The endoplasmic reticulum (ER) is the organelle where newly synthesized proteins enter the secretory pathway. Different physiological and pathological conditions may perturb the secretory capacity of cells and lead to the accumulation of misfolded and unfolded proteins. To relieve the produced stress, cells evoke an adaptive signalling network, the unfolded protein response (UPR), aimed at recovering protein homeostasis. Tumour cells must confront intrinsic and extrinsic pressures during cancer progression that produce a proteostasis imbalance and ER stress. To overcome this situation, tumour cells activate the UPR as a pro-survival mechanism. UPR activation has been documented in most types of human tumours and accumulating evidence supports a crucial role for UPR in the establishment, progression, metastasis and chemoresistance of tumours as well as its involvement in the acquisition of other hallmarks of cancer. In this review, we will analyse the role of UPR in cancer development highlighting the ability of tumours to exploit UPR signalling to promote epithelial-mesenchymal transition (EMT). The ER is responsible for a large number of metabolic processes, including folding and post-translational modification of secretory proteins. Cells must keep a balance between the protein synthetic load and their capacity to ensure that folding and post-translational modifications are correctly performed. Improper ER function causes misfolding of de novo synthesized proteins and their accumulation due to a stringent quality control. Proteins failing to pass this control are returned to the cytosol and targeted for degradation by the ER-associated degradation system (ERAD) ,2. UnderThe UPR is an adaptive mechanism evolved to relieve the ER stress restoring the metabolic and protein folding efficiency of the ER . ActivatPERK is a type I transmembrane protein that, upon ER stress, dimerizes trans-autophosphorylates resulting in the activation of its kinase domain and the phosphorylation of eukaryotic translation initiation factor 2 alpha subunit (eIF2α) . This phIRE1 is a type I transmembrane protein that contains two enzymatic activities on its cytosolic tail, a serine/threonine kinase and an endoribonuclease. In response to ER stress, IRE1 oligomerizes and trans-autophosphorylates undergoing a conformational change that activates its RNase domain. IRE1 RNase catalyses the unconventional splicing of an intron within XBP1 mRNA (XBP1u) shifting the XBP1 reading frame and producing a stable transcription factor known as XBP1s (thereafter XBP1) which promotes the transcription of genes involved in protein folding, ERAD, protein secretion and lipid synthesis ,10,11. AATF6 is a type II transmembrane protein that belongs to a family of transcription factors whose members contain a conserved bZIP domain on their cytosolic domain. Following ER stress ATF6 is transported from the ER to Golgi where it is cleaved by the proteases S1P and S2P . The relSeveral stresses both cell extrinsic and intrinsic may perturb the protein folding efficiency of the ER and lead to the accumulation of misfolded proteins, ER stress and UPR activation.EMT is a genetic and cellular programme that endows cells with mesenchymal features that ultimately facilitate motility  22,23].,23.22,23It is nowadays well established that most carcinoma cells highjack the EMT programme to progress towards malignancy. In carcinomas, environmental cues and the exchange of signals among tumour cells and their microenvironment are mostly responsible for EMT implementation and the acquisition of mesenchymal traits and malignant progression. In fact, EMT is involved in the invasion-metastasis cascade, provides tumour-initiating abilities and contributes to the dormant state of disseminated tumour cells, cancer cell resistance to chemotherapy and immune evasion ,23. The In cancer, increasing evidence indicates that the full implementation of the EMT programme, as defined in in vitro cellular models upon expression of an EMT-TF, would be a rare event. Besides, cells in different intermediate morphological states ranging from partially epithelial to quasi-mesenchymal are more likely to occur and be responsible for implementing different steps during tumour progression. Carcinoma cells undergoing EMT would thus show hybrid features and express a mixture of epithelial and mesenchymal markers, whereas incompletely losing their epithelial cell polarity. Indeed, these cells displaying partial EMT states have been documented in human cancers and are thought to be particularly plastic. These plastic EMT cells, while able to degrade and invade the surrounding stroma, may also, have given the signalling context, acquire stem cell-like traits, and become refractory to therapy and immune surveillance ,27,36. MTherefore, EMT endows subpopulations of cancer cells with a highly dynamic morphological plasticity tied to context-dependent functional abilities that facilitate malignant progression .Cancer cells are subjected to numerous intracellular and extracellular stresses that disturb ER homeostasis provoking ER stress and thus, UPR activation . During We will next describe the ER stressors that provoke UPR activation; linking how defined UPR signals modulate EMT .The UPR has been extensively studied in vitro by treating cells with chemical stressors such as thapsigargin, dithiothreitol or tunicamycin, which disturb calcium homeostasis, redox equilibrium or N-glycan synthesis, respectively. These drugs activate non-specifically the three arms of the UPR . Also, EEMT induction after UPR activation was first observed in vitro in rat alveolar epithelia cell lines upon treatment with the classical ER stressors tunicamycin and thapsigargin ,57. In bThe number of chemical stressors inducing EMT after UPR activation has been expanded by the recent finding that chemotherapeutic drugs activate ER stress and ultimately EMT . TreatmeHypoxia compromises ER protein folding leading to the activation of the UPR  whereas Hypoxia activates the PERK arm of the UPR to promote metastasis in human cervical tumoral cells . HypoxicOne consequence of the activation of the UPR in hypoxic tumours is an increase in autophagy. Through the liberation of amino-acids from long-lived proteins and the removal of damaged organelles, autophagy exerts a cytoprotective effect and helps cells to survive . Both th-/- mouse embryo fibroblasts decreased the production of VEGF-A after ER stress [Activation of the UPR also plays a role in tumour adaptation to hypoxic stress by promoting angiogenesis. Angiogenesis is regulated by the secretion of soluble factors such as VEGF-A. Several examples implicate the IRE1-XBP1 axis in this process. In TNBC, XBP1 expression is required for HIF1α-mediated VEGF-A production and vessel formation under hypoxia, and xenografts derived from cells transfected with XBP1 shRNA displayed reduced angiogenesis . Also, IR stress . In addiR stress . More reR stress ,68.The information about the axis hypoxia-UPR-EMT is still scarce, however several recent findings suggest that such a relationship exists. Gastric cancer cells under hypoxic conditions suffer EMT and the PERK and ATF6 arms of the UPR are activated. Moreover, knockout of PERK, ATF4 or ATF6 hinders the induction of EMT by hypoxia . In TNBCIntimately related with the insufficient vascularization of the tumour is the nutrient stress starvation suffered by tumour cells. The UPR participates in the rewiring of tumour metabolism by selectively activating catabolic pathways—we have already mentioned the induction of autophagy—but also biosynthetic pathways. In human tumour tissues, and several tumour cell lines, blocking UPR by silencing PERK or ATF4 significantly reduces the production of angiogenesis mediators induced by glucose deprivation . RecentlAnother recently published study has described that the availability of asparagine controls metastasis in breast cancer, at least in part through the modulation of EMT. Limiting asparagine by asparagine dietary restriction or by knocking down asparagine synthetase reduces the metastatic capacity of the primary tumour and, by contrast, an increase in dietary asparagine or enforced asparagine synthetase expression promotes metastatic progression. Interestingly, asparagine synthetase is a target of the PERK-eIF2α-ATF4 branch of the UPR and ATF4 knockdown mimics the phenotype caused by asparagine synthetase knockdown . One plaCancer cells and melanoma in particular require an exogenous supply of glutamine since it is commonly depleted in tumours. Interestingly, it has been recently demonstrated that glutamine starvation is responsible for an upregulation of ATF4 in melanoma cells . In thisDeprivation of essential amino acids also causes activation of the UPR but, in this case, the arms activated are the IRE1 and ATF6 . In thisMalignant transformation by the activation of oncogenes is associated with an increased cell proliferation that imposes severe pressures on cellular processes such as an excessive demand for protein synthesis in the ER. When the folding capacity of the ER is exceeded, cancer cells trigger the UPR activating PERK, IRE1 and/or ATF6-dependent signalling pathways as shown in different types of cancer  79,80,8,82. The In conclusion, the activation of the UPR upon oncogenic signalling is well documented but, in these cases, activation of the UPR pro-survival response is apparently unrelated to EMT induction.Tumour cells accumulate increased levels of reactive oxygen species (ROS) that lead to the general phenomenon of oxidative stress. ROS are produced intracellularly by both enzymatic and non-enzymatic reactions in different cellular compartments during cell growth . Cancer This protective function of the UPR against oxidative stress has an important impact during the first steps of tumour invasion, when an abnormal activation of EMT causes tumour cell detachment and acquisition of an invasive and migratory phenotype. In this situation, PERK activation favours survival upon ECM detachment  and alleCumulative data suggest that oxidative stress governs several phases of tumour progression  includinDifferent studies support a role for proteostasis imbalance in cancer development raising the possibility that alterations in the UPR itself may contribute to tumorigenesis ,100. ConSequencing the genome of different types of human cancers exposed somatic mutations in IRE1. In fact, ERN1, the gene coding for IRE1, was found to rank fifth among all the genes coding for human protein kinases carrying driver mutations across various human cancers . In a reNevertheless, although mutations in the distinct components of the UPR are present on different tumour types, knowledge on their role in cancer biology is still very limited.Overexpression of individual proteins, not necessarily involved in signalling pathways driving tumour progression, can also provoke UPR activation and subsequent EMT induction.One early report showed that enhanced expression of the protease inhibitor SERPINB3 promotes tumorigenesis and EMT via the PERK and ATF6 arms of the UPR which lead to NFκB activation and subsequent expression of the pro-tumorigenic cytokine IL6 .SNAI1, SNAI2, TCF3 and ZEB2 activating their expression [More recently, it has been reported that the overexpression of LOXL2 also causes UPR activation and subsequent EMT induction . LOXL2 ipression  (Figure pression . Remarkapression ,111.The information reviewed above relative to the cooperation between the UPR and EMT during tumour progression comes mainly from data derived from experiments performed using cell lines and mouse models. Concerning clinical samples, thorough reviews have recently addressed the evidences of ER stress in human tumours by examining the expression levels of UPR signalling components ,81. As aThere are few studies to date that analyse in human cancer biopsies markers of EMT along with UPR activation markers. These studies would help to elucidate the prognostic value of both programmes in terms of patient outcome, therapy choice and/or treatment response. Recent works addressing both UPR and EMT pathways in clinical samples are summarized in In this regard, the expression of an EMT signature is strongly correlated with ATF4 expression in datasets covering breast, colon, gastric, lung, and metastatic sites from patient tumour samples . In coloA direct connection between ER stress and UPR activation with malignancy has been formally established in different cancer settings ,46,47,81Cancer cells undergoing UPR activation and prone to EMT may sustain malignant progression since they are able to migrate and invade, display tumour-initiating properties and resilience in foreign microenvironments while they become skilled to evade drug therapy and immune surveillance. Nevertheless, the UPR can also be a pro-apoptotic signalling pathway. When cells are subjected to a chronic ER stress or cannot resolve the stress, the UPR directs the cells to apoptosis . This faThe involvement of ER stress and the activation of the UPR in cancer initiation and progression is now supported both by in vitro analyses and data from clinical samples ,43,46,81In conclusion, the development of drugs targeting ER stress in particularly susceptible cells such as those in different EMT states might constitute a promising cancer therapy.


In 2010, the World Health Assembly set three milestones for measles prevention to be achieved by 2015: 1) increase routine coverage with the first dose of measles-containing vaccine (MCV1) among children aged 1 year to ≥90% at the national level and to ≥80% in every district; 2) reduce global annual measles incidence to less than five cases per million population; and 3) reduce global measles mortality by 95% from the 2000 estimate (WHO and the United Nations Children’s Fund (UNICEF) use data from administrative records and vaccination coverage surveys reported annually by 194 countries to estimate coverage with MCV1 and MCV2 delivered through routine immunization services.Estimated MCV2 coverage increased globally from 15% in 2000 to 67% in 2017, largely because of an increase in the number of countries providing MCV2 nationally from 98 (51%) in 2000 to 167 (86%) in 2017 . Three cDuring 2017, approximately 205 million persons received supplementary doses of measles-containing vaccine (MCV) during 53 supplementary immunization activities (SIAs)In 2017, 189 (97%) countries conducted measles case-based surveillance in at least part of the country, and 191 (98%) had access to standardized quality-controlled testing through the WHO Global Measles and Rubella Laboratory Network. However, surveillance was weak in many countries, and fewer than half of the countries reporting surveillance indicators  achieved the sensitivity indicator target of two or more discarded measles and rubella,Countries report the aggregate number of incident measles casesGenotypes of viruses isolated from measles cases were reported by 76 (59%) of the 129 countries that reported at least one measles case in 2017. Among the 24 recognized measles virus genotypes, 11 were detected during 2005–2008, eight during 2009–2014, six in 2015, and five in 2016 and 2017, excluding those from vaccine reactions and cases of subacute sclerosing panencephalitis, a fatal progressive neurologic disease caused by persistent measles virus infection ; thus, all six regions now have RVCs. In September 2016, the AMR RVC declared the region free of endemic measles  countries provided MCV2. In 2017, MCV1 and MCV2 coverage in WPR was ≥94%, and measles incidence in this region was at an all-time low. The increasing number of countries verified as having achieved measles elimination indicates progress toward global interruption of measles virus transmission.Despite this progress, however, the 2015 global milestones have not been achieved; global MCV1 coverage has stagnated for nearly a decade; global MCV2 coverage is only at 67% despite steady increases; and SIA quality was inadequate to achieve ≥95% coverage in several countries. Since 2016, measles incidence has increased globally and in five of the six WHO regions. Furthermore, as of July 2018, endemic measles has been reestablished in Venezuela because of the sustained transmission of measles virus for >12 months; the remaining 34 AMR countries continue to maintain their measles elimination status, but the ongoing outbreak in Venezuela has led to measles virus importations and outbreaks in bordering AMR countries. In addition, the measles resurgence in Europe has likely led to reestablished endemic measles in some EUR countries. These outbreaks highlight the fragility of gains made toward global and regional measles elimination goals. Continuing to increase MCV1 and MCV2 coverage is critical to both the achievement and sustainability of the global and regional measles elimination goals. Meanwhile, conducting high quality SIAs that reach unvaccinated and undervaccinated children will prevent future outbreaks that are costly in terms of morbidity and mortality and are disruptive to immunizations service delivery. It is important to have high-performing surveillance for early detection of outbreaks; and when outbreaks do occur, thorough outbreak investigations are needed to better understand and address the underlying causes of the outbreak and why children are being missed by immunization delivery systems.The findings in this report are subject to at least three limitations. First, SIA administrative coverage data might be biased by inaccurate reports of the number of doses delivered, doses administered to children outside the target age group, and inaccurate estimates of the target population size. Second, large differences between the estimated and reported incidence indicate variable surveillance sensitivity, making comparisons between countries and regions difficult to interpret. Finally, the accuracy of estimates from the measles mortality model is affected by biases in all model inputs, including country-specific measles vaccination coverage and measles case-based surveillance data.Monitoring progress toward measles elimination goals could be improved by establishing updated indicators. For example, the WHO Strategic Advisory Group of Experts on Immunization recently approved country classifications, and updates to the framework for the verification of measles elimination will standardize monitoring of countries’ progress toward verified elimination (Strengthening routine immunization and continuing to conduct high-quality SIAs will help achieve global and regional measles elimination goals, improve overall vaccination coverage and equity, and assist in attaining universal health coverage. It is important that countries continue to strengthen case-based surveillance and increase MCV1 and MCV2 coverage and that immunization partners continue to raise the visibility of measles elimination goals and secure political commitment to these goals and sustained investments in health systems.In 2012, the World Health Assembly endorsed the Global Vaccine Action Plan; as a result, countries in all six World Health Organization regions have adopted goals for elimination of measles by 2020.During 2000–2017, annual reported measles incidence decreased 83%, and annual estimated measles deaths decreased 80%. Since 2000, global measles elimination efforts have prevented an estimated 21.1 million deaths. However, measles elimination milestones have not been met, and three regions are experiencing a large measles resurgence.To make further progress, case-based surveillance needs to be strengthened, and coverage with the first and second dose of measles-containing vaccine needs to increase; moreover, it is important to maintain political commitment, and secure substantial, sustained investments.


The glossopharyngeal nerve (GPN) is a rarely considered cranial nerve in imaging interpretation, mainly because clinical signs may remain unnoticed, but also due to its complex anatomy and inconspicuousness in conventional cross-sectional imaging. In this pictorial review, we aim to conduct a comprehensive review of the GPN anatomy from its origin in the central nervous system to peripheral target organs. Because the nerve cannot be visualised with conventional imaging examinations for most of its course, we will focus on the most relevant anatomical references along the entire GPN pathway, which will be divided into the brain stem, cisternal, cranial base  and cervical segments. For that purpose, we will take advantage of cadaveric slices and dissections, our own developed drawings and schemes, and computed tomography (CT) and magnetic resonance imaging (MRI) cross-sectional images from our hospital’s radiological information system and picture and archiving communication system.The glossopharyngeal nerve is one of the most hidden cranial nerves.• It conveys sensory, visceral, taste, parasympathetic and motor information.• Radiologists’ knowledge must go beyond the limitations of conventional imaging techniques.• The nerve’s pathway involves the brain stem, cisternal, skull base and cervical segments.• Systematising anatomical references will help with nerve pathway tracking.•  The glossopharyngeal nerve (GPN) or IX cranial nerve is one of the most unattended cranial nerves in imaging examinations. It is not depicted for most of its course and, when affected, clinical manifestations are usually not characteristic. In fact, the GPN and vagus nerve interact so closely that an isolated GPN dysfunction might not be clinically discriminated if vagus nerve function is not impaired , except Previous radiological publications have not been especially focused on the anatomic relationships of cranial nerves or on the GPN. Those reviews have placed more emphasis on diseases that may affect the cranial nerves , 4, 6–8.The GPN conveys: 1) sensory afferents (retroauricular region), visceral afferents  and taste afferents (posterior third of the tongue); 2) parasympathetic afferents (carotid sinus baroreceptors and carotid body chemoreceptors) and efferents (parotid gland); and (3) motor efferents (stylopharyngeus muscle) [ sensory  parasympThe images retrieved and included in this manuscript were acquired in a multislice spiral computed tomography (CT), GE LightSpeed VCT 64  and a GE Signa HDx 1.5T MRI scanner . All images corresponded to clinical examinations performed according to our standard cranial, skull base and cervical CT and MRI protocols  or the sphenopetrosal junction , 23 Fig and 7. IImmediately after leaving the jugular foramen, the GPN is located medial to the internal jugular vein and behind the internal carotid artery Figs.  and 8. NOnce the GPN is anterior to the vascular structures, it leaves the close relationship with the internal carotid artery approximately at the level of the soft palate  and runsAt approximately the point where the stylopharyngeus muscle merges with the constrictor muscles, the GPN enters the pharynx between the upper and middle constrictors . At thisOnce in the pharynx, the GPN splits into pharyngeal branches, which contribute to the pharyngeal plexus of the vagus, and the lingual branch , 14, 25.All anatomic references are summarised in Fig. Muscle denervation can be the most visible sign of neural injury, especially in larger muscles , 28, 29.The novelty of this article is the specific focus on the glossopharyngeal nerve (GPN) in cross-sectional imaging, which systematises the anatomical relationships of the main trunk and its branches. It will help radiologists obtain insights into a cranial nerve that, unlike many of its counterparts, remains beyond the observer’s attention due to its inconspicuous clinical signs and scarce visibility in normal and pathological clinical images.


Recent advancement in the field of molecular cancer research has clearly revealed that abnormality of oncogenes or tumor suppressor genes causes tumor progression thorough the promotion of intracellular metabolism. Metabolic reprogramming is one of the strategies for cancer cells to ensure their survival by enabling cancer cells to obtain the macromolecular precursors and energy needed for the rapid growth. However, an orchestration of appropriate metabolic reactions for the cancer cell survival requires the precise mechanism to sense and harness the nutrient in the microenvironment. Mammalian/mechanistic target of rapamycin (mTOR) complexes are known downstream effectors of many cancer-causing mutations, which are thought to regulate cancer cell survival and growth. Recent studies demonstrate the intriguing role of mTOR to achieve the feat through metabolic reprogramming in cancer. Importantly, not only mTORC1, a well-known regulator of metabolism both in normal and cancer cell, but mTORC2, an essential partner of mTORC1 downstream of growth factor receptor signaling, controls cooperatively specific metabolism, which nominates them as an essential regulator of cancer metabolism as well as a promising candidate to garner and convey the nutrient information from the surrounding environment. In this article, we depict the recent findings on the role of mTOR complexes in cancer as a master regulator of cancer metabolism and a potential sensor of nutrients, especially focusing on glucose and amino acid sensing in cancer. Novel and detailed molecular mechanisms that amino acids activate mTOR complexes signaling have been identified. We would also like to mention the intricate crosstalk between glucose and amino acid metabolism that ensures the survival of cancer cells, but at the same time it could be exploitable for the novel intervention to target the metabolic vulnerabilities of cancer cells. Proliferating cells require not only adenosine triphosphate (ATP) as an essential energy source, but also intracellular building blocks including nucleotides, fatty acids, and proteins, and a reprogrammed metabolism could serve to support the synthesis of macromolecules ,2. The WCancer cells take up a large amount of amino acids and glucose from the extracellular environment as a carbon and nitrogen source for protein and nucleotide synthesis ,8. In thHere, we focus on mammalian/mechanistic target of rapamycin (mTOR) complexes, essential regulator of cell proliferation and metabolism, as a potential key player to play a role in sensing nutrients to drive the intracellular tumor-promoting signaling cascade through metabolic reprogramming and epigenetic shift, and a key node which should be therapeutically targeted as a new mode of treatment to interfere with cancer cell metabolism.The hallmarks of cancer are composed of six biological capabilities, which are acquired during the multistep evolution of human neoplasms . The comGenetic mutations to constitutively activate phosphoinositide 3-kinase (PI3K)-Akt-mTOR signaling are reported to reprogram cellular metabolism and tumorigenesis, including receptor tyrosine kinase (RTK) amplification and mutations, phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit alpha isoform (PIK3CA) mutation and phosphatase and tensin homolog deleted from chromosome 10 (PTEN) loss . Among tThe structures of mTORC1 and mTORC2 are characterized by sharing some components: they share the catalytic mTOR subunit, as well as mammalian lethal with sec-13  ,27, the Oncogenes and tumor suppressors are a key determinant in controlling cancer metabolism ,42,43. ImTORC1 is an evolutionarily conserved multi-protein complex that coordinates a network of signaling cascades and functions as a key mediator of protein translation, gene transcription, and autophagy ,50,51. mLeucine, one of the essential amino acids in human cells, mainly induces the recruitment of mTORC1 to the lysosomal membrane and its subsequent activation; that is, mTORC1 is activated in response to the level of leucine . LeucineRecent studies also demonstrate the intriguing interaction of amino acid metabolism and mTORC1 signaling through the ubiquitin signaling systems, displaying that, in response to amino acids, the KLHL22 E3 ubiquitin ligase promotes K48-linked polyubiquitination, enabling mTORC1 signaling to promote tumorigenesis and aging . AnotherCancer cells convert the majority of glucose into lactate even under ample oxygen (the Warburg effect), the products of which could be used as carbon-containing precursors for the macromolecule production by rapidly proliferating cells. This intriguing phenomenon necessitates the presence of “glucose sensor” in cancer cells to precisely catch the information on the glucose in the microenvironment for appropriately responding to the environment and ensuring their survival. In recent years, several fascinating reports have been published, referring to the relationship between mTORC2, metabolic reprogramming, and nutrient sensing in cancer cells. mTORC2 is activated on the high-glucose extracellular condition via acetylation of Rictor, the main component of mTORC2 in human GBM cells  Figure . This isUnlike mTORC1, which works as an amino acid sensor, the role of mTORC2 as a sensor of amino acid has yet to be clarified. However, using an unbiased proteomic screen, our recent work unraveled that mTORC2 could suppress the activity of the cystine-glutamate antiporter, system Xc transporter-related protein (xCT) via inhibitory phosphorylation of serine 26 of xCT’s N terminus cytosolic domain  Figure . These rAn unexpected implication for cancer cell metabolism comes from these recent studies regarding the sensing mechanism of glucose and amino acid by mTORC2. Exogenous nutrients including glucose and acetate have been reported to activate mTORC2 to phosphorylate its downstream substrates as aforementioned , and thiMany enzymes that play important roles in epigenetic gene regulation utilize intermediary metabolites as co-substrates yielded by cellular metabolic reprogramming . Indeed,Acetylation on the N-terminal lysine tail of histones leads to the neutralization of positively charged lysine with an open chromatin configuration facilitating transcription. On the other side of the coin, deacetylation of histones is associated with condensed chromatin and reduction of transcriptional activity. Acetyl-CoA is the substrate used to modify histone tails, and can be produced through a variety of metabolic pathways. Its primary generation sources are through the conversion of pyruvate from glycolysis and citrate from the TCA cycle. Intriguingly, some of the processes seem to be governed by the RTK/PI3K/Akt/mTOR pathway, thus possibly linking their metabolic processes to epigenetic status. Recent studies demonstrated that dynamic translocation of mitochondrial pyruvate dehydrogenase complex (PDC) to the nucleus provides a pathway for nuclear acetyl-CoA synthesis required for histone acetylation and epigenetic regulation . InteresIn addition to histone acetylation, histone methylation is also important in defining the epigenetic state of chromatin as well as the methylation of DNA itself. A methyl-donor SAM derived from methionine is utilized by methyltransferases; thus, its metabolism can profoundly affect the DNA and histone methylation status. Methionine adenosyltransferase (MAT) is an essential enzyme responsible for SAM biosynthesis, and the function and subclass switch of MAT can be associated with PI3K/Akt signaling, affecting global DNA methylation and cell survival in cancer . RecentlSomatic mutation of the NADP(+)-dependent enzyme isocitrate dehydrogenase (IDH) is frequently found in cancer, and is shown to acquire a neomorphic enzymatic activity that converts α-ketoglutarate (α-KG) to 2-hydroxyglutarate (2-HG) . OncometUnderstanding the complex role of mTOR in regulating signal transduction is critical to developing more effective therapies to target metabolic reprogramming in cancer. Distinctly, rapamycin treatment  leads to the release of Akt suppression (hence activation of mTORC2), due to the loss of negative feedback for attenuating PI3K signaling . The PI3d-ribonucleoside (AICAR) effectively blocks the growth of EGFR-activated GBM primarily by inhibiting lipogenesis [mTOR also plays a critical role in integrating cellular metabolism with signal transduction. mTORC1 has emerged as a critical effector downstream of the tumor suppressor liver kinase B1 (LKB1). LKB1 is thought to suppress tumor formation by negatively regulating mTORC1 signaling through adenosine monophosphate (AMP)-activated protein kinase (AMPK). A study by our group demonstrated that the AMPK activator, 5-aminoimidazole-4-carboxamide-1-β-ogenesis  (Figure ogenesis . Furtherogenesis . Targetiogenesis . Recent ogenesis . Furtherogenesis . Additioogenesis  (Figure There have traditionally been plenty of the epidemiological studies endeavoring to reveal the statistic link between cancer incidence and metabolic factors including obesity, diabetes mellitus, and Western-type life style and diet. However, the relationship was not usually solid, partly because most of the studies did not touch on the tumor genotype. Future studies will be necessary to plan the mechanistic types of studies that will untangle the interaction between nutrients, metabolism, and cancer biology on a genetic and molecular basis, which will eventually reveal the impact of nutrient and metabolism on tumor pathogenesis, aggressiveness, and response/resistance to treatment. Thus, mTOR-dependent metabolism should be evaluated in the specific context of genotype-defined and nutrient/environment-restricted conditions.We have proposed the questions on mTOR-dependent metabolism in cancer, which should be tackled in the future for achieving the goal of developing novel therapeutic and preventive strategies against cancer with facilitated metabolism. In order to determine whether accelerated cellular metabolism is not only a consequence reprogrammed by oncogenic signaling but also potentially affected by a specific tumor microenvironment, it will be necessary to regulate diet and nutrient levels in genotyped human and mouse tumor models to assess the impact of metabolism on signal activation, tumor progression, and response/resistance to treatment. The critical point here is to recognize that the status of specific nutrients in the tumor microenvironment are not merely a consequence of the diet but rather established by the intricate interaction between diet uptake, de novo/salvage synthesis, and cellular utilization . Thus, dIn addition, to test a hypothesis that mTOR complexes are a key node to integrate growth factor receptor signaling with nutrient availability, influencing tumor growth and response to treatment, it will be necessary to study the role of mTOR and its modulation by nutrients in various cancer types. Furthermore, to test the hypothesis that glucose or amino acid-derived intermediates including acetyl-CoA and SAM directly contribute to tumor growth and drug resistance, genetic studies will be needed particularly to confirm the hypothesized importance of histone acetylation, histone methylation, and DNA methylation and to determine if persistent mTOR signaling is sufficient to maintain tumor growth and cause drug resistance through metabolic reprogramming and subsequent epigenetic shifts, for example, by examining the impact of elevated acetyl-CoA and SAM levels on enhancer activation and transcriptional reprogramming.The intricate orchestration of responses that enable cancer cells to meet their demands in a completely cell-autonomous fashion defines the specificity of metabolic reprogramming in cancer. A promising and less toxic therapeutic strategy for patients with cancer will be achieved by the development of inhibitors that target cancer-specific signal transduction and metabolism. However, that goal is unlikely to be achieved until the impact of cancer-causing driver mutations on metabolic reprogramming and epigenetic regulation are deeply comprehended, including the flexible ways in which tumor cells appropriately sense the nutrient status in microenvironment and adapt to changing conditions, so as to coordinately sustain the constitutive activation of downstream effectors necessary for tumor cell proliferation and survival. We have herein summarized the recent literature, clearly pointing out an unanticipated important role for both mTOR complexes in sensing essential nutrients including glucose and amino acids via cancer metabolic reprogramming, where they integrate aberrant signaling activities into biochemical reactions and potential transcriptional regulations that drive tumor progression. We have also highlighted an emerging role for mTORC2 in linking aerobic glycolytic metabolism with amino acid metabolism, which could potentially be a key mechanism to exquisitely tilt the balance between dichotomic cellular events essential for tumor cell survival including cell proliferation and cell protection from oxidative stress. Future endeavors should be directed to understand how driver mutations in cancer rewire intracellular signaling cascades to translate biochemical metabolic reactions into global epigenetic ensembles . Coopera


Phenylhydrazine (PHZ) induced anemia and was shown to have harmful effects on the male reproductive system. To investigate the protective effect of vitamin C (Vit C) on sperm parameters quality, in vitro fertilization potential and embryonic development in a mouse model of hemolytic anemia induced by PHZ.n = 8/each) were randomly classified into four groups. Group I (control) received normal saline, Group II (PHZ) received 8 mg/100 gr body weight PHZ as initial dose, continued by 6 mg/100 gr intraperitoneally every 48 hr, Group III (Vit C) received Vit C , and group IV (PHZ + Vit C) received PHZ and Vit C. After 35 days, sperm quality parameters, the percentage of sperm with DNA damage and in vitro fertilization outcomes up to blastocyst stage were evaluated.Thirty-two NMRI adult male mice (pppp = 0.002), but it could not improve the percentage of two cell embryos and blastocyst production.A significant (The data from this study indicated that Vit C decreased the adverse effects of PHZ on the quality of sperm parameters and in vitro fertilization rate, but it is insufficient to restore the in-vitro embryonic development and fertility potential. Anemia is a common and serious world public health affecting people of all ages and is related to a high risk of morbidity and mortality, especially in developing countries (1). Exposure to some chemicals and drugs have been associated with red blood cell (RBC) destruction and causing hemolytic anemia. Phenylhydrazine (PHZ), due to its toxic effects on RBC, is a useful agent in experimental model study of hemolytic anemia (2). This compound was first prescribed at the end of the nineteenth century as an antipyretic drug and is now well-known for its ability to RBC hemolysis in animals and humans inducing hemolytic anemia (3).Results of a previous study indicated that PHZ adversely affected the male reproductive system in mice that suggested to induce oxidative damage. It was indicated that administration of PHZ in mice for 35 days caused decrease in the epididymal sperm parameters quality . It was also shown that PHZ through the formation of lipid peroxidation causes DNA damage in sperms (4). Amer and colleagues reported that PHZ elevates reactive oxygen species (ROS) and lipid peroxidation and alleviates glutathione (5). These effects may have been reversed following antioxidant administration.Oxidative stress is a major cause of reproductive failure, and it has been proven that male germ cells may be prone to oxidative damage due to the high amount of polyunsaturated fatty acids and low capacity for DNA repair (6). Other investigators have indicated that damage in testicular tissue is associated with lipid peroxidation and they suggested that high production of ROS may play a major role in inducing testicular degeneration, resulting in infertility (7). Thus, administration of compounds with antioxidant properties can help the body to combat against pathological status caused by ROS or free radicals.Further studies have shown that antioxidants scavenge ROS are produced by leukocytes and protect sperm DNA from fragmentation and can also improve semen quality in persons exposed to oxidative stress . Vitamin C (Vit C) or ascorbic acid is one of the major antioxidant compounds in biological systems (9). Supplementation of Vit C can protect the blood-testis barrier destruction induced by cadmium through the inhibition of oxidative stress (10). Talebi and colleagues indicated that Vit C as a potent antioxidant alleviates adverse effects of diabetes on the sperm parameters, chromatin maturity and apoptosis in mice (11). Vit C deficiency in guinea pigs caused great degeneration of the seminiferous tubules and reduced the weight of the reproductive system, including testis and accessory sex organs, and Vit C supplementation reversed this reduction (12).Considering the positive effects of Vit C on the male reproductive system, the current study was conducted to evaluate the protective effects of Vit C on sperm parameters and IVF potential (embryonic development) in male mice exposed to PHZ-induced hemolytic anemia.n = 8): group I – control group, received intraperitoneal (IP) normal saline ; group II – PHZ group, received PHZ  at the dose of 8 mg/100 g/body weight/IP at first, followed later at the dose of 6 mg/100 g every 48 hr; group III – Vit C group, received Vit C  daily at the dose of 10mg/kg/IP; and group IV – PHZ + Vit C group, received PHZ and Vit C at the same dose. The treatment time for all the groups was 35 days.Thirty-two adult male NMRI mice (25–27 gr) were maintained in standard animal house conditions . The doses of PHZ and Vit C were selected according to previous studies . During the study, the animals were accessed to food and water ad libitum. After 35 days, the mice were euthanized with an overdose of ketamine . Then, the caudal portion of both epididymides of each mouse was transferred and minced in a petri dish containing 1 mL of human tubal fluid medium  preheated at 37After diluting, sperms were counted using a Neubauer slide under a light microscope. The motility of sperm was expressed by the percentage of motile sperm of the total sperm counted (15). For sperm viability, 20 µL of eosin solution was added into the same value of sperm sample and then 20 µL of nigrosine solution was added and smear prepared; after drying the slides, the percentage of unstained colorless alive sperms and red stained dead sperms were determined. The percentage of sperm morphology was evaluated using the slides stained by aniline blue (4). To determine all percentages , 200 sperms were counted per slide.AB stains lysine-rich histones and is considered as a marker of sperm chromatin evaluation used to detect sperm nucleus maturity. Briefly, dried smears from sperm solution were fixed for 30 min in 3% glutaraldehyde. The smears were stained with 5% AB for 7 min. In this test, normal mature sperms were pale and abnormal immature sperms were dark blue in color. Under a light microscope using 100AO is a fluorescent staining that is used to evaluate chromatin integrity and determine damage to DNA. Indeed, this staining detects double- and single-stranded regions in sperm chromatin. Briefly, air-dried sperm smear slides were fixed for 2 h in Carnoy's fixative (methanol/acetic acid 1:3). Then, the slides dried at room temperature were stained with AO solution for 7 min and dried again. The slides were examined using a fluorescent microscope with 100At the end of the study (35 days), the male mice were prepared for IVF. The authors used 60 adult female (10 wk. old) mice to obtain enough oocytes for IVF assay. To induce superovulation and collect mature oocytes from oviducts, each female was intraperitoneal injected with 10 IU pregnant mare`s serum gonadotropin hormone  and followed after 48 hr with IP injecting of 10 IU human chorionic gonadotropin hormone (hCG). About 12–14 hr after hCG injection, female mice were sacrificed and then the fallopian tubes were removed and transferred to a drop of HTF-BSA medium previously equilibrated in an incubator .pThe data of IVF assay were analyzed by two proportional test using Minitab software . Other results were examined by One-Way ANOVA using SPSS 16 software (USA). All results were shown as means pp = 0.002, respectively). The results also showed that sperm counts in group IV (PHZ + Vit C) were significantly . In addition, the sperm motility of the mice receiving PHZ + Vit C was significantly (p = 0.007) greater than those of the mice receiving only PHZ.Compared to group I (control) (35 ppp = 0.023) lower than group I (control). However, sperm viability in group IV (PHZ + Vit C) was significantly (p =0.006) greater than group II (PHZ) (Table I).The comparison of the percentage of alive sperm in group II (PHZ) with group I (control) indicated that it was significantly (pp = 0.02) compared to group II (PHZ).Table I also shows the analysis of sperm morphology data. Compared to group I (control), the percentages of sperm with normal morphology in groups II (PHZ) and IV (PHZ + Vit C) were significantly low (pppThe percentage of sperm with DNA damage in group II (PHZ) was significantly higher than groups I (control) and III (Vit C) . The percentage of two cell embryos formation in group II compared to group I was significantly reduced  was significantly lower than groups I (control) and IV (PHZ + Vit C),  improved them. The authors' findings also revealed that PHZ administration had a detrimental effect on IVF outcomes and embryonic development, and treatment with Vit C reverses only fertilization rate but is insufficient to restore in vitro embryonic growth.Recent reports have indicated that infertility is considered to be a major clinical problem, and it has been reported in one in four couples . Oxidative stress is one of the main causes of infertility in men (19). Overproduction of ROS in sperm induces nuclear DNA fragmentation, lipid peroxidation that leads to cell death . Due to the high amount of polyunsaturated fatty acids in the spermatozoa and also the lack of ability for DNA repair, they are sensitive to ROS elevation. Thus, the increased ROS amount has been associated with declined sperm parameters quality and quantity .In agreement with our findings, Mozafari and colleagues showed that sperm parameters' quality and quantity significantly decreased in mice treated with PHZ compared to control mice. They showed that PHZ increased the level of malondialdehyde (MDA) in testicular tissue in mice. MDA is an indicator of the degree of lipid peroxidation and causes testis tissue damages (4).Prior studies also demonstrated that short-term administration of rats with PHZ resulted in hemolysis anemia with decreased RBC and hemoglobin (Hb) . Hemolysis or declined levels of RBC induced by PHZ leads to overproduction of ROS and lipid peroxidation. Thus, oxidative damage plays a major role in the pathogenesis of PHZ-induced testicular damage. To protect sperm from ROS, it is important to use antioxidative defense mechanisms in the testis (24). Considering the effect of PHZ on the antioxidant state, the authors hypothesized that Vit C as a potent antioxidant could prevent the PHZ-dependent damages in mice sperm parameters and fertility potential.These findings revealed that PHZ significantly reduced the sperm count and motility. The decrease of sperm count is largely related to the onset of testicular damage, and reduction in sperm motility is mainly dependent on oxidative stress damage. Thus, a decrease in sperm motility is the first sign of elevated ROS level . This increase in ROS level affects sperm enzymatic content and enhances phospholipids peroxidation, which eventually leads to a decrease in cell membrane fluidity and sperm motility (27). However, supplementation with Vit C resulted in a notable improvement in sperm count and motility in mice received PHZ. Observations have indicated that PHZ-treated mice showed a significant increase in the percentage of sperm with abnormal morphology. In contrast to our findings, in an experimental study in diabetic rats, it was indicated that treatment with Vit C reverses only testicular damage but is not able to restore sperm motility and fertility (28).In order to find out how PHZ induce sperm parameters impairment, it should be considered that during spermatogenesis process a part of sperm cytoplasm must be phagocytosis and removed by Sertoli cells . Remaining of these cytoplasmic droplets in the middle piece of sperm impair sperm maturation. Therefore, it is logical to hypothesize that the adverse effects of PHZ on sperm maturation, during spermatogenesis, exert partly through impairing the normal physiological function of Sertoli cells, which may happen due to PHZ-induced oxidative stress damage. On the other hand, Vit C-treated mice revealed a significant reduction in the percentage of abnormal sperm. Therefore, it can be postulated that Vit C recovers Sertoli cells physiologic activity by improving the antioxidant state. These findings in PHZ-induced low sperm count were in accordance with a previous study (4). This reduction in sperm count may also be associated with a decrease in production of testosterone hormone due to damage to Leydig and Sertoli cells following PHZ supplementation (4).Regarding the AO staining test, which can detect abnormal single-stranded DNA from normal double-stranded sperms, it can be assumed that PHZ elevates denaturation of sperm DNA strands and co-treatment with Vit C reduces sperm DNA damage induced by PHZ, probably through a reduction in ROS production. In accordance with the current results, in a prior study, it was shown that the addition of Vit C to semen and prepared spermatozoa improved sperm parameters quality and DNA integrity following verification (31).About AB test, it should be considered that there are some different reports on the use of vitamins to improve sperm chromatin abnormalities. Silver and colleagues demonstrated that the administration of Vit C and E did not have any positive effects on sperm chromatin maturity or condensation (32). This controversy may be due to the different mechanisms of infertility in the studies and the administered doses.In vitro fertilization and embryonic development in the PHZ-treated group was significantly lower than the control group and the percentage of embryonic arresting was also higher. Studies indicated that there is a positive correlation between sperm parameters quality and fertilization rate, embryonic growth and high incidence of birth defects both in vivo and in vitro . In the present study, Vit C was only able to ameliorate the side effect of PHZ on fertilization rate. Thus, the findings of the present study showed that Vit C supplementation could not improve the percentage of two cell embryos, blastocyst and embryo arresting in mice co-treated with PHZ. Considering these findings, it can be postulated that Vit C significantly but not completely protects against the PHZ-induced testicular damage. The present study is the first report on the relationship between sperm parameters and embryonic development impairments and Vit C supplementation in PHZ-induced male reproductive system disorders. Thus, to compare these findings with other studies, the authors did not find a similar study. However, they were not able to explain why Vit C can only improve the fertilization rate. This may be because the Vit C dose used in this study was not sufficient to recover the embryonic development completely.The testis is sensitive to different stressors including hyperthermia, inflammation, radiation and also exposure to toxic agents that induce apoptosis of germ cells. Sertoli cells in testicular tissue have the main role in supporting normal spermatogenesis by creating a special environment that induces germ cells differentiation and also controls the entry of nutrients, hormones and other agents into seminiferous tubules (35). Thus, Sertoli cells are important in creating blood-testis-barrier and normal physiologic function of the male reproductive system is dependent on the maintenance of this selective physiologic barrier (35).The mechanism by which Vit C enters the adluminal compartment of seminiferous tubules to enter the developing germ cells was an unsolved problem for many years. So it is obvious that for Vit C to reach the adluminal part of seminiferous tubules, it must pass through Sertoli cells by passive diffusion or facilitative transporters (36). Angulo and colleagues explained this problem by showing that Sertoli cells express two functional active Vit C transporters that give the ability to Sertoli cells to metabolize and regulate the delivery of Vit C to germ cells. Thus, Sertoli cells have a major role in controlling Vit C concentration in the adluminal part of seminiferous tubules (37).The data obtained from this study suggest that Vit C as a potent antioxidant can attenuate the detrimental effects of PHZ on sperm parameters and DNA integrity and improve in vitro fertilization rate but is insufficient to restore the in vitro embryonic development and fertility potential.There are no conflicts of interest to declare.


HOX genes are master regulators of cell identity and cell fate during embryonic development. They play a crucial role in embryonic stem cell differentiation into specific lineages and their expression is maintained in adult stem cells along differentiation hierarchies. Aberrant HOX gene expression is found in several cancers where they can function as either oncogenes by sustaining cell proliferation or tumor-suppressor genes by controlling cell differentiation. Emerging evidence shows that abnormal expression of HOX genes is involved in the transformation of adult stem cells into cancer stem cells. Cancer stem cells have been identified in most malignancies and proved to be responsible for cancer initiation, recurrence, and metastasis. In this review, we consider the role of HOX genes in normal and cancer stem cells and discuss how the modulation of HOX gene function could lead to the development of novel therapeutic strategies that target cancer stem cells to halt tumor initiation, progression, and resistance to treatment.Stem cells are undifferentiated cells that have the unique ability to self-renew and differentiate into many different cell types. Their function is controlled by core gene networks whose misregulation can result in aberrant stem cell function and defects of regeneration or neoplasia.  HOX genes encode evolutionarily conserved transcription factors that are expressed under temporal and spatial control to establish patterning and morphogenesis in the vertebrate embryo. They play a critical role during development, and either loss or gain of function of HOX genes can lead to homeotic transformation and abnormal formation of body structures [HOX genes are organized in four paralogous gene clusters  coding for 39 genes. Their expression is tightly controlled by their clustered organization so that their activation in the posterior part of the primitive streak occurs by a process of temporal and spatial collinearity depending on their location within the cluster. The position of HOX genes within the cluster corresponds to their positional expression along the anterior-posterior axis, with expression following the direction from the 3’ side (anterior) to the 5’ (posterior). Their temporal expression is also coordinated so that HOX genes positioned 3’ in the cluster are expressed earlier than those at the 5’ [HOX gene transcription are CDX genes. These are direct targets of Wnt, RA, and FGF and therefore integrate signaling pathway networks to regulate HOX gene expression [ructures . In humat the 5’ . In addit the 5’ ,5. Imporpression .HOX gene expression. These include their chromosomal cluster organization, cis-regulatory elements (enhancers and lncRNA), histone modifications, chromosome boundaries, and 3D chromatin conformation [HOX genes is achieved by methylation of Histone H3 residues by Trithorax (TrxG) and Polycomb (PcG) group proteins, which establish the active H3K4me3 and repressive H3K27me3 marks, respectively. These epigenetic modifiers create bivalent domains and a poised state in the pre-gastrulating embryo by transcriptional control of developmental genes. In the bivalent state, H3K27me3 is dominant over the H3K4me3 mark, with genes controlled by these marks being repressed as default [HOX genes following a temporal and special collinearity [HOX genes in a lineage-specific manner [HOX code and epigenetic memory of differentiated cells [HOX genes. HOTAIR, HOTAIRM1, and HOTTIP are lncRNA located in the cluster, and they have been shown to configure the epigenetic profile of the HOX loci by association with PcG and TrxG proteins and to control gene expression either in cis or in trans [HOXC cluster and required for the silencing of HOXD genes through interaction with the PRC2 complex and for the establishment of the repressive H3K27me3 mark [HOXA cluster that plays a role in regulating the expression of 3’ HOXA genes by preventing the accessibility of the lysine-specific demethylase 6A (UTX) and by delaying the expression of central genes in the cluster. The 5’ HOXA genes are instead regulated by the lncRNA HOTTIP, which is located upstream of HOXA13 [HOX-coded transcription factors can act as either transcriptional activators or repressors of target genes. These consist of factors that regulate diverse biological processes and include a network of transcription factors, signaling molecules, components of signaling pathways, and “realizator” genes that are directly involved in cell differentiation and tissue organization [Different structural and epigenetic parameters have been described contributing to the collinearity of ormation . During inearity ,9. At gac manner . The silc manner ,12. Thered cells . Togethein trans . For insme3 mark . On the f HOXA13 . As a renization ,17. The nization ,19 and tHOX gene homeotic function, but also for regulation of organogenesis. In this process, the function of HOX genes is to control cell proliferation, differentiation, migration, and apoptosis [HOX gene expression profiles in each body region are then sustained in the adult so that cell and tissue characteristics are maintained according to a specific “HOX code” [The concerted actions of HOX proteins and their cofactors are responsible not only for poptosis . The resOX code” .HOX genes is to regulate cell specification and tissue differentiation. During embryogenesis, the expression of HOX genes maintains positional information, and it is epigenetically inherited so that cells can maintain a memory of their identity during the lifetime of an organism. A significant demonstration of the role played by HOX genes in the regulation of cell identity is provided by their expression pattern in adult human fibroblasts. In these cells, HOX gene expression can predict their original embryonic position along the developmental axes [HOXA and HOXD genes reflects the location along the proximal and distal axis of the limbs, whereas HOXC genes correlate with the anterior and posterior axis of the trunk. HOXB genes are instead associated with fibroblasts of non-dermal origin [HOX gene expression profiles in smooth and skeletal muscle cells [At the cellular level, the primary role of tal axes ,22. For le cells ,24.HOX genes play a crucial role in stem cells both in embryonic and in adult tissues by controlling lineage specification, differentiation, and tissue maturation [The expression of HOX proteins in different tissues, or in the same tissue at different anatomical locations, maintains correct cell identities during differentiation. Indeed, it has been demonstrated that turation .HOX genes are not expressed before gastrulation, and they are not transcribed in embryonic stem cells (ESC) derived from the inner cell mass of a blastocyst [HOX genes in ESC are regulated by bivalent chromatin domains so that they are poised for activation after differentiation. As a result, the coverage of the polycomb complex PRC2 in ESC is not as extensive as in differentiated cells with silenced HOX gene expression [HOX genes in their undifferentiated state, with HOX gene activation being induced upon differentiation [HOX lineage specifier genes, whilst PcG proteins maintain cell identity through transcriptional repression of HOX genes that are specific to other lineages [HOX gene cluster during gastrulation [Cdx2 [astocyst . ESC arepression . ESC prentiation . At thislineages ,30. In trulation . This meon [Cdx2  and in hon [Cdx2 .HOX gene expression during ESC differentiation. RA treatment of ESC can significantly increase the induction of neural differentiation and upregulate the expression of HOX genes [HOX genes which respond to RA signaling in a collinear fashion, with genes at 3’ responding to the signal more rapidly than those at the 5’ of the cluster [HOX gene transcription. Indeed, knockdown of RA receptor γ in mouse ESC impairs differentiation and reduces the expression of Hoxa and Hoxb cluster genes as well as the expression of their cofactors Pbx1 and Meis1 [HOX gene regulatory regions during ESC differentiation via recruitment of the H3K27me3 demethylase UTX-KDM6A [HOX genes are involved in the control of cell fate specification and differentiation of ESC into different lineages. For instance, inducible expression of Hoxb1 in mouse ESC can induce the differentiation and expansion of posterior neural stem/progenitor cells [HOXB4 acts as a master regulator of hematopoietic differentiation in both mouse and human pluripotent stem cells [Hoxb4 can in fact regulate multiple transcription factors involved in hematopoiesis and chromatin modifiers, thus playing a role in establishing the epigenetic landscape of the developing hematopoietic stem/progenitor cells [Hox6 gene paralogues  control the differentiation of mouse ESC into insulin producing pancreatic cells [HOX genes during embryonic development is mirrored in ESC and their expression allows the control of ESC differentiation to the three germ layers.RA is another key inducer of OX genes . RA resp cluster ,36. RA rnd Meis1 . ImportaTX-KDM6A ,38,39. Sor cells . On the em cells ,42,43. Hor cells . Finallyic cells . TherefoHOX gene expression during development and differentiation into adult tissues provides a mechanism for imposing cell identity and fate restriction. This information is maintained in adult stem cells (ASC) and along their differentiation hierarchies. This mechanism can be clearly observed in mesenchymal stem cells (MSC). These cells, isolated from different tissues including the bone marrow and adipose tissue, can differentiate to fat, bone, and cartilage. Despite their phenotypic similarity, MSC derived from different tissues present a profile of HOX gene expression that mirrors that of their developmental origin. The topographical specificity of HOX genes in MSC is also maintained after differentiation, indicating that HOX genes play a role in the specification of MSC identity [HOX gene expression. HOXA9, HOXB7, HOXC10 and HOXD6 are expressed in UC-MSC, whereas BM-MSC express HOXB7 and HOXD6 [The positional identity provided by identity ,46. Indend HOXD6 . HOX gene expression can also distinguish stem cell populations of functionally distinct tissues and influence their differentiation potential. For instance, the HOX code of skeletal stem/progenitor cells (SSC) can affect their differentiation into resident tissue osteoblasts. These stem cells show a HOX gene expression profile based on their embryonic origin. Therefore, mouse SSC in the tibia maintain their embryonic identity being of mesodermal origin and expressing Hoxa1. On the other hand, those in the mandibular originate from the neural crest and do not express HOX genes. This positional memory has been shown to affect the differentiation and regeneration potential of SSC when ectopically transplanted as the original HOX code is retained upon transplantation [HOX genes, and therefore they retain the potential to differentiate into three germ layers similarly to ESC [Patterns of antation . A simily to ESC .HOX genes also play a critical role in lineage restriction, as shown during the differentiation of the hematopoietic system. In this lineage, HOXA9, HOXB3, and HOXB4 are expressed in hematopoietic stem cells (HSC) which can differentiate into myeloid cells predominantly expressing HOXA genes, and erythroid and lymphoid cells expressing HOXB and HOXC genes, respectively [HOX genes are also involved in the differentiation of other lineages. For example, HOXA10 mediates osteogenic differentiation [Hoxa3 and Hoxd3 induce differentiation of endothelial cells and angiogenesis in the mouse [HOX genes play a dominant role in neurogenesis and HOXB4 can drive neural differentiation in the neural tube, whereas Hoxb1 is essential for driving mouse neural stem cells to hindbrain [HOX genes in the regulation of terminal differentiation and tissue maturation. For instance, HOXA5 and HOXD10 are required for maintaining differentiated endothelial cells in a mature and quiescence state [Hoxa2 and Hoxb1 regulate the maturation of facial nerves in the mouse [HOX gene expression during terminal differentiation, final tissue maturation can also require downregulation of HOX gene expression. This is the case for mature bone marrow cells that require downregulation of HOX genes specifying the hematopoietic lineage in order to terminally differentiate [HOX code in maintaining ASC function and restriction of differentiation programs. ectively ,50. HOX ntiation , whereashe mouse . In the indbrain . Finallyce state . Similarhe mouse . Althougrentiate . AltogetHOX gene expression in stem cells and their differentiated progenies is critical for normal tissue homeostasis. Consequently, alteration of HOX gene expression can play a critical role in the development of cancer [HOX genes are frequently deregulated in cancer and many studies have shown they can function as tumor modulators by playing either an oncogenic or a tumor suppressive role [HOX genes that are normally expressed in undifferentiated cells drives oncogenesis, whereas downregulation of HOX genes that are normally expressed in differentiated tissues results in the abolition of their function as tumor suppressors [HOX clusters is found in different cancers, including alteration of HOXA genes in breast and cervical cancers, HOXB in colon cancer, HOXC in prostate and lung cancers, and HOXD in colon and breast cancer [HOX genes in cancer is therefore complex, with mechanisms of deregulation of specific HOX genes differing among cancer types [The fidelity of f cancer . HOX genive role . Indeed,pressors . An altet cancer ,59. The er types .HOX genes in maintaining cell identity limits the ability of cells to transition between different phenotypes. However, research on HOX genes during regeneration and wound healing in lower organisms have highlighted a role for these transcription factors in dedifferentiation and in shifting cells between metastable states. Therefore, aberrant HOX gene expression can lead to loss of differentiation and increased cell plasticity in the context of regeneration [The role of neration .In cancer, this type of phenotypic plasticity can drive cells to re-acquire self-renewal and a stem cell phenotype, thus leading to the formation of cancer stem cells (CSC) and tumor initiation . CSC areHOX gene expression is common in cancer and can contribute to CSC plasticity [HOXC8 mediated by MIR-196 and DNA methylation induces a CSC phenotype in normal mammary stem cells, and it results in their increased self-renewal, impaired differentiation, and augmented tumorigenic potential [HOXD9 and HOXA10 in glioma CSC induces cell proliferation and survival [HOXB cluster genes driving the expansion of the malignant HSC pool [HOX-derived lncRNA can also influence the function of CSC [HOXD10 whilst sustaining the expression of genes inducing EMT in breast CSC [HOX cluster, has also been shown to be involved in CSC function. In pancreatic cancer, HOTTIP enhances CSC properties by induction of HOXA9 and activation of the Wnt pathway [Aberrant epigenetic regulation of asticity . By screotential . A similsurvival ,72. SimiHSC pool . Epigenen of CSC . HOTAIR n of CSC , and it east CSC . HOTAIR east CSC ,78,79. H pathway .HOX genes in CSC transformation. For instance, HOXA4 and HOXA9 are enriched during the transformation of colon CSC, and their expression contributes to sustained CSC self-renewal [HOXA9 has also been shown to sustain the self-renewal of HSC in acute myeloid leukemia (AML) and the NUP98-HOXA9 fusion protein induces long-term proliferation and impaired differentiation of leukemic CSC [HOXB3 has been shown to sustain the proliferation and drug resistance of breast CSC [HOXA5 and RA signaling in mammary cells leads to loss of the epithelial phenotype and acquisition of CSC characteristics [HOXA5 plays a similar role in colon cancer, as silencing of HOXA5 by Wnt signaling maintains the pool of CSC and reactivation of HOXA5 by RA treatment induces loss of the CSC  and PI3K/Akt (phosphoinositide 3-kinase/protein kinase B) signaling pathways in breast and gastric cancer, respectively [HOX genes have a direct effect on cell cycle progression by inducing expression of Cyclin D1, like HOXB7 in colorectal cancer [HOXA9 in leukemia [HOXB7 can induce resistance to cytotoxic drugs [HOXA5 results in resistance to apoptosis induced by TP53 in breast cancer [The deregulation of ll death . For exaectively ,90. Othel cancer  and HOXAleukemia . HOX genic drugs ,94, and t cancer .HOX genes is invasion and metastasis. Several HOX genes are involved in the regulation of EMT, a process involved in metastatic spread and acquisition of CSC characteristics. This role has been demonstrated for HOXB7 which promotes EMT in breast cancer [HOXA10 whose silencing induces EMT in endometrial and lung cancer [Another important CSC characteristic regulated by t cancer , and forg cancer ,97. HOX genes in the regulation of both normal and CSC function. Therefore, HOX genes may represent novel cancer biomarkers for targeted therapy and prediction of drug response and prognosis. Although targeting HOX genes represents an exciting opportunity, many challenges remain. First, transcription factors are not easily “druggable”. Targeting these proteins is difficult, mainly due to their intracellular or nuclear localization and their interaction with different cofactors [Therapeutic targeting of CSC is a growing area of research, with drug discovery programs aimed at finding specific therapies that can eradicate this tumor initiating and disseminating cell population. Current evidence supports an important role played by ofactors . This liHOX genes. This approach is also particularly important when considering the functional versatility of HOX genes. As previously discussed, the same HOX gene can play different roles by functioning either as an oncogene or a tumor suppressor in different cancer types. In addition, some HOX genes with conserved patterning function present a high degree of redundancy, and therefore the inhibition of one gene can be overcome by expression of a paralogous gene or another gene in the same cluster [HOX genes in a specific cellular context. The inhibition of HOX–PBX complexes has already been achieved by using the antagonist peptide HXR9 which can induce apoptosis in a number of cancers, including solid tumors of the breast, prostate, ovarian, kidney, skin, lung, and blood tumors such as myeloma and AML [Another possibility would be to interfere with targets downstream of the  cluster ,100. Tar and AML .HOXA5 and HOXD10 are expressed in normal epithelial and endothelial cells in the breast and their silencing is associated with CSC expansion and cell migration. Restoring the expression of both HOX genes could therefore be more effective and limit plasticity by targeting both CSC and angiogenesis [Although these approaches seem promising, therapeutic targeting of CSC remains a clinical challenge, mainly due to their inherent plasticity that is influenced by the tumor microenvironment. Therefore, strategies aimed at targeting the microenvironment as well as CSC could prove effective. For example, ogenesis ,103,104.HOX gene expression in CSC and affect their function by reducing self-renewal and promoting differentiation [HOX gene expression. RA can induce terminal differentiation of CSC, and it is currently used as standard of care for the treatment of acute promyelocytic leukemia [HOXC8 expression in breast CSC [HOXA5 in colon and breast CSC [Epigenetic therapies could also be instrumental for targeting ntiation . This apleukemia . We haveeast CSC , and otheast CSC ,86.HOX gene expression could also inform cancer prognosis given that cancers that are enriched with CSC are also associated with a worse patient outcome. An association between HOX expression and poor prognosis has already been established for breast cancer and lung adenocarcinoma (HOXB7), renal clear cell carcinoma (HOXC11), mesothelioma (HOXB4), oral squamous cell carcinoma (HOXD13), gastric cancer (HOXC6), thyroid cancer (HOXC10), bladder cancer (HOXB13), and AML (HOXA9) [HOX gene profiles in CSC defines breast cancer molecular subtypes, suggesting that their expression could be used as a blueprint for defining the cell of origin of different types of cancer and inform clinical decisions [ (HOXA9) . In addi (HOXA9) . We haveecisions .HOX genes are considered master regulators of cell fate determination during embryonic patterning. Positional and cell-specific HOX identities established during development are maintained during adulthood and enable tissue homeostasis. A specific HOX code is concealed in adult stem cells, which allows their self-renewal and differentiation potential. This code is then resolved during differentiation into different lineages, resulting in tissue-specific expression of HOX genes. In cancer, erasure of the HOX code causes a loss of cell identity and acquisition of a transformed phenotype and CSC characteristics (HOX genes in the function of normal and cancer stem cells, but the molecular mechanisms by which HOX gene deregulation alters stem cell function are still not fully known. Therefore, large scale genomic and proteomic approaches are needed to elucidate the function of HOX genes and their mode of action in regulating stem cells in normal and cancerous tissues. Given the critical role of HOX genes in lineage specification and acquisition of cell identity, HOX profiles could allow the development of new diagnostic and prognostic tools for personalized clinical management of cancer patients. In addition, they could be used to devise targeted therapies that could eliminate the so far elusive CSC.eristics . Current


Papio anubis), as a model for vertical transfer of ZIKV during pregnancy. Four mid-gestation, timed-pregnant baboons were inoculated with the French Polynesian ZIKV isolate (104 ffu). This study specifically focused on the acute phase of vertical transfer. Dams were terminated at 7 days post infection , 14 dpi (n = 2) and 21 dpi (n = 1). All dams exhibited mild to moderate rash and conjunctivitis. Viremia peaked at 5–7 dpi with only one of three dams remaining mildly viremic at 14 dpi. An anti-ZIKV IgM response was observed by 14 dpi in all three dams studied to this stage, and two dams developed a neutralizing IgG response by either 14 dpi or 21 dpi, the latter included transfer of the IgG to the fetus (cord blood). A systemic inflammatory response  was observed in three of four dams. Vertical transfer of ZIKV to the placenta was observed in three pregnancies (n = 2 at 14 dpi and n = 1 at 21 dpi) and ZIKV was detected in fetal tissues in two pregnancies: one associated with fetal death at ~14 dpi, and the other in a viable fetus at 21 dpi. ZIKV RNA was detected in the fetal cerebral cortex and other tissues of both of these fetuses. In the fetus studied at 21 dpi with vertical transfer of virus to the CNS, the frontal cerebral cortex exhibited notable defects in radial glia, radial glial fibers, disorganized migration of immature neurons to the cortical layers, and signs of pathology in immature oligodendrocytes. In addition, indices of pronounced neuroinflammation were observed including astrogliosis, increased microglia and IL6 expression. Of interest, in one fetus examined at 14 dpi without detection of ZIKV RNA in brain and other fetal tissues, increased neuroinflammation (IL6 and microglia) was observed in the cortex. Although the placenta of the 14 dpi dam with fetal death showed considerable pathology, only minor pathology was noted in the other three placentas. ZIKV was detected immunohistochemically in two placentas (14 dpi) and one placenta at 21 dpi but not at 7 dpi. This is the first study to examine the early events of vertical transfer of ZIKV in a NHP infected at mid-gestation. The baboon thus represents an additional NHP as a model for ZIKV induced brain pathologies to contrast and compare to humans as well as other NHPs.Zika virus (ZIKV) infection during pregnancy in humans is associated with an increased incidence of congenital anomalies including microcephaly as well as fetal death and miscarriage and collectively has been referred to as Congenital Zika Syndrome (CZS). Animal models for ZIKV infection in pregnancy have been developed including mice and non-human primates (NHPs). In macaques, fetal CZS outcomes from maternal ZIKV infection range from none to significant. In the present study we develop the olive baboon , with the most severe pathology being fetal microcephaly. Developing model organisms that faithfully recreate Zika infection in humans is critical for future development of treatments and preventions. In our present study, we infected Olive baboons at mid-gestation with Zika virus and studied the acute period of viremia and transfer of Zika virus to the fetus during the first three weeks after infection to better understand the timing and mechanisms of transfer of ZIKV across the placenta, leading to CZS. We observed Zika virus transfer to fetuses resulting in fetal death in one pregnancy and in a second pregnancy, significant damage to the frontal cortex of the fetal brain at a critical period of neurodevelopment in primates. Thus, the baboon provides a promising new non-human primate model to further compare and contrast the consequences of Zika virus infection in pregnancy to humans and other non-human primates. Flaviviridae family, genus Flavivirus, which includes dengue (DENV), West Nile (WNV), yellow fever (YFV), and Japanese encephalitis virus (JEV) ).Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay was performed using a commercially available kit from Trevigen . Briefly, fixed frozen fetal cortical sections were washed with PBS and incubated with Cytonin for 30 min, washed, and quenched before labeling with biotin-labeled dUTP. The labeling reaction was stopped by adding stop buffer. The tissue sections were then incubated with HRP-conjugated streptavidin for 10 min, washed, and immersed in DAB solution for color development. Sections were counterstained with methyl green before mounting. A positive and a negative control sections were included in the protocol. Apoptotic cells are stained with dark brown color.


Pharmacological targeting of KIT in gastrointestinal stromal tumours has dramatically changed the clinical outcome of this disease. Tyrosine kinase inhibitors are the cornerstone of this improvement, but resistance occurs through secondary KIT mutations. Studies aimed at improving our understanding of the molecular basis of sensitivity and resistance will soon allow us to further tailor our therapeutic strategies. Imatinib has been so effective in providing long-term disease control and prolonging overall survival in patients with KIT-mutated GISTs2 that time to definitive failure of imatinib is now recognised as a novel endpoint in clinical trials, in both adjuvant and advanced settings.3 In fact, imatinib continues to provide disease control in 10% of patients at the 10-year landmark,2 supporting the hypothesis that a subgroup of patients remain sensitive to imatinib despite the drug-selective pressure. In the majority of cases, however, GISTs eventually develop resistance to imatinib due to the emergence of subclones harbouring secondary KIT mutations.The start of the twenty-first century witnessed a paradigm shift in medical oncology, with the identification and pharmacological targeting of activating mutations in the tyrosine kinase KIT of gastrointestinal stromal tumours (GISTs) by imatinib.5 most likely due to the heterogeneity of secondary mutations in imatinib-resistant GISTs. A relationship between specific secondary KIT mutations and sensitivity to TKIs has previously been proposed using transfected animal cell models,6 but never validated using patient-derived GIST cells. In this issue of the British Journal of Cancer, Serrano et al.7 report on the activity of nine TKIs that have either been approved or are under clinical investigation as KIT inhibitors for GISTs, against imatinib-resistant GIST cell lines with different secondary KIT mutations. Secondary KIT mutations are known to arise most commonly in exons 13/14  or exons 17/18 , whereas primary KIT mutations predominantly affect the juxtamembrane domain encoded by exon 11. Among the approved agents, sunitinib showed marked activity against KIT exon 11 mutations coupled with a secondary mutation in exon 13, whereas regorafenib was only active against KIT exon 11 mutations coupled with secondary mutations in exon 17 or exon 18; both drugs were active against KIT exon 11 mutations coupled with an exon 14 mutation  of 22.1 months has been reported in a small phase II trial that investigated this TKI in GIST patients with KIT exon 17 mutations,9 compared to a median PFS of 4.8 months in the phase III registration trial, which included patients irrespective of their secondary mutations.6The above-described translational findings are clinically relevant. The poor activity of sunitinib against GISTs that harbour secondary mutations involving the KIT activation loop (exons 17 and 18) was discovered soon after its approval.7 also showed in vitro that rapid alternation of sunitinib and regorafenib is more effective than monotherapy using either drug. The hypothesis that selective pressure using specific TKIs might favour the expansion of different subclones is valid, and deserves to be further explored in a prospective trial (NCT02164240). Nonetheless, treatment with sunitinib and regorafenib might also eventually lead to the re-expansion of imatinib-sensitive clones. Indeed, we recently showed that rechallenge with imatinib after combined treatment with sunitinib and regorafenib in advanced GIST is associated with clinically significant disease control rates.10Importantly, using polyclonal cultures with different mutations, which more closely mimic the clinical heterogeneity of imatinib-resistant GIST, Serrano et al.As we begin to better understand the relationship between specific KIT mutations and the activity of different TKIs, we must also understand the relevance of these findings outside of clinical trials. Re-biopsy of imatinib-progressive disease to evaluate secondary mutations is uncommon in most centres and would only be clinically relevant in the presence of oligoprogressive disease. Although research in this field is active, using circulating tumour DNA sequencing as a surrogate source to provide a comprehensive record of all secondary KIT mutations that are simultaneously present in a single patient still requires further validation, both technically and clinically.7 was correctly based might soon profoundly change. In fact, the ongoing VOYAGER trial (NCT03465722) is investigating the novel TKI avapritinib (formerly known as BLU-285) against regorafenib as a third- or fourth-line treatment. DCC-2618, another promising TKI, will be compared with sunitinib as second-line therapy in the planned INTRIGUE trial (NCT03673501). These potential changes to the therapeutic sequence might result in the development of novel unrecognised secondary KIT mutations, due to the selective pressure of novel TKIs. Nevertheless, the work by Serrano et al. provides us with a translational research framework to assess the next generation of TKIs and the best therapeutic sequences and/or rotations to minimise the emergence of resistant clones, in order to simultaneously improve our understanding of GIST molecular biology and patient outcomes.It is also important to acknowledge that the therapeutic sequence on which the paper by Serrano et al.


Pheochromocytomas (PCCs) and paragangliomas (PGLs) are rare chromaffin cell tumors (PPGLs) that at times raise significant challenges in clinical recognition, diagnosis, and therapy and when undiagnosed could associate with severe morbidity. Recent discoveries in PPGL genetics propelled our understanding in the pathophysiology of tumorigenesis and allowed the application of functional classification of pathogenetically distinct groups of PPGLs. This also resulted in a qualitative change in our approach to clinical assessment, diagnosis, and therapy of different subgroups of PPGLs. Establishment of the fact that mutations in multiple components of the PHD–VHL–HIF-2α pathway associate with pseudohypoxia-driven tumorigenesis allowed us not only to better understand the effect of this phenomenon but also to more deeply appreciate the value of functional abnormalities in the physiologic tissue oxygen-sensing mechanism. Mutations in the tricarboxylic acid cycle–related genes opened an additional window into understanding the physiology of one of the basic cellular metabolic pathways and consequences of its disruption. Mutations in the kinase signaling–related genes allow the PPGL field to join a massive innovative process in therapeutic advances in current oncology. New pathophysiologically distinct groups of mutations will widen and deepen our understanding of additional pathways in PPGL tumorigenesis and hopefully introduce additional diagnostic and therapeutic approaches. All of these developments are tremendously important in our understanding of both the normal physiology and pathophysiology of PPGLs and are strong tools and stimuli in the development of modern approaches to all components of medical management. Though once thought of as being mostly benign and biochemically active, these tumors could show a wide spectrum of cellular and biochemical de-differentiation, including aggressive metastatic course and biochemical silence. Despite being anatomically distinct, PCCs and PGLs display a common pathologic basis and frequently are referred to as pheochromocytoma/paraganglioma tumors (PPGLs), the term we will use throughout this article. The incidence and prevalence of PPGLs are based on older literature, which in part paralleled our understanding of the field and could be misleading to some degree. Overall, it is stated that the annual incidence of PCC is between two and eight cases per million, whereas in patients with hypertension, the prevalence of PPGL varies between 0.2 and 0.6%. It is important to remember that in patients with incidentally radiologically discovered adrenal masses, 5% will have PCCs (reviewed inPheochromocytomas (PCCs) and paragangliomas (PGLs) are chromaffin cell tumors derived from adrenal (PCC) or extra-adrenal sympathetic or parasympathetic paraganglia (PGL)5.Recent years have tremendously changed our understanding of each and every aspect of PPGL biology—genetics, clinical and biochemical behavior, pathogenesis, diagnosis, and treatment—which to a major degree also propelled our understanding of PPGL-related and general pathophysiology of tumorigenesis. The PPGL field has also undergone a significant transformation in recent years. In older times, pheochromocytoma was seen as a mostly sporadic and benign disease, usually seen by endocrinology and surgery. Hereditary cases were relatively rare and included MEN2, neurofibromatosis type 1, and von Hippel–Lindau syndromes. Sympathetic paragangliomas were usually managed by the anatomically related  surgical specialties with significantly more frequent involvement of medical oncology and endocrinology on an “as needed basis”. Parasympathetic silent head-and-neck paragangliomas were seldom seen as part of the PPGL syndrome and were managed by ear, nose, and throat (ENT) and oncology. With increasing understanding of the high heritability of PPGL and similarity in pathology of seemingly discrete conditions, the field has transformed into all-inclusive and comprehensive medicine approach-based multispecialty management. We now know that PPGLs represent the highest hereditary-driven endocrine condition—up to 40% of cases are related to mutations in 15 well-established driver genes and a growing number of disease-modifying genes and these numbers are expected to growAs our understanding of PPGLs deepens, it seems that current anatomic  or standard staging  classifications fell short of providing sufficient insight into pathogenesis, clinical presentation, and (perhaps more importantly) prognostic value with respect to malignant potential, response to therapy, and possible recurrences. Functional, pathogenesis-based classification does allow a better understanding of actual tumorigenesis, expected biochemical profiles, tumor location, and malignant potential. The more recently formulated group of disease-modifying genes is expected to expand and possibly explain tumor formation in a growing number of apparently mutation-free cases .This functional classification of currently established susceptibility genes consists of four major groups: the pseudohypoxia group, the kinase signaling group, the Wnt signaling group, and the disease-modifying gene group.This group can be further subdivided to the following subgroups:VHL), Elegans homologs (EGLN1–3), and endothelial PAS domain protein 1 (EPAS1) gene.VHL, expressing both germline and somatic mutation patterns, shows mutation in up to 100% of patients withVHL-related PPGL. The product ofVHL is a von Hippel–Lindau tumor suppressor protein (pVHL), which serves as a component of an E3 ubiquitin ligase complex. The mechanism of tumorigenesis in this condition is related to the generation of the pseudoxypoxic metabolic state related to the stabilization of the hypoxia-inducible factor (HIF) , which will bind to the core DNA RCTCG sequence at the hypoxia-responsive elements (HREs) to activate transcription of multiple genes that could associate with tumor formation, including glucose transporter 1 (GLUT1), vascular endothelial growth factor (VEGF), and many others.Historically, this group was the first window into our understanding of the pathophysiology of the development of PPGLs. It represents 15 to 20% of PPGLs, which are hereditary in 25% of cases, and includes tumors associated with mutations in von Hippel–Lindau tumor suppressor gene  subunitsA,B,C, andD;SDHAF2 assembly factor forSDH; and fumarate hydroxylase (FH). These tumors represent about 10 to 15% of all PPGLs, are close to 100% hereditary, and functionally represent a tumor suppressor gene mutation-related tumorigenesis. SDH is a dual-functional mitochondrial enzyme, which converts succinate to fumarate in tricarboxylic acid (TCA), and participates as a complex II in the respiratory electron transfer within the mitochondrial membrane. FH subsequently converts fumarate to malate within the same TCA cycle (reviewed inMH2)—the enzyme that converts malate to oxaloacetate further down the TCA—that associate with PGL in a single family12. The exact mechanism of tumorigenesis is thought to be related to oncogenic properties of accumulated TCA metabolites—succinate, fumarate, and malate—by inhibiting enzymes involved in cell signaling and chromatin maintenance13. Succinate and fumarate can inhibit PDH, thus activating the HIF-related tumorigenesis pathway. In addition, both can drive epigenomic tumorigenesis through dose-dependent DNA hypermethylation by inhibition of α-ketoglutarate–dependent oxidative demethylation10.These are germline truncating mutations in succinate dehydrogenase (RET) proto-oncogene and H-ras GTPase proto-oncogene (HRAS)—and several tumor suppressors: neurofibromin 1 (NF1), transmembrane protein 127 (TMEM127), and MYC-associated factor X (MAX). These mutations are associated with the activation of kinase signaling pathways: RAS-RAF-MEK and PI3K-AKT-mTOR forRET,HRAS,NF1, andTMEM127 and MYC-MAX forMAX.This group comprises the majority of PPGLs (50 to 60%) and is driven by either germline or somatic mutations in up to 20% of cases. It includes oncogenes—rearranged during transfection  genes are frequently found in human cancers and function as activators of cell growth and survival. Of multiple group members, only Harvey (H-ras) and Kirsten (K-ras) were shown to associate with PPGL formation.Rat sarcoma viral oncogene  and oncogene mastermind-like transcriptional coactivator 3 (MAML3).Tumors in this group occur in 5 to 10% of PPGLs and in most cases are related to somatic mutations in tumor suppressor cold shock domain-containing E1  and prolyl hydroxylase activators (R59949 and KRH102053) that promote HIF hydroxylation, thus restoring VHL-driven recognition and rapid degradation. These could switch the pseudoxypoxic signal and potentially disrupt the tumorigenesis process at the actual pathogenetic point15. The possibility of multiple additional clinical presentations and novel mutations will further boost our ability to more deeply understand and affect the pathogenesis of this fascinating condition. One example would be a recent report of PPGL associated with cardiac dysplasia16. There are still plenty of questions waiting to be answered in relation to the particular mechanisms of tumorigenesis and possible interventions early in the course of the disease to prevent possible adverse outcomes. This will definitely improve with new knowledge and experience.Recent advances in our understanding of the pathogenesis of PPGL had significantly changed our approach to the condition. The change in classification moved different forms of PPGLs into pathogenesis-driven groups, which allowed a better understanding of major disease-related features: clinical presentation, biochemical approach, and predisposition to malignant course and possible differences in therapy and follow-up. It had also brought back some old concepts—like Warburg effect (tumorigenesis related to accelerated glycolysis)—that fit well into the model of pseudohypoxia-driven disease. Understanding differential pathogenetic pathways in different forms of PPGL allowed a better design of biochemical tests with a recent use of metabolomics through high-end nuclear magnetic resonance spectroscopy as a differential tool on a biochemical level