Idiopathic and heritable pulmonary arterial hypertension form a rare but molecularly heterogeneous disease group. We aimed to measure and validate differences in plasma concentrations of proteins that are associated with survival in patients with idiopathic or heritable pulmonary arterial hypertension to improve risk stratification.In this observational cohort study, we enrolled patients with idiopathic or heritable pulmonary arterial hypertension from London , Giessen , and Paris . Blood samples were collected at routine clinical appointment visits, clinical data were collected within 30 days of blood sampling, and biochemical data were collected within 7 days of blood sampling. We used an aptamer-based assay of 1129 plasma proteins, and patient clinical details were concealed to the technicians. We identified a panel of prognostic proteins, confirmed with alternative targeted assays, which we evaluated against the established prognostic risk equation for pulmonary arterial hypertension derived from the REVEAL registry. All-cause mortality was the primary endpoint.20 proteins differentiated survivors and non-survivors in 143 consecutive patients with idiopathic or heritable pulmonary arterial hypertension with 2 years' follow-up (cohort 1) and in a further 75 patients with 2·5 years' follow-up (cohort 2). Nine proteins were both prognostic independent of plasma NT-proBNP concentrations and confirmed by targeted assays. The functions of these proteins relate to myocardial stress, inflammation, pulmonary vascular cellular dysfunction and structural dysregulation, iron status, and coagulation. A cutoff-based score using the panel of nine proteins provided prognostic information independent of the REVEAL equation, improving the C statistic from area under the curve 0·83  to 0·91  and improving reclassification indices without detriment to calibration. Poor survival was preceded by an adverse change in panel score in paired samples from 43 incident patients with pulmonary arterial hypertension in cohort 3 (p=0·0133). The protein panel was validated in 93 patients with idiopathic or heritable pulmonary arterial hypertension in cohort 4, with 4·4 years' follow-up and improved risk estimates, providing complementary information to the clinical risk equation.A combination of nine circulating proteins identifies patients with pulmonary arterial hypertension with a high risk of mortality, independent of existing clinical assessments, and might have a use in clinical management and the evaluation of new therapies.National Institute for Health Research, Wellcome Trust, British Heart Foundation, Assistance Publique-Hôpitaux de Paris, Inserm, Université Paris-Sud, and Agence Nationale de la Recherche. Idiopathic and heritable pulmonary arterial hypertension constitute a rare disease group characterised by an imbalance in endothelial-derived vasoactive factors, inflammation, and structural remodelling of pulmonary vessels.Evidence before this studyWe searched PubMed for relevant articles  with search terms including “pulmonary arterial hypertension”, “prognostic”, “proteomics”, and “biomarker”. Several studies report single biomarkers for pulmonary arterial hypertension, usually derived from other diseases, but none have undertaken unbiased screening of large numbers of plasma markers and related these to outcomes in pulmonary arterial hypertension. The prognostication of pulmonary arterial hypertension remains poor. Pulmonary arterial hypertension is diagnosed at cardiac catheterisation. Thereafter a combination of exercise capacity , patient-reported symptoms , echocardiography, and circulating NT-proBNP concentrations—captured in prognostic equations such as the REVEAL score—are used to follow disease progression, response to treatment, and make clinical management decisions. Not all these measurements are made at each visit and some  are subject to confounding factors. Better, non-invasive, and objective methods of assessment are needed that can be deployed in the clinical setting.Added value of this studyWe did an unbiased screen of 1129 proteins measured in plasma samples collected on routine clinic visits. Measurement of circulating concentrations of nine proteins in combination predicted survival, which outperformed traditional clinical assessments. A prognostic score on the basis of plasma concentrations of the nine proteins was validated in independent cohorts from three countries  and is relevant to both incident and prevalent cases of pulmonary arterial hypertension. An increase in the panel score over time is associated with increased mortality.Implications of all the available evidenceThe guidelines for prognostication in pulmonary arterial hypertension recommend the use of only one blood biomarker, BNP or NT-proBNP, and consideration of a multitude of clinical measures, which when formalised into risk equations perform only moderately well in predicting outcomes. These data suggest that a panel of nine proteins, which report on different pathogenic mechanisms linked to pulmonary arterial hypertension, can be used to stratify patients according to risk and assess response to treatment better than existing clinical tools. Further investigation of the pathways represented in the protein panel might also offer new insights for the development of novel therapies.The existing management guidelines include the measurement of plasma brain natriuretic peptide (BNP) or N-terminal proBNP (NT-proBNP) concentrations, an indicator of right ventricular function, in the assessment of patients with pulmonary arterial hypertension.Proteomics offers an unbiased approach to identifying and quantifying multiple biomarkers representative of disease processes. Mass spectrometry-based proteomic analysis of lung tissue, plasma, and cultured cells from patients with pulmonary arterial hypertension has identified a small number of dysregulated proteins.17We used a SomaScan array to measure concentrations of 1129 proteins in plasma to identify and validate circulating proteomic signatures that predict survival of patients with idiopathic or heritable pulmonary arterial hypertension.In this multicentre, observational cohort study, we identified and analysed four cohorts of patients with idiopathic or heritable pulmonary arterial hypertension from three expert centres recognised internationally as centres of excellence for pulmonary arterial hypertension diagnosis and management in London , Giessen , and Paris . The diagnostic criteria for idiopathic or heritable pulmonary arterial hypertension over the course of this study were stable: raised mean pulmonary artery pressure of more than 25 mm Hg, with pulmonary capillary wedge pressure less than 15 mm Hg (and pulmonary vascular resistance [PVR] >3 Wood units) at rest with exclusion of known associated diseases. The guidelines quoted are internationally agreed. Samples from 25 healthy controls were also collected at Hammersmith Hospital for comparison of proteomic and alternative assay measurements.Patients were not fasting and were sampled at their routine clinical appointment visits . Peripheral venous blood samples were collected using EDTA (edetic acid) for cohorts 1, 2, and 3 or sodium citrate Vacutainer tubes  for cohort 4, immediately put on ice, centrifuged  within 30 min of collection, and plasma aliquots were stored at −80°C until required. The plasma samples underwent one freeze–thaw cycle to aliquot 120 μL for the SomaScan assay and provide other aliquots for NT-proBNP and targeted assays. Clinical data were collected within 30 days of blood sampling and biochemical data were collected within 7 days of blood sampling.All-cause mortality was the primary endpoint. In a secondary analysis, lung transplantation or death was used as a composite endpoint.U test and Kruskal–Wallis test for categorical variables.We present differences in protein concentrations by subtraction of log relative fluorescence units. We assessed the association between patient characteristics and biomarkers by Spearman's rank test or Mann–Whitney We did survival analyses using time from sampling to death or censoring. We compared the protein concentrations of survivors and non-survivors  with Mann–Whitney tests to maximise the power of the protein validation analysis. We used random sample analysis to assess robustness of differences between survivors and non-survivors: we repeated Mann–Whitney analyses 18 times in both discovery and validation (cohorts 1 and 2), each time removing one patient out of six patients in three randomised blocks, with each sample left out of three analyses.We tested two panel scoring systems. One system was based on a simple count of proteins indicating risk on the basis of receiver operating characteristic (ROC) cutoffs, and the second was a Cox regression model, in which predicted hazard based on continuous biomarker concentrations was calculated on the basis of fitting to the discovery cohort.For assessment of discrimination, we used ROC curves to compare prognostic discriminatory power of biomarkers. Kaplan–Meier plots illustrated events (deaths) in relation to biomarker levels and predicted risk in Cox models, assessed by the log-rank test. We fitted the simple panel score and REVEAL equation The funders of the study had no role in the study design, data collection, data analysis, data interpretation, or writing of the report. The corresponding author had full access to all the data in the study and had the final responsibility for the decision to submit for publication.Cohorts 1 and 2 were censored on May 15, 2014, cohort 3 on May 21, 2015, and cohort 4 on June 1, 2014. We assessed patients for eligibility between Oct 25, 2011, and Aug 13, 2013, for cohort 1, between Oct 24, 2002, and June 22, 2011, for cohort 2, between Aug 27, 2003, and Nov 19, 2012, for cohort 3, and between June 2, 2003, and Dec 23, 2011, for cohort 4 . At the Concentrations of 134 proteins were associated with overall survival in cohort 1 . 40 of tWe investigated whether the 20 prognostic proteins offered an improvement in risk estimation in addition to the only prognostic protein biomarker currently in use in pulmonary arterial hypertension, namely NT-proBNP. 14 of 20 proteins were each prognostic independent of NT-proBNP in Cox models with death as the primary endpoint —interleukin-1 receptor-like 1 (IL1R1/ST2), tissue inhibitors of metalloproteinases (TIMP-1 and TIMP-2), plasminogen, apolipoprotein-E (ApoE), erythropoietin (EPO), complement factor H and factor D, and insulin-like growth factor binding protein-1 (IGFBP-1). The measurements for these nine proteins allowed us to derive threshold protein concentrations associated with survival , and we We used the prognostic thresholds for each of these nine proteins to produce a protein panel score for each patient, whereby each protein indicating risk  added 1 to a patient's score. This calculation produced scores ranging from 0 to 9 for each patient and discriminated non-survivors in discovery  and validation . The simplified scoring of each protein based on a cutoff performed as well as an equation using continuous protein concentrations, which was also derived in cohort 1 and tested in cohort 2 . Although changes in the concentrations of any individual protein, including NT-proBNP, were not associated with outcome, an increasing panel score was prognostic , validation (cohort 2), and random sampling analysis (cohorts 1 and 2), which gave us confidence in our approach. In the final analyses, a panel of nine proteins provided information independent of the most up-to-date risk equation incorporating many clinical variables. The protein components of this panel report on different pathways recognised in the pathophysiology of pulmonary arterial hypertension and collectively are more informative than when used individually. ST2, secreted in response to stretching myocardiocytes,Effective clinical management of patients with idiopathic or heritable pulmonary arterial hypertension requires the early recognition of patients who are failing to respond to treatment and need alternative or additional targeted therapies. The existing assessment of patients is dependent on subjective reporting of wellbeing to assign functional class, functional tests such as the 6-min walk test, which lacks specificity, and the availability of data from echocardiography.This study represents a comprehensive analysis of the circulating proteome in patients with idiopathic or heritable pulmonary arterial hypertension, leading to the discovery of a panel of proteins capable of predicting mortality more accurately than established measurements. Although the number of proteins assayed represents a broad range of proteins with disparate functions, the proteins studied are limited by the aptamers developed for the assay. Pulmonary arterial hypertension is a rare disease, and the numbers of patients in this study preclude the analysis of interactions between proteins that might improve prognostication. The importance of changes in protein concentrations over time and in response to various therapeutic strategies requires further prospective study, with assessments of the effect of interval between samples. The heterogeneity of pulmonary arterial hypertension and of the patients studied in these cohorts  means that the proteins and biological pathways identified in this study might not always be the most important in each individual with pulmonary arterial hypertension. We did the analyses on independent cohorts of patients recruited at three distinct international centres of expertise. The differences in occurrence of death were primarily because of different durations of follow-up and preclusion of long-term survivors in cohort 1 (2011–13) from cohort 2 (2002–11). The effect of different therapies on the concentration of the proteins was not studied and would require sampling of the same subjects before and after initiation of specific therapies at set timepoints. Fasting and dietary status and the method of blood sampling are known to affect proteomic measurements, but we did not study this here. We validated proteomic measurements with alternative experimental methods and similar results were observed in both EDTA-preserved and sodium citrate-preserved plasma. Blood samples were collected alongside routine clinical plasma samples, showing the practical deployment of this protein panel in a clinical setting. The cost of these blood protein measurements would be relatively low compared with more complex clinical procedures. The development of a dedicated assay to measure all nine proteins together would simplify its clinical use.The prognostic nine-protein panel score powerfully selects subgroups of patients that are likely to have events (death or transplantation), which could be beneficial for targeting aggressive therapeutic strategies or maximising the power of clinical trials. The proteins identified warrant mechanistic evaluation in addition to examination in other distinct disease groups, including other forms of pulmonary hypertension and cardiovascular disease.


Editorial on the Research TopicUnderstanding Crohn’s Disease: Immunity, Genes and MicrobesCrohn’s disease (CD) is a chronic debilitating syndrome, associated with considerable morbidity and resulting in elevated public health costs every year. CD and ulcerative colitis (UC) are the two major forms of inflammatory bowel disease (IBD), which has long been characterized as an exacerbated inflammatory response to common antigenic stimuli in the gut due to immune dysregulation. Although both CD and UC share some common patterns, they are distinct diseases. In fact, while UC is characterized by a diffuse mucosal inflammation involving mainly the rectum and adjacent colonic tissue, CD is a transmural inflammatory disease that may involve any part of the gastrointestinal tract, from the mouth to the anus, although in most cases the terminal ileum is affected. Etiology of IBD, particularly CD, has been long debated and is likely to involve the contribution of multiple factors, making its study challenging. Among those contributing factors are genetic inheritance, epigenetic mechanisms, infection with particular pathobionts, and the gut microbiota in general. This topic aimed at bringing together contributions covering aspects related primarily to CD etiology and immunopathology, helping to drive forward a more comprehensive understanding of this challenging syndrome.Loddo and Romano point out the importance of genetic traits in susceptibility to IBD. The authors discuss the success of next-generation sequencing in the investigation of rare monogenic susceptibility variants, implicated in early-onset and very early-onset IBD. They also address the importance of epigenetic mechanisms, such as DNA methylation, in linking environmental stress and gene expression and discuss the use of microRNAs as biomarkers and therapeutical targets in IBD. The first genetic variant conferring susceptibility to ileal CD was located in the nucleotide oligomerization domain 2 (NOD2) gene  exhibited deficient binding to receptor-interacting protein kinase 2, an adaptor protein implicated in NOD2 signaling. They found that impairment of NOD2 signaling shown by variants containing CD-susceptibility polymorphisms did not correlate with deficient RIP2 binding, concluding that the causes for impairment are multifactorial.Genetic inheritance is an important predisposing factor for IBD. In a comprehensive review, D2) gene , a gene Di Giovangiulio et al. contributed with a comprehensive review on the neuromodulation of the gut immune system. Indeed, the cross talk between the nervous system and the gut immune system can shape the mucosal immune response to gut antigens and contribute to gut homeostasis. It is widely accepted that T cell function, particularly the interplay between regulatory and effector T cells, is of major importance in the maintenance of gut tolerance to intestinal antigens. Sarrabayrouse et al. discuss the important role played by a unique human subset of Treg cells, the IL-10-producers CD4CD8αα cells present in the intestinal mucosa. These cells are induced by clostridial bacteria to suppress T cell proliferation in a process independent on Foxp3 expression, since CD4CD8αα subset does not express this transcription factor. In a mini-review, Omenetti and Pizarro discuss the dynamic balance between intestinal Th17 and Treg cells, the plasticity allowing interdifferentiation and the participation of the gut microbiome in driving the differentiation toward each phenotype. Mucosal-associated invariant T cells (MAIT cells) are a non-conventional T cell subset possibly playing a role in CD  on gut microbiota composition, leading to dysbiosis and consequently to disease onset. Sechi and Dow mini-review addresses the long debated participation of Mycobacterium avium subsp. paratuberculosis (MAP) in CD etiology, discussing MAP exposure, human genetic susceptibility to mycobacterial infection and MAP possible involvement in CD, and other human inflammatory diseases.In recent years, evidence is accumulating in favor of a central role of gut microbiota in CD pathogenesis. Three reviews included in this topic highlight the relevance of gut microbiota composition in health and disease. Ferguson discusses the beneficial effects of directed nutritional therapy for CD patients, according to the genetic background and presence of particular susceptibility polymorphisms. Although data on the benefits of particular nutrients already exist, systems biology approaches would allow to validate efficacy of particular diet constituents to CD patients. This would help to drive nutritional therapy into the primary therapy in CD, integrated with currently accepted approaches.Finally, in line with increasing recognition of the role played by nutrition in disease prophylaxis and therapy, I hereby wish to manifest my appreciation to all the authors that participated in this research topic. Their articles significantly contributed to a more comprehensive view on CD immunopathology and associated factors, helping to improve current understanding of this debilitating disease.The author confirms being the sole contributor of this work and approved it for publication.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The interferon (IFN) response is a critical component of the innate immunity antiviral pathways in mammalians. IFN signaling results in increased expression of cellular factors that block key steps in the viral replication cycle. Many IFN-induced antiviral factors act through decreasing viral entry, replication, transcription, translation, packaging and release. However, these effects are also deleterious for the viability of the cell, which necessitates a tight control over the magnitude and duration of the IFN response. This is partially achieved through the IFN-mediated activation of negative regulatory factors that help in termination of the IFN response and return to a normal homeostatic state. Such built-in negative regulatory mechanisms are frequently hijacked by viruses such as the Hepatitis C virus (HCV) to increase viral replication and productive infections. We and others have shown that long non-coding RNAs (lncRNAs) play prominent roles in regulation of the IFN response. Activation of the IFN cascade alters the expression of a large number of lncRNAs, many of which are directly induced by the JAK/STAT pathway and thus, resemble the well-studied protein-coding interferon-stimulated genes (ISGs). While only a handful of IFN- and virally induced lncRNAs have been characterized, recent studies have identified several lncRNAs that act as positive or negative regulators of expression of ISGs during the IFN response. A number of such regulatory lncRNAs have multiple ISG targets, while others act on a single neighboring ISG. Another group of studied lncRNAs act further upstream and regulate the expression of IFN genes or factors that sense the presence of viral genome or replication products. The large number of unstudied IFN- and virally induced lncRNAs makes it highly likely that future studies will reveal a much greater share for this class of transcripts in regulation of the antiviral response. In addition to their physiological roles, the expression of such lncRNAs is frequently modulated by virally encoded factors to interfere with the antiviral response and promote viral replication, thus making them ideal targets for therapeutic intervention. High throughput transcriptome studies in the last decade have led to the discovery of 1000s of novel RNA molecules that do not appear to code for functional peptides. These transcripts, named the long non-coding RNAs (lncRNAs), can be found in both eukaryotes and prokaryotes. However, they are especially abundant in more complex eukaryotes including animals and plants . InteresDespite intense research in recent years, the sheer number and diversity of lncRNAs have limited our understanding of the scope of function of lncRNAs in higher eukaryotes. Evidence emerging from existing research indicate that the expression of many lncRNAs is strongly dependent on the cell type and cellular state and is tightly controlled by various cellular signals . Thus, iNonetheless, data from the small number of lncRNAs currently studied indicate their involvement in diverse aspects of cellular function . EmerginFigure 1). Genes coding for lncRNAs frequently overlap protein-coding genes or other lncRNA genes in the sense or antisense orientation with the lncRNA initiating from a different promoter. In many cases, such promoters are located within or near the 3′ UTR of protein-coding genes   . Such intergenic vicinal lncRNAs can potentially affect the expression of their nearby genes via transcriptional interference or epigenetic regulation  . Data frigure 1) . Anotherigure 1) . Finallygulation . It shougulation .As mentioned above, current evidence point to the involvement of lncRNAs in virtually every aspect of cellular function. Several pioneering studies in recent years have provided evidence for the critical role of lncRNAs in regulation of diverse aspects of the immune response, including both innate and adaptive immunity . While tFigure 2)  signaling cascade. IFNs are traditionally divided into three major groups . While binding distinct receptors, type I  and type II (IFN-γ) IFNs show extensive overlap in their downstream signaling cascades and regulated genes . Similarigure 2) . ActivatFigure 2). Next, together with NF-κB and ATF-2/c-jun, they nucleate the formation of active transcriptional complexes through interactions with several transcription factors and regulatory proteins such as the transcriptional cofactor (CREB)-binding protein (CBP)/p300 , while TLR3 and TLR7/TLR8 induce signaling via TRIF and MyD88, respectively . The actBP)/p300 . This, iBP)/p300 . These cFigure 2)  (IFN-α and -β bind to the IFN-α/β receptor (IFNAR) in an autocrine and paracrine manner, triggering the induction of the Janus kinase/signal transducers and activators of transcription (JAK/STAT) signaling cascade (igure 2) . The outigure 2) . In a hiigure 2) . Once acigure 2) .The final outcome of the IFN signaling is the induction of ISGs, many of which exert antimicrobial activity via regulation of different steps of cellular gene expression in addition to direct antimicrobial effects. For example, several ISGs including IFIT-1, IFIT-2, IFITM3, ISG15, ISG20, RNase L, PKR, viperin and BST2/Tetherin are known to have antiviral activity . Thus, aWhile the IFN cascade is mainly activated in response to the presence of exogenous viral RNAs, a group of long non-coding cellular RNAs, namely the retrotransposon-derived transcripts, also activate the IFN response . It has A number of high throughput transcriptomic analyses in both human and mouse have revealed the presence of strong changes in the expression of lncRNAs during the IFN response . In the The results obtained in the murine models were closely analogous to those obtain using human cells. In one such study, primary human hepatocytes from five donors of different ages and genders were subjected to high throughput transcriptome analysis before and at three time points of 3, 9, and 24 h after treatment with IFN-α . SimilarA third set of high throughput studies in HuH7 human hepatocytes focused on changes in gene expression observed at later time points (after 72 h) of treatment with a high dose of IFN-α2 using both microarray analysis  and RNA-trans via interacting with WDR5, a subunit of the MLL/SET1 histone H3 lysine 4 methyltransferase complex, potentially recruiting the complex to the IFNG locus to change its methylation state . One such RNA, which was identified in primary human hepatocytes by As mentioned above, high throughput transcriptomic studies have revealed the presence of a large number of lncRNAs that show differential expression in response to IFN stimulation  was described in a study focusing on genes differentially expressed in response to influenza virus H1N1 infection in A549 human alveolar epithelium cell line . NRAV orAnother lncRNA with a negative regulatory function in the IFN response is EGOT (Eosinophil Granule Ontogeny Transcript). EGOT is a structured polyadenylated nuclear lncRNA conserved, at least, in all placental mammals . InteresConsidering the rather small number of lncRNAs that have been studied in the context of the IFN response, the discovery of the negative regulatory roles of NRIR, NRAV and EGOT on the transcriptional induction of ISGs suggests that lncRNAs may play a prominent role in negative feedback loops controlling the IFN response and possibly other signaling cascades in the immune response. Interestingly, several protein factors involved in negative regulation of IFN response have been identified . DefininWhile the above described lncRNAs had a negative regulatory impact on the IFN response, a recent study has provided evidence for lncRNA-mediated positive regulation of the IFN cascade. A screen for IRF3-dependent genes in HuS immortalized human hepatocytes led to the identification of an annotated but unstudied lncRNA (LOC100506895/AC011738.4/ENST00000436105.1) that was down-regulated in an IRF3-dependent manner after poly(I:C) treatment . This trWhile the lncRNAs described above regulated a number of ISG targets, another IFN-induced lncRNA named BISPR (BST2 IFN-Stimulated Positive Regulator) seems to affect the expression of a single target gene. Independent studies from two groups  identifiHepatitis C (HCV) is a deadly virus that affects ∼2% of the world’s population (60–170 million people) . About 5In spite of the efforts of many groups, an effective vaccine to prevent HCV infection has not yet been developed. Recently, several inhibitors that target different viral proteins have been approved for the treatment of HCV infection . The effThe seven genotypes of HCV share similar characteristics. HCV viral particle is enveloped, small (40–80 nm) and encloses the genome coated by the core protein . The genThe majority of HCV virions are transported in the blood embedded into very low or low-density lipoproteins (VLDLs and LDLs) formed by triglycerides, apolipoproteins and cholesterol or phospholipids . This coThe new viral genomes can be used for translation of new viral proteins, replication to produce new viral genomes or packaging into new viral particles. Assembly takes place close to lipid droplets (LDs) bound to ER membranes. NS5A molecules from the replication complex bind simultaneously to the newly synthesized RNA and core protein, helping the interaction between core protein and viral RNA which will lead to formation of a nucleocapsid bound to a LD . The envFigure 2). While type III IFNλ signaling has a special impact on HCV infection  (A paradigm for an ISG that promotes HCV replication is ISG15 . ISG15 iigure 2) . Therefoigure 2) .The combined action of PKR and the ISGylation and ubiquitination pathways in HCV-infected cells results in decreased cap-dependent protein translation and increased protein modification that leads to protein malfunction and destabilization. Under such a protein-hostile environment it must be challenging to maintain proper cell functionality and virus replication. Therefore, it is conceivable that both cells and viruses have evolved to achieve functionality under these conditions through the expression of functional non-coding RNAs, which are at least partially immune to protein-hostile conditions .Hepatitis C virus infection causes a deregulation in the expression of the non-coding genome, which may function either to facilitate or to block viral viability. This has been best studied for miRNAs. Several cellular miRNAs have been described to target the HCV genome to help or prevent viral replication or to regulate the expression of cellular factors required for the virus cell cycle or the antiviral response. Generally, virus replication-related factors induce proviral and reduce antiviral miRNAs while the innate immune response plays the opposite effect. The interplay of HCV infection and miRNAs has been reviewed elsewhere , 2015.In contrast, the world of infection-induced lncRNAs is widely unexplored and HCV-related lncRNAs are not an exception. The few studies performed clearly indicate that, similar to what has been described for miRNAs, several cellular lncRNAs are deregulated in response to HCV replication or to the antiviral response induced by viral infection . The funStudies performed so far to identify HCV-deregulated cellular lncRNAs have employed infected cultured cells or liver tissue from infected patients. In the latter case, it is difficult to establish whether the deregulation of the lncRNAs indeed results purely from HCV infection, as the evaluated tissue has also developed liver cirrhosis and/or HCC. Comparison of the level of lncRNAs in these tissues versus healthy liver identifies the lncRNAs that are deregulated by HCV infection, liver cirrhosis or HCC alone or in all possible combinations. In line with this, some of the lncRNAs deregulated in HCV-infected livers have been shown to play a role in the development of liver cirrhosis and/or HCC . This haInfection with HCV or expression of the core protein in cultured cells has led to the identification of additional HCV-induced oncogenic lncRNAs such as PVT1, CASC15 and HOTAIR . The indOn the other hand, several lncRNAs have been described that are upregulated both in HCV-infected cells and in cells treated with IFN or PAMPs such as poly(I:C), or when cells are infected with viruses different than HCV. Therefore, they can be considered to be lncRNAs upregulated by the antiviral response.As described above, several IFN-induced lncRNAs have been identified after comparing the transcriptome of cells treated with IFN and controls . This isSTAT3 transcription factor, a negative regulator of the IFN pathway, can be induced by IFN, growth factors, stress, several cytokines such as IL6 and by HCV infection . HCV corTable 2). These lncRNAs were identified by transcriptome analysis of cultured liver cells with and without HCV infection and were named CSRs, after HCV Stimulated lncRNAs.Finally, a group of lncRNAs are induced 3–30 fold by IFN or PAMPs such as poly(I:C) and up to 100 times more by HCV infection   but not DNA viruses (adenovirus or HBV). Existing data suggest that the expression of EGOT (see above) is upregulated through sensing of HCV viral RNA in the cytoplasm. Therefore, increased levels of EGOT are detected shortly after infection even when UV inactivated non-replicative viruses are used . Later iAs expected from a negative regulator of the IFN response (see above), EGOT depletion leads to decreased levels of viral genome and proteins and viral titers in HCV and SFV-infected cells , likely In vitro studies on the role of lncRNAs in the IFN response in the absence of pathogens point to the presence of a strong perturbation in the expression of the non-coding transcriptome following IFN stimulation. From the very small number of studies on the role of lncRNAs in the antiviral response, it is evident that changes in the expression of this class of cellular effectors do play a critical role in negative regulation of the downstream steps of the IFN response. Together with proteins that act as negative regulators, this class of lncRNAs create a complex, decentralized regulatory network with overlapping and likely partially redundant functions which allows for extreme fine tuning of the magnitude and duration of the IFN response. However, in the presence of pathogens such as HCV, such negative feedback loops can favor viral replication and in some cases are actively hijacked by the virus to help in viral survival. There are two possible scenarios that can explain this from an evolutionary perspective. One possibility is that the co-evolution of the IFN response and pathogens has tipped in favor of viral survival due to the faster rate of viral evolution, with the viruses gaining the ability to exploit the immune response. Alternatively, it is plausible that at least in the case of some chronic infections, such a reduction in the magnitude of the immune response may be beneficial for the organism through limiting the damage incurred on the infected tissue. Future studies, by providing a more complete picture of the interplay of lncRNAs and viral infections, will shed additional light on the highly complex interplay of the antiviral response and pathogens.SV and PF wrote and reviewed the manuscript. PF built the table and SV the figures.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Metastatic cardiac tumors are far more common than primary tumors. Although the hematogenous spread of osteosarcoma is well known, the imaging findings of cardiovascular involvement by osteosarcoma are seldom reported and can be difficult to recognize.A 21-year-old man of African descent presented to our center complaining of shortness of breath, awareness of heart beats, easy fatigability, swelling of lower limbs, and left-side chest discomfort for the past 6 months getting worse for the last 3 months prior to his third readmission. In 2004 he was admitted with the diagnosis of osteosarcoma of his left calcaneus bone confirmed by bone biopsy and treated at an oncology center with several cycles of radiotherapy and chemotherapy; he was declared cured after 5 years of annual clinical and radiological skeletal survey follow-ups. In the current admission, a physical examination revealed bilateral lower limb swelling, pansystolic murmur on the left side of his sternum at fourth intercostal space (tricuspid area) grade three, hepatomegaly with a liver span of 17 cm, and a positive fluid test and shifting dullness.This case report presents a 21-year-old man with relapsed osteosarcoma manifesting as metastatic lesions to his right ventricle encroaching on his interventricular septum, which was identified by transthoracic/transesophageal echocardiography computed tomography scan and cardiac magnetic resonance imaging. Although hematogenous spread of osteosarcoma is well known, the imaging findings of cardiovascular involvement by osteosarcoma are seldom reported and can be difficult to recognize. The enhanced resolution of modern computed tomography (CT) and magnetic resonance imaging (MRI) scanners may lead to better detection of cardiovascular involvement .The antemortem diagnosis of cardiac metastases in osteogenic sarcoma has been rarely documented whereas a prevalence as high as 20% is seen at autopsy, suggesting that cardiac involvement is a late stage complication. As the survival times for primary osteosarcoma continue to improve, late stage complications will undoubtedly become more prevalent in the future .A 21-year-old man of African descent presented to our center complaining of shortness of breath, awareness of heart beats, easy fatigability, swelling of lower limbs, and left-side chest discomfort for the past 6 months getting worse the last 3 months prior to this third readmission. Such symptoms started gradually from 2015, approximately 6 years after he had received a declaration that his osteosarcoma had been cured. In 2004 he was admitted with the diagnosis of osteosarcoma of his left calcaneus bone, confirmed by bone biopsy, and treated at an oncology center with several cycles of radiotherapy and chemotherapy; he was declared cured after 5 years of annual clinical and radiological skeletal survey follow-ups. In the current admission, a physical examination revealed bilateral lower limb swelling, pansystolic murmur on the left side of his sternum at fourth intercostal space (tricuspid area) grade three, hepatomegaly with a liver span of 17 cm, and a positive fluid test and shifting dullness. Biochemical investigations noted elevated levels of brain natriuretic peptide. An electrocardiogram demonstrated an abnormal analysis. A chest X-ray showed cardiomegaly with increased interstitial pulmonary markings and pleural effusion  class III heart failure which he presented on admission. He was discharged on 15 December 2016 in a fair condition with NYHA class II and booked in a follow-up clinic by the time of drafting this manuscript.Noncardiac tumors may invade the heart by means of lymphatic or hematogenous dissemination, local extension, or a transvenous route. However, catheter-based biopsy is not suitable for cardiac tumors. Therefore, it is extremely important to accurately determine the nature of the cardiac mass before considering surgical options . Our pat18F-fluorodeoxyglucose (18F-FDG) positron emission tomography (PET)/CT which could have revealed the morphological features of the cardiac tumor and glucose uptake; these could have been significant indicators of tumor malignancy and the whole body metastasizes [A limitation is that our center does not perform stasizes . MetastaThis report presents a 21-year-old man with relapsed osteosarcoma manifesting as metastatic lesions to his RV encroaching on his interventricular septum, which was identified by transthoracic/transesophageal echocardiography CT scan and cardiac MRI.


Vitamin D is unique in being generated in our skin following ultraviolet radiation (UVR) exposure. Ongoing research into vitamin D must therefore always consider the influence of UVR on vitamin D processes. The close relationship between vitamin D and UVR forms the basis of the “vitamin D–folate hypothesis”, a popular theory for why human skin colour has evolved as an apparent adaption to UVR environments. Vitamin D and folate have disparate sensitivities to UVR; whilst vitamin D may be synthesised following UVR exposure, folate may be degraded. The vitamin D–folate hypothesis proposes that skin pigmentation has evolved as a balancing mechanism, maintaining levels of these vitamins. There are several alternative theories that counter the vitamin D–folate hypothesis. However, there is significant overlap between these theories and the now known actions of vitamin D and folate in the skin. The focus of this review is to present an update on the vitamin D–folate hypothesis by integrating these current theories and discussing new evidence that supports associations between vitamin D and folate genetics, UVR, and skin pigmentation. In light of recent human migrations and seasonality in disease, the need for ongoing research into potential UVR-responsive processes within the body is also discussed. Vitamin D is of ongoing research interest, stemming in part from our relatively recent discovery of vitamin D receptors (VDRs) in almost every system in the body. Vitamin D elicits the majority of its functions through binding to these receptors, which act as transcription factors that modulate gene expression by binding to vitamin D response elements on target genes . The maiConsiderable research on vitamin D has focused on elucidating the non-calcemic roles of vitamin D and the extent to which VDRs influence human biology. Discovered roles involve the regulation of cell proliferation and differentiation, hormone secretion, and immune responses by vitamin D ,10,11,12The close connection of vitamin D with UVR and degree of pigmentation is the basis of the “vitamin D–folate hypothesis”, which aims to explain the evolution of human skin pigmentation . VitaminThe vitamin D–folate hypothesis is one of the main theories potentially explaining the evolution of human skin pigmentation. It is apparent that human skin tones arose as an adaptation to our UVR climate, with individuals of the darkest pigmentation having an origin in high-UVR areas near to the equator and lighter-skinned populations arising in low-UVR regions closer to the poles. This pattern arose as a result of two clines in skin pigmentation, whereby our early human ancestors firstly evolved to have darker skin pigmentation while residing in Africa but then evolved to lose their pigmentation when out-of-Africa migrations occurred . The vitVitamin D and folate are linked by their disparate sensitivities to UVR. UVR, on the one hand, stimulates the production of vitamin D in the skin, but, on the other hand, it may cause folate degradation through the absorption of UVR by folates or the oxidation of folates via free radicals following UVR exposure ,19,20,21This hypothesis is plausible, given that in maintaining levels of vitamin D and folate via skin pigmentation processes, the widespread action of these nutrients in maintaining reproductive success would have been preserved ,27. The There are several alternative theories that counter the vitamin D–folate hypothesis. The skin mutagenesis and skin barrier hypotheses are two prominent theories proposed as explanations for the evolution of darker skin pigmentation ,24, and The skin mutagenesis theory proposes that skin pigmentation arose as a mechanism to protect against the development of skin cancers . This hyThe skin barrier hypothesis proposes that highly pigmented skin arose as a barrier that protected against multiple environmental stresses . UVR expThis hypothesis is proposed as a discrete theory to the vitamin D–folate hypothesis. However, vitamin D and folate exert an array of functions that regulate the skin as a barrier against environmental stresses, having roles not only in the development of skin structures, but also in defense mechanisms that protect against UVR, heat, and microbial stresses.3 synthesis and also possessing all enzymes needed to metabolise inert vitamin D to its active form, calcitriol [The role of vitamin D in the skin is an area that has received significant research. The skin is a hub of vitamin D activity, as skin keratinocytes are unique in being the only cells in the body capable of both producing and metabolising vitamin D; being our primary site of vitamin Dlcitriol . VitaminVitamin D regulates many processes involved in the development of the stratum corneum, the outmost layer of our skin. This is a highly permeable layer that expresses multiple elements of our adaptive and innate immune system, operating as a barrier against extensive water loss and microbial invasion . VitaminFolate may have a role in melanogenesis by regulating the production and stabilisation of tetrahydrobiopterin ,54,55,56The metabolic conservation theory proposes that the depigmentation of ancestral humans can be explained by a need to draw resources away from melanin production and towards other metabolic processes . MelanogThe conservation theory is therefore not entirely isolated from the vitamin D–folate hypothesis. It is not a stretch to suggest that the protection of the vitamin D and folate status via pigmentation acted as the predominant pressure for the evolution of contrasting skin types at the equator and near the poles, as the likelihood of a deficiency in these nutrients would be highest in these environments. The occurrence of intermediate skin types that display facultative pigmentation in central European and Asian populations would have allowed for adequate vitamin D production. However, the primary “driver” may have been a need to restrict melanin production and channel these resources into responding to increased energy needs associated with colder climates. However, this is not to say the importance of vitamin D in intermediate UVR environments would have been obsolete. Notably, functions of vitamin D and folate in vasodilation/vasoconstriction outlined above, as well as roles in adipocyte biology, may have been important in maintaining energy and temperature homeostasis in increasingly colder climates.Energy stored in adipose tissue can be utilised to maintain cellular functions in cases of increased energy needs or to fuel adaptive thermogenesis in response to cold stress . When enPGC1α, PPARα, UCP1, SIRT1, and AMPK involved in mitochondrial biogenesis and thermoregulation [The roles of vitamin D in regulating fatty acid β-oxidation, energy metabolism, and the formation of brown adipose tissue are indicated by studies employing VDR-null mice models ,64. Thesgulation . The rolgulation . Notablygulation ,67,68. IThe vitamin D–folate hypothesis was first proposed by Branda and Eaton in the 1970s . This hyVDR variants and ethnicity [VDR variant Fok1 has a lower frequency in African populations compared to European/Asian populations [Cdx2, being the highest in African populations and the lowest in Europeans [VDR gene that display strong latitudinal clines [VDR variant alleles  and latitude (as a surrogate measure of UVR exposure) [Bsm1, Fok1, and Taq1 variants are associated to increases in cytokine production [VDR expression [VDR genotypes that facilitated vitamin D modulated immune responses in these environments. Conversely, the occurrence of variant alleles for Apa1, Fok1, and Bsm1 is increased at higher latitudes (associated with lower UVR levels) and is associated with increases in serum vitamin D levels [Apparent differences are consistently reported between the frequency of common thnicity . For exaulations , with thuropeans . These el clines  and prevxposure) . Ancestroduction , immune oduction , and VDRpression , respectD levels ,79. In tCYP2R1 and GC for predicting UVB-induced vitamin D concentrations [GC variants are reported to account for 10% of racial differences in circulating vitamin D levels [Multiple studies support the idea that genetic variation in vitamin D metabolism genes influences the vitamin D status . Notablytrations . SimilarD levels . These gD levels ,82. MTHFR-C677T variant, which results in a thermo-labile form of MTHFR [MTHFR-C677T, MTHFR-A1298C, TYMS 28bp 2R>3R, and SHMT-C1420T) [MTHFR-C677T variant, closely linked to aberrant folate-dependent processes, is the lowest in darker-skinned populations [Several relationships are reported between UVR and folate metabolism genes ,84,85. T-C1420T) . These sulations . CollectThe explanation for the evolution of skin colour is likely a complex integration of our current predominant theories . This review provides key examples of overlap between the theories proposed as alternatives to the vitamin D–folate hypothesis and actions of vitamin D and folate in countering environmental stress.The vitamin D–folate paradigm proposes that skin pigmentation evolved as a balancing mechanism to maintain levels of two key vitamins in human health: vitamin D and folate. In maintaining levels of these vitamins, the roles of these nutrients during reproduction would be preserved. This review discusses a likely mutually beneficial relationship between vitamin D, folate, and skin pigmentation that updates and further extends the vitamin D–folate hypothesis . The proThe vitamin D–folate hypothesis and related theories propose that skin pigmentation evolved to regulate the biological effect of differing UVR levels in different regions of the globe . HoweverA significant motivator for understanding the relationship between skin pigmentation and UVR-responsive processes is the potential health consequences of this mismatch. As a consequence of migration patterns, many darkly pigmented individuals now reside in areas of low UVR, and individuals with lighter skin tones are exposed to heightened levels of UVR . These iThe current recommendations for vitamin D and folate are based on requirements needed to prevent deficiency diseases. However, our greatest motivator to further examine the relationship between UVR and vitamin D and folate systems is perhaps the accumulating evidence linking these vitamins to the risk and onset of many current chronic diseases, such as cardiovascular diseases, diabetes, and several cancers ,89. Nota


Intron retention has long been an exemplar of regulated splicing with case studies of individual events serving as models that provided key mechanistic insights into the process of splicing control. In organisms such as plants and budding yeast, intron retention is well understood as a major mechanism of gene expression regulation. In contrast, in mammalian systems, the extent and functional significance of intron retention have, until recently, remained greatly underappreciated. Technical challenges to the global detection and quantitation of transcripts with retained introns have often led to intron retention being overlooked or dismissed as “noise”. Now, however, with the wealth of information available from high-throughput deep sequencing, combined with focused computational and statistical analyses, we are able to distinguish clear intron retention patterns in various physiological and pathological contexts. Several recent studies have demonstrated intron retention as a central component of gene expression programs during normal development as well as in response to stress and disease. Furthermore, these studies revealed various ways in which intron retention regulates protein isoform production, RNA stability and translation efficiency, and rapid induction of expression via post-transcriptional splicing of retained introns. In this review, we highlight critical findings from these transcriptomic studies and discuss commonalties in the patterns prevalent in intron retention networks at the functional and regulatory levels. Alternative splicing (AS) is a widespread process, affecting the vast majority of human genes  , alternative 3′ terminal exons, and alternative 5′ exons. In addition, many complex ASEs involve combinations of these simple events  or other structural features that can activate or repress translational initiation efficiency.IR in the main ORF can result in PTCs leading to IR-NMD, or possibly production of truncated proteins.IR in the main ORF can maintain reading frame allowing production of pairs of protein isoforms.If the intron is more than ~55 nt into the 3′ UTR, where splicing would lead to NMD, IR can stabilize the RNA by avoiding NMD.cis-elements that affect the stability or translational efficiency of the mRNA.IR in the 3′ UTR can introduce Intron retention is most often associated with down-regulation of gene expression via NMD (IR-NMD) . This means that precautions need to be taken to ensure that observed IR products are indeed derived from processed RNA. This involves the use of routine controls, such as omission of reverse-transcriptase to rule out genomic DNA as the source template, and the use of oligo dT selection of RNA for priming of cDNA synthesis to ensure that poly-adenylated RNA is being analyzed. Use of cytoplasmic polyA+ RNA can help to reduce the signal from nascent RNA, but at the expense of missing functionally important nuclear-retained RNA species (see in the following). Many IR products are much longer than their spliced counterparts, meaning that it is not always possible to obtain single-reads that unambiguously cover both exon–intron junctions as well as the entire intron. Nevertheless, a range of approaches have been used to identify and profile intron retention using next generation sequencing (NGS)  in recognition of their dual nature and also of the ambiguous terminology that has previously been applied to such events. The majority of exitrons, with length in multiples of 3 nt, maintain the same reading frame upon retention or splicing, although some alter the reading frame on the downstream side. Exitrons often encode intrinsically disordered protein regions and are enriched for short linear peptide motifs and residues subject to various post-translational modifications , possibly as part of a feedback loop. This allows for the NXF1 IR isoform to be transported to the cytosol and translated into a C-terminal truncated sNXF1 protein that serves as a cofactor to its full-length counterpart. sNXF1 has recently been shown to be expressed in hippocampal and cortical neurons, localizing in cytoplasmic granules suggestive of functionality in the cytosolic export of the other intron containing mRNA . Here, the repressive effect of IR upon translation is not via uORFs, but appears to be related to the resultant longer, more structured 5′ UTR that confers EIF4EBP sensitivity. Splicing of the intron removes 117 nt from the 5′ UTR and the shorter YY2 isoform is not subject to EIF4EBP-mediated repression. This leads to the upregulation of YY2 and differentiation into the cardiovascular lineage  expression by the RNA binding protein sex-lethal (SXL). SXL protein binds to the MSL2 5′ UTR inhibiting the splicing of a resident intron  and Grm5 (mGluR5) were differentially regulated by IR between the cerebellum and the cerebrum highlighting a role for IR in synaptic plasticity , defined as unspliced introns in otherwise fully spliced polyA+ mRNA from mouse ES cells  expression in platelets. Unspliced IL1β and TF pre-mRNAs are transcribed in megakaryocytes and persist through to anucleate platelets, where they can be spliced upon platelet activation . More commonly in human genes, splice site mutations result in exon skipping reflecting initial recognition of splice site pairs across an exon (exon definition) which would be followed later on by cross-intron spliceosome assembly Berget . Whethercis mutations will also occur predominantly where intron definition operates. Indeed, many investigations of mammalian IR have noted common shared features, including short intron length and higher GC content, which are also associated with intron definition  drugs that redirect the splicing of specific cassette exons of the Dystrophin (Eteplirsen) and SMN2 (Nusinersen) genes, respectively, received FDA approval late in 2016. AONs can also be employed to modulate IR in different contexts and channel decay or protection mechanisms that enable control over mRNA and consequently protein expression. Careful design of AONs to block splice sites or mutations that induce IR (Yadegari et al.


The advent of genomics has led to the identification of specific “driver” mutations in oncogenic kinases, and the development of targeted small molecule inhibitors to block their tumor-driving functions. These specific inhibitors have been a clinical success, and often significantly prolong the lives of individuals with cancer. Inevitably, however, the treated tumors recur as resistance to these targeted therapies develops. Here, we review the major mechanisms by which a cancer cell can evade targeted therapy, focusing on mechanisms of resistance to kinase inhibitors in lung cancer. We discuss the promising concept of rational upfront polytherapy in lung cancer, which involves concurrently targeting multiple proteins in critical signaling pathways in a cancer cell to prevent or delay resistance. EGFR (~11%) and BRAF (~7%), and activating gene rearrangements involving ALK and ROS1 (1–2%), all of which encode protein kinases, and result in hyperactivation of downstream signaling pathways that drive cell growth, proliferation, and survival.A significant fraction of lung adenocarcinomas harbor activating-mutations in targetable oncogenes. These include mutations in 10 These targeted drugs function as ATP-competitive inhibitors. Additionally, inhibitors of kinases that are activated downstream of these oncoproteins have been developed for use as either monotherapy, or in combination with inhibitors of the upstream oncoprotein. The MEK1/2 inhibitor trametinib is one such drug—it inhibits MAPK pathway activation by binding to and blocking MEK in an allosteric fashion. All of these inhibitors have shown efficacy over conventional chemotherapies in patients harboring the cognate genetic driver kinase.The identification of these driver kinases has led to the clinical use of small molecule kinase inhibitors that suppress these oncoproteins—erlotinib, gefinitib, afatinib, osimertinib for mutant EGFR, vemurafenib and dabrafenib for mutant BRAF, and crizotinib, ceritinib, alectinib for ALK and/or ROS1 gene rearrangements.Unfortunately, the initial clinical response to targeted kinase inhibitors is almost always temporary, as acquired resistance to these drugs invariably develops. Many mechanisms of resistance to each targeted therapy have been identified, but can be generally categorized into three predominant classes Fig. : (1) tho11 The effect of the gatekeeper amino acid substitution is to prevent kinase-inhibitor binding while allowing retention of the ability of the kinase to bind ATP. More recently, data demonstrating that gatekeeper mutants can retain sensitivity to structurally similar but irreversible inhibitors suggest that steric hindrance may not explain the gatekeeper mechanism of resistance in all cases, and instead that the function of a gatekeeper mutation could be to bind ATP more strongly to decrease the ability of the ATP-competitive kinase inhibitor to bind in the pocket.12 Which of these mechanisms is responsible for gatekeeper mutation-mediated inhibitor resistance may depend on the kinase in question.Small molecule kinase inhibitors bind to their target through non-covalent bonds within the ATP-binding pocket. Cancer cells can develop resistance to specific small molecule kinase inhibitors by mutating a so-called “gatekeeper” residue within the pocket. This residue is often small in the native oncoprotein with the secondary resistance-associated mutation resulting in a bulky amino acid substitution. How gatekeeper mutations cause resistance to small molecule inhibitors remains incompletely understood. Initial studies showed that the gatekeeper mutation both creates a residue that cannot a hydrogen bond with the inhibitor, and sterically hinders inhibitor binding in the pocket, while leaving the pocket’s ATP-binding affinity unchanged.14 Gatekeeper mutations have similarly been identified in cancers that become resistant to ALK inhibitors (L1196M). Additionally, other “on-target” mutations in EGFR and ALK have been described, which are found at other residues within the ATP-binding domain and cause targeted inhibitor resistance.17 While the gatekeeper threonine mutation has been identified as an in vitro cause of BRAF inhibitor resistance, this mutation has not been seen in patients.18 This may be because many BRAF inhibitors induce paradoxical activation of wild-type RAF.19 Thus, upregulation of wild-type RAF or RAS signaling can activate the same pathways that are downstream of mutant BRAF. These signaling events may obviate the selective pressure to acquire resistance to so called “paradox activating” RAF inhibitors by mutation of the driver oncogene itself.The gatekeeper T790M mutation in EGFR is found in ~50% of EGFR-mutant patients who develop resistance to EGFR inhibition.20The prevalence of on-target mutations as a mechanism of resistance has led to the creation of new inhibitors that can inhibit both the original oncoprotein and its resistance-associated mutated form. While this strategy does improve patient survival in the short term, mechanisms of resistance emerge to these inhibitors as well.V600E mutant lung adenocarcinoma, our group identified that a switch from expression of a full-length BRAFV600E to a shorter splice variant was capable of mediating resistance to the BRAF inhibitor vemurafenib.21 This splice variant is able to dimerize and activate downstream MAPK pathway signaling despite presence of the RAF inhibitor. This mechanism of resistance was observed initially in BRAF-mutant melanomas, indicating conservation across different tumor histologies.22Other alterations in the driver oncogene can lead to resistance as well. Studying BRAFV600E was identified as a driver of resistance in inhibitor-insensitive melanomas.23 Intriguingly, loss of the initial driver oncogene and replacement with a new driver, also known as “oncogene swap”, has recently been described as a cause of targeted inhibitor resistance.24 In this instance, a cancer cell loses the targeted driver oncogene, and upregulates a new oncogene, such that oncogenic signaling driving cell survival is mediated by the new oncogene, and the inhibitor of the initial oncogene no longer has a substantial impact on cancer cell survival.Finally, changes in the level of the targeted driver oncogene can also cause inhibitor resistance. By increasing the levels of the driver oncogene, an inhibitor appears to be less potent, because there will be fewer molecules of inhibitor per molecule of oncoprotein. Upregulation of BRAFOne way that cancer cells can evolve resistance to a targeted therapy without altering the target oncogene is by upregulating expression and/or activation of a protein that signals through the same signaling pathway. In this way, despite continued suppression of the initial driver oncoprotein by the small molecule inhibitor, critical parallel signaling persists under the control of the newly upregulated protein activity.MET in the setting of EGFR mutant lung cancer was one of the earliest examples of this type of mechanism of resistance.27 Mutant EGFR heterodimerizes with ErbB-3 to activate the PI3K/Akt signaling pathway. Upon inhibition of EGFR with a targeted inhibitor, this pathway is suppressed. MET amplification leads to the reactivation of this pathway by forming a MET-ErbB-3 heterodimer.28 Thus, despite continuous suppression of EGFR by the inhibitor, a critical downstream signaling pathway is reactivated, and resistance to the inhibitor emerges. Upregulated expression of the MET ligand HGF, leading to hyperactivation of MET, has been found to drive resistance to EGFR inhibition through a similar mechanism as MET amplification.30 Clinical trials are underway to test the effect of dual inhibition of MET and EGFR to overcome this mode of resistance.31 Additionally, ErbB-3 blocking antibodies are currently under clinical development.32 Amplification of the ERBB family member HER2 has also been identified as a possible mechanism of resistance to EGFR inhibitors by similarly activating ErbB3 and downstream PI3K signaling.33 Inhibition of this key signaling pathway downstream of the activation node is another way to overcome resistance.34Amplification of wildtype EGFR-mutant and EML4-ALK positive lung cancer.37 Overexpression of the tyrosine kinase AXL, which is able to engage multiple downstream signaling nodes also activated by EGFR, was identified by our group and others as a driver of EGFR inhibitor resistance.38 Similar findings were observed in ALK gene rearrangement positive lung adenocarcinoma.39Upregulation or activation of non-ERBB family member receptor tyrosine kinases to reactivate downstream signaling pathways, and cause resistance has also been identified. IGF-1R pathway activation can mediate resistance in both V600E mutant lung adenocarcinoma.21 Combinatorial inhibition of multiple kinases could be a therapeutic possibility in cases where activation of alternative receptor tyrosine kinases is responsible for driving resistance.Similarly, resistance to Raf inhibition can result from upregulated levels of EGF, which activates EGFR, and drives downstream pathway activation in BRAFCancer cells can become resistant to targeted inhibitors by amplifying or acquiring activating mutations in pathway genes downstream of the driver oncogene. Thus, despite continued silencing of an oncoprotein’s activity by its inhibitor, there is persistent pathway activation downstream of the reactivated pathway node.BRAF mutations drive resistance to targeted inhibition of EGFR, while KRAS amplification leads to ALK kinase inhibitor resistance in EML4-ALK-drive lung cancer.41 Similarly, downregulation of genes that negatively regulate the MAP kinase pathway have also been implicated in resistance. Decreased expression of the phosphatase DUSP6 leading to rescue of phospho-ERK and reactivation of MAPK signaling was observed in ALK inhibitor-resistant cell lines and patient samples.40 Similarly, downregulation of NF1, leading to reduced levels of the Ras-GTPase activating protein neurofibromin, and thus increased Ras activity, has been identified as a driver of resistance to EGFR inhibition in lung cancer.42Activating mutations in or amplification of genes involved in MAP kinase pathway signaling have been identified as a mechanism of resistance to targeted inhibitors of several driver oncogenes. EGFR and PIK3CA mutations are seen in patients with acquired resistance to EGFR inhibition, and are correlated with a poor response to EGFR-targeted therapy; furthermore, PIK3CA mutants can drive resistance to EGFR inhibitor in vitro.44 These mutations lead to increased phosphorylation of PIP2, generating the second messenger PIP3 and activating AKT. Similarly, homozygous loss of PTEN, which normally acts to dephosphorylate PIP3 and suppress this signaling pathway, has also been identified as a mechanism of EGFR inhibitor resistance in vitro in lung and other cancers.46 However, the clinical significance of these mutations with regard to inhibitor sensitivity remains controversial. While concurrent mutations in the PI3K pathway with EGFR and KRAS mutations are associated with a poorer prognosis overall, PI3K mutations do not alter sensitivity or clinical responses to targeted inhibitors in EGFR-mutant lung cancer, suggesting reactivation of this pathway may not actually be a cause of targeted inhibitor resistance in patients.47Along with signaling through MAPK, the PI3K pathway is another major signaling pathway that is commonly hyperactivated downstream of driver oncogenes. Several lines of evidence suggest that downstream reactivation of this pathway may be a mechanism of acquired resistance to targeted inhibitors. First, concurrent 49 Co-treatment of cells with EGFR and NFκB inhibitors prevents the emergence of this resistance in pre-clinical models.49Besides reactivation of critical pro-growth pathways downstream of a driver oncogene, activation of pro-survival signaling networks has also been implicated in targeted inhibitor resistance. Our group discovered that, upon inhibition of EGFR, an NFκB-containing complex is rapidly recruited, and activates a downstream pro-survival signaling pathway that limits EGFR inhibitor-mediated cell death.52 In some tumors, YAP levels are elevated and drive transcription of downstream targets, including BCL2L1, which encodes the anti-apoptotic BCL-xL protein. These tumors are more resistant to MAPK pathway inhibitors because, even without pro-survival MAPK pathway signaling, these tumors maintain activation of alternative pro-survival signaling pathways through YAP activation.Activation of pro-survival genes also limits response to MAPK pathway inhibitors in BRAF and RAS-driven tumors. Our group and others identified the YAP pathway as a key mediator of response to MAPK pathway inhibition in these tumors.50These mechanisms of resistance are distinct from those discussed earlier, as they involve engagement of pathways separate from those activated by the driver oncogene prior to inhibitor treatment. In both cases described here, combined inhibition of the driving oncogenic pathway and the resistance-mediating pro-survival pathway is an effective way to overcome targeted inhibitor resistance.53 Intriguingly, IGF-1R signaling was found to be required for establishment of this drug-resistant cell population, and KDM5A upregulation was linked to IGF-1R signaling. This finding suggests that upregulation of IGF-1R could contribute to inhibitor resistance through multiple mechanisms, both by reactivating critical signaling pathways (as discussed earlier) and by altering a cell’s epigenetic landscape.Global epigenetic changes have been observed in response to small molecule inhibitor treatment. Sharma and colleagues found that the H3K4 histone demethylase KDM5A was upregulated in inhibitor-resistant EGFR-mutant cells, and this overexpression was required for drug resistance.BIM, a pro-apoptotic protein, as a mediator of intrinsic targeted inhibitor resistance in EGFR-driven lung cancer and chronic myeloid leukemia.54 Normally, MAP kinase pathway signaling activated by the driver oncogene suppresses BIM and it’s pro-apoptotic function. Upon treatment with a targeted inhibitor, this suppression is released, restoring the pro-death function of BIM. However, some patients harbor a polymorphism in BIM that lacks the critical pro-apoptotic BH3 domain, thus rendering BIM ineffective in driving apoptosis upon oncogene inhibition and causing intrinsic resistance to the targeted inhibitor. In these cases, Ng et al. suggest BH3 mimetics in combination with targeted oncogene inhibition may be efficacious in combating intrinsic resistance.Germline polymorphisms in pro-survival signaling pathways have also been implicated in targeted inhibitor resistance. Ng and colleagues identified a common polymorphism in 56One non-specific mechanism of small molecule inhibitor resistance is upregulation of drug efflux pumps. Expression of these pumps has long been associated with resistance to a variety of cytotoxic cancer therapies, as they reduce intracellular drug concentration. Some small molecule tyrosine kinase inhibitors are targets of efflux pumps, and in some cases, expression of specific drug transporters have been reported to induce drug resistance to targeted inhibitors of EGFR and ALK.38 EMT is observed as a morphologic change in cells, decreased epithelial markers (like E-cadherin) and upregulated mesenchymal markers (like vimentin), as well as increased migration and invasion properties of cells. However, whether EMT is directly responsible for acquired resistance or is an associated but not causal change remains controversial. For example, induction of EMT by TGFB signaling is associated with ALK inhibitor resistance in an EML4-ALK-positive cell line, and knockdown of the mesenchymal marker vimentin restores sensitivity in an EML4-ALK-acquired resistance model that displays EMT.57 However, other groups have found that reversal of the EMT phenotype in ALK inhibitor-resistant EML4-ALK lines can be induced despite continued resistance to the targeted inhibitor.58 Thus, whether or not EMT is a driver of resistance or just a consequence associated with the functional resistance mechanism remains to be elucidated.The EMT has been observed in association with acquired resistance to targeted inhibitors in a variety of oncogene-driven cancers, including EML4-ALK and EGFR mutant lung adenocarcinoma.61 Interestingly, these tumors retain expression of the original mutant oncogene, and do not appear to have acquired any of the resistance mechanisms discussed above. Analysis of these small-cell-transformed tumors revealed loss of RB1, a hallmark of small cell lung cancer.63 Loss of RB1 may be the alteration that induces this non-small-cell to small-cell transformation, resulting in a molecular switch from dependence on the original driver oncogene to a different survival program regulated by RB1 loss.64Finally, histologic transformation of non-small cell lung adenocarcinoma to small cell carcinoma is occasionally observed as a mechanism of resistance to multiple driver oncogene-targeted therapies, including inhibitors of EGFR and EML4-ALK.10 While much research over the past decade has focused on identifying mechanisms mediating acquired resistance to targeted inhibitors, there is still much to be learned about what other factors might be involved in preventing an initial complete response and supporting residual disease. This population of tumor cells that never completely responds to therapy likely functions as a transition state culminating eventually in a drug-resistant tumor (acquired resistance). Therefore, understanding the mechanisms involved in the survival of this subpopulation of residual tumor cells is critical for progress.Most patients will have an incomplete response to a given targeted therapy—that is, their tumors will not completely regress, and they will have some degree of residual disease from which acquired resistance subsequently emerges.68 Another possibility is that the treatment of tumor cells with a targeted inhibitor induces a drug-tolerant population of cells from which resistance can then emerge based on the stochastic presence of genetic variation due to the underlying mutation rate in replicating cells.68 A third hypothesis is that targeted inhibition of an oncogene itself somehow induces epigenetic or genetic changes, leading to resistance. Likely, any or all modes of resistance emergence may occur in an individual patient, depending on the intratumoral heterogeneity present prior to treatment and the characteristics of the cancer type and inhibitor used. However, if critical signaling pathways can be predicted and targeted in a patient in an upfront manner, the emergence of resistance through either mechanism described here may be delayed or prevented entirely, offering a promising approach to combat the heterogeneity and adaptiveness of most cancers.How resistance emerges in a tumor remains unclear. One possibility is that certain resistance mechanisms exist in a subset of tumor cells prior to treatment with a targeted agent. These pre-existing resistant cells are then selected for upon introduction of the targeted inhibitor. Many tumors show a great amount of intratumoral heterogeneity prior to therapy, and there is evidence of the existence of tumor cells harboring resistance mutations prior to the treatment with targeted inhibitors.40 The development of clinical trials to test this upfront therapy in patients is underway. Similar findings were observed in EGFR mutant lung adenocarcinoma preclinical models and individuals with BRAF V600E lung adenocarcinoma.70Despite initial frustrations over the inevitable emergence of resistance to targeted kinase inhibitors, new insights into understanding these mechanisms of resistance provide hope for the development of more durably effective treatment strategies. The emergence of resistance can suggest which pathway(s) are critical for the survival of cells driven by specific oncoproteins, suggesting the potential of upfront combinatorial inhibition of the driver oncoprotein and the crucial pathway further downstream. Preclinical studies from our group have demonstrated that upfront treatment of EML4-ALK positive lung tumors with both an ALK inhibitor and a MAPK pathway inhibitor can significantly delay or even prevent onset of resistance.72 Early pre-clinical and clinical data suggest it may be critical to combat resistance by treating it before it truly emerges—once a tumor has recurred on a macroscopic level, it appears to be less sensitive to inhibitors targeting its resistance mechanism as when a tumor is initially co-treated with inhibitors targeting both its driver oncogene and a common resistance mechanism.74 This clinical experience exemplifies the importance of early low-level detection of residual disease and tumor recurrence using liquid biopsies (and where feasible on-treatment tumor biopsies) so resistance can be addressed prior to the appearance of macroscopic disease, and of understanding the signaling pathways that are most critical to tumor survival to allow for upfront polytherapy to prevent resistance from ever appearing.10So far in this review, we have covered the myriad ways a cancer cell can develop resistance to targeted therapies. But how can the knowledge of how cancer cells evade inhibitors translate into real benefits for patients? Now that many pathways of resistance have been identified, these specific resistance mechanisms can be monitored for in the patient once they start inhibitor treatment. In many cases, additional drugs are available that can be combined with the initial inhibitor to overcome emerging resistance. The fact that tumor cells shed their DNA, which can be detected in the blood of patients, has recently begun to be harnessed to effectively obtain serial biopsies of a patient’s disease over the course of treatment. In this way, physicians can easily monitor for the emergence of genetic alterations associated with resistance to inhibitors, and potentially deploy combination therapies to short-circuit the emergence of resistance before it is fully established in the patient.While this review mostly focuses on resistance to targeted therapies in lung cancer, the mechanisms can be broadly applicable to other tumor types as well. First, many other tumors that harbor the same driver oncogenes discussed here, are targetable with the same small molecule inhibitors, and show similar patterns of resistance mechanisms. Second, identification of resistance mechanisms that may emerge, and then upfront treatment of patients with combinations of inhibitors to delay or prevent this resistance from occurring is a concept that is generalizable to virtually any tumor type. Better understanding of how the tumor cells escape from the detrimental effects of targeted therapies holds promise for our ability to prioritize, and deploy synergistic drug combinations that chronically control and potentially eliminate the ability of the tumor to evolve full resistance. While drug combinations may show clinical toxicity, prioritizing combinations of agents with synergistic anti-tumor effects could offer a therapeutic window. Alternatively, sequential or alternating drug schedules could be tested, where appropriate, to mitigate clinical toxicity and maintain anti-tumor efficacy.Altogether, the path to chronic cancer control is clearer and brighter due to the increasing understanding of the biological basis of resistance, and the arrival and adoption of emerging technologies that allow us to chart the molecular course of cancer evolution in individual patients during treatment.


STIL gene expression has been associated with either insufficient brain development  or cancer, two conditions resulting from perturbations in cell cycle and chromosomal segregation. This review describes the recent advances on STIL functions in the control of centriole duplication and mitotic spindle integrity, and discusses how pathological perturbations of its finely tuned expression result in chromosomal instability in both embryonic and postnatal situations, highlighting the concept that common key factors are involved in developmental steps and tissue homeostasis.Cell division and differentiation are two fundamental physiological processes that need to be tightly balanced to achieve harmonious development of an organ or a tissue without jeopardizing its homeostasis. The role played by the centriolar protein STIL is highly illustrative of this balance at different stages of life as deregulation of the human  STIL is a cell cycle-regulated protein specifically recruited at the mitotic centrosome to promote the duplication of centrioles in dividing cells.Complete loss of STIL results in no centrosomes, no cilia, and is not compatible with life.By contrast, residual or increased expression of STIL is viable but alters the centriole duplication process leading to either impaired or excessive centrosome formation.Genetic mutations in human STIL result in either residual expression or stabilization of STIL at the centrosome both leading to mitotic spindle defects and primary microcephaly (MCPH7).Abnormally high expression of STIL in differentiated tissues triggers centrosomal amplification and is associated with an increased metastatic potential in multiple cancers.Centrosome amplification is seen both in cancer and in MCPH phenotype. How is the context important in determining the phenotype?Is presence of STIL in the centrosome important in determining cell fate?STIL has several binding partners. To what extent is the STIL phenotype due to the independent functions of these binding partners?STIL gene that encodes a regulatory protein necessary for centriole biogenesis and is expressed in many cell types. The structure and function of STIL is described followed by an account of two phenotypes that have been associated with STIL dysfunction, autosomal recessive primary microcephaly (MCPH), and cancer. Reasons for these different phenotypes are discussed in relation with centriole duplication and mitotic checkpoints that operate in different cellular contexts to deal with aneuploidy and chromosomal instability.The developing brain appears particularly sensitive to centrosome dysfunction, which is also associated with a wide range of cancers. The centrosome is a cytoplasmic organelle built around microtubule-based core components called centrioles. This review focuses on the STIL gene was initially identified in a common chromosomal rearrangement in T-cell acute lymphoblastic leukemia and named SCL/TAL1 Interrupting Locus (SIL/STIL)1. It is predicted to encode five isoforms. Isoform1  is a 1288-amino-acid protein; isoform2 has 1287 amino acids (NP_001269865.1) and differs from isoform1 by a missing serine875; isoforms3–5  have several amino acids missing but their significance is unknown. STIL contains conserved regions and interacts with several proteins  mediates the binding of the centriole protein SAS-62. At the C terminus is the KEN box involved in Anaphase-Promoting-Complex/Cyclosome (APC/C)-mediated degradation of STIL5. The C terminus of STIL can also interact with conserved components of the Hedgehog signaling such as Suppressor-of-fused homolog (SUFU)6 and GLI17.The human 8. The centrosome has two centrioles orthogonal to each other, surrounded by proteins constituting the pericentriolar material (PCM). Both the centriolar core and the PCM nucleate microtubules, which are important for positioning the mitotic spindles and imparting polarity and asymmetry to the cell. During cell division the centrioles duplicate only once; STIL and its interactors are central to this process, ensuring the fidelity of this unique duplication, thereby minimizing chromosome instability9. STIL transiently associates with PLK4 and SAS-6 to form the core module for centriole duplication10. PLK4 is related to the Polo-kinase family of serine/threonine kinases and initiates centriole formation12, while SAS-6, a coiled-coil protein that self-assembles into the ninefold symmetry gives the carthweel its shape14. STIL and PLK4 are initially maintained at low levels in non-dividing and differentiated cells15: STIL forms dimers or tetramers through its CC domain and is degraded in the cytoplasm by APC/C and its co-activator CDC20, through recognition of the KEN motif5. STIL level in the cytoplasm rises in G1 because its degradation by APC/C is prevented by the absence of CDC205. PLK4 recruits STIL at the base of the parental centriole through the CC domain and phosphorylates the STAN domain at the late G1/G1-S transition18. Phosphorylated STIL then promotes its binding to the C-terminal region of SAS-6 and recruits it to the outside wall of the centriole17. This core module along with CPAP, also recruited by STIL to the centriole, starts the assembly of the cartwheel and centriole duplication20. At early S phase, the centrosome-associated protein ROTATIN was recently shown to associate with STIL and contribute to building full-length centrioles21. At S–G2 transition, the centrioles are duplicated but remain close to each other, potentially preventing reduplication. Remarkably, CDK1, transiently expressed at late G2 and during M phase, competes with PLK4 for binding to the CC domain of STIL, preventing PLK4 from recruiting STIL until mitosis is completed22. The spindle assembly checkpoint, a highly conserved mechanism also called mitotic checkpoint, prevents the degradation of CDK1 during mitosis by the inhibition of APC/C-CDC20 until all chromosomes are properly segregated23. CDK1 is thought to trigger progressive dissociation of STIL and SAS-6 from early mitotic centrosomes, thereby initiating cartwheel disassembly during M phase and ensuring that centriole biogenesis occurs only once before cell divides5. Upon activation, APC/C-CDC20 degrades CDK1 for entry into anaphase and again marks STIL for degradation until next the G1 phase. Thus, by prometaphase there is no STIL at the centrosome and by anaphase there is no STIL in the cytoplasm either until upon mitotic exit APC/C is blocked and STIL levels builds up once again in G1  and the BrainCloud database (http://braincloud.jhmi.edu) show its expression pattern during brain development. The association of STIL with cell proliferation is borne out by its expression pattern during fetal stages. At 15 postconceptional weeks STIL is strongly expressed in the ventricular and subventricular zones of the forebrain, the ganglionic eminence and the rostral migratory stream, and less expressed in intermediate zone, subplate, cortical plate, marginal zone, and subgranular layer of the forebrain. This pattern persists at 21 postconceptional weeks, although the expression is reduced in the subventricular zone. The expression of PLK4, SAS-6, and CPAP also broadly shows this pattern of expression, although the expression of SAS-6 and STIL diverges in some regions of the cortical plate. In the cerebellum, STIL, PLK4, and SAS-6 but not CPAP are expressed in the external granule layer and regions of the rhombic lip. However, none of these genes are expressed in the transient Purkinje cell cluster, the ventricular matrix zone of the cerebellum or in the migratory streams of the hindbrain. Microdissection experiments at different developmental stages confirmed that STIL expression is high in the ventricular zone and low in the subplate and cortical zones (http://www.brainspan.org/). Further, microdissected diencephalon did not have STIL (http://www.blueprintnhpatlas.org/). This suggests that (i) the structural components of the cartwheel STIL-SAS-6-PLK4 is by no means obligatory for centrosome duplication in all cells and (ii) STIL is present in specific populations of dividing cells.in utero. Most of them are autosomal recessive and include (i) isolated forms called MicroCephaly Primary Hereditary (MCPH), (ii) forms associated with growth retardation, called microcephalic dwarfism. Most of STIL mutations identified in patients are associated with a MCPH phenotype and STIL is recognized as MCPH740. However, a few patients exhibit criteria of microcephalic dwarfism, as short stature has been reported41. So far, eight mutations in STIL have been described in 37 patients all showing severe microcephaly (−4 to −10 S.D.). Five mutations are splice43, deletion, nonsense43, or duplication44 that predict the production of truncated proteins, while three mutations are missense substitutions46 . Despite the different kinds of mutations, the various domains affected  is surprising since genetic ablation of STIL is embryonically lethal early during embryogenesis in mice and fish47. This suggests that STIL mutations in human do not result in a complete loss-of-function, or are partially compensated by other genes. The first hypothesis is supported by the findings that two C-terminal nonsense mutations , which result in the loss of the KEN box43, do not affect the centrosomal localization of STIL nor its functionality but rather abolish its degradation by APC/C in late mitosis5. Removal of the KEN box thus causes a strong accumulation of the mutant protein, resulting in centrosome amplification5. Alternatively, microcephaly in MCPH7 patients can result from a decrease of STIL levels. This was illustrated by rescue experiments in U2OS cells showing that the pGly717Glu mutation induces a reduced but non-null activity of STIL on centriole duplication45. Another mutation, inducing exon 5 skipping and frameshift in the STIL sequence, is likely close to a null mutation as all domains are predicted to be lost42. However exon skipping was only partial in the patient analyzed, suggesting some residual activity of STIL, and providing an explanation for the fact that this mutation is compatible with life and the idea that MCPH7 microcephaly can result from a decreased STIL activity. Therefore, both accumulation and impairment of STIL protein levels during cell cycle affect centriole regulation and result in microcephaly  is indirectly diagnosed by a head circumference smaller than the age-specific and gender-adjusted mean by more than 2 standard deviations (S.D.s) at birth. Primary microcephaly refers to hereditary microcephalies already detectable 22. Considering the central role of STIL in maintaining centrosome integrity in highly proliferating cells, STIL has been found upregulated in several cancers of bad prognosis, including lung cancer, colon carcinoma, prostate adenocarcinoma36, and ovarian cancers48. Moreover, STIL expression is associated with an increased metastatic potential in multiple cancers49. As expected, STIL upregulation is associated with a high histopathological mitotic index in tumors36 and presumably affects the formation of mitotic spindles, as well as SHH signaling and the function of its interactors.Centrosomes are essential for chromosomal stability, and abnormalities of their number, or structure affect cell division50. Loss of this control leads to spindle defects, especially in highly polarized cells, where misoriented spindles influence daughter cell positioning, possibly causing a disruption in tissue morphology51. Several tumor suppressor genes such as APC, or VHL, are important for stabilizing microtubules and maintaining spindle orientation51. Similarly, overexpression of genes such as STIL could act as oncogenes and lead to cancer by promoting spindle defects, although direct evidence of a link between oncogene activation and spindle misorientation is lacking. STIL overexpression, which results in supernumerary centrosomes, could lead to cancer in inducing chromosomal instability52.Although cells can proliferate and microtubule can nucleate in their absence, centrosomes are obligatory for controlling spindle orientation53, a large body of literature has implicated the SHH pathway in development of various cancers including a subset of medulloblastomas55. STIL overexpression in pancreatic adenocarcinoma was shown to de-repress GLI1 from SUFU-mediated control and this phenotype was reversed when STIL was downregulated6. Thus, during the process of carcinogenesis, the increased expression of STIL promotes the transcriptional activity of GLI1, which is no longer regulated quantitatively. GLI1 upregulates genes that promote sustained proliferation, cell death resistance, stemness, angiogenesis, and genomic instability, which are hallmarks of cancer56. Therefore, increase in STIL expression leading to uncontrolled GLI1 de-repression likely represents a crucial step toward cancer progression.The role of STIL in promoting SHH signaling could be another pathway to account for its association with cancer. Following the initial observation that SHH overexpression results in the development of basal cell carcinomas in miceSTIL expression levels is compatible with life but triggers severe patterning phenotypes during development, including brain growth. Thus, microcephaly caused by STIL mutations can result from either too less centrosome duplication or too much centrosome amplification  protein and MCPH1 deletions have been identified as a risk for breast cancer63. Centrosome amplification is commonly seen following DNA damage and can arise as part of the DDR64. The serine-threonine kinase CHK1 plays a major role in DDR by halting cell division in G2/M until repair proteins are recruited at lesion sites. In this context, MCPH1 deficiency potentiates CHK1 activity and increases centrosome amplification65. Thus, compromising mitotic checkpoints results in cell division in the presence of abnormal centrosomes65. How CHK1 overactivation leads to centrosome amplification is not fully understood, but it is thought to activate CDK2, a cyclin-dependent kinase required for centrosome duplication, likely through activating its phosphorylation67. WD repeat-containing protein62 (WDR62), whose deficiency is associated with MCPH270, is required for maintaining spindle and centrosome integrity. Its overexpression, coincident with centrosome amplification, is also seen in lung adenocarcinomas and ovarian cancers72. WDR62 interacts physically during the cell cycle with the abnormal spindle-like microcephaly-associated protein (ASPM), whose deficiency causes the most frequent primary microcephaly (MCPH5)73. Here too, increased ASPM levels cause tumor growth and are seen in medulloblastomas while its reduction causes a decrease in tumor proliferation74. CDK5RAP2 (MCPH3) is also involved in the DDR; it functions by arresting cells before mitotic entry due to imparied centrosomes but also interacts with BUB1 and MAD2, which are important in spindle activation checkpoint76. Interestingly, a correlation between centrosomal abnormalities, aneuploidy, and cytogenic risk profile is seen in acute myeloid leukemia, where gene expression profiling has revealed the differential expression of genes encoding centrosomal and mitotic spindle proteins77. Among these are pericentrin, a scaffold protein that anchors many other proteins at the centrosome78 and NuMA, which associates with dynein and microtubules to create localized pulling forces, thus regulating the correct assembly and positioning of the mitotic spindle79. The role of such factors points to mitotic centrosomal abnormalities as important component of cancer progression.Seventeen MCPH loci have been described so far80, pediatric medulloblastoma81, and breast tumors82. Its transient overexpression leads to centrosome overduplication and in a P53−/− background that inhibits apoptosis, it results in aneuploidy and spontaneous skin cancer61. Another recent study has convincingly shown that PLK4 overexpression leads to spontaneous tumors in several organs83. It remains to be seen whether this phenotype is dependent on STIL expression. STIL binds to PLK4 in the cytoplasm and therefore STIL expression levels could impact PLK4 cytoplasmic activity17, which functions to remodel the cytoskeleton and may be important for cancer invasion and metastasis as its depletion is correlated with an increase in E-cadherin expression and less metastasis84. CYCLIN B is also frequently found elevated in primary breast cancer, esophageal squamous cell carcinoma, laryngeal squamous cell carcinoma, and colorectal carcinoma89 and its expression indicates a bad prognosis and is correlated with the malignancy of gynecological cancers90. Downregulation of STIL decreases CDK1/CYCLIN B activity, prevents G2–M transition, and causes inhibition of tumor growth in vivo91. Conversely, increasing STIL could promote CDK1/CYCLIN B activity and indirectly participate in CYCLIN B-dependent proliferation in tumor cells. Absence of STIL also results in an upregulation of Chfr, and lowers PLK1, resulting in the activation of the CDC25c phosphatase. This pathway could thus control the entry of cells into mitosis independent of its obligatory role in centriole duplication33.STIL could also have an indirect effect on cancer, as a downstream effector of PLK4. Increased PLK4 expression has been reported in malignancies such as colorectal cancerSTIL mutations in MCPH show that centrosomes and cilia are essential for normal brain development. During evolution, one way that the cortex has undergone expansion is likely linked to mechanisms controlling spindle orientation and keeping the balance between asymmetric and symmetric cell divisions93. A change in the nature of spindle orientation control has been proposed to account for the population of subventricular zone progenitors in human development and based on the strong expression of STIL in the ventricular zone, one may speculate that STIL is important for controlling spindle orientation in giving rise to subventricular zone’s cells94. Deficiency of several centrosomal proteins results in MCPH, and centrosomal proteins may thus be a key to discovering the mechanism of expansion of cortical area38. In addition, the involvement of STIL in cancer shows that the result of spindle defects can be vastly different depending on the stage of development and the tissue involved that probably is partly due to differential responses to mitotic checkpoint mechanisms. STIL intersects several pathways, and lowering its level could block the effects of PLK4 overexpression, CDK1/CYCLIN B activation, and GLI1 signaling, and reduce proliferation even in the presence of aneuploidy. Thus, centrosomal proteins may be a new class of targets for cancer.