Tumor Suppressor Gene In Cancer Research Articles

Andrographolide suppresses the malignancy of pancreatic cancer via alleviating DNMT3B-dependent repression of tumor suppressor gene ZNF382

BackgroundPancreatic ductal adenocarcinoma (PDAC) is a highly aggressive cancer type that urgently requires effective therapeutic strategies. Andrographolide, a labdane diterpenoid compound abundant in Andrographis paniculata, has anticancer effects against various cancer types, but its anticancer activity and mechanism against PDAC remain largely uncharacterized. PurposeThis study explores novel drug target(s) and underlying molecular mechanism of andrographolide against PDAC Study design and methodsThe malignant phenotypes of PDAC cells, PANC-1 and MIA PaCa-2 cells were measured using MTT, clonogenic assays, and Transwell migration assays. A PDAC xenograft animal model was used to evaluate tumor growth in vivo. Western blot, immunofluorescence and immunohistochemistry were used for measuring protein expression. The TCGA database was analyzed to evaluate promoter methylation status, gene expression, and their relationship with patient survival rates. RT-qPCR was used for detecting mRNA expression. Reporter assays were used for detecting signal transduction pathways. Promoter DNA methylation was determined by sodium bisulfite treatment and methylation-specific PCR (MSP). The biological function and role of specific genes involved in drug effects were measured through gene overexpression. ResultsAndrographolide treatment suppressed the proliferation and migration of PDAC cells and impaired tumor growth in vivo. Furthermore, andrographolide induced the mRNA and protein expression of zinc finger protein 382 (ZNF382) in PDAC cells. Overexpression of ZNF382 inhibited malignant phenotypes and cancer-associated signaling pathways (AP-1, NF-κB and β-catenin) and oncogenes (ZEB-1, STAT-3, STAT-5, and HIF-1α). Overexpression of ZNF382 delayed growth of PANC-1 cells in vivo. ZNF382 mRNA and protein expression was lower in tumor tissues than in adjacent normal tissues of pancreatic cancer patients. Analysis of the TCGA database found the ZNF382 promoter is hypermethylated in primary pancreatic tumors which correlates with its low expression. Furthermore, andrographolide inhibited the expression of DNA methyltransferase 3 beta (DNMT3B) and increased the demethylation of the ZNF382 promoter in PDAC cells. Overexpression of DNMT3B attenuated the andrographolide-suppressed proliferation and migration of PDAC cells. ConclusionOur finding revealed that ZNF382 acts as a tumor suppressor gene in pancreatic cancer and andrographolide restores ZNF382 expression to suppress pancreatic cancer, providing a novel molecular target and a promising therapeutic approach for treating pancreatic cancer.

Effect of SIRT7 on inhibiting the epithelial-mesenchymal transformation in pancreatic cancer cells and related mechanism

Objective: To investigate the effect and mechanism of SIRT7 in epithelial mesenchymal transformation (EMT) of pancreatic cancer cells. Methods: The pancreatic cancer cells were divided into siControl, siSIRT7, over-expression SIRT7, siSIRT7+siCOL4A1, and siSIRT7+siSLUG groups using siRNA or plasmid transfection. The proliferation, migration and invasion of pancreatic cancer cells were detected by EdU, wound healing assay and Transwell experiments, respectively. The expression of EMT and cancer stem cell (CSC) markers were detected by quantitative real-time reverse transcription polymerase chain reaction assay (qRT-PCR) and western blot. RNA sequencing (RNA-seq) in SIRT7 knockdown PANC-1 cells was performed to explore the signaling pathways and target genes regulated by SIRT7. Then the target genes directly regulated by SIRT7 were identified with quantitative chromatin immunoprecipitation experiment (q-ChIP) and chromatin immunoprecipitation polymerase chain reaction (ChIP-PCR). The expressions of SIRT7 and target genes were detected by immunohistochemical (IHC) in pancreatic cancer tissues, and the correlation between SIRT7 and target gene expression was analyzed using TCGA dataset. The correlation between expression of SIRT7 or target genes and survival was analyzed on KM-plotter website. Finally, GeneMANIA, STRING and ENCORI were used to predict SIRT7-related proteins and miRNAs. Results: EdU assay showed that the cell proliferation rates in SIRT7-overexpressed PANC-1 [(19.33±0.35)%] and BxPC-3 cells [(17.00±1.89)%] were lower than those in the control group [(31.60±1.37)% and (24.33±0.78)%, respectively, P＜0.05]. The proliferation rates of SIRT7-knockdown PANC-1 [(23.94±1.00)% and (27.08±0.97)%] and BxPC-3 cells [(22.00±1.86)% and (25.96±1.61)%] were higher than those of the siControl group [(11.80±1.86)% and (13.42±1.39)%, respectively, P＜0.05]. In PANC-1 cells, the wound healing assay showed that the relative migration rate of SIRT7-overexpression cells [(76.67±2.74)%] was lower than that of control cells [(100.00±2.13)%, P＜0.05]; the relative migration rate of cells with SIRT7 knockdown [(134.22±4.08)% and (199.82±9.20)%, respectively] was higher than that of siControl group [(102.24±3.13)%, P＜0.05]. Compared with the control group, SIRT7 overexpression decreased the number of migrated BxPC-3 cells (45.66±1.69 vs 28.33±2.62, P＜0.05); while SIRT7 knockdown increased these numbers (65.66±2.86 and 82.00±2.94 versus 33.00±0.81, P＜0.01). Transwell experiment revealed that the number of invaded cells in SIRT7 overexpression groups (16.33±2.05 and 34.66±1.69) was lower than that control groups (54.33±4.64 and 58.66±5.90, P＜0.05); with SIRT7 knockdown, the numbers of invaded PANC-1 (63.66±2.49 and 69.33±3.29) and BxPC-3 cells (134.33±3.09 and 181.66±4.02) were higher than those in control groups (35.33±2.49 and 42.00±0.81, P˂0.05). Also, SIRT7 knockdown decreased the expressions of epithelial markers and increased the expressions of mesenchymal and CSC markers. RNA-seq analysis showed that SIRT7 was involved in regulating a variety of cancer-related signaling pathways, including the pancreatic cancer pathway and the EMT pathway. Furthermore, SIRT7 could directly bind to the promoter regions of target genes, such as COL4A1 and SLUG. SIRT7 was negatively correlated with the expression and function of COL4A1 and SLUG in pancreatic cancer cells. The expressions of SIRT7, COL4A1, SLUG and SOX2 were verified in pancreatic cancer tissues by IHC. Finally, SIRT7 was predicted to be associated with many proteins and miRNAs based on GeneMANIA, STRING, and ENCORI online tools. Conclusions: SIRT7 can inhibit the EMT of pancreatic cancer cells through transcriptionally inhibiting the expression of target genes, such as COL4A1 and SLUG. Thus, SIRT7 may serve as a potential tumor suppressor gene in pancreatic cancer.

NTRK3 exhibits a pro-oncogenic function in upper tract urothelial carcinomas.

Neurotrophic receptor tyrosine kinase 3 (NTRK3) has pleiotropic functions: it acts not only as an oncogene in breast and gastric cancers but also as a dependence receptor in tumor suppressor genes in colon cancer and neuroblastomas. However, the role of NTRK3 in upper tract urothelial carcinoma (UTUC) is not well documented. This study investigated the association between NTRK3 expression and outcomes in UTUC patients and validated the results in tests on UTUC cell lines. A total of 118 UTUC cancer tissue samples were examined to evaluate the expression of NTRK3. Survival curves were generated using Kaplan-Meier estimates, and Cox regression models were used for investigating survival outcomes. Higher NTRK3 expression was correlated with worse progression-free survival, cancer-specific survival, and overall survival. Moreover, the results of an Ingenuity Pathway Analysis suggested that NTRK3 may interact with the PI3K-AKT-mTOR signaling pathway to promote cancer. NTRK3 downregulation in BFTC909 cells through shRNA reduced cellular migration, invasion, and activity in the AKT-mTOR pathway. Furthermore, the overexpression of NTRK3 in UM-UC-14 cells promoted AKT-mTOR pathway activity, cellular migration, and cell invasion. From these observations, we concluded that NTRK3 may contribute to aggressive behaviors in UTUC by facilitating cell migration and invasion through its interaction with the AKT-mTOR pathway and the expression of NTRK3 is a potential predictor of clinical outcomes in cases of UTUC.

Abstract 1808: Discovery of highly selective novel MTA-cooperative PRMT5 inhibitors for the treatment of cancers

Abstract Protein arginine methyltransferase 5 (PRMT5), a type II PRMT catalyzing the formation of symmetric dimethylation of arginine residues on histone and non-histone proteins, regulates many biological pathways in mammalian cells, including cell growth and differentiation. Methylthioadenosine phosphorylase (MTAP) is required for the methionine salvage pathway, deletion of MTAP leads to the accumulation of inhibitory PRMT5 cofactor methylthioadenosine (MTA). MTAP gene is adjacent to and frequently co-deleted with CDKN2A gene, the most commonly deleted tumor suppressor gene in human cancers. The increase in MTA significantly reduced PRMT5 activity in MTAP-deficient cancer cells, rendering them more vulnerable to PRMT5 inhibition than normal cells. Selective Inhibitors of PRMT5-MTA complex (MTA-cooperative) are supposed to exhibit an increased therapeutic index compared to first generation PRMT5 inhibitors for the treatment of MTAP-deleted (MTAP-del) cancer patients. Guided by computation-aided drug design (CADD), binding mode analysis of known PRMT5 inhibitors has led to the design and synthesis of a novel series of MTA-cooperative PRMT5 inhibitors. Many compounds within this series were found to inhibit PRMT5/MEP50 complex with single digit nM of IC50 in enzymatic assay. Cell based activity of these compounds was assessed by measuring the symmetric demethylarginine (SDMA) and 10-day cell proliferation assays. Several compounds exhibited low double-digit nM of cellular potency in HCT116 MTAP-del cells, the inhibition of SDMA and anti-proliferation activities in HCT116 MTAP-del cells over HCT116 MTAP-WT cells have excellent selectivity (80 - 400 fold). Further optimization of in vitro and in vivo pharmacokinetic properties has yielded candidate compounds suitable for evaluation of pharmacodynamic effect in the LU99 MTAP-del NSCLC xenograft model. Tumor growth inhibition (TGI) was observed in a dose-dependent manner, accompanied by a reduction of PRMT5-mediated SDMA levels in both the tumor and bone marrow. In summary, we have identified compounds with novel scaffolds that are potent and highly selective MTA-cooperative PRMT5 inhibitors. Further development of the second-generation PRMT5 inhibitors for treatment of cancers is also planned. Citation Format: Wen-Lian Wu, Taishan Hu, Zhilin Deng, Honghai Li, Quanrong Shen, Lei Zhang, Xiaochu Ma, Peihua Sun, Cindy Cheng, Fang Liu, Xin Chen, Ye Hua, Bryan Huang. Discovery of highly selective novel MTA-cooperative PRMT5 inhibitors for the treatment of cancers [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 1808.

HAND2-AS1 Promotes Ferroptosis to Reverse Lenvatinib Resistance in Hepatocellular Carcinoma by TLR4/NOX2/DUOX2 Axis.

Lenvatinib resistance causes less than 40% of the objective response rate. Therefore, it is urgent to explore new therapeutic targets to reverse the lenvatinib resistance for HCC. HAND2-AS1 is a critical tumor suppressor gene in various cancers. Here, we investigated the role of HAND2-AS1 in the molecular mechanism of lenvatinib resistance in HCC. It was found that HAND2-AS1 was lowly expressed in the HepG2 lenvatinib resistance (HepG2-LR) cells and HCC tissues and associated with progression-free intervals via TCGA. Overexpression of HAND2-AS1 (OE-HAND2-AS1) decreased the IC50 of lenvatinib in HepG2-LR cells to reverse lenvatinib resistance. Moreover, OE-HAND2-AS1 induced intracellular concentrations of malondialdehyde (MDA) and lipid ROS and decreased the ratio of glutathione to glutathione disulfide (GSH/GSSG) to promote ferroptosis. A xenograft model in which nude mice were injected with OE-HAND2-AS1 HepG2-LR cells confirmed that OE-HAND2-AS1 could reverse lenvatinib resistance and decrease tumor formation in vivo. HAND2-AS1 promoted the expression of ferroptosis-related genes (TLR4, NOX2, and DUOX2) and promoted ferroptosis to reverse lenvatinib resistance by increasing TLR4/ NOX2/DUOX2 via competing endogenous miR-219a-1-3p in HCC cells. Besides, patients with a low HAND2-AS1 level had early recurrence after resection. These findings suggested that HAND2-AS1 may be a potential therapeutic target and an indicator of early recurrence for HCC.

NOLC1 was identified as a tumor suppressor gene in thyroid cancer and correlated with prognosis by bioinformatics.

Thyroid cancer (THCA) is the most common endocrine malignancy, mainly affecting women's unilateral glandular lobes. However, for relapsed and distant metastasis of THCA patients, the existing early diagnosis and treatment methods were still insufficient, and a new method was urgently needed to diagnose and treat them. Nucleolar and coiled-body phosphoprotein 1 (NOLC1) was one of the most phosphorylated proteins in the cell, which was located mainly in the nucleolus. In addition, more and more studies have confirmed that NOLC1 plays a crucial role in various pathological processes, such as the occurrence and progression of cancer and viral infection. A previous study showed that NOLC1, as a member of RNA-binding protein, was significantly correlated with the prognosis of THCA patients. However, further exploration of NOLC1 in THCA is limited. To further explore the role of NOLC1 in THCA, we conducted expression and survival prognosis analysis of NOLC1 using multiple databases. We also evaluated the correlation between NOLC1 gene expression and clinical characteristics of THCA patients. Furthermore, we analyzed the relationship between NOLC1 and other genes, followed by enrichment analysis to investigate its metabolic pathways and molecular metabolism processes. Additionally, we examined the association between immune cell infiltration in tumor microenvironment and NOLC1. Notably, through vitro experiments, we confirmed the tumor suppressive effect of NOLC1 on the proliferation and migration of human THCA cells, providing evidence for clinical diagnosis of THCA. Furthermore, we confirmed the tumor suppressive effect of NOLC1 in vivo xenograft assay. To sum up, our results suggest that NOLC1 is a tumor suppressor gene for THCA.

Development of a Genetically Engineered Mouse Model Recapitulating LKB1 and PTEN Deficiency in Gastric Cancer Pathogenesis.

The LKB1 and PTEN genes are critical in gastric cancer (G.C.) development. LKB1, a robust tumor suppressor gene, encodes a serine/threonine kinase that directly triggers the activation of AMPK-an integral cellular metabolic kinase. The role of the LKB1 pathway extends to maintaining the stability of epithelial junctions by regulating E-cadherin expression. Conversely, PTEN, a frequently mutated tumor suppressor gene in various human cancers, emerges as a pivotal negative regulator of the phosphoinositide 3-kinase (PI3K) signaling pathway. This study is set to leverage the H+/K+ ATPase Cre transgene strain to precisely target Cre recombinase expression at parietal cells within the stomach. This strategic maneuver seeks to selectively nullify the functions of both LKB1 and PTEN in a manner specific to the stomach, thereby instigating the development of G.C. in a fashion akin to human gastric adenocarcinoma. Moreover, this study endeavors to dissect the intricate ways in which these alterations contribute to the histopathologic advancement of gastric tumors, their potential for invasiveness and metastasis, their angiogenesis, and the evolving tumor stromal microenvironment. Our results show that conditional deletion of PTEN and LKB1 provides an ideal cancer microenvironment for G.C. tumorigenesis by promoting cancer cell proliferation, angiogenesis, and metastasis.

Regional bias of tumor suppressor gene mutations of STARD8 and WNK2 in colon cancers

StAR-related lipid transfer domain protein 8 (STARD8), encoding a Rho-GTPase-activating protein, and WNK2, encoding a serine/threonine kinase are candidate tumor suppressor genes (TSGs) in human cancers. Inactivation of these genes that would promote cancer pathogenesis is largely unknown in colon cancer (CC). Our study addressed to address whether STARD8 and WNK2 genes are mutated in CC. STARD8 and WNK2 genes possess mononucleotide repeats in their exons, which could be the targets for frameshift mutations in cancers with high microsatellite instability (MSI-H). By single-strand conformation polymorphism (SSCP) analysis, we analyzed the repeated sequences in 140 CCs (95 CCs with MSI-H and 45 CCs with stable MSI (MSS)). By DNA sequencing, we found that five MSI-H CCs (5/95: 5.3%) harbored the frameshift mutations, whereas MSS CCs (0/45) did not. In addition, we detected regional heterogeneous frameshift mutations of these genes in four (25%) of 16 MSI-H CCs. In immunohistochemistry for WNK2, WNK2 expression in the MSI-H CCs was significantly lower than that in the MSS CCs. Our results for the mutation and expression indicate that STARD8 and WNK2 genes are altered at various levels (frameshift mutation, expression, and regional heterogeneity) in MSI-H CCs, which might play a role in the pathogenesis by inactivating their TSG functions.

RNA-Binding Protein RBM5 Plays an Essential Role in Acute Myeloid Leukemia By Activating the Oncogenic Protein HOXA9

Acute myeloid leukemia (AML) represents a type of malignant hematological disease that indicates poor outcomes in children and adults. HOXA9 overexpression is observed in 50-70% of human AML and positively correlates with poor patient outcomes. Leukemia subtypes with hallmark overexpression of HOXA9 include but are not limited to those carrying KMT2A gene rearrangements (KMT2A-r), NPM1c mutations, NUP98-fusions and EZH2 mutation. Accumulating genetic evidence also indicates that HOXA9 dysregulation is sufficient and necessary for leukemic transformation, and loss of function of HOXA9 and downstream pathways consequently impaired leukemia maintenance. Although HOXA9 is not an ideal direct candidate for therapeutic interventions, uncovering the novel regulators of HOXA9 might elucidate the HOXA9 regulation mechanism that could be exploited to identify novel therapeutic targets in HOXA9-driven leukemias. In this study, we performed genome-wide CRISPR/Cas9 screens in the HOXA9-endogenous reporter acute leukemia cells and unbiasedly identified that candidate genes are significantly enriched in RNA binding and RNA splicing regulation. An RBP protein, RBM5, was among the top essential genes. Initially, RBM5 was reported as a tumor suppressor gene in lung cancer. Thus far, the function of the encoding protein RBM5 in blood cancer remained largely unknown. To further test how RBM5 expression might correlate with AML development, we analyzed the public RNA-seq data from the TCGA clinical cancer sample datasets, RBM5 is highly expressed in AML, indicating a plausible role in AML oncogenesis. Functional validation by two individual sgRNAs against different exons of RBM5 confirmed the selectively survival essential of RBM5 in AML cell lines. Ectopic expression of sgRNA-resistant RBM5 cDNA ultimately rescued the cellular phenotype upon RBM5 loss. We utilized an alternative loss-of-function approach, RNA interference (RNAi), to further examine the functional requirement of RBM5, Two independent shRNAs targeted RBM5 mRNA in three AML cells lines. RBM5 loss significantly impaired cell growth, reduced colony numbers and triggered notable myeloid differentiation. We further transplant MOLM13 cells targeted with shRBM5 into NSG mice, and observed that RBM5 shRNAs-treated animals lagged progressively more behind with much less splenomegaly and reduced leukemia cell infiltration in bone marrow, spleen, and peripheral blood compared to the wild-type control, suggesting that RBM5 is required for AML progression in vivo. More importantly, Suppression of RBM5 in primary AML cells significantly reduced colony numbers and induced myeloid differentiation, as demonstrated by the maturation markers CD11b and CD14. In addition, the domain dropout CRISPR screen revealed that the DNA-binding domain C2H2 is required for the gene function. Integrative analysis further suggested that HOXA9 promises the direct transcriptional target of RBM5 as demonstrated by functional rescue assay and HOXA9 partially restore the gene transcriptional defect in RBM5 null leukemia cells. In summary, our innovative research reported that a new RNA-binding protein, RBM5, is required for the survival of AML through non-canonical transcriptional regulation via a complex regulatory mechanism. These data support RBM5 as a promising AML therapeutic target in future studies.

In Vivo Intra-Uterine Delivery of TAT-Fused Cre Recombinase and CRISPR/Cas9 Editing System in Mice Unveil Histopathology of Pten/p53-Deficient Endometrial Cancers.

Phosphatase and TENsin homolog (Pten) and p53 are two of the most frequently mutated tumor suppressor genes in endometrial cancer. However, the functional consequences and histopathological manifestation of concomitant p53 and Pten loss of function alterations in the development of endometrial cancer is still controversial. Here, it is demonstrated that simultaneous Pten and p53 deletion is sufficient to cause epithelial to mesenchymal transition phenotype in endometrial organoids. By a novel intravaginal delivery method using HIV1 trans-activator of transcription cell penetrating peptide fused with a Cre recombinase protein (TAT-Cre), local ablation of both p53 and Pten is achieved specifically in the uterus. These mice developed high-grade endometrial carcinomas and a high percentage of uterine carcinosarcomas resembling those found in humans. To further demonstrate that carcinosarcomas arise from epithelium, double Pten/p53 deficient epithelial cells are mixed with wild type stromal and myometrial cells and subcutaneously transplanted to Scid mice. All xenotransplants resulted in the development of uterine carcinosarcomas displaying high nuclear pleomorphism and metastatic potential. Accordingly, in vivo CRISPR/Cas9 disruption of Pten and p53 also triggered the development of metastatic carcinosarcomas. The results unfadingly demonstrate that simultaneous deletion of p53 and Pten in endometrial epithelial cells is enough to trigger epithelial to mesenchymal transition that is consistently translated to the formation of uterine carcinosarcomas in vivo.

IRF1 suppresses colon cancer proliferation by reducing SPI1-mediated transcriptional activation of GPX4 and promoting ferroptosis

IRF1 is a tumor suppressor gene in colon cancer. This study aimed to explore the potential regulation of IRF1 on the ferroptosis of colon cancer and the mechanisms underlying its regulation of GPX4 transcription. IRF1 interacting transcription factors regulating GPX4 transcription were predicted and validated. The role of the IRF1/SPI1-GPX4 axis on the ferroptosis of colon cancer cells was explored. Results showed that IRF1 overexpression reduced GPX4 transcription, increased reactive oxygen species (ROS) and lipid ROS accumulation, and enhanced erastin-induced colon cancer cell growth in vitro and in vivo. SPI1 could directly bind to the GPX4 promoter (−414 to −409) and activate its transcription. IRF1 could bind to SPI1 and suppress its transcriptional activating effects on GPX4 expression. SPI1 overexpression reduced ROS and lipid ROS accumulation and increased colon cancer cell viability and colony formation upon erastin induction. These trends were reversed by IRF1 overexpression. In conclusion, this study revealed a novel oncogenic mechanism of SPI1 by reducing erastin-induced ferroptosis in colon cancer. IRF1 interacts with SPI1 and suppresses its transcriptional activating effect on GPX4 expression. Through this mechanism, IRF1 can enhance erastin-induced ferroptosis of colon cancer. The IRF1/SPI1-GPX4 axis might play a crucial role in modulating ferroptosis in colon cancer and might serve as a potential therapeutic target in the future.

MiRNA-19b-3p downregulates the endothelin B receptor in gastric cancer cells to prevent angiogenesis and proliferation.

MiR19b-3p acts as a tumor suppressor gene in various cancers, but its function in gastric cancer is unknown. This study investigated the role of miR19b-3p in angiogenesis and the proliferation of human gastric cancer cells targeting ETBR expression. Cell proliferation in SGC-7901 cells, cell transfection, luciferase reporter assay, detection of endothelin B receptor mRNAs by RT-qPCR, and a Western blot assay were carried out. RT-qPCR expression analysis showed a prominent (p<0.01) downregulation of miR19b-3p in SGC-7901 cells, which was inversely correlated with a substantial increase (p<0.01) in the endothelin B receptor (ETBR). However, overexpression of miR19b-3p in SGC-7901 cells with its mimic (p<0.01) resulted in the loss of cell viability in the MTT assay. This effect was reversed (p<0.01) by the inhibitor. Western blot analysis revealed that ETBR was significantly (p<0.01) decreased by miR19b-3p overexpression compared with that of the negative control or its inhibitor. Based on bioinformatics tools and luciferase reporter assays, we found that miR19b-3p interacts with the 3'untranslated region (3'UTR) of ETBR. Restoring miR19b-3p overexpression with its mimic led to downregulation of ETBR in gastric cancer cells (SGC-7901), which significantly (p<0.01) decreased the expression of vascular endothelial growth factor A (VEGF-A). These findings were considerably reversed by miR19b-3p inhibitors (p<0.01). The results indicated that miR19b-3p exerts its molecular action by targeting ETBR at the post-transcriptional level by regulating angiogenesis and proliferation by overexpressing miR19b-3p as a potential treatment target for gastric cancer.

The deleted in oral cancer (DOC1 aka CDK2AP1) tumor suppressor gene is downregulated in oral squamous cell carcinoma by multiple microRNAs

Cyclin-dependent kinase 2-associated protein 1 (CDK2AP1; also known as deleted in oral cancer or DOC1) is a tumor suppressor gene known to play functional roles in both cell cycle regulation and in the epigenetic control of embryonic stem cell differentiation, the latter as a core subunit of the nucleosome remodeling and histone deacetylation (NuRD) complex. In the vast majority of oral squamous cell carcinomas (OSCC), expression of the CDK2AP1 protein is reduced or lost. Notwithstanding the latter (and the DOC1 acronym), mutations or deletions in its coding sequence are extremely rare. Accordingly, CDK2AP1 protein-deficient oral cancer cell lines express as much CDK2AP1 mRNA as proficient cell lines. Here, by combining in silico and in vitro approaches, and by taking advantage of patient-derived data and tumor material in the analysis of loss of CDK2AP1 expression, we identified a set of microRNAs, namely miR-21-5p, miR-23b-3p, miR-26b-5p, miR-93-5p, and miR-155-5p, which inhibit its translation in both cell lines and patient-derived OSCCs. Of note, no synergistic effects were observed of the different miRs on the CDK2AP1–3-UTR common target. We also developed a novel approach to the combined ISH/IF tissue microarray analysis to study the expression patterns of miRs and their target genes in the context of tumor architecture. Last, we show that CDK2AP1 loss, as the result of miRNA expression, correlates with overall survival, thus highlighting the clinical relevance of these processes for carcinomas of the oral cavity.

Relationship between the Reduced Expression of Zinc Finger Protein 668 in Bladder Cancer and Its Invasiveness.

The zinc finger protein 668 (ZNF668) gene encodes a Kruppel C2H2-type zinc-finger protein with 16 C2H2-type zinc fingers. The ZNF668 gene functions as a tumor suppressor gene in breast cancer. We histologically analyzed ZNF668 protein expression in bladder cancer and examined mutations of the ZNF668 gene in 68 cases of bladder cancer. In bladder cancer, the ZNF668 protein was expressed in the nuclei of cancer cells. In bladder cancer with submucosal and muscular infiltration, the expression of ZNF668 protein was significantly lower than that without submucosal and muscular infiltration. Eight heterozygous somatic mutations were detected in exon3 in five cases, and five of the mutations resulted in amino acid sequence mutations. Mutations resulting in amino acid sequence alterations also resulted in lower ZNF668 protein expression in bladder cancer cell nuclei, but no significant association with bladder cancer infiltration was detected. Decreased ZNF668 expression in bladder cancer was associated with submucosal and muscle invasion of cancer cells. Somatic mutations resulting in amino acid mutations in ZNF668 were found in 7.3% of the bladder cancer cases.

Alterations and Co-Occurrence of C-MYC, N-MYC, and L-MYC Expression are Related to Clinical Outcomes in Various Cancers.

MYC, also known as an oncogenic reprogramming factor, is a multifunctional transcription factor that maintains induced pluripotent stem cells (iPSCs). Although MYC is frequently upregulated in various cancers and is correlated with a poor prognosis, MYC is downregulated and correlated with a good prognosis in lung adenocarcinoma. MYC and two other MYC family genes, MYCN and MYCL, have similar structures and could contribute to tumorigenic conversion both in vitro and in vivo. We systematically investigated whether MYC family genes act as prognostic factors in various human cancers. We first evaluated alterations in the expression of MYC family genes in various cancers using the Oncomine and The Cancer Genome Atlas (TCGA) database and their mutation and copy number alterations using the TCGA database with cBioPortal. Then, we investigated the association between the expression of MYC family genes and the prognosis of cancer patients using various prognosis databases. Multivariate analysis also confirmed that co-expression of MYC/MYCL/MYCN was significantly associated with the prognosis of lung, gastric, liver, and breast cancers. Taken together, our results demonstrate that the MYC family can function not only as an oncogene but also as a tumor suppressor gene in various cancers, which could be used to develop a novel approach to cancer treatment.

TP53 and RB1 alterations characterize poor prognostic subgroups in pediatric acute myeloid leukemia.

Pediatric acute myeloid leukemia (AML) is a poor prognostic subtype of pediatric leukemia. However, the detailed characteristics of many genetic abnormalities are yet to be established in this disease. Although TP53 and RB1 are established as representative tumor suppressor genes in various cancers, alterations of these two genes, especially RB1, have not been characterized in pediatric AML. We performed next-generation sequencing in 328 pediatric AML patients from the Japanese AML-05 trial to ascertain TP53 and RB1 alterations, and their prognostic implications. We identified seven patients with TP53 alterations (2.1%) and six patients with RB1 alterations (1.8%). These alterations were found in only patients without RUNX1::RUNX1T1, CBFB::MYH11, or KMT2A rearrangements. TP53 and RB1 were frequently co-deleted with their neighboring genes PRPF8 and ELF1, respectively. Patients with TP53 alterations had significantly lower 5-year overall survival (OS; 14.3% vs. 71.4%, p < 0.001) and lower 5-year event-free survival (EFS; 0% vs. 56.3%, p < 0.001); similarly, patients with RB1 had significantly lower 5-year OS (0% vs. 71.8%, p < 0.001) and lower 5-year EFS (0% vs. 56.0%, p < 0.001) when compared to patients without these alterations. In gene expression analyses, oxidative phosphorylation, glycolysis, and protein secretion were upregulated in patients with TP53 and/or RB1 alterations. Additionally, Kaplan-Meier analysis revealed that high expressions of SLC2A5, KCNAB2, and CD300LF were related to poor OS of non-core-binding factor AML patients (p < 0.001, p = 0.001, and p = 0.021, respectively). This study will contribute to the development of risk-stratified therapy and precision medicine in pediatric AML.

Neuroglobin inhibits pancreatic cancer proliferation and metastasis by targeting the GNAI1/EGFR/AKT/ERK signaling axis

Pancreatic cancer is an extremely aggressive malignancy with a very disappointing prognosis. Neuroglobin (NGB), a member of the globin family, has been demonstrated to have a significant role in a variety of tumor forms. The possible role of NGB as a tumor suppressor gene in pancreatic cancer was investigated in this work. Information from the public dataset TCGA combined with GTEx was used to analyze the finding that NGB was commonly downregulated in pancreatic cancer cell lines and tissues, correlating with patient age and prognosis. The expression of NGB in pancreatic cancer was investigated via RT-PCR, qRT-PCR, and Western blot experiments. In-vitro and in-vivo assays, NGB elicited cell cycle arrest in the S phase and apoptosis, hindered migration and invasion, reversed the EMT process, and suppressed cell proliferation and development. The mechanism of action of NGB was predicted via bioinformatics analysis and validated using Western blot and co-IP experiments revealed that NGB inhibited the EGFR/AKT/ERK pathway by binding to and reducing expression of GNAI1 and p-EGFR. In addition, pancreatic cancer cells overexpressing NGB showed increased drug sensitivity to gefitinib (EGFR-TKI). In conclusion, NGB inhibits pancreatic cancer progression by specifically targeting the GNAI1/EGFR/AKT/ERK signaling axis.

Abstract 6023: Plasma methylome landscape across the spectrum of prostate cancer progression, and differentially methylated region (DMR) based biomarkers of hormonal therapy failure in metastatic hormone-sensitive prostate cancer

Abstract Background: DNA silencing of gene expression can affect tumorigenesis, treatment outcomes and prognosis. Epigenetic silencing of tumor suppressor genes in prostate cancer detected in plasma at different stages of progression and its effect on clinical outcomes has not been determined. In a cohort study across different stages of prostate cancer (PC) progression, we performed plasma cell-free DNA (cfDNA)-based epigenomic profiling for determining the landscape of methylation and Differentially Methylated Region (DMR) classifiers and the potential impact on clinical outcomes in metastatic hormone-sensitive prostate cancer (mHSPC) state. Methods: Plasma was collected prospectively in a longitudinal cohort study of 108 PC patients (pts) of which 24 had non-metastatic Local PC; 28 had mHSPC; 56 had mCRPC. cfDNA was used for enzymatic methylation sequencing (EM-seq) of a targeted panel of genes (N=441) implicated in prostate cancer biology. Methylation status at CpG islands of these genes were examined using Twist targeted cfDNA methylation assay. The Bismark program was used for methylation calls. Temporal trends of differentially methylated regions (DMRs) were identified in different states of progression by comparing the methylation status of target genes in localized PC, mHSPC and mCRPC states using a False Discovery Rate (FDR-q-value) of 0.2 or less. DMRs uniquely associated with mHSPC state were identified and determination of genes with DMRs in pts experiencing early failure of androgen deprivation therapy (ADT)-based therapies was performed. Early failure was defined as progression on ADT within 12 months. Results: We identified 304/441 genes that were differentially methylated between localized PC and advanced PC (mHSPC and mCRPC) (FDR&amp;lt;0.2), of which 237 genes differed between mHSPC and mCRPC (p&amp;lt;0.05) states. mHSPC pts were serially profiles before and after 3 months of ADT-based therapies. All 28 pts experienced a PSA response at 3-months and 112/237 genes showed significant change in methylation status in these serial samples taken before/after 3 months of ADT-based therapies. The median follow up of the cohort was 18 months (range 10-24). 6/28 mHSPC pts experienced early ADT failure (&amp;lt;12 months; median 10 months). 8/112 genes were significantly associated with early failures and included DMRs in RB1, FOXA1, SOX11. GSC, ALDH1L1, SCGB3A1, FLT4, and PTPRN2. Conclusions. A targeted cfDNA plasma liquid-biopsy based epigenomic profiling of PC cancer related genes was able to capture significant differences in DMRs across different states of cancer progression. Candidate differentially expressed CpG methylome gene biomarkers detected in plasma also identified biomarkers associated with early ADT-based treatment failure in mHSPC. Citation Format: Manish Kohli, Jodie Wong, Yijun Tian, Amir Bitaraf, Claire Hanson, Matt Larsen, Jennifer Lloyd, Julia Batten, Neeraj Agarwal, Umang Swami, Benjamin Maughan, Sumati Gupta, Jonathan Tward. Plasma methylome landscape across the spectrum of prostate cancer progression, and differentially methylated region (DMR) based biomarkers of hormonal therapy failure in metastatic hormone-sensitive prostate 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 6023.

LncRNA-BC069792 suppresses tumor progression by targeting KCNQ4 in breast cancer

BackgroundBreast cancer is the most common malignant tumor that threatens women's health. Attention has been paid on the study of long- non-coding RNA (lncRNA) in breast cancer. However, the specific mechanism remains not clear.MethodsIn this study, we explored the role of lncRNA BC069792 in breast cancer. In vitro and in vivo functional experiments were carried out in cell culture and mouse models. High-throughput next-generation sequencing technology and real-time fluorescence quantitative PCR technology were used to evaluate differentially expressed genes and mRNA expression, Western blot and immunohistochemical staining were used to detect protein expression. RNA immunoprecipitation assay and dual-luciferase activity assay were used to evaluate the competing endogenous RNAs (ceRNA), and rescue and mutation experiments were used for verification.ResultsWe found that lncRNA BC069792 was expressed at a low level in breast cancer tissues, and significantly decreased in breast cancer with high pathological grade, lymph node metastasis and high Ki-67 index groups. Moreover, BC069792 inhibited the proliferation, invasion and metastasis of breast cancer cells in vitro and in vivo. Mechanically, BC069792 acts as a molecular sponge to adsorb hsa-miR-658 and hsa-miR-4739, to up-regulate the protein expression of Potassium Voltage-Gated Channel Q4 (KCNQ4), inhibits the activities of JAK2 and p-AKT, and plays a role in inhibiting breast cancer growth.ConclusionsLncRNA BC069792 plays the role of tumor suppressor gene in breast cancer and is a new diagnostic index and therapeutic target in breast cancer.

G-quadruplex-enhanced circular single-stranded DNA (G4-CSSD) adsorption of miRNA to inhibit colon cancer progression.

Chromosomal heterogeneity leads to the abnormal expression and mutation of tumor-specific genes. Drugs targeting oncogenes have been extensively developed. However, given the random mutation of tumor suppressor genes, the development of its targeted drugs is difficult. Our early research revealed that artificial circular single-stranded DNA (CSSD) can restore multiple tumor suppressor genes to inhibit tumor malignant progression by adsorbing miRNA. Here, we improved CSSD to a fully closed single-stranded DNA with G quadruplex DNA secondary structure (G4-CSSD), which made G4-CSSD with higher acquisition rate and decreased degradation. The Cancer Genome Atlas (TCGA) and Human Protein Atlas database were used to predict tumour suppressor genes in colon cancer. Cellular and animal experiments were performed to validate the role of G4-CSSD in cancer cell progression. In colon cancer, we observed the simultaneous low expressions of chloride channel accessory 1 (CLCA1), UDP-GlcNAc:betaGal beta-1,3-N-acetylglucosaminyltransferase 6 (B3GNT6) and UDP glucuronosyltransferase family 2 member A3 (UGT2A3), which indicated an favourable prognosis. After repressing miR-590-3p with G4-CSSD590, the upregulation of CLCA1, B3GNT6 and UGT2A3 inhibited the proliferation and metastasis of colon cancer cells. This study may provide basis for new treatment methods for colon cancer by restoration of tumor suppressor genes.