Expression In Tumor Tissues Research Articles

TRIM47-CDO1 axis dictates hepatocellular carcinoma progression by modulating ferroptotic cell death through the ubiquitin‒proteasome system

Hepatocellular carcinoma (HCC) is the predominant form of liver cancer, characterized by high morbidity and mortality rates, as well as unfavorable treatment outcomes. Tripartite motif-containing protein 47 (TRIM47) has been implicated in various diseases including tumor progression with the activity of E3 ubiquitin ligase. However, the precise regulatory mechanisms underlying the involvement of TRIM47 in HCC remain largely unexplored. Here, we provide evidence that TRIM47 exhibits heightened expression in tumor tissues, and its expression is in intimate association with clinical staging and patient prognosis. TRIM47 promotes HCC proliferation, migration, and invasion as an oncogene by in vitro gain- and loss-of-function experiments. TRIM47 knockdown results in HCC ferroptosis induction, primarily through CDO1 involvement to regulate GSH synthesis. Subsequent experiments confirm the interaction between TRIM47 and CDO1 dependent on B30.2 domain, wherein TRIM47 facilitates K48-linked ubiquitination, leading to a decrease in CDO1 protein abundance in HCC. Furthermore, CDO1 is able to counteract the promotional effect of TRIM47 on HCC biological functions. Overall, our research provides novel insight into the mechanism of TRIM47 in CDO1-mediated ferroptosis in HCC cells, highlighting its value as a potential target candidate for HCC therapeutic approaches.

Identifying SLC2A6 as the novel protective factor in breast cancer by TP53-related genes affecting M1 macrophage infiltration.

The high heterogeneity of breast cancer (BC) caused by pathogenic gene mutations poses a challenge to immunotherapy, but the underlying mechanism remains unknown. The difference in the infiltration of M1 macrophages induced by TP53 mutations has a significant impact on BC immunotherapy. The aim of this study was to develop a TP53-related M1 macrophage infiltration molecular typing risk signature in BC and evaluate the biological functions of the key gene to find new immunotherapy biomarkers. Weighted correlation network analysis (WGCNA) and negative matrix factorization (NMF) were used for distinguishing BC subtypes. The signature and the nomogram were both constructed and evaluated. Biological functions of the novel signature gene SLC2A6 were confirmed through in vitro and in vivo experiments. RNA-Sequencing and protein profiling were used for detecting the possible mechanism of SLC2A6. The results suggested that four BC subtypes were distinguished by TP53-related genes that affect M1 macrophage infiltration. The signature constructed by molecular typing characteristics could evaluate BC's clinical features and tumor microenvironment. The nomogram could accurately predict the prognosis. The signature gene SLC2A6 was found to have an abnormally low expression in tumor tissues. Overexpression of SLC2A6 could inhibit proliferation, promote mitochondrial damage, and result in apoptosis of tumor cells. The HSP70 family member protein HSPA6 could bind with SLC2A6 and increase with the increased expression of SLC2A6. In summary, the risk signature provides a reference for BC risk assessment, and the signature gene SLC2A6 could act as a tumor suppressor in BC.

Subcellular expression of MTA1, HIF1A and p53 in primary tumor predicts aggressive triple negative breast cancers: a meta-analysis based study.

The prevalence of TNBC in India is higher compared to western countries. There is a multitude of biomarkers associated with different clinical outcomes of TNBC with contradictory reports. Identification of a set of specific biomarkers from the very many number of proteins reported in the literature to predict prognosis of TNBC is an urgent clinical need. A systematic review of key molecular biomarkers in cohort studies that have been investigated for their role in breast cancer prognosis was conducted. The Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) methodology was followed. A meta-analysis was used to evaluate their pooled hazard ratio (HR) and the corresponding 95% confidence interval (95% CI) statistically. Immunohistochemical characterization of the meta-analyzed markers were performed in a cohort of 200 retrospective TNBC and 100 non TNBC patient tissues. Kaplan-Meier plot were used to evaluate disease free survival (DFS), and overall survival (OS). Cox regression models were used to evaluate predictors of DFS and OS. Using a meta-analytical approach, we consolidated the biomarker signatures associated with survival outcomes in breast cancers. The promising markers that emerged for the prediction of DFS and OS included E-Cadherin, Survivin, p53, MTA1, HIF1A, CD133, Vimentin and CK5/6. Evaluation of these markers in tumor tissue revealed that subcellular localization of p53, MTA1 and HIF1A had a significant association in predicting TNBC prognosis. Kaplan Meier plot revealed that p53 (OS p = 0.007, DFS p = 0.004), HIF 1A (OS p = 0.054, DFS p = 0.009) and MTA1 (OS p = 0.043, DFS = p = 0.001) expression in the primary tumor tissue were found to be significantly correlated with poor OS and DFS, whereas expression of Survivin (DFS p = 0.024) and E Cadherin (DFS p = 0.027) correlated with DFS alone in TNBC. Univariate analysis revealed that p53, HIF1A and MTA1 could be independent prognostic markers. Our study suggests cytoplasmic over expression of HIF1A, nuclear over expression of MTA1 and mutated p53 in the primary tumor tissue of TNBC have significance as markers predicting survival of TNBC patients.

ESCO2’s oncogenic role in human tumors: a pan-cancer analysis and experimental validation

PurposeEstablishment of sister chromatid cohesion N-acetyltransferase 2 (ESCO2) is involved in the mitotic S-phase adhesins acetylation and is responsible for bridging two sister chromatids. However, present ESCO2 cancer research is limited to a few cancers. No systematic pan-cancer analysis has been conducted to investigate its role in diagnosis, prognosis, and effector function.MethodsWe thoroughly examined the ESCO2 carcinogenesis in pan-cancer by combining public databases such as The Cancer Genome Atlas (TCGA), Genotype-Tissue Expression Project (GTEx), UALCAN and Tumor Immune Single-cell Hub (TISCH). The analysis includes differential expression analysis, survival analysis, cellular effector function, gene mutation, single cell analysis, and tumor immune cell infiltration. Furthermore, we confirmed ESCO2’s impacts on clear cell renal cell carcinoma (ccRCC) cells’ proliferative and invasive capacities in vitro.ResultsIn our study, 30 of 33 cancer types exhibited considerably greater levels of ESCO2 expression in tumor tissue using TCGA and GTEx databases, whereas acute myeloid leukemia (LAML) exhibited significantly lower levels. Kaplan-Meier survival analyses in adrenocortical carcinoma (ACC), kidney chromophobe (KICH), kidney renal clear cell carcinoma (KIRC), kidney renal papillary cell carcinoma (KIRP), brain lower grade glioma (LGG), liver hepatocellular carcinoma (LIHC), lung adenocarcinoma (LUAD), mesothelioma (MESO), and pancreatic adenocarcinoma (PAAD) demonstrated that tumor patients with high ESCO2 expression have short survival periods. However, in thymoma (THYM), colon adenocarcinoma (COAD) and rectum adenocarcinoma (READ), ESCO2 was a favorable prognostic factor. Moreover, ESCO2 expression positively correlates with tumor stage and tumor size in several cancers, including LIHC, KIRC, KIRP and LUAD. Function analysis revealed that ESCO2 participates in mitosis, cell cycle, DNA damage repair, and other processes. CDK1 was identified as a downstream gene regulated by ESCO2. Furthermore, ESCO2 might also be implicated in immune cell infiltration. Finally, ESCO2’S knockdown significantly inhibited the A498 and T24 cells’ proliferation, invasion, and migration.ConclusionsIn conclusion, ESCO2 is a possible pan-cancer biomarker and oncogene that can reliably predict the prognosis of cancer patients. ESCO2 was also implicated in the cell cycle and proliferation regulation. In a nutshell, ESCO2 is a therapeutically viable and dependable target.

THOC7-AS1/OCT1/FSTL1 axis promotes EMT and serves as a therapeutic target in cutaneous squamous cell carcinoma

BackgroundTHOC7-AS1 and FSTL1 expression are frequently upregulated in cutaneous squamous cell carcinoma (cSCC). However, their molecular biological mechanisms remain elusive and their potential as therapeutic targets needs urgent exploration.MethodsHuman tissue samples were used to evaluate clinical parameters. In vitro and in vivo experiments assessed biological functions. Quantitative PCR, western blot, immunohistochemistry, immunocytochemistry, immunoprecipitation, RNA fluorescence in situ hybridization, RNA pull-down, RNA immunoprecipitation, silver staining, chromatin immunoprecipitation, dual luciferase reporter assays etc. were utilized to explore the molecular biological mechanisms.ResultsWe found FSTL1 is an oncogene in cSCC, with high expression in tumor tissues and cells. Its elevated expression closely associates with tumor size and local tissue infiltration. In vitro and in vivo, high FSTL1 expression promotes cSCC proliferation, migration and invasion, facilitating malignant behaviors. Mechanistically, FSTL1 interacts with ZEB1 to promote epithelial-to-mesenchymal transition (EMT) in cSCC cells. Exploring upstream regulation, we found THOC7-AS1 can interact with OCT1, which binds the FSTL1 promoter region and promotes FSTL1 expression, facilitating cSCC progression. Finally, treating tumors with THOC7-AS1 antisense oligonucleotides inhibited cSCC proliferative and migratory abilities, delaying tumor progression.ConclusionsThe THOC7-AS1/OCT1/FSTL1 axis regulates EMT and promotes tumor progression in cSCC. This study provides clues and ideas for cSCC targeted therapy.

Abstract LB329: Interleukin6 as a Potential Prognostic Marker and Therapeutic Target in Oral Squamous Cell Carcinoma

Abstract This study investigates the role of interleukin (IL)-6 in oral squamous cell carcinoma (OSCC), evaluating its potential as a prognostic marker and therapeutic target. This study involved 95 untreated OSCC patients, with pre-surgical serum IL-6 levels measured by Electro chemiluminescence immunoassay (ECLIA). Patients were categorized into high (78 patients) and low (17 patients) IL-6 groups based on a cutoff value of 8 pg/mL.A strong correlation was observed between higher IL-6 levels and lower overall survival (P<0.0001) and disease-free survival (P=0.0283). Serum IL-6 levels also showed a strong positive correlation with certain blood test markers such as neutrophil and lymphocyte fractions and C-reactive protein (CRP), and a negative correlation with albumin levels. These findings suggest a link between systemic inflammation, malnutrition, and the Inflammation-based prognostic score (IBPS).Further analysis through microarray assessed the gene expression in primary tumor tissues, revealing 513 genes with more than a two-fold change in the high IL-6 group compared to controls. Gene Ontology analysis indicated increased metabolic activity in the high IL-6 group and suppressed inflammation and immune-related molecules in the low IL-6 group.Immunohistochemical staining for IL-6 in tumor tissues showed its presence mainly in the stroma, particularly in cancer-associated fibroblasts (CAFs) identified by vimentin and α-SMA positivity. Both OSCC cell lines and CAFs established from OSCC tissues demonstrated high IL-6 secretion, especially in cell lines derived from lymph node metastases. Furthermore, cell lines from primary, lymph node, and lung metastases exhibited high IL-6 secretion, with HRAS mutations also identified. Knockdown of HRAS using siRNA reduced IL-6 secretion.Finally, in vivo experiments using an anti-IL-6 receptor antibody showed that blocking IL-6 signaling reduced the proliferation of OSCC cell lines.The study concludes that IL-6 is a useful prognostic and therapeutic target in OSCC. The wide applicability of IL-6 testing and the clinical use of anti-human IL-6R antibody drugs, combined with aggressive nutritional intervention, suggest a potential paradigm shift in OSCC treatment. Citation Format: Hiroyuki Goda, Tomoko Adachi, Koh-ichi Nakashiro, Nobuyuki Kuribayashi, Daisuke Uchida. Interleukin6 as a Potential Prognostic Marker and Therapeutic Target in Oral Squamous Cell Carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB329.

Abstract LB146: Multi Omics analyses identified novel loci and genes for lung cancer risk among European Descendants

Abstract Introduction Genome-wide association studies (GWAS) have identified >60 genomic loci for lung cancer risk. However, causal genes and the underlying biological mechanisms for most of these loci remain unknown. Therefore, we conducted a multi-omics study to identify lung cancer susceptibility genes. Method We first conducted a transcriptome-wide association study (TWAS) using S-PrediXcan framework. Whole transcriptome data from adjacent-normal lung tissue samples and genomic data from 304 European-ancestry lung cancer patients were used to build genetic prediction models for expression levels of protein-coding genes and lincRNAs. The prediction models were then applied to GWAS meta-analysis results for lung cancer with 39,363 cases and 621,480 controls of European descents from TRICL-ILCCO, the UK Biobank, and FinnGen. COJO was used to assess whether those significant genes were independent of proximal (±1.5 Mb) GWAS-identified risk variants. To replicate our TWAS-identified genes, we used data from GTEx version 8 normal lung tissue (N=444) to build imputation models and then run association analyses. We acquired gene (N=107) and protein expression data (N=111) from tumor and matched adjacent-normal lung tissues from European-ancestry participants in the Clinical Proteomic Tumor Analysis Consortium. Paired Wilcoxon rank sum tests were then conducted to assess whether identified genes and their corresponding proteins were expressed differentially. Results Out of 18,850 genes (read count >6 and TPM >0.1 in at least 20% samples), 6,754 protein-coding genes and 802 lincRNAs were predicted reliably using genetic variants (R>0.1 and P<0.05). Among them, genetically proxied expression levels of 24 genes located at 15 loci were significantly associated with lung cancer risk at a Bonferroni adjusted P <0.05. Four genes were located at four novel loci, including SERF1A at 5q13.2, SMIM33 at 5q31.2, MUC5B at 11p15.5, and RASSF10 at 11p15.2. Six putative candidate genes, namely H4C13, HCP5B, STN1, COPS2, SKIV2L, and LINC00243 resided in previously GWAS-identified lung cancer loci but have not been reported. Of these six genes, SKIV2L (Padjust=1.49e-5) and LINC00243 (Padjust=3.47e-5) were independently associated with lung cancer risk even after adjusting for GWAS-identified variants. Among 24 identified genes, 16 showed a consistent significant association with lung cancer risk (p<0.05) based on the prediction models built using GTEx data. Of ten unreported or novel genes, MUC5B, SMIM33, SERF1A, STN1, COPS2, and LINC00243 showed differential gene expression at P <0.05 and consistent association direction with TWAS results. Three proteins, STN1, COPS2, and SKIV2L, showed differential expression in tumor and adjacent-normal tissue at P<0.05, which is consistent with our TWAS direction. Conclusion Our findings offer novel insights into lung cancer carcinogenesis by uncovering new genes and loci. Citation Format: Shuai Xu, Yaohua Yang, Tianying Zhao, Jiajun Shi, Jie Ping, Wanqing Wen, Hui Cai, Xingyi Guo, Ran Tao, Xiao-Ou Shu, Wei Zheng, Jirong Long, Qiuyin Cai. Multi Omics analyses identified novel loci and genes for lung cancer risk among European Descendants [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB146.

Unlocking Overexpressed Membrane Proteins to Guide Breast Cancer Precision Medicine.

Breast cancer (BC) is a prevalent form of cancer affecting women worldwide. However, the effectiveness of current BC drugs is limited by issues such as systemic toxicity, drug resistance, and severe side effects. Consequently, there is an urgent need for new therapeutic targets and improved tumor tracking methods. This study aims to address these challenges by proposing a strategy for identifying membrane proteins in tumors that can be targeted for specific BC therapy and diagnosis. The strategy involves the analyses of gene expressions in breast tumor and non-tumor tissues and other healthy tissues by using comprehensive bioinformatics analysis from The Cancer Genome Atlas (TCGA), UALCAN, TNM Plot, and LinkedOmics. By employing this strategy, we identified four transcripts (LRRC15, EFNA3, TSPAN13, and CA12) that encoded membrane proteins with an increased expression in BC tissue compared to healthy tissue. These four transcripts also demonstrated high accuracy, specificity, and accuracy in identifying tumor samples, as confirmed by the ROC curve. Additionally, tissue microarray (TMA) analysis revealed increased expressions of the four proteins in tumor tissues across all molecular subtypes compared to the adjacent breast tissue. Moreover, the analysis of human interactome data demonstrated the important roles of these proteins in various cancer-related pathways. Taken together, these findings suggest that LRRC15, EFNA3, TSPAN13, and CA12 can serve as potential biomarkers for improving cancer diagnosis screening and as suitable targets for therapy with reduced side effects and enhanced efficacy.

DNMT3A promotes glioma growth and malignancy via TNF-α/NF-κB signaling pathway.

DNMT3A is the main molecule responsible for DNA methylation in cells. DNMT3A affects the progression of inflammation, degenerative diseases, and malignant tumors, and exhibits significant aberrantly expression in tumor tissues. Transcriptome data and relevant clinical information were downloaded from The Cancer Genome Atlas (TCGA), Chinese Glioma Genome Atlas (CGGA), and Gene Expression Omnibus (GEO) datasets. Differential expression analysis and prognostic analysis were conducted based on above statistics. We constructed a clinical prognostic model and identified DNMT3A as an independent prognostic factor to accurately predict patient prognosis. Differential gene enrichment analysis revealed that DNMT3A affects the progression of glioma through multiple pathways, among which the tumor necrosis factor-α (TNF-α)/nuclear factor-kappa B (NF-κB) pathway shows a strong correlation. Immunological analysis also revealed a certain correlation between DNMT3A and tumor immunity. We demonstrated through gene editing that DNMT3A can affect the release of TNF-α in cells, thereby affecting the progression of glioma. Functional experiments have also demonstrated that DNMT3A plays a crucial role in tumors. RNA-sequencing and survival analyses of lower-grade glioma (LGG) patients in TCGA, CGGA, and GEO cohorts showed that high DNMT3A expression correlated with poor prognosis of LGG patients. Univariate and multivariate Cox regression analyses showed that DNMT3A expression was an independent prognostic indicator in LGG. The prognosis prediction nomogram with age, World Health Organization (WHO) grading, and DNMT3A expression showed reliable performance in predicting the 1-, 3-, and 5-year overall survival (OS) of LGG patients. Functional enrichment analysis, gene set enrichment analysis (GSEA), and ESTIMATE algorithm analyses showed that DNMT3A expression was associated with the tumor infiltration of immune cells and predicted response to immunotherapy in two immunotherapy cohorts of pan-cancer patients. Furthermore, short hairpin RNA (shRNA)-mediated knockdown of DNMT3A in the LGG cell lines suppressed proliferation, migration, and invasion of LGG cells by downregulating the TNF-α/NF-κB signaling pathway. Our data showed that DNMT3A was a potential prognostic biomarker in glioma. DNMT3A promoted proliferation and malignancy of LGG cells through the TNF-α/NF-κB signaling pathway. DNMT3A is a promising therapeutic target for treating patients with LGG.

MicroRNA expression as a prognostic biomarker of tongue squamous cell carcinoma (TSCC): a systematic review and meta-analysis

BackgroundRecent studies have indicated that microRNA (miRNA) expression in tumour tissues has prognostic significance in Tongue squamous cell carcinoma (TSCC) patients. This study explored the possible prognostic value of miRNAs for TSCC based on published research.MethodsA comprehensive literature search of multiple databases was conducted according to predefined eligibility criteria. Data were extracted from the included studies by two researchers, and HR results were determined based on Kaplan‒Meier curves according to the Tierney method. The Newcastle‒Ottawa Scale (NOS) and GRADE (Grading of Recommendations Assessment, Development, and Evaluation) pro-GDT were applied to assess the quality of all studies. Publication bias was estimated by funnel plot, Egger’s rank correlation test and sensitivity analysis.ResultsEleven studies (891patients) were included, of which 6 reported up-regulated miRNAs and 7 mentioned down-regulated miRNAs. The pooled hazard ratio (HR) from the prognostic indicator overall survival (OS) was 1.34 (1.25–1.44), p < 0.00001, indicating a significant difference in miRNA expression between TSCC patients with better or worse prognosis.ConclusionMiRNAs may have high prognostic value and could be used as prognostic biomarkers of TSCC.

Synthesis and Evaluation of [18F]AlF-NOTA-c-DVAP: A Novel PET Probe for Imaging GRP78 in Cancer.

GRP78, a member of the HSP70 superfamily, is an endoplasmic reticulum chaperone protein overexpressed in various cancers, making it a promising target for cancer imaging and therapy. Positron emission tomography (PET) imaging offers unique advantages in real time, noninvasive tumor imaging, rendering it a suitable tool for targeting GRP78 in tumor imaging to guide targeted therapy. Several studies have reported successful tumor imaging using PET probes targeting GRP78. However, existing PET probes face challenges such as low tumor uptake, inadequate in vivo distribution, and high abdominal background signal. Therefore, this study introduces a novel peptide PET probe, [18F]AlF-NOTA-c-DVAP, for targeted tumor imaging of GRP78. [18F]AlF-NOTA-c-DVAP was radiolabeled with fluoride-18 using the aluminum-[18F]fluoride ([18F]AlF) method. The study assessed the partition coefficients, stability in vitro, and metabolic stability of [18F]AlF-NOTA-c-DVAP. Micro-PET imaging, pharmacokinetic analysis, and biodistribution studies were carried out in tumor-bearing mice to evaluate the probe's performance. Docking studies and pharmacokinetic analyses of [18F]AlF-NOTA-c-DVAP were also performed. Immunohistochemical and immunofluorescence analyses were conducted to confirm GRP78 expression in tumor tissues. The probe's binding affinity to GRP78 was analyzed by molecular docking simulation. [18F]AlF-NOTA-c-DVAP was radiolabeled in just 25 min with a high yield of 51 ± 16%, a radiochemical purity of 99%, and molar activity within the range of 20-50 GBq/μmol. [18F]AlF-NOTA-c-DVAP demonstrated high stability in vitro and in vivo, with a logD value of -3.41 ± 0.03. Dynamic PET imaging of [18F]AlF-NOTA-c-DVAP in tumors showed rapid uptake and sustained retention, with minimal background uptake. Biodistribution studies revealed rapid blood clearance and excretion through the kidneys following a single-compartment reversible metabolic model. In PET imaging, the T/M ratios for A549 tumors (high GRP78 expression), MDA-MB-231 tumors (medium expression), and HepG2 tumors (low expression) at 60 min postintravenous injection were 10.48 ± 1.39, 6.25 ± 0.47, and 3.15 ± 1.15% ID/g, respectively, indicating a positive correlation with GRP78 expression. This study demonstrates the feasibility of using [18F]AlF-NOTA-c-DVAP as a PET tracer for imaging GRP78 in tumors. The probe shows promising results in terms of stability, specificity, and tumor targeting. Further research may explore the clinical utility and potential therapeutic applications of this PET tracer for cancer diagnosis.

PD-L1 as a Urine Biomarker in Renal Cell Carcinoma-A Case Series and Proof-of-Concept Study.

Renal cell carcinoma (RCC) is among the most lethal urologic malignancies once metastatic. Current treatment approaches for metastatic RCC (mRCC) involve immune checkpoint inhibitors (ICIs) that target the PD-L1/PD-1 axis. High PD-L1 expression in tumor tissue has been identified as a negative prognostic factor in RCC. However, the role of PD-L1 as a liquid biomarker has not yet been fully explored. Herein, we analyze urine levels of PD-L1 in mRCC patients before and after either ICI therapy or surgical intervention, as well as in a series of patients with treatment-naïve RCC. The mid-stream urine of patients with mRCC (n = 4) or treatment-naïve RCC, i.e., prior to surgery from two centers (cohort I, n = 49: cohort II, n = 29) was analyzed for PD-L1 by ELISA. The results from cohort I were compared to a control group consisting of patients treated for non-malignant urologic diseases (n = 31). In the mRCC group, urine PD-L1 levels were measured before and after tumor nephrectomy (n = 1) or before and after ICI therapy (n = 3). Exosomal PD-L1 in the urine was analyzed in selected patients by immunoblotting. A strong decrease in urine PD-L1 levels was found after tumor nephrectomy or following systemic treatment with ICIs. In patients with treatment-naïve RCC (cohort I), urine PD-L1 levels were significantly elevated in the RCC group in comparison to the control group (median 59 pg/mL vs. 25.7 pg/mL, p = 0.011). PD-L1 urine levels were found to be elevated, in particular, in low-grade RCCs in cohorts I and II. Exosomal PD-L1 was detected in the urine of a subset of patients. In this proof-of-concept study, we show that PD-L1 can be detected in the urine of RCC patients. Urine PD-L1 levels were found to correlate with the treatment response in mRCC patients and were significantly elevated in treatment-naïve RCC patients.

Enhancing Specific Fluorescence In Situ Hybridization with Quantum Dots for Single-Molecule RNA Imaging in Formalin-Fixed Paraffin-Embedded Tumor Tissues.

Single-molecule fluorescence in situ hybridization (smFISH) represents a promising approach for the quantitative analysis of nucleic acid biomarkers in clinical tissue samples. However, low signal intensity and high background noise are complications that arise from diagnostic pathology when performed with smFISH-based RNA imaging in formalin-fixed paraffin-embedded (FFPE) tissue specimens. Moreover, the associated complex procedures can produce uncertain results and poor image quality. Herein, by combining the high specificity of split DNA probes with the high signal readout of ZnCdSe/ZnS quantum dot (QD) labeling, we introduce QD split-FISH, a high-brightness smFISH technology, to quantify the expression of mRNA in both cell lines and clinical FFPE tissue samples of breast cancer and lung squamous carcinoma. Owing to its high signal-to-noise ratio, QD split-FISH is a fast, inexpensive, and sensitive method for quantifying mRNA expression in FFPE tumor tissues, making it suitable for biomarker imaging and diagnostic pathology.

The significance of m6A RNA methylation regulators in diagnosis and subtype classification of HBV-related hepatocellular carcinoma.

In recent years, abnormal m6A alteration in hepatocellular carcinoma (HCC) has been a focus on investigating the biological implications. In this study, our objective is to determine whether m6A modification contributes to the progression of HBV-related HCC. To achieve this, we employed a random forest model to screen top 8 characteristic m6A regulators from 19 candidate genes. Subsequently, we developed a nomogram model that utilizes these 8 characteristic m6A regulators to predict the prevalence of HBV-related HCC. According to decision curve analysis, patients may benefit from the nomogram model. The clinical impact curves exhibited a robust predictive capability of the nomogram models. Additionally, consensus molecular subtyping was employed to identify m6A modification patterns and m6A-related gene signature. The quantification of immune cell subsets was accomplished through the implementation of ssGSEA algorithms. PCA algorithms were developed to compute the m6A score for individual tumors. Two distinct m6A modification patterns, namely cluster A and cluster B, exhibited significant correlations with distinct immune infiltration patterns and biological pathways. Notably, patients belonging to cluster B demonstrated higher m6A scores compared to those in cluster A, as determined by the m6A score metric. Furthermore, the expression of IGFBP3 proteins was validated through immunofluorescence, revealing their pronounced lower expression in tumor tissues. In summary, our study underscores the importance of m6A modification in the advancement of HBV-related HCC. This research has the potential to yield novel prognostic biomarkers and therapeutic targets for the identification of HBV-related HCC.

The Novel SSTR3 Agonist ITF2984 Exerts Antimitotic and Proapoptotic Effects in Human Non-Functioning Pituitary Neuroendocrine Tumor (NF-PitNET) Cells.

Somatostatin receptor ligands (SRLs) with high affinity for somatostatin receptors 2 and 5 (SSTR2 and SSTR5) are poorly efficacious in NF-PitNETs, expressing high levels of SSTR3. ITF2984 is a pan-SSTR ligand with high affinity for SSTR3, able to induce SSTR3 activation and to exert antitumoral activity in the MENX rat model. The aim of this study was to test ITF2984's antiproliferative and proapoptotic effects in NF-PitNET primary cultured cells derived from surgically removed human tumors and to characterize their SSTR expression profile. We treated cells derived from 23 NF-PitNETs with ITF2984, and a subset of them with octreotide, pasireotide (SRLs with high affinity for SSTR2 or 5, respectively), or cabergoline (DRD2 agonist) and we measured cell proliferation and apoptosis. SSTR3, SSTR2, and SSTR5 expression in tumor tissues was analyzed by qRT-PCR and Western blot. We demonstrated that ITF2984 reduced cell proliferation (-40.8 (17.08)%, p < 0.001 vs. basal, n = 19 NF-PitNETs) and increased cell apoptosis (+41.4 (22.1)%, p < 0.001 vs. basal, n = 17 NF-PitNETs) in all tumors tested, whereas the other drugs were only effective in some tumors. In our model, SSTR3 expression levels did not correlate with ITF2984 antiproliferative nor proapoptotic effects. In conclusion, our data support a possible use of ITF2984 in the pharmacological treatment of NF-PitNET.

Abstract 5674: Characterizing Cellular Heterogeneity and Identifying Potential Biomarkers in Esophageal Squamous Cell Carcinoma (ESCC) Using Single-Cell RNA Sequencing

Abstract Esophageal squamous cell carcinoma (ESCC) exhibits significant cellular heterogeneity, rendering it susceptible to developing radioresistance and recurring tumors. Single-cell RNA sequencing (scRNA-seq) is an advanced technique that allows for the exploration of distinct gene expression profiles in individual cells. To investigate cellular heterogeneity in ESCC, scRNA-seq analysis was performed on tumor tissues from ten ESCC patients, as well as adjacent non-malignant esophageal tissues. The cellular composition revealed 19 distinct cell clusters, further categorized into seven cell populations: 69.31% Fibroblast, 15.36% T cell, 5.30% Monocytes, 5.26% Endothelial cells, 2.68% plasma cell, 1.91% Epithelial cells, and 0.18% ambiguous. Notably, Fibroblast and Endothelial cells were more prevalent in non-malignant tissues, while the remaining cell types were enriched in tumor tissues. Differential gene expression analysis identified 49 genes with higher expression in tumor cells and 37 genes with lower expression. Gene Set Enrichment Analysis (GSEA) revealed the top three most relevant GO Biological Processes pathways: positive regulation of immune response, regulation of T cell activation, and positive regulation of cell migration. Furthermore, in post-treatment follow-up, four patients were observed to develop metastasis within one year. Comparative analysis between metastatic and non-metastatic patients unveiled 9 Differentially Expressed Genes (DEGs), including KRT6A and AKR1B10, which exhibited specific expression in Epithelial cells and were highly expressed in tumor tissues. Additionally, IGLC2, IGHG4, and IGHG1 demonstrated specific expression in plasma cells, with elevated expression in tumor tissues. These findings suggest that KRT6A, AKR1B10, IGLC2, IGHG4, and IGHG1 may serve as potential biomarkers for predicting post-chemotherapy metastasis in ESCC patients. Citation Format: Tzu-Hung Hsiao, Chih-Hung Lin, Ting-Shuan Wu, Li-Wen Lee, Chung-Ping Hsu. Characterizing Cellular Heterogeneity and Identifying Potential Biomarkers in Esophageal Squamous Cell Carcinoma (ESCC) Using Single-Cell RNA Sequencing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5674.

Abstract 3770: Comparative analysis of single cell expression and digital spatial profiling in FFPE tumor tissues

Abstract Formalin Fixed Paraffin Embedded (FFPE) tissues are a staple in clinical diagnostics associated with solid tumor. Next Generation Sequencing (NGS) approaches characterize genomes, epigenomes, transcriptomes, and proteomes from a single sample source. FFPE blocks from several different tumor tissue types were serially sliced into FFPE slides. Individual slides were then utilized to explore the genome, epigenome, single cell RNA-Seq (scRNA-Seq), and Digital Spatial Profiling (DSP). Here we present a comparative analysis of two transcriptomic modalities within one FFPE tissue sample: fixed-cell scRNA-Seq of the transcriptome and digital spatial profiling of the whole transcriptome. Independent analysis of scRNA-Seq and DSP identified common trends as well as distinct differences within the same sample. Comparative analysis within sample between modalities identified statistically significant differences, driven by local tumor microenvironment, but also highlighted commonalities between methods. Cross sample comparisons showed striking differences, as indicated by tumor type. These studies highlight the strengths of each assay individually, as well as the power of a dual-assay approach for comprehensive analysis of the tumor and its microenvironment. Conclusions from this standalone analysis were further aggregated into downstream multiomics analyses, including proteomics analysis, illustrating the expanding landscape of information that can be extracted from FFPE derived tissue and the potential for novel discovery and diagnostic power integrating these complex data types holds. Citation Format: Anthony Moore, Andrea O'Hara, Pranay Vishwanath, Laure Turner, Haythem Latif, Chris Mozdzierz, Ginger Zhou. Comparative analysis of single cell expression and digital spatial profiling in FFPE tumor tissues [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3770.

Abstract 2083: Discovery of ETX-19477, a novel and selective PARG inhibitor with high potency against tumors with underlying replication stress

Abstract DNA damage response (DDR) is an important cellular process that is regulated by poly ADP ribosylation (PARylation). PARP1 binds to DNA break sites and then catalyzes PARylation, which leads to recruitment of repair factors and initiation of base-excision and single-strand break (SSB) repair. Removal of PAR chains is performed by PARG and is also critical for successful completion of DNA repair. Suppression of PAR chain hydrolysis via PARG depletion or inhibition leads to defective single-strand break repair, reduced kinetics of repair, and hypersensitivity to certain DNA damaging agents. Cancer cells demonstrate high levels of replication stress, characterized as the slowing or stalling of replication forks during DNA replication. Replication stress generates DNA SSBs and exposes unligated Okazaki fragments which activate PARP1. PARP1 catalyzed PARylation slows replication fork progression as a protective mechanism, initiates damage repair, and promotes fork reversal. PAR chain removal, via PARG activity, enables completion of damage repair, replication fork restart, and recovery from replication stress. Under conditions of replication stress, PARP inhibition can result in unrestrained fork progression and the occurrence of DNA double strand breaks from fork runoff. In contrast, PARG inhibition causes prolonged slowing or stalling of replication fork progression, which can lead to replication catastrophe and cancer cell death. Because of these opposing roles, PARG inhibitors show distinct pharmacology as compared to PARP inhibitors in certain cancer cells including cancer cells that are BRCA wild type, homologous recombination proficient, and PARP inhibitor insensitive or resistant. ETX-19477 is a novel inhibitor of PARG that demonstrates low nM, on-target activity in cells as measured by the accumulation of PAR chains. ETX-19477 induces death and/or inhibits the proliferation of multiple cancer cell types including ER+HER2- breast, serous and mucinous ovarian, lung, and gastric cancers and induces pan-nuclear γH2AX expression, a hallmark of replication catastrophe. Preclinical studies demonstrate that ETX-19477 is orally bioavailable with favorable pharmacokinetics. Oral treatment of mice with ETX-19477 is well tolerated and shows robust dose-dependent in vivo anti-tumor activity in breast and ovarian cell-line derived and patient-derived xenograft models. Efficacy is observed with either once or twice daily dosing and with a discontinuous dosing regimen. In mouse xenograft models, induction of γH2AX expression in tumor tissue correlates with tumor growth inhibition. Preclinical data show ETX-19477 demonstrates potent and specific anti-proliferative effects by exposing underlying existing replication stress vulnerabilities in cancer cells across multiple tumor types. ETX-19477 will be advanced into clinical trials in 2024. Citation Format: John P. Holleran, Tamara S. Rodems, Shweta Sharma, Angelina M. Santini, Cindy Wuerz, Juping Liu, Lu Tian, Joseph Dosch, Joy Nader, Richard Rivera, Shyama Herath, Joseph Marakovits, Christopher McBride, Ryan Stansfield, Amy Salyards, James M. Veal, Jeffrey A. Stafford, Gretchen Bain. Discovery of ETX-19477, a novel and selective PARG inhibitor with high potency against tumors with underlying replication stress [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2083.

Lactate dehydrogenase A is implicated in the pathogenesis of B-cell lymphoma through regulation of the FER signaling pathway.

Lactate dehydrogenase A (LDHA) is highly expressed in various tumors. However, the role of LDHA in the pathogenesis of B-cell lymphoma remains unclear. Analysis of data from The Cancer Genome Atlas (TCGA) and Genotype-Tissue Expression (GTEx) databases revealed an elevated LDHA expression in diffuse large B-cell lymphoma (DLBC) tissues compared with normal tissues. Similarly, our results demonstrated a significant increase in LDHA expression in tumor tissues from the patients with B-cell lymphoma compared with those with lymphadenitis. To further elucidate potential roles of LDHA in B-cell lymphoma pathogenesis, we silenced LDHA in the Raji cells (a B-cell lymphoma cell line) using shRNA techniques. Silencing LDHA led to reduced mitochondrial membrane integrity, adenosine triphosphate (ATP) production, glycolytic activity, cell viability and invasion. Notably, LDHA knockdown substantially suppressed in vivo growth of Raji cells and extended survival in mice bearing lymphoma (Raji cells). Moreover, proteomic analysis identified feline sarcoma-related protein (FER) as a differential protein positively associated with LDHA expression. Treatment with E260, a FER inhibitor, significantly reduced the metabolism, proliferation and invasion of Raji cells. In summary, our findings highlight that LDHA plays multiple roles in B-cell lymphoma pathogenesis via FER pathways, establishing LDHA/FER may as a potential therapeutic target.

Abstract 3482: Single-cell transcriptome analysis reveals that PLK4 signature genes are highly associated with cancer proliferation and hotspot mutations in non-small cell lung cancer (NSCLC)

Abstract Background: Polo-like kinase 4 (PLK4) is a member of the polo-like kinase family known for its precise regulation during the cell cycle and its key role in centriole duplication, which is essential for proper cell division. Dysregulation of PLK4 is associated with abnormal centriole number, genomic instability and cancer development. In our previous study, we proposed PLK4 as a potential therapeutic target and biomarker for LUAD patients with TP53 mutations by bulk RNA-seq analysis and confirmed its association with drug resistance. In this study, we analyzed the expression of PLK4 and its association with cell cycle at the cellular level by scRNA-seq in NSCLC data. Methods: In a previous study, we analyzed NSCLC data from The Cancer Genome Atlas (TCGA) database using the UCSC Xena data portal. In this study, we analyzed E-MTAB-6653 dataset from the single cell expression atlas(https://www.ebi.ac.uk/gxa/sc/home). Results: In the previous study, we demonstrated that PLK4 expression is higher in primary tumors compared to normal tissues in NSCLC. Higher PLK4 expression in patients is associated with an unfavorable prognosis. Using genes highly correlated with PLK4, we confirmed a significant association between PLK4 and TP53 mutation by statistical analysis. Using the Drug Gene Interaction database, we observed an association between the drug resistance-related genes PBK and BIRC5 with PLK4, and these genes were also correlated with TP53 mutation. In this study, we investigated the association between PLK4 and the cell cycle using single cell RNA-seq. First, we performed data pre-processing to selectively isolate NSCLC cells. Using a set of 87 PLK4 signature genes obtained from previous research, we stratified the samples into PLK4 high and low groups based on the expression of these genes. We then analyzed cell cycle related genes in both groups and observed a higher expression of G2/M phase related genes in the PLK4 high group compared to the PLK4 low group. We also examined the expression of PLK4 signature genes based on pathological differences and found higher expression in tumor tissue compared to normal tissue. There was also an increasing expression gradient from the tumor margin to the tumor core. Finally, using Gene Ontology analysis of both groups, we confirmed the upregulation of genes associated with transcription and translation, as indicated by the GO terms obtained. Conclusion: In a previous study, the association between PLK4 expression, TP53 mutation and drug resistance was identified by bulk RNA-seq. In the current study, we used single cell RNA-seq to verify the correlation between PLK4 and cell cycle in NSCLC. In addition, we observed that the expression of PLK4 signature genes increased towards the tumor core based on pathological differences. Citation Format: Youngtaek Kim, Joon Yeon Hwang, YoungJoon Park, Dong Kwon Kim, Kwangmin Na, Seul Lee, Sujeong Baek, Seong-san Kang, Seung Min Yang, Mi Hyun Kim, Heekyung Han, Chai Young Lee, Yu Jin Han, Min Hee Hong, Jii Bum Lee, Sun Min Lim, Kyoung-Ho Pyo, Byoung Chul Cho. Single-cell transcriptome analysis reveals that PLK4 signature genes are highly associated with cancer proliferation and hotspot mutations in non-small cell lung cancer (NSCLC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3482.