SETD8 inhibits ferroptosis in pancreatic cancer by inhibiting the expression of RRAD.

BACKGROUND: As an oncogene, SETD8 can promote tumour growth and tumour cell proliferation. This study aims to reveal the relationship between SETD8 and ferroptosis in pancreatic cancer and its role in pancreatic cancer to provide a possible new direction for the comprehensive treatment of pancreatic cancer. METHODS: The downstream targets were screened by RNA sequencing analysis. Western blot, Real-time Quantitative PCR (qPCR) and immunohistochemistry showed the relationship between genes. Cell proliferation analysis and cell metabolite analysis revealed the function of genes. Chromatin immunoprecipitation (CHIP) assays were used to study the molecular mechanism. RESULTS: The potential downstream target of SETD8, RRAD, was screened by RNA sequencing analysis. A negative correlation between SETD8 and RRAD was found by protein imprinting, Real-time Quantitative PCR (qPCR) and immunohistochemistry. Through cell proliferation analysis and cell metabolite analysis, it was found that RRAD can not only inhibit the proliferation of cancer cells but also improve the level of lipid peroxidation of cancer cells. At the same time, chromatin immunoprecipitation analysis (CHIP) was used to explore the molecular mechanism by which SETD8 regulates RRAD expression. SETD8 inhibited RRAD expression. CONCLUSIONS: SETD8 interacts with the promoter region of RRAD, which epigenetically silences the expression of RRAD to reduce the level of lipid peroxidation in pancreatic cancer cells, thereby inhibiting ferroptosis in pancreatic cancer cells and resulting in poor prognosis of pancreatic cancer.

RRAD inhibits aerobic glycolysis, invasion, and migration and is associated with poor prognosis in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is one of the most prevalent and lethal cancer worldwide. However, the mechanism underlying the HCC development remains unclear. Ras-related associated with diabetes (RRAD) is a small Ras-related GTPase which has been implicated in metabolic disease and several types of cancer, yet its functions in HCC remain unknown. A tissue microarray constructed by 90 paired HCC tissues and adjacent non-cancerous liver tissues was used to examine the protein levels of RRAD, and the messenger RNA (mRNA) expression of RRAD was also detected in a subset of this cohort. The prognostic significance of RRAD was estimated by the Kaplan-Meier analysis and Cox regression. The glucose utilization assay and lactate production assay were performed to measure the role of RRAD in HCC glycolysis. The effect of RRAD in HCC invasion and metastasis was analyzed by transwell assays. Our results suggested that the expression of RRAD was downregulated in HCC tissues compared to the adjacent non-tumorous liver tissues both in mRNA and protein levels and lower RRAD expression served as an independent prognostic indicator for the survival of HCC patients. Moreover, RRAD inhibited hepatoma cell aerobic glycolysis by negatively regulating the expression of glucose transporter 1 (GLUT1) and hexokinase II (HK-II). In addition, RRAD inhibition dramatically increased hepatoma cell invasion and metastasis. In conclusion, our study revealed that RRAD expression was decreased in HCC tumor tissues and predicted poor clinical outcome for HCC patients and played an important role in regulating aerobic glycolysis and cell invasion and metastasis and may represent potential targets for improving the treatment of HCC.

Ras-induced epigenetic inactivation of the RRAD (Ras-related associated with diabetes) gene promotes glucose uptake in a human ovarian cancer model.

RRAD (Ras-related associated with diabetes) is a small Ras-related GTPase that is frequently inactivated by DNA methylation of the CpG island in its promoter region in cancer tissues. However, the role of the methylation-induced RRAD inactivation in tumorigenesis remains unclear. In this study, the Ras-regulated transcriptome and epigenome were profiled by comparing T29H (a Ras(V12)-transformed human ovarian epithelial cell line) with T29 (an immortalized but non-transformed cell line) through reduced representation bisulfite sequencing and digital gene expression. We found that Ras(V12)-mediated oncogenic transformation was accompanied by RRAD promoter hypermethylation and a concomitant loss of RRAD expression. In addition, we found that the RRAD promoter was hypermethylated, and its transcription was reduced in ovarian cancer versus normal ovarian tissues. Treatment with the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine resulted in demethylation in the RRAD promoter and restored RRAD expression in T29H cells. Additionally, treatment with farnesyltransferase inhibitor FTI277 resulted in restored RRAD expression and inhibited DNA methytransferase expression and activity in T29H cells. By employing knockdown and overexpression techniques in T29 and T29H, respectively, we found that RRAD inhibited glucose uptake and lactate production by repressing the expression of glucose transporters. Finally, RRAD overexpression in T29H cells inhibited tumor formation in nude mice, suggesting that RRAD is a tumor suppressor gene. Our results indicate that Ras(V12)-mediated oncogenic transformation induces RRAD epigenetic inactivation, which in turn promotes glucose uptake and may contribute to ovarian cancer tumorigenesis.

Ras-related associated with diabetes genes for biomarker-based therapeutics in cancer: A comparative evolutionary genomic study.

OBJECTIVES: To compare Ras-related associated with diabetes (RRAD) across different species and to identify specific biomarkers for cancer therapy. METHODS: The study involves comparing the coding sequences, genes, messenger ribonucleic acid (RNA), non-coding RNA, open reading frame, short- and long-sequence repeats, and transcription factors of RRAD genes from 82 species. Various tools and software are employed for these comparisons, and evolutionary analysis was carried out to understand the gene's evolutionary history. The data are classified based on forward and reverse sequences. RESULTS: Our analysis indicates that ACTG1 may function as a downstream effector of RRAD, offering potential avenues for diabetes and cancer treatments. By collecting RRAD sequences from 82 species and carrying out comparative genomics, this study provides diverse strategies for developing biomarker-based therapeutics. Furthermore, it suggests using RRAD in other organisms as a model for studying the knockdown effects of specific sequence sets. The study presents RRAD sequences from 82 organisms across different families, contributing to a diverse knowledge base for identifying drug-designing biomarkers. CONCLUSION: This research offers insights into the potential of RRAD as a therapeutic target in various organisms and highlights the importance of biomarker identification in drug development.

Friend or Foe: Regulation, Downstream Effectors of RRAD in Cancer.

Ras-related associated with diabetes (RRAD), a member of the Ras-related GTPase superfamily, is primarily a cytosolic protein that actives in the plasma membrane. RRAD is highly expressed in type 2 diabetes patients and as a biomarker of congestive heart failure. Mounting evidence showed that RRAD is important for the progression and metastasis of tumor cells, which play opposite roles as an oncogene or tumor suppressor gene depending on cancer and cell type. These findings are of great significance, especially given that relevant molecular mechanisms are being discovered. Being regulated in various pathways, RRAD plays wide spectrum cellular activity including tumor cell division, motility, apoptosis, and energy metabolism by modulating tumor-related gene expression and interacting with multiple downstream effectors. Additionally, RRAD in senescence may contribute to its role in cancer. Despite the twofold characters of RRAD, targeted therapies are becoming a potential therapeutic strategy to combat cancers. This review will discuss the dual identity of RRAD in specific cancer type, provides an overview of the regulation and downstream effectors of RRAD to offer valuable insights for readers, explore the intracellular role of RRAD in cancer, and give a reference for future mechanistic studies.

Pharmacological or genetic inhibition of LTCC promotes cardiomyocyte proliferation through inhibition of calcineurin activity.

Cardiomyocytes (CMs) lost during ischemic cardiac injury cannot be replaced due to their limited proliferative capacity, which leads to progressive heart failure. Calcium (Ca2+) is an important signal transducer that regulates key cellular processes, but its role in regulating CM proliferation is incompletely understood. A drug screen targeting proteins involved in CM calcium cycling in human embryonic stem cell-derived cardiac organoids (hCOs) revealed that only the inhibition of L-Type Calcium Channel (LTCC), but not other Ca2+ regulatory proteins (SERCA or RYR), induced the CM cell cycle. Furthermore, overexpression of Ras-related associated with Diabetes (RRAD), an endogenous inhibitor of LTCC, induced CM cell cycle activity in vitro, in human cardiac slices, and in vivo. Mechanistically, LTCC inhibition by RRAD induces the cell cycle in CMs by modulating calcineurin activity and translocating Hoxb13 to the CM nucleus. Together, this represents a robust pathway for regenerative strategies.

NEAT1_2/RRAD/EHF Positive Feedback Loop Facilitates Aerobic Glycolysis in Papillary Thyroid Cancer Cells.

Papillary thyroid carcinoma (PTC) is the most prevalent endocrine-related malignancy. In spite of the good prognosis, a more aggressive disease can develop in some PTC patients, leading to poor survival. Nuclear paraspeckle assembly transcript 1 (NEAT1) enhances tumorigenesis; however, the relationship between NEAT1_2 and glycolysis in PTC has not been identified. The expressions of NEAT1_2, KDM5B, Ras-related associated with diabetes (RRAD), and EHF were determined by quantitative reverse transcription polymerase chain reaction and immunocytochemistry. The effects of NEAT1_2, KDM5B, RRAD, and EHF on PTC glycolysis were ascertained employing in vitro as well as in vivo experiments. Chromatin immunoprecipitation (ChIP), RNA binding protein immunoprecipitation, luciferase reporter assays, and co-immunoprecipitation were utilized to analyze the binding abilities among NEAT1_2, KDM5B, RRAD, and EHF. Overexpression of NEAT1_2 was associated with glycolysis in PTC. NEAT1_2 could activate glycolysis by regulating the expression of RRAD in PTC. NEAT1_2 mediated H3K4me3 modification at the promoter of RRAD by recruiting KDM5B. RRAD further negatively regulated glycolysis by binding and regulating the subcellular location of the transcription factor EHF. EHF could activate the transcription of NEAT1_2, hexokinase 2, and pyruvate kinase M2, thereby forming the NEAT1_2/RRAD/EHF feedback loop. Our study revealed that the NEAT1_2/RRAD/EHF positive feedback loop facilitated glycolysis in PTC, which might avail meaningful insight for PTC management.

PURPL and NEAT1 Long Non-Coding RNAs Are Modulated in Vascular Smooth Muscle Cell Replicative Senescence.

Cellular senescence is characterized by proliferation and migration exhaustion, senescence-associated secretory phenotype (SASP), and oxidative stress. Senescent vascular smooth muscle cells (VSMCs) contribute to cardiovascular diseases and atherosclerotic plaque instability. Since there are no unanimously agreed senescence markers in human VSMCs, to improve our knowledge, we looked for new possible senescence markers. To this end, we first established and characterized a model of replicative senescence (RS) in human aortic VSMCs. Old cells displayed several established senescence-associated markers. They stained positive for the senescence-associated ß-galactosidase, showed a deranged proliferation rate, a dramatically reduced expression of PCNA, an altered migratory activity, increased levels of TP53 and cell-cycle inhibitors p21/p16, and accumulated in the G1 phase. Old cells showed an altered cellular and nuclear morphology, downregulation of the expression of LMNB1 and HMGB1, and increased expression of SASP molecules (IL1ß, IL6, IL8, and MMP3). In these senescent VSMCs, among a set of 12 manually selected long non-coding RNAs (lncRNAs), we detected significant upregulation of PURPL and NEAT1. We observed also, for the first time, increased levels of RRAD mRNA. The detection of modulated levels of RRAD, PURPL, and NEAT1 during VSMC senescence could be helpful for future studies on potential anti-aging factors.

RAD-Deficient Human Cardiomyocytes Develop Hypertrophic Cardiomyopathy Phenotypes Due to Calcium Dysregulation.

Ras associated with diabetes (RAD) is a membrane protein that acts as a calcium channel regulator by interacting with cardiac L-type Ca2 + channels (LTCC). RAD defects can disrupt intracellular calcium dynamics and lead to cardiac hypertrophy. However, due to the lack of reliable human disease models, the pathological mechanism of RAD deficiency leading to cardiac hypertrophy is not well understood. In this study, we created a RRAD -/- H9 cell line using CRISPR/Cas9 technology. RAD disruption did not affect the ability and efficiency of cardiomyocytes differentiation. However, RAD deficient hESC-CMs recapitulate hypertrophic phenotype in vitro. Further studies have shown that elevated intracellular calcium level and abnormal calcium regulation are the core mechanisms by which RAD deficiency leads to cardiac hypertrophy. More importantly, management of calcium dysregulation has been found to be an effective way to prevent the development of cardiac hypertrophy in vitro.

Tumor cells containing the African-Centric S47 variant of TP53 show increased Warburg metabolism.

Mutations in the TP53 tumor suppressor gene remain a hallmark of human cancer. In addition to mutation of TP53, single nucleotide polymorphisms (SNPs) in this gene can have a profound impact on p53 function, and can affect cancer risk as well as other p53 functions. Wild type (WT) p53 contains a proline at amino acid 47, but approximately 1% of African-Americans express a p53 allele with a serine at amino acid 47 (Pro47Ser, hereafter S47). In a mouse model for this variant, mice expressing S47 are predisposed to spontaneous cancers. The S47 variant also is associated with increased pre-menopausal breast cancer risk in African American women. We recently reported that S47 tumor cells are resistant to the majority of cytotoxic chemotherapeutic agents, but show increased sensitivity to a subset of anti-cancer agents, compared to tumors with WT p53. In this work, we report on another potentially promising therapeutic vulnerability of S47 tumors. We find that S47 tumors show decreased mitochondrial metabolism, along with increased dependency on glycolysis. S47 tumor cells also show increased sensitivity to the glycolytic poison 2-deoxy-glucose. We propose that the altered metabolism in S47 tumor cells may be yet another potentially-actionable therapeutic vulnerability to exploit in cancer-prone individuals with this genotype.

HPV 16 E6/E7 up-regulate the expression of both HIF-1α and GLUT1 by inhibition of RRAD and activation of NF-κB in lung cancer cells.

Chronic infection of HPV16 E6/E7 is frequently associated with lung cancers, especially in non-smokers and in Asians. In our previous studies, we found that HPV16 E6/E7 up-regulated HIF-1α at protein level and further up-regulated GLUT1 at both protein and mRNA levels in well-established lung cancer cell lines. In one of our further mechanism study, the results demonstrated that HPV16 E6/E7 up-regulated the expression of GLUT1 through HPV-LKB1-HIF-1α-GLUT1 axis. However, there are multiple pathways involved in HPV16 E6/E7 regulation of HIF-1α expression. In current study, using double directional genetic manipulation in well-established lung cancer cell lines, we showed that both E6 and E7 down-regulated the expression of RRAD at both protein and mRNA levels. Like LKB1, RRAD is one of the cancer suppressor genes. The loss of RRAD further activated NF-κB by promoted cytoplasmic p65 translocated to nucleus, and up-regulated the expression level of the p-p65 in nucleus. Furthermore, p-p65 up regulated HIF-1α and GLUT1 at both protein and mRNA levels. Thus, we proposed HPV16 E6/E7 up-regulated the expression of GLUT1 through HPV-RRAD-p65- HIF-1α- GLUT1 axis. In conclusion, we demonstrated for the first time that E6 and E7 promoted the expression of HIF-1α and GLUT1 by relieving the inhibitory effect of RRAD which resulted in the activation of NF-κB by promoting cytoplasmic p65 translocated to nucleus, and up-regulated the expression of the p-p65 in nucleus in lung cancer cells. Our findings provided new evidence to support the critical role of RRAD in the pathogenesis of HPV-related lung cancer, and suggested novel therapeutic targets.

RRAD inhibits the Warburg effect through negative regulation of the NF-κB signaling.

Cancer cells preferentially use aerobic glycolysis to meet their increased energetic and biosynthetic demands, a phenomenon known as the Warburg effect. Its underlying mechanism is not fully understood. RRAD, a small GTPase, is a potential tumor suppressor in lung cancer. RRAD expression is frequently down-regulated in lung cancer, which is associated with tumor progression and poor prognosis. Recently, RRAD was reported to repress the Warburg effect, indicating that down-regulation of RRAD expression is an important mechanism contributing to the Warburg effect in lung cancer. However, the mechanism by which RRAD inhibits the Warburg effect remains unclear. Here, we found that RRAD negatively regulates the NF-κB signaling to inhibit the GLUT1 translocation and the Warburg effect in lung cancer cells. Mechanically, RRAD directly binds to the p65 subunit of the NF-κB complex and inhibits the nuclear translocation of p65, which in turn negatively regulates the NF-κB signaling to inhibit GLUT1 translocation and the Warburg effect. Blocking the NF-κB signaling largely abolishes the inhibitory effects of RRAD on the translocation of GLUT1 to the plasma membrane and the Warburg effect. Taken together, our results revealed a novel mechanism by which RRAD negatively regulates the Warburg effect in lung cancer cells.

Brahma-related gene 1 inhibits proliferation and migration of human aortic smooth muscle cells by directly up-regulating Ras-related associated with diabetes in the pathophysiologic processes of aortic dissection.

OBJECTIVE: To elucidate the mechanisms of Brahma-related gene 1 (Brg1) involvement in the pathophysiologic processes of aortic dissection. METHODS: Seventeen dissecting, 4 dilated, and 10 healthy human aorta samples were collected. Expression of Brg1 in the medium of aorta was evaluated by quantitative real-time polymerase chain reaction, Western blot, and immunohistochemical staining, respectively. The regulation effect of Brg1 on proliferation and migration of human aortic smooth muscle cells (HASMCs) was analyzed in 3 ways: using cell counting, a migration chamber, and a wound scratch assay. A polymerase chain reaction array was used for screening potential target genes of Brg1. A chromatin immunoprecipitation assay was adopted for direct deoxyribonucleic acid-protein binding detection. RESULTS: Expression levels of Brg1 were increased in aortic dissection and aortic dilation patients. In vitro results indicated that overexpression of Brg1 inhibited proliferation and migration of HASMCs. The candidate proliferation- and migration-related Brg1 target gene found was Ras-related associated with diabetes (RRAD), expression levels of which were enhanced in dissecting aortic specimens. The direct regulation effect of Brg1 on RRAD was verified by chromatin immunoprecipitation assay results. Furthermore, down-regulating RRAD significantly alleviated the suppression effects of Brg1 on proliferation and migration of HASMCs. CONCLUSIONS: Our study illustrated that Brg1 inhibited the proliferation and migration capacity of HASMCs, via the mechanism of direct up-regulation of RRAD, thus playing an important role in the pathophysiologic processes of aortic dissection.

Aberrant methylation of the Ras-related associated with diabetes gene in human primary esophageal cancer.

BACKGROUND/AIM: Ras-related associated with diabetes (RRAD), a member of the Ras-related GTPase superfamily, is frequently methylated in several human cancers, though its methylation profile remains unclear in esophageal cancer. MATERIALS AND METHODS: We examined RRAD promoter hypermethylation using real-time quantitative methylation-specific PCR in 229 primary human esophageal tissues of contrasting histological types. RESULTS: RRAD hypermethylation showed highly discriminative receiver-operator characteristic curve profiles, clearly distinguishing esophageal squamous cell carcinoma (ESCC) from esophageal adenocarcinoma (EAC) or normal esophagus (NE) (p<0.01 and p<0.01, respectively). RRAD normalized methylation values were significantly higher in ESCC (0.0242) than in NE (0.0057, p<0.05) or EAC (0.0139, p<0.01). RRAD hypermethylation frequency was also significantly higher in ESCC (23.1%) than in NE (0%, p<0.05) or EAC (5.4%, p<0.05). CONCLUSION: Promoter hypermethylation of RRAD is a frequent, tissue-specific event in ESCC, and is uncommon in EAC. The aberrant methylation of RRAD may be involved in the pathogenesis of a subset of ESCC, but not in EAC.