Alterations In Signaling Pathways Research Articles

Inhibitory effects of estrogenic endocrine disrupting chemicals on fin regeneration in zebrafish are dependent on estrogen receptors.

For fish and other aquatic organisms, disrupting their capacity for repair and regeneration will reduce their quality of life and survivorship in the wild. Studies have shown that 17α-ethinylestradiol (EE2), a synthetic estrogenic endocrine disrupting chemical (EEDC), can inhibit caudal fin regeneration in larval zebrafish following fin amputation. However, whether the inhibitory effects of EE2 are dependent on estrogen receptor (ER) remains unknown. Therefore, in this study, amputated zebrafish larvae were exposed to the ER agonist EE2 alone and in combination with the ER antagonist ICI 182,780 (ICI), and the change in regenerative capacity was determined. The inhibition of fin regeneration caused by EE2 alone (100ng/L) was ameliorated after combination with ICI (30-300μg/L), and these changes in regeneration-related signaling and the immune system corresponded with morphological observations, implying that the effects of EE2 on regeneration were possibly initiated by the activation of ER. Furthermore, the role of ER was confirmed with a natural ligand of ER, namely, 17β-estradiol (E2), and as expected, the effects of E2 (10, 100 and 1000ng/L) paralleled those of EE2. In conclusion, EEDCs can disrupt the regenerative capacity in zebrafish, possibly due to the binding and activation of ERs and the consequent alteration of signaling pathways that regulate fin regeneration and immune competence. Given that EEDCs appear to be ubiquitous in the aquatic environment, the risk of these chemicals might be readdressed regarding their potential effects on tissue repair and regeneration.

Microsatellite-stable tumors with high tumor mutational burden in association with tumor response to immune checkpoint inhibitor therapy across solid tumors and correlation with specific oncogenic alterations.

2634 Background: High tumor mutational burden (TMB-H) has shown promise as a predictive biomarker in certain tumors, but broad applicability across tumors is unclear. In 2020, pembrolizumab was approved for treatment refractory metastatic or unresectable solid tumors with TMB ≥10 mutations/megabase based on exploratory analysis of the KEYNOTE-158 study. Growing evidence suggest limitations of TMB-H across tumors. Our aim is to evaluate outcomes of patients (pts) with advanced microsatellite-stable (MSS) solid tumors with TMB ≥10 mutations/megabase treated with immune checkpoint inhibitors (ICIs) and identify genetic alterations that predict poor response to therapy. Methods: Pts treated between 01/2015 to 12/2020 at Robert H Lurie Cancer Center, Northwestern University, with ICIs and TMB ≥10 mutations/megabase on next generation sequencing by FoundationOne CDx, Tempus xT or Guardant360 CDx platforms were identified. Tumors other than carcinomas and microsatellite instability were excluded. Radiologic response was assessed using iRECIST criteria. Responders (CR/PR) were compared with non-responders (SD/PD). Subgroup analyses were conducted based on tumor type, genetic alterations within signaling pathways. Survival curves were calculated using Kaplan-Meier method. Multivariate analyses were performed to determine impact of clinical variables on response. Results: Of 119 pts, median age was 68 yrs, and 61% of pts were male. 15 tumor types were represented, most commonly melanoma (33%), NSCLC (28%), small cell lung carcinoma (5.9%), colorectal carcinoma (5%), and pancreatobiliary carcinoma (5%). When evaluating for efficacy (n = 106), ORR was 36% with CR 14%. Median PFS and OS overall were 10.9 mo and 29.9 mo. Lack of exposure to previous therapy was associated with response (p = 0.039). PD-L1 status, specific ICI regimen, age, and sex did not have prognostic significance. When comparing melanoma/NSCLC (n = 66) and other tumor types (n = 40) there was no significant difference in response (p = 0.3). When comparing response by specific gene mutations, TP53 (p = 0.026) and PIK3CA (p = 0.025) were associated with worse response. Similarly, ROS1 alteration trended to poorer response (p = 0.057). When evaluating genetic pathways, mutations within TP53 pathway were associated inferior response (p = 0.018). There was no significant difference in RTK/RAS, NRF2, PI3K, TGFβ, WNT, MYC, Cell Cycle, and Notch pathways. Conclusions: Growing evidence shows limitations of TMB-H as a predictive biomarker for ICI therapy in MSS tumors. Our data suggests alterations within TP53 and its signaling pathway, PIK3CA mutations, and likely ROS1 alterations are associated with non-responders in MSS TMB-H solid tumors. Further analysis of tumor-specific cohorts and genetic alterations will be presented.

Characterization and impact of canonical Wnt Signaling Pathway (WSP) alterations on outcomes of metastatic prostate cancer.

5053 Background: Recurrent alterations in the WSP have been identified in patients with advanced prostate cancer. Aberrant Wnt signaling has been implicated in disease progression and hormonal resistance in prostate cancer. We utilized a multi-institutional real-world dataset to characterize molecular alterations in the canonical WSP in men with prostate cancer, and correlate that with overall survival (OS). Methods: Prostate cancer patients who underwent tissue-based DNA and RNA sequencing utilizing a commercially available CLIA-certified assay (Caris Life Sciences) were investigated. Next generation sequencing (NGS)/ whole exome (WES) and transcriptome sequencing (WTS) was performed on prostate cancer tissue derived from prostatic and/or metastatic sites. Patients with somatic activating mutations in CTNNB, pathogenic fusions in RSPO2, or inactivating mutations in APC or RNF43 were characterized as having aberrant canonical Wnt signaling (WSP-MT). A subset analysis was conducted in metastatic prostate cancer (mPCa) samples with microsatellite stable (MSS) tumors excluding RNF43 (G569fs*) mutations. Comparative analyses were done using Fisher-Exact or X2 tests, and significance was determined by adjusted p value using Benjamini-Hochberg correction (q < 0.05). OS was obtained from insurance claims data and calculated using Kaplan-Meier estimates. Enriched mRNA transcripts were identified as those with an adjusted p value < 0.001, logFC > 1.5, and (-)log10 FDR > 20. Results: Overall, 21.8% (n = 715/3283) of samples were classified as WSP-MT, of which 297 (41.5%) were from primary tumor samples and 418 (58.5%) were from metastatic sites. WSP-MT were highest in metastases to CNS (54.5%, n = 12/22), liver (43.8%, n = 91/208) and lung (42.3%, n = 91/208). Compared to WSP wild-type, WSP-MT tumors had a greater frequency of dMMR/MSI-H status (18% vs. 2%, q < 0.001). In the subset of mPCa, MSS tumors (WSP-MT excluding RNF43 (G569fs*) mutations, n = 318; WSP wild-type n = 951), WSP-MT were enriched for mutations in SPOP (14.3% vs. 6.9%, q = 0.004). In these tumors, WSP-MT samples were most enriched for mRNA expression of CST1, NKD1, AXIN2, and ZNRF3 (all known canonical WSP activators). OS was inferior in mPCa WSP-MT patients compared to WSP wild-type (HR 0.49, 95% CI 0.37-0.64, p < 0.0001). Relative to WSP wild-type cases, OS was shorter from time of enzalutamide/abiraterone/apalutamide start (HR 0.25, 95% CI 0.15-0.43, p = 0.0001), and from first-line taxane chemotherapy (HR 0.47, 95% CI 0.21-1.02, p = 0.052) in pts with WSP-MT. Conclusions: Canonical WSP alterations are enriched in metastatic tumors, with highest prevalence in visceral (CNS, liver, lung) metastases. Clinical outcomes in WSP-mutant cancers are inferior with both hormonal and chemotherapies, heralding an urgent need to develop novel therapeutic strategies for such patients.

Comparative analysis of microsatellite instability-high (MSI-H) BRAF V600E-mutated versus MSI-H BRAFwild type colorectal cancers (CRC), including tumor microenvironment (TME), associated genomic alterations, and immunometabolomic biomarkers.

3066 Background: The BRAFV600E mutation is associated with the hypermethylator phenotype CIMP, which can also lead to the MSI-H phenotype. BRAFV600E mutation and MSI-H/dMMR status seem to be biologically intertwined; however, the impact of coexisting BRAFV600E mutations on the TME and immunometabolomic features of MSI-H/dMMR CRC tumors is not well characterized. Methods: A retrospective review of deidentified records of patients with MSI-H/dMMR CRC tumors was conducted using next-generation sequencing data (Tempus |xT assay: DNA-seq of 595-648 genes at 500x coverage, and full transcriptome RNA-seq). Several immune markers of tumor immunogenicity in BRAF wild-type ( BRAFwt) vs. V600E-mutated (BRAFV600E) tumors were assessed, including tumor mutational burden (TMB), neoantigen tumor burden (NTB, ScanNeo), PD-L1 expression, immune infiltration, and canonical immuno-metabolomic pathways (82 geneset signatures). Results: A total of 459 MSI-H/dMMR CRC tumors were analyzed, of which 123 (27%) tumors harbored BRAFV600Emutations, and 336 (73%) were BRAFwt. MSI-H/dMMR BRAFV600E tumors were more frequently identified in females (69% vs. 55%; P= 0.01), non-Hispanic or non-Latino (100% vs. 73%; P =0.001), and older patients (median age: 76 yrs vs. 62 yrs; P< 0.001). Compared to BRAFWT, BRAFV600E tumors exhibited significantly higher TMB (Median: 49 mut/MB vs. 36 mut/MB; P < 0.001) and were more frequently associated with TMB-High status (> 10 mut/MB; 100% vs. 95%; P = 0.008); however, no significant differences were observed with tumor NTB, immune score, or T cell infiltration (CD4 or CD8). NK cell infiltration was higher in the BRAFV600Ecohort (< 0.001). When compared to BRAFWT tumors , BRAFV600E tumors harbored a greater frequency of mutations in MSH6(42% vs. 20%), B2M (33% vs. 16%), BRCA2 (31% vs. 12%), ATM (23% vs. 12%), and TP53 (30% vs. 19%) but a lower frequency of MSH2 (3.3% vs. 11%), all P< 0.05. Pathway enrichment analysis identified 10 significantly altered signaling pathways, most of which related to stromal/immune cell signaling and metabolism. Five were upregulated among BRAFV600E tumors: glycerophospholipid, galactose, cyclin-dependent cell signaling; Nucleotide, and TH1 inflammation. Five pathways were downregulated (Wnt, Notch, TH17 inflammation, amino sugar, and cancer stem cell signaling). Conclusions: MSI-H/dMMR BRAFV600E CRCs undergo broad metabolic reprogramming (e.g., glycerophospholipidgalactose, nucleotide). A rise in lipid metabolism, particularly with NK inflammation, suggests that BRAFV600Emutated tumors may be associated with a non-classical immune component. BRAFV600E and BRAFwt CRCs exhibited similar NTB and T cell infiltration, suggesting that both are likely to benefit from immune checkpoint inhibitors.

Long-lasting and Sex-dependent Effects of Postweaning Swimming Exercise on Social Dominance in Adult Mice

Increasing evidence has shown that early life events exert long-lasting effects on brain function and mental diseases. Exercise has been proven to have many positive effects on behaviors, such as reducing anxiety- and depression-like behaviors and alleviating cognitive impairment. However, the long-lasting and even short-term effects of regular swimming exercise on social dominance remain unclear. The purpose of this study was to investigate the potential effects of postweaning swimming exercise on social dominance and metabolic adaptation in adult mice. Three-week-old mice performed 1 h of swimming exercise in warm water for 4 weeks. A series of behavioral tests, such as the social dominance test (SDT), open field test (OFT), and forced swim test (FST), were conducted. Behavioral test results showed that both male and female mice in the swimming group had a higher rank than those in the sedentary group in the SDT of early adulthood, while only female mice in the swimming group maintained the social dominance in late adulthood. There was no difference between the swimming and sedentary groups in anxiety- and depression-like behaviors. Metabolomics analysis showed that there were alterations in particular metabolites and signaling pathways after one month of swimming exercise, including sphingolipid metabolism, neuroactive ligand–receptor interaction and caffeine metabolism. In conclusion, our results provide the first evidence that postweaning swimming exercise has long-lasting and sex-dependent effects on social dominance, which may be caused by metabolic adaptation.

Photoprotective effects of Sargassum thunbergii on ultraviolet B-induced mouse L929 fibroblasts and zebrafish

BackgroundChronic exposure to ultraviolet B (UVB) causes a series of adverse skin reactions, such as erythema, sunburn, photoaging, and cancer, by altering signaling pathways related to inflammation, oxidative stress, and DNA damage. Marine algae have abundant amounts and varieties of bioactive compounds that possess antioxidant and anti-inflammatory properties. Thus, the objective of this study was to investigate the photoprotective effects of an ethanol extract of Sargassum thunbergii.MethodsSargassum thunbergii phenolic-rich extract (STPE) was prepared, and its activity against UVB damage was evaluated using L929 fibroblast cells and zebrafish. STPE was extracted and purified by 40% ethanol and macroporous resin XDA-7. Reactive oxygen species (ROS) and antioxidant markers, such as superoxide dismutase (SOD), catalase (CAT) activities, and malondialdehyde (MDA) content were analyzed. The effect of STPE on UVB-induced inflammation was determined by inflammatory cytokine gene and protein expression. The expression of signaling molecules in the Nuclear Factor KappaB (NF-κB) pathway was determined by western blotting. DNA condensation was analyzed and visualized by Hoechst 33342 staining. In vivo evaluation was performed by tail fin area and ROS measurement using the zebrafish model.ResultsThe total polyphenol content of STPE was 72%. STPE reduced ROS content in L929 cells, improved SOD and CAT activities, and significantly reduced MDA content, thereby effectively alleviating UVB radiation-induced oxidative damage. STPE inhibited the mRNA and protein expression of TNF-α, IL-6, and IL-1α. STPE reversed DNA condensation at concentrations of 20 and 40 μg/mL compared with the UVB control. Moreover, STPE inhibited NF-κB signaling pathway activation and alleviated DNA agglutination in L929 cells after UVB irradiation. Additionally, 1.67 μg/mL STPE significantly increased the tail fin area in zebrafish, and 0.8–1.6 μg/mL STPE effectively eliminated excessive ROS after UVB radiation.ConclusionsSTPE inhibited UVB-induced oxidative stress, inflammatory cytokine expression, and DNA condensation via the downregulation of the NF-κB signaling pathway, suggesting that it prevents UVB-induced photodamage, and has potential for clinical development for skin disease treatment.

Transcriptome analysis reveals high tumor heterogeneity with respect to re-activation of stemness and proliferation programs.

Significant alterations in signaling pathways and transcriptional regulatory programs together represent major hallmarks of many cancers. These, among all, include the reactivation of stemness, which is registered by the expression of pathways that are active in the embryonic stem cells (ESCs). Here, we assembled gene sets that reflect the stemness and proliferation signatures and used them to analyze a large panel of RNA-seq data from The Cancer Genome Atlas (TCGA) Consortium in order to specifically assess the expression of stemness-related and proliferation-related genes across a collection of different tumor types. We introduced a metric that captures the collective similarity of the expression profile of a tumor to that of ESCs, which showed that stemness and proliferation signatures vary greatly between different tumor types. We also observed a high degree of intertumoral heterogeneity in the expression of stemness- and proliferation-related genes, which was associated with increased hazard ratios in a fraction of tumors and mirrored by high intratumoral heterogeneity and a remarkable stemness capacity in metastatic lesions across cancer cells in single cell RNA-seq datasets. Taken together, these results indicate that the expression of stemness signatures is highly heterogeneous and cannot be used as a universal determinant of cancer. This calls into question the universal validity of diagnostic tests that are based on stem cell markers.

Correlation of Immunological and Molecular Profiles with Response to Crizotinib in Alveolar Soft Part Sarcoma: An Exploratory Study Related to the EORTC 90101 "CREATE" Trial.

Alveolar soft part sarcoma (ASPS) is a rare subtype of soft tissue sarcoma characterized by an unbalanced translocation, resulting in ASPSCR1-TFE3 fusion that transcriptionally upregulates MET expression. The European Organization for Research and Treatment of Cancer (EORTC) 90101 “CREATE” phase II trial evaluated the MET inhibitor crizotinib in ASPS patients, achieving only limited antitumor activity. We performed a comprehensive molecular analysis of ASPS tissue samples collected in this trial to identify potential biomarkers correlating with treatment outcome. A tissue microarray containing 47 ASPS cases was used for the characterization of the tumor microenvironment using multiplex immunofluorescence. DNA isolated from 34 available tumor samples was analyzed to detect recurrent gene copy number alterations (CNAs) and mutations by low-coverage whole-genome sequencing and whole-exome sequencing. Pathway enrichment analysis was used to identify diseased-associated pathways in ASPS sarcomagenesis. Kaplan–Meier estimates, Cox regression, and the Fisher’s exact test were used to correlate histopathological and molecular findings with clinical data related to crizotinib treatment, aiming to identify potential factors associated with patient outcome. Tumor microenvironment characterization showed the presence of PD-L1 and CTLA-4 in 10 and 2 tumors, respectively, and the absence of PD-1 in all specimens. Apart from CD68, other immunological markers were rarely expressed, suggesting a low level of tumor-infiltrating lymphocytes in ASPS. By CNA analysis, we detected a number of broad and focal alterations. The most common alteration was the loss of chromosomal region 1p36.32 in 44% of cases. The loss of chromosomal regions 1p36.32, 1p33, 1p22.2, and 8p was associated with shorter progression-free survival. Using whole-exome sequencing, 13 cancer-associated genes were found to be mutated in at least three cases. Pathway enrichment analysis identified genetic alterations in NOTCH signaling, chromatin organization, and SUMOylation pathways. NOTCH4 intracellular domain dysregulation was associated with poor outcome, while inactivation of the beta-catenin/TCF complex correlated with improved outcome in patients receiving crizotinib. ASPS is characterized by molecular heterogeneity. We identify genetic aberrations potentially predictive of treatment outcome during crizotinib therapy and provide additional insights into the biology of ASPS, paving the way to improve treatment approaches for this extremely rare malignancy.

Hepatic Hedgehog Signaling Participates in the Crosstalk between Liver and Adipose Tissue in Mice by Regulating FGF21.

The Hedgehog signaling pathway regulates many processes during embryogenesis and the homeostasis of adult organs. Recent data suggest that central metabolic processes and signaling cascades in the liver are controlled by the Hedgehog pathway and that changes in hepatic Hedgehog activity also affect peripheral tissues, such as the reproductive organs in females. Here, we show that hepatocyte-specific deletion of the Hedgehog pathway is associated with the dramatic expansion of adipose tissue in mice, the overall phenotype of which does not correspond to the classical outcome of insulin resistance-associated diabetes type 2 obesity. Rather, we show that alterations in the Hedgehog signaling pathway in the liver lead to a metabolic phenotype that is resembling metabolically healthy obesity. Mechanistically, we identified an indirect influence on the hepatic secretion of the fibroblast growth factor 21, which is regulated by a series of signaling cascades that are directly transcriptionally linked to the activity of the Hedgehog transcription factor GLI1. The results of this study impressively show that the metabolic balance of the entire organism is maintained via the activity of morphogenic signaling pathways, such as the Hedgehog cascade. Obviously, several pathways are orchestrated to facilitate liver metabolic status to peripheral organs, such as adipose tissue.

How Phosphofructokinase-1 Promotes PI3K and YAP/TAZ in Cancer: Therapeutic Perspectives.

Simple SummaryWe propose that PFK1 promotes a positive feedback loop with PI3K/AKT and YAP/TAZ signaling pathways in cancer cells. Therefore, targeting PFK1 (or its product F-1,6-BP) could improve the efficacy of PI3K and YAP/TAZ inhibitors currently tested in clinical trials. To this aim, we suggest the use of citrate, which is a physiologic and potent inhibitor of PFK1.PI3K/AKT is one of the most frequently altered signaling pathways in human cancers, supporting the activation of many proteins sustaining cell metabolism, proliferation, and aggressiveness. Another important pathway frequently altered in cancer cells is the one regulating the YAP/TAZ transcriptional coactivators, which promote the expression of genes sustaining aerobic glycolysis (such as WNT, MYC, HIF-1), EMT, and drug resistance. Of note, the PI3K/AKT pathway can also regulate the YAP/TAZ one. Unfortunately, although PI3K and YAP inhibitors are currently tested in highly resistant cancers (both solid and hematologic ones), several resistance mechanisms may arise. Resistance mechanisms to PI3K inhibitors may involve the stimulation of alternative pathways (such as RAS, HER, IGFR/AKT), the inactivation of PTEN (the physiologic inhibitor of PI3K), and the expression of anti-apoptotic Bcl-xL and MCL1 proteins. Therefore, it is important to improve current therapeutic strategies to overcome these limitations. Here, we want to highlight how the glycolytic enzyme PFK1 (and its product F-1,6-BP) promotes the activation of both PI3K/AKT and YAP/TAZ pathways by several direct and indirect mechanisms. In turn, PI3K/AKT and YAP/TAZ can promote PFK1 activity and F-1,6-BP production in a positive feedback loop, thus sustaining the Warburg effect and drug resistance. Thus, we propose that the inhibition of PFK1 (and of its key activator PFK2/PFKFB3) could potentiate the sensitivity to PI3K and YAP inhibitors currently tested. Awaiting the development of non-toxic inhibitors of these enzymes, we propose to test the administration of citrate at a high dosage, because citrate is a physiologic inhibitor of both PFK1 and PFK2/PFKFB3. Consistently, in various cultured cancer cells (including melanoma, sarcoma, hematologic, and epithelial cancer cells), this “citrate strategy” efficiently inhibits the IGFR1/AKT pathway, promotes PTEN activity, reduces Bcl-xL and MCL1 expression, and increases sensitivity to standard chemotherapy. It also inhibits the development of sarcoma, pancreatic, mammary HER+ and lung RAS-driven tumors in mice without apparent toxicities.

JUN and PDGFRA as Crucial Candidate Genes for Childhood Autism Spectrum Disorder.

Autism spectrum disorder (ASD) is a complex neurodevelopmental disorder, characterized by marked genetic heterogeneity. In this study, two independent microarray datasets of cerebellum of ASD were integrative analyzed by NetworkAnalyst to screen candidate crucial genes. NetworkAnalyst identified two up-regulated genes, Jun proto-oncogene (JUN) and platelet derived growth factor receptor alpha (PDGFRA), as the most crucial genes in cerebellum of ASD patients. Based on KEGG pathway database, genes associated with JUN in the cerebellum highlight the pathways of Th17 cell differentiation and Th1 and Th2 cell differentiation. Genes associated with PDGFRA in the cerebellum were found enriched in pathways in EGFR tyrosine kinase inhibitor resistance and Rap1 signaling pathway. Analyzing all differentially expressed genes (DEGs) from the two datasets, Gene Set Enrichment Analysis (GSEA) brought out IL17 signaling pathway, which is related to the expression of JUN and PDGFRA. The ImmuCellAI found the elevated expression of JUN and PDGFRA correlating with increased Th17 and monocytes suggests JUN and PDGFRA may regulate Th17 cell activation and monocytes infiltrating. Mice model of maternal immune activation demonstrated that JUN and PDGFRA are up-regulated and related to the ASD-like behaviors that provide insights into the molecular mechanisms underlying the altered IL17 signaling pathway in ASD and may enable novel therapeutic strategies.

Severe Muscle Deconditioning Triggers Early Extracellular Matrix Remodeling and Resident Stem Cell Differentiation into Adipocytes in Healthy Men.

Besides the loss of muscle mass and strength, increased intermuscular adipose tissue (IMAT) is now a well-recognized consequence of muscle deconditioning as experienced in prolonged microgravity. IMAT content may alter the muscle stem cell microenvironment. We hypothesized that extracellular matrix structure alterations and microenvironment remodeling induced by fast and severe muscle disuse could modulate fibro-adipogenic progenitor fate and behavior. We used the dry immersion (DI) model that rapidly leads to severe muscle deconditioning due to drastic hypoactivity. We randomly assigned healthy volunteers (n = 18 men) to the control group (only DI, n = 9; age = 33.8 ± 4) or to the DI + thigh cuff group (n = 9; age = 33.4 ± 7). Participants remained immersed in the supine position in a thermo-neutral water bath for 5 days. We collected vastus lateralis biopsies before (baseline) and after DI. 5 days of DI are sufficient to reduce muscle mass significantly, as indicated by the decreased myofiber cross-sectional area in vastus lateralis samples (−18% vs. baseline, p < 0.05). Early and late adipogenic differentiation transcription factors protein levels were upregulated. Platelet-derived growth Factors alpha (PDGFR⍺) protein level and PDGFR⍺-positive cells were increased after 5 days of DI. Extracellular matrix structure was prone to remodeling with an altered ECM composition with 4 major collagens, fibronectin, and Connective Tissue Growth Factor mRNA decreases (p < 0.001 vs. baseline). Wearing thigh cuffs did not have any preventive effect on the measured variable. Our results show that altered extracellular matrix structure and signaling pathways occur early during DI, a severe muscle wasting model, favoring fibro-adipogenic progenitor differentiation into adipocytes.

Recent discoveries in microbiota dysbiosis, cholangiocytic factors, and models for studying the pathogenesis of primary sclerosing cholangitis.

Primary sclerosing cholangitis (PSC) is a cholangiopathy caused by genetic and microenvironmental changes, such as bile homeostasis disorders and microbiota dysbiosis. Therapeutic options are limited, and proven surveillance strategies are currently lacking. Clinically, PSC presents as alternating strictures and dilatations of biliary ducts, resulting in the typical “beaded” appearance seen on cholangiography. The pathogenesis of PSC is still unclear, but cholangiocytes play an essential role in disease development, wherein a reactive phenotype is caused by the secretion of neuroendocrine factors. The liver–gut axis is implicated in the pathogenesis of PSC owing to the dysbiosis of microbiota, but the underlying mechanism is still poorly understood. Alterations in cholangiocyte responses and related signalling pathways during PSC progression were elucidated by recent research, providing novel therapeutic targets. In this review, we summarise the currently known underlying mechanisms of PSC pathogenesis caused by the dysbiosis of microbiota and newly reported information regarding cholangiocytes in PSC. We also summarise recently reported in vitro and in vivo models for studying the pathogenesis of PSC.

Transcriptomic Evidence of the Immune Response Activation in Individuals With Limb Girdle Muscular Dystrophy Dominant 2 (LGMDD2) Contributes to Resistance to HIV-1 Infection.

LGMDD2 is a rare form of muscular dystrophy characterized by one of the three heterozygous deletions described within the TNPO3 gene that result in the addition of a 15-amino acid tail in the C-terminus.TNPO3 is involved in the nuclear import of splicing factors and acts as a host cofactor for HIV-1 infection by mechanisms not yet deciphered. Further characterization of the crosstalk between HIV-1 infection and LGMDD2 disease may contribute to a better understanding of both the cellular alterations occurring in LGMDD2 patients and the role of TNPO3 in the HIV-1 cycle. To this regard, transcriptome profiling of PBMCs from LGMDD2 patients carrying the deletion c.2771delA in the TNPO3 gene was compared to healthy controls. A total of 545 differentially expressed genes were detected between LGMDD2 patients and healthy controls, with a high representation of G protein-coupled receptor binding chemokines and metallopeptidases among the most upregulated genes in LGMDD2 patients. Plasma levels of IFN-β and IFN-γ were 4.7- and 2.7-fold higher in LGMDD2 patients, respectively. An increase of 2.3-fold in the expression of the interferon-stimulated gene MxA was observed in activated PBMCs from LGMDD2 patients after ex vivo HIV-1 pseudovirus infection. Thus, the analysis suggests a pro-inflammatory state in LGMDD2 patients also described for other muscular dystrophies, that is characterized by the alteration of IL-17 signaling pathway and the consequent increase of metallopeptidases activity and TNF response. In summary, the increase in interferons and inflammatory mediators suggests an antiviral environment and resistance to HIV-1 infection but that could also impair muscular function in LGMDD2 patients, worsening disease evolution. Biomarkers of disease progression and therapeutic strategies based on these genes and mechanisms should be further investigated for this type of muscular dystrophy.

Potential Biomarkers of Skin Melanoma Resistance to Targeted Therapy-Present State and Perspectives.

Simple SummaryAround 5–10% of advanced melanoma patients progress early on anti-BRAF targeted therapy and 20–30% respond only with the stabilization of the disease. Presumably, these patients could benefit more from first-line immunotherapy. Resistance to BRAF/MEK inhibitors is generated by genetic and non-genetic factors inherent to a tumor or acquired during therapy. Some of them are well documented as a cause of treatment failure. They are potential predictive markers that could improve patients’ selection for both standard and also alternative therapy as some of them have therapeutic potential. Here, a summary of the most promising predictive and therapeutic targets is presented. This up-to-date knowledge may be useful for further study on implementing more accurate genetic/molecular tests in melanoma treatment.Melanoma is the most aggressive skin cancer, the number of which is increasing worldwide every year. It is completely curable in its early stage and fatal when spread to distant organs. In addition to new therapeutic strategies, biomarkers are an important element in the successful fight against this cancer. At present, biomarkers are mainly used in diagnostics. Some biological indicators also allow the estimation of the patient’s prognosis. Still, predictive markers are underrepresented in clinics. Currently, the only such indicator is the presence of the V600E mutation in the BRAF gene in cancer cells, which qualifies the patient for therapy with inhibitors of the MAPK pathway. The identification of response markers is particularly important given primary and acquired resistance to targeted therapies. Reliable predictive tests would enable the selection of patients who would have the best chance of benefiting from treatment. Here, up-to-date knowledge about the most promising genetic and non-genetic resistance-related factors is described. These are alterations in MAPK, PI3K/AKT, and RB signaling pathways, e.g., due to mutations in NRAS, RAC1, MAP2K1, MAP2K2, and NF1, but also other changes activating these pathways, such as the overexpression of HGF or EGFR. Most of them are also potential therapeutic targets and this issue is also addressed here.

A Presurgical-Window Intervention Trial of Isothiocyanate-Rich Broccoli Sprout Extract in Patients with Breast Cancer.

Dietary isothiocyanates (ITCs) from cruciferous vegetables have shown potent anti-breast cancer activities in preclinical models, but their anticancer effects in vivo in breast cancer patients remain elusive. A proof-of-principle, presurgical window of opportunity trial is conducted to assess the anticancer effects of dietary ITCs in breast cancer patients. Thirty postmenopausal breast cancer patients are randomly assigned to receive ITC-rich broccoli sprout extract (BSE) (200µmol ITC per day) or a placebo for 2 weeks. Expression of biomarkers related to ITCs functions are measured in breast cancer tissue specimens at pre- and post-interventions using immunohistochemistry staining. First morning urine samples are collected at both timepoints for proteomic analysis. Overall, the study shows high compliance (100%) and low toxicity (no grade 4 adverse event). Trends of increase in cleaved caspase 3 and tumor-infiltrating lymphocytes (TILs) and trends of decrease in Ki-67 and nuclear to cytoplasm ratio of estrogen receptor (ER)-α are observed in the BSE arm only, consistent with the significantly altered signaling pathways identified in urinary proteomic analysis. Anticancer activities of ITCs are observed in breast cancer patients, supporting the potential beneficial roles of ITC-containing cruciferous vegetables in breast cancer prognosis.

MO365: Single-Cell Profiling Analysis Reveals the Transcriptomic Landscape Of Kidneys in Patients With Acute Kidney Injury

Abstract BACKGROUND AND AIMS Acute kidney injury (AKI) is a common clinical syndrome associated with considerable mortality and healthcare costs. Up to now, the underlying pathological mechanisms of AKI are still unclear, and specific strategies for early diagnosis and treatment of AKI are lacking. In this research, single-cell RNA sequencing (scRNA-seq) was applied to kidney biopsies of patients with AKI to elucidate novel molecular mechanisms and intercellular communications. METHOD The transcriptomes of human kidney specimens obtained from subjects with AKI and renal transplant healthy living donors were defined by scRNA-seq technology. Differentially expressed genes (DEGs) and cell clusters of the kidney were determined. Gene ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were performed, and cell–cell crosstalk communication analysis among distince cell subtypes were conducted. RESULTS A total of 16 distinct cell clusters were determined in kidney from subjects of AKI and control. Proximal tubule cells of AKI had up-regulation of novel pro-apoptotic genes including USP47, RASSF4, EBAG9, IER3 and NUB1, which have not been reported previously. Further, proximal tubule cells highly expressed endoplasmic reticulum stress related genes (PDIA6, ATF6, HSPA5 and DNAJC3), autophagy related genes (S100A11, CLDN1, TMBIM6, RB1CC1 and VMP1) and genes enriched in RIG-1 signaling. PALM3 was overexpressed in proximal tubule cells of AKI at both mRNA and protein levels, which is involved in cell adhesion, TLR4 signaling and infammation response. DEGs overexpressed in other tubular cells were primarily enriched in NOD-like receptor signaling, estrogen signaling, interleukin-12-mediated signaling and IL-17 signaling. Glomerular cells such as EC upregulated DEGs participating in cell adhesion, apoptosis and chemotaxis. Overexpressed genes in kidney-resident immune cells including macrophages, NK-T cells, monocytes and dendritic cells suggested abnormal regulation related with leukocyte activation, chemotaxis, cell adhesion and complement activation. In addition, the ligand-receptor interactions analysis revealed cell–cell communication contributing to adhesion, recruitment and infiltration of inflammatory cells into the kidneys, and cytokine production in the process of AKI. CONCLUSION Together, this study reveals distinct cell-specific transcriptional landscapes of kidney in AKI patients, also altered signaling pathways and potential cell-cell crosstalk in development of AKI. These data may provide new insights into the pathogenesis and potential therapeutic strategies of AKI.

Dysregulation of CD8+ T cell function in the setting of altered cytokine signaling due to inborn errors of immunity

Abstract A key outcome of chronic antigenic stimulation in the setting of sustained inflammation is the development of CD8+ T cell exhaustion. However, the relative roles of inflammation and antigen stimulation on T cell dysfunction remain unclear. Inborn errors of immunity (IEIs) provide an opportunity to study the effect of genetic alterations in signaling pathways on immune function. Here, we focus on an IEI resulting from genetically amplified cytokine signaling: gain-of-function mutations in STAT3 (STAT3 GOF). STAT3 signals downstream of multiple inflammatory cytokines (e.g., IL-6 and IL-27) and we used STAT3 GOF as a genetic mimic of chronic inflammation. To assess how amplified STAT3 signaling impacts immune cell subset distribution and activation status, PBMCs from STAT3 GOF patients and age-matched healthy controls were profiled via mass cytometry (CyTOF). This revealed altered states of CD8+ T cell activation including increased expression of immunoregulatory receptors (e.g., PD-1 and CD39). STAT3 GOF CD8+ T cells also showed impaired cytokine production and reduced proliferative capacity. Bulk RNA sequencing of naïve STAT3 GOF CD8+ T cells identified an exhausted-like transcriptional profile. Quantitation of metabolic function shows alterations in glucose use and oxidative phosphorylation in STAT3 GOF. Our studies suggest that inflammation alone may directly impair CD8+ T cell function. Understanding the mechanisms by which inflammation impacts T cell function may identify components of T cell dysregulation that can be targeted therapeutically in these rare patients, as well as those with more common inflammatory diseases (e.g., obesity) with shared aspects of T cell dysfunction. Supported by CHOP Research Institute, The Primary Immune Deficiency Treatment Consortium (PIDTC) and Immune Deficiency Foundation (IDF).

Chimeric Cytokine Targeting NKG2D and IL-2 Receptor Augments CD8+ T Cell-Metabolic Fitness and Improves Immunotherapy

Abstract T cell mediated immunotherapy has gained credible traction for cancer treatment. To date experimental and clinical strategies have relied on antibody-mediated co-stimulatory blockade, or the administration of high doses of naturally occurring or engineered cytokines to increase T cell activation. Cytokine therapy is by limited undesirable off-target side effects as well as terminal differentiation and exhaustion of chronically stimulated T cells. Here we describe functionality of a unique chimeric cytokine by design, called OMCPmutIL-2, binds with high affinity to the cytotoxic lymphocyte-defining immunoreceptor NKG2D but signals through the common γ-chain cytokine receptor, thus eliminating IL-2Ra mediated off-target side effects. In addition to precise activation of cytotoxic T cells due to redirected binding we describe that OMCPmutIL-2 results in superior activation of both human and murine CD8+ T cells by improving their survival, decreasing exhaustion, and improving memory cell generation. This functional improvement is the result altered signal transduction based on the reorganization of surface membrane lipid rafts resulting in JAK-mediated phosphorylation of the T cell receptor (TCR) rather than canonical STAT/AKT signaling intermediates activated by other common γ-chain cytokines. This altered signaling pathway increases CD8+ T cell response to low affinity antigens, activates Nuclear Factor of Activated T Cells (NFAT) transcription factors, and enhances mitochondrial biogenesis with improved T cell survival, cytotoxicity, and memory cell generation. OMCPmutIL-2 thus outperforms other common γ-chain cytokines as both a reagent for T cell expansion and in vivo CD8+ T cell-based immunotherapy. Supported by grant I01 BX002299

Involvement of ethylene in glutamate-mediated tomato fruit resistance to Alternaria alternata

Recent progress showed the potential of glutamate to regulate stress responses by affecting phytohormones homeostasis, but much remains to be explored. To decipher the role of ethylene in glutamate-induced resistance to Alternaria alternata, ethephon application, ethylene perception inhibitor (1-MCP) experiment and ethylene measurement were pursued. Concurrently, temporal alteration in ethylene biosynthesis and signaling pathways were transcriptionally analyzed in tomato fruit. Our data exhibited that ethylene conferred tomato fruit susceptibility rather than resistance to Alternaria alternata. The enhanced resistance in glutamate treatment was related to decreased ethylene production during the early stages of interaction, accompanied by lower expression levels of SlACS1, SlACS2 and SlACO1 relative to the control. Interestingly, 1-MCP induced amounts of resistance similar to that observed in glutamate treatment, while ethylene receptors (SlETR3 and SlETR4), mitogen-activated protein kinase SlMPK3 and ethylene response factor SlERF1 were down-regulated by glutamate during infection. Collectively, antagonizing the ethylene biosynthesis or signaling might play a pivotal role in glutamate-induced resistance to the necrotrophic pathogen Alternaria alternata.