Ras Signaling Research Articles

Enrichment of RedoxifibromiR miR-21-5p in Plasma Exosomes of Hypertensive Patients with Renal Injury

Several microRNAs (miRNAs) emerged as powerful regulators of fibrotic processes, “fibromiRs”, and can also influence the expression of genes responsible for the generation of reactive oxygen species, “redoximiRs”. We aimed to investigate whether plasma exosomes from hypertensive and diabetes patients are enriched in fibromiRs and redoximiRs using deep sequencing technology and their association with relevant signalling pathways implicated in oxidative stress and fibrogenesis by GO terms and KEGG pathways. RNA-Seq analysis from P-EXO identified 31 differentially expressed (DE) miRNAs in patients compared to controls, of which 77% are biofluid specific. The majority of the exosomal DE miRNAs were identified as fibromiRs (55%) or redoximiRs (26%). One of the most representative miRNAs identified was miR-21-5p, of which levels in P-EXO were increased by 3.83-fold change (p < 0.0001) in hypertensive patients with albuminuria and were highly associated (r Spearman = 0.64, p < 0.0001). In addition, P-EXO miR-21-5p had a high accuracy in discriminating renal damage (AUC = 0.82, p < 0.0001). Bioinformatic analysis revealed that miR-21-5p regulates key pathways in the context of organ fibrosis, such as chemokine, Ras, and MAPK signalling. Additionally, in vitro studies showed an increase in P-EXO miR-21-5p levels after TGF-β1 damage and oxidative stress. This novel study found an enrichment of fibromiRs and redoximiRs in P-EXO from hypertensive/diabetic patients with renal dysfunction. miR-21-5p, such as a RedoxifibromiR, has a significant accuracy for discriminating renal damage and is closely related with relevant signalling pathways implicated in fibrogenesis in podocytes.

Study on mechanism of Spatholobi Caulis in the treatment of the hand-foot skin reaction induced by targeted drug therapy based on network pharmacology and molecular docking: An observational study.

Based on network pharmacology and molecular docking methods, this study explored its active compounds and confirmed its potential mechanism of action against Hand-foot skin reaction induced by tumor-targeted drugs. Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform and UniProt Database were used to obtain the active ingredients and target proteins of Spatholobi Caulis. All hand-foot skin reaction (HFSR)-related targets were obtained with the help of the Human Gene Database, Online Mendelian Inheritance in Humans (OMIM), DisGeNET and DrugBank databases. Cytoscape 3.7.1 software was used to construct the active ingredient-target visualization network of Spatholobi Caulis, and the common target network of Spatholobi Caulis and HFSR. And through the BisoGenet plug-in in Cytoscape, PPI network topology analysis was performed. The Metescape database and online mapping tool platform were used for Gene Ontology (GO) function and Kyoto Encyclopedia of Genes Genomes (KEGG) pathway enrichment analysis to identify the key pathways of Spatholobi Caulis. Finally, Autodock Vina software and PyMol 2.5 software were used for molecular docking verification. There were 24 active components of Spatholobi Caulis and 244 target genes, 1635 disease-related target genes, 51 common target genes of Spatholobi Caulis and Hand-foot skin reaction, and key targets included NTRK1, epidermal growth factor receptor (EGFR), APP, TP53, HSP90AB1, HSP90AA, CUL3, etc. GO functional analysis involved a total of 66 molecular functions, 39 cellular components, and 817 biological processes. KEGG pathway analysis found 154 related signaling pathways, mainly enriched in Pathways in cancer, Human cytomegalovirus infection, Kaposi arcoma-associated herpesvirus infection, panic cancer, EGFR tyrosine kinase inhibitor resistance, P13K-Akt signaling pathway, Proteoglycans in cancer, HIF-1 signaling pathway and Ras signaling pathway, etc. Molecular docking results showed that luteolin, the active component of Spatholobi Caulis, had a high affinity with EGFR. Medicagol, the active components of Spatholobi Caulis, is proved in the Hand-foot skin reaction induced by lung cancer targeted therapy by regulating multiple signaling pathways through EGFR. It is confirmed that the treatment of Hand-foot skin reaction has the characteristics of multi-component, multi-target and multi-pathway regulation.

Network pharmacology and molecular docking approach to explore the potential mechanisms of Xuefu Zhuyu Capsule in coronary heart disease

Xuefu Zhuyu Capsule (XFZYC) plays a pivotal role in treating coronary heart disease (CHD) because of its potent clinical effects and fewer side effects. However, the possible pharmacological effect on CHD is limited. Thus, this research systematically analyzes XFZYC and CHD through network pharmacology technology. We identified 139 active compounds and 127 overlapped target genes in 11 herbs of XFZYC by using network pharmacology, and these 127 genes regulated the major signaling pathways related to CHD. The analysis of protein–protein interaction networks demonstrated that 30 important gene targets, such as interleukin-6, signal transducer and activator of transcription 3, protein kinase B1, mitogen activated protein kinases 3, cellular tumor antigen p53, vascular endothelial growth factor A, transcription factor p65, and proto-oncogene c-Fos, participated in the regulation of 79 major signaling pathways related to CHD. On this basis, we found that protein kinase B1, cellular tumor antigen p53, and transcription factor p65, which played a role in multiple regulatory pathways of the network, were also regulated by more than 3 compounds and expressed in at least 4 herbs. Molecular docking showed that XFZYC had a good binding potential with the overlapped protein targets. Gene Ontology enrichment analysis revealed that the active targets involved a various of biological process, cellular component, and molecular function, which included the key ones such as positive regulation of transcription from RNA polymerase II promoter, plasma membrane, and protein binding. T cell receptor signaling pathway, programmed death-ligand 1 expression and programmed death cell receptor-1 checkpoint pathway in cancer, FOXO signaling pathway, Ras signaling pathway, interleukin-17 signaling pathway, and VEGF signaling pathway, which were selected from Kyoto Encyclopedia of Genes and Genomes, were closely related to XFZYC in the treatment of CHD. XFZYC has a potential pharmacological effect on CHD, which provides the value for further study of XFZYC’s therapeutic effect on CHD.

Roles of WDR12 and MRTO4 genes in colorectal cancer

Colorectal cancer refers to malignant tumors occurring in the walls of the colon or rectum. The roles of WD Repeat Domain 12 (WDR12) and mitochondrial ribosome-associated tumor suppressor 4 (MRTO4) genes in colorectal cancer remain unclear. The colorectal cancer dataset GSE113513 configuration file was downloaded from the gene expression omnibus database generated from GPL15207. Differentially expressed genes screening, functional enrichment analysis, gene set enrichment analysis, Weighted Gene Co-expression Network Analysis, construction and analysis of protein–protein interaction networks, survival analysis, and gene expression heatmap plotting were conducted. Comparative toxicogenomics database analysis was performed to find diseases most relevant to core genes. TargetScan was used to screen miRNAs regulating core genes. A total of 3106 differentially expressed genes were identified. According to gene ontology analysis, they mainly enriched in organic acid metabolic processes, condensed chromosome kinetochore, oxidoreductase activity, and cell cycle. In Kyoto encyclopedia of genes and genomes analysis, they primarily concentrated in the cell cycle, TGF-β signaling pathway, Jak-STAT signaling pathway, PI3K-Akt signaling pathway, Ras signaling pathway, TNF signaling pathway, p53 signaling pathway, NF-kB signaling pathway, and WNT signaling pathway. Weighted Gene Co-expression Network Analysis with a soft thresholding power set to 12 generated 29 modules. The protein–protein interaction network identified 6 core genes (DDX27, NAT10, WDR12, DKC1, MRTO4, and NOP56). Survival analysis showed core genes (POSTN, MYH11, LUM, COL6A3, and COL4A1) as risk factors. Gene expression heatmap revealed high expression of core genes (WDR12 and MRTO4) in colorectal samples. Comparative toxicogenomics database analysis linked core genes (WDR12 and MRTO4) with local tumor infiltration, bowel obstruction, abdominal pain, and colorectal neoplasms. WDR12 and MRTO4 genes are highly expressed in colorectal cancer, potentially influencing its progression.

Syngap1 and the development of murine neocortical progenitor cells.

SYNGAP1 is a major regulator of synaptic plasticity through its interaction with synaptic scaffold proteins and modulation of Ras and Rap GTPase signaling pathways. SYNGAP1 mutations in humans are often associated with intellectual disability, epilepsy, and autism spectrum disorder. Syngap1 heterozygous loss-of-function results in impaired LTP, premature maturation of dendritic spines, learning disabilities and seizures in mice. More recently, SYNGAP1 was shown to influence cortical neurogenesis and the proliferation of progenitors in human organoids. Here, we show that the absence or haploinsufficiency of Syngap1 does not influence the properties of neocortical progenitors and their cellular output in mice. This discrepancy highlights potential species-specific or methodological differences and raises important questions about the broader applicability of SYNGAP1's role in neurogenesis.

Comprehensive analysis of selenoprotein gene expression and prognostic value in ovarian cancer.

To comprehensively analyze the expression and prognostic value of selenoprotein in ovarian cancer (OV). GEPIA and cBioPortal were used to analyze selenoprotein expression and mutations and copy number variations. Kaplan-Meier plotter and the tumor immune estimation resource were used to evaluate the impact of these genes on clinical prognosis and their correlation with tumor immune infiltration. Compared with normal tissues, the expression of iodothyronine deiodinase 3 (DIO3), glutathione peroxidase 4, SECISBP2, SELM, and SELP was decreased in the four gynecological malignancies. In OV, selenoprotein had the highest number of mutations (309) and mutation frequency (52.91%), whereas the lowest was observed in endometrial cancer (29.72%). DIO3, selenoprotein O (SELO), and selenoprotein T (SELT) are significantly related to the prognosis of OV. Immune infiltration analysis showed that DIO3 was associated with tumor-associated macrophages, SELO with CD4+ T-cells and monocytes, and SELT with T-cells. Enrichment analysis revealed that DIO3 is mainly involved in inflammatory immune responses and the Ras signaling pathway, SELO is primarily related to innate immune responses, and SELT is closely associated with mitochondrial oxidative phosphorylation. This study explored the expression characteristics of 25 selenoprotein in patients with gynecological malignancies and found that DIO3, SELO, and SELT were significantly associated with the prognosis and clinical features of OV, which are potential therapeutic targets.

Identification of Transcriptional Regulators and Signaling Pathways Mediating Postnatal Rumen Growth and Functional Maturation in Cattle.

The rumen plays an essential role in the physiology and health of ruminants. The rumen undergoes substantial changes in size and function from birth to adulthood. The cellular and molecular mechanisms underlying these changes are not clear. This study was aimed to identify the transcription factors and signaling pathways mediating these changes in cattle. We found that the ratios of the emptied rumen, reticulum, omasum, and abomasum to body weight in adult steers were 4.8 (P < 0.01), 3.1 (P < 0.01), 6.0 (P < 0.01), and 0.8 (P = 0.9) times those in neonatal calves, respectively. The length of rumen papillae and the thickness of rumen epithelium, tunica mucosa and submucosa, tunica muscularis, and tunica serosa increased 7.4-, 2.0-, 3.0-, 2.9-, and 4.6-fold (P < 0.01 for all), respectively, from neonatal calves to adult steers. However, the density of rumen papillae was lower in adult steers than in neonatal calves (P < 0.05). The size of rumen epithelial cells was not different between neonatal calves and adult steers (P = 0.57). RNA sequencing identified 2,922 genes differentially expressed in the rumen between neonatal calves and adult steers. Functional enrichment analyses revealed that organ development, blood vessel development, Ras signaling, and Wnt signaling were among the functional terms enriched in genes downregulated in adult steers vs. neonatal calves and that fatty acid metabolism, immune responses, PPAR signaling, and Rap1 signaling were among those enriched in genes upregulated in adult steers vs. neonatal calves. Serum response factor (SRF), interferon regulatory factor 4, and purine-rich single-stranded DNA-binding protein alpha were among the major candidate transcription factors controlling the expression of genes upregulated, while TCF4, inhibitor of DNA binding 4, and snail family transcriptional repressor 2 were among those controlling the expression of genes downregulated in adult steers vs. neonatal calves. Taken together, these results suggest that the rumen grows by increasing the number, not the size, of cells from birth to adulthood, that the absorptive, metabolic, immune, and motility functions of the rumen are acquired or significantly enhanced during the postnatal life, and that the changes in rumen size and function from birth to adulthood are mediated by many candidate transcription factors, including SRF and TCF4, and many candidate signaling pathways, including the PPAR and Wnt signaling pathways.

Discovery and validation of Hsa-microRNA-3665 promoter methylation as a potential biomarker for the prognosis of esophageal squaous cell carcinoma.

Methylation of microRNA (miRNA) promoters associated with diseases is a common epigenetic mechanism in the development of various human cancers. However, its relationship with prognosis in esophageal squamous cell carcinoma (ESCC) remains unclear. This study aims to explore the association between the methylation level of has-miR-3665 promoter and prognosis in ESCC. Human miRNA data were downloaded from miRbase, and we identified CpG islands of these human miRNAs by genomics browser analysis. MiRNA methylation levels were detected by methylation-specific high-resolution melting. Gene ontology (GO), and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were used to explore the molecular mechanism of hsa-miR-3665. Cox regression analysis was used to investigate prognostic factors. The overall survival rate was predicted by a nomogram. We found that 88 human miRNAs had promoter methylatio, of which 15 miRNAs were found to be epigenetically regulated in ESCC cells compared with their normal counterparts, including hsa-miR-3665. Meanwhile, hsa-miR-3665 expression was significantly lower in ESCC tumour tissue than in adjacent tissue (P = 0.03). GO and KEGG analyses demonstrated that the target genes are involved in protein transport, transcription regulator activity, MAPK and RAS signaling pathway. High hsa-miR-3665 promoter methylation levels were associated with a poor prognosis (HR = 3.89, 95% CI 1.11 ~ 13.55). Moreover, a nomogram incorporating the hsa-miR-3665 methylation level and clinical factors presented a good performance for predicting survival in the training and validation tests, with C-indices of 0.748 and 0.751, respectively. High hsa-miR-3665 promoter methylation levels may be a potential biomarker for the progression of ESCC.

Bile molecular landscape provides pathological insight and classifies signatures predictive of carcinoma of gall bladder.

Carcinoma of gall bladder (CAGB) has poor prognosis. Molecular analysis of bile could classify indicators of CAGB. Bile samples (n=87; training cohort) were screened for proteomics and metabolomics signatures of cancer detection. In bile, CAGB showed distinct proteomic (217up-, 258 downregulated) and metabolomic phenotype (111 up-, 505 downregulated, p<0.05, FC>1.5, FDR<0.01) linked to significantly increased inflammation (coagulation, arachidonic acid, bile acid) and alternate energy pathways (pentose-phosphate pathway, amino-acid, lipid metabolism) and decreased glycolysis, cholesterol metabolism, PPAR, RAS and RAP1 signaling, oxidative phosphorylation, and others compared to GS or HC (p<0.05). Bile proteins/metabolites signatures showed significant correlation (r2>0.5, p<0.05) with clinical parameters. Metabolite/protein signature-based probability of detection for CAGB (cancer) was >90% (p<0.05) with AUC>0.94. Validation of top four metabolites: Toluene, 5,6-DHET, Creatine, and Phenylacetaldehyde in separate cohorts (n=80; bile [T1] and paired plasma [T2]) showed accuracy (99%) and sensitivity/specificity (>98%) for CAGB detection. Tissue validation showed bile 5,6 DHET positively correlated with tissue PCNA (proliferation), and Caspase-3 linked to cancer development (r2>0.5, p<0.05). In conclusion, the bile molecular landscape provides critical molecular understanding and outlines metabolomic indicator panels for early CAGB detection.

Clinical and Preclinical Activity of EGFR Tyrosine Kinase Inhibitors in Non-Small-Cell Lung Cancer Harboring BRAF Class 3 Mutations.

Patients with tumors harboring BRAF class 3 mutations lack targeted therapies. These mutations are characterized by low/absent BRAF kinase domain activation and are believed to amplify already active RAS signaling, potentially triggered by receptor tyrosine kinases like EGFR. Two patients with BRAF class 3-mutated metastatic non-small-cell lung cancer (NSCLC) were treated with erlotinib at our Institution after failure of standard therapies. Two cell lines were established from patients with BRAF class 3-mutated NSCLC, and their sensitivity to EGFR tyrosine kinase inhibitors (EGFR-TKIs) was assessed using EGFR-mutated, BRAF class 1 and 2-mutated, and KRAS-mutated NSCLC cell lines as controls. Patient 1, a 60-year-old male with BRAFD594N-mutated NSCLC, achieved complete response to erlotinib after progression on first- and second-line chemotherapy. Patient 2, a 60-year-old female with BRAFD594G-mutated NSCLC, achieved partial response to erlotinib after progression on first-line chemoimmunotherapy. High baseline phosphorylated EGFR values and reduced EGFR activation following erlotinib were observed in BRAF class 3-mutated and EGFR-mutated cell lines, but not in BRAF class 1-mutated, BRAF class 2-mutated, or KRAS-mutated lines. Erlotinib inhibited 2-dimensional growth in BRAF class 3-mutated cell lines (IC50 6.33 and 7.11 µM) and in the BRAF class 2-mutated cell line (IC50 5.51 µM), albeit at higher concentrations than in EGFR-mutated lines, whereas it showed no effect on BRAF class 1-mutated (IC50, >25 µM) or KRAS-mutated (IC50, >25 µM) lines. These findings were corroborated by 3-dimensional and sphere formation assays. In the Cancer Cell Line Encyclopedia, BRAF class 3-mutated NSCLC cell lines showed greater sensitivity to EGFR-TKIs compared with BRAF class 2-mutated and KRAS-mutated lines. BRAF class 3 mutations in NSCLC may identify a novel targetable population sensitive to EGFR-TKIs.

The efficacy of plasma exosomal miRNAs as predictive biomarkers for PD-1 blockade plus chemotherapy in gastric cancer.

The response of gastric cancer (GC) patients to first-line programmed cell death 1 (PD-1) blockade and S-1 plus oxaliplatin (SOX) chemotherapy varies considerably, and the underlying mechanisms driving this variability remain elusive. Exosomal microRNAs (miRNAs or miRs) have emerged as potential biomarkers for efficacy prediction due to their roles in GC biology and stable expression in serum. In this study, we aimed to identify biomarkers to predict patients' response to anti-PD-1 therapy and further elucidate the potential mechanisms by which these exosomal miRNAs modulate the immune response in GC. Serum exosomes were extracted from 11 GC patients (five in the primary cohort and six in the validation cohort) treated with SOX and camrelizumab (a PD-1 inhibitor). High-throughput sequencing was performed to identify miRNA expression profiles, after which hierarchical clustering and a differential expression analysis were conducted. Functional enrichment analyses of the target genes for the significantly upregulated miRNAs were performed using the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases. The validation of the candidate miRNAs was carried out by quantitative polymerase chain reaction (qPCR) in an independent cohort. MiRNA sequencing identified 3,083 miRNAs, of which 74 (42 upregulated and 32 downregulated) were differentially expressed between the responders and non-responders. The GO and KEGG pathway analyses of the top 20 upregulated miRNAs indicated that the target genes were significantly involved in transcription regulation, cytoplasmic processes, and protein binding, and that key pathways included the PI3K-AKT, MAPK, RAP1, and RAS signaling pathways. Consistent with the sequencing findings, the qPCR validation results showed significant differences in the expression levels of miRNA451a and miRNA142-5p between the responders and non-responders. This study identified specific plasma exosomal miRNAs in GC patients that differ between responders and non-responders to PD-1 monoclonal antibody therapy combined with chemotherapy. These miRNAs could serve as predictive biomarkers, paving the way for precision medicine and personalized therapy in the treatment of GC.

Differential analysis of microRNAs in plasma exosomes in patients with cerebral ischemic stroke.

The high incidence, disability, mortality, and recurrence rates of cerebral infarction impose a heavy burden on both the Chinese and global populations. It is essential for the early diagnosis, prevention, and protection against brain cell injury. To identify differentially expressed microRNAs (miRNAs) in plasma exosomes of patients with cerebral ischemic stroke, determine relevant biomarkers, and explore their potential signaling pathways. High-throughput sequencing was used to detect the expression of plasma exosomal miRNAs in patients with cerebral ischemic stroke and in a control group. Gene Ontology, Kyoto Encyclopedia of Genes and Genomes enrichment, and target gene network analyses were performed to investigate the target genes and signaling pathways of the differentially expressed miRNAs. The sequencing results identified 95 differentially expressed miRNAs, with 40 upregulated and 55 downregulated miRNAs. Among these, hsa-miR-1303, hsa-miR-125b-1-3p, and hsa-miR-548ab were significantly upregulated in the stroke group and downregulated in the normal control group, whereas hsa-miR-1289 was downregulated in the stroke group and upregulated in the normal group. Gene Ontology, Kyoto Encyclopedia of Genes, and genomes enrichment analyses indicated that the differentially expressed miRNAs and their target genes were mainly concentrated in the PI3K-AKt, mitogen-activated protein kinase, calcium, Ras, Rap1, and cAMP signaling pathways. The expression of plasma exosomal hsa-miR-1303, hsa-miR-125b-1-3p, and hsa-miR-1289 was significantly different in stroke patients than in the control group. These miRNAs may be involved in various signaling pathways related to cerebral infarction, providing a reference for further experimental research.

Molecular characteristics and prognostic role of EPHA2 in human tumors via pan-cancer analysis.

The tyrosine kinase ephrin type-A receptor 2 (EPHA2) was remarkably elevated expressed in various tumors and plays a crucial role in cancer tumorigenesis and progression, while pan-cancer analyses are currently lacking. This study was designed to analyze the expression status and prognostic significance of EPHA2 in pan-cancer. By mining The Cancer Genome Atlas data, we performed a comprehensive and systematic characterization of EPHA2 across >10,000 samples of 33 types of cancer. EPHA2 expressions were substantially different in most of the normal control and tumor tissues, and it was considerably associated with the prognosis of tumor patients. EPHA2 gene modifications in malignant tumors were mainly missense mutations. There was a significant correlation between EPHA2 expression and cancer-associated fibroblasts in most The Cancer Genome Atlas cancers. Furthermore, functional enrichment analysis showed that the biological role of EPHA2 in tumors was mainly involved in some noticeably pro-oncogenic pathways, such as the Ras signaling pathway, PI3K-Akt signaling pathway, ErbB signaling pathway, MAPK signaling pathway, etc. This study provided the first pan-cancer analyses of EPHA2 in various tumors, and EPHA2 was potentially involved in many cancer types and can be developed as candidates for cancer diagnosis, prognosis, and therapeutic biomarkers. In addition, EPHA2 seemed to be a key modulator of the tumor immune microenvironment and might be a potential biomarker in predicting the immunotherapeutic efficacy for cancer patients.

Comprehensive analysis identifies endocrine fibroblast growth factors as promising prognostic markers for colorectal carcinoma

Endocrine fibroblast growth factors (eFGFs) play essential roles in cellular signaling processes, including development and differentiation, and are implicated in various cancers. However, their precise involvement in colon neoplasia and colon adenocarcinoma (COAD) remains incompletely understood. Here, we conducted a comprehensive investigation utilizing multiple databases to explore the multifaceted characteristics of eFGFs. Through integrated analyses of diverse databases, including TIMER2.0, UALCAN, OncoDB, cBioPortal, LinkedOmics, STRING, htfTarget, mirTarBase, circBank, and DGIdb, we explored eFGFs’ gene expression, DNA methylation, prognostic significance, genetic alterations, gene regulatory networks, functional analysis, and drug interactions in COAD patients. Our findings revealed elevated expression levels of eFGFs in COAD, with aberrant gene expression potentially linked to promoter methylation. Importantly, hypermethylation of FGF21 and FGF23 and downregulation of FGF23 correlated with poor survival outcomes in COAD patients. Functional analyses highlighted the involvement of eFGF genes in Ras signaling, PI3K-Akt signaling, and cancer pathways. Furthermore, we validated our findings through a cross-sectional study by quantitative real-time polymerase chain reaction (qRT-PCR), confirming significant overexpression of FGF21 in colon polyps compared to normal mucosa. Additionally, we observed elevated RNA expression of FGF21 and FGF23 in adenomatous polyps compared to hyperplastic polyps. This study sheds new light on the critical roles of eFGFs in COAD tumorigenesis and underscores their potential as promising prognostic markers for COAD, as well as discriminative markers for distinguishing high-risk from low-risk polyps. These findings provide valuable insights into the complex molecular mechanisms underlying colorectal neoplasia and offer potential avenues for targeted therapeutic strategies.

RAS signaling pathway is essential in regulating PIEZO1-mediated hepatic iron overload in dehydrated hereditary stomatocytosis.

PIEZO1 encodes a mechanoreceptor, a cation channel activated by mechanical stimuli. Gain-of-function (GoF) variants in PIEZO1 cause dehydrated hereditary stomatocytosis (DHS), or xerocytosis, a pleiotropic syndrome characterized by anemia and iron overload. DHS patients develop hepatic iron overload independent of the degree of anemia and transfusion regimen. PIEZO1-GoF variants suppress hepcidin expression in both hepatic cellular model and constitutive/macrophage-specific Piezo1-GoF mice model. Therefore, PIEZO1-GoF variants regulate hepcidin expression by a crosstalk between hepatocytes (HCs) and macrophages with a still unknown mechanism. Transcriptomic and proteomics analysis in the human hepatic Hep3B cells engineered for the PIEZO1-R2456H variant (PIEZO1-KI) revealed alterations in the actin cytoskeleton regulation, MAPK cascade, and RAS signaling. These changes mainly occur through a novel key regulator, RRAS, whose protein and mRNA levels are regulated by PIEZO1 activation and inhibition. This regulation was further confirmed in C57BL/6 mouse primary HCs treated with Yoda-1 and/or GsMTx-4. Indeed, PIEZO1-KI cells exhibited hyper-activated RAS-GTPase activity that is rescued by PIEZO1 inhibition, restoring expression of the hepcidin gene HAMP. A negative correlation between RAS signaling and HAMP regulation was confirmed by inhibiting RAS-GTPase and MEK1-2 activity. Conversely, rescued HAMP gene expression requires downregulation of RRAS, confirming negative feedback between RAS-MAPK and BMP/SMADs pathways in HAMP regulation. We demonstrated that PIEZO1-GoF variants influence the actin cytoskeleton organization by activating the hepatic RAS signaling system. Understanding the role of RAS signaling in regulating iron metabolism could pave the way for new therapeutic strategies in DHS and other conditions characterized by iron overload.

Abstract A041: Deciphering the crosstalk between cancer cells and stroma that drives EMT- coupled fibrosis

Abstract In the tumor microenvironment (TME) cancer cells engage dynamically with the stromal component in promoting tumor progression and metastasis formation that are accompanied by intratumor fibrosis. Extensive extracellular matrix (ECM) remodeling and stiffness boost fibrosis which co-occurs with poor immune infiltration. Very little is known about the cross-interactions between tumor cells and stroma. A better understanding of this dynamic relationship may advance the development of new therapies. Transforming growth factor (TGF)-β signaling in both cancer epithelial cells and stroma is crucial for the establishment of intratumor fibrosis that enhances tumor progression. We previously discovered that in cancer cells, TGF-β signaling synergizes with RAS signal to regulate classical EMT transcription factors (Snail, Zeb1) and fibrogenic factors (Il11, Pdgf, Ctgfb, Wisp and others). This synergistic program is required for metastasis formation in pancreatic and lung adenocarcinoma (LUAD) models. However, how this dual program is affecting the stromal population in vivo remains unknown. Additionally, the role of each TGF-β dependent fibrogenic factor in regulating the stroma is still unclear. Using metastasis in vivo assays, we tested the metastatic ability of LUAD cells wild-type in comparison to Knock-Out cells targeting each one of the TGF-β dependent fibrogenic factors. We found that these factors are drivers of metastasis. More interestingly, these fibrogenic factors have a preferential effect depending on the metastatic site, suggesting that survival of the cancer cells in a certain tissue is dependent on the stromal niche within the site, which will act in favor or not of their growth. We also deeply characterize the stroma of micro-metastatic lesions by using high-throughput multiplex immunofluorescence to analyze the whole tissue TME at single cell resolution and found that lack of any of these fibrogenic factors compromise the establishment of a robust ECM at the very beginning of the metastatic colonization, to favor immune infiltration and consequently lead to cancer cell clearance. Together, these findings shed light on the potential role of these specific fibrogenic factors in metastasis, how their absence impacts the TME and how targeting these factors could be beneficial in metastatic disease. Citation Format: Elena Spina, Liangji Li, Junho Lee, Joan Massaguè. Deciphering the crosstalk between cancer cells and stroma that drives EMT- coupled fibrosis [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr A041.

Abstract PR016: Dynamic chromatin landscapes of colorectal cancer development and cell-free DNA fragmentation

Abstract The stepwise progression of colorectal cancer (CRC) from healthy epithelium, to premalignant adenoma, to cancer is accompanied by genome-wide epigenetic reprogramming which may be reflected in circulating cell-free DNA (cfDNA). However, current analyses of these processes have been hampered by complex mixtures of cells in normal, adenoma and cancer tissue samples, as well as absence of suitable adenoma model systems. Cultured colorectal organoids, comprised of pure epithelial cells, could enable insights on the epigenetic changes leading to progression from healthy tissue to cancer. Such features could be instrumental in the early cancer detection using genome-wide analyses of cfDNA fragmentation. Using 43 patient-derived colorectal organoids derived from healthy, adenoma, and cancer tissues, as well as 10 freshly collected peripheral blood mononuclear cell (PBMC) control samples, we examined the chromatin landscape of tumor progression using transposase accessible chromatin analyses with next-generation sequencing. We defined a consensus set of nucleosome depleted regions for each disease state and identified loci with differential accessibility between PBMCs, colon healthy, colon adenoma, and colon cancer tissues. We analyzed these differential loci for fragmentation characteristics in the circulating cfDNA of 250 healthy individuals and 51 patients with metastatic CRC. Across all organoid samples, we identified &amp;gt;35 million nucleosome depleted regions that we coalesced into a consensus set of 62,708 regions. As 78% of these regions could be linked to genes, we assessed whether these signatures identify known pathways involved in colorectal cancer. Within these regions, we identified unique colon tumor-related signatures comprising genes of known and novel pathways, including those involved in WNT, hippo, and RAS signaling. While the majority of chromatin accessibility differences were shared between adenomas and cancers, we identified 895 chromatin changes in adenomas that were not present in either healthy or malignant tissues. In healthy individuals, we found that the cfDNA coverage was negatively correlated with accessibility of white blood cell accessibility loci (Spearman ρ= - 0.60) while in individuals with cancer, changes in chromatin accessibility were associated with independent circulating tumor DNA abundance by droplet digital PCR (R2=0.69, p&amp;lt;0.001). Chromatin accessibility profiles were correlated with genome-wide cell-free DNA fragmentation features and could be used to distinguish healthy individuals from those with colorectal cancer (AUC=0.97). These analyses are consistent with dynamic chromatin remodeling during colorectal tumor development and may provide an avenue for evaluating these through genome-wide analyses of cfDNA fragmentation. Citation Format: Nicholas A. Vulpescu, Zachariah H. Foda, Pieter H.A. Wisse, Christopher Cherry, Jaime E. Medina, Vilmos Adleff, Remond J.A. Fijneman, Robert B. Scharpf, Gerrit A. Meijer, Beatriz Carvalho, Victor E. Velculescu. Dynamic chromatin landscapes of colorectal cancer development and cell-free DNA fragmentation [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr PR016.

Identification and validation of miR-21 key genes in cervical cancer through an integrated bioinformatics approach

Cervical cancer is one of the most prevalent female reproductive cancers. miR-21 is a multi-target oncomiR that has shown its potential in regulating several cancers including colon, pancreatic, breast, prostate, ovarian, and cervical cancer. However, the signaling network of miR-21 remains underexplored, and only a limited number of miR-21 gene targets in cervical cancer have been reported. In this context, the present study was undertaken to evaluate the role of miR-21 in cervical cancer by combining in silico analysis with in vitro validation in cervical cancer cells. The miR-21 target genes were predicted using four different prediction tools: miRWalk, DIANA, miRDB, and TargetScan. A total of 113 overlapping target genes, common in at least three of the prediction tools, were shortlisted and subjected to functional enrichment analysis. The analysis predicted that JAK-STAT, MAPK, neurotrophin, and Ras signaling pathways are significantly (p≤0.05) targeted by miR-21. The MCODE plugin identified the potential cluster in the protein-protein interaction network based on the highest degree of connectivity. After GEPIA2 validation of all 20 hub genes, NTF3, LIFR, and IL-6R were shortlisted for validation in cervical cancer cell lines. The results showed that NTF3, LIFR, and IL-6R were significantly upregulated in the miR-21 knockdown CaSki cell lines in 6.27, 1.92 and 1.71 folds (p≤0.01), respectively. Similarly, in HeLa cell lines expression of NTF3, LIFR, and IL-6R were overexpressed in 4.06, 5.65, 2.42 folds (p≤0.001), respectively. Findings of the study was confirming the role of miR-21 in regulating the expression of these genes. Additionally, the knockdown of miR-21 significantly inhibited the secretion of matrix metalloproteinases by CaSki cells. These results highlight that miR-21 could be a potential therapeutic target for cervical cancer, although further preclinical and clinical studies are required to validate its role and efficacy.

The pro-oncogenic noncanonical activity of a RAS•GTP:RanGAP1 complex facilitates nuclear protein export

Canonical RAS signaling, including PI3K/AKT- and RAF/MEK-dependent activities, results mainly from RAS•GTP interaction with its effectors at the plasma membrane. Here, we identified a fundamental, oncogenic, noncanonical RAS•GTP activity that increases XPO1-dependent export of nuclear protein cargo into the cytoplasm and is independent of PI3K/AKT and RAF/MEK signaling. This RAS-dependent step acts downstream from XPO1 binding to nuclear protein cargo and is mediated by a perinuclear protein complex between RAS•GTP and RanGAP1 that facilitates hydrolysis of Ran•GTP to Ran•GDP, which promotes release of nuclear protein cargo into the cytoplasm. The export of nuclear EZH2, which promotes cytoplasmic degradation of the DLC1 tumor suppressor protein, is a biologically important component of this pro-oncogenic activity. Conversely, preventing nuclear protein export contributes to the antitumor activity of KRAS inhibition, which can be further augmented by reactivating the tumor suppressor activity of DLC1 or potentially combining RAS inhibitors with other cancer treatments.

CSIG-24. BRAIN MICROENVIRONMENT-DEPENDENT NEURODEVELOPMENTAL LINEAGE PLASTICITY PROMOTES ADAPTATION TO ONCOGENE INHIBITION IN MALIGNANT GLIOMAS

Abstract Phenotypic plasticity drives non-genetic tumor adaptation in response to various microenvironmental and therapeutic pressures, consequently promoting tumor heterogeneity and progression. In malignant gliomas, the intrinsic and extrinsic mechanisms underlying phenotypic lineage plasticity and its contribution to drug resistance remain unclear. The epidermal growth factor receptor (EGFR) is frequently altered in glioma and serves as a critical regulator of normal radial glia (RG) cells – multipotent progenitors that give rise to cortical neurons and glia in the developing brain. Here, through interrogation of a large panel of diverse glioblastoma (GBM) patient-derived gliomaspheres and orthotopic xenografts, we find that enrichment of RG-like cells is significantly correlated with responses to pharmacological ablation of EGFR, highlighting EGFR as a crucial lineage survival factor for a population of malignant RG-like cells in GBM. Single-cell RNA sequencing and quantitative proteomics reveal that adaptation to EGFR inhibition is linked to a temporal contraction in RG-like cells and concomitant increase in lineage-restricted neuronal and oligodendrocyte progenitor (NPC/OPC)-like states exclusively in the orthotopic brain environment. This lineage transition is coupled to heightened RAS signaling gene expression programs and sustained activation of MAPK signaling, despite robust and durable inhibition of EGFR activation. Furthermore, constitutively active MEK – but not AKT – is sufficient to rescue tumor proliferation under EGFRi, suggesting that the brain microenvironment modulates RAS-MAPK oncogenic signaling to drive lineage transitions following EGFR blockade. Collectively, these results highlight a critical link between oncogenic signaling and neurodevelopmental lineage plasticity in malignant gliomas, presenting a compelling therapeutic opportunity to block tumor cell state transitions for more durable tumor responses in the native glioma tumor microenvironment.