In this review, we discuss the role of oxidative stress in activating AF-triggered activities, regulating intracellular Ca2+ handling, and functional and anatomical reentry mechanisms, all of which are associated with AF initiation, perpetuation, and progression. Changes in the extracellular matrix, inflammation, ion channel expression and function, myofibril structure, and mitochondrial function occur during the early transitional stages of AF, opening a window of opportunity to target NOX and mitochondria-derived oxidative stress using isoform-specific NOX inhibitors and mitochondrial ROS scavengers, as well as drugs that improve mitochondrial dynamics and metabolism to treat persistent AF and its transition to permanent AF.Atrial fibrillation (AF) is the most common type of cardiac arrhythmia and its prevalence increases with age. The irregular and rapid contraction of the atria can lead to ineffective blood pumping, local blood stasis, blood clots, ischemic stroke, and heart failure. NADPH oxidases (NOX) and mitochondria are the main sources of reactive oxygen species in the heart, and dysregulated activation of NOX and mitochondrial dysfunction are associated with AF pathogenesis. NOX- and mitochondria-derived oxidative stress contribute to the onset of paroxysmal AF by inducing electrophysiological changes in atrial myocytes and structural remodeling in the atria. Because high atrial activity causes cardiac myocytes to expend extremely high energy to maintain excitation-contraction coupling during persistent AF, mitochondria, the primary energy source, undergo metabolic stress, affecting their morphology, Ca Atrial fibrillation (AF) is the most common cardiac arrhythmia in clinical practice and contributes significantly to cardiovascular morbidity and mortality ,2. In adAccording to the American Heart Association guidelines on AF management, AF can be classified as paroxysmal, recurrent, or permanent, depending on its duration . If a paFocal activity and reentry are two mechanisms that play crucial roles in the development of AF. The primary source of focal activity in the atrium is the pulmonary veins, and eliminating this activity through pulmonary vein isolation is considered the most effective therapy for non-permanent forms of AF . TriggerINaL), and increased L-type Ca2+ currents . EADs occur when voltage-gated sodium and L-type Ca2+ channels recover from their inactive state and reactivate during membrane repolarization. DADs, on the other hand, are associated with altered intracellular Ca2+ homeostasis. Spontaneous Ca2+ discharges from the sarcoplasmic reticulum (SR), caused by Ca2+ overload and dysfunction (leakiness) of ryanodine receptor 2 (RyR2), activating forward-mode action of the sodium-calcium exchanger (NCX). In this operating mode, one calcium ion is extruded from the cell in exchange for three sodium ions entering the cell. This inward sodium current (ITi) perturbs the membrane potential, causing DADs. If the DAD reaches the threshold for sodium current activation, it can trigger a non-physiological AP. EADs and DADs are both triggered activities that are important for initiating AF in the pulmonary veins. It is notable that triggered activity has also been observed in atrial myocytes isolated from lone AF patients (AF in patients younger than 60 years without coexisting heart disease) and in AF animal models. These findings suggest that triggered activity may play a significant role in the development of AF. Further investigation is necessary to determine whether triggered atrial myocyte activity is a cause of AF [AP prolongation is mainly caused by decreased repolarizing potassium currents or increased inward sodium currents, especially late sodium (se of AF . Ito) and/or decreased inward ICa,L can accelerate membrane repolarization and reduce the refractory period. Sodium current downregulation [Reentry is associated with abnormal conduction, as opposed to abnormal impulse formation. Reentry occurs when a propagating impulse fails to extinguish after the normal activation of the heart tissue and continues to re-excite the heart after the refractory period is completed. The reduced wavelength of the cardiac impulse is a determinant of reentry, which is a product of conduction time and refractory period ,11. Elecgulation , gap jungulation ,14, atrigulation ,16, and gulation ,18 are o2+ due to RyR2 dysfunction or Ca2+ overload, for instance, can result in the development of AF structural substrates like atrial enlargement [2+ entry during atrial tachycardia triggers calcium-dependent signaling that leads to atrial hypertrophy [Even though triggers and substrates are distinct, they are dynamic and can establish causal feedback mechanisms that favor AF progression. The mishandling of intracellular Caargement . Increasertrophy . Alteredertrophy ,21,22. T2+ handling transporters, and structural cellular elements and/or affecting the signaling pathways that modulate them. For this purpose, we conducted a comprehensive literature review on oxidative stress, particularly NADPH oxidases (NOXs) and AF pathogenesis, using the methods below. In this review, we will explore how ROS (reactive oxygen species) are an important intermediary in developing AF substrates and triggers by directly oxidizing membrane ion channels, intracellular CaWe systematically searched electronic databases, such as PubMed, Google Scholar, Scopus, and Web of Science, to identify relevant studies. Our primary search used a combination of keywords and Medical Subject Headings (MeSH) terms that included reactive oxygen species, oxidative stress, NADPH oxidases, mitochondrial oxidative stress, atrial fibrillation, aging, electrical remodeling, structural remodeling, tachypacing, reentry, cell signaling, and relevant synonyms. Our inclusion criteria consisted of human and animal studies published in English, original research articles, and systematic reviews that investigated the role of ROS in atrial fibrillation pathogenesis, published on or after January 1985. We summarized and analyzed the findings from the selected studies using a narrative synthesis approach.Cardiovascular cells produce ROS through various enzymes such as NOX, xanthine oxidase, cyclooxygenases, lipoxygenases, myeloperoxidase, uncoupled endothelial NO synthase, and mitochondrial respiratory chain complexes ,24. The 2•−) [The NOX enzyme family is the most significant and the only one whose sole function is ROS production in normal and pathological conditions. The enzyme family comprises seven isoforms, including NOX 1–5 and dual oxidases (DUOX) 1 and 2 . These e2•−) ,32. The 2•−; however, increased NOX1 expression and activation produce higher levels, indicating the role of cytosolic subunits in increasing ROS production [+ reabsorption, leading to hypertension, another AF risk factor [NOX1 constitutively generates low levels of Ooduction ,33. Optioduction ,35,36. Ooduction . Ang II k factor ,39. NOX1k factor , a drug k factor  and humak factor .2•− generation in SHRs. These effects were associated with inhibiting multiple signaling pathways, including TGF-β1/Smad2/3, NF-κB-P65, NOX1, NOX2, Kir2.1, Kv1.5, and Cx43. This suggests that NOXs may participate in macrophage recruitment and AF induction. Studies have shown a decrease in nitric oxide availability in the left atrium of swine with pacing-induced AF, indicating an oxidative stress-mediated degradation mechanism [2•− was observed in the left atrium and left atrial appendage, with both xanthine oxidase and NOX contributing to the increase [New-onset AF and early AF recurrence after ablation are associated with left ventricular diastolic dysfunction (DD) ,47,48,49echanism . Increasincrease . A changIn the heart, NOX2 and NOX4 are the most commonly found NOX isoforms. NOX2 is quiescent and found in association with p22phox in the plasma membrane. It can be activated by various stimuli such as growth factors, cytokines, G-protein coupled receptor agonists , and mechanical forces. This activation occurs through the binding and activation of cytosolic regulatory subunits  at the plasma membrane ,58. NOX2n = 4–6) had increased TGF-β expression, oxidative stress, and loss of myofibrils compared to those with sinus rhythm. The activation of NOX2 by endotoxins is implicated in the development of AF in patients with community-acquired pneumonia [2•− did not affect atrial electrical and structural properties, but instead, it moderately increased the probability of AF induction [+ current (KAchI) mediated by activation of PKCε, which increased in magnitude with increasing frequency of atrial tachypacing [KAchI activity could increase atrial vulnerability to tachyarrhythmia in chronic AF patients, thus sustaining the condition [The activation of NOX2 may cause AF, as observed in patients with paroxysmal or permanent AF . In the neumonia . Obstrucneumonia ,69. In mneumonia . Howeverneumonia . In casenduction . These snduction . A studyhypacing . This inondition ,75. ICa,L [2+ leak in left atrial cardiomyocytes [2+ leak in pressure-overloaded hearts by RyR2 oxidation in left atrial myocytes was mediated by activation and upregulation of NOX2 and NOX4, suggesting a mechanistic relationship between SR Ca2+ leak induced by stretch and AF. Similarly, activation of CaMKII-dependent diastolic SR Ca2+ leak is observed in the right atrial myocardium of AF patients [2+ overload, inhibiting AF development [Activation of the renin-angiotensin system has been linked to heart failure and ischemia-reperfusion-related arrhythmias. Angiotensin-converting enzyme (ACE) inhibitors and Ang II receptor blockers have been found to reduce AF ,77. ReduICa,L . OxidizeICa,L . An atrimyocytes . SR Ca2+patients  and indupatients ,84. Actielopment . However2O2), rather than O2•−, because of an E-loop within its structure that promotes the rapid dismutation of O2•− before it leaves the enzyme [Nox4 forms a heterodimer with p22phox, which ensures structural stability and catalytic activity, without requiring cytosolic subunits ,87,88. Ue enzyme . NOX4 ise enzyme . The acte enzyme . NOX4 ise enzyme ,93,94. Ie enzyme . Increase enzyme . Increase enzyme . Young tNox4 in cardiomyocytes can protect against chronic pressure overload by preserving myocardial capillary density [NOX4 is found in cardiomyocytes, fibroblasts, and microvascular endothelial cells in the heart ,103,104. density . The hea density . Additio density . 2O2 levels in the left atrial appendages of AF patients were more than double those in patients without AF. Additionally, H2O2 levels were found to be correlated with NOX4 levels, especially in patients with hypertension, which is a known risk factor for AF [2O2 were produced. This result implies that NOX4-derived H2O2 may play a crucial role in the development of AF  homogenates or isolated myocytes from 15 patients with AF . Dipenyleneiodonium (DPI), an inhibitor of flavin-containing oxidases or apocynin, a NOX inhibitor that blocks p47phox translocation, inhibited O2•− production in AF, suggesting a pathogenic role for NOX-derived O2•− in AF [2•− generated by NOX2 in left atrial appendages (LAA) and left atrial enlargement in AF patients [2•−, NADPH-stimulated O2•−, and ONOO− production, as reported by Casadei and colleagues [After cardiac surgery, paroxysmal AF (PAF), also known as postoperative AF or POAF, commonly occurs and typically peaks between days 2 and 4 following the operation . Clinica•− in AF . Similarpatients . The serpatients . Additiopatients . POAF palleagues . NaI expression, and atrial APD were decreased while Kv1.5 potassium channel expression and corresponding current (KurI), F2-isoprostanes, NOX2, and PKC-a/d expression, and atrial fibrosis significantly increased. A mitochondrial antioxidant restored NaI, Ca,LI, KurI, APD, reversed atrial fibrosis, and attenuated AF burden in diet-induced obese mice. Studies on animals have revealed that Rac1-dependent NOX-derived ROS plays a role in AF pathogenesis ,162,163.Oxidative stress in mitochondria contributes significantly to cardiac dysfunction in the pathogenesis of POAF ,167. In 2+ handling is impaired, mitochondrial membrane potential decreases, and ATP production is reduced [•− production increased, while eNOS expression and circulating NO levels decreased [Patients with persistent AF experience high atrial activity, leading to excessive energy consumption by cardiac myocytes to maintain excitation-contraction coupling, which can affect their structure and function. Mitochondria, the primary energy source, are also subject to metabolic stress. In individuals with AF, fragmented mitochondria are present in the atrial tissue. When HL-1 atrial myocytes are subjected to tachypacing, mitochondrial Ca reduced . Increas reduced . Additio reduced . In a shecreased .2+ leak have an increased susceptibility to AF due to mitochondrial dysfunction, ROS production, and atrial RyR2 oxidation [SCN5A have also been linked to AF in patients with congenital long QT syndrome and lone AF. Atrial myocytes from chronic AF patients showed a significant reduction in peak INa density and lower expression of Nav1.5, along with an increase in late INa [INaL- INa peak ratio, ranolazine was found to reverse proarrhythmic activity in atrial myocytes and improve diastolic function. Studies by Avula et al. reveal that expressing human mitochondrial catalase in mice overexpressing human F1759A NaV1.5 channels can reduce cardiac structural remodeling, spontaneous AF incidences, and pacing-induced after-depolarizations despite the heterogeneously prolonged atrial action potential [Marks and colleagues showed that mice with RyR2 mutations that cause intracellular Caxidation . In patixidation ,175. Genlate INa ,177,178.otential . Transcriptional oxidative stress response affects permanent AF pathophysiology. In their study, Kim et al. used cDNA microarrays to compare transcription levels of putative genes that might be involved in the oxidative stress response between control patients and patients with chronic atrial fibrillation AF . ResultsParoxysmal AF begins with short episodes that become persistent and eventually permanent over time. As AF advances from short episodes to a persistent state, the atria undergo progressive structural and functional changes that ensure its long-term persistence ,184. In 2•− production increases due to increased NOX expression and activity. Conversely, in cases where AF or atrioventricular block has been persistent, mitochondrial oxidases and uncoupled NOS activity in the right atrium are responsible for the biatrial increase in increase in O2•− production (due to a reduction in BH4 and/or an increase in arginase activity). It was found that Rac1 activity in RA samples from persistent AF patients was not increased. In addition, atorvastatin did not reduce O2•− production in a mevalonate-reversible manner, which indicates that statins may not be beneficial in secondary AF prevention. The group discovered that miR-31 upregulation in goats and patients with persistent AF depletes nNOS and reduces NOS activity due to mRNA decay and the translational repression of dystrophin, leading to the loss of nNOS in the sarcolemmal region [It is undeniable that oxidative stress plays a significant role in the development of AF. However, as AF progresses from paroxysmal to permanent, the source of ROS also changes. According to Riley et al., the mechanism responsible for NO-redox imbalance differs between atria, as well as the duration and substrate of AF . After tl region . nNOS isl region . nNOS-del region ,193 and l region  and prevl region . UpregulKCNJ3, KCNJ5), calcium channel genes (CACNA1C), or sodium channel genes (SCN5A), most notably when comparing control and transition samples. In contrast, others, such as HCN2 or KCNH7, have increased expression [RCAN1 and LGALS3BP, have a significant effect on its pathophysiology. Patients with persistent AF show significant DNA damage correlated with poly(ADP)-ribose polymerase activation, NAD+ depletion, and oxidative stress in CMs [Martins et al. studied persistent long-standing AF in sheep and discovered that the rate at which dominant frequency (DF) increases can predict when AF stabilizes and becomes persistent . Transcrpression . Some ofs in CMs . In patis in CMs . These d2O2 (2 mM) demonstrated induced ectopic firing and EADs, as well as shortened APD in PVs and the left atrium [2O2-induced PV burst firing and increased spontaneous rates. It has been observed that ectopic beats originating in pulmonary veins can lead to frequent AF paroxysms . By modit atrium . PretreaINaL and ICa,L currents. This occurrence is linked to oxidative stress and is the primary arrhythmogenic mechanism. In an isolated aged rat atria study, acute oxidative stress through H2O2 perfusion initiated spontaneous AF that was preceded by EADs in the left atrial epicardial appendages [INaL upregulation prevented and suppressed EAD-mediated triggered activity and spontaneous AF  and accelerate membrane repolarization during phases 1 and 2 of the AP [KurI is crucial for the excitability of atrial muscles as seen in patients with a Kv1.5 (E375X) loss-of-function variant who experience idiopathic AF [Human atrial myocytes express Kv1.5 channels that enable ultra-rapid potassium current . The increased function of SERCA results in a higher Ca2+ content in atrial myocytes than ventricular myocytes. The greater atrial SR Ca2+ content makes atrial myocytes more susceptible to spontaneous diastolic SR Ca2+ release when RyR2 channels are sensitized under pathological conditions [2+ signals start at the cell’s outer edge during E-C coupling [2+ flux properties of the atrial myocytes play a role in their efficient E-C coupling and their ability to sustain high excitation frequencies in AF. Excitation-contraction (E-C) coupling connects the electrical activation of cardiac muscle cells to their mechanical contraction . During traction . During ectively . The chawaveform ,208. Fornditions ,211,212.coupling ,213. Ove2+ handling properties of myocytes [2+ release [2+ release from the SR. At the same time, dithiothreitol reverses the effects of SH oxidizing agents [2+ leak in permeabilized isolated ventricular myocytes, further supporting the role of redox regulation of RyR2 in heart failure [SR membranes contain NOX, supporting the notion that oxidative stress plays a significant role in the Camyocytes . Confoca release . It is w release . Hydroxyg agents ,218. Oxi failure . Isolate failure . 2+ leak and a proarrhythmic phenotype in mice. RyR2 oxidation can lead to inhibition instead of activation. When SR membrane preparations were treated with a physiological concentration of NADH-activated NOX, Ca2+-induced Ca2+ release (CICR) was inhibited, with RyR2 single channel open probability significantly reduced [2+ release. An increase in RyR2 S-glutathionylation, induced by NOX, resulted in a preconditioning response [2+ release (SOICR) [Cardiomyocytes express all three NOS isoforms . Gonzale reduced . In permresponse . The fun (SOICR) . Low lev−/−FKBP12.6 mice have increased diastolic SR Ca2+ leak, spontaneous Ca2+ release, and greater vulnerability to pacing-induced AF [2+ leak and propensity for AF [2+ release and reduced the likelihood of atrial arrhythmia in isolated atrial myocytes. A disruption of RyR2-FKBP interaction caused by oxidative stress may also contribute to RyR2 leak and cardiac arrhythmia caused by palmitoyl-carnitine [2+-induced activation at low cytosolic Ca2+ levels. This increases spontaneous Ca2+ release during diastole [2+ releases in atrial myocytes from AF patients [2+ concentrations can lead to an inward Na+/Ca2+ exchange current, resulting in DADs, which may cause atrial arrhythmias [In patients with chronic AF, impaired diastolic closure of SR RyR2 is associated with increased serine 2808 phosphorylation of RyR2 and decreased binding levels of FKBP12.6 , which is an inhibitory subunit of RyR2 ,225. Atry for AF . The oxiarnitine . Mitochodiastole ,228. PLBpatients ,230. Durhythmias ,231.2+/CaM)-dependent protein kinase II) is more responsive to an elevation of Ca2+ [2+ leak in isolated atrial myocytes and makes mice more susceptible to AF induction. Mice overexpressing methionine sulfoxide reductase A, an enzyme that reduces oxidized CaMKII, and mice with oxidation resistant CaMKII were resistant to AF induction following Ang II infusion, suggesting CaMKII is a mechanistic link between oxidative stress and AF [2+ leak, abnormal mitochondrial structures in atrial cardiomyocytes, atrial fibrosis, and pacing-induced AF [In conditions of increased ROS, the activity of CaMKII  [ICa,L levels. Carnes and colleagues [ICa,L in atrial myocytes isolated from AF patients. In line with this, acute exposure of cardiomyocytes from the left atria to H2O2 led to a reduction ICa,L, and a shorter APD [ICa,L, highlighting the significance of LTCC downregulation in AF pathogenesis due to imbalances in ROS/NOS production [Functional reentry occurs when an impulse propagates around a refractory core without any underlying substrate or anatomical barriers . This is(Cav1.2) . Many cllleagues  discoverrter APD . Therefooduction .INa is an ionic mechanism that promotes reentry by impairing conduction [INa. Rat neonatal cardiomyocytes treated with NADH, a substrate for NOX, show a rapid decrease in INa, which can be prevented by superoxide dismutase [INa by modulating the expression of the major in the heart, NaV1.5 encoded by SCN5A. Ang II and H2O2 promote the binding of NF-kB to the cardiac Scn5a promoter, reducing Scn5a mRNA levels [Scn5a transcription [Scn5a promoter, and FoxO deletion upregulates Scn5a transcription [Scn5a promoter region [2O2 exposure reduces Nav1.5 channel expression and INa levels by retaining FoxO1 in the nucleus.In AF, the downregulation of nduction . Severalismutase . It is sismutase . OxidatiA levels . FoxO trcription . Studiescription . FoxO1 ir region . UpregulIK1 largely determines the resting membrane potential and the terminal repolarization of the cardiac action potential [IK1 is mediated by Kir2.x channels [IK1 can lead to electrical remodeling, which contributes to the maintenance of arrhythmia by reducing the refractory period [The inwardly rectifying potassium current otential . Specifichannels . In chroy period ,261. Mory period .2O2. This reduces the forward trafficking of Cx43 [2O2 exposure slowed cardiac excitation and decreased cardiac gap junction coupling in zebrafish. ROS can also directly target Cx43 and alter its distribution. In a mouse model of diabetes, there were increased levels of oxidized connexin 43 tyrosine sites, leading to Cx43 mislocalization and abnormal ventricular conduction patterns [Gap junctions are clusters of channels that connect adjacent cells for chemical and electrical communication. Atrial gap junctions comprise connexin proteins Cx40 and Cx43 . Changes of Cx43 . Moreovepatterns . In a slpatterns  , resulting in cardiac structural remodeling. According to Nakano et al., the expression and activity of MMPs are changed in fibrillating atria, leading to atrial structural remodeling and dilatation during AF . Activatmulation . Moreovemulation ,275. Moemulation .2+-activated calpains are responsible for degrading myofibrils in cases of permanent AF [2+ levels resulting from the ROS-induced ionotropic effect on LTCC during AF.In cases of permanent AF, oxidative stress can lead to structural remodeling that impacts the contractile machinery of atrial myocytes. Research has shown that tyrosine nitration and carbonylation of the myofibrils can cause dysfunction, rather than changes in composition . Studiesanent AF ,278. ThiA proteomic analysis was conducted on the tissue of LAA from patients with AF and those with sinus rhythm. The results indicated that mitochondrial dysfunction, oxidative phosphorylation, and Sirtuin signaling were the most affected pathways . An incrThe enlargement of the atrium is an independent risk factor for the development of AF ,281. ThiThe electrical and structural remodeling of the atria, as well as the initiation and progression of paroxysmal to permanent AF, are significantly impacted by oxidative stress from NOX activation and mitochondrial dysfunction. Mitochondrial dysfunction can be caused by NOX-derived ROS, which can damage mitochondrial DNA and protein complexes, leading to AF associated with aging. Uncoordinated responses to meet the energetic demands of AF can reduce ATP availability, negatively impacting calcium signaling and the contractile machinery of cardiomyocytes. This dysregulation can create heterogeneity in atrial contractility and electrophysiology, leading to persistent arrhythmia. Patients with permanent AF, heart failure, and sinus rhythm typically have dystrophic atrial myocytes with extensive sarcomere loss and DNA damage. Changes in extracellular matrix remodeling, inflammation, ion channels, myofibril structure, and mitochondrial function occur during the early transitional stages of AF but not later. Given the limited effectiveness of antiarrhythmic drugs for AF, new treatment options for pAF patients and inhibiting the transition from paroxysmal AF to permanent AF include isoform-specific NOX inhibitors, mitochondrial ROS scavengers, and drugs that target mitochondrial dynamics and metabolism.


The loss of contact inhibition is a key step during carcinogenesis. The Hippo–Yes-associated protein (Hippo/YAP) pathway is an important regulator of cell growth in a cell density–dependent manner. However, how Hippo signaling senses cell density in this context remains elusive. Here, we report that high cell density induced the phosphorylation of spectrin α chain, nonerythrocytic 1 (SPTAN1), a plasma membrane–stabilizing protein, to recruit NUMB endocytic adaptor protein isoforms 1 and 2 (NUMB1/2), which further sequestered microtubule affinity–regulating kinases (MARKs) in the plasma membrane and rendered them inaccessible for phosphorylation and inhibition of the Hippo kinases sterile 20–like kinases MST1 and MST2 (MST1/2). WW45 interaction with MST1/2 was thereby enhanced, resulting in the activation of Hippo signaling to block YAP activity for cell contact inhibition. Importantly, low cell density led to SPTAN1 dephosphorylation and NUMB cytoplasmic location, along with MST1/2 inhibition and, consequently, YAP activation. Moreover, double KO of NUMB and WW45 in the liver led to appreciable organ enlargement and rapid tumorigenesis. Interestingly, NUMB isoforms 3 and 4, which have a truncated phosphotyrosine-binding (PTB) domain and are thus unable to interact with phosphorylated SPTAN1 and activate MST1/2, were selectively upregulated in liver cancer, which correlated with YAP activation. We have thus revealed a SPTAN1/NUMB1/2 axis that acts as a cell density sensor to restrain cell growth and oncogenesis by coupling external cell-cell contact signals to intracellular Hippo signaling. Cells within multicellular organisms are able to sense cell-cell contact and their density to control the proper tissue morphogenesis and organ size , 2. WhenDrosophila Hippo, their downstream large tumor suppressor kinases LATS1 and LATS2 (LATS1/2), and regulator Salvador  and Mps one binder 1 (MOB1). MST1/2 phosphorylate and activate the MOB1-LATS1/2 complex, which then phosphorylates downstream effectors YAP and its paralog TAZ, followed by their degradation or sequestration in the cytoplasm by 14-3-3 proteins. At low cell density (LCD) with limited cell-cell contact, Hippo signaling activation is low, and YAP and TAZ are predominantly located in the nucleus, where they interact with TEAD transcription factors to regulate proproliferative genes, thereby enabling cell proliferation. In contrast, at high cell density (HCD) with extensive cell-cell contacts, the Hippo pathway is activated and YAP/TAZ retained cytosolically and degraded, leading to cell growth arrest. Several molecules including spectrins have been shown to be recruited to adherens or tight junctions and serve as regulators of the Hippo pathway  to regulate YAP through the Hippo kinases MST1/2 in response to cell density cues. Increased cell density gradually enhanced SPTAN1 phosphorylation at tyrosine 1176 (Tyr1176) to recruit NUMB1/2 through their PTB domain, and NUMB1/2 further sequestered the MST1/2 inhibitor microtubule affinity–regulating kinase (MARK) at the cell membrane. At LCD, SPTAN1 was dephosphorylated and NUMB1/2 were released from SPTAN1 into the cytoplasm, resulting in competition between MARK and WW45, a positive regulator of MST1/2, to phosphorylate and inhibit MST1/2. Importantly, LCD or double-KO of WW45 and NUMB resulted in strong inhibition of MST1/2 by MARK, which, in the liver, led to appreciable organ enlargement and rapid tumorigenesis. Moreover, NUMB3/4, with their truncated PTB domains that render them unable to interact with SPTAN1 and activate MST1/2, were preferentially upregulated in liver cancer, along with a reduction of phosphorylated SPTAN1 (p-SPTAN1), which correlated with YAP activation. Thus, selective upregulation of NUMB3/4 expression along with a reduction of p-SPTAN1 might be an important mechanism of suppression of MST1/2 activity, leading to loss of cell-cell contact inhibition of cell growth during the initiation and progression of cancer. Our findings demonstrate a critical role of the SPTAN1/NUMB axis in cell density sensing, shedding light on the essential role of NUMB isoform splicing in the loss of cell contact inhibition and the initiation of cancer.https://doi.org/10.1172/JCI168888DS1). Higher phosphorylation levels of MOB1 (p-MOB1), LATS1 (p-LATS1), and YAP (p-YAP), and lower levels of nuclear YAP in HepG2 cells grown at HCD were observed as compared with cells at LCD, suggesting that the Hippo/YAP signaling cascade was regulated by cell density in HepG2 cells  and NUMB2 (p66), and 2 NUMB-PTBS isoforms with short PTB domains, i.e., NUMB3 (p71) and NUMB4 (p65). We observed that NUMB1/2 were mainly located at the cell plasma membrane at HCD, whereas NUMB3/4 were constitutively located in the cytoplasm , but not NUMB-PTBs isoforms , dramatically enhanced Hippo/YAP signaling, promoted YAP nuclear exit, and inhibited cell proliferation in NUMB-deficient cells , but not the truncated PTB splicing short isoforms NUMB-PTBS , were able to associate with the middle region of SPTAN1, and this interaction was abolished when the PTB domain of NUMB1/2 was deleted  assays showed that NUMB1 did not directly bind to MST1 or MST2 . Mass sp control –45. Quan control , and co- control . Similar control . In addi control , suggest control . In addi control . It has  control . We foun control . In cont control , while k control , indicat control .fl/flNumb mice with adeno-associated virus serotype 8 (AAV8) expressing Cre recombinase under the TBG promoter (AAV-TBG-Cre) or with control viruses to generate hepatocyte-specific NUMB KO (NumbΔHep) or WT control (NumbCtr) mice, respectively. Surprisingly, we found that KO of NUMB in hepatocytes did not alter the liver size or the liver/BW ratio, nor did it alter the percentages of Ki67+ cells and CK19+ biliary/progenitor cells in periportal areas of the liver  or NUMB-deficient (NumbΔHeptdTomato) hepatocytes, respectively, and then treated these mice with the DDC diet. The results showed that NumbΔHeptdTomato livers had substantially more tdTomato-expressing CK19+ biliary/progenitor cells than did NumbWTtdTomato livers, indicating that a greater number of NUMB-deficient hepatocytes transdifferentiated into biliary/progenitor cells , WW45-KO (Ww45ΔHep), and control Ww45NumbCtr mice, respectively. Compared with hepatocytes from Ww45 NumbCtr and NumbΔHep mice, we observed enhanced interaction of MARK2 and MST1 -l-alanyl]-s-phenylglycine t-butyl ester (DAPT), an inhibitor of the γ-secretase complex that is essential for the generation of NICD  were located on the plasma membrane of cells grown at HCD, while the NUMB-PTBS isoforms  were persistently distributed in the cytoplasm  in liver might be an important mechanism contributing to the inactivation of Hippo signaling and lead to loss of contact inhibition and liver tumorigenesis , we examined 60 pairs of liver-derived tumorous and adjacent nontumorous tissues . The relytoplasm , we sougn of HCC . Since Mn of HCC , we obsen of HCC , MARK2 wn of HCC . These p tissues , B and Cll lines . Indeed,P at HCD , B and CP at HCD , B and CP at HCD , D–G. InP at HCD , H–J. SiP at HCD , K–N. Taigenesis .Cell density–dependent growth inhibition is crucial to restrain organ growth and tumorigenesis. The Hippo pathway senses cell density information to control tissue growth by regulating localization of the transcriptional regulator YAP/TAZ , 73–75. Drosophila, spectrins were proposed to regulate Hippo/YAP signaling by binding to Crb, Expanded, Merlin, and Kibra or by modulating cortical actomyosin activity in response to cell density, cell shape, or cortical tension  mainly through Hpo   . MST1/2  MST1/2) , 96. In and YSK1 , 98, so For a detailed description of all methods, see the fl/flYap and fl/flWw45 conditional-KO mice were previously described (tm14 (CAG-tdTomato)Hze/J (stock no. 007914), WT C57BL/6, and Tg (Alb-Cre) 21Mgn/J (stock no. 003574) mice were originally purchased from The Jackson Laboratory. Rbpi-floxed mice  were purchased from the Shanghai Model Organisms Center. fl/flNumb mice were generated by crossing tm1Zili Numbltm1Zili/NumbJ mice  with WT C57BL/6 mice. Athymic mice (BALB/c) were purchased from Charles River Laboratories.The escribed . B6.Cg-G11 genome copies of AAV. Specially, further treatments were administered at least 2 weeks after AAV-TBG-Cre injection. To induce chronic liver injury, mice were fed a diet supplemented with 0.1% DDC  for 4 weeks or another indicated duration. For DAPT treatment, mice were administered DAPT  or an equivalent volume of vehicle by intraperitoneal injection every other day for 6 or 12 weeks. To induce liver cancer, 2-week-old male C57BL/6 mice were treated with DEN  by intraperitoneal injection and were sacrificed at 8–9 months of age for liver analysis.For AAV infection, 4-week-old male or female mice received a tail vein injection of 1 × 10t test was used for comparisons between 2 groups. Survival data were analyzed using the Kaplan-Meier statistical method. The exact number of biological or experimental replicate numbers can be found in the figure legends, and the exact P values are indicated in the graphed data. A P value of less than 0.05 was considered statistically significant. Most experiments were carried out at least 3 times, and the findings of all key experiments were reliably reproduced.All statistical analyses were performed using GraphPad Prism 8 (GraphPad Software). The data are presented as the mean ± SD as indicated, and a 2-tailed, unpaired Student’s All mice were maintained under specific pathogen–free (SPF) conditions at the Xiamen University Laboratory Animal Center. All animal experiments were approved by the IACUC of Xiamen University  and were in strict accordance with good animal practices as defined by the Xiamen University Laboratory Animal Center. The use of human HCC samples and the relevant database were approved by the research ethics committee of Xiang’an hospital of Xiamen University and Zhongshan Hospital of Xiamen University and complied with all relevant ethics regulations. All tissue samples were collected in compliance with the informed consent policy.All data generated or analyzed during this study are included in this manuscript . Values for all data points found in graphs can be found in the Supplemental DZ and LC conceived the project with the input from WH, BZ, DS, YL and WZ. DS, YL, WZ, HG, YC, YH, Junhong LiL, YY, Z Lai, Z Li, HH, Jiaxin Li, Jinhuan Li, MC, CN, NW, ZZ, YX, YZ, HL, JT, QC, and LH performed experimental biological research. PL, WL, and ZP provided human HCC samples. RLJ provided mutant mice. DZ, LC, and DS co-wrote the manuscript, and all authors reviewed and revised the manuscript. The authorship order was assigned on the basis of relative contributions to the study, with the persons most involved in the research listed first and the most experienced contributors last. The authorship order of the co–first authors is based on the relative contributions of their input to the final version of the manuscript.


Nanoceria dissolution occurs in acidic environments. Nanoceria stabilization is a known problem even during its synthesis; in fact, a carboxylic acid, namely citric acid, is used in many synthesis protocols. Citric acid adsorbs onto nanoceria surfaces, limiting particle formation and creating stable dispersions with extended shelf life. To better understand factors influencing the fate of nanoceria, its dissolution and stabilization have been previously studied in vitro using acidic aqueous environments. Nanoceria agglomerated in the presence of some carboxylic acids over 30 weeks, and degraded in others, at pH 4.5 . Plants release carboxylic acids, and cerium carboxylates are found in underground and aerial plant parts. To further test nanoceria stability, suspensions were exposed to light and dark conditions, simulating plant environments and biological systems. Light induced nanoceria agglomeration in the presence of some carboxylic acids. Nanoceria agglomeration did not occur in the dark in the presence of most carboxylic acids. Light initiates free radicals generated by ceria nanoparticles. Nanoceria completely dissolved in the presence of citric, malic, and isocitric acid when exposed to light, attributed to nanoceria dissolution, release of Ce3+ ions, and formation of cerium coordination complexes on the ceria nanoparticle surface that inhibit agglomeration. Key functional groups of carboxylic acids that prevented nanoceria agglomeration were identified. A long carbon chain backbone containing a carboxylic acid group geminal to a hydroxy group in addition to a second carboxylic acid group may optimally complex with nanoceria. The results provide mechanistic insight into the role of carboxylic acids in nanoceria dissolution and its fate in soils, plants, and biological systems.Cerium atoms on the surfaces of nanoceria  can store or release oxygen, cycling between Ce Acetic, citric, lactic, succinic, and tartaric acid secreted from plant roots are known to complex with metals/metal oxides within the rhizosphere –13. CollNanoceria has been shown to accumulate and persist in rats and mice for 90 days and five months . A signi2 nanoparticles [Nanoceria dissolution or agglomeration is pH-dependent and influenced by carboxylic acids. Nanoceria dissolution in aqueous solution was shown to be pH-dependent with dissolution occurring at pH values less than 7 . Nanocerarticles . The shoarticles . Partialarticles –40. In aarticles . The molIn this study, nanoceria stability and dissolution in the presence of carboxylic acids was tested for up to 30 weeks. Sixteen carboxylic acids  [In the presence of light, the majority of the nanoceria dispersions changed color from light yellow to colorless (attributed to Ceto Ce3+) . Nanocer4+ to Ce3+. The citric acid sample exposed to light appears to be completely colorless after four weeks. No color change was present for the sample kept in the dark. A slight color change was noticeable for the control sample in water; however, a yellow tint was still present after four weeks, for both samples, exposed to dark and light.Nanoceria exposed to citric acid or DI water under light and dark conditions up to four weeks are shown in 4+ to Ce3+ during dissolution. The particles in citric acid exposed to light were completely dissolved after four weeks as indicated by DLS and transmission electron microscopy (TEM), shown in 3+ concentration increased as the particle size decreased, but only slightly. Figure 7 of Yokel et al. [3+ valence state, is apparent.The color change from yellow to colorless could also be representative of particle dissolution, due to the valence state change from Cel et al.  shows liDLS results of the 20 chemicals studied under the two conditions are shown in The results shown in Group one includes nanoceria exposed to citric, malic, and isocitric acid. For all three samples stored in the dark, there was an immediate reduction in hydrodynamic particle size, followed by slow dissolution over time. The solution color remained yellow for the experimental duration. For the samples exposed to light, there was an immediate reduction in hydrodynamic particle size accompanied by a color change from yellow to clear. The nanoceria particles decreased to 8.4, 7.3, and 10.9 nm in diameter within 250 h for citric, malic, and isocitric acid, respectively . Instead, approximately 5 nm features were observed at weeks 2, 4, and 8. These features were not stable and were altered by the high-intensity electron beam, confirming that they are not cerium oxides  do not contain a second carboxylic acid group. Furthermore, citric, malic, and isocitric acid molecules all have higher ratios of carboxylic acid and hydroxy groups to aliphatic carbon atoms  completely dissolved nanoceria when exposed to light and one (glyceric acid) prevented agglomeration throughout the entire experimental duration. Two acids (lactic and tartaric acid) stabilized nanoceria for an extended time before agglomeration. One acid (α-hydroxybutyric acid) prevented agglomeration for a short time, while the remaining two  led to immediate agglomeration upon exposure. This result indicates that while the carboxylic acid group geminal to a hydroxy group may be important, other functional groups may also play a role in stabilizing citrate-coated nanoceria. For instance, citric, malic, and isocitric acid all contain at least one more carboxylic acid group . The same is true for tartaric, tartronic, and dihydroxymalonic acid; however, the carbon chain is shorter for these molecules and does not contain any CH4+ to Ce3+. As nanoceria particles dissolve and become smaller, the surface oxidation state transitions from Ce4+ to Ce3+. Of the 16 carboxylic acids tested, nanoceria exposed to only three acids  fully dissolved in less than 1000 h when exposed to light. Glyceric acid prevented agglomeration, but nanoparticles were still present after 30 weeks of exposure. Nanoceria formed micrometer-sized particles in the remainder of the samples either immediately or between 300 and 2000 h of exposure. In the dark, particles were still present at the end of 30 weeks for all carboxylic acids and the controls. This indicates that light accelerates nanoceria dissolution and stabilizes some nanoceria–carboxylic acid complexes. Lactic acid dissolved the particles at a greater rate than any other acid in the dark. TEM imaging of nanoceria in citric and β-hydroxybutyric acid supported the DLS results. Relating the carboxylic acid composition  to the nanoceria dissolution rate provides insight into the molecular components of carboxylic acids that affect nanoceria dissolution or stable complexation. A long carbon chain backbone containing a carboxylic acid group geminal to a hydroxy group in addition to a second carboxylic acid group may optimally form complexes with nanoceria. The results provide mechanistic insight into the role of carboxylic acids in nanoceria dissolution and the fate of nanoceria in soils and plants. They suggest that lactic and other carboxylic acids accelerate nanoceria dissolution in the soil, releasing Ce3+, some of which is taken up by plant roots, is complexed to carboxylic acids, and persists as cerium carboxylates in aerial plant structures. Further determination of the cerium species in aerial plant structures is needed to determine if the cerium carboxylates are soluble/stable complexes, as predicted for cerium citrate, or precipitates, as suggested by the cerium carboxylate complexes in groups three to five above.Citric acid-coated nanoceria was exposed to 16 carboxylic acids, ascorbic acid, sodium nitrate, ammonium nitrate, and water at pH 4.5 in light and dark environments to simulate sunlight exposure to plants and within biological systems. The hydrodynamic particle size was determined by DLS over 30 weeks. For most light exposure conditions, a color change from yellow to colorless was observed, indicating a valence state transition from CeThe following chemicals, including their sources, purity, and CAS numbers, were used: citric acid monohydrate, Fisher, ACS grade, 5949-29-1; ᴅʟ-malic acid, Alfa Aesar, 98%, 6915-15-7; ᴅʟ-isocitric acid, trisodium salt hydrate, Acros Organics, 95%, 1637-73-6; ᴅʟ-lactic acid, TCI, >85%, 50-21-5; ᴅʟ-glyceric acid, TCI, 20% in water (ca. 2 mol/L), 473-81-4; ᴅʟ-tartaric acid, TCI, >99%, 133-37-9; glutaric acid, Acros Organics, 99% 110-94-1; tricarballylic acid, Alfa Aesar, 98%, 99-14-9; adipic acid, Sigma, 99%, 124-04-9; acetic acid, Sigma, ACS grade, 64-19-7; pimelic acid, Alfa Aesar; >98%, 111-16-0; succinic acid, TCI, >99%, 110-15-6; tartronic acid, Sigma, >97%, 80-69-3; sodium mesoxalate monohydrate, Chemodex, >98%, 31635-99-1; ᴅʟ-2-hydroxybutyric acid sodium salt, Alfa Aesar, >97%, 5094-24-6; ᴅʟ-3-hydroxybutyric acid sodium salt, Chem Impex Int’l Inc., 100.3%, 150-83-4/306-31-0; ascorbic acid, TCI, >99%, 50-81-7; ammonium nitrate, Fisher, ACS grade, 6484-52-2; sodium nitrate, VWR, ACS grade, 7631-99-4; sodium hydroxide, VWR, ACS grade, 1310-73-2; nitric acid, Sigma, ACS grade, 7697-37-2; and sodium azide, Sigma, 99.8%, 26628-22-8. Lacey carbon, 300 mesh, copper grids (product #01895) from Ted Pella, Inc. were used for electron microscopy.2. The product was dialyzed for 120 h at ten times the volume, changing the dialysate every 24 h, against 0.11 M iso-osmotic citric acid adjusted to pH 7.4. The nanoceria sol was then stored in the dark at 4 °C.Hexagonal nanoceria particles (4.2 ± 1.2 nm) (mean ± SD) were synthesized using a hydrothermal method  and extePrior to transmission electron microscopy (TEM) imaging, samples were sonicated for 10 min in a sonication bath. Lacey carbon, 300 mesh, copper grids were dipped into the solution for approximately 5 s and dried overnight at room temperature. TEM was used to obtain images of particles throughout the experiment’s duration. The Thermo Scientific Talos F200X instrument equipped with a SuperX G2 EDS detector was operated at an accelerating voltage of 200 keV. The TEM images were recorded on a Ceta CCD camera. DLS was performed using a Brookhaven 90Plus particle size analyzer. Three analysis runs of 5 min each were completed for each sample, and the average result of each run was analyzed and recorded. All samples were evaluated using the multimodal setting.2. Sodium hydroxide and nitric acid were used to adjust solution pH and sodium nitrate to adjust the osmolarity. In addition to the 16 carboxylic acids, ascorbic acid, studied by Muhammad et al. [3. Ammonium nitrate (20 mM) was also studied since it was used in the initial nanoceria synthesis. Further, DI water was used as a control at pH 6.5. As a bacteriostat, 0.02% sodium azide was added to each sample. Each of the 20 chemicals was studied under two conditions; one sample was placed next to the window for exposure to ambient laboratory light, including natural UV irradiation from the sun, and the other sample was kept in the dark, achieved by aluminum foil covering. Each condition was studied in at least two replicates.Nanoceria, 1 mg, was dispersed in 2 mL (500 µg/mL) of 0.11 M iso-osmotic aqueous media at pH 4.5 representative of acidic soil and lysosomes –58. Throd et al. , was incd et al. . This coDLS was employed to repeatedly determine the hydrodynamic particle size over 30 weeks (approximately 5040 h). The study of some samples was stopped early because of full dissolution. TEM and particle size/shape analyses were also carried out on citric acid and β-hydroxybutyric acid samples under light and dark conditions after 0, 1, 2, 4, and 8 weeks. High-angle annular dark-field scanning TEM and EDS were used to determine the elemental composition of some particles seen in TEM. In addition, camera photos were taken after 0, 1, 2, and 4 weeks of the samples exposed to citric acid and water under light and dark conditions.To identify the key functional groups that influence nanoceria agglomeration, the number of carboxylic acid groups and total number of carbons, aliphatic carbons, and hydroxy groups in each carboxylic acid were counted. The molecular ratios of the number of carboxylic acid groups to the total number of carbons and aliphatic carbons, and of the number of hydroxy groups to the total number of carbons and aliphatic carbons were calculated. These values and the position of the carboxylic acid groups in relation to the hydroxy groups were reviewed to suggest the molecular components of carboxylic acids required for stable complexation with nanoceria.Molecular structures of the carboxylic acids, electron microscopy images of nanoceria particles, quantitative DLS results supporting File 1Additional experimental data.


The cell division cycle 20 homologue (CDC20) is known to regulate the cell cycle. Many studies have suggested that dysregulation of CDC20 is associated with various pathological processes in malignant solid tumors, including tumorigenesis, progression, chemoradiotherapy resistance, and poor prognosis, providing a biomarker for cancer diagnosis and prognosis. Some researchers have demonstrated that CDC20 also regulates apoptosis, immune microenvironment, and tumor angiogenesis. In this review, we have systematically summarized the biological functions of CDC20 in solid cancers. Furthermore, we briefly synthesized multiple medicines that inhibited CDC20. We anticipate that CDC20 will be a promising and effective biomarker and therapeutic target for the treatment of human cancer. The entire cellular process is regulated by many factors, and ubiquitination plays a pivotal role in controlling diverse biological processes. The anaphase-promoting complex/cyclosome (APC/C), a multi-subunit E3 ubiquitin ligase enzyme, has been considered the major driving force governing these cellular processes. APC/C regulates cell cycle progression in both the M and G1 phases and promotes chromosome segregation. APC/C has a complex structure that consists of over 14 different subunits, together with one of the 2 co-activators, CDH1 and cell division cycle 20 homologue (CDC20). CDC20 includes 2 main segments: the C-terminal region containing the WD40 repeats and IR motif and the N-terminal region containing C-box, CRY-box, and KEN-box motifs. Remarkably, both CRY-box and KEN-box are important for CDC20 binding to APC/C as a substrate of CDH1. The CRY-box can lead to timely ubiquitination and destruction of CDC20. Two CDC20 regulators MAD2 and MAD3/ BUBR1 interact with CDC20 through different KEN motifs. Additional regulatory sites are located in the amino- and carboxy-terminal extensions and include the IR tail, C-box, and MAD2-intercating motif, whose function is to bind CDC20 to APC/C , replication of its DNA (synthesis: S), preparation to divide (Gap2: G2), and the mitosis phase (M).: G1, rep and CDC20 knockdown enhanced adipogenesis of bone marrow-derived stem cells by modulating β-catenin and might lead to a new therapeutic target for “fatty bone” and osteoporosis. High CDC20 expression in cardiomyocytes aggravates the hypertrophic response by targeting LC3 directly, and which could be a new therapeutic target for patients with hypertrophic heart disease. CDC20 is also spatially expressed in human menstrual cycles and is regulated by estrogen and progesterone, an experiment confirmed that the CDC20 inhibitor, Apcin, can reduce the proliferation and adhesion of human endometrial cells and inhibit embryo implantation in mice. In addition, many studies have demonstrated that mutations in human CDC20 could result in female infertility and male azoospermia.–12 Furthermore, CDC20 has been shown to play a crucial role in tumorigenesis, progression, and treatment of human malignant solid tumors , gastric carcinoma (GC), colorectal cancer (CRC), renal clear cell carcinoma, bladder urothelial carcinoma, prostate cancer, epithelial ovarian cancer, and cervical squamous carcinoma relative to matched non-cancerous tissues. Strikingly, CDC20 expression was also significantly higher in intestinal metaplasia, low-grade dysplasia, high-grade dysplasia, and early GC than in normal mucosa, and their expression levels were the highest in high-grade dysplasia, suggesting their roles in the early stage of gastric carcinogenesis. Furthermore, the expression of CDC20 and STK15, identified as CDC20-associated proteins, was enhanced by more than 2-fold in 58% of pancreatic cancer tissues.,24 Zhang et al found that CDC20 was overexpressed in smoking-related lung adenocarcinoma, indicating that the levels of CDC20 may correlate with smoking status. A study further demonstrated that hexavalent chromium (Cr [VI]) leads to cancer by altering CDC20 kinetochore localization and reducing the interaction between phosphorylated CDC20 and Mad2. Notably, CDC20 was upregulated in glioblastoma and decreased in low-grade glioma, indicating that additional studies are required to delineate the exact function of CDC20 in gliomas. Recent studies also revealed that speckle-type POZ protein interacts with CDC20 and promotes poly-ubiquitination and subsequent degradation of CDC20 in a degron-dependent manner (tumorigenesis). Therefore, CDC20 acts as an oncogene during tumorigenesis. In addition, multiple studies have indicated that CDC20 is overexpressed significantly along with poor prognosis in astrocytoma, NSCLC,,30 HCC, and prostate cancer. Aberrant expression of the GTF2E2/CDC20 signaling pathway is associated with glioblastoma pathogenesis and poor prognosis. Moreover, the expression of CDC20 was associated with the clinicopathological parameters of the tumors mentioned above. High expression of CDC20 was positively correlated with sex, poor tumor differentiation, large tumor size, lymph node invasion, distant metastasis, advanced TNM stage, and cytokine levels in breast cancer, HCC,,33 GC,,34 CRC, epithelial ovarian cancer. In addition, CDC20 overexpression was associated with advanced age, high grade, non-papillary growth pattern in bladder cancer patients, and Gleason score, vesicle invasion, and biochemical recurrence in prostate cancer, indicating that CDC20 expression is an independent prognostic factor.Many studies have demonstrated that CDC20 is abnormally expressed in various solid cancers, and aberrant expression of CDC20 is associated with tumorigenesis and poor prognosis. For example, CDC20 is upregulated in various types of tumors, including non-small cell lung cancer (NSCLC),These characteristics are consistent with CDC20 as an oncogene. These studies indicate that CDC20 can be used as an independent prognostic factor for human solid cancers Table . Therefo CDC20 maintains the proliferation and survival of glioma cells through degradation of p21CIPI/WAF1, and CDC20 inhibition induces cell cycle arrest in glioma cells. Furthermore, neurofibromatosis type 2 might restore spindle assembly checkpoint function by impairing the binding of APC/C and CDC20, thereby inhibiting meningioma proliferation. The CDC20-APC/SOX2 signaling axis controls the key biological properties of glioma cells, and targeting this pathway in glioblastoma may dispute the glioblastoma stem-like cell line state. In addition, hyperphosphorylation of CDH1 is a major mechanism that drives attenuated APC/CDC20 activity in the G1 phase of the cell cycle and reduces the viability of glioma cells. Furthermore, CDC20/APC/C/securing-dependent signaling is a key regulator of cell survival, and its disruption promotes premature senescence in normal lung cells. P53 suppressed cancer cell growth by indirectly regulating CDC20 expression, arrests cells at the G2/M phase, and reduces the colony-forming activity of lung cancer cells. Remarkably, CDC20 also promotes cell growth and migration in breast cancer cells, including triple-negative breast cancer cells. CDC20 regulates the growth of breast cancer cells via several pathways. REC8-CDC20 axis: REC8 decreases proliferation, migration, and invasion of breast cancer cells by inhibiting CDC20. AhR-CDC20 axis: AhR activation by 3-methylcholanthrene suppresses mammosphere formation by downregulating CDC20 expression in breast cancer cells. CDC20-SMAR1 axis: The tumor suppressor SMAR1 plays a crucial role in maintaining genomic stability and cell cycle progression, whereas CDC20 promotes proteasomal degradation of SMAR1. In HCC, CDC20 overexpression could also promote cell signal transduction and proliferation by altering the subcellular localization and distribution of phosphatidylcholine-specific phosphatase C (PC-PLC), and causing PC-PLC degradation through the ubiquitin-proteasome pathway. In addition, CDC20-mediated degradation of PHD3 stabilized HIF-1a and promoted HCC tumorigenesis. The depletion of endogenous PHD3 in CDC20 knockdown HCC cells greatly attenuated the decline in HIF-1a protein and restored the secretion of VEGF. The proliferation of p53 mutant HCC cells can also be regulated by CDC20 via the Bcl/Bax pathway. Additionally, Wnt/β-catenin signaling regulates cell proliferation by modulating the cell cycle and is negatively regulated by conductin/ axin2/axil, while conductin is degraded by CDC20, indicating that knockdown of CDC20 blocked Wnt/β-catenin signaling through conductin and attenuated colony formation of CRC cells. In GC, the synergy between CDC20 and MYBL2 induces the proliferation of GC cells, and this effect may have involved the Wnt/β-catenin signaling pathway. Furthermore, CBX4 mainly promotes CDC20 mRNA levels and notably enhances cell proliferation capacity, migration ability, and in vivo metastatic efficacy. In renal cancer, CDC20 promotes proliferation by increasing the protein levels of securin, cyclin B1, and cyclin A. In addition, CYP1B1 may promote renal cell proliferation, migration, and invasion, and inhibit apoptosis by inducing CDC20 expression. miR-182-5 overexpression inhibits cell proliferation by directly targeting MALAT-1, then the downregulation of MALAT-1 leads to a decrease in CDC20. In prostate cancer, L-type amino acid transport protein is expressed at all stages of prostate cancer. Inhibition of L-type amino acid transport suppresses tumor growth and cell cycle progression by downregulating M-phased cell cycle genes, including CDC20 and mTORC1, in prostate cancer. Another study found that COX2 inhibitors suppressed prostate cancer cell proliferation and arrested cell cycle progression by inhibiting CDC20. Moreover, NUSAP1 accelerated osteosarcoma cell proliferation and cell cycle progression by upregulating CDC20, whereas CDC20 silencing inhibited NUSAP1- induced cell proliferation and cell cycle in osteosarcoma cells. CDC20 also promotes the growth of other cancer cells, including pancreatic,,68 cervical,,70 and ovarian cancers.Multiple studies have demonstrated that CDC20 plays an important role in promoting and maintaining tumor cell proliferation. Dai et al CDC20 plays a critical role in FoxM1-related cell survival in glioma cells.In general, suppression of CDC20 inhibited cell proliferation by inducing G2/M phase arrest, anti-angiogenesis, and activating various signaling pathways Table . Therefo Hu et al also found that miR-1321 and miR-7515 impede NSCLC cell apoptosis by targeting CDC20. Furthermore, CDC20/APC/C/securing-dependent signaling is a key regulator of cell survival, and its disruption promotes premature senescence in normal lung cells and induces apoptosis in lung cancer. The tumor suppressor SMAR1 plays a crucial role in maintaining genomic stability and apoptosis, whereas CDC20 promotes proteasomal degradation of SMAR1 through K48-linked specific polyubiquitylation, and short hairpin RNA-mediated inactivation of CDC20 leads to significant stabilization of SMAR1. Furthermore, silencing CDC20 expression can activate cell apoptosis by altering the subcellular localization and distribution of PC-PLC and inhibiting PC-PLC degradation, PC-PLC is involved in the cell apoptosis.,71 CDC20 also coordinates with MYBL2 to inhibit cell apoptosis through the Wnt/β-catenin signaling pathway. Notably, CYP1B1 may inhibit apoptosis by inducing CDC20 expression. In addition, miR-182-5 overexpression inhibited cell apoptosis by directly targeting MALAT-1, leading to downregulation of CDC20. Other studies have further confirmed that CDC20 could induce apoptosis in prostate cancer and osteosarcoma.Several studies have reported that CDC20 is associated with apoptosis in various solid cancers. A previous study demonstrated that CDC20 inhibition could induce apoptosis in glioma.Based on the above, the expression level of CDC20 is associated with apoptosis of cancer cells, indicating that inhibiting apoptosis is one of the main mechanisms by which CDC20 promotes cancer cell growth Table . CDC20 r CDC20 is negatively regulated by miR-1321 and miR-7515 to suppress NSCLC cell migration and invasion. CDC20 also promotes the migration of triple-negative breast cancer cells, and CDC20 inhibitors would block cancer cell growth and migration. The function of CDC20 in breast cancer is regulated by REC8 and the A20/TNFAIP3-CDC20-ASP1 axis. REC8 decreases proliferation, migration, and invasion of breast cancer cells by inhibiting CDC20. The A20/TNFAIP3-CDC20-CASP1 axis, which includes inflammation-related genes found in TNBC, is associated with poor patient prognosis, cancer metastasis, and cytokines levels. Besides, CBX4 promoted CDC20 mRNA levels and notably enhanced cell migration ability and metastatic efficacy as a positive regulatory factor. The lncRNA SpRY-IT4 also promotes cell invasion by regulating CDC20 in pancreatic cancer cells. Furthermore, CYP1B1 may promote renal cell migration and invasion by inducing CDC20 expression. Suppression of CDC20 slao inhibited migration and resulted in G2/M phase arrest in wilms tumor cells by increasing the expression levels of securin, cyclin B1, and cyclin A. Moreover, several studies have shown that CDC20 knockdown can inhibit the invasion and metastasis of prostate cancer, osteosarcoma, cervical cancer, and ovarian cancer .Based on the above, CDC20 can directly promote the invasion and metastasis of solid cancers through various signaling pathways, including the REC8-CDC20, A20/TNFAIP3-CDC20-ASP1, CBX4-CDC20, and VHL/HIF-1α/STAT3 axes. Notably, CDC20 also promotes angiogenesis in tumor tissues by regulating EMT and VEGF expression, which is an important pathway promoting invasion and metastasis. Besides, CDC20 expression was significantly correlated with immune infiltration in HCC. There was a loss of association between CDC20 and Th1/Th17 cytokines, monocytes, CD4pos, and CD8pos T cells, as well as exhausted T cells. The positive correlation between the CDC20 gene and inhibitory checkpoint genes PD-1, CTLA4, and TIM-3 indicates that the CDC20 gene is linked with weakened antitumor immunity in HCC. Furthermore, CDC20 expression is positively correlated with the degree of infiltration of B cells, neutrophils, macrophages, and myeloid dendritic cells. In addition, CDC20 has been linked to the tumor mutation burden, immune checkpoint molecules, tumor microenvironment, and immunological infiltration. Therefore, CDC20 plays a critical role in the antitumor immunity system, such as the expression of cytokines, activation of immune cells, expression of immune checkpoint molecules, microenvironment , Withaferin A, and 1-L-MT , have been developed to kill cancer cells by delaying mitotic exit followed by inducing chromosome instability.,86,93 Bhuniya et al designed new apcin-based inhibitors by replacing the pyrimidine group with substituted thiazole-containing groups, which can improve antitumor activity. M2I-1 (MAD2 inhibitor-1) has been shown to disrupt the CDC20-MAD2 interaction, and consequently, the assembly of the mitotic checkpoint complex and increase the sensitivity of cancer cells to antimitotic drugs via MCL-1s. Moreover, a triterpene mixture extracted from the mushroom Poria cocos, polyporenic acid C, can downregulate the expression of CDC20 in some cancer cells and inhibit cancer metastasis. Zhang et al have identified a compound as “331” which upregulate miR-494 and downregulated CDC20 to induce cell death in glioma cells but not in astrocytes.Given the important oncogenic role of CDC20 in tumorigenesis, targeting CDC20 may interfere with mitosis, thereby inhibiting cancer proliferation. Therefore, we have summarized several CDC20 inhibitors with therapeutic effectiveness in suppressing tumor progression Table . Some inHowever, given the fluctuations in CDC20 expression during cell cycle progression, the adverse side effects of CDC20 inhibitors must be considered. CDC20 is also a crucial factor in cell cycle regulation in normal cells. Thus, targeting CDC20 may cause defects in the cell cycle of normal tissues. Therefore, it is necessary to fully understand the modulation of CDC20 expression and activity during the cell cycle.In summary, CDC20 acts as an oncogene through various pathways, including the promotion of EMT, suppression of apoptosis, inhibition of immunological infiltration, and signaling pathways. In addition, we briefly summarize the pathway for CDC20-degradation of its major downstream targets as well as the identified CDC20 upstream regulators, and the effects of CDC20 on solid tumors Fig. . Lastly,The authors are grateful to Dr Yinglin Yuan, School of Medicine, University of Electronic Science and Technology of China; for her great help and support during the progress of the work.Conceptualization: Guohui Xu.Data curation: Feng Xian, Caixia Zhao, Guohui Xu.Formal analysis: Feng Xian, Chun Huang, Guohui Xu, Jun Bie.Funding acquisition: Feng Xian, Guohui Xu.Investigation: Feng Xian, Chun Huang.Methodology: Feng Xian, Caixia Zhao, Chun Huang, Jun Bie.Resources: Feng Xian.Software: Feng Xian, Caixia Zhao.Supervision: Feng Xian.Validation: Feng Xian.Visualization: Feng Xian.Writing – original draft: Feng Xian, Guohui Xu.Writing – review & editing: Guohui Xu, Jun Bie.


Lycopene is a natural, red-colored pigment that is found in plants and has particularly robust antioxidant activity among the carotenoids. Plenty of research has highlighted its antioxidant, anti-inflammatory, anticancer, and antidiabetic properties. Emerging evidence has promoted the development of lycopene as a natural antioxidant feed supplement to enhance the health status and performance of pigs and poultry. This review aimed to summarize the latest research advances on lycopene in swine and poultry, to deepen our understanding of its biological functions and practical applications in livestock production.Oxidative stress and in-feed antibiotics restrictions have accelerated the development of natural, green, safe feed additives for swine and poultry diets. Lycopene has the greatest antioxidant potential among the carotenoids, due to its specific chemical structure. In the past decade, increasing attention has been paid to lycopene as a functional additive for swine and poultry feed. In this review, we systematically summarized the latest research progress on lycopene in swine and poultry nutrition during the past ten years (2013–2022). We primarily focused on the effects of lycopene on productivity, meat and egg quality, antioxidant function, immune function, lipid metabolism, and intestinal physiological functions. The output of this review highlights the crucial foundation of lycopene as a functional feed supplement for animal nutrition. Oxidative stress severely threatens the productivity and health status of farm animals, which results in huge economic losses for the livestock industry . In anim40H56), a red-colored carotenoid, is a natural pigment found in plants, mainly in fruits and vegetables such as tomatoes, carrots, watermelon, and guava [Lycopene  [Lycopene was first discovered as a red pigment in tomatoes by Millardet in 1876; 27 years later, it was named lycopene by Schunck in reference to the scientific name of tomato (ulentum) . Lycopenulentum) . It has ulentum) . Tomatoeulentum) . Most imulentum) . Microbiulentum) . Additioulentum) .40H56  challenge condition [1 challenge, as indicated by increased ADG  and decreased FCR (day22–42 and day1–42: 200 or 400 mg/kg lycopene) [Most importantly, lycopene has been demonstrated to exert growth-promoting effects on farm animals under stressful conditions, such as heat stress, and due to mycotoxin feed contamination ,55,56,57ondition . Moreoveycopene) . Under hycopene) . It shouCytc, TFAM, TFB1M, CS, COX1, MyHC1, MyHC IIa, MyHC IIx, and TNNI1 were up-regulated, while the mRNA level of MyHC IIb was down-regulated in the LD muscle by lycopene supplementation [MyHC, Cytc, myoglobin, slow-twitch fiber percentage, succinic dehydrogenase (SDH), and malate dehydrogenase (MDH) was increased, while fast MyHC, fast-twitch fiber percentage, and lactate dehydrogenase (LDH) activity were decreased in the LD muscle of finishing pigs fed lycopene diets (100 or 200 mg/kg lycopene) [SOD1, SOD2, CAT, GPX1, GST, GR, and Nrf2, and down-regulated Keap1 mRNA levels [Along with rapid economic development and livestock production technology improvement, the ever-increasing attention of consumers has been paid to meat and egg quality in place of meat and egg quantity ,59. The entation . In addiycopene) . Importaycopene) . As we kycopene) . An et aycopene) . LikewisA levels . SimilarA levels . HoweverA levels .Regarding egg quality, the study by Shevchenko et al. showed that supplementing High Line W36 laying hens’ diets with lycopene (20/40/60 mg/kg) for 90 days resulted in improved egg quality, as indicated by an increased carotenoid level and yolk color in fresh eggs or in eggs undergoing a 4 °C and 12 °C storage period . Orhan e2O2 and ROS levels in the placental tissues of sows [GPX1, GPX4, FABP4, SLPI, ANX1, and APOE in the placenta were also up-regulated by lycopene supplementation at 50 mg/kg [SOD1 and CAT were also found to be linearly affected by dietary lycopene supplementation  [2O2 levels in the serum and jejunum [NRF2, SOD2, CAT, GLUT2, GLUT5, CD36, CLDN1, and IL-22, along with the protein levels of NRF2, CD36, and CLDN1, and down-regulated the mRNA and protein levels of KEAP1 [The antioxidant potential of lycopene is one of its predominant biological characteristics, as has been proven in swine and poultry by several animal nutritionists. For instance, Sun et al. demonstrated that dietary 50 mg/kg lycopene supplementation during gestation increased total antioxidant capability (T-AOC) and GSH-Px activity and decreased the H of sows . Additio50 mg/kg . In fini50 mg/kg . The gen0 mg/kg) . Similar0 mg/kg) . Lycopenof KEAP1 . The autof KEAP1 . In an iof KEAP1 .SOD2, NQO1, and HO-1 [1 contamination commonly occurs in poultry production and seriously threatens the productivity and health of poultry [1 toxicity, and the addition of exogenous antioxidants (such as antioxidant polyphenols and carotenoids) in diets can effectively alleviate the adverse effects of AFB1 in poultry [1-contaminated diets in a 42-day feeding trial [1-contaminated feed elevated the hepatic mitochondrial antioxidant status, including greater mGSH, GSH-Px, MnSOD, and ATP levels, along with lower ROS and H2O2 levels, and reduced mitochondrial swelling [MnSOD, Trx2, TrxR2, Prx3, PGC-1α, NRF1, and TFAM [In a broiler-feeding trial by Wang et al., supplementing lycopene  to 1-day-old broilers for 42 days markedly elevated the serum antioxidant status, as indicated by increased T-AOC, GSH-Px, and SOD activities, and decreased MDA level on day 21 of the study . Moreoveand HO-1 . Feed AF poultry . It has  poultry . In an eng trial . The antng trial . Similarswelling . Additioand TFAM . El-Shesand TFAM . Mezbaniand TFAM . Sahin eand TFAM .21, 20, 40, or 80 mg/kg lycopene; day35, 80 mg/kg lycopene), T-AOC , GSH/GSSG , GSH-Px , and increased hepatic SOD, T-AOC, and GSH/GSSG  [In laying hens, as observed by Orhan et al., supplementation of 20 mg/kg lycopene as a purified powder or tomato powder for 84 days decreased serum MDA and cholesterol levels in Lohman LSL laying hens . Likewisycopene) . Amer etycopene) . Sun et ycopene) . TherefoIL-1β, IL-8, and TNF-α in the placenta were also down-regulated by lycopene supplementation [IL-1β in the jejunum, and up-regulated interleukin-10 (IL-10) mRNA levels in the duodenum and jejunum [Inflammation is reported to be the cost paid by livestock productivity, and it is the basis for allocating nutrient resources between growth and survival in livestock production . Inflammentation . In a re jejunum . Fachine jejunum .1 challenge conditions, as suggested by increased IL-10 levels at day 21 and day 42, and decreased the interferon-γ (IFN-γ) levels in jejunum at day 21 of the study [The immunomodulatory effects of lycopene have also been reported in poultry. In a study conducted by Shevchenko et al., feeding High Line W36 laying hens with lycopene  for 90 days markedly decreased the leukocytes and erythrocytes, but had no effects on the serum antibody titer of hens vaccinated against Newcastle disease, avian rhinotracheitis, egg drop syndrome, and infectious bronchitis . In a 42he study . Furtherhe study . Howeverhe study . TherefoAMPKα1, AMPKα2, Sirt1, PGC-1α, and up-regulated the protein levels of P-AMPK/AMPK, NRF1, CaMKKβ, Sirt1, PGC-1α in LD muscle of finishing pigs [Lycopene has been demonstrated to exert modulatory effects on lipid metabolism in swine and poultry. Most recently, Meng et al. reported that supplementing 50 mg/kg lycopene to weaned piglets for 28 days increased their serum total cholesterol (TC) level . In finiing pigs . Howevering pigs . The plaing pigs .AMPK-α, and down-regulated the mRNA levels of SREBP-1, FAS, and ACC in the livers of broilers, which are indicative of hepatic lipid metabolism [1-contaminated diets [In broilers, a study by Wan et al. suggested that the dietary inclusion of lycopene at 100 mg/kg for 42 days decreased the abdominal fat weight and abdominal fat percentage of broilers . These rtabolism . In an eed diets . A studyed diets . Additioed diets . Similared diets . Howevered diets .7,35, 20, 40, and 80 mg/kg lycopene), increased high-density lipoprotein cholesterol (HDLC) , and T3 level  in the serum of hens [PPARα, RARα, PGCα (40 and 80 mg/kg lycopene), RXRα , and down-regulated mRNA levels of FABP10 , FABP1 , and FATP4 (40 mg/kg lycopene). In the duodenum, the RARα mRNA level was up-regulated by the supplementation of 40 or 80 mg/kg lycopene. In the jejunum, lycopene up-regulated the mRNA levels of PPAPγ, RXRγ (40 and 80 mg/kg lycopene), and RXRα (20 and 40 mg/kg lycopene), and down-regulated the mRNA levels of FATP4 (40 and 80 mg/kg lycopene) [The regulatory functions of lycopene on fat deposition and lipid metabolism have also been reported in laying and breeding hens. Shevchenko et al. found that feeding High Line W36 laying hens with lycopene (20/40/60 mg/kg) for 90 days markedly increased the serum levels of glucose, creatinine, and ALT, and decreased the serum levels of cholesterol, AST, alkaline phosphatase at day 31 of the study; increased serum levels of glucose, cholesterol, and ALT, and decreased serum level of AST were recorded at day 61 of the study, and increased serum levels of glucose, creatinine, and cholesterol were recorded at day 91 of the study . Additio of hens . Tian etycopene) . Sun et ycopene) . TherefoThe intestinal tract is the main site of digestion and the absorption of feed nutrients in farm animals and is also an indispensable line of defense against exogenous pathogenic microorganisms and their toxins ,83,84. T1 challenge [21: 200, 400 mg/kg lycopene; day42: 200 mg/kg lycopene), and jejunum amylase and lipase activities , as well as ileum amylase activity (day42: 200 mg/kg lycopene) [1 challenge conditions, as suggested by reduced serum diamine oxidase concentrations, with up-regulated jejunum ZO-1 and Claudin-1 mRNA levels at day 42 of the experiment [1 challenge conditions, as suggested by decreased jejunum H2O2 (day42) and MDA levels , along with increased GSH, GST, and GR levels, as well as up-regulated mRNA levels of Nrf2, HO-1, Cu/ZnSOD, MnSOD, and CAT (day42) [Similar results have been reported in poultry studies. Sarker et al. reported that dietary supplementation with lycopene  improved the intestinal morphology of broilers undergoing an AFBhallenge . The autycopene) . Accordiperiment . Additioperiment . Similar (day42) . Hence, A summary of lycopene as a functional feed additive for swine, meat-producing, and egg-producing poultry is presented in Lycopene is widely considered an effective antioxidant that can capture oxygen free radicals, regulate the cellular expression of related transcription factors, enhance the activities of antioxidant enzymes, and protect cells from free radical damage. As a natural pigment and potent antioxidant, lycopene can be developed as a functional feed supplement for swine and poultry. Several pieces of research have revealed that lycopene has great potential to improve productivity, meat quality, and egg quality, as well as antioxidant activity, immune function, lipid metabolism, and intestinal physiological functions. In view of the current knowledge, lycopene could be supplemented as lycopene products  at 10–400 mg/kg in swine and poultry diets, while a higher supplemental dosage could adversely affect the productivity and health of animals. However, the research on lycopene to alleviate oxidative stress and promote productivity is still in the initial stages, and the research results have not been widely popularized. Therefore, future research directions should further explore the additive effects of lycopene on swine and poultry at different physiological phases and different rearing environments, and, finally, establish the optimum supplemental dosage for the productivity and health of swine and poultry. More importantly, further research is warranted to elaborate the specific mechanism of action of lycopene, which is of great significance in promoting the research and application of lycopene in animal nutrition, or even in human nutrition.


Medulloblastoma (MB) is the commonest primary malignant brain cancer. The current treatment of MB is usually surgical resection combined with radiotherapy or chemotherapy. Although great progress has been made in the clinical management of MB, tumor metastasis and recurrence are still the main cause of death. Therefore, definitive and timely diagnosis is of great importance for improving therapeutic effects on MB. In 2016, the World Health Organization (WHO) divided MB into four subtypes: wingless-type mouse mammary tumor virus integration site (WNT), sonic hedgehog (SHH), non-WNT/non-SHH group 3, and group 4. Each subtype of MB has a unique profile in copy number variation, DNA alteration, gene transcription, or post-transcriptional/translational modification, all of which are associated with different biological manifestations, clinical features, and prognosis. This article reviewed the research progress of different molecular pathology markers in MB and summarized some targeted drugs against these molecular markers, hoping to stimulate the clinical application of these molecular markers in the classification, diagnosis, and treatment of MB. TP53) mutant and TP53 wild-type], group 3, and group 4 , a tumor-suppressor gene encoding histone 3 lysine 27 (H3K27) demethylase, plays a vital part in determining cell fate and cell differentiation during development [KDM6A in MB. KDM6A mutations are enriched in group 4 MB and are identified with lower frequencies in SHH and group 3 MB [KDM6A copy-number loss was often found in female non-WNT/non-SHH MB patients [KDM6A gene mutation promotes tumorigenesis in a mouse model of MB [KDM6A in MB.elopment , 44, 71elopment  first reoup 3 MB , 23, 72.patients . Althougel of MB , providiKBTBD4 gene encodes a Kelch protein belonging to a family of ubiquitin-ligase adapters that facilitates the ubiquitination of target substrates. KBTBD4 gene insertions are located at a hotspot region (codons 308–313) and have been reported exclusively in non-WNT/non-SHH MBs [KBTBD4 mutations at the hotspot region [KBTBD4 mutations in MB.-SHH MBs , 48, 49.t region . This maGLI is the end effector of hedgehog (HH) signaling and promotes transcription of HH-target genes, which could regulate cell survival, invasion, and angiogenesis, as well as stem cell self-renewal and epithelial-mesenchymal transition [GLI2 amplification exists in SHH MB [TP53 loss (defined as SHH α subtype), predicting a worse prognosis in patients with this subtype of MB [ansition –79. It hn SHH MB , 25 and pe of MB . Additiope of MB , which ipe of MB .Chromosome abnormalities are often observed in MBs, particularly those classified as group 3 and group 4 subtypes . IsochroTNRC6C was significantly increased in all subgroups of MB, and can be used as a potential prognostic marker to predict clinical outcomes in patients with this tumor [TNRC6 proteins, including TNRC6A, TNRC6B, and TNRC6C, serve as scaffolding proteins within miRNA-induced silencing complex and therefore play an important role in miRNA-mediated gene silencing . Whole-gis tumor .MXI1 and IL8 was identified as a novel independent high-risk biomarker in the survival models of SHH and non-WNT/non-SHH MB patients [MXI1 is a negative regulator of the MYC family of proteins , and IL8patients . IncorpoLmx1A enhancer activity and expression are important for the identification of group 4 MB. Their chromatin immunoprecipitation (ChIP)-sequencing data supported Lmx1A as a master regulator of the transcription factor in the transcriptional program of group 4 MB.Lmx1A is a LIM homeobox transcription factor 1, alpha previously shown to function as a critical regulator of cell-fate determination in cerebellar development . Lin et PRDM6 gene expression was markedly upregulated in a subset of group 4 MB patients, due to “enhancer hijacking” induced DNA rearrangement [PRDM6 as an oncogene in this subtype of MB.PRDM6 belongs to the PRDM family of transcriptional repressors, a family that is essential for the growth of smooth muscle cells . It was angement . FurtherGFI1B is a paralog of GFI1. Both genes functioned as sustained natural apophyseal glides (SNAG) domain-containing zinc finger transcriptional repressors essential for a variety of developmental processes [GFI1 and GFI1B were identified as prominent oncogenes specifically activated in non-WNT/non-SHH MB, and somatic genomic rearrangements together with mutually exclusive activation of GFI1 and GFI1B were found in approximately one-third of group 3 MB patients [MYC gene amplification in promoting the malignant progression of MB [GFI1 and GFI1B are promising biomarkers for molecular typing and targeted therapy in non-WNT/non-SHH MB.rocesses . GFI1 anpatients . These opatients , 25, 92,ERBB4 is the only member of the ERBB receptor family with growth-inhibiting properties. According to the Cancer Cell Line Encyclopedia database, its messenger RNA (mRNA) expression is only present in a small fraction of tumor cell lines, whereas the other ERBB receptors are highly expressed in the majority of tumor cell lines [ERBB4 can in part be explained by the multiple ligands that can activate ERBB4, its numerous intracellular phosphorylation sites, the presence of alternative splice variants, the different intracellular signaling pathways affected, and the different downstream responses in different cell types and different disease stages. Using quantitative (phospho)-proteomics in primary human MBs, Forget et al. [ERBB4 promoted MB malignance and could serve as a therapeutic target in group 4 subtype.ll lines . The cont et al.  unraveleMYC amplification is a “hallmark” of MYC-active MB, but not all tumors of this type have MYC amplification [MYC-active MB cells to radiation [fication , 95. Arcfication  quantitaadiation , which omiRNAs are a class of endogenous non-coding small RNAs with a length of 18–22 basepair (bp) that regulate the expression of target genes by inducing mRNA degradation or translation inhibition . They coSurgical resection, combined with radiotherapy and chemotherapy, is still the main mode for the treatment of MB, but the efficacy is limited. Thanks to the advances in molecular pathological markers of MB, molecular targeted therapy is becoming a promising strategy to overcome this type of pediatric brain tumor. Here, we mainly documented the targeted therapy studies related to the molecule markers mentioned above.Thirty percent of MBs show hyperactivation of SHH signaling pathways , 106. ViSMO mutations [GLI transcription factors are critical mediators of the HH pathway, which is usually activated in SHH MB. Glioma-associated oncogene antagonist-61 (GANT61), the first GLI antagonist, has been proven to inhibit cell migration, invasion, and proliferation while enhancing cell apoptosis in human MB cells . Arsenicutations , 113.Alterations of the signaling pathway of the intracellular lipid kinase PI3K are known to play a crucial role in MB by regulating cellular growth, proliferation, and cell survival . It has CREBBP-CTNNB1 interacting antagonist, is currently being involved in phase I clinical trials of pancreatic cancer and hepatitis-C-virus-infected cirrhosis  [PRI-724, a 2195440) , and is 2195440) . InhibitMYC amplification [The CDK4/6-meidated signaling pathway has been recently identified as a druggable target for all non-WNT MBs . As an ification . It is cfication . In addification  reportedMYC signature activation is found to be associated with poor outcomes in group 3 MB [MYC-driven MB cells and xenografts [oup 3 MB , 53, whinografts , 122. AsDDX3X, KDM6A, and MLL2/3, have not yet emerged and require further research. Small-molecule drugs targeting various miRNAs are also a new option for the treatment of MB [Targeted drugs against other molecular markers, such as nt of MB , 123, buMYC amplification is specific for group 3 MB and predicts poor prognoses, and CDK6 amplification is a vital feature of group 4 MB. Other markers are needed to be more comprehensively studied to clarify their specific roles in MB, such as MLL2/3, TERT, KDM6A, KBTBD4, and ERBB4. It should also be noted that some molecular markers are interrelated or mutually exclusive. In pediatric MB, TP53 mutation often occurs simultaneously with GLI2 and MYCN amplification. SUFU mutation is mutually exclusive with PTCH1 and SMO mutation, and it mainly occurs in infant MB, while DDX3X, CREBBP, and TERT-promoter mutation is frequently found in adult MB.With the development of more efficient and accurate molecular biology technologies, molecular pathology markers have shown an increasingly important role in MB. As summarized in The advances in molecular pathology markers provide deep insights into the tumorigenesis mechanism and targeted therapy of MB. The molecular mechanism driving WNT and SHH MB has been deeply studied, and some drugs targeting SHH pathways  and PI3K pathways  are being tested in clinical trials. Other drugs targeting the WNT/SHH MB, e.g., WNT/β-catenin inhibitor and GLI inhibitor, have also been investigated extensively, but they are still in the preclinical stage. The non-WNT/non-SHH MB, accounting for more than two-thirds of all MBs, has the highest metastasis rate. The underlying mechanism of this category of MB is largely unknown, which limits the development of targeted drugs. MYC activation is found to be associated with poor outcomes in group 3 MB, while there has not been a clear path for targeted therapy. The CDK4/6 inhibitor, palbociclib, is the sole drug that has entered the clinical trial for the treatment of non-WNT/non-SHH MB, especially group 3 subtype. Chromosome abnormalities are often found in group 3 and group 4 MB, such as iso 17q, chromosome 17 gain, and chromosome 11 loss. The alteration in chromosomes can be easily detected by sequencing and provide useful information for the prognosis of non-WNT/non-SHH MB, but the drug targets are so far lacking due to the complexity and heterogeneity of this type of MB. Therefore, further investigations are needed to identify homogeneous subtypes within the non-WNT/non-SHH MB, which may simplify the development of targeted drugs.