Regulation Of Tumor Suppressor Genes Research Articles

Minimally Invasive and Emerging Diagnostic Approaches in Endometrial Cancer: Epigenetic Insights and the Promise of DNA Methylation

Endometrial cancer (EC) is the most common gynecological malignancy, with rising incidence and mortality rates. Key risk factors, including obesity, prolonged estrogen exposure, and metabolic disorders, underscore the urgent need for non-invasive, early diagnostic tools. This review focuses on the role of DNA methylation as a potential biomarker for early EC detection. Aberrant DNA methylation in the promoter regions of tumor suppressor genes can lead to gene silencing and cancer progression. We examine recent studies utilizing minimally invasive samples, such as urine, cervicovaginal, and cervical scrapes, to detect early-stage EC through DNA methylation patterns. Markers such as RASSF1A, HIST1H4F, GHSR, SST, and ZIC1 have demonstrated high diagnostic accuracy, with AUC values up to 0.95, effectively distinguishing EC from non-cancerous conditions. This review highlights the potential of DNA methylation-based testing as a non-invasive alternative to traditional diagnostic methods, offering earlier detection, better risk stratification, and more personalized treatment plans. These innovations hold the promise of transforming clinical practice by enabling more timely and effective management of endometrial cancer.

Activation of AMPK inhibits cervical cancer growth by hyperacetylation of H3K9 through PCAF

BackgroundDysregulation in histone acetylation, a significant epigenetic alteration closely associated with major pathologies including cancer, promotes tumorigenesis, inactivating tumor-suppressor genes and activating oncogenic pathways. AMP-activated protein kinase (AMPK) is a cellular energy sensor that regulates a multitude of biological processes. Although a number of studies have identified the mechanisms by which AMPK regulates cancer growth, the underlying epigenetic mechanisms remain unknown.MethodsThe impact of metformin, an AMPK activator, on cervical cancer was evaluated through assessments of cell viability, tumor xenograft model, pan-acetylation analysis, and the role of the AMPK-PCAF-H3K9ac signaling pathway. Using label-free quantitative acetylproteomics and chromatin immunoprecipitation-sequencing (ChIP) technology, the activation of AMPK-induced H3K9 acetylation was further investigated.ResultsIn this study, we found that metformin, acting as an AMPK agonist, activates AMPK, thereby inhibiting the proliferation of cervical cancer both in vitro and in vivo. Mechanistically, AMPK activation induces H3K9 acetylation at epigenetic level, leading to chromatin remodeling in cervical cancer. This also enhances the binding of H3K9ac to the promoter regions of multiple tumor suppressor genes, thereby promoting their transcriptional activation. Furthermore, the absence of PCAF renders AMPK activation incapable of inducing H3K9 acetylation.ConclusionsIn conclusion, our findings demonstrate that AMPK mediates the inhibition of cervical cancer growth through PCAF-dependent H3K9 acetylation. This discovery not only facilitates the clinical application of metformin but also underscores the essential role of PCAF in AMPK activation-induced H3K9 hyperacetylation.

Ultrasensitive Detection of Tumor Suppressor Gene Methylation by Piezoelectric Sensing Based on Enrichment of Transcription Activator-Like Effectors.

The detection of DNA methylation at cytosine/guanine dinucleotide (CpG) islands in promoter regions of tumor suppressor genes has great potential for early cancer screening, diagnosis, and prognosis monitoring. Nevertheless, achieving accurate, sensitive, cost-effective, and quantitative detection of target methylated DNA remains challenging. Herein, we propose a novel piezoelectric sensor (series piezoelectric quartz crystal (SPQC)) based on transcription activator-like effectors (TALEs) for detecting DNA methylation of Ras association domain family 1 isoform A (RASSF1A) tumor suppressor genes (R-5mC). The sensor employs TALEs-Ni magnetic beads to specifically recognize and separate the R-5mC, thereby improving the detection selectivity. The TALEs-Ni magnetic beads-R-5mC complex is sheared by a nucleic acid enzyme (DNAzyme) to release the single-stranded DNA (ST). ST initiates a catalyzed hairpin assembly (CHA) reaction on the surface of the electrode, which in turn triggers the hybridization chain reaction (HCR) and silver staining for enhanced detection sensitivity. The strategy exhibits a linear response in the detection of R-5mC in the range of 1 fM to 1 nM with a detection limit of 0.79 fM. R-5mC as low as 0.01% can be detected, even in the presence of large numbers of unmethylated DNA. The detection of R-5mC in circulating cell-free DNA (cfDNA) derived from clinical plasma specimens of lung cancer patients yielded satisfactory results.

Optimizing Droplet Digital PCR Assay for Precise Assessment of MEIS1 Gene Promoter Methylation

Abstract DNA methylation is characterized as a gene regulatory mechanism that involves the methylation of the 5-carbon (C5) position of cytosine, resulting in the formation of 5-methylcytosine. The analysis of aberrantly methylated cytosine-phosphate-guanine (CpG) dinucleotides, primarily in the promoter regions of tumor suppressor genes, can serve as promising prognostic and predictive markers of cancer development. Meis homeobox 1 (MEIS1) gene, crucial for cell growth and differentiation, exhibits dysregulation linked to various cancer types, acting as both a positive and negative regulator. The selection of an appropriate method for the evaluation of gene promoter methylation status is important for clinical implementation without biases regarding false positive and false negative outcomes. The study focuses on the optimization of a novel droplet digital PCR (ddPCR) assay for identifying the methylation status of MEIS1. Compared to traditional methods, ddPCR offers an increased sensitivity and specificity, presenting a promising tool for precise DNA methylation assessment with potential implications for cancer diagnostics and prognostics.

Abstract 3647: Digital spatial transcriptomic reveals different epithelial and immune cells signatures across endometriosis-related diseases: From simple ovarian endometriosis to ovarian cancer

Abstract Endometriosis represents a pressing medical concern, imposing a relevant burden on patients including potential malignant transformation into endometriosis-associated ovarian cancer (EAOC). Despite the process of cancer development remains unclear, immune cells abnormalities arose as putative biomarkers in the neoplastic progression. One-third of ovarian endometriosis cases exhibit PD-1/PD-L1 expression levels comparable to those observed in cancer. To substantiate the biological basis of these findings, this study aimed to elucidate the spatial transcriptomic profile of epithelial and immune components from different endometriosis-related conditions, along with their genetic interplay. Formalin-fixed paraffine embedded (FFPE) specimens from 4 ovarian endometriosis (OE) cases, two of which displayed elevated levels of PD-1/PD-L1 expression and were referred to as cancer-like endometriosis (CLE), 1 Clear Cell Ovarian Cancer (CCOC) and 1 Endometrioid Ovarian Cancer (EOC) were cut in 5um section for spatial transcriptomic analysis using GeoMX Human Whole Transcriptome Atlas (WTA) probe-set (>18000 genes). For all sections, the regions of interest (ROIs) were identified using a set of morphology markers: anti-human CD3, CD68 and PanCK antibodies. Spatial transcriptomic analysis revealed condition-specific gene enrichment of epithelial and immune cells across the different conditions. In spite of similar H&E findings, PanCKpos epithelial cells from CLE showed an increased expression of genes involved in stress response and defense regulation pathways (e.g. CXCL6, S100A9, and PECAM-1), when compared to OE, in which PanCKpos cells were enriched in DUSP1/2 and VIM. Interestingly, in CCOC, location-specific gene-enrichment was found in PanCKpos cells, with a significant up regulation of tumor suppressor genes (e.g. EGR-1, NR4A1) in normal-looking peri-tumoral epithelium compared to cancerous intra-tumoral PanCKpos cells. Similarly, in the immune compartment, spatial transcriptomic analysis revealed that CD3pos cells from CLE displayed an up-regulation of genes involved in IL-17 signaling, response to oxydative stress and complement activation (e.g. CXCL6, HMOX1, S100A9, and PECAM-1), when compared to OE. Moreover, in CCOC, CD68pos cells of the tumoral mass showed over-expression of tumor growth and progression markers (e.g. SPP1) compared to their counterpart in the outer area of the tumoral mass. In this first comprehensive spatial transcriptomic study of endometriosis-related diseases with GeoMx WTA, our findings uncovered remarkable differences in epithelial and immune cells among different stages and tissue locations. Importantly, CLE emerged as a transitional entity with a unique immune profile. Ongoing analysis of six additional samples will further refine our results. Citation Format: Valentina Iacobelli, Camilla Nero, Stefano Alivernini, Giulia Scaglione, Floriana Camarda, Francesca Sillano, Rita Trozzi, Alessia Piermattei, Barbara Tolusso, Giulia Mantini, Luciano Giacò, Gloria Anderson, Gian Franco Zannoni, Elisa Gremese, Anna Fagotti, Giovanni Scambia. Digital spatial transcriptomic reveals different epithelial and immune cells signatures across endometriosis-related diseases: From simple ovarian endometriosis to ovarian cancer [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 3647.

Abstract IA020: Targeting transposable elements with epigenetic modulators to reverse ovarian cancer immune evasion

Abstract Transposable elements (TEs) comprise the majority of the human genome. In most somatic tissues, TEs are silenced by DNA methylation and other epigenetic modifications. Tumors exhibit changes in DNA methylation including global loss of methylation at regions that are silenced for genome stability, like TEs, and gain of methylation at the promoter regions of tumor suppressor genes. We showed that low doses of DNMT inhibitors (DNMTis) upregulate immune signaling in solid tumor cell lines and patient samples. DNMTis activate type I interferon signaling by reducing methylation and increasing expression of endogenous retroviruses (ERVs), a type of TE, that activate dsRNA sensors. DNMTis plus HDACis (histone deacetylase inhibitors, which increase histone acetylation) increase TEs in a mouse model of ovarian cancer, activating interferon signaling and recruiting CD8+ T cells to kill cancer cells and sensitize to immune checkpoint blockade therapy. We performed a genome-wide analysis of the response to DNMTi, HDACi, and combination HDACi/DNMTi in four ovarian cancer cell lines (RNA-Seq, global methylome profiling, ATAC-Seq, and P53 chIP-Seq). The addition of HDACis to DNMTis augments the upregulation of specific ERVs and the resulting downstream interferon response in human ovarian cancer cell lines. Other TE species, including LINE1 and Alu elements, are increased by DNMTi and HDACi and may contribute to the interferon response. Alu elements, which can directly bind to the dsRNA sensor MDA5, are the most significantly demethylated TEs after DNMTi treatment. Importantly, we find considerable overlap between TEs upregulated by DNMTi in cell lines and TEs upregulated by DNMTi in ovarian cancer patients from a DNMTi clinical trial. We observed that ovarian cancer cell lines with TP53 mutations exhibited significantly fewer upregulated TEs with epigenetic therapy than wild-type TP53 cell lines. This observation was validated using isogenic cell lines; the CRISPR-edited TP53-mutant cell line had significantly higher baseline expression of TEs but upregulated fewer upon epigenetic treatment. p53 bound directly to TE DNA by chIP-Seq analysis and upregulated TEs in wild type but not mutant cell lines. In addition, our RNA-sequencing analysis indicated that A-to-I RNA editing of TEs by ADAR1 was increased after DNMTi treatment. This A-to-I editing makes TE RNA less immunogenic and inhibits the immune response to DNMTi. We find that combining ADAR1 knockdown and DNMTi treatment significantly increases type I interferon signaling, recruitment and activation of host immune cells, and survival in a murine model of ovarian cancer. These data thus give a comprehensive, genome-wide picture of TE chromatin and transcription-related changes in ovarian cancer after epigenetic treatment and implicate novel regulators (p53 and ADAR1) in response to epigenetic therapies for cancer. Citation Format: Katherine B. Chiappinelli. Targeting transposable elements with epigenetic modulators to reverse ovarian cancer immune evasion [abstract]. In: Proceedings of the AACR Special Conference on Ovarian Cancer; 2023 Oct 5-7; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_2):Abstract nr IA020.

Abstract 2345 Inhibition of Cervical Cancer Cell Function by Cannabinoid-Containing Product, BRF1A is Associated with Regulation of Tumor Suppressor Gene

Abstract 2345 Inhibition of Cervical Cancer Cell Function by Cannabinoid-Containing Product, BRF1A is Associated with Regulation of Tumor Suppressor Gene

EZH2-interacting lncRNAs contribute to gastric tumorigenesis; a review on the mechanisms of action.

Gastric cancer (GC) remains one of the deadliest malignancies worldwide, demanding new targets to improve its diagnosis and treatment. Long non-coding RNAs (lncRNAs) are dysregulated through gastric tumorigenesis and play a significant role in GC progression and development. Recent studies have revealed that lncRNAs can interact with histone-modifying polycomb protein, enhance Zeste Homolog 2 (EZH2), and mediate its site-specific functioning. EZH2, which functions as an oncogene in GC, is the catalytic subunit of the PRC2 complex that induces H3K27 trimethylation and epigenetically represses gene expression. EZH2-interacting lncRNAs can recruit EZH2 to the promoter regions of various tumor suppressor genes and cause their transcriptional deactivation via histone methylation. The interactions between EZH2 and this lncRNA modulate different processes, such as cell cycle, cell proliferation and growth, migration, invasion, metastasis, and drug resistance, in vitro and in vivo GC models. Therefore, EZH2-interacting lncRNAs are exciting targets for developing novel targeted therapies for GC. Subsequently, this review aims to focus on the roles of these interactions in GC progression to understand the therapeutic value of EZH2-interacting lncRNAs further.

Epigenetic modifications in solid tumor metastasis in people of African ancestry.

This review focuses on the critical role of epigenetic modifications in solid tumor metastasis, particularly in people of African ancestry. Epigenetic alterations, such as DNA methylation, histone modifications, alterations in non-coding RNAs, and mRNA methylation, significantly influence gene expression, contributing to cancer development and progression. Despite the primary focus on populations of European, American, and Asian descent in most cancer research, this work emphasizes the importance of studying the unique genetic and epigenetic landscapes of African populations for a more inclusive approach in understanding and treating cancer. Insights from this review have the potential to pave the way for the development of effective, tailored treatments, and provide a richer resource for understanding cancer progression and metastasis. Specific focus was placed on the role of DNA methylation, histone modifications, non-coding RNAs, and mRNA methylation in solid tumor metastasis, including how these modifications contribute to the regulation of tumor suppressor genes and oncogenes, influence cellular pathways and signaling, and interact with the immune system. Moreover, this review elaborates on the development of epigenetic-targeted therapeutic strategies and the current advances in this field, highlighting the promising applications of these therapies in improving outcomes for African ancestry populations disproportionately affected by certain types of cancer. Nevertheless, this work acknowledges the challenges that lie ahead, particularly the under-representation of African populations in cancer genomic and epigenomic studies and the technical complications associated with detecting subtle epigenetic modifications. Emphasis is placed on the necessity for more inclusive research practices, the development of more robust and sensitive methods for detecting and interpreting epigenetic changes, and the understanding of the interplay between genetic and epigenetic variations. The review concludes with an optimistic outlook on the future of epigenetic research in People of African ancestry, urging the concerted efforts of researchers, clinicians, funding agencies, and policymakers to extend the benefits of this research to all populations.

The differential regulation of tumor suppressor genes (SAMD9, SPRED1, TGFBI, DUSP6, CDX2, TP53) and MAPK/ERK signaling pathway in colorectal cancer

The differential regulation of tumor suppressor genes (SAMD9, SPRED1, TGFBI, DUSP6, CDX2, TP53) and MAPK/ERK signaling pathway in colorectal cancer

Functional loss of tumor suppressor genes detected by loss of heterozygosity, but not driver mutations, predicts aggressive lymph node status in papillary thyroid carcinoma

BackgroundRecognizing aggressive tumor biology is essential to optimizing patient management for papillary thyroid carcinomas (PTC). Aggressive lymph node (ALN) status is one feature that influences decision-making. We evaluated genomic deletions in regions of tumor suppressor genes, detected by loss of heterozygosity (LOH) analysis, to understand causal alterations linked to thyroid cancer aggressiveness and to serve as a molecular diagnostic biomarker for ALN status. MethodsWe analyzed 105 primary PTC enriched for patients with ALN (64% with, 36% without). We also analyzed 39 positive lymph nodes (79% with, 21% without ALN). LOH was determined using a panel of 25 polymorphic microsatellite alleles targeting 10 genomic loci harboring common tumor suppressor genes. Additionally, ThyGeNEXT® and ThyraMIR® assays were performed. ResultsLOH was detected in 43/67 primary PTC from patients with ALN status, compared with only 5/38 primary PTC without ALN (minimal metastatic burden) (P=0.0000003). This is further supported by post hoc analyses of paired primary and metastatic samples. Paired samples from patients with ALN are more likely to harbor LOH, compared to the ALN negative group (P=0.0125). Additionally, 12/31 paired samples from patients with ALN demonstrated additional or different LOH loci in metastatic samples compared to the primary tumor samples. No association was seen between ALN and mutational, translocation, or microRNA data. ConclusionsLOH detected in primary PTC significantly predicts ALN status. Analysis of paired primary and metastatic samples from patients with / without ALN status further supports this relationship. The acquisition of LOH at additional loci is common in lymph nodes from patients with ALN status. Simple summaryA subset of patients with papillary thyroid carcinoma (PTC) will develop recurrent disease. One known predictor of recurrence is the American Thyroid Association category “Aggressive Lymph Node” (ALN) disease, considering metastatic burden. Loss of heterozygosity (LOH) — chromosomal loss in regions of tumor suppressor genes — has yet to be investigated as a possible mechanism driving ALN status in PTC. The ability to predict ALN status prior to surgery can guide the extent of surgery and postoperative treatment options. We found that paired samples from patients with ALN are more likely to harbor LOH, compared to patients without ALN disease. 38% of patients with ALN demonstrated additional or different LOH loci in metastatic samples compared to the primary tumor samples. LOH complements current molecular analysis of thyroid cancer when searching for evidence of aggressive biology.

Hyper-methylation of ABCG1 as an epigenetics biomarker in non-small cell lung cancer.

Non-small cell lung cancer (NSCLC) is the most prevalent histological type of lung cancer and the leading cause of death globally. Patients with NSCLC have a poor prognosis for various factors, and a late diagnosis is one of them. The DNA methylation of CpG island sequences found in the promoter regions of tumor suppressor genes has recently received attention as a potential biomarker of human cancer. In this study, we report DNA methylation changes of the adenosine triphosphate (ATP)-binding cassette transporter G1 (ABCG1), which belongs to the ATP cassette transporter family in NSCLC patients. Our results demonstrate that ABCG1 is hyper-methylation in NSCLC samples, and these changes are negatively correlated to gene and protein expression. Furthermore, the expression of the ABCG1 gene is significantly associated with the survival time of lung adenocarcinoma (LUAD) patients; however, it did not show a correlation to overall survival (OS) of lung squamous cell carcinoma (LUSC) patients. Notably, we found ABCG1 methylation status at locus cg20214535 is strongly associated with the survival time and consistently observed hyper-methylation in LUAD samples. This novel finding suggests ABCG1 is a potential candidate for targeted therapy in lung cancer via this specific probe. In addition, we illustrate the protein-protein interaction (PPI) of ABCG1 with other proteins and the strong communication of ABCG1 with immune cells.

Abstract 6003: Study of secondary epimutations in cis of the promoter in germline tumor suppressor genes as a cause of hereditary breast and ovarian cancer

Abstract Cancer is a multifactorial and highly heterogeneous disease. Breast cancer (BC) is the main malignant neoplasm in women and has the highest incidence and mortality rates worldwide. About 10% of all BC cases are hereditary and its most common form is Hereditary Breast and Ovarian Cancer Syndrome (HBOC).In a study conducted by our research group in a cohort of 300 Mexican women with HBOC using a panel of 143 genes of susceptibility to different types of cancer (Quezada-Urban et al., 2018), we found that 74% of patients were negative for variants in the coding regions and only 15% of patients had at least one pathogenic variant in 18 different genes. In several types of hereditary cancer syndromes, it has been shown that germline genetic alterations such as single nucleotide variations, and indels in cis of the promoter region of tumor suppressor genes modify the methylation pattern and cause epigenetic silencing and the concomitant somatic loss of heterozygosity. These events are known as secondary cis epimutations and represent an alternative mechanism of molecular pathogenesis in patients with hereditary cancer syndromes. In this work we are investigating in germline gDNA the methylation profile in the promoter region of 18 tumor suppressor genes in 231 patients negative for pathogenic variants and 156 matched controls by using direct bisulfite massive parallel sequencing. So far, we have extracted gDNA and treated with sodium bisulfite from all patient and control samples. From the converted gDNA, we amplified the promoter region of the 18 study genes by PCR (9,675 endpoint PCR products). We have prepared a total of 387 genomic libraries from which we perform massive sequencing and bioinformatic analysis of 229 patients, 74 controls and 2 positive controls. Currently, we have found 4 patients with promoter hypermethylation in the RAD51C, POLH, EPCAM, and BRCA1 genes that can be generated by a secondary cis epimutation. We have found statistically significant differences in the methylation levels of the promoter regions of the BRCA1/2, PMS2, FANCI, MLH1, PALB2, POLH, ERCC3, ATM, MSH2, BRIP1, and EPCAM genes in patients compared with controls. This result suggests that low level basal methylation marks could be potential biomarkers of HBOC pathogenesis. Interestingly, our studies indicate that hypermethylation at the promoter of MSH2, PALB2, BRIP1, and EPCAM are associated with HBOC risk, and that specific hypermethylation at the CpG sites of MSH2 and PALB2 (cg47630224 and cg23652916, respectively), could be potential biomarkers in HBOC risk. Lastly, according to preliminary in silico results, the co-occurrence of site-specific hypermethylation in MSH2 and PALB2 is possibly due to the absence of interaction of the common transcription factor SP4 in a GC-box site. These findings expand our vision of the genetic and epigenetic mechanisms of susceptibility to this disease. Citation Format: Miguel Ruiz De La Cruz, Héctor Martínez Gregorio, Clara Estela Díaz Velásquez, Aldo Hugo De La Cruz Montoya, Fidel de la Cruz Hernández-Hernández, Felipe Vaca Paniagua. Study of secondary epimutations in cis of the promoter in germline tumor suppressor genes as a cause of hereditary breast and ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6003.

Genetic Characterization of Ovarian Tumor Tissues from Patients with Epithelial Ovarian Cancer in a Philippine Tertiary Hospital: A Descriptive Study.

This study identified genetic variations in ovarian tumor specimens from Filipino epithelial ovarian cancer (EOC) patients using next-generation sequencing. Genomic DNA was isolated from formalin-fixed paraffin-embedded ovarian specimens from 8 chemosensitive and 8 chemoresistant EOC patients. Targeted next-generation sequencing was done to identify mutations in hotspot regions of common oncogenes and tumor-suppressor genes. The mutations were cross-referenced with dbSNP and ClinVar databases to identify previously reported alterations, and potentially damaging variants were predicted using PolyPhen-2. Our study has identified 85 unique variants, 35 in chemosensitive EOC, 22 in chemoresistant EOC, and 28 in both. Chemosensitive EOC specimens had more exonic single nucleotide variants than chemoresistant EOC specimens. Of the 50 oncogenes and tumor suppressor genes, KDR gene had the most frequent variations in EOC patients. Two of the unique KDR variants identified were novel mutations. Thirty-nine unique protein-modifying genetic variants were identified in all specimens, the majority of which have been previously reported in dbSNP and ClinVar. This study was the first non-BRCA genetic analysis done on ovarian cancer in Filipino patients. Next-generation sequencing was able to identify previously reported alterations with known therapeutic implications which may benefit from targeted therapy instead of standard chemotherapy regimen.

Non-asbestiform elongate mineral particles and mesothelioma risk: Human and experimental evidence

The presentations in this session of the Monticello II conference were aimed at summarizing what is known about asbestiform and non-asbestiform elongate mineral particles (EMPs) and mesothelioma risks based on evidence from experimental and epidemiology studies. Dr. Case discussed case reports of mesothelioma over the last several decades. Dr. Taioli indicated that the epidemiology evidence concerning non-asbestiform EMPs is weak or lacking, and that progress would be limited unless mesothelioma registries are established. One exception discussed is that of taconite miners, who are exposed to grunerite. Drs. Mandel and Odo noted that studies of taconite miners in Minnesota have revealed an excess rate of mesothelioma, but the role of non-asbestiform EMPs in this excess incidence of mesothelioma is unclear. Dr. Becich discussed the National Mesothelioma Virtual Bank (NMVB), a virtual mesothelioma patient registry that includes mesothelioma patients' lifetime work histories, exposure histories, biospecimens, proteogenomic information, and imaging data that can be used in epidemiology research on mesothelioma. Dr. Bernstein indicated that there is a strong consensus that long, highly durable respirable asbestiform EMPs have the potential to cause mesothelioma, but there is continued debate concerning the biodurability required, and the dimensions (both length and diameter), the shape, and the dose associated with mesothelioma risk. Finally, Dr. Nel discussed how experimental studies of High Aspect Ratio Engineered Nanomaterials have clarified dimensional and durability features that impact disease risk, the impact of inflammation and oxidative stress on the epigenetic regulation of tumor suppressor genes, and the generation of immune suppressive effects in the mesothelioma tumor microenvironment. The session ended with a discussion of future research needs.

Abstract A027: The oncogenic mutation BRAF-V600E is associated with colorectal cancers expressing low levels of CFTR mRNA

Abstract Background and purpose: The cystic fibrosis transmembrane conductance regulator (CFTR) gene is a tumor suppressor in colorectal cancer (CRC). People with cystic fibrosis, caused by biallelic germline mutations in CFTR, have a higher risk developing CRC. We also found that loss of CFTR is implicated in sporadic CRC. In a study of 90 persons diagnosed with stage II CRC, the 23% of patients with lowest CFTR expression had 30% lower disease-free survival at 3 years. The cause of decreased CFTR expression in this group is not known. We analyzed the TCGA COADREAD study to identify associations between low expression of CFTR and cancer-causing genetic alterations. Of these alterations, only BRAF-V600E mutation correlated with low expression of CFTR. The BRAF-V600E mutation is found in ~10% of CRC and is associated with global promoter hypermethylation leading to down regulation of tumor suppressor genes. Accordingly, our hypothesis was that BRAF-V600E mutation contributes to low expression of CFTR via CFTR promoter DNA methylation. Methods: In the TCGA COADREAD database 283 primary colorectal cancers diagnosed at stages II, III and IV had full information needed for this study: CFTR mRNA expression, somatic mutation, overall survival (OS), and DNA methylation status. OS was visualized using Kaplan-Meier analysis. Association between CFTR expression and methylation was determined by Pearson correlation coefficient (PCC) analysis. Associations between mutations and CFTR expression or methylation were determined by Point Biserial correlation coefficient (PBS) analysis. Results: Kaplan-Meier analysis of the 283 cases showed that overall survival was worse in the 25% of cases with lowest CFTR expression (p=0.035). The BRAF-V600E mutation was significantly associated with lower CFTR expression (PBS r=-0.38, p<0.0001) and 81% of BRAF V600E mutations were found in the 25% of cases with lowest CFTR expression. In contrast mutations in APC and KRAS showed a positive association with CFTR expression (PBS r=0.25, p<0.0001; PBS r=0.13, p=0.028 respectively). The CFTR promoter is hypermethylated in several cancers and BRAF-V600E mutation is associated with aberrant promoter DNA methylation. We examined DNA methylation at 9 CpG sites in the CFTR promoter and found that low CFTR expression significantly associated with increased DNA methylation at these 9 sites (PCC for average of 9 sites r= -0.56, p=<0.001). The association between BRAF-V600E mutation and CFTR methylation was modest but significant (PBS r=0.16, p=0.0063). Conclusions: BRAF-V600E mutation was significantly associated with CRC cases expressing low levels of CFTR, a subset with poor overall survival, as was CFTR promoter methylation. BRAF-V600E showed a modest association with CFTR promoter methylation and so may play a role in down-regulation of CFTR expression. However, association of BRAF-V600E with CFTR promoter methylation was modest compared to its association with CFTR expression suggesting that BRAF-V600E mutation may play additional roles in CRC tumors expressing low levels of CFTR. Citation Format: Patricia Scott, Anna Prizment, Rahul Bhattacharya, Zachary Blankenheim, Nathan Pankratz, Timothy Starr, Robert Cormier. The oncogenic mutation BRAF-V600E is associated with colorectal cancers expressing low levels of CFTR mRNA [abstract]. In: Proceedings of the AACR Special Conference on Colorectal Cancer; 2022 Oct 1-4; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_1):Abstract nr A027.

Molecular Effects of Iodine-Biofortified Lettuce in Human Gastrointestinal Cancer Cells.

Considering the growing number of cancer cases around the world, natural products from the diet that exhibit potential antitumor properties are of interest. Our previous research demonstrated that fortification with iodine compounds is an effective way to improve the antioxidant potential of lettuce. The purpose of the present study was to evaluate the effect of iodine-biofortified lettuce on antitumor properties in human gastrointestinal cancer cell lines, gastric AGS and colon HT-29. Our results showed that extracts from iodine-biofortified lettuce reduce the viability and proliferation of gastric and colon cancer cells. The extracts mediated cell cycle arrest which was accompanied by inactivation of anti-apoptotic Bcl-2 and activation of caspases, as assessed by flow cytometry. However, extracts from lettuce fortified with organic forms of iodine acted more effectively than extracts from control and KIO3-enriched plants. Using quantitative PCR, we detected the increase in pro-apoptotic genes BAD, BAX and BID in AGS cells whereas up-regulation of cell cycle progression inhibitor CDKN2A and downregulation of pro-proliferative MDM2 in HT-29 cells. Interestingly, lettuce extracts led to down-regulation of pro-survival AKT1 and protooncogenic MDM2, which was consistent for extracts of lettuce fortified with organic form of iodine, 5-ISA, in both cell lines. MDM2 downregulation in HT-29 colon cancer cells was associated with RB1 upregulation upon 5-ISA-fortified lettuce extracts, which provides a link to the epigenetic regulation of tumor suppressor genes by RB/MDM2 pathway. Indeed, SEMA3A tumor suppressor gene was hypomethylated and upregulated in HT-29 cells treated with 5-ISA-fortified lettuce. Control lettuce exerted similar effects on RB/MDM2 pathway and SEMA3A epigenetic activation in HT-29 cells. Our findings suggest that lettuce as well as lettuce fortified with organic form of iodine, 5-ISA, may exert epigenetic anti-cancer effects that can be cancer type-specific.

CREPT Disarms the Inhibitory Activity of HDAC1 on Oncogene Expression to Promote Tumorigenesis.

Simple SummaryIt has been proposed that highly expressed HDAC1 (histone deacetylases 1) removes the acetyl group from the histones at the promoter regions of tumor suppressor genes to block their expression in tumors. We here revealed the underlying mechanism that HDAC1 differentially regulates the expression of oncogenes and tumor suppressors. In detail, we found that HDAC1 is unable to occupy the promoters of oncogenes but maintains its occupancy with the tumor suppressors due to its interaction with an oncoprotein, CREPT (cell cycle-related and expression-elevated protein in tumor).Histone deacetylases 1 (HDAC1), an enzyme that functions to remove acetyl molecules from ε-NH3 groups of lysine in histones, eliminates the histone acetylation at the promoter regions of tumor suppressor genes to block their expression during tumorigenesis. However, it remains unclear why HDAC1 fails to impair oncogene expression. Here we report that HDAC1 is unable to occupy at the promoters of oncogenes but maintains its occupancy with the tumor suppressors due to its interaction with CREPT (cell cycle-related and expression-elevated protein in tumor, also named RPRD1B), an oncoprotein highly expressed in tumors. We observed that CREPT competed with HDAC1 for binding to oncogene (such as CCND1, CLDN1, VEGFA, PPARD and BMP4) promoters but not the tumor suppressor gene (such as p21 and p27) promoters by a chromatin immunoprecipitation (ChIP) qPCR experiment. Using immunoprecipitation experiments, we deciphered that CREPT specifically occupied at the oncogene promoter via TCF4, a transcription factor activated by Wnt signaling. In addition, we performed a real-time quantitative PCR (qRT-PCR) analysis on cells that stably over-expressed CREPT and/or HDAC1, and we propose that HDAC1 inhibits CREPT to activate oncogene expression under Wnt signaling activation. Our findings revealed that HDAC1 functions differentially on tumor suppressors and oncogenes due to its interaction with the oncoprotein CREPT.

Abstract 6212: Multiomics analyses reveal multimodal Fusobacterium DNA damage perturbations in colorectal cancer

Abstract Fusobacterium nucleatum is an opportunistic bacterial pathogen and oncogenic microbe. To systematically investigate Fusobacterium-host interactions in colorectal cancers (CRCs), using host sequencing data, we developed a multi-omics PAThogen CHaracterization tool (PATCH) allowing for gene-level annotations. In 1,020 CRC transcriptomes and 28 genomes from The Cancer Genome Atlas (TCGA), we detected 9 Fusobacterium species and genes encoding 2,346 proteins, including carcinogenic virulence factors, oxidative stress chaperones, and immune response modulators. Fusobacterium was absent in tumor-adjacent normal tissue, however showed variable intra-tumor heterogeneity. PATCH-defined Fusobacterium-positive CRCs confirmed known and revealed novel perturbations in several host DNA damage responses (DDR) mechanisms, including deficiencies in telomere maintenance pathways, elevated cytosolic DNA/RNA sensing signaling, and mutations in genes involved in multiple oncogenic-related pathways. Moreover, PATCH uncovered Fusobacterium’s DNA integrated into the coding regions of tumor suppressor genes in the cancer genome and in non- coding regions significantly enriched in close proximity to Long Interspersed Elements (LINE-1), suggesting a potential rationale for LINE-1 hypomethylation in Fusobacterium-positive CRCs. Collectively, our analytical approaches have delineated Fusobacterium’s multimodal contribution to disrupting genomic stability, which ultimately could facilitate tailored treatment options targeting several DDR pathways in Fusobacterium-positive CRC. Citation Format: Radhika Kataria, Jelmar Quist, Sunjae Lee, Anargyros Megalios, Elinor Sawyer, Thomas Hardiman, Chang Liu, Katherine Kelly, Theo Portlock, Saeed Shoaie, Anita Grigoriadis. Multiomics analyses reveal multimodal Fusobacterium DNA damage perturbations in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6212.

3'untranslated regions of tumor suppressor genes evolved specific features to favor cancer resistance.

Cancer-related genes have evolved specific genetic and genomic features to favor tumor suppression. Previously we reported that tumor suppressor genes (TSGs) acquired high promoter CpG dinucleotide frequencies during evolution to maintain high expression in normal tissues and resist cancer-specific downregulation. In this study, we investigated whether 3'untranslated regions (3'UTRs) of TSGs have evolved specific features to carry out similar functions. We found that 3'UTRs of TSGs, especially those involved in multiple histological types and pediatric cancers, are longer than those of non-cancer genes. 3'UTRs of TSGs also exhibit higher density of binding sites for RNA-binding proteins (RBPs), particularly those having high affinities to C-rich motifs. Both longer 3'UTR length and RBP binding sites enrichment are correlated with higher gene expression in normal tissues across tissue types. Moreover, both features together with the correlated N6-methyladenosine modification and the extent of protein-protein interactions are positively associated with the ability of TSGs to resist cancer-specific downregulation. These results were successfully validated with independent datasets. Collectively, these findings indicate that TSGs have evolved longer 3'UTR with increased propensity to RBP binding, N6-methyladenosine modification and protein-protein interactions for optimizing their tumor-suppressing functions.