Expression Of Cancer-related Genes Research Articles

Interleukin-12 treatment reduces tumor growth and modulates the expression of CASKA and MIR-203 in athymic mice bearing tumors induced by the HGC-27 gastric cancer cell line

Gastric cancer (GC) is one of the most common malignant tumors in the digestive system and due to its poor prognosis, there is an increase in the demand for more effective anticancer therapies. Interleukins are potential anticancer agents which can modulate expression of cancer related genes and have therapeutic effects. Interleukin 12 (IL-12) exhibits potent anti-tumor, anti-angiogenic and anti-metastatic activities and represents the ideal candidate for tumor immunotherapy, due to its ability to activate both innate and adaptive immunities. The aim of this study was to evaluate the effect of IL-12 administration on GC tumor growth induced in the cancer xenograft nude mouse model. Tumor development was analyzed weekly and after 8 weeks, the animals were sacrificed for cytokine collection and analysis (IL-4, TNF-alfa, IL-2, INF-gamma, IL-12, IL-10, TGF-beta) by ELISA. The tumor cells were biopsied (sections) in the implanted areas of the animals that developed solid growth of the tumor (anatomopathological analysis was performed). We have also evaluated the expression CASK and miR203 expression, two related cell invasion factors, in the induced tumors after administration of 6n/kg IL-12. The development of tumor masses was observed in all groups of animals inoculated with HGC-27 neoplastic cells. In animals treated with 6n/kg IL-12, there was no tumor development confirmed by anatomopathological analysis. Changes in the levels of pro and anti-inflammatory cytokines were also observed in this study. Our results indicated that miR203 expression was elevated while CASK was downregulated. These results suggest that IL-12 treatment repress the tumor growth by induction of miR203 expression which in turn repress CASK expression.

Single-Nucleus RNA-Sequencing Reveals a MET+ Oligodendrocyte Subpopulation That Promotes Proliferation of Radiation-Induced Gliomas

PurposeRadiation-induced gliomas (RIGs) are fatal late complications of radiotherapy, with a median survival time of 6–11 months. RIGs demonstrate unique molecular landscape and may originate from a glial lineage distinct from that of primary malignancies or diffuse midline gliomas (DMGs). This study aimed to explore the intratumoral diversity within RIGs to uncover their cellular origin and characteristics, and enhance our understanding of this uncommon tumor type. Methods and MaterialsFormalin-fixed, paraffin-embedded samples were collected from two RIGs and two DMGs for single-nucleus RNA sequencing. A detailed analysis was conducted to assess intratumoral heterogeneity and cellular interactions, including gene set enrichment, pseudotime trajectory, and cell communication analyses. Immunofluorescence staining, proliferation assay, and RNA-seq analysis were also applied to validate our findings. ResultsOur analysis revealed distinct heterogeneity in oligodendrocytes between the DMG and RIG samples. A unique subpopulation of oligodendrocytes in RIGs, which was characterized by gene encoding mesenchymal-epithelial transition factor (c-MET) and therefore termed MET+ ODs, exhibited characteristics typical of cancer cells, such as increased mitotic activity, cancer-related gene expression, and extensive copy number variations. Cell communication studies indicated that MET+ ODs interact vigorously with G1/S and G2/M cycling cells via the neural cell adhesion molecule (NCAM) signaling pathway, potentially enhancing the proliferation of cycling malignant cells. Integrating our results with existing RNA-seq data further supported our hypothesis. The presence of MET+ ODs in RIGs was confirmed by immunostaining, and activation of the NCAM signaling pathway in vitro significantly promoted the proliferation of RIG tumor cells. Moreover, in-vitro radiation induced the transformation of oligodendrocytes to be more similar to MET+ ODs. ConclusionsRIGs are characterized by an oligodendrocyte composition distinct from that of DMGs. A specific subpopulation of MET+ ODs in RIGs may be crucial in tumorigenesis and promote the growth of malignant cells. Identifying MET+ ODs offers a valuable target for future clinical surveillance and therapeutic strategies.

Pan-cancer Analysis Reveals m6A Variation and Cell-specific Regulatory Network in Different Cancer Types.

As the most abundant messenger RNA (mRNA) modification in mRNA, N 6-methyladenosine (m6A) plays a crucial role in RNA fate, impacting cellular and physiological processes in various tumor types. However, our understanding of the function and role of the m6A methylome in tumor heterogeneity remains limited. Herein, we collected and analyzed m6A methylomes across nine human tissues from 97 m6A sequencing (m6A-seq) and RNA sequencing samples. Our findings demonstrate that m6A exhibits different heterogeneity in most tumor tissues compared to normal tissues, which contributes to the diverse clinical outcomes in different cancer types. We also found that the cancer type-specific m6A level regulated the expression of different cancer-related genes in distinct cancer types. Utilizing a novel and reliable method called "m6A-express", we predicted m6A-regulated genes and revealed that cancer type-specific m6A-regulated genes contributed to the prognosis, tumor origin, and infiltration level of immune cells in diverse patient populations. Furthermore, we identified cell-specific m6A regulators that regulate cancer-specific m6A and constructed a regulatory network. Experimental validation was performed, confirming that the cell-specific m6A regulator CAPRIN1 controls the m6A level of TP53. Overall, our work reveals the clinical relevance of m6A in various tumor tissues and explains how such heterogeneity is established. These results further suggest the potential of m6A for cancer precision medicine for patients with different cancer types.

Overview of the interplay between m6A methylation modification and non-coding RNA and their impact on tumor cells.

N6-methyladenosine (m6A) is one of the most common internal modifications in eukaryotic RNA. The presence of m6A on transcripts can affect a series of fundamental cellular processes, including mRNA splicing, nuclear transportation, stability, and translation. The m6A modification is introduced by m6A methyltransferases (writers), removed by demethylases (erasers), and recognized by m6A-binding proteins (readers). Current research has demonstrated that m6A methylation is involved in the regulation of malignant phenotypes in tumors by controlling the expression of cancer-related genes. Non-coding RNAs (ncRNAs) are a diverse group of RNA molecules that do not encode proteins and are widely present in the human genome. This group includes microRNAs (miRNAs), long non-coding RNAs (lncRNAs), circular RNAs (circRNAs), and PIWI interaction RNAs (piRNAs). They function as oncogenes or tumor suppressors through various mechanisms, regulating the initiation and progression of cancer. Previous studies on m6A primarily focused on coding RNAs, but recent discoveries have revealed the significant regulatory role of m6A in ncRNAs. Simultaneously, ncRNAs also exert their influence by modulating the stability, splicing, translation, and other biological processes of m6A-related enzymes. The interplay between m6A and ncRNAs collectively contributes to the occurrence and progression of malignant tumors in humans. This review provides an overview of the interactions between m6A regulatory factors and ncRNAs and their impact on tumors.

Clinicopathological features and cancer transcriptomic profiling of poorly cohesive gastric carcinoma subtypes.

Gastric poorly cohesive carcinoma (PCC) manifests with a diffuse pattern and diverse tumor cell morphologies, often indicating a more unfavorable prognosis. Recent consensus has reclassified PCC based on the proportion of signet-ring cells (SRCs) in tumors for research purposes. The two most distinct subtypes, poorly cohesive carcinoma not otherwise specified (PCC-NOS) and signet-ring cell carcinoma (SRCC), are characterized by less than 10% and more than 90% SRCs, respectively. However, research comparing the clinicopathological and transcriptomic differences between these subtypes remains limited. In this study, we conducted a comparative analysis of clinicopathological features in 55 advanced-stage PCCs, consisting of 43 PCC-NOS and 12 SRCC cases. Subsequently, 12 PCC-NOS and 5 SRCC cases were randomly selected for initial cancer-related gene expression profiling and pathway enrichment analysis using the GeoMx digital spatial profiler, followed by validation in a separate validation group comprising 16 PCC-NOS and 6 SRCC cases. These transcriptomic findings were then correlated with tumor morphology and clinicopathological data. PCC-NOS cases exhibited larger tumor size, a higher prevalence of pathological N3 disease, and a worse 1-year progression-free survival rate compared to SRCC cases. Clustering of PCC-NOS and SRCC was successfully achieved using the GeoMx Cancer Transcriptome Atlas. Among all studied genes, only MMP7 showed differential expression, with its overexpression significantly associated with the PCC-NOS subtype, increased perineural invasion, and earlier disease progression. Pathway analysis revealed significantly enriched pathways in PCC-NOS related to vesicle-mediated transport, adaptive immune systems, oncogenic signaling, and extracellular matrix organization, while SRCC displayed significant enrichment in pathways associated with respiratory electron transport and the cell cycle. In conclusion, this study compares and correlates clinicopathological features and transcriptomic data between PCC-NOS and SRCC at advanced stages, employing the latest consensus classification and a novel platform for analysis.

Molecular mechanisms of extracellular-ATP-mediated colorectal cancer progression: Implication of purinergic receptors-mediated nucleocytoplasmic shuttling of HuR.

One of the leading causes of cancer-related deaths worldwide is colorectal cancer (CRC). Extracellular ATP (e-ATP) and purinergic receptors (P2R) play a central role in CRC proliferation and progression. Human antigen R (HuR) is becoming more and more understood to be essential for the expression of genes linked to cancer. The current study demonstrates that ATP can mediate CRC (Caco-2 cells) progression via induction of HuR nucleocytoplasmic shuttling and subsequent expression of cancer-related genes, a consequence mostly mediated via the P2R receptor. It was also noted that suppression of HuR activity by using dihydrotanshinone I (DHTS) prevents cancer-related gene expression and subsequent CRC (Caco-2 cells) progression induced by ATP. The expression of cyclin A2/cyclin-dependent kinase 2 (CDK2), Bcl-2, ProT-α, hypoxia-inducible factor1-α (HIF1-α), vascular endothelial growth factor A (VEGF-A), transforming growth factor-β (TGF-β) and matrix metallopeptidase 9 (MMP-9) induced by ATP were highly reduced in the presence of either PPADS (non-selective P2R antagonist) or DHTS. In addition, e-ATP-induced Caco-2 cell proliferation as well as cell survival were highly reduced in the presence of either PPADS or DHTS or selective CDK-2 inhibitor (Roscovitine) or selective Bcl-2 inhibitor (ABT-263). Furthermore, it was found that MMP-9 is critical for Caco-2 cells migration induced by e-ATP as demonstrated by a clear reduction in cells migration in the presence of a selective MMP-9 inhibitor (Marimastat). Collectively, these data demonstrate that ATP through P2R activation can induce HuR nucleocytoplasmic shuttling that could be translated into an increase in cancer-related genes expression and subsequent, cell proliferation and progression.

Exploring non-coding RNA mechanisms in hepatocellular carcinoma: implications for therapy and prognosis.

Hepatocellular carcinoma (HCC) is a significant contributor to cancer-related deaths in the world. The development and progression of HCC are closely correlated with the abnormal regulation of non-coding RNAs (ncRNAs), such as microRNAs (miRNAs), long non-coding RNAs (lncRNAs), and circular RNAs (circRNAs). Important biological pathways in cancer biology, such as cell proliferation, death, and metastasis, are impacted by these ncRNAs, which modulate gene expression. The abnormal expression of non-coding RNAs in HCC raises the possibility that they could be applied as new biomarkers for diagnosis, prognosis, and treatment targets. Furthermore, by controlling the expression of cancer-related genes, miRNAs can function as either tumor suppressors or oncogenes. On the other hand, lncRNAs play a role in the advancement of cancer by interacting with other molecules within the cell, which, in turn, affects processes such as chromatin remodeling, transcription, and post-transcriptional processes. The importance of ncRNA-driven regulatory systems in HCC is being highlighted by current research, which sheds light on tumor behavior and therapy response. This research highlights the great potential of ncRNAs to improve patient outcomes in this difficult disease landscape by augmenting the present methods of HCC care through the use of precision medicine approaches.

Effects of miR-330 restoration on pancreatic cancer cells oncogenesis

BackgroundInappropriate expressions of various miRNAs have reported in different human malignancies. Evidence suggested that miR-330 may play as both onco-miR and/or tumor suppressor-miR in different cancers. In the present study, we evaluated effects of miR-330 on proliferation and migration of pancreatic cancer (PC) cells as well as underlying molecular mechanisms. DesignThe expression of miR-330 was evaluated in clinical tissue samples of patients with PC. Transfection of the PC cells (PANC-1) by miR-330 was conducted by pCMV vector. The cancer-related genes expression was investigated in mRNA and protein level following transfection of the PC cells. Furthermore, the PC cells viability, invasion, migration, mitochondrial membrane potential, apoptosis, autophagy, and cell cycle profile were investigated after transfection by miR-330. ResultsThe results indicated that expression of miR-330 downregulated in patients with PC. Stable increase of miR-330 expression after transfection in PC cells reduces viability, mitochondrial membrane potential, invasion, and migration. Further assessments demonstrated that upregulation of miR-330 increases apoptosis and autophagy percentage in the PC cells. Moreover, a cell cycle arrest was observed in G1, Sub-G1, and S phases following transfection of the PC cells. These findings can be explained by modified mRNA and protein expression of apoptosis- and metastasis-related genes. ConclusionOur study suggested that miR-330 acts as a tumor suppressor in PC cells, and revealed that upregulation of miR-330 may provide an effective therapeutic approach for overcoming progression and metastasis in patients with PC.

BRAF mutation, selected miRNAs and genes expression in primary papillary thyroid carcinomas and local lymph node metastases

Mutations in cancer-related genes are now known to be accompanied by epigenetic events in carcinogenesis by modification of the regulatory pathways and expression of genes involved in the pathobiology. Such cancer-related mutations, miRNAs and gene expression may be promising molecular markers of the most common papillary thyroid carcinoma (PTC). However, there are limited data on their relationships. The aim of this study was to analyse the interactions between BRAF mutations, selected microRNAs (miR-21, miR-34a, miR-146b, and miR-9) and the expression of selected genes (LGALS3, NKX2–1, TACSTD2, TPO) involved in the pathogenesis of PTC. The study cohort included 60 primary papillary thyroid carcinomas (PTC) that were classified as classical (PTC/C; n=50) and invasive follicular variant (PTC/F; n=10), and 40 paired lymph node metastases (LNM). BRAF mutation status in primary and recurrent/persistent papillary thyroid carcinomas was determined. The mutation results were compared both between primary and metastatic cancer tissue, and between BRAF mutation status and selected genes and miRNA expression in primary PTC. Furthermore, miRNAs and gene expression were compared between primary PTCs and non-neoplastic tissue, and local lymph node metastatic tumor, respectively. All studied markers showed several significant mutual interactions and contexts. In conclusion, to the best our knowledge, this is the first integrated study of BRAF mutational status, the expression levels of mRNAs of selected genes and miRNAs in primary PTC, and paired LNM.

Similar immune responses to alpha1-oleate and Bacillus Calmette-Guérin treatment in patients with bladder cancer.

The molecular content of urine is defined by filtration in the kidneys and by local release from tissues lining the urinary tract. Pathological processes and different therapies change the molecular composition of urine and a variety of markers have been analyzed in patients with bladder cancer. The response to BCG immunotherapy and chemotherapy has been extensively studied and elevated urine concentrations of IL-1RA, IFN-α, IFN-γ TNF-α, and IL-17 have been associated with improved outcome. In this study, the host response to intravesical alpha 1-oleate treatment was characterized in patients with non-muscle invasive bladder cancer by proteomic and transcriptomic analysis. Proteomic profiling detected a significant increase in multiple cytokines in the treatment group compared to placebo. The innate immune response was strongly activated, including IL-1RA and pro-inflammatory cytokines in the IL-1 family (IL-1α, IL-1β, IL-33), chemokines (MIP-1α, IL-8), and interferons (IFN-α2, IFN-γ). Adaptive immune mediators included IL-12, Granzyme B, CD40, PD-L1, and IL-17D, suggesting broad effects of alpha 1-oleate treatment on the tumor tissues. The cytokine response profile in alpha 1-oleate treated patients was similar to that reported in BCG treated patients, suggesting a significant overlap. A reduction in protein levels at the end of treatment coincided with inhibition of cancer-related gene expression in tissue biopsies, consistent with a positive treatment effect. Thus, in addition to killing tumor cells and inducing cell detachment, alpha 1-oleate is shown to activate a broad immune response with a protective potential.

The effect mechanism of ergosterol from the nutritional mushroom Leucocalocybe mongolica in breast cancer cells: Protein expression modulation and metabolomic profiling using UHPLC-ESI-Q

The ergosterol from mushrooms has gained significant ethnopharmacological importance in various cultures, including China, Japan, and Europe. This compound has been found to possess immune-boosting and anti-inflammatory properties, making it useful in the treatment of immune disorders. In this study, we focused on investigating the potential anticancer properties of ergosterol isolated from the edible mushroom Leucocalocybe mongolica in breast cancer cell lines. The ergosterol was purified and identified using advanced analytical techniques such as ESI-MS and NMR. We conducted cell proliferation assays on 4 T1 breast cancer cells to assess the cytotoxic effects of ergosterol. Furthermore, we analyzed the transcription levels of BAX, caspase-7, BCL-2, STAT-3, and PARP proteins using real-time PCR and Western blot analysis. Additionally, we employed non-targeted ultra-high-performance liquid chromatography and high-resolution mass spectrometry (UPLC-MS/MS) to study the potential mechanisms underlying the anticancer effects of ergosterol at the metabolomics level. The results demonstrated a significant reduction in cell viability and the induction of apoptosis upon treatment with ergosterol, especially at higher concentrations (P < 0.05). Moreover, ergosterol affected the expression of cancer-related genes, upregulating pro-apoptotic proteins such as BAX, caspase-7, and PARP, while downregulating the anti-apoptotic proteins BCL-2 and STAT-3 (P < 0.05). Western blot analysis confirmed these findings and provided further evidence of ergosterol's role in inducing apoptosis. Metabolomics analysis revealed substantial changes in pathways related to amino acid, antioxidant, and carbohydrate metabolism. In conclusion, our study demonstrates that ergosterol exhibits anticancer effects by inducing apoptosis and modulating metabolic pathways in breast cancer cells.

Perfluorooctanesulfonic Acid Alters Pro-Cancer Phenotypes and Metabolic and Transcriptional Signatures in Testicular Germ Cell Tumors.

The potential effects of poly- and perfluoroalkyl substances (PFAS) are a recently emergent human and environmental health concern. There is a consistent link between PFAS exposure and cancer, but the mechanisms are poorly understood. Although epidemiological evidence supporting PFAS exposure and cancer in general is conflicting, there is relatively strong evidence linking PFAS and testicular germ cell tumors (TGCTs). However, no mechanistic studies have been performed to date concerning PFAS and TGCTs. In this report, the effects of the legacy PFAS perfluorooctanesulfonic acid (PFOS) and the newer "clean energy" PFAS lithium bis(trifluoromethylsulfonyl)imide (LiTFSi, called HQ-115), on the tumorigenicity of TGCTs in mice, TGCT cell survival, and metabolite production, as well as gene regulation were investigated. In vitro, the proliferation and survival of both chemo-sensitive and -resistant TGCT cells were minimally affected by a wide range of PFOS and HQ-115 concentrations. However, both chemicals promoted the growth of TGCT cells in mouse xenografts at doses consistent with human exposure but induced minimal acute toxicity, as assessed by total body, kidney, and testis weight. PFOS, but not HQ-115, increased liver weight. Transcriptomic alterations of PFOS-exposed normal mouse testes were dominated by cancer-related pathways and gene expression alterations associated with the H3K27me3 polycomb pathway and DNA methylation, epigenetic pathways that were previously showed to be critical for the survival of TGCT cells after cisplatin-based chemotherapy. Similar patterns of PFOS-mediated gene expression occurred in PFOS-exposed cells in vitro. Metabolomic studies revealed that PFOS also altered metabolites associated with steroid biosynthesis and fatty acid metabolism in TGCT cells, consistent with the proposed ability of PFAS to mimic fatty acid-based ligands controlling lipid metabolism and the proposed role of PFAS as endocrine disrupters. Our data, is the first cell and animal based study on PFAS in TGCTs, support a pro-tumorigenic effect of PFAS on TGCT biology and suggests epigenetic, metabolic, and endocrine disruption as potential mechanisms of action that are consistent with the non-mutagenic nature of the PFAS class.

Abstract 7501: Gene expression profiling of rare cells captured from liquid biopsy using the genesis cell isolation system

Abstract Background: Targeting rare cell types such as circulating tumor cells (CTCs), considered to be metastatic precursors in cancer progression, may provide long-term clinical benefits by enhancing patient outcomes. The study of CTCs comes with technical challenges due to their heterogeneity and low cell numbers, leading to difficulties in genomic characterization. This study aims to overcome these technical challenges by developing a comprehensive gene expression profiling workflow using qPCR and RNA sequencing (RNA-Seq) on rare cells enriched with Genesis System with Celselect SlidesTM. Methods: To mimic CTCs in blood, we created a rare cell model sample by spiking A549 non-small cell lung cancer cells at concentrations of 50, 100, 500 and 1000 cells/ml in whole blood from healthy donors. The Genesis System, which can capture rare cells of 8µm-30µm, isolated 83.73% of spiked A549 cells in whole blood. Using the same blood samples without A549 spiked in, we enriched cells using the Genesis and it was used as a baseline for gene expression controls. High quality RNA was extracted from each of the enriched samples and used for both qPCR and RNA-Seq to detect the gene expression signature of the non-small cell lung cancer cells. Results: Our analyses revealed significant differential expression of cancer-related genes (p &amp;lt; 0.05) in all A549-spiked samples when compared to the blood-only controls. Notably, in the 50 cells/ml dilution, RNA-Seq analysis identified over 3,000 differentially expressed genes, encompassing protein-coding genes, long non-coding RNAs, and small non-coding RNAs. Among these, 759 genes were consistently present across all enriched samples, suggesting a common expression pattern. To investigate the potential utility of these genes as non-small cell lung cancer biomarkers, we constructed a custom qPCR array comprising 38 of these genes and pre-amplified their targets using 50pg of RNA from the enriched cell samples. Notably, six genes from this panel (MET, TOP2A, NF1, SPP1, GAPDH, and ANXA5) exhibited significant differential expression (+/- 4-fold, p&amp;lt;0.05) in the enriched samples. MET and TOP2A, in particular, are well-known biomarkers for non-small cell lung cancer. Conclusion: Our liquid biopsy workflow demonstrates the recovery of high-quality RNA from enriched rare cells, including CTCs. It enables comprehensive profiling of the tumor transcriptome, encompassing RNAs of various sizes, including miRNAs. Our approach has the potential to facilitate the identification of patient-specific biomarkers, enabling swift monitoring using preamplification and qPCR in advancement of targeted oncology therapy and research. Citation Format: Srikanth Perike, Angelica Olcott, Cissy Jiang, Nish Kumar, Nathan Knapp, Jennifer Placek, Candice Cox, Yoon-Tae Kang, Linda Lingelbach, Elizabeth Dreskin. Gene expression profiling of rare cells captured from liquid biopsy using the genesis cell isolation system [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 7501.

Abstract 748: Impact of oral supplementation of sulforaphane for 12 months on lung cancer-related gene expression in former smokers at high risk for lung cancer: Results from a randomized placebo-controlled phase II clinical trial

Abstract Introduction: Higher consumption of dietary sulforaphane (SFN), a phytochemical found in many cruciferous vegetables, has been shown to be associated with lower risk of lung cancer (LC). SF activates the NRF2-pathway, which initiates cellular detoxification, antioxidant, and anti-inflammatory responses. To date, no chemoprevention trial for LC has examined the impact of SFN on LC-related gene expression (GE) as an endpoint in nasal and bronchial tissue. The objective of our study was to examine whether oral intake of SF for 12 months impacts the GE of previously identified LC- and premalignant lesions (PML)-related genes in bronchial and nasal brushing samples. Methods: A randomized, placebo-controlled clinical trial (NCT03232138) was conducted in former smokers with ≥ 30 pack-years and quitting smoking for 1-10 years in Western Pennsylvania, USA. Forty-three subjects were randomly assigned to the SFN treatment (n = 21) or placebo group (n = 22) in 2018-2022. Each participant was instructed to take 4 Avmacol® or identical placebo capsules BID for 12 months. Each Avmacol® capsule contained 15 mg of glucoraphanin, which yielded a daily internal dose of 120 μmol SFN. Nasal swabs and bronchial brushings were obtained at baseline and after 12 months. Among the 37 participants who completed the study, RNA was extracted from their nasal swabs and bronchial brushings. We constructed single sample gene signature scores (GSS) using the GSVA algorithm for the following: 1) the NRF2-pathway, 2) genes associated with LC risk in bronchial biopsies, and 3) genes associated with PML. The genes in these sets were stratified by their up- or down-regulation in LC or PML. We evaluated the association between the interaction of SFN supplementation over time (pre-/post-treatment) on GSS using linear mixed models, adjusted for the fixed effects of age at baseline, RNA integrity number (RIN), packyears of smoking, and random effect of participant. Results: GE data was available for 31 participant pairs with bronchial and/or nasal samples. Within nasal tissue, SFN treatment increased both the GSSs of the NRF2-pathway (p=0.0089) and the downregulated LC-risk genes (p=0.0047), and decreased the upregulated LC-risk genes (p=0.012) over time, whereas SFN had no significant impact on GSSs of the NRF2-pathway and LC-risk genes in the bronchial tissue. No association between SFN treatment over time and the PML-related GSS was observed in nasal or bronchial tissue. Conclusion: Our initial analysis indicates that SFN treatment over 12 months impacted LC-associated GE in nasal tissue but not in bronchial tissue from former smokers. The discrepancy results may be due to small sample size and additional studies are warranted to confirm if SFN has direct impact on LC-risk related genes in bronchial tissue (NIH grant No. R01CA213123). Citation Format: Kathryn Demanelis, Avrum Spira, Yohana Kefella, Renwei Wang, Jennifer Adams-Haduch, Thomas W. Kensler, Jennifer E. Beane, David O. Wilson, Jian-Min Yuan. Impact of oral supplementation of sulforaphane for 12 months on lung cancer-related gene expression in former smokers at high risk for lung cancer: Results from a randomized placebo-controlled phase II clinical trial [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 748.

Molecular pathological approach to cancer epigenomics and its clinical application.

Careful microscopic observation of histopathological specimens, accumulation of large numbers of high-quality tissue specimens, and analysis of molecular pathology in relation to morphological features are considered to yield realistic data on the nature of multistage carcinogenesis. Since the morphological hallmark of cancer is disruption of the normal histological structure maintained through cell-cell adhesiveness and cellular polarity, attempts have been made to investigate abnormalities of the cadherin-catenin cell adhesion system in human cancer cells. It has been shown that the CDH1 tumor suppressor gene encoding E-cadherin is silenced by DNA methylation, suggesting that a "double hit" involving DNA methylation and loss of heterozygosity leads to carcinogenesis. Therefore, in the 1990s, we focused on epigenomic mechanisms, which until then had not received much attention. In chronic hepatitis and liver cirrhosis associated with hepatitis virus infection, DNA methylation abnormalities were found to occur frequently, being one of the earliest indications that such abnormalities are present even in precancerous tissue. Aberrant expression and splicing of DNA methyltransferases, such as DNMT1 and DNMT3B, was found to underlie the mechanism of DNA methylation alterations in various organs. The CpG island methylator phenotype in renal cell carcinomawas identified for the first time, and its therapeutic targets were identified by multilayer omics analysis. Furthermore, the DNA methylation profile of nonalcoholic steatohepatitis (NASH)-related hepatocellular carcinomawas clarified in groundbreaking studies. Since then, we have developed diagnostic markers for carcinogenesis risk in NASH patients and noninvasive diagnostic markers for upper urinary tract cancer, as well as developing a new high-performance liquid chromatography-based diagnostic system for DNA methylation diagnosis. Research on the cancer epigenome has revealed that DNA methylation alterations occur from the precancerous stage as a result of exposure to carcinogenic factors such as inflammation, smoking, and viral infections, and continuously contribute to multistage carcinogenesis through aberrant expression of cancer-related genes and genomic instability. DNA methylation alterations at the precancerous stages are inherited by or strengthened in cancers themselves and determine the clinicopathological aggressiveness of cancers as well as patient outcome. DNA methylation alterations have applications as biomarkers, and are expected to contribute to diagnosis, as well as preventive and preemptive medicine.

Region-specific DNA hydroxymethylation along the malignant progression of IDH-mutant gliomas.

The majority of low-grade isocitrate dehydrogenase-mutant (IDHmt) gliomas undergo malignant progression (MP), but their underlying mechanism remains unclear. IDHmt gliomas exhibit global DNA methylation, and our previous report suggested that MP could be partly attributed to passive demethylation caused by accelerated cell cycles. However, during MP, there is also active demethylation mediated by ten-eleven translocation, such as DNA hydroxymethylation. Hydroxymethylation is reported to potentially contribute to gene expression regulation, but its role in MP remains under investigation. Therefore, we conducted a comprehensive analysis of hydroxymethylation during MP of IDHmt astrocytoma. Five primary/malignantly progressed IDHmt astrocytoma pairs were analyzed with oxidative bisulfite and the Infinium EPIC methylation array, detecting 5-hydroxymethyl cytosine at over 850,000 locations for region-specific hydroxymethylation assessment. Notably, we observed significant sharing of hydroxymethylated genomic regions during MP across the samples. Hydroxymethylated CpGs were enriched in open sea and intergenic regions (p < 0.001), and genes undergoing hydroxymethylation were significantly associated with cancer-related signaling pathways. RNA sequencing data integration identified 91 genes with significant positive/negative hydroxymethylation-expression correlations. Functional analysis suggested that positively correlated genes are involved in cell-cycle promotion, while negatively correlated ones are associated with antineoplastic functions. Analyses of The Cancer Genome Atlas clinical data on glioma were in line with these findings. Motif-enrichment analysis suggested the potential involvement of the transcription factor KLF4 in hydroxymethylation-based gene regulation. Our findings shed light on the significance of region-specific DNA hydroxymethylation in glioma MP and suggest its potential role in cancer-related gene expression and IDHmt glioma malignancy.

Designing Potential Antisense Oligonucleotides (ASO) against Lung Cancer Associated microRNA and Studying their Interaction

Abstract: MicroRNAs (miRNAs) are an abundant class of small non-protein-coding RNAs that function as gene regulators. They regulate diverse biological processes and bioinformatics data indicates that each miRNA can control hundreds of gene targets, underscoring the potential influence of miRNAs on almost every genetic pathway. miRNA mutations or mis-expression correlate with various human cancers and miRNAs can function as tumor suppressors and oncogenes. miRNAs have been shown to repress the expression of important cancer-related genes and might prove useful in the diagnosis and treatment of cancer. In this context, we identified various miRNAs associated with the prevalent cancer phenotype NSCLC/SCLC and used the respective target miRNA sequences as templates for designing antisense oligonucleotides (ASOs). We investigated the interaction of this cognate oligonucleotides and target miRNAs to hypotheses the inhibitory potential of such oligonucleotides through molecular docking studies.

Genetic Analysis of Locally Advanced Well-Differentiated Thyroid Cancer: Clinical Prognostic Implications

Background and Objectives Despite the usually favorable prognosis of well-differentiated thyroid cancer (WDTC) following appropriate treatment, advanced T-staged WDTCs are associated with poor prognosis. This study focused on identifying genes associated with the prognosis of locally advanced WDTC by analyzing a The Cancer Genome Atlas cohort.Subjects and Method We analyzed the data of 501 patients with WDTC and classified them into two subgroups: pathological T4 stage (Cluster 1) or T1-3 stage (Cluster 2). We compared the mRNA expressions of thyroid cancer-related genes, and the somatic mutation frequencies of various cancer genes between the two subgroups.Results Cluster 1 included 23 patients (papillary=21/follicular-variant papillary thyroid cancer [FVPTC]=2) and Cluster 2 478 patients (papillary=371/FVPTC=100/others=7). Cluster 1 showed worse overall and disease-free survival than Cluster 2 (&lt;i&gt;p&lt;/i&gt;&lt;0.05 and &lt;i&gt;p&lt;/i&gt;=0.12, respectively). Patients with pT4 stage had about a 1.8-fold increased risk of overall recurrence or death. &lt;i&gt;MET, SERPINA1, TIMP1, PROS1, FN1, CDKN2A&lt;/i&gt;, and &lt;i&gt;CDKN2B&lt;/i&gt; were significantly elevated while &lt;i&gt;TG, DNAH9, TFF3, CRABP1, TPO, JAK2, KIT, KDR&lt;/i&gt;, and &lt;i&gt;NFE2L2&lt;/i&gt; were significantly lower in Cluster 1 (all, &lt;i&gt;p&lt;/i&gt;&lt;0.05 and adjusted &lt;i&gt;p&lt;/i&gt;&lt;0.05). A &lt;i&gt;TERT, EIF1AX&lt;/i&gt;, and &lt;i&gt;ATM&lt;/i&gt; showed significantly frequent somatic mutations in Cluster 1 when compared to Cluster 2. We also identified seven pathways related to 16 genetic markers.Conclusion Locally advanced WDTC presented 16 genetic alterations. We identified somatic mutations associated with local invasion transformation. Relevant pathways related to the 16 genetic markers could be therapeutic targets. Genetic analysis of locally advanced WDTC may be used to predict clinical applications in patient survival.

DDX5 enhances HIF-1 activity by promoting the interaction of HIF-1α with HIF-1β and recruiting the resulting heterodimer to its target gene loci.

Cancer cells acquire malignant characteristics and therapy resistance by employing the hypoxia-inducible factor 1 (HIF-1)-dependent adaptive response to hypoxic microenvironment in solid tumors. Since the underlying molecular mechanisms remain unclear, difficulties are associated with establishing effective therapeutic strategies. We herein identified DEAD-box helicase 5 (DDX5) as a novel activator of HIF-1 and found that it enhanced the heterodimer formation of HIF-1α and HIF-1β and facilitated the recruitment of the resulting HIF-1 to its recognition sequence, hypoxia-response element (HRE), leading to the expression of a subset of cancer-related genes under hypoxia. This study reveals that the regulation of HIF-1 recruitment to HRE is an important regulatory step in the control of HIF-1 activity. The present study provides novel insights for the development of strategies to inhibit the HIF-1-dependent expression of cancer-related genes.

Dissection of an impact of VDR and RXRA on the genomic activity of 1,25(OH)2D3 in A431 squamous cell carcinoma

BackgroundHuman skin is the natural source, place of metabolism, and target for vitamin D3. The classical active form of vitamin D3, 1,25(OH)2D3, expresses pluripotent properties and is intensively studied in cancer prevention and therapy. To define the specific role of vitamin D3 receptor (VDR) and its co-receptor retinoid X receptor alpha (RXRA) in genomic regulation, VDR or RXRA genes were silenced in the squamous cell carcinoma cell line A431 and treated with 1,25(OH)2D3 at long incubation time points 24 h/72 h. Extending the incubation time of A431 WT (wild-type) cells with 1,25(OH)2D3 resulted in a two-fold increase in DEGs (differentially expressed genes) and a change in the amount of downregulated from 37% to 53%. VDR knockout led to a complete loss of 1,25(OH)2D3-induced genome-wide gene regulation at 24 h time point, but after 72 h, 20 DEGs were found, of which 75% were downregulated, and most of them belonged to the gene ontology group “immune response”. This may indicate the existence of an alternative, secondary response to 1,25(OH)2D3. In contrast, treatment of A431 ΔRXRA cells with 1,25(OH)2D3 for 24 h only partially affected DEGs, suggesting RXRA-independent regulation. Interestingly, overexpression of classic 1,25(OH)2D3 targets, like CYP24A1 (family 24 of subfamily A of cytochrome P450 member 1) or CAMP (cathelicidin antimicrobial peptide) was found to be RXRA-independent. Also, immunofluorescence staining of A431 WT cells revealed partial VDR/RXRA colocalization after 24 h and 72 h 1,25(OH)2D3 treatment.Comparison of transcriptome changes induced by 1,25(OH)2D3 in normal keratinocytes vs. cancer cells showed high cell type specific expression pattern with only a few genes commonly regulated by 1,25(OH)2D3. Activation of the genomic pathway at least partially reversed the expression of cancer-related genes, forming a basis for anti-cancer activates of 1,25(OH)2D3. In summary, VDR or RXRA independent genomic activities of 1,25(OH)2D3 suggest the involvement of alternative factors, opening new challenges in this field.