In the last few years, non-coding RNAs (ncRNAs) have been a hot topic in cancer research. Many ncRNAs were found to regulate the apoptotic process and to play a role in tumor cell resistance to treatment. The apoptotic program is on the frontline as self-defense from cancer onset, and evasion of apoptosis has been classified as one of the hallmarks of cancer responsible for therapy failure. The B-cell lymphoma 2 (BCL-2) family members are key players in the regulation of apoptosis and mediate the activation of the mitochondrial death machinery in response to radiation, chemotherapeutic agents and many targeted therapeutics. The balance between the pro-survival and the pro-apoptotic BCL-2 proteins is strictly controlled by ncRNAs. Here, we highlight the most common mechanisms exerted by microRNAs, long non-coding RNAs and circular RNAs on the main mediators of the intrinsic apoptotic cascade with particular focus on their significance in cancer biology. Each program leads to the activation of a normally latent cysteine protease , which triggers a cascade of proteolytic events involving effector caspases 3 and 7 responsible for the execution phase of apoptosis, resulting in the progressive disassembly of cellular structures and ultimately engulfment by phagocytic cells.The word “apoptosis” was coined from Greek to address a programmed cell death, which contributes to the tissue homeostasis as the “falling off” of autumn leaves in a tree . The apoMitochondria are the cells’ energy factories, but also the organelles where suicidal signals converge and are executed. The intrinsic apoptotic program is more widely implicated as a barrier to cancer pathogenesis . It culmProteins belonging to the BCL-2 family are the key regulators of MOMP. This family consists of three groups of structurally-related proteins: the pro-survival BCL-2-like proteins , the pro-apoptotic multi-BH domain BAX/BAK proteins and the pro-apoptotic BH3-only proteins . In healthy cells, BAX and BAK are kept inactive by the interaction with pro-survival members ,5. In reEvasion of apoptosis has been classified as one of the hallmarks of cancer  and is aNoteworthy, some BCL-2 family proteins are involved in other cancer hallmarks besides apoptosis resistance. BCL-W expression was reported to be upregulated in the infiltrative types of gastric cancer  and in gIn a bi-transgenic mouse model engineered to spontaneously develop pancreatic neoplasia, BCL-XL viral transduction in hyperplastic or early dysplastic lesions increased tumor invasion and facilitated lymph node metastasis, without altering cancer cell survival or proliferation . BCL-XL-BCL-2 was reported to have a role in tumor onset, spread and therapy resistance that does not rely uniquely on the apoptotic pathway. Human lung epithelial cells chronically exposed to the carcinogenic hexavalent chromium Cr(VI) undergo transformation that strongly relies on BCL-2. Medan and colleagues showed that BCL-2 drives invasive and proliferative properties of the transformed cells, as well as their colony-forming and angiogenic activities . FurtherOverall, these results point out the involvement of BCL-2, BCL-W and BCL-XL in molecular events not related to the apoptotic cascade. These “other” cellular activities have been linked to the BH4 domain, which mediates direct interactions with specific proteins functionally implicated in distinct stages of tumor progression. Lately, recent efforts are aiming at developing new inhibitors directed against the BH4 domain, which is considered an appealing target due to its unique structure and crucial involvement in many cellular functions .BCL-2 family proteins are subjected to several layers of regulation that affect transcript levels and protein half-life, phosphorylation status and localization. Besides p53-mediated transcriptional activation of PUMA and NOXA following DNA damage , other tHere, we aim to outline the most relevant regulatory strategies involving ncRNAs that human cells evolved to control cell death and their implications in cancer biology .BCL-2 3′ UTR by competitive binding, thus stabilizing BCL-2 mRNA [MicroRNAs (miRNAs) are small (~22 nt) single-stranded non-coding RNAs responsible for post-transcriptional control of gene expression. miRNAs exert their regulatory function through Watson–Crick base pairing of their 5′ region (seed sequence) with the target mRNA, generally in the 3′ UTR . RegulatL-2 mRNA .The broad regulatory power of microRNAs relies on their ability to control whole pathways or even multiple complementary pathways by regulating the expression of different actors of the same set. Our group reported on the role of miR-197 and miR-663 in conferring apoptosis resistance in non-small cell lung (NSCL) cancer. Briefly, miR-197 behaves as a master regulator of the apoptotic response in p53 wt background by targeting NOXA and BMF . MiR-663Further, microRNAs can take part in complex regulatory circuitries, involving protein factors and other ncRNAs, where the activity of main effectors of pivotal cellular functions are finely modulated in response to intra- and extra-cellular stimuli. One striking example of such intriguing connections is the miR-34 family. MiR-34, a p53-induced microRNA, functions as a tumor suppressor by playing a role in cell cycle arrest, apoptosis and metabolic regulation ,57,58. mAs discussed above, pro-survival members of the BCL-2 family are involved in several hallmarks of cancer, spanning from invasion and metastasis to angiogenesis and DNA replication and repair . InteresMulti-drug resistance (MDR) is one of the major dilemmas in cancer therapy, due to mutations, changes in gene expression and drug-induced karyotypic alterations. The BCL-2 family is known to have a major role in tumor evasion of drug-induced cell death . MiRNAs Interestingly, the miR-221/222 cluster is one of the most commonly upregulated miRNAs in human solid and hematologic tumors, and it was found to target multiple BH3-only pro-apoptotic proteins. The role of the miR-221/222 cluster was studied in a model of dexamethasone-induced drug resistance in multiple myeloma (MM). By using the isogenic cell lines MM1R and MM1S, respectively resistant and sensitive to dexamethasone, the authors identified DNA copy number gains in MM1R cells that were associated with increased miR-221-222 expression and consequent downregulation of its target PUMA. Modulation of the miR-221/222 cluster alone was able to revert the resistance of MM1R cells and conversely to confer resistance to sensitive MM1S cells, thus demonstrating the key role of these microRNAs as mediators of resistance in MM. These data were consistent with observed overexpression of this miR cluster together with the decrease of PUMA in relapsed patients .Multiple regulatory mechanisms targeting BCL-2 family members involve a different class of non-coding RNAs called long non-coding RNAs (lncRNAs).LncRNAs have recently jumped on the non-coding RNA stage as crucial regulators of gene expression through a wide variety of molecular mechanisms. These transcripts >200 bp in length fold into evolutionarily-conserved secondary structures and exert their regulatory functions at different levels by recruiting chromatin modifiers or transcription factors on gene promoters, by affecting the assembly of protein complexes, by interfering with mRNA processing or behaving as microRNA sponges (competing endogenous RNAs (ceRNAs)) ,74. The Lnc_ASNR (apoptosis suppressing non-coding RNA) was identified through transcriptome profiling of different tumor specimens  where it was found upregulated. Further analyses unveiled that lnc_ASNR exerted a pro-survival role in tumor cells by promoting nuclear retention of the RNA binding protein AUF1 that in physiological conditions mediates BCL-2 mRNA degradation [MALAT1) behaves as a ceRNA targeting tumor suppressor miR-125b unveiled a negative feedback loop in bladder cancer [MALAT1 as a target of miR-125b overexpressed in bladder cancer [MALAT1 activation or interference, the authors demonstrated that MALAT1 induces miR-125b degradation through the Ago2 complex, resulting in concurrent upregulation of miR-125b target genes BCL-2 and MMP13. Accordingly, silencing of BCL-2 and MMP13 neutralized the anti-apoptotic and invasion-promoting effect of miR-125b inhibitor on MALAT1 knockdown cells, thus confirming the role of these proteins in the establishment of cancer phenotypes in a MALAT1-overexpressing background. Further, BCL-2 upregulation mediated by lncRNA HOTTIP  was found to underlie chemoresistance in small cell lung cancer (SCLC) [HOTTIP expression was increased in human SCLC chemoresistant cell lines and tissues compared to their chemosensitive counterparts. Noteworthy, the manipulation of HOTTIP expression affected the drug resistance of SCLC cell lines, measured as changes in the IC50. In addition, the combination of HOTTIP knockdown and administration of cisplatin and etoposide upon subcutaneous injection in mice of chemoresistant cells showed an additive effect in reducing tumor burden. Mechanistically, HOTTIP acts as a competing endogenous RNA by binding miR-216a, thus abolishing the miRNA-induced downregulation of BCL-2. As described for BCL-2, lncRNAs can post-transcriptionally upregulate the anti-apoptotic relatives of the BCL-2 family by acting as miRNA decoys. In breast cancer, high expression of BCL-W showed a positive correlation with lncRNA HOX transcript antisense RNA (HOTAIR) levels. Mechanistically, HOTAIR was demonstrated to titer away miR-206, thereby relying on the BCL-W transcript from miRNA-induced repression [RP11-436H11.5 competitive binding to miR-335-5p [radation . The finr cancer . In factr cancer . More rer (SCLC) . HOTTIP pression . A similR-335-5p .TUG1) provides an interesting example of epigenetic regulation of BAX transcription. TUG1 aberrant expression in serum samples from lung adenocarcinoma patients compared to healthy donors showed a correlation with tumor size, degree of differentiation, metastasis and TNM stage. RNA immunoprecipitation (RIP) and RNA pulldown assays in lung adenocarcinoma cells demonstrated the interaction between TUG1 and the histone methyltransferase EZH2. Chromatin immunoprecipitation (ChIP) analysis revealed that TUG1 mediates epigenetic silencing of BAX promoter through EZH2 recruitment and consequent H3K27 hypermethylation, hence conferring tumor cells’ protection against apoptotic triggers [LncRNA taurine upregulated 1  represent a novel class that has gained increasing consideration. These covalently-closed circular structures arise from either exon skipping or direct back-splicing. Exon skipping leads to a lariat that later on undergoes circularization, whereas direct back-splicing refers to the coupling of a donor splice site with an upstream unspliced acceptor site, which results in the circularization of the intervening RNA. CircRNAs are highly stable in vivo compared with linear RNAs, mainly localized in the cytoplasm and can be detected in exosomes . Recentlg miR-29 ,87. Circg miR-29 . Circ-Fog miR-29 . MechaniThe identification of the growing family of ncRNAs has broadened the vision of gene expression regulation, providing further layers of complexity that explain the capability of cells to rapidly adapt to external and internal stimuli . Moreove9 TargomiRs per week was well tolerated and was accompanied by early signs of antitumor activity. These encouraging data support additional studies of miR-16-based TargomiRs in combination with chemotherapy or immune checkpoint inhibitors [Besides MCL-1-mediated resistance, modulation of ncRNAs controlling the BCL-2 family, alone or in combination with other agents, could be explored as a therapeutic strategy against cancer . Unfortuhibitors . Further


Three-dimensional (3D) tissues are engineered by stacking cell sheets, and these tissues have been applied in clinical regenerative therapies. The optimal fabrication technique of 3D human tissues and the real-time observation system for these tissues are important in tissue engineering, regenerative medicine, cardiac physiology, and the safety testing of candidate chemicals. In this study, for aiming the clinical application, 3D human cardiac tissues were rapidly fabricated by human induced pluripotent stem (iPS) cell-derived cardiac cell sheets with centrifugation, and the structures and beatings in the cardiac tissues were observed cross-sectionally and noninvasively by two optical coherence tomography (OCT) systems. The fabrication time was reduced to approximately one-quarter by centrifugation. The cross-sectional observation showed that multilayered cardiac cell sheets adhered tightly just after centrifugation. Additionally, the cross-sectional transmissions of beatings within multilayered human cardiac tissues were clearly detected by OCT. The observation showed the synchronous beatings of the thicker 3D human cardiac tissues, which were fabricated rapidly by cell sheet technology and centrifugation. The rapid tissue-fabrication technique and OCT technology will show a powerful potential in cardiac tissue engineering, regenerative medicine, and drug discovery research. Recently, tissue engineering methodology and cell-based regenerative therapy have been progressing rapidly and are attracting attention worldwide –3. Our lμm in resolution)   were proNY, USA) , and layNY, USA) . The movIn this study two OCT systems [Proto 3(DT)  , 15, 20 https://doi.org/10.1155/2017/5341702. After detachment, the cardiac cell sheet with medium was transferred onto a polystyrene culture dish and was spread out on the dish by three manipulations using a pipette: (i) rotation of the dish, (ii) slow aspiration, and/or (iii) gentle dropping of culture medium as shown in previous reports [Human iPS cell-derived cardiac cells on a temperature-responsive culture surface beat spontaneously and synchronously as shown in Supplementary Movie 1 in Supplementary Material available online at  reports , and the reports , like th reports . Those rNext, after incubation , a second human iPS cell-derived cardiac cell sheet was layered onto the first cardiac cell sheet . After cNext, five-layered human iPS cell-derived cardiac cell sheets were observed by OCT. Just after the layering of a fifth cardiac cell sheet onto a four-layered cardiac cell sheet and centrifugation, the lower cell sheets beat synchronously . The movie shows the cross-section of a beating human iPS cell-derived cardiac cell sheet on a polystyrene culture surface just after transfer and centrifugation. Beating areas within the cell sheet are shown with green colors. Although just after transfer (Just after transfer) there were many spaces between the cell sheet and the culture surface, after centrifugation (Just after centrifugation) the spaces were hardly observed by OCT. The movie shows the beating phenomenon at real-time speed. An OCT system (IVS-2000) was used in this study.Supplementary Movie 3: Observation of double-layered human iPS cell-derived cardiac cell sheets by optical coherence tomography (OCT) at 15 min (Before synchronization) and 45 min (After synchronization) after centrifugation. The movie shows the cross-sectional observation of double-layered human iPS cell-derived cardiac cell sheets. Beating areas within the cell sheet are shown with green colors. The movie shows the phenomenon at real-time speed. An OCT system (IVS-2000) was used in this study.Supplementary Movie 4: Observation of five-layered human iPS cell-derived cardiac cell sheets by optical coherence tomography (OCT) just after the last centrifugation (Before synchronization) and at 60 min (After synchronization). The movie shows the cross-sectional observation of five-layered human iPS cell-derived cardiac cell sheets. Beating areas within the cell sheet are shown with green colors. The movie shows the phenomenon at real-time speed. An OCT system (IVS-2000) was used in this study.


This review is aimed at summarizing the current understanding of Tat in HIV-related cardiovascular diseases.The human immunodeficiency virus (HIV) is a major global public health issue. HIV-related cardiovascular disease remains a leading cause of morbidity and mortality in HIV positive patients. HIV Tat is a regulatory protein encoded by  The human immunodeficiency virus (HIV) is a major global public health issue. It has claimed more than 35 million lives so far. In 2016, 1 million people died from HIV-related causes globally . Cardiovgag, pol, env, tat, rev, nef, vif, vpr, and vpu. Tat stands for “trans-activator of transcription”, which is a small nuclear protein encoded by the tat gene in HIV-1. It consists of 86–101 amino acids depending on the subtype. During the process of HIV replication, Tat serves as a trans-activator that drastically enhances the efficiency of viral transcription m) and the electrophysiological activity of cardiomyocytes, which also aggravated hypoxia/re-oxygenation-induced cardiomyocyte injury through interference with the initiation of autophagy and the clearance of autophagic proteins [Latest research reveals that Tat has profound impacts on cardiomyocytes. Transfection of adenoviral-Tat impaired the uptake of mitochondrial Caproteins . It is wCACNA1C, that encodes the alpha 1c (Cav1.2) subunit of the L-type calcium channel [A study addressed the individual and combined effects of HIV-1 and methamphetamine on cardiac dysfunction using a transgenic mouse model of HIV infection. The results demonstrated that Tat and methamphetamine increased calcium uptake and promoted mitochondrial dysfunction in cardiomyocytes, this altered the DNA methylation and expression of  channel .+ current (IKr) channels encoded by human ether-a-go-go-related gene (hERG) [Corrected QT (QTc) prolongation is predictive of cardiovascular mortality. Frequent occurrence of QT interval prolongation have been found in HIV-positive patients , 27. Thee (hERG) . A studye (hERG) .+ current (Ito). Thus, it appears that HIV itself contributes to the delayed repolarization observed in the transgenic mice [90) and reduced IKr [Kr in hERG stably expressed HEK293 cells through the inhibition of hERG protein expression but with no effect on the hERG mRNA expression [Kr and IKs through sequestering membrane phosphatidylinositol--bisphosphate (PIP2), with no significant alteration in ICa-L [In HIV transgenic mice, cardiac repolarization is prolonged compared with wild-type littermates, which is at least partially attributed to the reduction of transient outward Knic mice . Subsequuced IKr . Furtherpression . Similarin ICa-L .2+ elevation in neurons was largely attributed to the promotion of lysosomal Ca2+ mobilization, Ca2+ release via inositol 1,4,5-trisphosphate-sensitive endoplasmic reticulum pools, and Ca2+ influx via transient receptor potential vanilloid type 2 (TRPV2) channels [In addition, Tat could affect both cardiomyocytes and neurons thus inducing bradycardia. Transgenic mice with myocardial overexpression of Tat exhibits a relative bradycardia phenotype, which is associated with reduced susceptibility of adrenergic responsiveness . Furtherchannels .HIV-seropositive patients are at higher risk for atherosclerosis than HIV-seronegative persons. This has been variably attributed to antiretroviral treatment, advanced immunodeficiency, and HIV-associated inflammation , 35. PriCarotid intima-media thickness (IMT), an indicator of generalized atherosclerosis, is higher in HIV patients than in age-matched control subjects, which is associated with classic coronary risk factors and with low CD4+ T cell count . Beyond Chronic immune activation and inflammation are risk factors of cardiovascular diseases. HIV-infection is also associated with increase in inflammation factors and immune activation. The inflammatory and coagulation biomarkers IL-6, hsCRP and D-dimer expression levels are associated with an increased risk of cardiovascular disease in HIV-infected individuals . The immThe inflammatory vascular environment is critical for the initiation and development of atherosclerosis. Tat induces vascular inflammation through the activation of nuclear factor-kappa B (NF-κB) in human vascular endothelial cells (HUVECs) and leads to the upregulation of inflammatory mediators, including IL-1β, MCP-1, VCAM-1 and E-selectin, which could be attenuated by estrogen . FurtherHIV infected patients have a greater incidence of pulmonary arterial hypertension (PAH) compared to the general population. Specifically, in comparison with the incidence of idiopathic PAH in the general population (1–2 per million), HIV-infected patients have a 2500-fold increased risk of developing PAH . The devHIV infection and intravenous drug abuse have been identified as common independent risk factors of PAH. For example, morphine potentiates HIV Tat protein-mediated apoptosis and proliferation of pulmonary endothelial cells, which may have led to strikingly more pronounced pulmonary vascular remodeling than exposed alone . The proTransgenic technology has made it possible to investigate the pathogenic mechanism of HIV in mice. HIV transgenic mice develops HIV-infection related complications. Some HIV encoded proteins, including Tat, Nef and Vpr, are important determinants for the pathogenicity of HIV , 58. TatAll these findings confirmed that Tat protein not only promotes the transcription of HIV but also participates in the pathogenesis of HIV-associated cardiovascular complications Fig. . It is aIn recent years, a randomized phase II exploratory clinical trial found that Tat immunization is safe, well tolerated and could induce anti-Tat antibodies in most patients. It promoted a durable and significant restoration of immune homeostasis and induced a significant reduction of blood proviral DNA in patients on effective combination anti-retroviral therapy regimen. Thus, Tat immunization represents a promising pathogenesis-driven intervention to intensify HAART efficacy . HIV-1 OBesides the conventional therapy involved in circumventing the effect of TAT in HIV- related cardiovascular diseases, there are also natural herbal extracts reported to possess anti-HIV properties. For instance, Curcumin is a natural herb extract, which has anti-HIV activity. A recent study revealed that curcumin causes inhibition and degradation of Tat which may be one of the major mechanisms behind its anti-HIV activity . Also, CThis review has been able to show the effects of Tat on HIV-related cardiovascular diseases. Further investigation is still needed to elucidate the molecular mechanism of Tat and HIV-related cardiovascular disorders which may suggest novel insights and therapeutic targets.


FH) cell differentiation and maintenance is strongly promoted during chronic infection. Thereby, the immune system promotes antibody responses, which bear less immune-pathological risk compared to cytotoxic and pro-inflammatory T cell responses. This emphasis on TFH cells contributes to tolerance of the chronic infection and is pivotal for the continued maturation and adaptation of the antibody response, leading eventually to the emergence of virus-neutralizing antibodies, which possess the potential to control the established chronic infection. However, sustained high levels of TFH cells can also result in a less stringent B cell selection process in active germinal center reactions, leading to the activation of virus-unspecific B cells, including self-reactive B cells, and to hypergammaglobulinemia. This dispersal of B cell help comes at the expense of a stringently selected virus-specific antibody response, thereby contributing to its delayed maturation. Here, we discuss these opposing facets of TFH cells in chronic viral infections.Chronic infections with non-cytopathic viruses constitutively expose virus-specific adaptive immune cells to cognate antigen, requiring their numeric and functional adaptation. Virus-specific CD8 T cells are compromised by various means in their effector functions, collectively termed T cell exhaustion. Alike CD8 T cells, virus-specific CD4 Th1 cell responses are gradually downregulated but instead, follicular T helper (T Non-or poorly cytopathic viruses like human immunodeficiency virus 1 (HIV-1), hepatitis B virus (HBV), and hepatitis C virus (HCV) in humans or lymphocytic choriomeningitis virus (LCMV) in mice can induce persistent infections employing several mechanisms to evade control by the immune system. Continuous high-level viral replication and therefore high viral burden in the host is a major factor leading to numeric reduction and functional impairment of virus-specific cytotoxic CD8 T cells and Th1 CD4 T cells, collectively termed T cell exhaustion [reviewed in Ref. ]. In thiFH) cells and cognate B cells -6  B cells (FH cells and cognate B cells is necessary to induce differentiation of TFH cells into GC TFH cells and to sustain their TFH phenotype (FH cells mediate affinity selection of B cells that have undergone proliferation and somatic hypermutation (SHM) by delivering survival signals via ICOS, CD40 ligand (CD40L), and the cytokine IL-21, depending on the affinity of the B cell for a given antigen -6 –26 whichfollicle –29. Ther B cells –33. Convhenotype –37, albehenotype . TFH cel antigen –41. TherFH cells accumulate during the persistent phase of viral infections with non- or poorly cytopathic viruses (FH population is most likely driven by follicular dendritic cell (FDC)-derived IL-6 signaling via signal transducer and activator of transcription (STAT)-3 -3 , 43, 45,(STAT)-3 . It woule Figure . Here, we Figure .FH cell differentiation is mediated in two steps. Priming of CD4 T cells that commit to the TFH cell lineage takes place in the T cell zone and is mediated by conventional DCs or monocyte-derived DCs -2, or P-selectin glycoprotein-1  upon priming  and CD40L, the immunoregulatory molecule PD-1, their hallmark cytokine IL-21, and the T cell adaptor protein SAP  cells. These are derived from thymus-derived T regulatory (Treg) cells, which adopt some TFH cell characteristics, like CXCR5 and Bcl-6 expression, to be able to migrate into B cell follicles. However, TFR cells lack expression of CD40L, IL-4, or IL-21 and have a higher expression of PD-1 and negatively regulate the GC response  and the light zone (LZ). In the DZ the cytokine CXCL12 is predominantly produced while the cytokine CXCL13 is predominantly produced in the LZ. Thereby, localization of B cells in DZ and LZ is controlled by differential expression of the chemokine receptors CXCR4 (migration into DZ) and CXCR5 (migration into LZ) . In the FH cells and FDCs are located , exhibit an abrogated antibody response and prolonged viral persistence (−/− (−/− (FH cell response upon (persistent) LCMV infection and are unable to eventually control the infection. Conversely, increasing the number of TFH cells by NK cell depletion accelerated viral clearance by improving the virus-specific antibody response , where the frequency of TFH cells positively correlated with the appearance of high-affinity SIV-specific antibodies in infected rhesus macaques (RM)  (CXCR5+CXCR3−PD-1+) were observed  HIV-1 infected individuals (FH cells in SIV-infected RMs (Similar correlations between Tues (RM) . These Tpression . Furthercted RMs . Also, iobserved , 91–98, tibodies . In HIV quencies . Howeverividuals , 99 or ected RMs .FH cells in the eventual emergence of virus-neutralizing antibodies during persistent viral infection is the high frequency of somatic mutations in the variable regions of these antibodies  to viral quasi species that emerge in vivo under selection pressures.Another indication implicating Ttibodies , 102. SHFH cells . It is t+/+ or Bcl6+/+ TFH cells is strictly required for the (late) emergence of LCMV-neutralizing antibodies. Using a novel in vivo experimental system allowed conditional depletion of specifically TFH cells or all LCMV-specific CD4 T cells during established persistent LCMV infection, after the initial establishment of the virus-specific IgG antibody response  were dispensable for maintaining overall LCMV-specific IgG titers and LCMV-specific IgG secreting plasma cells in spleen and bone marrow. By contrast, continued presence of LCMV-specific CD4 T cells was required to maintain overall LCMV-specific IgG titers as well as LCMV-specific IgG secreting plasma cells in bone marrow, suggesting that non-TFH LCMV-specific CD4 T cells are able to support an extra-follicular response to maintain the pool of LCMV-specific antibody-secreting cells and hence LCMV-specific IgG titers. However, sustained activity of TFH cells was strictly required for the development of LCMV-neutralizing antibodies by GC B cells  destruction of the lymphoid tissue architecture  structure as compared with fast progressing SIV-infected RMs with a less expanded TFH cell compartment  , 103. DuIn the context of HIV and SIV infection, B cell dysfunction was reported by a number of studies, characterized by loss of naïve and resting memory B cells, increases of activated B cells and tissue-like memory B cells, expansion of regulatory B cells, and altered functionality [reviewed in Ref. ].FH cells ineffective in delivering help to B cells. GC B cells isolated from HIV or SIV-infected individuals/animals displayed a higher expression of PD ligand 1 (PD-L1) as compared with B cells isolated from healthy donors. Therefore, TFH cells received more signals via PD-1 during HIV/SIV infections, which mediated downregulation of IL-21 and IL-4 expression, and at the same time had a negative impact on TFH cell survival and proliferation   . Furthertibodies . It has  and SIV , 126, al and SIV . Decreas in Ref. ].reg improved the LCMV-specific antibody response and viral clearance drastically  and exist as a so-called quasi species in the infected host , against which neutralizing antibodies are directed, e.g., the variable loops of HIV gp120 , 155 or indrance , 158–162indrance , 163–165indrance . In caseindrance . Interesindrance –171 wereFH cells to develop the necessary neutralization breadth. Factors curtailing the GC response as described earlier might well contribute to the impaired or delayed emergence of such bnab. Furthermore, to allow the continued development of bnab in the GC response, their evolution/selection has to occur against viral variants that do not undergo complete viral escape from these bnab precursors  and FcγR, as knockout mice still display hypergammaglobulinemia (via the BCR could occur concurrent with influenza hemagglutinin (HA). This led to the simultaneous presentation of MOG and HA on the MHC II surface molecules of MOG-specific B cells and subsequently their activation via HA-specific CD4 T cells (via this mechanism from the environment.The described B cell dysfunctions have been further linked to the delayed appearance of neutralizing antibodies and in the context of persistent LCMV infection have been shown to be dependent on CD4 T cell help to cognate B cells  signals , 207. Itulinemia . A recen T cells . This sc T cells . Another T cells . AssuminFH cells to dysregulated B cell responses, it has been shown before in settings of autoimmunity that prolonged maintenance of TFH cells, and therefore prolonged maintenance of GC B cells, is one cause for the emergence of autoreactive antibodies (FH numbers are increased; thereby permitting the survival of low affinity and self-reactive B cells (Regarding the contribution of Ttibodies , 209–213 B cells —a situatFH cells observed in LNs of infected individuals correlated with hypergammaglobulinemia and polyclonal B cell activation as well as the deletion of circulating memory B cells (FH cell numbers and at the same time B cell dysfunctions (FH cell population in persistent HIV and SIV infections and dysregulated B cell responses. Similarly, in persistent HBV infection, the frequency of cTFH cells correlated with the emergence of autoantibodies (Analogous, in HIV and SIV infection, the expansion of T B cells , 43, 215unctions , 200, whtibodies .FH accumulation and its impact on the antibody response that organ-specific differences might exist in specific persistent viral infections. This has been recently addressed in the context of SIV infection (FH responses have been analyzed in spleens of SIV-infected RMs (FH cell frequency in spleen was drastically reduced already in the acute phase of SIV infection as compared with healthy animals. This phenomenon was maintained in the persistent phase of SIV infection. In addition, TFH cells in spleen of SIV-infected RMs expressed less of the TFH-associated transcription factors Bcl-6 and c-Maf and instead upregulated transcription factors that counter-regulate TFH cell fate, i.e., Krüppel-like factor-2. This decrease in TFH cell frequency was further associated with reduced titers of SIV-specific IgG antibodies (FH frequencies were similar or elevated in LNs of these infected RM as compared with healthy animals and in accordance with previous reports (FH cells in the spleen of SIV-infected RMs occurred in the context of severe destruction of the splenic architecture (FH cells might have enhanced contact with cytotoxic CD8 T cells in the acute phase of infection, which might cause deletion of TFH cells in the spleen. Possibly, also differences in the recruitment of effector cells or different cytokine milieus in the LN and the spleen might influence the maintenance of TFH cells upon SIV infection.It also should be considered when discussing Tnfection . Most stcted RMs . In conttibodies . However reports , 215. Initecture . TherefoFH cell frequency and function have to be taken into consideration as together they might account for the outcome of the virus-specific antibody response.Therefore, organ-specific differences in TFH cells and cognate B cells often are hampered during persistent viral infections due to several factors. These include sustained increase of TFH cells, leading to non-specific B cell activation and hypergammaglobulinemia at the expense of virus-specific antibodies, destruction of the lymphoid tissue architecture, B cell exhaustion, skewed ratios of regulatory cells to TFH/GC B cells or in case of HIV/SIV infection TFH cells being directly infected. Due to these dysregulations, protective virus-specific antibody responses are delayed. Moreover, viruses use different mechanisms to evade recognition by antibodies using, e.g., variable loops or glycan shields to protect neutralizing epitopes. Furthermore, constant viral evolution leads to continued selection of escape variants upon exerted pressure by neutralizing antibodies, which fuels a molecular arms race between virus and the humoral immune response.Follicular T helper cell function and optimal interactions between TFH cells is essential for the induction of neutralizing, protective antibody responses upon persistent viral infection and that the eventual emergence of these antibodies can afford control of the persistent infection in absence of overt immunopathology.Nevertheless, it is clear that sustained activity of TFH cell function and interactions with cognate B cells as well as understanding the underlying mechanisms of the arms race between virus and humoral immune response might serve to improve the induction of neutralizing antibody responses and reduce B cell dysfunctions, thereby improving control of persistent viral infections.Therefore, targeting mechanisms that promote optimal TAll authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication.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.


The role of paracrine Hepatocyte Growth Factor (HGF) in the resistance to angiogenesis inhibitors (AIs) is hidden in xenograft models because mouse HGF fails to fully activate human MET. To uncover it, we compared the efficacy of AIs in wild-type and human HGF knock-in SCID mice bearing orthotopic human colorectal tumors. Species-specific HGF/MET signaling dramatically impaired the response to anti-angiogenic agents and boosted metastatic dissemination. In cell-based assays mimicking the consequences of anti-angiogenic therapy, colorectal cancer cells were completely resistant to hypoxia but extremely sensitive to nutrient deprivation. Starvation-induced apoptosis could be prevented by HGF, which promoted GLUT1-mediated glucose uptake, sustained glycolysis and activated autophagy. Pharmacological inhibition of GLUT1 in the presence of glucose killed tumor cells as effectively as glucose deprivation, and this effect was antagonized by HGF. Concomitant targeting of GLUT1 and HGF potently suppressed growth and dissemination of AI-resistant human tumors in human HGF knock-in SCID mice without exacerbating tumor hypoxia. These data suggest that stroma-derived HGF protects CRC cells against glucose starvation-induced apoptosis, promoting resistance to both AIs and anti-glycolytic agents. Combined inhibition of glucose metabolism and HGF/MET signaling (‘anti-METabolic therapy’) may represent a more effective CRC treatment compared to utterly blocking tumor blood supply. KRAS- or BRAF-mutant tumors [Metastatic colorectal cancer (CRC) is one of the most common tumors in both men and women, and the second leading cause of cancer deaths worldwide . The curt tumors . These pt tumors .Angiogenesis inhibitors (AIs), and in particular bevacizumab, achieved encouraging results in advanced CRC , 6. HoweThe biological mechanisms underlying resistance to AIs are not fully understood. Tumor hypoxia consequent to vessel pruning has been identified as a major cause of therapeutic failure and relapse . It has HGF is a pleiotropic cytokine of mesenchymal origin that controls cell proliferation, motility, differentiation, and survival . Its higIn this study we hypothesized that HGF/MET signaling could be responsible not only for allowing CRC cells to physically escape anti-angiogenic therapy, but also for rewiring their metabolism in response to vessel pruning. To investigate this possibility, we exploited the inability of mouse HGF to fully activate human MET –28. In fHGF gene has been replaced by homologous recombination with a cDNA encoding human HGF. Since human HGF activates both human and mouse MET, these animals are vital and fertile, and thanks to the SCID background, they support the growth of human tumor xenotransplants [HGF gene promoter, hHGF KI SCID mice display fully physiological HGF expression levels [We recently described the use of human HGF knock-in (hHGF KI) SCID mice for studying the role of stroma-derived HGF in the resistance to MET inhibitors . In thisnsplants . In contnsplants , hHGF KIn levels .KRAS or BRAF mutations into wild-type (WT) and hHGF KI SCID mice, and compared the efficacy of different anti-angiogenic drugs in these two strains. To determine the effect of HGF on tumor metabolism, we compared the expression of glucose transporters in WT and hHGF KI SCID mice subjected to anti-angiogenic therapy. Finally, to dissect the mechanism by which HGF affects the cellular response to angiogenesis inhibition, we simulated the environmental conditions set by anti-angiogenic therapy in culture, and tested the impact of HGF on both classical biological parameters  and cellular metabolic functions .To unmask the role of micro-environment-derived HGF in the response of CRC to AIs, we orthotopically injected human CRC cells bearing KRAS (HCT-116) or BRAF (HT-29). HCT-116 and HT-29 cells, which displayed HGF-dependent MET activation  . Tumor g6 Figure  and HT-26 Figure  tumors g6 Figure . In both6 Figure , as dete6 Figure , but res6 Figure . These dBioluminescence analysis of isolated organs revealed that metastatic dissemination was markedly HGF-dependent. In fact, livers and lungs extracted from hHGF KI SCID mice bearing HCT-116 Figure  or HT-29KRAS or BRAF, including HCT-116 and HT-29, were dramatically more sensitive to glucose deprivation compared to cells with WT KRAS and BRAF, and their survival in low glucose could be rescued by HGF. Oxygen deprivation had little effect on survival, regardless of genetic status. Altogether, these data suggest that KRAS- and BRAF-mutant CRC cells rely on glucose much more strictly than they depend on oxygen for survival, and that HGF extends the permissive range of glucose concentration.By pruning tumor vessels, anti-angiogenic therapy increases tumor hypoxia and deprives cancer cells of nutrients, dramatically reducing glucose availability , 33. In Increased MET expression and HGF-dependent invasion under hypoxia has been described years ago and has been subsequently confirmed by other studies conducted in several model systems, including glioblastoma and pancreatic cancer –14. To eSLC2A1 gene  and low (0.5 g/L) glucose culturing conditions Figure , accompae Figure , resultie Figure . AltogetGrowing evidence points at autophagy as one of the mechanisms that cancer cells adopt to escape the metabolically challenging conditions set by anti-angiogenic therapy . To inveThe data presented so far suggest that HGF promotes a metabolic ‘salvage’ program allowing CRC cells to avoid an energetic crash when facing glucose shortage. We reasoned that pharmacological blockade of HGF/MET signaling could prevent this adaptive process and overcome acquired resistance to AIs. To test this hypothesis, we orthotopically micro-injected HCT-116-luc and HT-29-luc cells into hHGF KI SCID mice as described above, and then randomly assigned tumor-bearing animals to the following arms: no treatment; 15 mg/kg bevacizumab; 10 mg/kg tivozanib; 40 mg/kg JNJ-38877605  ; 20 mg/k50 at 1.0 g/L glucose of 6.7 μmol/L and 11.2 μmol/L, respectively. HGF significantly protected HCT-116 and HT-29 cells against GLUT1 inhibition, increasing the EC50 by approximately one log . This analysis was extended to the same CRC cell panel used above for oxygen vs. glucose dependency. Consistent with their extraordinary vulnerability in low glucose conditions, KRAS- and BRAF-mutant CRC cells were significantly more sensitive to GLUT1 blockade compared to cells with WT KRAS and BRAF  of the bis-hydroxybenzoate compound WZB-117, an irreversible blocker of GLUT1 , in the KRAS/BRAF-mutant CRC cells, and that environmental HGF protects CRC cells against metabolic stress through GLUT1 upregulation. We therefore reasoned that selective targeting of GLUT1 would have been a cleaner and more effective therapeutic approach compared to generalized cutting of tumor blood supply, and that neutralization of HGF would have overcome micro-environment-mediated resistance to glucose uptake blockade. To test this hypothesis, we subjected hHGF KI SCID mice bearing orthotopic HCT-116-luc and HT-29-luc tumors to the following treatments: control; 20 mg/kg ficlatuzumab; 10 mg/kg WZB-117; 10 mg/kg WZB-117 plus 20 mg/kg ficlatuzumab. Tumor growth and metastatic dissemination was determined as described above. The results of this analysis provided relevant feedback on our work hypothesis. First, WZB-117 inhibited colorectal tumor growth much more effectively than AIs in the same conditions  rather than to ‘starve’ it (by hijacking its access to glucose). But is oxygen deprivation the real biological mechanism underlying the anti-tumor effect of AIs? Or on the contrary, is it a trigger for the malignant adaptation of cancer cells, leading to acquired resistance and disease progression?KRAS/BRAF-mutant cells: while hypoxia increased their invasiveness and aggressiveness, prolonged hypoglycemia was completely incompatible with their survival. Altogether, these data point at nutrient rather than oxygen dependency as the real Achille's heel of KRAS- and BRAF-mutant tumors.The cell-based experiments aimed at dissecting the biological effects of anti-angiogenic therapy on CRC cells provide support for the latter hypothesis. In fact, all CRC cells tested, regardless of their genetic status, were relatively resistant to hypoxia, which had a mere cytostatic effect, but more sensitive to both glucose deprivation and GLUT1 blockade, which caused apoptosis. This dichotomy was particularly evident in KRAS- or BRAF-mutant tumors may be a safer and more effective strategy compared to utterly impairing tumor angiogenesis.By pruning tumor blood vessels, AIs do deprive cancer cells of nutrients, thus in principle targeting this dependency, but at the same time cause tumor hypoxia, which represents a major stimulus for invasion and resistance. The data presented here suggest that, when inhibiting tumor angiogenesis, the benefits of reducing glucose availability are negatively compensated by the adverse effects of tumor hypoxia. In contrast, selective blockade of glucose metabolism achieves tumor starvation -which is what counts from a therapeutic viewpoint- without altering tumor oxygenation. Based on these results, it could be inferred that targeting glucose metabolism in CRC patients bearing KRAS- and BRAF-mutant tumors that compare the efficacy and safety of metabolism-targeting agents with that of angiogenesis inhibitors. Also, control of tumor metabolism by HGF may bring new life to the many HGF/MET inhibitors that are currently struggling to find an application in oncology. Possibly, the combined use of anti-metabolic agents and HGF/MET-targeted drugs (‘anti-METabolic therapy’) may be considered in order to unmask metabolic addictions and, most importantly, to prevent or delay the occurrence of acquired resistance.A variety of metabolism-targeting drugs are about to reach the clinic, including inhibitors of glucose uptake and utilization . The avaAll tumor and stroma cell lines used in this study were purchased from the American Type Culture Collection (ATCC) and cultured as indicated by the provider. See online Suppl. Methods for more information on culturing conditions. Genetic identity of each cell line was confirmed by short tandem repeat profiling using Cell ID System (Promega).in vivo and ex vivo bioluminescence analysis, and immunohistochemical examination of tumors.All animal protocols were approved by the Animal Research Ethical Committee, Fondazione Piemontese per la Ricerca sul Cancro - ONLUS, Candiolo, Italy, and by the Italian Ministry of Health. WT SCID CB17 SCID mice were purchased from Charles River. The hHGF KI SCID strain was obtained from AVEO Pharmaceuticals and has been described previously . OrthotoTM ADP analyzer (Beckman Coulter). Cell cycle analysis was determined as described [Cells were cultured in standard conditions until they reached 60% confluency, and then incubated in progressively lower concentrations of oxygen (21-0.1%) or glucose (4.5-0.01 g/L) in the absence or presence of 50 ng/ml human recombinant HGF (R&D Systems). After 48 hours, cells were processed for apoptosis and cell cycle analysis. For apoptosis analysis, cells were stained with a solution containing anti-annexin V antibodies and propidium iodide (eBioscience). The percentage of annexin V/propidium iodide-positive cells was determined by flow cytometry using a CyAnescribed . For hypGlucose and lactate concentration in the medium was determined using commercial kits (both from BioVision). For glucose uptake analysis, HCT-116 and HT-29 cells were pre-cultured in 4.5 g/L or 0.5 g/L glucose in the presence or absence of 50 ng/ml recombinant human HGF (R&D Systems) for 24 hours, and then incubated with 10 μmol/L 2-NBDG (Life Technologies) in glucose-free medium for 30 minutes. The 2-NBDG reaction was stopped by washing cells with pre-chilled PBS. Glucose uptake was quantified by measuring the fluorescent intensity of cells in the FL-1 channel using a CyAnTM ADP analyzer (Beckman Coulter). For glycolytic capacity analysis, the extracellular acidification rate (ECAR) was determined using a Seahorse XF96 analyzer (Seahorse Bioscience) as described . See onlp value ≤ 0.05 was considered significant. In all Figures, values are expressed as mean of biological replicates ± SEM. Sample size is indicated in each Figure legend.Statistical significance was determined using a homoscedastic, two-tailed Student's t-test. A


Although significant levels of side effects are often associated with their use, microtubule-directed agents that primarily target fast-growing mitotic cells have been considered to be some of the most effective anti-cancer therapeutics. With the hope of developing new-generation anti-mitotic agents with reduced side effects and enhanced tumor specificity, researchers have targeted various proteins whose functions are critically required for mitotic progression. As one of the highly attractive mitotic targets, polo-like kinase 1 (Plk1) has been the subject of an extensive effort for anti-cancer drug discovery. To date, a variety of anti-Plk1 agents have been developed, and several of them are presently in clinical trials. Here, we will discuss the current status of generating anti-Plk1 agents as well as future strategies for designing and developing more efficacious anti-Plk1 therapeutics. However, one of the major shortcomings of these MT-targeting agents has been severe and dose-limiting side effects that arise as the consequence of indiscriminately disrupting widespread MT functions, not only in actively dividing mitotic cells but also in non-dividing interphase cells. Thus, over the past decade, a high level of interest has been drawn to targeting a variety of mitosis-specific proteins in order to develop agents that can specifically disrupt the mitotic progression of highly proliferative cancer cells. These proteins include protein kinases (polo-like kinase 1 [Plk1]2 and Aurora A3), motor proteins (CENP-E5 and Eg56), DNA-damage checkpoint proteins (Chk1 and Chk27), and components of the ubiquitin proteasome pathway (APC/Cdc20 and the proteasome9). Among these endeavors, anti-Plk1 drug discovery has reached an advanced stage of development that merits reflection on its progress. In this short review, we will summarize recent advances and future directions toward developing therapeutics against one of the most appealing anti-cancer drug targets, Plk1.For several decades, anti-microtubule (MT) drugs such as taxanes and vinca alkaloids have been effectively used against a wide range of cancers, including solid and hematological malignancies10. Plk1 is composed of theN-terminal catalytic domain and theC-terminal non-catalytic polo-box domain (PBD)  and plays a key role at multiple stages of mitotic progressionn (PBD) . The coo24 . In addition, several other ATP-competitive inhibitors, such as GSK461364 31, MLN0905 33, RO3280 35, NMS-P937 37, and TAK-960 38 have shown only limited efficacy with more-than-acceptable dose-limiting toxicity in diverse preclinical/clinical trials. Dose-limiting toxicity arises mainly from non-specific activity of the inhibitors39. In fact, one of the common problems associated with the currently available Plk1 ATP-competitive inhibitors is their low degree of selectivity against other kinases24, including two that are closely related, Plk2 and Plk3, with possible tumor-suppressor function41. Therefore, improving Plk1 specificity is likely one of the most pressing concerns to address in order to accomplish better clinical outcomes with fewer toxicological problems.Targeting the catalytic activity of a protein kinase has been the predominant method of generating kinase inhibitors. Accordingly, a large number of ATP-competitive inhibitors directed against the catalytic activity of Plk1 have been developed and tested under various preclinical and clinical settings24 . Among t39. Therefore, for a given kinase, having a better understanding of the exact nature of the ATP-binding site  could be crucial for overcoming lack of specificity associated with non-allosteric ATP-competitive inhibitors.Generating ATP-competitive inhibitors aimed at inhibiting the catalytic activity of a protein kinase is a widely used approach, in part, because isolating small molecules that block the catalytic activity of a kinase is relatively straightforward. However, owing to the highly conserved nature of the ATP-binding pocket, a large fraction of this class of inhibitors show a significant level of cross-reactivity with various other kinases24  that are somewhat selective against non-Plks 43.Further development of Plk1 ATP-competitive inhibitors can be achieved through structure-guided medicinal chemistry targeting unique residues lining the ATP-binding pocket. Comparative analyses of the X-ray co-crystal structures of the catalytic domains of Plk1–3 revealed that the overall shapes of their ATP-binding pockets are similar, and the residues forming the pockets are mostly analogous to one another. Nevertheless, both F58 and R134 residues are unique to Plk1, whereas R57, L132, and R136 residues found in Plk1 and one of Plk2 and Plk3 ATP-binding pockets are semi-specific to Plk124 . These oon-Plks . This fi44. Such work highlights what can be achieved using structure-based drug design to take advantage of small differences in protein-binding sites. An additional effort to target the Plk1-specific F58 and R134 residues that lie at the mouth of the core ATP-binding pocket  that are important for achieving kinase selectivity . Volaser50. However, subsequent studies showed that it possesses rather poor anti-Plk1 activity and specificity and inhibits multiple kinases53. In a broad sense, the anti-PBD agents described below can be classified as target-specific allosteric inhibitors, which inhibit Plk1 function by binding to a specific site distant from the ATP-binding pocket.Another potentially appealing strategy toward achieving target-specific allosteric inhibition of Plk1 could be to block its essential substrate-binding interface. In principle, this may be approached by understanding the nature of the Plk1-substrate binding interactions that are critical for proper mitotic progression. ON01910, a substituted benzyl styryl sulfone, was initially reported as a non-ATP-competitive inhibitor of Plk1 that was thought to interfere with the substrate-binding sitecis-acting phosphorylation of bound substrates by theN-terminal catalytic domain54. A large body of evidence suggests that the Plk1 PBD contains a well-defined protein–protein interaction (PPI) pocket that can be inhibited by short peptides or peptide mimetics24. Intriguingly, inhibition of Plk1 PBD function alone is sufficient for effectively imposing mitotic arrest and apoptotic cell death in cancer cells but not in normal cells20. This suggests that targeting the PBD may serve as a promising alternative for developing anti-Plk1 therapeutics. It should be noted, however, that not all of Plk1’s substrates require a prior interaction with the PBD before being phosphorylated by itsN-terminal catalytic domain54. Therefore, inhibiting PBD function represents a target-restricted strategy that is designed to antagonize a subset of Plk1 functions dysregulated in cancer54.The PBD is a functionally essential domain that binds to the enzyme’s physiological substrates and mediates the39, PBD inhibitors target a structurally unique domain found in only four proteins (Plk1−3 and Plk5)24. This greatly diminishes the likelihood of facing unwanted non-specific cross-reactivities. Another potential advantage of targeting the PBD is that while ATP-competitive inhibitors can abrogate all Plk1-dependent biochemical processes indiscriminately in both cancer and normal cells, PBD inhibitors interfere in only a subset of Plk1 functions that require a PBD-mediated biochemical step. Furthermore, since the Plk1 PBD can interact with a variety of structurally diverse phosphoepitope-containing proteins54, it is reasonable to speculate that anti-PBD agents could potentially be optimized so that they selectively inhibit a subset of PBD-dependent interactions, which are enriched in biochemically rewired, Plk1-addicted cancer cells24.Unlike ATP-competitive inhibitors, whose specificities must be obtained against more than 500 other cellular kinases55, several high-affinity peptide-derived inhibitors, including 4j  an adjoining Pro-binding region that engages portions of the bound ligandN-proximal to the Ser-p-Thr dipeptide motif55, and 3) a hydrophobic channel, which is capable of boosting the binding affinity ~500–1,000-fold without compromising Plk1 PBD specificity59. However, despite the fact that these peptide-based inhibitors exhibit extremely high affinity and specificityin vitro, their utility in cellular contexts is greatly decreased by poor membrane permeability and limited bioavailability. Efforts to increase cellular uptake have been directed at the reduction of anionic charge through charge masking and prodrug protection60, macrocyclization62, and reducing peptide character63.Since the initial discovery of a pentameric p-Thr-containing peptide, PLHSpT, as a specific Plk1 PBD-binding ligandding 4j , have belk1 PBD . These a64, poloxipan65, purpurogallin (PPG)66, T52167, Cpd16168, and (−)-epigallocatechin69 , are thought to be some of the common artifacts that lead to the isolation of pan assay interference compounds (PAINS)73 (as discussed in depth in a recent review74).Paralleling the development of peptidomimetics as described above are efforts to generate anti-Plk1 PBD agents that have yielded a wide range of small molecule inhibitors, including thymoquinone (TQ) and poloxinchin69 . As expeEven though currently available peptide-derived and small molecule inhibitors have provided valuable information in understanding the nature of Plk1 PBD-dependent interactions and their cellular functions, it remains uncertain whether they can be used as leads for developing anti-PBD agents. Therefore, while these inhibitors are being further developed, additional endeavors could be considered in parallel in the hope of generating a new class of inhibitors with more druggable properties. Identifying promising leads possessing outstanding biochemical selectivity (i.e. specificity and affinity) and druggable physicochemical properties  will likely be a critical step for successful lead optimization and ultimate discovery of anti-Plk1 PBD therapeutic agents.56 that collectively form a “Y-shaped” binding pocket   . Future 75. In addition, structural elaboration of hydrophobic channel-binding functionality and improvement of the interactions with Plk1-specific residues found in the surroundings of the hydrophobic channel have significantly increased overall ligand affinity and selectivity77. These findings suggest that the binding affinity and specificity of each of the three binding modules are determined independently of one another. Since a ligand capable of simultaneously binding to two or more binding modules must additionally conform to the exact geometrical arrangement of these modules, anti-Plk1 PBD agents generated through the Y-shaped, three-binding-module platform (Recent work has demonstrated that suitably designed p-Thr mimetic derivatives optimized for the phosphoepitope-recognition module can achieve several-fold enhancement in PBD-binding affinitylatform  may likeN-terminal catalytic domain and theC-terminal PBD. The primary challenge facing currently available ATP-competitive inhibitors of Plk1 appears to be their dose-limiting toxicities. Although this hurdle can be conquered in principle by improving specificity or generating potentially more specific allosteric inhibitors as discussed above, considering extensive efforts made to date, this advancement may not be easily accomplished unless a breakthrough occurs. In this regard, it is quite intriguing to define the PBD as an alternative target for anti-Plk1 drug discovery. In addition to the superb binding specificity that PPI inhibitors can bring, Plk1 PBD inhibitors can provide an opportunity to selectively interfere with cancer-cell-enriched Plk1 PBD-binding targets. If developed, Plk1 PBD inhibitors with superior specificity can be used not only as a single therapeutic agent but also as an agent easily amenable for combination therapy with other anti-cancer therapeutics.Given the significant level of side effects associated with conventional MT-targeting anticancer therapeutics, developing an inhibitor against a mitosis-specific and cancer-cell-addicted target such as Plk1 may represent a promising strategy for the generation of a cancer-cell-specific therapeutic agent. One of the unique molecular features of Plk1 is that it offers, within one molecule, two independent drug targets—theSignificantly, the use of multiple inhibitors targeting different regions within one molecule is considered an effective way to eliminate drug resistance and to diminish the side effects of a high dose of a single agent. Therefore, using a Plk1 PBD inhibitor in combination with an ATP-competitive inhibitor, such as volasertib, could present a great treatment regimen that may achieve the maximum drug efficacy of both inhibitors while keeping volasertib at the highest acceptable dose that does not cause nonspecific cytotoxicity. Considering the success of anti-mitotic therapeutics and the remarkable advantages of targeting Plk1, there is ample reason to believe that further developing anti-Plk1 therapeutics may prove to be a worthwhile endeavor in the fight against cancer.


We describe the development and application of a suite of modular tools for high-resolution detection of proteins and intracellular protein complexes by electron microscopy (EM). Conditionally stable GFP- and mCherry-binding nanobodies  are characterized using a cell-free expression and analysis system and subsequently fused to an ascorbate peroxidase (APEX) enzyme. Expression of these cassettes alongside fluorescently labelled proteins results in recruitment and stabilisation of APEX, whereas unbound APEX nanobodies are efficiently degraded by the proteasome. This greatly simplifies correlative analyses, enables detection of less-abundant proteins, and eliminates the need to balance expression levels between fluorescently labelled and APEX nanobody proteins. Furthermore, we demonstrate the application of this system to bimolecular complementation (‘EM split-fluorescent protein’), for localisation of protein–protein interactions at the ultrastructural level. The use of enzymatic tags such as the ascorbate peroxidase (APEX) for electron microscopic detection of proteins is changing electron microscopy (EM) in the same way that the use of GFP and related proteins caused a revolution in light microscopy. We previously developed expression plasmids encoding GFP-binding peptide (or nanobody) fused to APEX, which allows EM localisation of GFP-tagged proteins in vivo. Here, we have generated conditionally stable GFP- and mCherry-binding nanobodies fused to APEX. Using co-transfection of these APEX nanobodies with fluorescent-tagged constructs, we recruit APEX and detect the tagged proteins by electron microscopy. As unbound conditionally stable nanobodies are efficiently degraded by the proteasome, the signal to noise ratio is dramatically reduced. This enables detection of less abundant proteins and eliminates the need to balance expression levels between fluorescent-labelled and APEX nanobody constructs. Furthermore, and perhaps most exciting, is our application of this method to bimolecular fluorescence complementation—in which two tagged proteins interact—allowing the detection and localisation of protein-protein interactions in EM. Rapid and reliable protein localisation is critical for the functional characterisation of any protein of interest (POI). Traditionally, this has been achieved through antibody-mediated methods or tagging with a fluorescent protein, such as GFP. The recent emergence of nanobodies  has allowed the development of new biotechnological tools based on the detection of epitopes in living cells ,2, althoWe have previously generated expression plasmids that encode a GFP-nanobody/binding peptide (GBP) for high-resolution detection of GFP-tagged proteins by electron microscopy. To achieve this, we genetically fused the GBP nanobody to the well-characterized soybean-derived enzyme APEX. When APEX–GBP is expressed in the presence of any GFP-tagged POI, its localisation can be determined by transmission EM following processing ,6.Here, we have developed and characterized a new suite of APEX/nanobody-mediated tools. As GFP and mCherry are the most broadly used fluorescent proteins in cell biology, we used cell expression to screen a library of putative mCherry-binding peptides (ChBPs) by single-molecule coincidence detection. We demonstrate the utility of a single mCherry nanobody for high-resolution, EM-based analysis of protein distribution and use this probe for correlative analyses. Furthermore, we generate conditionally stable (cs) nanobodies for both GFP and mCherry fused to APEX and show that degradation of unbound cs nanobodies by the proteasomal system reduces background APEX signals and results in an increased signal-to-noise ratio. Finally, we show that the new suite of APEX nanobody tools opens up entirely new avenues for EM localisation through the application of the csAPEX-nanobody system to bimolecular fluorescence complementation, allowing the detection and localisation of intracellular protein-protein interactions at the ultrastructural level.Leishmania tarentolae cell-free lysate ) resulted in the accumulation of electron density in the cytoplasm of transfected cells  cells with APEX-ChBP and three different subcellular markers: (i) mCherry to denote the cytoplasm, (ii) mCherry-Cavin1 to denote caveolae on the plasma membrane (PM), and (iii) 2xFYVE-mCherry to denote early endosomes. Co-expression of the soluble APEX-ChBP and mCherry . Any unbound APEX nanobody will produce a diffuse cytosolic pool that will hinder detection of the POI and reduce the signal-to-noise ratio, particularly for low-abundance antigens. Recent work using the GBP nanobody has shown that manipulation of specific conserved residues produces a cs protein that is rapidly degraded by the proteasomal system in the unbound state . We usedTo confirm efficient degradation of our new, conditionally stable csAPEX-ChBP via the proteasomal pathway, we used the well-established proteasome inhibitor MG132 . Cells e1–155), Cavin3 fused to the C-terminal fragment (Cavin3-mVenus156–239), and our csAPEX-GBP construct, schematically represented in Bimolecular fluorescence complementation (BiFC) is a technique for testing pairwise protein-protein interactions in fixed or living cells by genetically tagging candidates with different halves of a “split” fluorescent protein . If thesIn summary, we have utilised cell-free expression and single-molecule analysis to screen a number putative ChBP for association with mCherry-tagged Caveolin-1. The single nanobody we identify is a selective, high-affinity binder of the mCherry tag, lacks detectable self-aggregation or cross-reactivity with GFP, can be linked to APEX for high-resolution analysis of mCherry-tagged proteins in cell culture systems, and is compatible with correlative light and EM. We have also employed conditional stabilisation of both GFP and mCherry binding nanobodies fused to APEX2 which results in the generation of an APEX reaction product only when bound to their target fluorescent proteins. By degrading unbound APEX-BP protein, this modification facilitates an improved signal-to-noise ratio and circumvents any potential oversaturation of the APEX-BP vector. Finally, we have coupled the csAPEX-GBP system with bimolecular fluorescence complementation. This now allows direct visualisation of intracellular protein–protein interactions at the ultrastructural level, far beyond the resolution of light microscopy. This system is immediately applicable (without any new cloning steps) to any system in which the fluorescent split GFP system has been used. Unlike labelling on sections, APEX methods are compatible with 3D EM methods  such as Single-molecule spectroscopy was performed as previously described . BrieflyBHK cells were passaged in Dulbecco’s Modified Eagle Medium (Gibco) supplemented with 10% Fetal Bovine Serum and L-Glutamine. Cells were seeded onto 35-mm culture dishes (TPP), transfected with Lipofectamine 3000 as per the manufacturer’s instructions and processed for EM 24 h later. For bimolecular fluorescence complementation experiments, an 8-h incubation in 50 μM cyclohexamide prior to processing was used to reduce background staining.2O2 for 30 min at room temperature. Cells were post-fixed with 1% OsO4 for 2 min to provide contrast. Cells were then washed in water and serially dehydrated in increasing percentages of ethanol before serial infiltration with LX112 resin in a BioWave microwave (Pelco). Resin was polymerised to hardness at 60°C overnight. Ultrathin sections were cut on an ultramicrotome (UC6: Leica) and imaged at 80 kV on a JEOL1011 transmission electron microscope. Sections were not post-stained.EM was performed exactly as described previously ,6. BriefCells were grown on 35-mm gridded MatTek dishes  and co-transfected with nls-mCherry and APEX-ChBP. Live cell imaging was performed on an EVOS FL epifluorescent microscope (ThermoFisher Scientific) at 10x and 20x magnification. Cells were processed as described above with the following exceptions. Post-polymerisation, the flat-embedded cells were removed from the dish and the region of interest was trimmed using the now-imprinted grid coordinates on the block face. Ultrathin sections were cut, placed on a slot grid, and imaged on a Tecnai 12 transmission electron microscope fitted with a 4K x 4K LC1100 camera (Direct Electron) at 120 kV under the control of SerialEM. Low-magnification  montages were acquired at a binning of 1 and stitched together using the Blendmont program in IMOD. Correlation of light and EM images was performed using Photoshop (Adobe Inc.).L. tarentolae expression were constructed by PCR subcloning from the original templates [Split mVenus constructs were made by first removing the Fos and Jun inserts from pcs_kmVenus1-155_FosLZ135-171 and pcs_kmVenus156-239_JunLZ253-289 using EcoRV/SpeI. Human Cavin1 and Cavin3 open reading frames were amplified by PCR using the primer tags forward 5′-AGCGGCGGCGGCTCTGATATC-3′ and reverse 5′-ACAAGAAAGCTGGGTACTAGT-3′ and subcloned using infusion (BD). The series of ChBP-GFP expression vectors for emplates , mCherryemplates , Cavin2-emplates , pCSDESTemplates , pDEST-Templates , APEX2-GS1 FigLeishmania lysate with mCherry tagged Caveolin1. A) ChBP1, B) ChBP2, C) ChBP3, D) ChBP4, E) ChBP6, F) ChBP8. Left-hand panels show GFP intensity through the confocal volume determined by single-molecule counting over time. Middle panels show simultaneous detection of coincidence of ChBP-GFP and mCherry-Cav1 over time. Right-hand panels show plots of the coincidence ratio between red and green channels. Only ChBP2 demonstrated a lack of self-aggregation/cross-reactivity with GFP , equivalent detection of red and green signal intensity over time , and a 1:1 coincidence ratio of GFP to mCherry . Data underlying all middle panels is available in Six putative ChBPs were GFP-tagged and co-expressed in cell free (TIF)Click here for additional data file.S2 Figp < 0.0001. See also A) Quantitation of the effect of GFP presence on stabilisation of the conditionally stable APEX-GBP. Co-transfection of GFP with the csAPEX-GBP construct results in greater than 40% of cells with cytoplasmic density, compared to approximately 5% with transfection of csAPEX-GBP alone. Chi squared, (TIF)Click here for additional data file.S1 Data(XLSX)Click here for additional data file.S2 Data(XLSX)Click here for additional data file.


Radial endobronchial ultrasound (R-EBUS) is one important diagnostic approach in non-small cell lung cancers (NSCLC). However, the small samples obtained from R-EBUS-guided transbronchial biopsies are sometimes insufficient for pathological and molecular diagnosis. Herein, we investigated the suitability of R-EBUS-guided bronchial brushing specimens for NSCLC diagnosis and EGFR genotyping. We enrolled 941 consecutive patients with peripheral pulmonary lesions who underwent R-EBUS. Cytology-positive brushing specimens from non-squamous NSCLC patients were tested for EGFR mutations. Non-squamous NSCLC was diagnosed in 624 patients (66.3%). Positive cytology was documented in the brushing samples of 376 patients (60.3%). Higher diagnostic yields were obtained in patients exhibiting bronchus signs on chest tomography, and those with R-EBUS probe located within the lesion. EGFR genotyping was successfully performed in 363 samples (96.5% of cytology-positive brushing samples). EGFR genotyping concordance between brushing specimens and matched tissue samples was 88.7% . Furthermore, 144 non-squamous NSCLC patients (23.1%) with failed pathological diagnosis or EGER sequencing by R-EBUS-guided transbronchial biopsy required repeat biopsies. However, it was achieved successfully from the brushing specimens of 57 patients (39.6%). In conclusion, for patients with peripheral lung cancer, R-EBUS-guided bronchial brushing could provide an additional sampling method for diagnosis and EGFR genotyping. EGFR), as well as that of EGFR-tyrosine kinase inhibitors (TKIs), introduced a new era of precision medicine in lung cancer treatment2. Clinical studies demonstrated the significant treatment efficacy of targeted therapy in patients with sensitizing EGFR mutations5. As a result, current guidelines for the diagnosis and treatment of patients with advanced non-small cell lung cancer (NSCLC) recommend that EGFR genotyping be performed in tumors with non-squamous histologies7.The discovery of mutations in the epidermal growth factor receptor -guided transthoracic needle biopsy or surgical resection, to obtain additional tissue samples for molecular testing. Hence, patients undergoing repeat invasive procedures are exposed to additional risks, and treatment may be delayed as a result.In many patients with NSCLC, especially those with tumors of non-squamous histologies, tumors often present with peripheral pulmonary lesions (PPLs) that impede access to the target tumor by conventional bronchoscopy. Indeed, these lesions present a frequently encountered challenge to pulmonologists. With the development of radial endobronchial ultrasound (R-EBUS), which dramatically improved the visualization and localization of PPLs, the diagnostic yield of transbronchial biopsy has improved13. However, no cohort studies have been performed to investigate the role of R-EBUS-guided bronchial brushing in cytopathological diagnosis and EGFR analysis. Therefore, in the present study, we investigated the performance of R-EBUS-guided bronchial brushing in both the cytopathological diagnosis of and EGFR mutation detection in peripheral non-squamous NSCLC.Our previous pilot study showed that RNA-based sequencing of waste bronchial brushing specimens may be a feasible method for multi-gene analysis14. The cutoff date for data collection was November 31, 2016. This study was approved by the Institutional Review Board of National Taiwan University Hospital. Written informed consent was obtained from all patients before undergoing bronchoscopic procedures. All methods were performed in accordance with the relevant guidelines and regulations.This study was conducted at National Taiwan University Hospital, a tertiary referral center. Consecutive patients with PPLs who were referred for R-EBUS between September 2010 and December 2015 were enrolled (n = 941). A PPL was defined as a lesion surrounded by lung parenchyma with no endobronchial abnormalities detected by conventional bronchoscopy. Computed tomography-based findings, including tumor location, size, and presence of a bronchus sign  were documented. The treatment responses of patients with advanced NSCLC who were administered first-line EGFR-TKIs  were recorded based on the Response Evaluation Criteria in Solid Tumors, version 1.1Conventional bronchoscopy  was initially performed to examine the trachea and bronchi. R-EBUS was then performed using an endoscopic ultrasound center  and a 20-MHz radial ultrasonic probe . The R-EBUS probe position was recorded as within or adjacent to the target tumor. After a lesion was located, the radial probe was withdrawn from the working channel of the bronchoscope, and the R-EBUS procedure consisting of transbronchial biopsy, bronchial brushing, and bronchial washing was then performed.15. The bronchial brushing cytology slides were examined by a board-certified cytopathologist. EGFR mutation analysis was then performed on tumor cell-containing bronchial brushing specimens obtained from patients diagnosed with non-squamous NSCLC.Each specimen obtained by bronchial brushing was first smeared onto slides. Air-dried smears and those fixed in 95% ethyl alcohol were prepared for routine evaluation. Next, the brushing head was removed and dipped into TRI reagent solution , and specimens were stored as described previously16. The RT-PCR amplicons were purified and sequenced with a BigDye Terminator Sequencing Kit . Sequencing products underwent electrophoresis on an automated ABI PRISM 3700 genetic analyzer (Applied Biosystems). Both the forward and reverse sequences obtained were analyzed, and chromatograms were examined manually.Reverse transcription-polymerase chain reaction (RT-PCR) on RNA extracted from the brushing specimens was performed with the Qiagen One-Step RT-PCR Kit  using previously reported conditions and primersEGFR mutation detection. EGFR mutations were analyzed using standard methods at our institution, including either direct sequencing or matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS), as previously described19.Matched histological specimens, including biopsy specimens and surgical tissues, were used for EGFR mutations were determined by calculating Cohen’s kappa coefficient. Progression-free survivals after treatment with EGFR-TKIs were analyzed using the Kaplan-Meier method. All analyses were conducted using SPSS for Windows, version 15.0 . A two-tailed P-value of <0.05 was considered statistically significant.For univariate analysis, categorical variables were analyzed using the chi-squared test or Fisher’s exact test as appropriate. For multivariate analysis, variables with P-values of <0.10 were incorporated into the multivariate logistic regression model to identify independent factors. Intra-individual agreements between the different methods used to detect EGFR mutation analysis.A total of 941 consecutive patients who underwent R-EBUS were enrolled in this study. The final diagnoses of these patients, established either by R-EBUS or other diagnostic procedures, are shown in Fig. Factors associated with the diagnostic yield of non-squamous NSCLC samples are shown in Table EGFR genotyping was achieved in bronchial brushing samples from 363 of 376 patients (96.5%), while samples from 13 patients (3.5%) failed EGFR amplification and sequencing because of insufficient material. EGFR mutations were detected in 216 patient samples; among them, 99 (38.5%) were exon 19 deletions, 84 (23.9%) were L858R, and 33 (9.4%) were other uncommon mutations.Successful EGFR genotyping of brushing specimens was successful, 284 had matched samples available for EGFR testing using standard testing methods. Specifically, 204 specimens were genotyped using MALDI-TOF-MS, whereas 80 were analyzed with Sanger sequencing. Comparison of EGFR testing results between the bronchial brushing and matched histological samples , negative transbronchial biopsy results (n = 107), or inadequate material (n = 31). Among patients requiring repeat biopsies, the R-EBUS brushing specimens of 57 patients (39.6%) provided positive cytology and successful EGFR sequencing results  required repeat biopsies for pathological diagnosis or EGFR mutation profiles were obtained from the bronchial brushing specimens of 142 cases. Tumors from 121 patients harbored EGFR mutations, whereas 21 possessed wild-type EGFR. All patients for whom tumor EGFR mutation status was obtained were then treated with first-line EGFR-TKIs. Among these patients, those with EGFR mutations exhibited improved disease response  and disease control rates , as well as prolonged progression-free survival , compared to those without EGFR mutations.Of the 419 patients with advanced (stage IIIB/IV) non-squamous NSCLC enrolled in this study, EGFR mutation analysis in patients with peripheral non-squamous NSCLC, thus allowing patients to avoid more invasive procedures.Our findings support a role for R-EBUS-guided bronchial brushing samples in the cytological diagnosis and EGFR mutation analysis in predicting the treatment efficacy of EGFR-TKIs in patients with NSCLC5. The reliable harvesting of samples for pathological diagnosis and EGFR testing is critical for patients with advanced non-squamous NSCLC. Marked progress in diagnostic bronchoscopy has been achieved in the last decade, and R-EBUS is one of the most important of these advances20. Previous studies have demonstrated that EBUS-guided bronchial brushing improved the diagnostic yields of samples from patients with PPLs22. However, few studies have investigated whether R-EBUS specimens are conducive for molecular diagnostics. Guiser et al. demonstrated that the molecular diagnosis of R-EBUS specimens was feasible for approximately 80% of patients with peripheral lung cancer23. However, the majority of these specimens were biopsy, rather than bronchial brushing, specimens, and the results from EGFR mutation analyses of brushing specimens were not compared with those from patient-matched histological samples.Several previous studies have demonstrated the importance of 26. Our previous pilot study demonstrated the potential utility of EBUS-guided brushing specimens in molecular diagnostics13. However, the factors that predict diagnostic yields of R-EBUS-guided bronchial brushing samples were not assessed because of the relatively small population size. Therefore, in the current study, we investigated the feasibility of using R-EBUS-guided brushing specimens for the cytological diagnosis and EGFR mutation analysis of tumors in patients with peripheral non-squamous NSCLC.Previous studies have investigated the utility of cytological specimens for molecular testing, but focused on fine-needle aspiration and pleural effusion analysis rather than brushing cytology specimens21. Moreover, we found that the presence of a bronchus sign on chest CT, as well as the localization of the R-EBUS probe within the lesion, were independent factors that improved the diagnostic yield of R-EBUS-guided bronchial brushing specimens. Previous studies have shown that the location of the tumors may influence the diagnostic yield of R-EBUS-guided transbronchial biopsies28. To our knowledge, our study is the first to investigate the factors that predict the diagnostic yield of R-EBUS-guided bronchial brushing specimens.In the present study, the utility of R-EBUS brushing samples for molecular diagnostics was determined. We observed that the diagnostic yields of specimens from R-EBUS-guided transbronchial biopsy, brushing cytology, and both techniques combined were 68.4%, 60.3%, and 78.4%, respectively, consistent with the results of previous studiesEGFR mutation detection was performed successfully in 96.5% of the 363 R-EBUS-guided brushing specimens that contained tumor cells. We observed good concordance between the EGFR mutation status of brushing-derived vs. patient-matched histological specimens (kappa = 0.745). Using RT-PCR, we also identified rare mutations that were not detected with standard DNA-based methods29. Moreover, 142 advanced NSCLC patients with EGFR-mutant brushing specimens exhibited better treatment responses and progression-free survival compared to patients with EGFR-wild-type specimens. The treatment efficacy of EGFR-TKIs was similar to that observed in previous studies5.EGFR mutational profiles of the brushing samples and matched biopsy/resection specimens were noted. First, some patients with EGFR-wild-type biopsy specimens harbored EGFR mutations in their R-EBUS brushing specimens. The sensitivity of mutation assessment may be affected by interference from non-tumor cells in the biopsy specimens. Using an RT-PCR-based system for mutation detection may overcome discrepancies caused by heterogeneous specimens15. Second, some patients with a single mutation in their biopsy specimens harbored uncommon, complex mutations in their R-EBUS brushing specimens. The RT-PCR-based method with Sanger sequencing identified these uncommon, complex mutations, which is important for predicting the treatment efficacy of EGFR-TKIs30. Third, some EGFR mutations detected in biopsy specimens were not detected in the corresponding R-EBUS brushing specimens, potentially due to the limited number of tumor cells in the latter sample type. Moreover, tumor spatial heterogeneity may also lead to discrepancies in EGFR mutation status32.Some discrepancies between the EGFR mutation analysis. The simultaneous use of both techniques enabled successful pathological diagnosis and EGFR genotyping following a single procedure, averting the need for additional invasive procedures.In this study, 144 patients required repeat invasive procedures because of negative biopsy results or insufficient samples for EGFR mutation analysis. Among them, 76 patients (52.8%) had stage IV non-squamous NSCLC with various distant metastases, including pleura, pericardium, bone, brain, liver, adrenal metastases, and so on. Because of tumor size and location, some of these metastases were more difficult and invasive to approach compared with R-EBUS. In this context, acquisition of R-EBUS-guided bronchial brushing samples and the associated increased diagnostic yields facilitated 37; however, recent studies showed variable reliability and sensitivity when detecting mutations using this method; cost and technical problems were also considerations. Cytological/histological specimens and liquid biopsy may be considered as complementary methods of molecular testing.Advances in the molecular diagnosis of lung cancer include “liquid biopsy”, by which genotyping is performed on circulating tumor DNA in the plasmaEGFR mutation status were not obtained exclusively from R-EBUS transbronchial biopsies. Second, the study was conducted at a single institution; multi-center studies remain under consideration. Third, testing for other mutations, such as ALK translocation, was not performed in this study and should be investigated in further work. Finally, advances in bronchoscopy techniques, including guide sheath and electromagnetic navigation bronchoscopy, can increase sample diagnostic yield without causing additional complications, such as pneumothorax40. As such, the utility of these techniques to molecular diagnostics, together with R-EBUS-guided bronchial brushing, requires further investigation.Our results should be interpreted in light of the limitations of our study. First, the matched specimens for comparing The datasets that were generated and/or analyzed during the current study are available from the corresponding author on reasonable request.


Ewing Sarcoma is an aggressive, oncofusion-driven, malignant neoplasm of bone and soft tissue affecting predominantly children and young adults. Seeking to identify potential novel therapeutic targets/agents for this disease, our previous studies uncovered microRNAs regulated by EWS/Fli1, the most common oncofusion, with growth modulatory properties. In the present study, we sought to identify EWS/Fli1-repressed, growth suppressive, microRNAs potentially amenable to replacement in Ewing Sarcoma cells. Eight microRNAs  were selected for evaluation as EWS/Fli1-repressed and underexpressed in Ewing Sarcoma cells, and reported to be growth suppressive in other pediatric or/and adult cancers. The selected miRs, and appropriate non-targeting controls, were introduced into two different Ewing Sarcoma cell lines (A673 and SK-ES-1), and effects on growth were examined using a high and low-density growth assay. MiR-193b was growth inhibitory in both assays and cell lines. In subsequent analyses, we found that stable overexpression of miR-193b also inhibits anchorage-independent growth in both A673 and SK-ES-1 cells. We further show that miR-193b negatively regulates expression of the ErbB4 oncogene in A673 and SK-ES-1 cells, and that depletion of ErbB4 is itself inhibitory to anchorage-independent growth in the same cell lines. Together, our studies show that the EWS/Fli1-repressed miR-193b is growth suppressive in Ewing Sarcoma, and identify ErbB4 as a target gene and candidate mediator of this growth suppression. Ewing Sarcoma is a cancer of bone and soft tissue predominantly affecting the pediatric age group . It is aMicroRNAs (miRs) play an important role in the regulation of gene expression in normal physiology and disease . MiRs arSeeking to identify new EWS/Ets-driven oncogenic pathways with targeting potential in Ewing Sarcoma, our laboratory previously performed global miR profiling upon silencing of the EWS/Fli1 oncoprotein . This stThe origin and culture of the Ewing Sarcoma cell lines A673 and SK-ES-1, and human Mesenchymal Stem Cells  have been previously described . The EwiTransient ectopic microRNA (miR) expression was achieved using transfection of miR mimics, or non-targeting negative control mimic , using Lipofectamine 2000, as previously described . For stashRNA-mediated gene expression silencing via lentiviral delivery was performed as previously described . The conFor MTT assays, one day following miR transfection, cells were counted and replated at a density of 5,000 cells/well in 96-well plates. On subsequent days 1, 3 and 5, cell growth was measured using an MTT assay, as described . For cloQuantification of miR-193b levels in Ewing Sarcoma cell lines and MSCs was performed using RT-qPCR, as previously described . PrimersProtein extract preparation, SDS-PAGE, immunoblotting and detection were performed as previously described . The priFor 3’UTR activity assays, Ewing Sarcoma cells were transiently cotransfected using Lipofectamine 2000 with a psiCHECK-2 reporter construct containing the wild-type ErbB4 3’UTR miR-193b site or the mutated site (introduced via standard cloning techniques followed by sequence verification), and miR-193b mimic, non-targeting negative control mimic, or no nucleic acid (Mock group). Reporter activity was determined 48 hours later, as described previously .Using global microRNA (miR) profiling, our laboratory previously identified miRs with altered expression as a function of EWS/Fli1 levels in Ewing Sarcoma, including miRs that were upregulated and others that were downregulated by the EWS/Fli1 oncoprotein . We hypoThe selected miRs  were then subjected to a functional miniscreen using inhibition of growth as the readout. Specifically, miRs were introduced into two different Ewing Sarcoma cell lines (A673 and SK-ES-1) using transient transfection of miR mimics (and corresponding non-targeting negative control mimics), and effects on cell growth were monitored using two different assays: an MTT assay (measuring growth under standard moderate-density culture conditions), and a clonogenic assay (measuring colony initiation and growth under low-density plating conditions). Each assay was repeated four independent times to ascertain reproducibility of effects. The results, plotted as fractional growth relative to mock-transfected control cells, are shown in Given our finding of a growth suppressive phenotype effected by miR-193b in two different cell lines and growth assays, we focused our further studies on this miR. To expand on our observation of miR-193b repression by EWS/Fli1 , we compTo gain insight into potential mechanisms of growth inhibition by miR-193b in Ewing Sarcoma, we queried miR target prediction algorithms, including the pathway-focused algorithm DIANA-mirPath. This analysis identified the ErbB4 oncogene as one of the top candidates for regulation by miR-193b. Interestingly, ErbB4 had previously been identified as a suppressor of anoikis in Ewing Sarcoma , a phenoTo determine whether downregulation of ErbB4 levels contributes to the growth suppressive phenotype of miR-193b, we stably depleted ErbB4 in A673 and SK-ES-1 cells using lentivirally delivered shRNAs. Compared to control cells (transduced with non-targeting scrambled control shRNA), stable depletion of ErbB4 with two different targeting shRNAs inhibited anchorage-independent growth in soft agar in both cell lines . Thus, nOur studies identify miR-193b as a new growth-suppressive microRNA in Ewing Sarcoma, and provide evidence in support of ErbB4 as a mediator of this growth inhibition. As shown in our previous  and currMiR-193b has been identified as a modulator of cancer phenotypes in a number of other contexts. In the vast majority of published literature, miR-193b is reported to be downregulated and to elicit anti-cancer action upon ectopic expression, including inhibition of cell/tumor growth and metastasis. Such action as a tumor/metastasis suppressor miR has been shown in a number of adult epithelial malignancies, including cancers of the breast , prostatOur studies identify ErbB4 as a relevant miR-193b target in Ewing Sarcoma. Previous work of others has uncovered an important role for ErbB4 in suppression of anoikis , a phenoIn closing, our studies identify miR-193b as a new growth-suppressive miR in Ewing Sarcoma, and ErbB4 as a relevant candidate target potentially contributing to its growth-suppressive effects. Both molecules are tractable pharmacologic targets, and their therapeutic manipulation could be of interest in Ewing Sarcoma and other cancers.


The roseoloviruses, human herpesvirus (HHV)-6A, HHV-6B, and HHV-7, can cause severe encephalitis or encephalopathy. In immunocompetent children, primary HHV-6B infection is occasionally accompanied by diverse clinical forms of encephalitis. Roseolovirus coinfections with heterologous viruses and delayed primary HHV-7 infection in immunocompetent adults result in very severe neurological and generalized symptoms. Recovery from neurological sequelae is slow and sometimes incomplete. In immunocompromised patients with underlying hematological malignancies and transplantation, frequent single or simultaneous reactivation of roseoloviruses elicit severe, lethal organ dysfunctions, including damages in the limbic system, brain stem, and hippocampus. Most cases have been due to HHV-6B with HHV-6A accounting for 2–3%. The most severe manifestation of HHV-6B reactivation is post-transplantation limbic encephalitis. Seizures, cognitive problems, and abnormal EEG are common. Major risk factors for HHV-6B-associated encephalitis include unrelated cord blood cell transplantation and repeated hematopoietic stem cell transplantation. Rare genetic disorders, male gender, certain HLA constellation, and immune tolerance to replicating HHV-6 in persons carrying chromosomally integrated HHV-6 might also predispose an individual to roseolovirus-associated brain damage. At this time, little is known about the risk factors for HHV-7-associated encephalitis. Intrathecal glial cell destruction due to virus replication, overexpression of proinflammatory cytokines, and viral mimicry of chemokines all contribute to brain dysfunction. High virus load in the cerebrospinal fluid, hippocampal astrogliosis, and viral protein expression in HHV-6B-associated cases and multiple microscopic neuronal degeneration in HHV-7-associated cases are typical laboratory findings. Early empirical therapy with ganciclovir or foscarnet might save the life of a patient with roseolovirus-associated encephalitis. Viral encephalitis is a medical emergency. It is an aseptic inflammatory process of the brain parenchyma associated with clinical evidence of brain dysfunction, significant morbidity, and mortality . This phenotype could be unmasked and/or exacerbated by HHV-6B infection, potentially contributing to clinical deterioration. Mutations in POLG have recently been identified in a number of neurological syndromes including encephalopathy and seizures , tiredness, sudden onset of dizziness, and comatous state . As with other viruses that utilize the olfactory route, the presence of HHV-6 in limbic tissues can lead to limbic encephalitis -beta upregulation found in degenerating neurons of the pyramidal cell layer suggests a possible association with persistent glial-tropic HHV-7 infection and chronic neuroinflammatory conditions via glio-neuronal communication ; however, infection remains asymptomatic , experimental HHV-6A infection accelerated simian immunodeficiency virus (SIV) infection and the progression of pathological conditions, but no neurological symptoms have been described  resulted in neurological symptoms including motor weakness and sensory abnormalities, while infection with HHV-6B remained asymptomatic. HHV-6A and HHV-6B DNA could be occasionally detected in the brain of animals and MRI revealed hyperintense lesions in the brain of one animal. Intranasal HHV-6A infection rarely induced seroconversion or manifested in disease. Neurological symptoms observed with intravenous injection might be due to the immune response raised against HHV-6 rather than to the direct effect of HHV-6  have faced reactivation of both HHV-6 and HHV-7 more often than those not irradiated  or drug rash with eosinophilia and systemic symptoms (DRESS). HDAC inhibitor trichostatin A, hydrocortisone, and 12-O-tetradecanoyl-13-acetate (TPA) have all been shown to reactivate ciHHV-6 in vitro  can show abnormalities prior to neuroimaging. Several abnormalities, especially in the temporal lobe, have been described in both acute or reactivated HHV-6 and ciHHV-6-associated encephalitis, including slow wave activity in primary HHV-6B or HHV-7 infection-associated encephalitis consistent with brain stem involvement. EEG normalization parallels antiviral treatment and clinical improvement  of the patients’ cells can locate ciHHV-6 at chromosomes  of HHV-6 DNA in CSF have been detected in many of the patients with HHV-6 encephalitis/encephalopathy during primary infection  with higher sensitivity is commonly used to detect roseoloviruses in CSF and brain materials  can inhibit replication of both HHV-6A (strain GS) and HHV-6B (strains Z29 and HST) in cell cultures (Bonnafous et al. Roseoloviruses are important pathogens in immunocompetent young people and immunocompromised individuals, especially in the transplantation setting, and cause serious and lethal complications including CNS disorders. The scale of the contribution of Roseolovirus infection to diverse neurological diseases has not been appreciated in the past (Steiner et al.