cancers. This review provides a brief overview of the FSCN1 role in various cancers with emphasis on gynecological malignancies. We also discuss fascin interactions with other genes and oncoviruses through which it might induce cancer development and progression.Fascin is an actin-binding protein that is encoded by the  FSCN1 pmembrane ,6,7,8. Icription ,11,12,13FSCN  present in humans as well as other vertebrates. Of the three isoforms, FSCN1 is expressed during development in the mesenchymal and nervous tissues and is the most extensively studied form of fascin [FSCN2 is expressed in hair and retinal cells [FSCN3 is present in the testis and developing spermatozoa [FSCN-2 and -3 are homologous to FSCN-1 by 56% and 29%, respectively.There are three isoforms of f fascin . On the al cells ,16, whilrmatozoa . Both FSFSCN1 expression, in transformed epithelial cells and carcinomas, it is highly expressed [FSCN1 is associated with increased metastatic potential and poor prognosis, indicating its role as a candidate potential biomarker and therapeutic target [FSCN1 and its underlying mechanisms in order to elucidate its multiple roles in the onset and progression of gynecological cancer.While in normal tissues there is low or lack of xpressed . In diffc target . Here weFSCN1 (Gene ID: 6624) encodes a distinct 493 amino acid polypeptide and is located on the short arm of chromosome 7, encompassing ~14 kb of DNA including 5 exons. In humans, the FSCN1 to consist of four tandem β-trefoil domains  organized as two twisted lobes , each of which includes six 2-stranded β-hairpins [FSCN1 play a role in actin binding; while the actin-binding site (ABS)-1 involves the β-trefoil 1 and 4, ABS-2 includes residues from β-trefoil 1 and 2, and ABS-3 involves β-trefoil 3 [FSCN1 directly via β-trefoil 2, plausibly inhibiting ABS-1 [FSCN1 activity [FSCN1 consists of another phosphorylation site, Ser274; mutation of Ser274 to alanine is also involved in inhibiting fascin-bundling activity; however, Ser274 does not lie in the region of ABS1-3 [FSCN1 (between amino acids 241–250) [FSCN1 at K247/K250 stimulates bundle assembly [X-ray crystal structure and sequence analyses revealed hairpins . Studiesrefoil 3 . Microtung ABS-1 . The ABSactivity ,23. In af ABS1-3 ,24,25. Oassembly . FSCN1 is expressed at comparatively low levels in normal cells as compared to malignant cells. Therefore, the distinct roles of FSCN1 in both cell types are important to review. Physiologically, FSCN1 is an actin-bundling protein and crosslinks actin filaments via the three binding sites [FSCN1 plays a role in the formation and stabilization of several cellular protrusions  [FSCN1 also regulates focal adhesion dynamics in multiples types of cells and is partially dependent on the canonical actin-bundling function of FSCN1 [FSCN1 controls cellular processes including cell migration, neurite cone extension, and dendrite formation [FSCN1 is highly expressed; FSCN1-dendritic cells aid in effectual interaction and present antigens to T cells, thus playing a vital role in adaptive and innate immunity [As mentioned in the section above, ng sites ,7,21. FSlopodia) ,7,21 whilopodia) ,10,11. Aof FSCN1 ,12. Moreormation ,28,29. Dimmunity .FSCN1 binds to microtubule cytoskeleton and regulates focal adhesion dynamics and cell migration [FSCN1 and microtubule interaction leads to stability of cell adhesion and reduces cell migration [FSCN1 interacts with nesprin-2, a nuclear envelope protein to promote nuclear deformation and mobility [FSCN1 is present in the nucleus and controls histone methylation and gene transcription by interaction with H3K4me3, the H3K4 methyltransferase core subunit RbBP5 form [FSCN1 regulates extracellular vesicle release [On the other hand, igration . Interruigration . During mobility . NeverthBP5 form . Nonethe release . FSCN1 is expressed during mouse embryonic development; however, its expression patterns are widely conserved in human tissues [FSCN1 also plays an important role in delamination during border cell migration by modifying the localization of E-cadherin in the border cells [FSCN1 is significantly expressed during development. In adults, FSCN1 is absent or at low levels in normal epithelial cells, and its expression is limited to the neuronal, endothelial, mesenchymal, dendritic, and immune cells [FSCN1 is largely expressed in the nervous systems  [ tissues ,28. In Der cells . During ne cells . During c cells) ,34,35. FSCN1 expression levels are highly elevated [FSCN1 overexpression is documented in the majority of cancers, including ovarian [FSCN1 activation during the onset and progression of cancer are still understated. Although mutations or amplification of FSCN1 gene are not common, hypomethylation of FSCN1 promoter has been found in normal epithelium and cancer cells alike [FSCN1 in cancer.When epithelial cells undergo the transformation, elevated . FSCN1 o ovarian , breast  ovarian , colon [ ovarian , pancrea ovarian ,41, glio ovarian , melanom ovarian , leukemi ovarian , lymphom ovarian , and eso ovarian ; howeverls alike , indicatFSCN1 in cancer was first described in breast cancer by Grothey et al. [FSCN1 induced aggressive phenotype. Several additional investigations also reported the role of FSCN1 in other types of cancer and its association with an aggressive phenotype, poor prognosis, and short survival [FSCN1 can promote cancer progression through both canonical and non-canonical pathways by triggering cancer proliferation, migration, invasion, and metastasis [The role of y et al. , where tsurvival ,36,48,49survival . In thistastasis ,36.FSCN1 promotes cancer progression via inducing cancer cell growth, proliferation, migration, invasion, and metastasis [FSCN1 in cell growth and proliferation. While upregulated expression of FSCN1 was found to be correlated with enhanced cell proliferation in different cancer cell lines [FSCN1-transduced cancer cells [FSCN1 expression to provoke cell proliferation [FSCN1 expression on MDA-MB-231 cell proliferation. Similarly, in the non-small lung cancer cell line, A549, it was reported that enhanced FSCN1 has a light impact on cell proliferation [FSCN1 could enhance cell proliferation via the YAP/TEAD signaling pathway [FSCN1 leads to the inhibition of the A549 cell growth and proliferation via the MAPK signaling pathway [There are several functions by which tastasis ,36. In vll lines ,52,53, fer cells ,55. Whilferation , other iferation  and Heinferation  did not feration ; however pathway .FSCN1 contributions to cancer progression is the actin-bundling function [FSCN1 also facilitates mechano-transduction by interaction with the LINC complex, which is responsible for cell invasion and migration [FSCN1 within the nucleus to control nucleolar size and morphology [FSCN1 also regulates oxidative phosphorylation of the mitochondria and metabolic stress resistance, thus inducing cancer metastasis [On the other hand, one of the principal functions of function . Alteratfunction ,60. FSCNigration ,61,62. Trphology , in addirphology ,65, chrorphology . Thus, drphology ,67. Additastasis .FSCN1 in inducing cancer cell migration by promoting filopodia formation and epithelial-mesenchymal transition (EMT) [FSCN1 induces filopodia and invadopodia stability and formation [FSCN1-induced migration and invasion. In mouse and zebrafish models, FSCN1 expression was found to induce colon tumor invasion [FSCN1 expression to induce cancer cell metastasis to the lung [FSCN1 was linked with metastasis in pancreatic [FSCN1 expression provoked lung metastasis in osteosarcoma cells [FSCN1 could promote cancer progression by inducing chemoresistance in cancer cells as well as controlling metabolism and contributing to a de-differentiated and more stem-like state [Moreover, in vitro data also demonstrated the role of on (EMT)  in varioon (EMT) , oral [5on (EMT) , hypophaon (EMT) , osteosaon (EMT) , and panon (EMT) . FSCN1 iormation . In addiormation ,69, thusinvasion ,72. Simithe lung . In nudencreatic , renal [ncreatic , and colncreatic . Additioma cells . Furtherke state . FSCN1 promoter regions, thus regulating FSCN1 expression. It was reported that FSCN1 transcriptional activity is controlled by the promoter region (−219/+114); in breast and colon cancer, CREB and aryl hydrocarbon receptors (AhRs) bind with the −219/+114 promoter region [FSCN1 expression and promoting invasion  interact and bind to the r region . On the r region  reportedinvasion . Furthern cancer ,80,81  are known to bind to the 3′ untranslated region (UTR) of FSCN1 and regulate its expression in several human cancer tissues and cell lines, including breast, lung, liver, colon, cervix, prostate, pancreatic, hepatocellular, esophageal, and nasopharyngeal [FSCN1 expression, thus stimulating cancer cell growth, proliferation, migration, and invasion along with the inhibition of apoptosis [FSCN1 is directly targeted by several miRNAs in different cancers. Thus, miRNA-24 targets FSCN1 in nasopharyngeal and prostate cancer [FSCN1 is targeted by miRNA-143 in esophageal [FSCN1 expression [FSCN1 expression via the inhibition of AMPK and activation of mTOR signaling [Similar to transcription factors regulating aryngeal ,100,101.poptosis ,100,101.e cancer ,102, FSCophageal  and miRNophageal ,105, respression . Chen etignaling . FSCN1 via viral oncogenesis (FSCN1 was reported in Epstein-Barr virus (EBV)-induced lymphoblastoid cell lines [FSCN1 in lymphocytes via NF-κB signaling, contributing to lymphocyte migration and invasion [FSCN1-negative Hodgkin’s lymphoma-derived cell lines also show upregulated FSCN1 levels [FSCN1 expression [FSCN1 expression and was associated with lymph node metastasis and higher proliferation index of the cancer cells [FSCN1 can help in EBV release. Moreover, the transmission of EBV to epithelial cells is dependent on NF-κB signaling [FSCN1 induction. LMP1 of EBV can induce expression of FSCN1 at both mRNA and protein levels in lymphocytes [FSCN1 levels and the invasive ability of EBV-transformed lymphoblastoid cells. Moreover, the study showed that the knockdown of FSCN1 by two different small hairpin RNAs reduced invasion of lymphocytes [FSCN1 is reported to induce cell migration [FSCN1 expression was reported in LMP1-positive samples and was associated with moderately to poorly differentiated adenocarcinomas [FSCN1 in LMP1-positive cancers to provoke cancer progression  was found to restore β-catenin accompanied by the downregulation of FSCN1 expression pattern, thus inhibiting cell invasion ability of these cancer cell lines [FSCN1 in cervical squamous cell carcinoma and found an association between FSCN1 overexpression and HPV positivity [FSCN1 was also reported by our group [FSCN1 overexpression in human colorectal cancer. These studies clearly show that human oncoviruses can deregulate the expression patterns of FSCN1, thereby promoting cancer progression , primary human cancer-associated fibroblasts and primary human omental mesothelial cells reduced metastasis . TMA anaA recent study examined the effect of curcumin against FSCN1 in the ovarian cancer cell line SKOV3 and found curcumin to inhibit STAT3 via the JAK/STAT3 signaling pathway. Notably, the inhibition of STAT3 also led to FSCN1 activity inhibition . In addiSimilar to ovarian cancer, studies were performed to detect FSCN1 expression in uterine cancer. Uterine carcinosarcoma cases were assessed for FSCN1 expression using IHC; while FSCN1 was absent in benign cases, it was present in both malignant epithelial and mesenchymal elements of uterine carcinosarcomas. This finding was associated with a more aggressive phenotype (advanced stage and large tumor size) and a poor outcome . AdditioIn spite of the confirmed role of FSCN1 in several human carcinomas, there has been a limited number of investigations pertaining to the presence and role of FSCN1 in cervical cancers. In this context, Stewart et al. analyzed FSCN1 expression by IHC in in situ and invasive adenocarcinoma of the endocervix and found FSCN1 overexpression to occur during the development and progression of some endocervical neoplasms, indicating the role of FSCN1 in tumor invasion . Koay etFSCN1 expression using PCR and immunohistochemistry (IHC), revealing its increased expression compared to normal tissues. A systematic review and meta-analysis of studies analyzing the relevance of FSCN1 in five different carcinomas  by IHC reported that FSCN1 correlates with a high risk of disease progression in breast and colorectal cancers [Several previous studies analyzed  cancers . They al cancers . Of the gastrointestinal cancers, esophageal cancers has the worst prognosis. FSCN1 is involved in the pathogenesis and metastasis of esophageal carcinoma; high FSCN1 expression increases gradually from the normal to the invasive form and correlates with cell proliferation, lymph node invasion, metastasis, and high tumor stage ,155,156.While lymphocytes, myeloid, and plasma cells stain negative for FSCN1, in human hematologic malignancies including HIV-related lymphoid hyperplasia, Reed-Sternberg cells, Hodgkin’s lymphoma, Castleman’s disease, and other lymphoid hyperplasia, FSCN1 is overexpressed ,175. A sFSCN1 to induce cell migration, invasion, and metastasis, FSCN1 is a potential candidate molecule for anti-cancer or anti-metastatic therapy in human cancers [FSCN1 are being developed and include miRNA, siRNA, shRNA, small molecule inhibitors, and nanobodies [Due to the ability of  cancers ,115,178.nobodies ,188,189.FSCN1 in cancer cell lines reduce cell growth, proliferation, migration, and invasion [FSCN1 knockdown by siRNA leading to decreased cell migration and metastasis, respectively [In multiple types of cancers, in vitro studies demonstrated that overexpression of miRNAs targeting invasion ,95,97,99ectively ,182,183.ectively . While mectively . Multiplectively ,193,194.ectively . On the ectively . Howeverectively . In soliectively ,197. Morectively . Since iectively ,198. It ectively . Finallyectively . Experiments using inhibitory nanobodies against FSCN1 protein showed disruption of the FSCN1/actin-bundling . AlthougFSCN1-induced tumors [FSCN1 promoter and reducing its expression, thus acting as a plausible anti-metastatic therapeutic agent [FSCN1 and block its activity, thereby reducing cell migration, invasion, and tumor metastasis [FSCN1 activity [FSCN1-induced tumors. A novel small molecule compound G2 and its derived analogs  were tested and displayed anti-metastatic properties along with enhanced response and survival in in vivo models by blocking FSCN1 activity [Likewise, small molecule inhibitors can reduce tumor cell migration and invasion and help pave the way against d tumors ,185,186.d tumors . Bestatiic agent . Other stastasis ,185. In activity , thus inactivity ,186. Recactivity . While nactivity . Prior tactivity . Moreoveactivity . Proposeactivity . Table 2FSCN1 can stimulate cancer cell migration and metastasis, there are several limitations in developing therapeutic targets against FSCN1. While FSCN1 is not expressed in adult epithelial tissues, it is normally expressed in other adult non-epithelial tissues [FSCN1 inhibitors may have negative side effects. Specifically, the inhibition of FSCN1 may cause neuronal, kidney, endocrine, wound healing, and immune defects.Since  tissues , raisingFSCN1 is regulated by several signaling pathways  and is overexpressed in various human carcinomas including gynecological cancers; however, understanding the exact molecular mechanisms underlying FSCN1 deregulation and interaction with other genes and oncoviruses, especially in gynecological cancers, is still nascent. Although several studies have indicated a potential diagnostic utility of FSCN1, its therapeutic role as an anti-cancer target is still under investigation. We believe that further studies are needed, including the development of conditional transgenic and/or knockout animal models, to determine the role of FSCN1 targeting as a potential therapeutic route for gynecological carcinomas.


NRAS-initiated JMML is heterogeneous and adequate management ranges from watchful waiting to allogeneic hematopoietic stem cell transplantation (HSCT). Upfront azacitidine in KRAS patients can achieve long-term remissions without HSCT; if HSCT is required, a less toxic preparative regimen is recommended. Germline CBL patients often experience spontaneous resolution of the leukemia or exhibit stable mixed chimerism after HSCT. JMML driven by PTPN11 or NF1 is often rapidly progressive, requires swift HSCT and may benefit from pretransplant therapy with azacitidine. Because graft-versus-leukemia alloimmunity is central to cure high risk patients, the immunosuppressive regimen should be discontinued early after HSCT.Juvenile myelomonocytic leukemia (JMML) is a rare pediatric leukemia characterized by mutations in five canonical RAS pathway genes. The diagnosis is made by typical clinical and hematological findings associated with a compatible mutation. Although this is sufficient for clinical decision-making in most JMML cases, more in-depth analysis can include DNA methylation class and panel sequencing analysis for secondary mutations.  PTPN11, NRAS, KRAS, NF1, or CBL) [JMML is a pediatric leukemia with shared features of myelodysplastic and myeloproliferative neoplasms, usually manifesting during early childhood with leukocytosis, thrombocytopenia, pronounced monocytosis, splenomegaly, immature precursors on peripheral blood (PB) smear, and bone marrow (BM) blast count below 20% ,2,3. Its or CBL) . For mos or CBL) ,7. Clini or CBL) ,9. In thThe Ras pathway is a sequence of kinases in the cell that serves as a chain of communication between extracellular mitogens and the cell nucleus . ExternaPTPN11, NRAS, KRAS, NF1, or CBL), resulting in net hyperactivation of the Ras-GTP-GDP loop, are present in hematopoietic cells of >90% of children diagnosed with JMML [Genetic mutations in specific Ras pathway components , SOS1, RAF1, KRAS, BRAF, NRAS or other members of the RAS pathway [PTPN11 mutations is not identical in JMML and NS/MPD [Noonan syndrome (NS), the most common rasopathy with an incidence of 1 in 1000–2500 children , bears c pathway ,44,45. C pathway . Althoug pathway . Althougd NS/MPD , there id NS/MPD .SETBP1, JAK3, SH2B3, or ASXL1 genes. Not infrequently, the secondary mutations affect the Ras pathway itself (“Ras double mutants”). In addition, a role for subclonal mutations in the Polycomb Repressive Complex 2 network was highlighted [Systematic exome sequencing studies revealed that JMML is generally characterized by a paucity of somatic mutations in the neoplastic clone when compared to most other types of cancer . Howeverhlighted . Severalhlighted ,49. Furthlighted ,49.RRAS mutations [CCDC88C-FLT3 fusion responsive to sorafenib was described in a pediatric patient with clinical features of JMML and monosomy 7 [ALK [ROS1 [FIP1L1-RARA [HCMOGT-1-PDGFRB [NDEL1-PDGFRB [NUP98-HOXA11 [Less than 10% of JMML cases are negative for the five canonical driver mutations. Rarely, these children harbor germline or somatic activating utations ,50. Recenosomy 7 . Other fy 7 [ALK ,53, ROS1LK [ROS1 ,53, FIP11L1-RARA , HCMOGT-1-PDGFRB , NDEL1-P1-PDGFRB , and NUP8-HOXA11 . Althoug8-HOXA11 ,53.JMML occurs in 1.2 children per million per year, accounting for 2% of pediatric hematopoietic malignancies . One hal9 G/L in all but exceptional cases [The PB smear typically shows mild to pronounced leukocytosis with monocytosis without a significantly increased number of blasts (median 2% myeloblasts) ,64. Immaal cases . Bone maal cases .PTPN11, KRAS, NRAS, and CBL genes, and a meticulous search for features of NF-1 including family history. Genetic analysis of NF1 can be added, but it is laborious, and the interpretation of findings is not always straightforward. On cytogenetics, two-thirds of cases exhibit a normal karyotype. Monosomy 7 is the most frequent aberration [PTPN11 and KRAS mutations, but rarely with NF1, NRAS, or CBL. A traditional hallmark of clonogenic JMML cells is their hypersensitivity to GM-CSF in vitro [The combination of young age, splenomegaly, skin lesions, appearance of myeloid and erythroid precursors in the PB, and/or elevated levels of HbF should prompt the pediatric oncologist to suspect JMML and initiate specific tests. First of all, this involves the molecular analysis of driver mutations in the erration ,53,65, oin vitro ,67. Howein vitro . Similarin vitro . It was in vitro .NRAS and KRAS mutations as in JMML [Bacterial  and vira in JMML , but the in JMML ,76, high in JMML .LIN28B [The genetic subtypes discussed above account for the phenotypic diversity of JMML only incompletely. For example, long-identified prognostic parameters, such as age of the patient, sex, platelet count, or elevated levels of fetal hemoglobin ,40,59, dLIN28B . In addiLIN28B ,82.The first study examining the role DNA methylation changes in a large European series of 127 children with JMML revealed CpG island hypermethylation of a candidate gene set in up to half of the cases . ImportaPTPN11 mutation and older children, both known factors for inferior clinical course, whereas the low-methylation group was enriched for patients with NS/MPD, CBL cases, and young children with somatic NRAS mutations. The group with intermediate hypermethylation was characterized by somatic KRAS mutations and occurrence of monosomy 7 [NF1 or PTPN11 mutations, secondary mutations, LIN28B overexpression and AML-like expression profile [Extending these findings to a genome-wide scope using array-based methods, study groups in Europe , Japan  and Unitnosomy 7 . The Japnosomy 7 . In addi profile . The Nor profile . Interes profile ,88, agai-Level of fetal hemoglobin -PTPN11, NF1, KRAS, NRAS, CBL)Panel sequence analysis of the five primordial Ras pathway genes -Where available, the following additional information will aid in clinical decision-making, though it is not indispensable for adequate management in most cases:-DNA methylation class-SETBP1, JAK3, RRAS, RRAS2, and ASXL1; other targets are rare.Panel sequence analysis of recurrent secondary mutations. The assessment should include With extensive molecular diagnostic work-up of JMML established in major international study groups and large centers around the world, it has become evident that there can no longer be a uniform one-size-fits-all approach for this disorder . The autPTPN11- or NF1-driven JMML. On the other hand, a group of patients exist who are clinically well and have low HbF. Here, spontaneous clinical regression of the disease can occur in the long run without therapy. The search for an unrelated stem cell donor may be deferred in these cases. In between these two ends of the spectrum, the prospective identification of patients who benefit from watchful waiting is the real challenge. Factors suggesting surveillance without therapy include infant age, clinical status, age-appropriate levels of HbF, and low methylation class [The disease course in this group is remarkably heterogeneous. In older children with severe thrombocytopenia, increased HbF and high methylation class, a rapidly progressive course with a considerable risk of recurrence after HSCT is to be expected, likening the disorder to on class ,89,90. HNRAS or KRAS mutation to be detected in a suspected JMML case. Most of these children have additional syndromic features of the rasopathy spectrum [It is very rare for a germline spectrum ,92 or cospectrum . Anecdotspectrum ,95. BecaKRAS-JMML are typically very young, often infants. Concurrent monosomy 7 in the neoplastic clone is often observed (see below). Autoimmune phenomena  should be searched for, and a diagnostic differentiation from RALD [KRAS-driven JMML tends to be aggressive, requiring rapid intervention. In the past, long-term survival without HSCT has not been reported for this group, but the picture is now changing with the introduction of azacitidine. KRAS-mutated JMML responds particularly well to low-dose azacitidine with long-lasting clinical and molecular remissions [2/day on five consecutive days or 75 mg/m2/day on seven consecutive days, repeated every 28 days; due to instability, immediate intravenous or subcutaneous application of the cold reconstituted solution must be observed. Azacitidine has a favorable toxicity profile in children with JMML, mainly including lower-grade cytopenias, gastrointestinal discomfort, and infections [KRAS-mutated JMML with a low risk profile.Children diagnosed with rom RALD ,96,97 shmissions ,99. Regifections ,98. It i2/day, to be adjusted according to clinical course) and/or cytarabine (40 mg/m2/day × 5 days) [2/day × 5 days) and fludarabine (30 mg/m2/day × 5 days) may also be considered [PTPN11 mutation.This is the numerically largest group that also carries the highest risk of rapid progression and early death. As no curative chemotherapy regimen is known, expeditious planning of allogeneic HSCT is mandatory. Within this group, risk factors for an unfavorable course are: age at diagnosis ≥2 years, significantly increased level of HbF, presence of secondary mutations, and/or high methylation class. In these cases, the aim should be to perform HSCT within a period of 3 months after diagnosis (recommendations for implementation see below). Low-dose azacitidine is the preferable option for upfront therapy until HSCT in non-high risk cases, with the goal to achieve a more favorable biological status of the leukemia. In many affected children, this treatment leads to a clinical partial remission or at least sufficient disease control until HSCT ,99,100 H 5 days) . If a pansidered . Howevernsidered . It is nnsidered . Until mPTPN11 cause Noonan syndrome, a condition of the rasopathy spectrum predisposing to a myeloproliferative disorder imitating JMML. For this reason, genetic diagnostics in suspected cases of JMML must always be carried out in both hematopoietic and non-hematopoietic material. Cultivated fibroblasts from a skin biopsy are ideal; hair follicles are less invasive, but more challenging to analyze. An oral mucous membrane swab has a high probability of contamination with hematopoietic cells, even if it is macroscopically not bloody, and should therefore only be scored if the result is negative.Germline mutations in PTPN11 should not just encompass the hotspot exons 3 and 13 because Noonan syndrome mutations may also be found in exons 4 or 8. The spectrum of somatic PTPN11 mutations in nonsyndromic JMML and germline PTPN11 mutations in Noonan-associated MPD overlaps to a great extent but not completely [In cases of suspected JMML in very young patients or with clinical evidence of Noonan syndrome, the diagnostic test for mpletely .The distinction between non-syndromic JMML and Noonan-associated MPD is important, as the latter is usually self-limiting. However, these patients must be monitored carefully, as there can be relevant clinical compromise from cell infiltrates, making it necessary to begin cytoreductive therapy. In individual cases, a clonal development towards a bona fide neoplastic disease is possible ,102,103.NF1 usually confirms the clinical diagnosis in children with JMML/NF-1 [NF1-inactivating heterozygous variation in the germline of the patient that arose de novo (~50% of cases) or was inherited. These lesions are often, but not always, deletions or truncating missense mutations reported as recurrent aberrations in the NF-1 literature. In addition, the neoplastic clone exhibits somatic loss of heterozygosity at the NF1 locus or an independent second NF1 mutation, leading to biallelic NF1 inactivation [NF1 lesion, but driven by an unrelated event, may be present. With careful work-up, however, such dilemmas are rare.If not already recognized in the patient, the syndrome can usually be diagnosed clinically and/or through family history at the time of onset of JMML. In younger children, only café-au-lait spots but not the other typical signs of neurofibromatosis may be present. Six or more café-au-lait spots as stipulated in the NIH criteria are noted in the majority of children, but in exceptional cases there may be none or fewer. Genetic analysis of MML/NF-1 . The typtivation ,104,105.PTPN11 mutation. Consistent with this recommendation, JMML/NF-1 cases almost always have an intermediate or high methylation profile [JMML on the basis of NF-1 manifests more frequently at an older age than the other groups and typically does not involve a drastically reduced platelet count. Some children initially show little clinical impairment. However, long-term survival without HSCT has not yet been observed in this group, so that proceeding to transplant and inception of therapy with azacitidine is advisable as in patients with somatic  profile .CBL mutations in JMML is a heterozygous missense point mutation in CBL exons 8 or 9 in the germline, accompanied by uniparental isodisomy of the 11q chromosome arm as a somatic event in hematopoietic cells, leading to loss of heterozygosity [CBL-JMML have massive organ enlargement and may require splenectomy for symptom relief. Most patients do not require swift HSCT but can be managed with watchful waiting; many of these experience spontaneous resolution of the myeloproliferation. The homozygous CBL-mutant status in hematopoietic cells may persist until adulthood even in the absence of hematologic abnormalities [CBL inactivation in five patients with a clinical course that required HSCT [The typical configuration of zygosity ,36,37. Mmalities . Patientmalities ,36. It ired HSCT , again iNF1 gene in the absence of clinical NF-1 features [ALK, ROS1, or FLT3 [Suspected cases of JMML with negative panel sequencing for all five primordial genes and no clinical evidence of NF-1 are called “quintuple-negative” or “all-negative”. In a third of these cases, in-depth multimodal genetic analysis uncovered a driving role of the features . In othe or FLT3 ,53. SomeKRAS mutations, in contrast to an association between monosomy 7 and PTPN11 or NF1 in the high methylation group, and the absence of monosomy 7 in patients with low methylation pattern [The significance of monosomy 7 for the biology of JMML is unclear, and there seems to be no association with clinical features or relevance for outcome . Interes pattern . The mec pattern . This co pattern .2/d on day 1) [Busulfan-based myeloablative conditioning regimens are commonly chosen and achieve 55–73% OS with a moderate 10–15% rate of transplant-related mortality but significant probability of leukemia relapse in the order of 25–35% ,111,112.n day 1) . In an an day 1) . Matchedn day 1) . Matchedn day 1) ,116,117.n day 1) ,112,118.n day 1) .The North American group noted better post-HSCT outcome of patients with JMML who experienced molecular response to pretransplant chemotherapy , similarBeside the leukemia biology factors discussed above, the way the transplant procedure is handled significantly influences the risk of relapse. It is likely that it is not so much the conditioning regimen but rather the establishment of a graft-versus-leukemia effect that is decisive for the success of allogeneic HSCT in JMML ,110,111.NF1 or somatic PTPN11 mutation, and high DNA methylation define a patient group whose risk of JMML recurrence after HSCT is even higher than 50%, bringing up the question of post-transplant prophylaxis. On the basis of favorable data for other myeloid neoplasms [2/day for five consecutive days, every 28 days) plus donor lymphocyte infusions . However, we emphasize that there are no systematic data for this approach in JMML.Age at diagnosis ≥2 years, eoplasms ,130,131 PTPN11-driven JMML to trametinib [Despite the prominent role of the Ras/MAPK network, attempts to target this complex signal cascade have shown limited therapeutic benefit in JMML ,132. Theametinib . BCL2 inametinib ,135 and ametinib ,137. Witametinib ,139,140.


ACTA2, ACTR10, CAPG, CORO1C, KRT17, and PALLD in cytoskeleton, CDH5, CLSTN1, ITGA10, ITGAX, ITGB7, ITGA8, FAT4, ITGAE, CDH10, ITGAM, ITGB6, and CDH18 in adhesion, COL8A2, FBN1, LAMB3, and LAMA2 in ECM, ADAM22, ADAM28, MMP11, and MMP24 in MMP. Prognosis prediction formulas with the gene expression values and the Cox regression model clearly divided survival curves of the subgroups in each status. Furthermore, collagen genes contributed to gene network formation in glasso, suggesting that the ECM balance controls the PCNSL microenvironment. Finally, the comprehensive balance of morphology and microenvironment enabled prognosis prediction by a combinatorial expression of 8 representative genes, including KRT17, CDH10, CDH18, COL8A2, ADAM22, ADAM28, MMP11, and MMP24. Besides, these genes could also diagnose PCNSL cell types with MTX resistances in vitro. These results would not only facilitate the understanding of biology of PCNSL but also consider targeting pathways for anti-cancer treatment in personalized precision medicine in PCNSL.Dysregulation of cell morphology and cell-cell interaction results in cancer cell growth, migration, invasion, and metastasis. Besides, a balance between the extracellular matrix (ECM) and matrix metalloprotease (MMP) is required for cancer cell morphology and angiogenesis. Here, we determined gene signatures associated with the morphology and microenvironment of primary central nervous system lymphoma (PCNSL) to enable prognosis prediction. Next-generation sequencing (NGS) on 31 PCNSL samples revealed gene signatures as follows:  The interaction of cytoskeleton with proteins involved in cancer progression or regression contributes to tumor initiation and progression, or anticancer mechanisms , which aPrimary central nervous system lymphoma (PCNSL) is an aggressive lymphoma of the brain with poor prognoses, which is classified as diffuse large B-cell lymphoma (DLBCL), a type of non-Hodgkin’s lymphoma (NHL) . DLBCLs 2+-binding glycoprotein are potential biomarkers in PCNSL . Th. Th28]. Gene expression patterns were clustered into subgroups using a two-way hierarchical method using the JMP built-in module (SAS Institute) .Pairs plot analysis was performed to estimate the distribution of single variables and relationships between two variables, using the scatterplot package in R .Gene associations in module hub networks among variables were analyzed with the graphical lasso estimation of Gaussian graphical models, a sparse inverse covariance matrix using a lasso (L1) penalty, using the glasso package in R .Samples were randomly divided into training data and test data at a ratio of 3:1. A regression equation was estimated for the training data with the Cox regression, and a time-dependent ROC analysis was carried out for the test data with the regression score and area under the curve (AUC). The 2-year and 5-year survivals were evaluated. The process was repeated 10,000 times, and the average of AUC was calculated using the timeROC package in R, as described .Statistical analyses were performed using R, Bioconductor , JMP10 , ECM and MMP were slightly correlated , and the others were weakly correlated with no significance   , consist−5)  . The resin vitro. The two MTX-resistant PCNSL cell lines, TK-MTX and HKBML-MTX, derived from TK and HKBML, respectively, were examined for the 204 genes focused on this study. Differential expression genes (DEGs) in MTX-resistant PCNSL cells compared with control PCNSL cells were designated by |log2(fold change)| > 1. DEGs were divided into the four patterns. Thirteen genes indicated similar expression patterns in TK-MTX and HKBML-MTX , CDH2 (N-cadherin), CDH3 (P-cadherin), CDH11, CDH12, and LTF (lactoferrin) are considered potential biomarkers of CNS tropism in adhesion, migration, and inflammatory response [FN1, in addition to LMO2, BCL2, BCL6, CCND2 (cyclin D2), and SCYA3 is effective in estimating OS using polymerase chain reaction and microarray in DLBCL [laminin receptor 2 is a PCNSL-specific gene detected by principal component analysis on DNA arrays in a small sample size [FBN1 is observed at 23% in NHL [MMP19 is highly expressed in the MTX-resistant PCNSL cell lines, HKBML-MTX and TK-MTX in vitro [Immunohistochemistry and immunoelectron microscopy in PCNSL, secondary CNSL, and systemic DLBCL have demonstrated that response . Perivasresponse . Therefoin DLBCL . In addiple size . Further% in NHL . Althoug% in NHL ,22. MMP1in vitro –32.KRT17, CDH10, CDH18, COL8A2, ADAM22, ADAM28, MMP24, and MMP11, selected at mixed statuses, clearly divided survival curves of the subgroups in the Kaplan-Meier estimation. A glasso model revealed genetic interaction with module hub networks. However, status correlation analyses with glasso and pairs plot did not connect the morphology status with the microenvironment status. Interestingly, survival tree analyses demonstrated poor prognoses of the subgroups with CFL2lowACTA2high (cytoskeleton), FBN1highCOL9A1low (ECM), and ADAM28highMMP19high (MMP). The result of ADAM28highMMP19high was consistent with the results of ADAM28high and MMP19high in univariate analysis, suggesting that it is important to estimate OS with combinatorial expression of the determinant factors in multivariate analysis. Moreover, the tree analysis also proposed the possibility that COL9A1 would be a stabilizer for FBN1 and that MMP19 would be an accelerator for ADAM28, to modulate the expression of the determinant genes for PCNSL survival. Besides, a part of genes examined were also suggestive of clonal expression in MTX-resistant PCNSL cells including TK-MTX and HKBML-MTX, and personalized diagnosis and precision medicine, so called tailored medicine in PCNSL. Therefore, these results suggest a possibility that immunohistochemistry of the cell morphology and ECM proteins on the clinical samples could be applied diagnosis of PCNSL cell-types, such as TK-type or HKBML-type, and prognosis prediction in the PCNSL patients. These results are limited due to the small sample size and the status of interest, but the results described above would help understand cancer morphology and microenvironment with patient survival and develop de novo molecular target therapy in PCNSL.In this study, the gene expression associated with cancer morphology and microenvironment was assessed using univariate and multivariate analyses of the RNA-seq data and clinical information in PCNSL. Univariate analysis detected a correlation between differential expression of the genes related to cancer morphology and microenvironment and patient survivals with poor progoses in PCNSL. From the random forests and Cox hazard analyses, a combinatorial expression of the significant 8 genes, including S1 Figa) Cytoskeleton. (b) Cell adhesion. (c) Extracellular matrix (ECM). (d) Matrix metalloprotease (MMP). Numbers in the parentheses indicated the numbers of the genes.Heat maps were drawn with the two-way clustering method. Gene expression with IQR > 0.1 were enrolled. ((PDF)Click here for additional data file.S2 Figa) CDH10. (b) CLSTN1. (c) MMP15. (d) COL7A1. (e) ITGA10. (f) ITGAL. (g) ITGB7. (h) KRT17. HR; hazard ratio, OS; overall survival. Log-rank test; p < 0.1.((PDF)Click here for additional data file.S3 Figa) Cytoskeleton. (b) Cell adhesion. (c) Extracellular matrix (ECM). (d) Matrix metalloprotease (MMP). The gene symbols with the top variables by the random forests survival analysis are presented in the graphs.Random forests survival analyses selected top variables in each category. Top variable selected were shown in graph. ((PDF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.S3 Table(PDF)Click here for additional data file.S4 Table(PDF)Click here for additional data file.S5 Table(PDF)Click here for additional data file.S1 Appendix(XLSX)Click here for additional data file.


Trachydoras paraguayensis (2n=56),Trachydoras steindachneri (2n=60), Ossancorapunctata (2n=66) and Platydoras hancockii whosekaryotypes showed a multiple NOR system. The large thorny catfishes, such asPterodoras granulosus, Oxydoras niger andCentrodoras brachiatus share several karyotype features,with subtle variations only regarding their heterochromatin distribution. On theother hand, a remarkable karyotypic variability has been reported in thefimbriate barbells thorny catfishes. These two contrasting karyoevolutiontrajectories emerged from a complex interaction between chromosomerearrangements  and mechanismsof heterochromatin dispersion. Moreover, we believe that biological features,such as microhabitats preferences, populational size, low vagility and migratorybehavior played a key role during the origin and maintenance of chromosomediversity in Doradinae subfamily.Doradinae (Siluriformes: Doradidae) is the most species-rich subfamily amongthorny catfishes, encompassing over 77 valid species, found mainly in Amazon andPlatina hydrographic basins. Here, we analyzed seven Doradinae species usingcombined methods  in order to scrutinize the processes that mediated the karyotypediversification in this subfamily. Our ancestral reconstruction recovered that2n=58 chromosomes and simple nucleolar organizer regions (NOR) are ancestralfeatures only for Wertheimerinae and the most clades of Doradinae. Someexceptions were found in  Cytogenetic studies have provided valuable information about the evolutionary trendsand relationships in a range of vertebrate species, such as amphibians , reptileDespite the paucity of studies involving this kind of evolutionary approach in fish,analysis combining cytogenetic data and reconstruction of ancestral features haveemerged in recent years . TherefoWithin Neotropical Siluriformes, Doradidae stands out as one of the most diverse andrepresentative families, with over 96 species , commonlPterodoras granulosus Valenciennes, 1821,Oxydoras niger Valenciennes, 1821, Centrodorasbrachiatus Cope, 1872, Megalodoras uranoscopusEigenmann & Eigenmann, 1888, Lithodoras dorsalis Bleeker, 1862,Astrodoradinae (15 species), and Doradinae (78 species) . The later, 1862. On the Tenellus . The latTenellus .Anadoras sp. “araguaia” andTrachydoras. paraguayensis Eigenmann & Ward 1907 (2n=56chromosomes), and Ossancora punctata Kner, 1853 (2n=66chromosomes), the highest diploid number in the family to date. Additionally, aconsiderable cytogenetic variability is also observed in the structural level ,supernumerary chromosomes, as seen in Ossancora punctata,Pterodoras granulosus and Platydoras armatulusValenciennes, 1840 and a unique ZZ/ZW sex chromosome system in Tenellustrimaculatus Boulenger, 1898  number 11399-1. Allprocedures and experiments used in this study were approved, performed inaccordance with all relevant guidelines and fulfill the rules of the EthicsCommittee for Animal Use of the Londrina State University (Protocol: 60/2017).The individuals were properly identified by morphological criteria andsubsequently deposited in the Museum of Zoology of the State University ofLondrina (MZUEL), available online via SpeciesLink .All individuals were treated with an intraperitoneal injection of 2 mL (1 mL/50g) body weight) of bacterial lysate Broncho-vaxom (7 mg/mL), to trigger aninflammatory response and hence increase the number of renal cells in mitoticdivision . Themitin situ hybridization (FISH) was performedaccording to Prochilodus argenteus Spix & Agassiz, 1829 and NORpattern using Mesquite software . For thaus, 1766 . This stus, 1766 . Werecous, 1766  performeThe ancestral state was inferred using Maximum Likelihood analysis and Markovmodel 1 state (Mk1), which considers that all changes are equally possible. Thecytogenetic data used in the reconstruction were obtained from the literature, includiPlatydoras hancockii: Valenciennes 1840: had 2n=58chromosomes (26m + 14sm + 18st-a) ( 18st-a) .Heteroc 18st-a) . The FIS 18st-a)  box.Centrodoras brachiatus: had 2n=58 chromosomes (22m + 16sm + 20st-a) ( 20st-a) . C-bandi 20st-a) . The FIS 20st-a)  box.Pterodoras granulosus: had 2n=58 chromosomes (22m + 16sm + 20st-a) ( 20st-a) . Heteroc 20st-a) . The FIS 20st-a)  box.Oxydoras niger: had 2n=58 chromosomes (22m + 16m + 20st-a)( 20st-a)G. C-band 20st-a)H. FISH a 20st-a) box.Hemidoras stenopeltis Steindachner 1881: had 2n=58 chromosomes (34m + 16sm + 8a) (sm + 8a) A. The hesm + 8a) B. The 18sm + 8a)  boxed.Trachydoras steindachneri Perugia 1897: had 2n=60 chromosomes (30m + 14sm + 16a) (m + 16a) . C-bandim + 16a) . FISHrem + 16a)  boxed.Ossancora punctata: had the karyotype and heterochromatin pattern previously described byDoras + Ossancora andTrachydoras clades have a greater 2n variability reportedin its analyzed species; hence, increasing the studies in other species of thesegenera is required prior to reconstructing their likely ancestral 2n withaccuracy is not significantly high and sufficient to confirm this hypothesis. Only onespecies from Astrodoradinae has cytogenetic data available; therefore,insufficient samples to define the pattern of NORs for this subfamily . On the other hand, simple NORs was confirmed as an ancestralcondition with high support values  in Wertheimerinae. SimpleNORs was defined as an ancestral trait in most clades of Doradinae, except forthe basal clades  and a part of the apical ones , Table 4The origin of the current karyotype diversity in Doradidae has been assigned tonumerical (Robertsonian translocations), structural (pericentric inversions) anddifferent mechanisms of repetitive DNA dispersion . The hypAcanthodoras and Agamyxis will be required toconfirm or refute the ancestral karyotype hypothesis previously claimed for thegroup.The reconstruction analysis of ancestral characters based on the likelihood methodand Markov MK1 model imply that none of the evaluated characteristics (diploidnumber and NORs) had sufficient support values to be confirmed as plesiomorphicconditions for Doradidae. In fact, the hypothesis of 2n=58 chromosomes and simpleNORs as ancestral states is applicable solely to Wertheimerinae and part ofDoradinae clades, groups in which most of the cytogenetic studies are concentrated.Therefore, this would be the reason that led some authors to attempt to defineancestral conditions for the whole family. The uncertainty of the ancestral patternsfor Doradidae is a reflect of the paucity of karyotype data in the basal-mostclades. Cytogenetic studies in Astrodoradinae, as well as inPlatydoras,Rhinodoras, Pterodoras,Oxydoras and Centrodoras, where all thespecies have 2n=58, however, most of them has variable chromosomal morphology  and Ossancora punctata with2n=66 chromosomes, which possibly arose due to four centric fissions and multiplepericentric inversions from an ancestral karyotype composed by 58 chromosomes . In Auchenipteridae, 5S rDNA sites distribution pattern has also been usefulto characterize species of Tatia . Our results combined, shed light on the karyotype diversification of Doradinae, themost representative subfamily among thorny catfishes. Our cytogenetic analyses andreconstruction of ancestral states brought important new insights into evolutionarypathways traced by doradids, providing thus, two striking evolutionary trajectories:low variation and conservatism of several chromosomal features in large thornycatfishes (non-fimbriate barbells) and remarkable diversity in tiny species fromfimbriate barbells group, often mediated by dynamic behaviors and complexevolutionary processes, still far from being fully solved. However, the availabledata suggest that the main mechanisms responsible for the current karyotypevariability are: pericentric inversions , chromos


Aberrant DNA methylation is associated with multiple malignancies, including lung cancer. Differences in methylation patterns have been observed across lung cancer subtypes, the mutational status of cancer driver genes, and various epidemiological factors. This review summarizes current knowledge of DNA methylation related to lung cancer, providing a foundation for improving prevention, diagnosis, and treatment strategies for this lethal disease.KRAS, EGFR, and TP53), and major epidemiological risk factors . Understanding the mechanisms of DNA methylation regulation and their associations with various risk factors can provide further insights into carcinogenesis, and create future avenues for prevention and personalized treatments. In addition, we also highlight outstanding questions regarding DNA methylation in lung cancer to be elucidated in future studiesLung cancer is the major leading cause of cancer-related mortality worldwide. Multiple epigenetic factors—in particular, DNA methylation—have been associated with the development of lung cancer. In this review, we summarize the current knowledge on DNA methylation alterations in lung tumorigenesis, as well as their associations with different histological subtypes, common cancer driver gene mutations (e.g.,  Lung cancer is the second most common malignancy and the leading cause of cancer-related mortality worldwide, with more than 2.2 million newly diagnosed cases and 1.9 million deaths in 2020 , and theTP53 and RB [TP53 and CDKN2A are frequently mutated in LUSC, LUAD is commonly disrupted with KRAS; EGFR; TSGs TP53, KEAP1, STK11, and NF1 mutations [MALAT1 and HOTAIR, have been reported to be overexpressed in lung cancer [SHOX2 and RASSF1A double gene methylation displayed much higher specificity (>90%) and sensitivity (>70%) for early lung cancer detection than traditional cytological method [RASSF1A and RARβ2; SHOX2 and PTGER4; or p16 and RARβ2 are feasible biomarkers for early diagnosis [DAL-1, EPHB6, HS3ST2, TMEM88 and MGMT, and decreased methylation of ELMO3, are linked to higher rates of metastasis in NSCLC [Multiple genetic and epigenetic alterations have been attributed to the development and progression of different lung cancer subtypes. SCLC is often characterized with concomitant inactivation of both tumor suppressor genes (TSGs) utations . Epigeneutations . MicroRNg cancer . Among tg cancer ). Changeg cancer ,11,12,13g cancer , smokingg cancer , sex [16g cancer ,18. In ag cancer ,20,21. Dg cancer ). For inl method ,24. Otheiagnosis ,26,27,28in NSCLC ,32,33,34APC, HOXA9, RARβ2 and RASSF1A could assist in better defining lung cancer subtypes (SCLC vs. NSCLC) and stage to develop corresponding treatment plans [IGFBP-3 methylation suggests cisplatin-resistant cells [RASSF1A methylation might indicate a better response towards gemcitabine in NSCLC [MGMT methylation [Treatment options for lung cancer patients, including surgery, chemotherapy, radiotherapy, immunotherapy, and targeted therapy, are determined by various factors such as tumor histology, stage, and molecular features . While snt plans . DNAm want cells , high RAin NSCLC . Furtherhylation .In this review, we summarize major findings on DNAm alterations in lung cancers, focusing on their mechanisms in lung tumorigenesis and specific associations with different lung histological subtypes, mutational status, and major epidemiological factors. Finally, we will discuss major outstanding questions that remain to be investigated in the field.DNAm is characterized by the covalent addition of a methyl group to the carbon-5 position of a cytosine base (5mC) by DNA methyltransferase enzymes (DNMTs). In mammals, the modification preferentially occurs at genomic regions enriched with CpG dinucleotide contexts, called CpG islands (CGIs), frequently found close to or within gene promoter regions. Aberrant genome-wide DNA hypomethylation and local promoter-specific hypermethylation have been observed in most tumors, even at early stages of malignancy . While aThere are five known DNMTs in humans, each exhibiting different specificity towards methylated and unmethylated DNA: DNMT1, DNMT2, DNMT3A, DNMT3B, and DMNT3L. DNMT1 preferentially acts at hemimethylated CpG sites and is responsible for methylation maintenance on daughter DNA strands after DNA replication . In contDNMT3A in a mouse model promotes LUAD tumor growth and progression [Overexpression of DNMTs has been frequently observed in lung cancer with upregulation of DNMT1, and is consistently and independently associated with poor prognosis ,47,48. Bgression . It is ugression . The spegression .Mechanistically, there are different ways in which aberrant DNMT activity might contribute to lung carcinogenesis. As higher DNMT expression has been shown to positively correlate with cell proliferation, DNMT dysregulation is believed to disrupt the cell cycle. A study from Sato et al. suggests that overexpression of DNMTs reflects increased cell proliferation in human lung cancers . DNMTs aIn addition, TSGs could be silenced by DNAm through increased DNMT activity. This could occur by direct and/or indirect inhibition. Direct inhibition involves direct blockage of the interaction between methylated DNA and methylation-sensitive TFs. Alternatively, methylated DNA recruits the m5CpG-binding domain (MBD), which contain proteins to form a complex that blocks the binding of TFs to DNA . IndirecRASSF1A and GADD45β promoters in NSCLC cell lines [FHIT and WWOX, resulting in tumorigenicity inhibition in both in vivo and in vitro settings [Given the role of DNMTs in lung tumorigenesis, many DNMT inhibitors (DNMTis) have been developed and tested for lung and other cancers. Commonly used DNMTis, such as 5-Azacytidine  and its derivative 5-Aza-2′-deoxycytidine , are nucleoside analogs, and work by forming covalent bonds with DNMTs, inactivating these enzymes . Promotell lines . Antisenll lines . MiR-29ssettings . Howeversettings . TherefoABE1, HOXA9, HOXA7, OGG1, TIMP, and XRCC1) and their inactivation [TET (Ten–Eleven Translocation) enzymes catalyze the oxidation of 5mC, leading to the reversal of DNAm, which was initially thought to be an irreversible epigenetic event. The family of TET proteins consists of TET1, TET2, and TET3, which have the same catalytic activity but differ in their domain architectures . Althougtivation ,71. In ctivation . In addiSNCG associated with upregulation was observed in multiple solid tumors, including lung cancer [SNCG was later found to be involved in cancer cell migration and invasion [MAGE genes were upregulated in 70–85% of NSCLC tumors and carcinogen-damaged lung epithelial cells, and such upregulation correlates with loss of methylation [MAGEs’ overexpression is associated with poor prognosis and is required for tumor growth and metastases in lung cancer [MAGEs might also contribute to the initiation and maintenance of cancer stem cells [Like many other cancers, extensive global hypomethylation in lung tumors is seen specifically at repetitive sequences, including SINEs, LINEs, subtelomeric repeats, and segmental duplications ,73. Hypog cancer . SNCG wainvasion . An earlhylation . MAGEs’ g cancer . In addiem cells .HRAS may contribute to gene loss [Alternatively, hypomethylation in tumors might induce genomic instability by reactivating retrotransposon elements and/or chromosome rearrangements. A study in NSCLC showed that hypomethylation at the 3′ tandem repeat region of ene loss . Daskaloene loss . In concene loss . These rene loss . The comCASP8, DAPK, TNFRSF6, DR4, DR5) [CDKN2A/p16, PTEN, RASSF1A) [MGMT, MLH1, MSH2) [APC, RARβ-2, RUNX3, SHOX2) [CDH1, CDH13, TSLC1) [While reports and mechanistic details on hypomethylation are limited, there is plethora of studies on the targets and roles of hypermethylation in lung cancers. The variations in results reported across different studies are due to the disparity in designs, materials , heterogeneity (varied lung cancer histology types), and techniques  . NeverthR4, DR5) ,89,90,91RASSF1A) ,94,95,961, MSH2) ,98, regu, SHOX2) ,101,102,, TSLC1) ,104  , further, TSLC1) . UNC5D was hypermethylated while downstream introns were hypomethylated in lung cancer, suggesting a potential complex interplay of the two opposite mechanisms to regulate gene expression [Interestingly, there have been reports of both cancer-specific hyper- and hypomethylation in the same gene. For instance, the promoter of potential TSG pression . Furtherpression . Althougpression ,108, theWhile many of the reported genes are subjected to altered methylation in lung cancer in general, some of them are specifically associated with different lung cancer subtypes, smoking status, and molecular alterations. Understanding the specific associations of DNAm in individual lung cancer subgroups could help to elucidate distinct underlying mechanisms, provide useful biomarkers for diagnosis and prognosis, and guide personalized treatments.APC, CDH13, and CDKN2A/P16 were more frequently hypermethylated in NSCLC than in SCLC [CASP8, TNFRSF6/Fas, and TRAIL-R1/DR4) were found to be methylated in SCLC cell lines and tumors, but not in NSCLC samples [With most lung cancer cases being NSCLC, many DNAm patterns described in lung cancer are dominated by those from this histological group. Despite accounting for only 10–15% of cases, SCLC is characterized by rapid proliferation and metastasis, and high lethality, suggesting the need for a better understanding of this subtype. To the best of our knowledge, there have not been any studies systematically comparing genome-wide DNAm profiles between NSCLC and SCLC. Comparing methylation results from independent studies could be challenging due to various technical and analytical variations. Early studies examining selected genes showed that the promoters of multiple genes are hypermethylated in both SCLC and NSCLC  with dif in SCLC ,110,113. samples . NEUROD1, HAND1, ZNF423, REST, and homeobox genes [A global methylation study in primary SCLC tumors identified 73 gene targets enriched at neuroendocrine-specifying TF binding sites, including ox genes . The autox genes . EZH2 haox genes . In addiox genes , as prevox genes . The resox genes . AlthougBCL2 oncogene and downregulation of TSGs RB1 and TCF21 in SCLC [BCL2 or RB1 dysregulation by methylation in NSCLC, TCF21 has been established to be silenced via hypermethylation in this subtype [CDKN2A, FHIT, DAPK, and RASSF1A). A recent genome-wide study identified 20 potential hypermethylated driver genes  and 13 methylation-driver non-coding RNAs in NSCLC [A correlation between methylation and gene expression could help to pinpoint cancer driver events in each subtype. DNAm was found to be one of the primary mechanisms responsible for upregulation of  in SCLC ,126. Alt subtype ,129. Earin NSCLC . FurtherRASSF1A methylation level in SCLC and large cell neuroendocrine carcinomas than in NSCLC, suggesting it could be a potential marker for differential diagnosis [APC, HOXA9, RARβ2, and RASSF1A) across lung cancer subtypes in circulating cell-free DNA (cfDNA) [HOXA9 and RASSF1A to have higher methylation levels in SCLC than NSCLC among 129 samples, displaying relatively high specificity and accuracy (>79%) but modest sensitivity . With larger, independent cohorts and systematic exploration of useful markers from genome-wide methylation, DNAm analysis from circulating cfDNA could be proven useful in clinical settings. Using DNAm markers for differential diagnosis is also a current active area of research. Walter et al. observed a significantly higher iagnosis . In a re (cfDNA) . They foMGMT promoter indicates that SCLC patients might benefit from treatment with the alkylating agent temozolomide, but patients with NSCLC would not [RASSF1A methylation has been shown in NSCLC but it is unclear in SCLC. NSCLC patients with methylated RASSF1A respond better to gemcitabine compared to patients with wild-type tumors [RASSF1A have better survival when treated with paclitaxel compared to gemcitabine [Interestingly, methylation patterns have also shown distinct associations with prognosis and treatment responses for different subtypes. For instance, hypermethylation at the ould not . The clie tumors . In addicitabine . Unsupercitabine . Howevercitabine . ANK1, APC, CCND2, CDH13, GATA3, KCNH5, LINE-1, RARβ, RASSF1, and RUNX3 are significantly higher in LUAD than in LUSC [AGTR1, CDKN2A/P16, DAPK, HOXA9, MLH1, SHOX2, SFMTB2, SFRP4, TIMP3, TGIF, and ZIC4, are more often observed in LUSC compared to LUAD [CTSE and SLC5A7 also displaying opposite expression patterns [LUAD and LUSC are two histopathological subtypes that compose the majority of NSCLC. They are believed to have different cells of origins with distinct molecular characteristics, gene expression profiles , and the in LUSC ,137,138, to LUAD ,141,142. to LUAD . Howeverpatterns . These gpatterns . TherefoGAS1, FAM78B, and DOTL1) [CDKN2A and MGMT), and three hypermethylated genes , when comparing LUAD to LUSC, might be able to distinguish the two subtypes [CLDN1, TP63, TPX5, TCF21, ADHFE1, and HNF1B) as potential DNAm markers for LUSC diagnosis compared to a non-tumor lung [HOXA9 promoter in LUSC than in LUAD, but the biomarker only displayed 55.2% sensitivity and 74.3% specificity for LUSC detection [Immunohistochemical staining of resected tumors is often used for differential diagnosis between LUAD and LUSC, but the sensitivity for even the most widely used marker, TTF-1, is only 62% . In an ed DOTL1) . Howeversubtypes . Neverthmor lung . Howeveretection . Therefoetection . ERBB2 and ZEB2 being especially predictive. In addition to the traditional histological classification, gene expression divides NSCLC to epithelial- and mesenchymal-like phenotypes with distinct responses to EGFR inhibitors  . Walter HOXA2 and HOXA10 LUSC have worse prognoses than those with wild-type LUSC [FAM83A could indicate a poorer prognosis [GRK6 could promote cell migration and invasion [There is limited information on the specific associations of DNA prognosis markers in LUAD and LUSC. In an exploratory analysis, Heller et al. observed that patients with methylated ype LUSC . In LUADrognosis , while hinvasion . HoweverKLF6, TERT, MSH5, ACTA2, GATA3, and VTI1A) and hypermethylation (STK3A and CHRNA5) of genes related to lung cancer susceptibility [AHRR (cg05575921), 6p21.33 (cg06126421), and F2RL3 (cg03636183) is associated with tobacco smoking and higher lung cancer risk [It is well established that tobacco smoking is associated with higher risk for lung cancer (between 15- to 25-fold in comparison to never-smokers). Previous studies have suggested that smoking is highly related to SCLC development, with only 2.5% of SCLC cases occurring among non-smokers , in conttibility , suggestcer risk . Baglietcer risk . Interescer risk .p16, APC, MGMT, ANK1, MTHFR) [MGMT promoter methylation is more common in never-smokers than in smokers [RUNX3 was more frequently methylated in non-smokers [KRAS mutations). Tobacco smoking has been shown to be associated with an increased DNAm of specific genes in somatic analyses of lung cancers  ,159,160. smokers ,162. Alt-smokers . Mechani-smokers ,164. In -smokers . Further-smokers . They hyCDH13 being more methylated in non-smokers compared to smokers in advanced NSCLC [IRF8, IHH, LGALS4, IL18BP, and VTN), but only LGALS4 as downregulated in smokers compared to never-smokers LUAD [LGALS4 was validated as hypermethylated and downregulated as seen previously [MYO1G, and ZFP28 [MYO1G and cytochrome p450 family genes  in former smokers. However, the author acknowledged that these results should be interpreted with caution given the modest sample size and cell type heterogeneity. Moreover, the differences might reflect epigenetic changes due to cancer progression rather than tobacco smoke exposure.There have been limited studies systematically comparing DNAm profiles in lung cancers from ever- and never-smokers, and most of the differences observed have been modest. Using a 30-gene panel, Pesek et al. observed ed NSCLC . An earlers LUAD . Similarers LUAD , but noneviously . Even theviously , its connd ZFP28 . These CKRAS mutations occur in 20–40% of NSCLC, with a higher prevalence in LUAD vs. LUSC (32% vs. 4%), Western vs. Asian (26% vs. 11%), smokers vs. non-smokers (30% vs. 10%), and almost never occurring in SCLC [KRAS mutations and methylated p16, as well as a higher overall methylation index in LUAD [KRAS mutations were associated with an epigenetic subgroup in LUAD with higher methylation [KRAS mutations did not appear to drive this subgroup, but more complex molecular mechanisms may be involved. In contrast, a genome-wide comparison between KRAS-mutated tumors and wild-type tumors only discovered two hypomethylated CpGs corresponding to PFDN1 and MAEA genes [KRAS mutations lack RASSF1A promoter methylation [KRAS mutations in isogenic lung cancer cell lines resulted in stochastic DNAm changes in genes enriched in development and differentiation [ in SCLC ,171. An  in LUAD . In conchylation , reminishylation . HoweverEA genes . Intereshylation , but sephylation ,175. In ntiation . EGFR mutation is much higher in NSCLC never-smokers compared to smokers (49.3% vs. 21.5%), females compared to males (43.7% vs. 24.0%), LUAD compared to non-LUAD (38.0% vs. 11.7%), and Asians compared to non-Asians (38.4% vs. 15–20%) [EGFR-mutated NSCLCs, a comprehensive understanding of the genetic and epigenetic profiles would be essential to fully exploit the treatment strategy. An early study showed that EGFR-mutated tumors had lower methylation indexes and were more often associated with unmethylated p16 and CDH13 [APC, CDKN2B, ESR1, and VHL in patients with EGFR mutations [EGFR is significantly hypomethylated in LUAD tumors with EGFR mutations compared to those without [EGFR and gene expression in both mutated and wild-type EGFR tumors, suggesting that EGFR could be epigenetically regulated. In fact, methylation at the EGFR promoter has been suggested to contribute to EGFR-TKI resistance [The frequency of  15–20%) . With thnd CDH13 . Pesek eutations . A study without . In addisistance .TP53 mutations and DNAm in lung cancer is lesser known. Bjaanæs et al. identified 2026 hypomethylated and 349 hypermethylated CpGs corresponding to 834 unique genes in LUAD TP53-mutated tumors compared to wild-type tumors [TP53-mutated breast cancer such as AFF3 [TP53 mutations were suggested to affect global DNAm through DNMT1 overexpression in lung cancer, and increased genomic instability, but how this is linked to the changes observed, largely of hypomethylation, is unclear. Interestingly, TP53 mutations were also associated with hypomethylation in basal-like breast cancer [The specific association between e tumors . Althoug as AFF3 . TP53 mut cancer , and CIMt cancer .MGMT and GSTP1 for cases from the USA and Australia than in those from Japan and Taiwan [FOXK2, PBX1, FNDC7, FUBP3) differentially between Native Hawaiian smokers vs. white/Japanese American smokers, which might explain some of the lung-cancer-risk variations [F2RL3 and GPR15 to have similar methylation patterns in African American and Caucasian populations, suggesting common molecular mechanisms of epigenetic modification induced by environmental exposures across different populations [Epidemiological studies have shown that lung cancer risks differ across ethnic/racial and gender groups, with African Americans and Native Hawaiians, and females vs. males, having the highest risks ,185,186.d Taiwan   play a major role in altering DNAm patterns in lung cancer, creating a window of opportunity for targeted drug development and treatment. However, as highlighted in our review, further studies are required to reconcile and elucidate the conflicting reports regarding their specific roles . We suspect that the paradox might be attributed to various factors, including mutational driver status, model systems, and histological subtypes.In addition, there remains a knowledge gap in how different DNMT isoforms specifically contribute to lung cancer development. For instance, DNMTB has over 30 different isoforms with ∆DNMT3B4-del being the most abundant isoform in NSCLC . In thisThe dynamic remodeling of DNAm is essential for lung development and cell fate decisions as stem cells exit pluripotency . Many stTERT upregulation through DNA hypermethylation requires further investigation of similar phenomena [Most studies so far have focused on describing hypermethylated promoters and downregulated target genes. However, the notable example of henomena . Althoughenomena ,108; thuAlthough cell lines provide a useful model to study processes that can be observed in tumors, several studies have revealed that DNAm data from cell lines might not be representative of those from primary tumors. For instance, a global analysis indicated that cell lines are much more heavily methylated compared to primary tumors . In concAlthough different lung cancer subtypes  have distinct genetic and molecular profiles, there have been limited direct comparisons of the DNAm epigenome between them. Future studies with a large sample size encompassing various lung cancer pathological entities would be required to systematically characterize the differences in their DNAm landscapes. The knowledge would be essential to understand the underlying mechanisms driving tumorigenesis of different subtypes, identify biomarkers for accurate differential diagnosis, and develop effective personalized treatments.Lung study [While in lung cancers from smokers, tobacco-smoking carcinogens can provide a “fertile ground” for oncogenic mutations that drive tumor development  even in ng study , many ofRecent studies have observed congruent genomic and DNAm evolutionary trajectories in lung cancer ,201 as wRecent advances in single-cell DNA-methylation (sc-DNAm) profiling technologies have enabled the examination of cell-of-origins and cell type heterogeneity in cancers . It is i