Bioinformatics Analysis Research Articles

Metabolomic Effects of Liraglutide Therapy on the Plasma Metabolomic Profile of Patients with Obesity

Background: Liraglutide, a long-acting glucagon-like peptide-1 receptor agonist (GLP1RA), is a well-established anti-diabetic drug, has also been approved for the treatment of obesity at a dose of 3 mg. There are a limited number of studies in the literature that have looked at changes in metabolite levels before and after liraglutide treatment in patients with obesity. To this end, in the present study we aimed to explore the changes in the plasma metabolomic profile, using liquid chromatography-high resolution mass spectrometry (LC-HRMS) in patients with obesity. Methods: A single-center prospective study was undertaken to evaluate the effectiveness of 3 mg liraglutide therapy in twenty-three patients (M/F: 8/15) with obesity, mean BMI 40.81 ± 5.04 kg/m2, and mean age of 36 ± 10.9 years, in two groups: at baseline (pre-treatment) and after 12 weeks of treatment (post-treatment). An untargeted metabolomic profiling was conducted in plasma from the pre-treatment and post-treatment groups using LC-HRMS, along with bioinformatics analysis using ingenuity pathway analysis (IPA). Results: The metabolomics analysis revealed a significant (FDR p-value ≤ 0.05, FC 1.5) dysregulation of 161 endogenous metabolites (97 upregulated and 64 downregulated) with distinct separation between the two groups. Among the significantly dysregulated metabolites, the majority of them were identified as belonging to the class of oxidized lipids (oxylipins) that includes arachidonic acid and its derivatives, phosphorglycerophosphates, N-acylated amino acids, steroid hormones, and bile acids. The biomarker analysis conducted using MetaboAnalyst showed PGP (a21:0/PG/F1alpha), an oxidized lipid, as the first metabolite among the list of the top 15 biomarkers, followed by cysteine and estrone. The IPA analysis showed that the dysregulated metabolites impacted the pathway related to cell signaling, free radical scavenging, and molecular transport, and were focused around the dysregulation of NF-κB, ERK, MAPK, PKc, VEGF, insulin, and pro-inflammatory cytokine signaling pathways. Conclusions: The findings suggest that liraglutide treatment reduces inflammation and modulates lipid metabolism and oxidative stress. Our study contributes to a better understanding of the drug’s multifaceted impact on overall metabolism in patients with obesity.

MiR-30c-5p inhibits esophageal squamous cell carcinoma progression by repressing the PI3K/AKT signaling pathway.

Esophageal squamous cell carcinoma (ESCC) is one of the most common malignant tumors, with high incidence and poor prognosis. Revealing mechanisms of ESCC progression and developing new therapeutic targets remains crucial. The aim of this study was to elucidate the molecular mechanism of miR-30c-5p in regulating the malignant progression of ESCC. TCGA, GEO, and other datasets were used to analyze the differential expression of miR-30c-5p in ESCC and adjacent tissues, and its impact on prognosis. Then the effects of miR-30c-5p on the proliferation, migration, and invasion of TE-1 and Eca9706 cells were investigated through proliferation experiments, transwell and wounding healing assays. The regulatory mechanism of miR-30c-5p on the PI3K/AKT signaling pathway and its interaction in cancer progression were investigated through Western blots, dual-luciferase reporter assay, and rescue experiments. miR-30c-5p was significantly downregulated in ESCC tissue and represented a poor prognosis. miR-30c-5p mimic significantly inhibited the proliferation, migration, and invasion ability of ESCC, while miR-30c-5p inhibitor significantly promoted tumor cell progression. Through bioinformatic analysis and experimental results, miR-30c-5p interacted directly with PIK3CA mRNA and inhibited subsequent signaling pathway activation. PIK3CA activator could eliminate the inhibitory effects of miR-30c-5p mimic on the progression of ESCC, while PIK3CA inhibitors could rescue the promoting effect of miR-30c-5p inhibitor group cells. In summary, we found that miR-30c-5p inhibited the proliferation, invasion and migration of ESCC by inhibiting PI3K/AKT signaling pathway for the first time, and this study is expected to provide a novel insight and potential therapeutic target for managing ESCC.

Decoding recurrent pregnancy loss: insights from comparative proteomics studies.

Recurrent pregnancy loss (RPL) represents a common disorder that affects up to 2% of the women aiming at childbirth with long-term consequences on family and society. Factors contributing to it in more than half of the cases are still unknown. Comparative proteomic analysis can provide new insights into the biological pathways underlining the pathogenesis of RPL. Until now, chorionic villi, decidua, placenta, endometrium, and maternal blood from women with RPL have been analyzed by comparative proteomics studies. In this review, we aimed to provide a critical evaluation of the published comparative studies of RPL on human samples, gathered by systematic literature search using PubMed and Google Scholar databases. We provide a detailed overview of the analyzed materials, proteomics platforms, proposed candidate biomarkers and altered pathways and processes linked with RPL. The top, most identified and validated biomarker candidates from all studies are discussed, followed by bioinformatics analysis of the available high-throughput data and presentation of common altered processes and pathways in RPL. Finally, future directions aimed at developing new and efficient therapeutic strategies are discussed as well.

Unlocking the Door for Precision Medicine in Rare Conditions: Structural and Functional Consequences of Missense ACVR1 Variants.

Rare diseases and conditions have thus far received relatively less attention in the field of precision/personalized medicine than common chronic diseases. There is a dire need for orphan drug discovery and therapeutics in ways that are informed by the precision/personalized medicine scholarship. Moreover, people with rare conditions, when considered collectively across diseases worldwide, impact many communities. In this overarching context, Activin A Receptor Type 1 (ACVR1) is a transmembrane kinase from the transforming growth factor-β superfamily and plays a critical role in modulating the bone morphogenetic protein signaling. Missense variants of the ACVR1 gene result in modifications in structure and function and, by extension, abnormalities and have been predominantly linked with two rare conditions: fibrodysplasia ossificans progressiva and diffuse intrinsic pontine glioma. We report here an extensive bioinformatic analyses assessing the pool of 50,951 variants and forecast seven highly destabilizing mutations (R206H, G356D, R258S, G328W, G328E, R375P, and R202I) that can significantly alter the structure and function of the native protein. Protein-protein interaction and ConSurf analyses revealed the crucial interactions and localization of highly deleterious mutations in highly conserved domains that may impact the binding and functioning of the protein. cBioPortal, CanSAR Black, and existing literature affirmed the association of these destabilizing mutations with posterior fossa ependymoma, uterine corpus carcinoma, and pediatric brain cancer. The current findings suggest these deleterious nonsynonymous single nucleotide polymorphisms as potential candidates for future functional annotations and validations associated with rare conditions, further aiding the development of precision medicine in rare diseases.

Unravelling stress granules in the deep cold: Characterisation of TIA-1 gene sequence in Antarctic fish species

Stress granules (SGs) are cytoplasmic foci lacking membranes, comprising non-translating messenger ribonucleoproteins, translational initiation factors, and additional proteins. Their formation is crucial for rapidly modulating gene expression in response to adverse environmental conditions, such as pollution and infections. Limited research has focused on investigating the molecular components of SGs in fish, with minimal exploration in Antarctic fish. This study characterises for the first time the transcript sequences of one key protein component of SGs, TIA-1 (T-cell intracellular antigen 1), in two Antarctic endemic fish species, i.e. Trematomus bernacchii and Chionodraco hamatus. The mRNA-binding protein TIA-1 acts as a post-transcriptional regulator of gene expression and its aggregation leads to the formation of SGs in response to cellular damage. The in vitro and bioinformatic analyses of the TIA-1 gene sequences of these two species highlighted interesting peculiarities, which include the transcription of alternatively spliced isoforms unique to the notothenioid lineage, potentially unlocking further insights into their unique adaptations to extreme environmental conditions. This is the first study to analyze tia-1 expression levels in different tissues of Antarctic fish species. Our key findings indicate that the TIA-1 gene is expressed at particularly high levels in the liver and spleen of C. hamatus, as well as in the heart and skeletal muscle of T. bernacchii. This suggests that those tissues play a significant role in the stress response mechanisms of the studied species. This study provides novel insights into the molecular adaptations of Antarctic fish, highlighting the potential importance of TIA-1 in their response to environmental stressors. The unique features of TIA-1 identified in these species may offer broader implications for understanding how Antarctic fish regulate gene transcriptions in their extreme environments.

Characterization of Shy1, the Schizosaccharomyces pombe homolog of human SURF1

Cytochrome c oxidase (complex IV) is the terminal enzyme in the mitochondrial respiratory chain. As a rare neurometabolic disorder caused by mutations in the human complex IV assembly factor SURF1, Leigh Syndrome (LS) is associated with complex IV deficiency. In this study, we comprehensively characterized Schizosaccharomyces pombe Shy1, the homolog of human SURF1. Bioinformatics analysis revealed that Shy1 contains a conserved SURF1 domain that links to the biogenesis of complex IV and shares high structural similarity with its homologs in Saccharomyces cerevisiae and humans. Our study showed that Shy1 is required for the expression of mtDNA-encoded genes and physically interacts with structural subunits and assembly factors of complex IV. Interestingly, Rip1, the subunit of ubiquinone-cytochrome c oxidoreductase or cytochrome bc1 complex (complex III), can also co-immunoprecipitate with Shy1, suggesting Shy1 may be involved in the assembly of the mitochondrial respiratory chain supercomplexes. This conclusion is further corroborated by our BN-PAGE analysis. Unlike its homologs, deletion of shy1 does not critically disrupt respiratory chain assembly, indicating the presence of the compensatory mechanism(s) within S. pombe that ensure mitochondrial functionality. Collectively, our investigation elucidates that Shy1 plays a pivotal role in the sustainability of the regular function of mitochondria by participating in the assembly of complex IV in S. pombe.

Predictive action of oncomiR in suppressing TP53 signaling pathway in hypoxia-conditioned colon cancer cell line HCT-116.

Hypoxia-induced heterogeneity in colorectal cancer (CRC) significantly impacts patient survival by promoting chemoresistance. These conditions alter the regulation of miRNAs, key regulators of crucial processes like proliferation, apoptosis, and invasion, leading to tumor progression. Despite their promising potential as diagnostic and therapeutic targets, the underlying mechanisms by which miRNAs influence hypoxia-mediated tumorigenesis remain largely unexplored. This study aims to elucidate the action of miRNAs in HCT-116 colorectal cancer stem cells (CSCs) under hypoxia, providing valuable insights into their role in tumor adaptation and progression. MiRNA expression was determined using Nanostring nCounter, and bioinformatic analysis was performed to explain the molecular pathway. A total of 50 miRNAs were obtained with an average count of ≥ 20 reads for comparative expression analysis. The results showed that hypoxia-affected 36 oncomiRs were increased in HCT-116, and 14 suppressor-miRs were increased in MSCs. The increase in miRNA expression occurred consistently from normoxia to hypoxia and significantly differed between mesenchymal stem cells (MSCs) and HCT-116. Furthermore, miR-16-5p and miR-29a-3p were dominant in regulating the p53 signaling pathway, which is thought to be related to the escape mechanism against hypoxia and maintaining cell proliferation. More research with a genome-transcriptome axis approach is needed to fully understand miRNAs' role in adapting CRC cells and MSCs to hypoxia. Further research could focus on developing specific biomarkers for diagnosis. In addition, anti-miR can be developed as a therapy to prevent cancer proliferation or inhibit the adaptation of cancer cells to hypoxia.

An Integrated Approach of Network Pharmacology, Bioinformatics, Molecular Docking, and Experimental Verification Uncovers Prunellae Spica as the potential Medicine of Prognosis Improvement for Oral Squamous Cell Carcinoma.

Prunellae Spica (PS), the spike from Prunella vulgaris L., is a traditional Chinese medicine that can treat Oral Squamous Cell Carcinoma (OSCC), whereas its molecular mechanisms and effects on the prognosis of patients remain unclear. Our study aimed to identify potential anti-OSCC targets of PS and explore its mechanisms and effects on prognosis through network pharmacology, bioinformatics analysis, molecular docking, and in vitro cell assays. Sixty-two potential targets of 11 active anti-OSCC ingredients of PS were identified, with Quercetin, the core ingredient of PS, exhibiting the most significant number of OSCC-related targets. GO analysis indicated that the primary biological processes involved in OSCC treatment by PS were the cellular response to nitrogen compound, response to xenobiotic stimulus, and cellular response to organonitrogen compound. KEGG analysis revealed that Pathways in cancer were the top highly enriched signaling pathway in the treatment of OSCC by PS. DisGeNET analysis is mainly about Lip and Oral Cavity Carcinoma. More importantly, 6 of the 62 targets were markedly related to prognosis. Molecular docking revealed high affinities between the key component and the prognosis-related target proteins. Treatment of OSCC cell line SCC-25 with Quercetin could inhibit malignant biological behaviors, such as cell proliferation, colony formation, invasion, and migration, as well as affect the targets related to prognosis and promote autophagy. Overall, these results suggest that PS plays a significant role in treating and improving the prognosis of OSCC by directly influencing various processes in OSCC.

HCMV detection in Asian gastric cancer RNA-seq data sets and clinical validation in Indian GC patients reveals the HCMV-GC specific gene signatures.

Gastric cancer (GC) prevalence is very high in the Asian population. Oncogenic viruses play a crucial role in the progression of different types of cancers. Through reanalysis of clinical RNA-seq data sets derived from Asian GC patients, this study identified the presence of human cytomegalovirus (HCMV) in Asian GC tumors, next to the well-studied association of EBV. Clinical recruitment of the Indian GC cohort and screening for HCMV presence identified a 14.28% occurrence, similar to that observed in the bioinformatics analysis. A combinatorial approach of rank-based meta-analysis and ranking of groups based on an expectation-maximization algorithm identified that the upregulated LINC02864 and MAGEA10 correlated with poor survival of GC patients and downregulated tumor suppressor genes enriching for gastric acid secretion pathway to be associated with HCMV-positive GC patients, revealing the progressive role of HCMV infection in GC. Genes that discriminate between different stages of GC were identified through feature selection implemented in a machine-learning approach. LTF and KLK10 expressions were found to be specifically dysregulated by HCMV and can also indicate the GC stages. The results of this study will guide future studies to identify the functional role of these genes in the HCMV-associated GC.IMPORTANCENearly 75% of gastric cancer (GC) cases reported globally are from the Asian population. Most existing public databases, such as TCGA, comprise only a fractional portion of data derived from Asian ancestry. This study identified EBV and human cytomegalovirus (HCMV)'s higher detection in GC patients. The presence and role of EBV associated with GC are well-known, and the observation of HCMV prompted us to validate the findings in a small cohort of 40 Indian GC patients. We observed a 14.28% occurrence of HCMV in the Indian cohort, similar to that observed from next-generation sequencing. A combinatorial approach of rank-based meta-analysis and ranking of groups based on an expectation-maximization algorithm identified that the upregulated LINC02864 and MAGEA10 correlated with poor survival of GC patients and downregulated tumor suppressor genes enriching for gastric acid secretion pathway to be associated with HCMV-positive GC patients, revealing the progressive role of HCMV infection in GC.

Huopuxialing Decoction: A Promising Candidate for Precancerous Lesions of Gastric Cancer Treatment Based on Bioinformatics and Experimental Verification.

The Precancerous Lesion of Gastric Cancer (PLGC) is an early stage in the development of gastric cancer. The clinical application of HPXLD has been found to be effective in treating PLGC, but the mechanism of how HPXLD acts on PLGC is still unclear. The objectives of this study were to reveal the molecular mechanism of how HPXLD can be used to treat PLGC and investigate this mechanism through bioinformatics and experimental validation. PLGC-associated target genes were identified through bioinformatics analysis. A rat model of PLGC was induced using N-methyl-N'-nitro-N-nitrosoquanidine (MNNG) in combination with ranitidine, hot saline, ethanol, and intermittent fasting, with interventions by HPXLD. The pathological alterations in gastric mucosa were assessed through Hematoxylin-eosin staining (HE). Immunohistochemistry (IHC) and Western blot analyses were employed to evaluate the changes in expression levels of inflammation-related proteins. After conducting bioinformatics analysis, it was found that there were 23 HPXLDPLGC crossover genes, which were significantly enriched in the IL-17 signaling pathway, TNF signaling pathway, and NF-kappa B signaling pathway. The results of HE showed that HPXLD was effective in improving gastric mucosal histopathological changes. Additionally, the IHC results demonstrated that HPXLD was able to downregulate the expression of IL-6, COX-2, MCP- 1, and MMP-9. Furthermore, Western blot analysis revealed that HPXLD was able to downregulate the expressions of IL-6, IL-17RA, ACT1, NF-κB, and TNF-α. HPXLD has been shown to improve PLGC by reducing the expression of inflammation- related proteins. This suggests that HPXLD may potentially be a treatment option for PLGC.

Systematic analysis and functional verification of citrus ascorbate peroxidases reveal that CsAPX01 and CsAPX02 negatively regulate citrus bacterial canker through the hydrogen peroxide regulation

Ascorbate peroxidases (APXs) are antioxidant enzymes that play vital roles in redox homeostasis in plants. Citrus is susceptible to infection by Xanthomonas citri subsp. citri (Xcc), resulting in citrus bacterial canker (CBC). The present study used bioinformatic and expression analyses to investigate the APX family in Citrus sinensis. Bioinformatic research revealed the chromosomal locations, phylogeny, gene structure, promoter elements, functional domains, conserved motifs, and most likely physicochemical properties of the sequences. Six APXs clustered in three groups were identified, with each protein containing a single peroxidase domain. The promoter regions contained a variety of transcription factor-binding and hormone-response components. Xcc infection induced different CsAPX01 and CsAPX02 expressions in the CBC-susceptible Wanjincheng and CBC-resistant Kumquat varieties. Subcellular localization and transient expression showed that CsAPX01 and CsAPX02 were expressed in the cytoplasm and nucleus and had hydrogen peroxide (H2O2)-scavenging activity. Virus-induced gene silencing (VIGS) of CsAPX01 and CsAPX02 resulted in strong resistance to CBC and H2O2 bursts without effects on the plant phenotype. The current study focused on investigating and characterizing the citrus APX family. It was found that CsAPX01 and CsAPX02 exacerbated CBC by altering the balance of H2O2. These findings emphasize the importance of APXs in enhancing plant resistance to pathogens.

Deciphering the Protective Role of HIF-1α Downregulation on HIBD through the MALAT1/miR-140-5p/TGFBR1/NF-κB Signaling Pathway.

Hypoxic-ischemic brain damage (HIBD) in neonates is a substantial cause of mortality and neurodevelopmental impairment, with the exact molecular mechanisms still being elucidated. The involvement of HIF-1α, MALAT1, miR-140-5p, TGFBR1, and the NF-κB signaling pathway in such injury cascades is of increasing research interest due to their pivotal roles in cellular and pathological processes. This study aimed to explore how HIF-1α regulates the MALAT1/miR-140-5p/TGFBR1/NF-κB signaling axis to participate in the molecular mechanisms of HIBD in neonatal rats. Utilizing bioinformatic analyses and a suite of experimental approaches, the study delineated interactions and regulatory relationships among the molecules. Knockdown of HIF-1α was shown to mitigate brain tissue damage in a neonatal HIBD rat model through the MALAT1/miR-140-5p/TGFBR1/NF-κB signaling axis, revealing a protective effect achieved by inhibiting hippocampal neuron apoptosis and potentially guiding the way toward therapeutic interventions in HIBD. This study implicates the HIF-1α mediated regulation of the MALAT1/miR-140-5p/TGFBR1/NF-κB signaling axis in the pathological development of HIBD, offering insights into novel potential interventional strategies.

Modern technology-based exploration of mechanism of traditional Chinese medicine in prevention and treatment of gastric cancer

This review comments on the article “To explore the mechanism of Yigong San anti-gastric cancer and immune regulation”. We are interested that the article applied network pharmacology and bioinformatics techniques to elucidate the mechanism of action of Yigong Sang, a traditional Chinese medicine (TCM), in the treatment of gastric cancer (GC). The mechanism of action of Yigong Sang in the treatment of GC has not yet been elucidated because it is composed of multiple Chinese medicines with multiple components and multiple targets. The emergence of network pharmacology and bioinformatics analysis helps explain the mechanism of action of TCM in preventing and treating GC, and provides a possibility for TCM to transform from empirical to evidence-based medicine. This is of great significance for the application of TCM in oncology, new drug development, formula optimization, and the improvement of clinical efficacy.

Abstract PR-15: Identifying synergistic combinations with KRAS inhibition in PDAC

Abstract Background: To date, targeted therapies have largely failed in eliciting sustained responses in advanced pancreatic ductal adenocarcinoma (PDAC). KRAS-targeted therapies have the potential to drastically transform clinical management. Emerging data indicate that only a subset of patients respond, and that acquired resistance is common. Here, we used a combination of in vivo CRISPR activation (CRISPRa) screening and in vitro/preclinical models to identify synergistic combinations with KRAS inhibition (KRASi) in pancreatic and lung cancer. Material and Methods: We used our CRISPRa-competent PPKS (P53(F/F), Kras(LSL-G12D/+), R26(LSL-SAM)) model of adenocarcinoma to conduct targeted screening for drivers of resistance to MRTX133. Our library consisted of ∼450 frequently mutated and amplified putative oncogenes across human adenocarcinoma based on TCGA data. Mice were treated with 1e8 TU of lentiviral library through nasal instillation. After 6 weeks, treatment was initiated with 3 mg/kg MRTX1133 daily, 5 days/week (i.p.). Mice were sacrificed after 4 weeks of treatment and the guide distribution determined through bioinformatic analysis. Genes statistically significantly enriched (FDR<0.05) in the treatment group were determined. Cell lines with acquired resistance to MRTX1133 derived from the KPC PDAC model and the KPP lung adenocarcinoma (LUAD) model were generated using long-term, low-dose treatment. Resistance was confirmed with MTT drug response assays. In vitro combinatorial drug response was determined using a 72h, 96-well MTT assay, and clonogenic response using a 2 week, 384-well FACS assay. Mice were treated with 20 mg/kg MRTX1133 and/or 20mg/kg Temuterkib once daily, 7 days/week (i.p.) for 2 weeks. Results: We performed targeted in vivo CRISPRa screening for drivers of resistance to KRASi in an autochthons model of lung adenocarcinoma (LUAD) using a library targeting 452 genes commonly amplified or mutated in cancer. We identified a set of genes that were specifically enriched in at least 50% of tumors treated with MRTX1133, including MAPK1/ERK2. We also generated 16 cell lines with acquired resistance to MRTX1133, derived from KPC PDAC or KPP LUAD tumors. Transcriptomic analysis identified a large set of genes differentially expressed in cell with acquired resistance relative to controls. GSEA indicated that reactivation of the MAPK cascade as a common trait in resistant cells. To test if ERK inhibition synergizes with KRASi, we quantified the combinatorial response to MRTX1133 and Temuterkib, a specific inhibitor of ERK1 and ERK2, in a set of KPC and KPP derived cell lines, revealing significant synergy and reduced clonogenic potential in a majority of cell lines. A majority of cell lines with acquired resistance to MRTX1133 remained sensitive to Temuterkib. Finally, we tested combinatorial treatment of MRTX1133 and Temuterkib in a syngenetic subcutaneous model of PDAC, which showed at minimum additive effect. In conclusion we have identified ERK inhibition as a promising combination with KRAS inhibition in KRAS-mutant PDAC and LUAD. Citation Format: Fredrik I Thege, Amber Hoskins, Sonja M Woermann, Anirban Maitra. Identifying synergistic combinations with KRAS inhibition in PDAC [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr PR-15.

GNAS, not a Highly Mutated Gene, Has Prognostic Significance and Carcinogenic Effects in Osteosarcoma

Background/Aims: Osteosarcoma is a prevalent and aggressive primary malignant bone tumor affecting children and adolescents. Despite advancements in sequencing technologies, there remains a lack of reliable prognostic biomarkers and effective targeted therapies for osteosarcoma. This study focuses on identifying key prognostic genes, particularly the role of GNAS, in osteosarcoma progression. Methods: Bioinformatics analyses were performed on osteosarcoma datasets from the Gene Expression Omnibus (GEO). Differential gene expression analysis, weighted correlation network analysis (WGCNA), and survival analysis identified potential prognostic hub genes. The expression and function of these genes were validated through immunohistochemistry and animal experiments. Specifically, the role of GNAS was investigated through siRNA-mediated knockdown in osteosarcoma cell lines and nude mice models. Results: Five hub genes (PROP1, GNAS, CYP4F2, LHX3, CNGB1) were identified as significantly related to osteosarcoma prognosis. Among these, GNAS was found to be highly expressed in osteosarcoma tissues compared to normal tissues. Immunohistochemical analysis confirmed the elevated expression of GNAS in osteosarcoma samples. GNAS mutation analysis revealed a low mutation rate in osteosarcoma, suggesting its oncogenic role is independent of mutational status. Animal experiments demonstrated that knocking down GNAS significantly inhibited tumor growth and induced apoptosis in osteosarcoma cells. Conclusion: GNAS is highly expressed in osteosarcoma and associated with poor prognosis, acting as an oncogene in osteosarcoma progression. Targeting GNAS could be a potential therapeutic strategy for osteosarcoma. Further studies on GNAS-related signaling pathways may provide deeper insights into the molecular mechanisms driving osteosarcoma malignancy.

Abstract B082: Oncogenic KRAS-mediated epigenetic reprogramming is altered by loss of Activating Transcription Factor 3

Abstract Introduction: Over 90% of PDAC patients harbor an activating KRAS mutation, the most common form being KRASG12D. However, additional genetic mutations and environmental events, including chronic or hereditary pancreatitis, are needed for KRAS mutations to lead to PDAC. Our laboratory showed Activating Transcription Factor 3 (ATF3) was required for repressing genes that stabilize the mature acinar cell phenotype and for KRASG12D-driven progression to advanced PanIN lesions. However, ATF3 contributes to gene activation and repression and has been linked to numerous transcription factors and epigenetic mediators, and how ATF3 affects PDAC progression remains unknown. We hypothesize that KRASG12D promotes ATF3-driven histone acetylation in precursor PanINs lesions. Methods: C57Bl/6l mice allowing for acinar-inducible KRASG12D combined with (Ptf1acreERTKRASG12D) or without (Ptf1acreERTKRASG12DAtf3-/-; APK) Atf3 deletion were treated with tamoxifen to induce KRASG12D. After 10 days, mice were treated with cerulein to induce injury and PanIN progression. Mice were sacrificed after two weeks and 3D organoid lines developed. RNA-seq and chromatin immunoprecipitation for H3K27 acetylation (H3K27ac) followed by sequencing (ChIP-seq) was performed. Bioinformatic analysis was used to identify differentially enriched pathways. Levels of acetyl-CoA, the substrate for acetylation, was compared between lines along with levels of H3K27ac, H3K4me1, H3K4me3, and H3K9me3 through western blots. Results and Discussion: KRASG12D expression promoted epigenetic reprogramming in PanINs, including dysregulation of H3K27ac enrichment. ChIP-seq results showed the absence of ATF3 reduces H3K27 acetylation preferentially at gene promoters and pathway analysis showed differential enrichment and activation of KRAS signaling. Mechanistically, ATF3-deleted mice have lower acetyl-CoA levels, linking ATF3 to metabolic pathways as a possible mechanism for gene regulation. Comparing genes with an ATF3-dependent acetylation pattern to a curated list of KRAS-targeted genes identified SMARCC2 (BAF170), a SWI/SNF complex subunit, and KDM1B, a histone demethylase affecting H3K4, suggesting a potential role for ATF3 in widespread epigenetic regulation. Public sequencing data from PDAC patients, supports these findings as ATF3 expression is positively correlated with expression of several SWI/SNF complex subunits and demethylases. Conclusion: This study highlights a role for ATF3- mediated epigenetic dysregulatio. In oncogenic KRAS-dependent PDAC. Targeting this ATF3- mediated epigenetic landscape could be a promising new therapeutic avenue. Citation Format: Fatemeh Mousavi, Christopher L Pin, Parisa Shooshtari. Oncogenic KRAS-mediated epigenetic reprogramming is altered by loss of Activating Transcription Factor 3 [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr B082.

Abstract C037: Characterization of cysteine dependency implicates glutathione metabolism as a critical adaptation axis in pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma (PDAC) is a deadly cancer that is highly malignant and lethal. The treatment options are limited, contributing to its high mortality rate. PDAC is known to have profound metabolic alterations that enable its growth and survival. Here, we screened for amino acid dependency in PDAC cell lines and found that the cells cluster into cysteine highly dependent, moderately dependent, and independent cells. In the highly dependent cells, cysteine deprivation reduces viability by ∼90%, which could be rescued by glutathione (GSH) supplementation. Metabolomics revealed that cysteine deprivation led to GSH depletion, suggesting an impairment in the GSH pathway machinery in these cells. Consistently, most metabolite intermediates in the GSH pathway are downregulated in the cysteine-reliant PDAC cell lines compared to cell lines that resist cysteine deprivation, and those cells showed higher sensitivity to oxidative stress induction. Bioinformatics analysis of multiple PDAC tumor gene expression datasets indicated that the GSH pathway is strongly upregulated in PDAC and correlates with proliferation, metastasis, and oncogenic KRAS upregulation. Using CRISPR/Cas9, we found that the knockout of GSH genes enhanced sensitivity to oxidative stress. Our data implicate the GSH pathway as a critical nexus to be targeted to overcome metabolic adaptation in PDAC. Citation Format: Chiamaka J Ezeh, Matthew H Ward, Chesta Jain, Verodia Charlestin, Daeho D Kim, Andrew Yang, Maya Nassif, Nneka Mbah, Peter Sajjakulnukit, Anthony Andren, Li Zhang, Marina Pasca di Magliano, Costas A Lyssiotis, Zeribe C Nwosu. Characterization of cysteine dependency implicates glutathione metabolism as a critical adaptation axis in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr C037.

Circ_0001947 encapsulated by small extracellular vesicles promotes gastric cancer progression and anti-PD-1 resistance by modulating CD8+ T cell exhaustion

BackgroundWhile small extracellular vesicles (sEVs)-derived circular RNAs (circRNAs) have been emerged as significant players in cancer, the function and underlying mechanism of sEVs-derived circRNAs in anti-cancer immunity remain unclear.MethodsGastric cancer (GC)-derived circRNAs were identified using RNA-seq data from GEO datasets and quantitative reverse transcription polymerase chain reaction (qRT-PCR), RNA immunoprecipitation, dual-luciferase assay, and bioinformatics analysis were performed to investigate the regulatory axis. Transwell assay, wound healing assay, cell counting kit-8 (CCK-8) assay, and xenograft models were used to evaluate its role in GC progression in vivo and in vitro. The delivery of specific circRNAs into sEVs were verified through electron microscopy, nanoparticle tracking analysis (NTA) and fuorescence in situ hybridization (FISH). Flow cytometric analysis and immunohistochemical staining were conducted to find out how specific circRNAs mediated CD8+ T cell exhaustion and resistant to anti-programmed cell death 1 (PD-1) therapy.ResultsWe identified that circ_0001947, packaged by GC-derived sEVs, was obviously elevated in GC and was associated with poor clinical outcome. High circ0001947 level augmented the proliferation, migration, and invasion of GC cells. Mechanistically, circ0001947 sponged miR−661 and miR−671−5p to promote the expression of CD39, which further facilitated CD8+ T cell exhaustion and immune resistance. Conversely, blocking circ_0001947 attenuated CD8+ T cell exhaustion and increased the response to anti-PD−1 therapy.ConclusionsOur study manifested the therapeutic potential of targeting sEVs-transmitted circ_0001947 to prohibit CD8+ T cell exhaustion and immune resistance in GC.

Genome-wide analysis of the WRKY family in Nicotiana benthamiana reveals key members regulating lignin synthesis and Bemisia tabaci resistance

WRKY transcription factors (TFs) play a pivotal role in plant resistance against stress. Lignin is a plant component that is of great importance in many industrial and agricultural disciplines. However, how WRKYs regulate plant lignin synthesis pathways and defense against insects is largely unknown. Here, we investigate WRKYs in Nicotiana benthamiana, a model plant and biofactory widely used worldwide, elucidating their responses to the Bemisia tabaci whitefly, a globally significant agricultural pest, and their functions in regulating plant defense by regulating lignin synthesis. Firstly, we identified a total of 118 NbWRKYs in the N. benthamiana genome and performed necessary bioinformatics analyses. Then, we studied nine whitefly-inducible NbWRKYs and found that they were localized in the nucleus, significantly expressed in leaf tissues, and affected the phytohormone pathways. Meanwhile, NbWRKY21, NbWRKY45, NbWRKY81, and NbWRKY117 were proved to be involved in defense against whiteflies. Additionally, through DNA-TFs interaction experiments, we demonstrated that the WRKYs activated the expression of key genes—Cinnamate-4-hydroxymate (C4H), 4-coumarate-CoA ligase (4CL), and Cinnamoyl-CoA reductase (CCR)—in the lignin synthesis pathway. Specifically, NbWRKY81 activated the NbC4H and Nb4CL, while NbWRKY45 promoted NbCCR, by binding to their promoters, affecting whitefly feeding behavior, and therefore enhancing whitefly defense. Our findings contribute to a comprehensive knowledge of the WRKYs in plant defense against herbivore insects by regulating lignin synthesis.

Dual client binding sites in the ATP-independent chaperone SurA

The ATP-independent chaperone SurA protects unfolded outer membrane proteins (OMPs) from aggregation in the periplasm of Gram-negative bacteria, and delivers them to the β-barrel assembly machinery (BAM) for folding into the outer membrane (OM). Precisely how SurA recognises and binds its different OMP clients remains unclear. Escherichia coli SurA comprises three domains: a core and two PPIase domains (P1 and P2). Here, by combining methyl-TROSY NMR, single-molecule Förster resonance energy transfer (smFRET), and bioinformatics analyses we show that SurA client binding is mediated by two binding hotspots in the core and P1 domains. These interactions are driven by aromatic-rich motifs in the client proteins, leading to SurA core/P1 domain rearrangements and expansion of clients from collapsed, non-native states. We demonstrate that the core domain is key to OMP expansion by SurA, and uncover a role for SurA PPIase domains in limiting the extent of expansion. The results reveal insights into SurA-OMP recognition and the mechanism of activation for an ATP-independent chaperone, and suggest a route to targeting the functions of a chaperone key to bacterial virulence and OM integrity.