Estrogen-related receptor alpha promotes thyroid tumor cell survival via a tumor subtype-specific regulation of target gene networks.

Mortalin (encoded by HSPA9) is a mitochondrial chaperone often overexpressed in cancer through as-yet-unknown mechanisms. By searching different RNA-sequencing datasets, we found that ESRRA is a transcription factor highly correlated with HSPA9 in thyroid cancer, especially in follicular, but not C cell-originated, tumors. Consistent with this correlation, ESRRA depletion decreased mortalin expression only in follicular thyroid tumor cells. Further, ESRRA expression and activity were relatively high in thyroid tumors with oncocytic characteristics, wherein ESRRA and mortalin exhibited relatively high functional overlap. Mechanistically, ESRRA directly regulated HSPA9 transcription through a novel ESRRA-responsive element located upstream of the HSPA9 promoter. Physiologically, ESRRA depletion suppressed thyroid tumor cell survival via caspase-dependent apoptosis, which ectopic mortalin expression substantially abrogated. ESRRA depletion also effectively suppressed tumor growth and mortalin expression in the xenografts of oncocytic or ESRRA-overexpressing human thyroid tumor cells in mice. Notably, our Bioinformatics analyses of patient data revealed two ESRRA target gene clusters that contrast oncocytic-like and anaplastic features of follicular thyroid tumors. These findings suggest that ESRRA is a tumor-specific regulator of mortalin expression, the ESRRA-mortalin axis has higher significance in tumors with oncocytic characteristics, and ESRRA target gene networks can refine molecular classification of thyroid cancer.

Whole Genome Sequencing Analysis of Soybean Diversity Across Different Countries and Selection Signature of Korean Soybean Accession.

Soybean is an important agricultural crop known for its high protein and oil content, contributing to essential nutritional and health benefits for humans. Domesticated in China over 5000 years ago, soybean has since adapted to diverse environments and spread worldwide. This study aimed to investigate the genomic characteristics and population structures of 2,317 publicly available soybean whole-genome sequences from diverse geographical regions, including China, Korea, Japan, Europe, North America and South America. We used large-scale whole-genome sequencing data to perform high-resolution analyses to reveal the genetic characteristics of soybean accessions. Soybean accessions from China and Korea exhibited landrace characteristics, indicating higher genetic diversity and adaptation to local environments. On the other hand, soybean accessions from Japan, the European Union, and South America were found to have low genetic diversity due to artificial selection and breeding for agronomic traits. We also identified key variants and genes associated with the ability to adapt to different environments. In Korean soybean accessions, we observed strong selection signals for isoflavone synthesis, an adaptive trait critical for improving soybean adaptability, survival, and reproductive success by mitigating environmental stress. Identifying specific genomic regions showing unique patterns of selective sweeps for genes such as HIDH, CYP73A11, IFS1, and CYP81E11 associated with isoflavone synthesis provided valuable insights into potential adaptation mechanisms. Our research has significantly improved our understanding of soybean diversity at the genetic level. We have identified key genetic variants and genes influencing adaptability, laying the foundation for future advances in genomics-based breeding programs and crop improvement efforts.

CILP2 is a potential biomarker for the prediction and therapeutic target of peritoneal metastases in colorectal cancer.

Peritoneal metastases (PM) in colorectal cancer (CRC) is associated with a dismal prognosis. Identifying and exploiting new biomarkers, signatures, and molecular targets for personalised interventions in the treatment of PM in CRC is imperative. We conducted transcriptomic profiling using RNA-seq data generated from the primary tissues of 19 CRC patients with PM. Using our dataset established in a previous study, we identified 1422 differentially expressed genes compared to non-metastatic CRC. The profiling demonstrated no differential expression in liver and lung metastatic CRC. We selected 12 genes based on stringent criteria and evaluated their expression patterns in a validation cohort of 32 PM patients and 84 without PM using real-time reverse transcription-polymerase chain reaction. We selected cartilage intermediate layer protein 2 (CILP2) because of high mRNA expression in PM patients in our validation cohort and its association with a poor prognosis in The Cancer Genome Atlas. Kaplan-Meier survival analysis in our validation cohort demonstrated that CRC patients with high CILP2 expression had significantly poor survival outcomes. Knockdown of CILP2 significantly reduced the proliferation, colony-forming ability, invasiveness, and migratory capacity and downregulated the expression of molecules related to epithelial-mesenchymal transition in HCT116 cells. In an in vivo peritoneal dissemination mouse knockdown of CILP2 also inhibited CRC growth. Therefore, CILP2 is a promising biomarker for the prediction and treatment of PM in CRC.

Neutrophil diversity is associated with T-cell immunity and clinical relevance in patients with thyroid cancer.

Neutrophil heterogeneity is involved in autoimmune diseases, sepsis, and several cancers. However, the link between neutrophil heterogeneity and T-cell immunity in thyroid cancer is incompletely understood. We investigated the circulating neutrophil heterogeneity in 3 undifferentiated thyroid cancer (UTC), 14 differentiated thyroid cancer (DTC) (4 Stage IV, 10 Stage I-II), and healthy controls (n = 10) by transcriptomic data and cytometry. Participants with UTC had a significantly higher proportion of immature high-density neutrophils (HDN) and lower proportion of mature HDN in peripheral blood compared to DTC. The proportion of circulating PD-L1+ immature neutrophils were significantly increased in advanced cancer patients. Unsupervised analysis of transcriptomics data from circulating HDN revealed downregulation of innate immune response and T-cell receptor signaling pathway in cancer patients. Moreover, UTC patients revealed the upregulation of glycolytic process and glutamate receptor signaling pathway. Comparative analysis across tumor types and stages revealed the downregulation of various T-cell-related pathways, such as T-cell receptor signaling pathway and T-cell proliferation in advanced cancer patients. Moreover, the proportions of CD8+ and CD4+ T effector memory CD45RA+ (TEMRA) cells from peripheral blood were significantly decreased in UTC patients compared to DTC patients. Finally, we demonstrated that proportions of tumor-infiltrated neutrophils were increased and related with poor prognosis in advanced thyroid cancer using data from our RNA-seq and TCGA (The Cancer Genome Atlas) data. In conclusion, observed prevalence of circulating immature high-density neutrophils and their immunosuppressive features in undifferentiated thyroid cancers underscore the importance of understanding neutrophil dynamics in the context of tumor progression in thyroid cancer.

Transcriptomic characteristics according to tumor size and SUVmax in papillary thyroid cancer patients.

The SUVmax is a measure of FDG uptake and is related with tumor aggressiveness in thyroid cancer, however, its association with molecular pathways is unclear. Here, we investigated the relationship between SUVmax and gene expression profiles in 80 papillary thyroid cancer (PTC) patients. We conducted an analysis of DEGs and enriched pathways in relation to SUVmax and tumor size. SUVmax showed a positive correlation with tumor size and correlated with glucose metabolic process. The genes that indicate thyroid differentiation, such as SLC5A5 and TPO, were negatively correlated with SUVmax. Unsupervised analysis revealed that SUVmax positively correlated with DNA replication(r = 0.29, p = 0.009), pyrimidine metabolism(r = 0.50, p < 0.0001) and purine metabolism (r = 0.42, p = 0.0001). Based on subgroups analysis, we identified that PSG5, TFF3, SOX2, SL5A5, SLC5A7, HOXD10, FER1L6, and IFNA1 genes were found to be significantly associated with tumor aggressiveness. Both high SUVmax PTMC and macro-PTC are enriched in pathways of DNA replication and cell cycle, however, gene sets for purine metabolic pathways are enriched only in high SUVmax macro-PTC but not in high SUVmax PTMC. Our findings demonstrate the molecular characteristics of high SUVmax tumor and metabolism involved in tumor growth in differentiated thyroid cancer.

Small RNA-modulated anaerobic respiration allows bacteria to survive under antibiotic stress conditions.

Despite extensive knowledge of antibiotic-targeted bacterial cell death, deeper understanding of antibiotic tolerance mechanisms is necessary to combat multi-drug resistance in the global healthcare settings. Regulatory RNAs in bacteria control important cellular processes such as cell division, cellular respiration, metabolism, and virulence. Here, we investigated how exposing Escherichia coli to the moderately effective first-generation antibiotic cephalothin alters transcriptional and post-transcriptional dynamics. Bacteria switched from active aerobic respiration to anaerobic adaptation via an FnrS and Tp2 small RNA-mediated post-transcriptional regulatory circuit. From the early hours of antibiotic exposure, FnrS was involved in regulating reactive oxygen species levels, and delayed oxygen consumption in bacteria. We demonstrated that bacteria strive to maintain cellular homeostasis via sRNA-mediated sudden respiratory changes upon sublethal antibiotic exposure.

Systematic identification of a synthetic lethal interaction in brain-metastatic lung adenocarcinoma.

Metastatic lung adenocarcinoma (LuAC) presents a significant clinical challenge due to the short latency and the lack of efficient treatment options. Therefore, identification of molecular vulnerabilities in metastatic LuAC holds great importance in the development of therapeutic drugs against this disease. In this study, we performed a genome-wide siRNA screening using poorly and highly brain-metastatic LuAC cell lines. Using this approach, we discovered that compared to poorly metastatic LuAC (LuAC-Par) cells, brain-metastatic LuAC (LuAC-BrM) cells exhibited a significantly higher vulnerability to c-FLIP (an inhibitor of caspase-8)-depletion-induced apoptosis. Furthermore, in vivo studies demonstrated that c-FLIP knockdown specifically inhibited growth of LuAC-BrM, but not the LuAC-Par, tumors, suggesting the addiction of LuAC-BrM to the function of c-FLIP for their survival. Our in vitro and in vivo analyses also demonstrated that LuAC-BrM is more sensitive to c-FLIP-depletion due to ER stress-induced activation of the c-JUN and subsequent induction of stress genes including ATF4 and DDIT3. Finally, we found that c-JUN not only sensitized LuAC-BrM to c-FLIP-depletion-induced cell death but also promoted brain metastasis in vivo, providing strong evidence for c-JUN's function as a double-edged sword in LuAC-BrM. Collectively, our findings not only reveal a novel link between c-JUN, brain metastasis, and c-FLIP addiction in LuAC-BrM but also present an opportunity for potential therapeutic intervention.

Unraveling the role of the mitochondrial one-carbon pathway in undifferentiated thyroid cancer by multi-omics analyses.

The role of the serine/glycine metabolic pathway (SGP) has recently been demonstrated in tumors; however, the pathological relevance of the SGP in thyroid cancer remains unexplored. Here, we perform metabolomic profiling of 17 tumor-normal pairs; bulk transcriptomics of 263 normal thyroid, 348 papillary, and 21 undifferentiated thyroid cancer samples; and single-cell transcriptomes from 15 cases, showing the impact of mitochondrial one-carbon metabolism in thyroid tumors. High expression of serine hydroxymethyltransferase-2 (SHMT2) and methylenetetrahydrofolate dehydrogenase 2 (MTHFD2) is associated with low thyroid differentiation scores and poor clinical features. A subpopulation of tumor cells with high mitochondrial one-carbon pathway activity is observed in the single-cell dataset. SHMT2 inhibition significantly compromises mitochondrial respiration and decreases cell proliferation and tumor size in vitro and in vivo. Collectively, our results highlight the importance of the mitochondrial one-carbon pathway in undifferentiated thyroid cancer and suggest that SHMT2 is a potent therapeutic target.

CYB5R3 functions as a tumor suppressor by inducing ER stress-mediated apoptosis in lung cancer cells via the PERK-ATF4 and IRE1α-JNK pathways.

Cytochrome b5 reductase 3 (CYB5R3) is involved in various cellular metabolic processes, including fatty acid synthesis and drug metabolism. However, the role of CYB5R3 in cancer development remains poorly understood. Here, we show that CYB5R3 expression is downregulated in human lung cancer cell lines and tissues. Adenoviral overexpression of CYB5R3 suppresses lung cancer cell growth in vitro and in vivo. However, CYB5R3 deficiency promotes tumorigenesis and metastasis in mouse models. Transcriptome analysis revealed that apoptosis- and endoplasmic reticulum (ER) stress-related genes are upregulated in CYB5R3-overexpressing lung cancer cells. Metabolomic analysis revealed that CYB5R3 overexpression increased the production of nicotinamide adenine dinucleotide (NAD+) and oxidized glutathione (GSSG). Ectopic CYB5R3 is mainly localized in the ER, where CYB5R3-dependent ER stress signaling is induced via activation of protein kinase RNA-like ER kinase (PERK) and inositol-requiring enzyme 1 alpha (IRE1α). Moreover, NAD+ activates poly (ADP-ribose) polymerase16 (PARP16), an ER-resident protein, to promote ADP-ribosylation of PERK and IRE1α and induce ER stress. In addition, CYB5R3 induces the generation of reactive oxygen species and caspase-9-dependent intrinsic cell death. Our findings highlight the importance of CYB5R3 as a tumor suppressor for the development of CYB5R3-based therapeutics for lung cancer.

Hypoxia stabilizes SETDB1 to maintain genome stability.

Von Hippel-Lindau (VHL) is a tumor suppressor that functions as the substrate recognition subunit of the CRL2VHL E3 complex. While substrates of VHL have been identified, its tumor suppressive role remains to be fully understood. For further determination of VHL substrates, we analyzed the physical interactome of VHL and identified the histone H3K9 methyltransferase SETBD1 as a novel target. SETDB1 undergoes oxygen-dependent hydroxylation by prolyl hydroxylase domain proteins and the CRL2VHL complex recognizes hydroxylated SETDB1 for ubiquitin-mediated degradation. Under hypoxic conditions, SETDB1 accumulates by escaping CRL2VHL activity. Loss of SETDB1 in hypoxia compared with that in normoxia escalates the production of transposable element-derived double-stranded RNAs, thereby hyperactivating the immune-inflammatory response. In addition, strong derepression of TEs in hypoxic cells lacking SETDB1 triggers DNA damage-induced death. Our collective results support a molecular mechanism of oxygen-dependent SETDB1 degradation by the CRL2VHL E3 complex and reveal a role of SETDB1 in genome stability under hypoxia.

Epigenetic regulation of SMAD3 by histone methyltransferase SMYD2 promotes lung cancer metastasis.

Epigenetic alterations, especially histone methylation, are key factors in cell migration and invasion in cancer metastasis. However, in lung cancer metastasis, the mechanism by which histone methylation regulates metastasis has not been fully elucidated. Here, we found that the histone methyltransferase SMYD2 is overexpressed in lung cancer and that knockdown of SMYD2 could reduce the rates of cell migration and invasion in lung cancer cell lines via direct downregulation of SMAD3 via SMYD2-mediated epigenetic regulation. Furthermore, using an in vitro epithelial-mesenchymal transition (EMT) system with a Transwell system, we generated highly invasive H1299 (In-H1299) cell lines and observed the suppression of metastatic features by SMYD2 knockdown. Finally, two types of in vivo studies revealed that the formation of metastatic tumors by shSMYD2 was significantly suppressed. Thus, we suggest that SMYD2 is a potential metastasis regulator and that the development of SMYD2-specific inhibitors may help to increase the efficacy of lung cancer treatment.

Single Cell Analysis of Human Thyroid Reveals the Transcriptional Signatures of Aging.

The thyroid gland plays a critical role in the maintenance of whole-body metabolism. However, aging frequently impairs homeostatic maintenance by thyroid hormones due to increased prevalence of subclinical hypothyroidism associated with mitochondrial dysfunction, inflammation, and fibrosis. To understand the specific aging-related changes of endocrine function in thyroid epithelial cells, we performed single-cell RNA sequencing (RNA-seq) of 54 726 cells derived from pathologically normal thyroid tissues from 7 patients who underwent thyroidectomy. Thyroid endocrine epithelial cells were clustered into 5 distinct subpopulations, and a subset of cells was found to be particularly vulnerable with aging, showing functional deterioration associated with the expression of metallothionein (MT) and major histocompatibility complex class II genes. We further validated that increased expression of MT family genes are highly correlated with thyroid gland aging in bulk RNAseq datasets. This study provides evidence that aging induces specific transcriptomic changes across multiple cell populations in the human thyroid gland.

The EDN1/EDNRA/ß­arrestin axis promotes colorectal cancer progression by regulating STAT3 phosphorylation.

Endothelin receptor A (EDNRA) has been reported to play various crucial physiological roles and has been shown to be associated with the pathology of several diseases, including colorectal cancer (CRC). However, the molecular mechanisms of EDNRA in the development of human CRC have not been fully elucidated to date. In this context, the present study was performed to investigate biological functions and novel downstream signaling pathways affected by EDNRA, during CRC progression. First, using public data repositories, it was observed that the EDRNA expression levels were markedly increased in CRC tissues, as compared to normal tissues. Patients with CRC with an increased EDNRA expression exhibited a significantly decreased survival rate in comparison with those with a lower EDNRA expression. Furthermore, a positive correlation between the levels of EDNRA and its ligand, EDN1, was found in CRC tissues. The ectopic expression of EDNRA or its ligand, EDN1, promoted, whereas the silencing of EDNRA or EDN1 decreased cell proliferation and migration in vitro. To elucidate the signaling pathways involved in the regulation of EDNRA expression in CRC cells, a phosphokinase array analysis was performed, and it was observed that the knockdown of EDNRA substantially suppressed the phosphorylation of signal transducer and activator of transcription 3 (STAT3) in CRC cells. Of note, STAT3 silencing simultaneously decreased EDN1 and EDNRA expression, with the expression of EDN1 and/or EDNRA appearing to be directly regulated by binding STAT3 to their promoter region, according to chromatin immunoprecipitation and promoter assays, ultimately indicating a positive feedback loop in the expression of EDNRA and EDN1. It was also observed that treatment with an EDNRA antagonist (macitentan), alone or in combination with cisplatin, suppressed cell growth and migration ability, and induced cell apoptosis. Collectively, these data suggest a critical role of the EDN1/EDNRA signaling pathway in CRC progression. Thus, the pharmacological intervention of this signaling pathway may prove to be a potential therapeutic approach for patients with CRC.

Utility of a Molecular Signature for Predicting Recurrence and Progression in Non-Muscle-Invasive Bladder Cancer Patients: Comparison with the EORTC, CUETO and 2021 EAU Risk Groups.

To evaluate the utility of different risk assessments in non-muscle-invasive bladder cancer (NMIBC) patients, a total of 178 NMIBC patients from Chungbuk National University Hospital (CBNUH) were enrolled, and the predictive value of the molecular signature-based subtype predictor (MSP888) and risk calculators based on clinicopathological factors (EORTC, CUETO and 2021 EAU risk scores) was compared. Of the 178 patients, 49 were newly analyzed by the RNA-sequencing, and their MSP888 subtype was evaluated. The ability of the EORTC, MSP888 and two molecular subtyping systems of bladder cancer (Lund and UROMOL subtypes) to predict progression of 460 NMIBC patients from the UROMOL project was assessed. Cox regression analyses showed that the MSP888 was an independent predictor of NMIBC progression in the CBNUH cohort (p = 0.043). Particularly in patients without an intravesical BCG immunotherapy, MSP888 significantly linked with risk of disease recurrence and progression (both p < 0.05). However, the EORTC, CUETO and 2021 EAU risk scores showed disappointing results with respect to estimating the NMIBC prognosis. In the UROMOL cohort, the MSP888, Lund and UROMOL subtypes demonstrated a similar capacity to predict NMIBC progression (all p < 0.05). Conclusively, the MSP888 is favorable for stratifying patients to facilitate optimal treatment.

Study on the use of Nanostring nCounter to analyze RNA extracted from formalin-fixed-paraffin-embedded and fresh frozen bladder cancer tissues.

Formalin-fixed paraffin-embedded (FFPE) tissue is the most common source of archived material for genomic medicine. However, FFPE tissue is suboptimal for high-throughput analyses, such as RNA sequencing, because the quality of nucleic acids in FFPE tissues is low. We compared RNA-seq with the nCounter system to evaluate use of FFPE tissue for genomic medicine. Twelve fresh frozen bladder cancer samples were analyzed by both RNA sequencing and nCounter, and matched FFPE samples, by nCounter. Gene-expression values obtained by these two platforms were compared by calculating Pearson correlation coefficients for each sample (across the set of matched genes) and for each matched gene (across the set of samples). For each sample, gene-expression levels measured by RNA sequencing highly correlated with those measured by nCounter (all Pearson's R > 0.8, P < 0.0001), as seen by hierarchical clustering. RNA sequencing results for fresh frozen tissues positively correlated with nCounter results for FFPE tissues (R ranged from 0.675 to 0.873, all P < 0.0001). Correlation and hierarchical-clustering analyses of nCounter data from the two specimens demonstrated a strong positive correlation between each group (R ranged from 0.779 to 0.977, all P < 0.0001). Our findings suggest that the nCounter system is useful for assaying archived-FFPE samples and that the gene-expression signatures obtained from FFPE samples represent those from fresh frozen tissues.

Downregulation of SETD5 Suppresses the Tumorigenicity of Hepatocellular Carcinoma Cells.

Hepatocellular carcinoma (HCC) is an aggressive and incurable cancer. Although understanding of the molecular pathogenesis of HCC has greatly advanced, therapeutic options for the disease remain limited. In this study, we demonstrated that SETD5 expression is positively associated with poor prognosis of HCC and that SETD5 depletion decreased HCC cell proliferation and invasion while inducing cell death. Transcriptome analysis revealed that SETD5 loss downregulated the interferon-mediated inflammatory response in HCC cells. In addition, SETD5 depletion downregulated the expression of a critical glycolysis gene, PKM (pyruvate kinase M1/2), and decreased glycolysis activity in HCC cells. Finally, SETD5 knockdown inhibited tumor growth in xenograft mouse models. These results collectively suggest that SETD5 is involved in the tumorigenic features of HCC cells and that targeting SETD5 may suppress HCC progression.

YAP1 activation is associated with the progression and response to immunotherapy of non-muscle invasive bladder cancer.

BACKGROUND: Despite the availability of several treatments for non-muscle-invasive bladder cancer (NMIBC), many patients are still not responsive to treatments, and the disease progresses. A new prognostic classifier can differentiate between treatment response and progression, and it could be used as a very important tool in patient decision-making regarding treatment options. In this study, we focused on the activation of Yes-associated protein 1 (YAP1), which is known to play a pivotal role in tumour progression and serves as a factor contributing to the mechanism of resistance to various relevant therapeutic agents. We further evaluated its potential as a novel prognostic agent. METHODS: We identified YAP1-associated gene signatures based on UC3-siYAP1 cells (n=8) and NMIBC cohort (n=460). Cross-validation was performed using 5 independent bladder cancer patient cohorts (n=1006). We also experimentally validated the changes of gene expression levels representing each subgroup. FINDINGS: The 976-gene signature based on YAP1-activation redefined three subgroups and had the benefits of Bacillus Calmette-Guérin (BCG) treatment in patients with NMIBC (hazard ratio 3.32, 95% CI 1.29-8.56, p = 0.01). The integrated analysis revealed that YAP1 activation was associated with the characterization of patients with high-risk NMIBC and the response to immunotherapy. INTERPRETATION: This study suggests that YAP1 activation has an important prognostic effect on bladder cancer progression and might be useful in the selection of immunotherapy. FUNDING: A funding list that contributed to this research can be found in the Acknowledgements section.