PVT1 Research Articles

P1305 Prediction of extraintestinal manifestations in inflammatory bowel disease using clinical and genetic variables with machine learning

Abstract Background Patients with inflammatory bowel diseases (IBD) may develop extraintestinal manifestations (EIM) that increase their morbidity. We investigated clinical and genetic factors associated with EIM, analyzing an IBD cohort between 2019-2024. We designed predictive models for EIM using machine learning techniques. Methods A total of 414 IBD patients were recruited (314 UC and 100 with CD). The presence of EIM was assessed, and clinical differences between the groups with and without EIM were analyzed using Chi-square tests and Mann-Whitney U tests. In a subset of 227 genotyped patients, genetic variants associated with EIM were identified, and a polygenic risk score (PRS) was calculated. Logistic regression (LR) and Random Forest (RF) predictive models were constructed. Furthermore, univariate/multivariate regression analyses were conducted to confirm associations with EIM. Results Clinical data from 414 patients were analysed, of which 29% (120) had EIM (Figure 1). Of these, 69% (83) had ulcerative colitis (UC). Meanwhile, 294 (71%) did not have EIM, of whom 231 (78.6%) had UC. Significant differences were observed between the groups with and without EIM: median age (45 vs. 52 years, p=0.01), BMI (26 vs. 27, p=0.006), and ESR (9 vs. 10, p=0.01). Categorical variables like family history of IBD (p=0.02) and anti-TNF use (p=0.01) were significant. In genotyped group, 50 of 200 genetic variants were linked to EIM, with 2 significantly increasing EIM risk: rs44107871-TC (PVT1 gene), three-genotype model; OR = 1.89, CI = 1.06-3.41, p-value = 0.03 and rs9936833-C (LINC917, FENDR gene) recessive model OR 2.29, CI = 1.16-4.55, p-value = 0.02, dominant-C model OR 1.86, CI 1.01-3.49, p-value = 0.04, additive-CC model OR = 3.01, CI 1.36-6.80 (p=0.007). The PRS was not discriminative, with an AUC of 0.67 and an accuracy of 0.56. In contrast, the LR and RF models better differentiated groups with and without EIM (Figure 2). Both in EIM univariate and multivariate analyses, significant associations were found with the variables rs9936833TT (univariate: OR=0.33, 95% CI=0.15-0.74, p=0.007; multivariate: OR=0.25, 95% CI=0.10-0.61, p=0.003), family history of IBD (univariate: OR=3.42, 95% CI=1.52-7.76, p=0.002; multivariate: OR=3.86, 95% CI=1.59-9.37, p=0.002), use of anti-TNF (univariate: OR=3.18, 95% CI=1.14-7.19, p=0.005; multivariate: OR=5.18, 95% CI=1.96-13.7, p=0.0009), age (univariate OR: 1.02, 95% CI 1.0-1.04, p=0.03, multivariate OR=1.03, 95% CI 1.00-1.06, p=0.01). Conclusion About third of IBD patients had EIM, as reported. Variants rs9936833 and rs4410871 were linked to higher EIM risk. While PRS had limits, LR and RF models predicted EIM well. Family history of IBD, rs9936833-CC, age, and anti-TNF use were significant risk factors. References Khrom M, Long M, Dube S, et al. Comprehensive Association Analyses of Extraintestinal Manifestations in Inflammatory Bowel Disease. Gastroenterology. 2024;167(2):315-332. doi:10.1053/j.gastro.2024.02.026 Gordon H, Burisch J, Ellul P, et al. ECCO Guidelines on Extraintestinal Manifestations in Inflammatory Bowel Disease. J Crohns Colitis. 2024;18(1):1-37. doi:10.1093/ecco-jcc/jjad108

The Emerging Roles of CircPVT1 in Cancer Progression.

CircRNA is stable due to its ring structure and is abundant in humans, which not only exists in various tissues and biofluids steadily but also plays a significant role in the physiology and pathology of human beings. CircPVT1, an endogenous circRNA, has recently been identified from the PVT1 gene located in the cancer risk region 8q24. CircPVT1 is reported to be highly expressed in many different tumors, where it affects tumor cell proliferation, apoptosis, invasion, and migration. We summarize the biosynthesis and biological functions of circPVT1 and analyze the relationship between circPVT1 and tumors as well as its significance to tumors. Further, it's noteworthy for the diagnosis, treatment, and prognosis of cancer patients. Therefore, circPVT1 is likely to become an innovative tumor marker.

Overcoming Breast Cancer Treatment Resistance with Optimizing Sonodynamic Therapy and Radiation Sensitizers on lncRNA PVT1 and miR-1204 Expression.

Acoustic cavitation is a foundational mechanism in ultrasound therapy, primarily through inertial cavitation resulting from microbubble collapse. Sonodynamic therapy, with inertial acoustic cavitation threshold and low-dose radiation in the presence of sensitizers, may provide significant effects for cancer treatment, potentially overcoming resistance encountered with single therapies. MCF7 breast cancer cells were subjected to sonodynamic therapy either alone or combined with ionizing radiation, gold nanoparticles coated with apigenin, and methylene blue. Several parameters were evaluated, including reactive oxygen species (ROS) generation and colonization. Additionally, the investigation included assessing the long non-coding RNA (lncRNA) PTV1 with miRNA1204 and related genes using Real-Time PCR. Sonodynamic therapy at a mechanical index of 0.31 as acoustic cavitation threshold increased intracellular ROS. Combining sonodynamic therapy and 2Gy X-ray radiation with methylene blue and gold nanoparticles coated with apigenin significantly decreased plating efficiency (4.44±1.69), and survival fraction (2.75±1.98) compared with control (Ctrl.) (98.77±4.49) and (97.59± 2.94), respectively. This was associated with a marked increase in ROS with a mean fluorescence intensity of 20576.2 ± 4.6 (>4.5 times). The combined treatment also increased p53 expression and decreased the expression of PVT1, miR-1204, and related genes. Sonodynamic therapy in inertial acoustic cavitation threshold, combined with ionizing radiation in the presence of biocompatible nanoparticles, could enhance the therapeutic effects on the miR-1204, derived from lncRNA PVT1, that functions as an oncogenic microRNA in breast cancer. This approach has the potential to overcome treatment resistance encountered with single therapies.

LncRNA PVT1 silencing inhibits gastric cancer cells’ progression via enhancing chemosensitivity to paclitaxel

Gastric cancer (GC) is one of the leading causes of cancer-related deaths worldwide because of its high morbidity and the absence of effective therapies. Even though paclitaxel is a powerful anticancer chemotherapy drug, recent studies have indicated its ineffectiveness against GC cells. Long non-coding RNA (lncRNA) PVT1 has a high expression in GC cells and increases the progression of tumors via inducing drug resistance. In the present study, the effects of the siRNA-mediated lncRNA PVT1 gene silencing along with paclitaxel treatment on the rate of apoptosis, growth, and migration of AGS GC cells were investigated. AGS cells were cultured and then transfected with siRNA PVT1 using electroporation. The MTT test was used to examine the effect of treatments on the viability of cultured cells. Furthermore, the flow cytometry method was used to evaluate the impact of treatments on the cell cycle process and apoptosis induction in GC cells. Finally, the mRNA expression of target genes was assessed using the qRT-PCR method. The results showed that lncRNA PVT1 gene suppression, along with paclitaxel treatment, reduces the viability of cancer cells and significantly increases the apoptosis rate of cancer cells and the number of cells arrested in the G2/M phase compared to the control group. Based on the results of qRT-PCR, combined treatment significantly decreased the expression of MMP3, MMP9, MDR1, MRP1, Bcl-2, k-Ras, and c-Myc genes and increased the expression of the Bax gene compared to the control group. The results of our study showed that lncRNA PVT1 gene targeting, together with paclitaxel treatment, induces apoptosis, inhibits growth, alleviates drug resistance, and reduces the migratory capability of GC cells. Therefore, there is a need for further investigations to evaluate the feasibility and effectiveness of this approach in vivo in animal models.

Exploration of PVT1 as a biomarker in prostate cancer.

Prostate cancer is a malignant tumor originating from the prostate gland, significantly affecting patients' quality of life and survival rates. Public data was utilized to identify differentially expressed genes (DEGs). Weighted gene co-expression network analysis was constructed to classify gene modules. Functional enrichment analysis was performed through Kyoto Encyclopedia of Genes and Genomes and gene ontology annotations, with results visualized using the Metascape database. Additionally, gene set enrichment analysis evaluated gene expression profiles and related pathways, constructed a protein-protein interaction network to predict core genes, analyzed survival data, plotted heatmaps and radar charts, and predicted microRNAs for core genes through miRTarBase. Two prostate cancer datasets (GSE46602 and GSE55909) were analyzed, identifying 710 DEGs. Gene ontology and Kyoto Encyclopedia of Genes and Genomes analyses revealed that DEGs were primarily involved in organic acid metabolism and the P53 signaling pathway. Gene set enrichment analysis and Metascape analyses further confirmed the significance of these pathways. After constructing the weighted gene co-expression network analysis network, 3 core genes (DDX21, NOP56, plasmacytoma variant translocation 1 [PVT1]) were identified. Survival analysis indicated that core genes are closely related to patient prognosis. Through comparative toxicogenomics database and miRNA prediction analysis, PVT1 was considered to play a crucial role in the development of prostate cancer. The PVT1 gene is highly expressed in prostate cancer and has the potential to become a diagnostic biomarker and therapeutic target for prostate cancer.

Abstract 473: PVT1 exon 9 overexpression induces AR null phenotype and cellular reprogramming in prostate cancer

Abstract Plasmacytoma variant translocation 1 (PVT1) is an oncogenic long-noncoding RNA (lncRNA) gene located directly downstream of MYC on chromosome 8q24. PVT1 is an oncogene overexpressed in prostate cancer (PCa) and can promote oncogenesis by altering gene expression directly or acting as a noncoding RNA sponge. Beyond functioning as a lncRNA, PVT1 gene encodes six microRNAs that each have independent functions. Our group identified overexpression of PVT1 exon 9 in PCa samples derived from men of African Ancestry and overexpression may explain why this population group experiences more aggressive disease. We overexpressed PVT1 exon 9 in RWPE1 prostate epithelial cells (RWPE1_ex9) and found enhanced migration and invasion compared to empty vector (RWPE1_ev). Implantation of RWPE1_ex9 cells into mice led to the formation of tumors with the neuroendocrine PCa (NEPC) phenotype that was histopathologically confirmed. RNA-sequencing analysis of PVT1 exon 9 overexpression showed significant (p-value < 0.05) upregulation of 141 genes. The majority of pathways impacted by PVT1 exon 9 overexpression were interferon type 1 signaling (RSAD2, CMPK2), developmental NOTCH signaling (HES7), cargo-transport (STX16) and tumor microenvironment promoting factors (HS3ST3A1). We found loss of androgen receptor (AR) expression at mRNA and protein levels in RWPE1_ex9 cells, a hallmark of NEPC, which further supports our hypothesis that PVT1 exon 9 overexpression plays a role in NEPC development. Validation of RNA-sequencing confirmed upregulation of RSAD2, CMPK2, STX16, HES7 and HS3ST3A1 within RWPE1_ex9 cells compared to RWPE1_ev and RWPE1 wild-type, suggestive of cellular reprogramming of prostate epithelial cells. We further analyzed expression profile in a novel circulating tumor cell model of castration-resistant 22RV1 cells (C22OH) and found enhanced expression of PVT1 exon 9, suppression of AR, phosphorylation of AR serine residue 308 and enhanced CDK11 activity compared to the primary 22RV1 cell line T22OH suggestive of a cell-cycle mediated regulatory mechanism. We further found enhanced expression of chromogramin A in the C22OH cell line, a marker of NEPC. These results suggest PVT1 exon 9 overexpression may play a critical role in NEPC development from normal epithelial cells via cellular reprogramming as well as facilitating a transition to NEPC from a castration resistant phenotype. PVT1 exon 9 may be a useful predictive biomarker that can identify populations which are likely to have disease progression from castration resistance to NEPC and further highlights the need to identify therapeutic strategies to target PVT1 exon 9 overexpression. Citation Format: Rachel E. Sexton-Bonacci, Chinedum Udekwu, Baris Boylu, Olorunseun O. Ogunwobi. PVT1 exon 9 overexpression induces AR null phenotype and cellular reprogramming in prostate 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 473.

Transcriptional activation of the Myc gene by glucose in β-cells requires a ChREBP-dependent 3-D chromatin interaction between the Myc and Pvt1 genes

ObjectiveAll forms of diabetes result from insufficient functional β-cell mass. Thus, achieving the therapeutic goal of expanding β-cell mass requires a better mechanistic understanding of how β-cells proliferate. Glucose is a natural β-cell mitogen that mediates its effects in part through the glucose-responsive transcription factor, carbohydrate response element binding protein (ChREBP) and the anabolic transcription factor, MYC. However, mechanistic details by which glucose activates Myc at the transcriptional level are poorly understood. MethodsHere, siRNA was used to test the role of ChREBP in the glucose response of MYC, ChIP and ChIPseq to identify potential regulatory binding sites, chromatin conformation capture to identify DNA/DNA interactions, and an adenovirus was constructed to expresses x-dCas9 and an sgRNA that specifically disrupts the recruitment of ChREBP to a specific targeted ChoRE. ResultsWe found that ChREBP is essential for glucose-mediated transcriptional induction of Myc, and for increases in Myc mRNA and protein abundance. Further, ChIPseq revealed that the carbohydrate response element (ChoRE) nearest to the Myc transcriptional start site (TSS) is immediately upstream of the gene encoding the lncRNA, Pvt1, 60,000 bp downstream of the Myc gene. Chromatin Conformation Capture (3C) confirmed a glucose-dependent interaction between these two sites. Transduction with an adenovirus expressing x-dCas9 and an sgRNA specifically targeting the highly conserved Pvt1 ChoRE, attenuates ChREBP recruitment, decreases Myc-Pvt1 DNA/DNA interaction, and decreases expression of the Pvt1 and Myc genes in response to glucose. Importantly, isolated and dispersed rat islet cells transduced with the ChoRE-disrupting adenovirus also display specific decreases in ChREBP-dependent, glucose-mediated expression of Pvt1 and Myc, as well as decreased glucose-stimulated β-cell proliferation. ConclusionsThe mitogenic glucose response of Myc is mediated via glucose-dependent recruitment of ChREBP to the promoter of the Pvt1 gene and subsequent DNA looping with the Myc promoter.

Dissecting transcription of the 8q24-MYC locus in prostate cancer recognizes the equilibration between androgen receptor direct and indirect dual-functions

BackgroundAndrogen receptor (AR) activation and repression dual-functionality only became known recently and still remains intriguing in prostate cancer (PCa). MYC is a prominent oncogene that functionally entangles with AR signaling in PCa. Further exploration of AR regulatory mechanisms on MYC gene transcription bears clinical and translation significance.MethodsBioinformatics analysis of PCa cell line and clinical RNA-Seq and ChIP-Seq (chromatin immunoprecipitation-sequencing) datasets to anchor interactions of AR and MYC transcriptional networks. ChIP-qPCR and 3C (chromosome conformation capture) analyses to probe MYC distal regulation by AR binding sites (ABSs). CRISPR/Cas9-mediated genome-editing to specify functions of ABS within the 8q24-MYC locus on androgen-mediated MYC transcription. Global FoxA1 and HoxB13 distribution profiling to advance AR transcriptional mechanisms.ResultsHere we recognize AR bi-directional transcription mechanisms by exploiting the prominent 8q24-MYC locus conferring androgen hyper-sensitivity. At ~ 25 Kb downstream of the MYC gene, we identified an undefined ABS, P10. By chromatin analyses, we validated androgen-dependent spatial interaction between P10 and MYC-Promoter (MYC-Pro) and temporal epigenetic repression of these MYC-proximal elements. We next designed a CRISPR/Cas9-mediated double genomic knock-out (KO) strategy to show that P10-KO slightly lessened androgen-elicited MYC transrepression in LNCaP-AR cells. In similar genomic editing assays, androgen-mediated MYC repression became slightly deepened upon KO of P11, an ABS in the PVT1 gene locus highly enriched in AR-binding motifs and peaks. We also investigated multiple ABSs in the established PCAT1 super-enhancer that distally interacts with MYC-Pro for transactivation, with each KO pool consistently shown to relieve androgen-elicited MYC repression. In the end, we systemically assessed androgen effects in the 8q24-MYC locus and along PCa genome to generalize H3K27ac and BRD4 re-distribution from pioneer factors (FoxA1 and HoxB13) to AR sites.ConclusionTogether, we reconciled these observations by unifying AR dual-functions that are mechanistically coupled to and equilibrated by co-factor redistribution.Graphical

Identification of LncPVT1 and CircPVT1 as prognostic biomarkers in human colorectal polyps

LncPVT1 and CircPVT1 are isoforms for the PVT1 gene and are associated with cancer progression and carcinogenesis. Our study investigated the expression of LncPVT1 and CircPVT1 in colon adenoma polyps. 40 tissues of colorectal polyps and 40 normal-adjacent tissues (NATs) were taken. The expression of LncPVT1 and CircPVT1 was evaluated through qRael-Time PCR. The relation between expression and features of clinicopathological was explored. The ceRNA network was constructed by LncPVT1 and CircPVT1 and predicted miRNAs and miRNAs targets. Further, hub nodes in this network were determined using the cytoHubba package. Over-expressed LncPVT1 and CircPVT1 were differentiated in polyp and NATs. The expression level of LncPVT1 and CircPVT1 were significantly higher in adenoma polyps than in hyperplastic polyps. The area under the curve of the ROC estimate for the LncPVT1 and CircPVT1 was 0.74 and 0.77, respectively. A positive correlation was observed between the LncPVT1 expression and CircPVT1. Three miRNAs, including hsa-miR-484, hsa-miR-24-3p, hsa-miR-423-5p, and CircPVT1, were detected as ceRNA hub nodes. In this study, expression profiles of LncPVT1 and CircPVT1 were significantly higher in precancerous polyps. In addition, based on our in silico analysis, LncPVT1, CircPVT1/miR-484, miR-24-3p, miR-423-5p/PLAGL2 axis might be involved in colon cancer development. LncPVT1 and CircPVT1 can be prescribed as warning problems as potential prognostic biomarkers in patients with pre-CRC colon polyps.

LncRNA PVT1 promotes bladder cancer progression by forming a positive feedback loop with STAT5B

BackgroundPlasmacytoma Variant Translocation 1 (LncRNA PVT1) and signal transducer and activator of transcription 5B (STAT5B) play important roles in various cancers, but their interaction in bladder cancer (BC) remains unclear. PurposeWe aimed to explore the interaction between lncRNA PVT1 and STAT5B in BC tumorigenesis and find potential drugs for BC. MethodsThe association of the expression of lncRNA PVT1 and STAT5B to the prognosis of BC patients was evaluated via bioinformatic analysis. Loss- and gain-of-function assays were performed to determine the biological functions of lncRNA PVT1 and STAT5B. Quantitative real time polymerase chain reaction, Western blot, immunohistochemistry, and immunofluorescence were used to detect lncRNA PVT1 and STAT5B expression. Fluorescence in situ hybridization, RNA pull-down and RNA immunoprecipitation assays were conducted to determine the regulatory effect of lncRNA PVT1 on STAT5B. The transcriptional effect of STAT5B on lncRNA PVT1 gene was determined using luciferase reporter assay, chromatin immunoprecipitation and DNA-affinity precipitation assays. Connectivity Map analysis was used to screen anticancer drugs. ResultsLncRNA PVT1 and STAT5B enhance the expression of each other and promote the malignant phenotypes in BC, including cell viability and invasion. lncRNA PVT1 stabilizes STAT5B by decreasing ubiquitination, enhances STAT5B phosphorylation, and promotes the translocation to the nucleus of STAT5B to trigger further carcinogenesis activities. In the nucleus, STAT5B activates the transcription of lncRNA PVT1 by binding directly to its promoter region, leading to a positive feedback. Tanespimycin effectively abated the oncogenic effect. ConclusionsWe first identified the lncRNA PVT1/STAT5B positive feedback loop for bladder carcinogenesis, and found a potentially effective drug for BC.

1728-P: Transcriptional Activation of Myc by Glucose Requires Recruitment of ChREBP to the Pvt1 Promoter Regulating Proliferation in ß-Cells

Myc and Carbohydrate Response Element (ChoRE) Binding Protein (ChREBP) are glucose-responsive transcriptional regulators of β-cell proliferation. Myc requires ChREBP to drive glucose-mediated proliferation in β-cells, yet no ChoREs are present in Myc’s promoter. In primates and rodents, the Myc gene and the long non-coding RNA, PVT1 gene are syntenic, separated by approximately 60 kbp. Mutations in this loci are associated with malignancies and complications of diabetes but are unexplored in β-cells. In INS-1 cells, Myc and PVT1 mRNAs were induced in response to glucose by 3.5±0.02and 2.9±0.1 fold, respectively, and Myc protein levels increased by 4-fold ±0.17. Silencing of ChREBPβ significantly blocked the glucose response of Myc and Pvt1. ChREBP ChIPseq revealed a single ChoRE located approximately 100 bp upstream of the Pvt1 transcription start site and is highly conserved in primates and rodents. By Chromosome Conformation Capture (3C), we demonstrated that high glucose increased interaction between the Pvt1 and Myc promoters by a factor of 3.5±0.4. Using adenoviral delivery of CRISPR-xCas9-sgRNA targeting the Pvt1 ChoRE, we specifically attenuated ChREBP recruitment by 2.8±0.8 fold, while other known ChoREs were unaffected. We demonstrated by 3C that the chromatin looping in response to glucose was abolished by the Pvt1 ChoRE disruptor. Both in primary rat islets and in INS-1 cells, disruption of Pvt1’s ChoRE resulted in complete and significant blockage of Pvt1 and Myc mRNA upregulation in response to glucose, with no effect on other ChoRE-containing genes. Finally, disruption of the ChoRE on the Pvt1 promoter eliminated glucose-mediated proliferation of primary rat β-cells (a decrease of 9.8±1.9 fold on Ki67+ Ins+ staining). In conclusion, we identify a novel mechanism driving the ChREBP-dependent activation of the Myc and Pvt1 genes and proliferation of β-cells. Disclosure L.S.Katz: None. P.Wang: None. G.Brill: None. P.Zhang: None. J.Haldeman: Employee; Janssen Research & Development, LLC. C.B.Newgard: Advisory Panel; Eli Lilly and Company, Novo Nordisk, AstraZeneca. A.F.Stewart: None. A.Garcia-ocana: Consultant; Sun Pharmaceutical Industries Ltd. D.Scott: None. Funding National Institutes of Health (R01DK130300, P30DK020541)

Abstract 4723: DNA G-quadruplex facilitates MYC enhancer-promotor interaction

Abstract Background: G-quadruplexes (G4) are nucleic acid secondary structures formed in guanine-rich regions of DNA or RNA. The biological significance of G4 has been attributed to the regulation of gene expression, and more recently, to the stabilization and generation of enhancer-promotor interaction. Since G4 are enriched in the promoter region of the proto-oncogene MYC, we sought to investigate the role of G4 in stabilizing the enhancer-promoter loop using the locus 8q24 (hg38) 127,700,000-128,500,000 that contains the MYC gene as a model. Methods: Two different cell lines were used, the spontaneously immortalized, non-oncogenic human epidermal keratinocyte (HaCaT), in which the enrichment of G4 in the MYC promoter was demonstrated, and the primary normal human epidermal keratinocyte (nHEK) that is depleted of G4 in MYC promoter. Both cell lines were exposed to the G4 selective stabilizer 360-A drug and the half-maximal inhibitory concentration (IC50) could be calculated. Gene expression was accessed by RT-qPCR and DNA methylation by Methylation-Sensitive High Resolution Melting (MS-HRM). Chromatin Conformation Capture (3C-qPCR) technique was performed interrogating the MYC gene (chr8:127,733,798-127,744,729) and the putative enhancer in the lncRNA PVT1 gene (chr8:127,914,509-127,922,393) to compare the frequency of promoter-enhancer interaction between the two cell lines. Results: The HaCaT cells showed interaction between the MYC promoter and the distal region. The nHEK cells that are not enriched for G4 showed no promoter interaction with the distal region. The use of 360-A in IC50 did not change the results. The gene expression analysis of the MYC showed higher expression in HaCaT cells compared to nHEK. There was no difference in DNA methylation in the MYC promoter between the two cell lines. Conclusions: The data suggest an important role of G4 for the interaction of the MYC promoter and downstream enhancer. Additional analyzes of other genomic regions enriched for G4 should be performed to confirm the mechanism. Citation Format: Dieila Giomo De Lima, Gustavo Narvaes Guimarães, Bianca Caroline Bianco, Ana Paula De Souza, Aline Cristiane Planello. DNA G-quadruplex facilitates MYC enhancer-promotor interaction. [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 4723.

HERV-K (HML-2) insertion polymorphisms in the 8q24.13 region and their potential etiological associations with acute myeloid leukemia.

Human endogenous retroviruses (HERVs) are LTR retrotransposons that are present in the human genome. Among them, members of the HERV-K (HML-2) group are suspected to play a role in the development of different types of cancer, including lung, ovarian, and prostate cancer, as well as leukemia. Acute myeloid leukemia (AML) is an important disease that causes 1% of cancer deaths in the United States and has a survival rate of 28.7%. Here, we describe a method for assessing the statistical association between HERV-K (HML-2) transposable element insertion polymorphisms (or TIPs) and AML, using whole-genome sequencing and read mapping using TIP_finder software. Our results suggest that 101 polymorphisms involving HERV-K (HML-2) elements were correlated with AML, with a percentage between 44.4 to 56.6%, most of which (70) were located in the region from 8q24.13 to 8q24.21. Moreover, it was found that the TRIB1, LRATD2, POU5F1B, MYC, PCAT1, PVT1, and CCDC26 genes could be displaced or fragmented by TIPs. Furthermore, a general method was devised to facilitate analysis of the correlation between transposable element insertions and specific diseases. Finally, although the relationship between HERV-K (HML-2) TIPs and AML remains unclear, the data reported in this study indicate a statistical correlation, as supported by the χ2 test with p-values < 0.05.

Upregulation of the c-MYC oncogene and adjacent long noncoding RNAs PVT1 and CCAT1 in esophageal squamous cell carcinoma

BackgroundAll cell types express long non-coding RNAs (lncRNAs), which have the potential to play a role in carcinogenesis by altering the levels of their expression. Squamous cell carcinoma of the esophagus (ESCC) is a deadly disease with a poor prognosis and a high frequency of lymphatic metastases. Understanding the functional role and signaling pathways of two neighboring lncRNAs, CCAT1 and PVT1, in this oncogene’s pathogenesis may help us determine ESCC. Furthermore, it is still unclear whether these lncRNAs are linked to the clinicopathological characteristics of patients with ESCC.MethodsFor this study, we used biopsy from the Imam Khomeini Cancer Institute’s tumor bank in Tehran, Iran to obtain 40 ESCC tumor samples and their normal margin counterparts. The expression levels of the CCAT1, PVT1, and c-MYC genes were assessed using quantitative Real-Time RT-PCR. Additionally, demographic data and clinical-pathologic characteristics, such as tumor grade, tumor stage, lymph node, and metastasis, were taken into consideration. Graphpad prism version 8 was used for bioinformatics analyses.ResultsComparing ESCC tissues to non-tumor tissues, we found significant upregulation of PVT1, CCAT1, and c-MYC. Patients with ESCC who had increased PVT1 expression also had higher rates of advanced stage and lymph node metastasis, whereas increased CCAT1 expression was only linked to advanced stage and wasn’t associated with lymph node metastasis. In predicting ESCC, CCAT1 (p < 0.05) was found to be an important factor. Overall survival was reduced by c-MYC and PVT1 overexpression (p < 0.001), according to Kaplan-Meier analysis. PVT1, CCAT1, and c-MYC were found to interact with 23 miRNAs with high and medium score classes, as shown in a bioinformatics study. We summarized the experimentally proven interactions between c-MYC, PVT1, and CCAT1 and other miRNAs, lncRNAs, and proteins.ConclusionThis is the first report that CCAT1, PVT1 and c-MYC have been found to be up-regulated simultaneously in ESCC. It is possible that these genes may be involved in ESCC as a result of these findings. Therefore, as consequence, more research is needed to determine whether or not these lncRNAs play an oncogenic role in ESCC development and progression, as well as the regulatory mechanisms that control them.

The role of the long non-coding RNA gene PVT1 polymorphism in the development of multiple sclerosis

Long non-coding RNA locus PVT1 produces multiple regulatory RNA molecules via alternative splicing, and therefore is a promising object for the development of targeted therapies of different disorders. Performing associationanalysis of genetic variants rs4410871 andrs759648 at the PVT1 locus with multiple sclerosis (MS) in the ethnic group of Russians, Tatars, and Bashkirs from the Volga-Ural region of Russia, we established an association between rs759648*С and MS in the group of Tatars (OR=1.42, PBonf0.046), confirmed by the results of meta-analysisin the three ethnic groups (OR=1.28, P = 0.015). Haplotypic analysis has revealed an association of the rs759648*С/rs4410871*С haplotype with MS in the total study group (χ2=6.083, Рperm =0.012), whilers4410871*C/C + rs759648* C/C combination conferred an increased risk of MS (OR = 2.71, Pperm = 0.027) in this groupaccording to the data yielded by the multilocus analysis. The results of our analysis indicate the significance of the rs759648 polymorphism located near the DNA sequence producing miR-1208. Further studies will help to elucidate the molecular mechanisms of the involvement of PVT1 gene in the etiopathogenesis of MS.

Transcriptome Analyses Identify Deregulated MYC in Early Onset Colorectal Cancer.

Despite a global decrease in colorectal cancer (CRC) incidence, the prevalence of early-onset colorectal cancer (EOCRC), or those occurring in individuals before the age of 50, has steadily increased over the past several decades. When compared to later onset colorectal cancer (LOCRC) in individuals over 50, our understanding of the genetic and molecular underpinnings of EOCRCs is limited. Here, we conducted transcriptomic analyses of patient-matched normal colonic segments and tumors to identify gene expression programs involved in carcinogenesis. Amongst differentially expressed genes, we found increased expression of the c-MYC proto-oncogene (MYC) and its downstream targets in tumor samples. We identified tumors with high and low differential MYC expression and found patients with high-MYC tumors were older and overweight or obese. We also detected elevated expression of the PVT1 long-non-coding RNA (lncRNA) in most tumors and found gains in copy number for both MYC and PVT1 gene loci in 35% of tumors evaluated. Our transcriptome analyses indicate that EOCRC can be sub-classified into groups based on differential MYC expression and suggest that deregulated MYC contributes to CRCs that develop in younger patients.

Transcriptome-wide analysis of glioma stem cell specific m6A modifications in long-non-coding RNAs

The interest in chemical RNA modifications is rapidly growing in the field of molecular biology. Dynamic and reversible alterations of N6-methyladenosine (m6A) RNA modification are responsible for a platter of structural and functional changes in healthy and cancerous cell states. Although many studies reported the link between tumor initiation/progression and m6A modulators, there are few studies exploring transcriptome-wide m6A profile of non-coding RNAs. The aim of current study was to identify glioma stem cell (GSC) specific m6A landscape of long non-coding RNAs (lncRNAs) applying MeRIP-seq approach. MeRIP-seq analysis assigned 77.9% of m6A peaks to mRNAs and 8.16% to lncRNAs. GSCs and differentiated cells showed 76.4% conservation of m6A peaks, while 19.4% were unique to GSCs. Seven novel GSC-specific m6A modified lncRNAs were identified: HRAT92, SLCO4A1-AS1, CEROX1, PVT1, AGAP2-AS1, MIAT, and novel lncRNA gene ENSG00000262223. Analysis disclosed a strong negative correlation between lncRNAs m6A modification rate and expression. MeRIP-seq analysis revealed m6A modifications on previously reported glioma-associated lncRNAs: LINC000461, HOTTIP, CRNDE, TUG1, and XIST. Moreover, current study disclosed that most highly m6A modified lncRNAs primarily contain m6A modifications close to 3′ and 5′ ends. Our results provide basis and insight for further studies of m6A modifications in non-coding transcriptome of GSCs.

CD8+ T-cell Immune Surveillance against a Tumor Antigen Encoded by the Oncogenic Long Noncoding RNA PVT1.

CD8+ T cells recognize peptides displayed by HLA class I molecules on cell surfaces, monitoring pathologic conditions such as cancer. Advances in proteogenomic analysis of HLA ligandomes have demonstrated that cells present a subset of cryptic peptides derived from noncoding regions of the genome; however, the roles of cryptic HLA ligands in tumor immunity remain unknown. In the current study, we comprehensively and quantitatively investigated the HLA class I ligandome of a set of human colorectal cancer and matched normal tissues, showing that cryptic translation products accounted for approximately 5% of the HLA class I ligandome. We also found that a peptide encoded by the long noncoding RNA (lncRNA) PVT1 was predominantly enriched in multiple colorectal cancer tissues. The PVT1 gene is located downstream of the MYC gene in the genome and is aberrantly overexpressed across a variety of cancers, reflecting its oncogenic property. The PVT1 peptide was recognized by patient CD8+ tumor-infiltrating lymphocytes, as well as peripheral blood mononuclear cells, suggesting the presence of patient immune surveillance. Our findings show that peptides can be translated from lncRNAs and presented by HLA class I and that cancer patient T cells are capable of sensing aberrations in noncoding regions of the genome.

MicroRNA-1205 Regulation of FRYL in Prostate Cancer.

High mortality rates of prostate cancer (PCa) are associated with metastatic castration-resistant prostate cancer (CRPC) due to the maintenance of androgen receptor (AR) signaling despite androgen deprivation therapies (ADTs). The 8q24 chromosomal locus is a region of very high PCa susceptibility that carries genetic variants associated with high risk of PCa incidence. This region also carries frequent amplifications of the PVT1 gene, a non-protein coding gene that encodes a cluster of microRNAs including, microRNA-1205 (miR-1205), which are largely understudied. Herein, we demonstrate that miR-1205 is underexpressed in PCa cells and tissues and suppresses CRPC tumors in vivo. To characterize the molecular pathway, we identified and validated fry-like (FRYL) as a direct molecular target of miR-1205 and observed its overexpression in PCa cells and tissues. FRYL is predicted to regulate dendritic branching, which led to the investigation of FRYL in neuroendocrine PCa (NEPC). Resistance toward ADT leads to the progression of treatment related NEPC often characterized by PCa neuroendocrine differentiation (NED), however, this mechanism is poorly understood. Underexpression of miR-1205 is observed when NED is induced in vitro and inhibition of miR-1205 leads to increased expression of NED markers. However, while FRYL is overexpressed during NED, FRYL knockdown did not reduce NED, therefore revealing that miR-1205 induces NED independently of FRYL.

Abstract 2387: Targeting PVT1 exon 9 transcript is not pro-apoptotic but induces claudin 4 expression and inhibits migration in triple negative breast cancer cells

Abstract Triple negative breast cancer (TNBC) is a lethal molecular subtype of invasive breast cancer (BC) that is ER-, PR- and HER2-. Claudin low (CL) TNBC, a distinct molecular subtype of TNBC, has the worst prognosis compared to other BC subtypes. Aberrant expression of claudin proteins disrupts the function of tight junctions. Consequently, inducing epithelial-to-mesenchymal transition (EMT) in cancers, an important factor for enhanced motility and metastasis. Therefore, understanding the molecular mechanisms that modulate claudin expression in TNBC is crucial. The 8q24 genomic locus is an important cancer susceptibility locus that is associated with poor clinical outcomes in cancer due to common amplification events that occur in many malignant diseases. This locus contains the PVT1 gene, which encodes a long noncoding RNA (lncRNA) that has been implicated in multiple cancers including BC. PVT1 consists of 12 exons that are alternatively spliced to generate lncRNAs. Although previous research has implicated PVT1 as an important player in BC, the underlying molecular mechanisms of PVT1 in CL TNBC is unknown. To examine the role of PVT1 in CL TNBC, we assessed PVT1 expression in T47D (ER+), MDA MB 231 (CL) and MDA MB 468 (claudin high (CH)) BC cells. We observed that PVT1 exons 4A, 4B, and 9 are significantly overexpressed in CL MDA MB 231 and significantly underexpressed in CH MDA MB 468 in comparison to T47D cells, suggesting a protumorigenic role of PVT1 in CL TNBC. We analyzed the functional consequences of siRNA targeting of PVT1 exon 9 expression in CL TNBC cells. siRNA targeting of PVT1 exon 9 expression in the MDA MB 231 CL TNBC cells led to a significant reduction in migration and the re-expression of claudin 4. To determine the role of PVT1 on EMT, we assessed the expression of EMT markers (vimentin, fibronectin, and E-cadherin) in MDA MB 231 cells. We observed no changes in the expression of EMT markers when PVT1 exon 9 is knocked down. However, our data show that mesenchymal markers are more highly expressed in MDA MB 231 cells in comparison to MDA MB 468 cells. Further, knockdown of PVT1 exon 9 did not induce apoptosis in MDA MB 231 cells as assessed by caspase 3 and caspase 9 expression. Taken together, our data indicate that PVT1 exon 9 regulates claudin expression and migration in CL TNBC, and may have implications for clinical outcomes in TNBC. Citation Format: Fayola A. Levine, Olorunseun O. Ogunwobi. Targeting PVT1 exon 9 transcript is not pro-apoptotic but induces claudin 4 expression and inhibits migration in triple negative breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2387.