RASSF1A Levels Research Articles

Novel Circulating Hypermethylated RASSF1A ddPCR for Liquid Biopsies in Patients With Pediatric Solid Tumors.

PURPOSELiquid biopsies can be used to investigate tumor-derived DNA, circulating in the cell-free DNA (cfDNA) pool in blood. We aimed to develop a droplet digital polymerase chain reaction (ddPCR) assay detecting hypermethylation of tumor suppressor gene RASSF1A as a simple standard test to detect various pediatric tumor types in small volume blood samples and to evaluate this test for monitoring treatment response of patients with high-risk neuroblastoma.METHODSWe developed a ddPCR assay to sensitively detect tumor-derived hypermethylated RASSF1A DNA in liquid biopsies. We tested this assay in plasma of 96 patients with neuroblastoma, renal tumors, rhabdomyosarcoma, or Hodgkin lymphoma at diagnosis and in cerebrospinal fluid of four patients with brain tumors. We evaluated the presence of hypermethylated RASSF1A in plasma samples during treatment and follow-up in 47 patients with neuroblastoma treated according to high-risk protocol and correlated results with blood mRNA–based and bone marrow mRNA–based minimal residual disease detection and clinical outcomes.RESULTSThe total cfDNA level was significantly higher in patients with metastatic neuroblastoma and nephroblastoma compared with healthy adult and pediatric controls. Hypermethylated RASSF1A was present in 41 of 42 patients with metastatic neuroblastoma and in all patients with nephroblastoma, with the median percentage of 69% and 21% of total RASSF1A, respectively. Hypermethylated RASSF1A levels decreased during therapy and recurred at relapse.CONCLUSIONOur findings demonstrate the value of ddPCR-based detection of hypermethylated RASSF1A as a circulating molecular tumor marker in neuroblastoma. Our preliminary investigation of RASSF1A hypermethylation detection in circulating cfDNA of other pediatric tumor entities demonstrates potential as a pan-tumor marker, but requires investigation in larger cohorts to evaluate its use and limitations.

Dynamic Changes of Fetal-Derived Hypermethylated RASSF1A and Septin 9 Sequences in Maternal Plasma.

DNA methylation has a tissue-specific feature, and placenta has distinct methylation patterns from peripheral blood cells. Although fetal/placental-derived cell free DNA (cfDNA) in the maternal blood has been reported in recent decades, systematic exploration of dynamic changes of the placental epigenetic signatures across gestation is lacking. The primary goal of this study was to characterize prenatal and postnatal methylation levels of placental-sourced RASSF1A and Septin 9 sequences in maternal plasma. Here, we used a quantitative methylation-sensitive PCR (qMS-PCR) assay to check the methylation status of RASSF1A and Septin 9 in placental tissues of pregnant women and plasma samples from non-pregnant individuals. Then, we examined the methylation levels of the two targets in maternal plasma from expectant women at different gestational ages and postdelivery. Hypermethylated RASSF1A and Septin 9 were identified in placental samples but undetectable in peripheral blood of healthy non-pregnant women. Further, hypermethylated RASSF1A sequence was found in all three trimesters of pregnancy except for early gestation (8weeks). Moreover, methylation scores of the two targets increased as pregnancy progressed. In addition, hypermethylated RASSF1A sequence was detectable in maternal plasma from 12h (one case) to 24h postdelivery (three cases) in 18 pregnant women. Our data on the variation of fetal-sourced methylated RASSF1A levels in maternal plasma in relation to gestational age provide a useful basis for improving the reliability of the methylation assay for non-invasive prenatal diagnosis (NIPD) in clinical practice.

Transcriptomic expression levels of the VHL, TIMP-3, and RASSF1A genes in renal tumors.

In this study, we aimed to investigate the relation between the mRNA expression levels of VHL, TIMP-3 and RASSF1A genes, and the histopathological and clinical characteristics of patients with renal tumors. Radical nephrectomy specimens of cases presented without neoadjuvant treatment were confirmed to be cancerous, non-cancerous, benign, and healthy after removal from separate localizations. A total of 69 patients with kidney tumors (138 tissue samples) were included in the study group. RNA isolation, reverse transcriptase PCR (RT-PCR), and quantitative real time PCR (qPCR) were performed, and the GAPDH gene was used to normalize mRNA levels. In the RCC cancerous tissue, TIMP-3 levels increased 1.3 times and RASSF1A levels increased 1.4 times compared to the corresponding levels in non-cancerous tissues, and there was no statistically significant difference in these values. On the other hand, VHL gene expression levels in cancerous tissue were 2.8 times higher than in matched adjacent non-cancerous tissues (p < 0.05). In the case of oncocytomas, TIMP-3 levels were found to be 3.2 times higher, RASSF1A levels 3.8 times higher, and VHL levels 2.2 times lower than the corresponding levels in healthy tissues (p < 0.05). The roles of VHL, TIMP-3, and RASSF1A mRNA expression in contributing to the development of renal tumors could not be clearly established. Further studies are therefore required to elucidate the mechanisms underlying renal tumors.

PML/RARα-Regulated miR-181a/b Cluster Targets the Tumor Suppressor RASSF1A in Acute Promyelocytic Leukemia.

In acute promyelocytic leukemia (APL), all-trans retinoic acid (ATRA) treatment induces granulocytic maturation and complete remission of leukemia. microRNAs are known to be critical players in the formation of the leukemic phenotype. In this study, we report downregulation of the miR-181a/b gene cluster in APL blasts and NB4 leukemia cells upon ATRA treatment as a key event in the drug response. We found that miR-181a/b expression was activated by the PML/RARα oncogene in cells and transgenic knock-in mice, an observation confirmed and extended by evidence of enhanced expression of miR-181a/b in APL patient specimens. RNA interference (RNAi)-mediated attenuation of miR-181a/b expression in NB4 cells was sufficient to reduce colony-forming capacity, proliferation, and survival. Mechanistic investigations revealed that miR-181a/b targets the ATRA-regulated tumor suppressor gene RASSF1A by direct binding to its 3'-untranslated region. Enforced expression of miR-181a/b or RNAi-mediated attenuation of RASSF1A inhibited ATRA-induced granulocytic differentiation via regulation of the cell-cycle regulator cyclin D1. Conversely, RASSF1A overexpression enhanced apoptosis. Finally, RASSF1A levels were reduced in PML/RARα knock-in mice and APL patient samples. Taken together, our results define miR-181a and miR-181b as oncomiRs in PML/RARα-associated APL, and they reveal RASSF1A as a pivotal element in the granulocytic differentiation program induced by ATRA in APL.

RASSF1A in maternal plasma as a molecular marker of preeclampsia

This study aimed to quantitate cell free (cf) and cell free fetal (cff) DNA in maternal plasma by determining RASSF1A levels before and after enzyme digestion in women who subsequently developed preeclampsia (PE) and compare them with uncomplicated pregnancies. Twenty-four samples from pregnant women who developed PE and 48 samples from women with uncomplicated pregnancies were analysed. Blood samples were obtained at 11-13 weeks. cfDNA was determined by quantifying RASSF1A using qRT-PCR. A second qRT-PCR was performed following methylation-sensitive enzyme digestion by BstUI, to quantitate hypermethylated RASSF1A sequences of fetal origin. ACTB gene was used as control to confirm complete enzyme digestion. cfDNA and cffDNA levels were significantly increased in women who developed PE as compared with uncomplicated pregnancies (median cfDNA: 9402 vs 2698, median cffDNA: 934.5 vs 62, respectively). Following operating characteristic curve analysis, cut-off values of 7486 Εq/mL for cfDNA and 512 Εq/mL for cffDNA were chosen, which provided a sensitivity of 75% and 100% and specificity of 98% and 100%, respectively, to identify women at risk for PE. The study demonstrates potential use of cfDNA and cffDNA in maternal plasma as markers for the early prediction of women at risk for PE.

RASSF1A and the Taxol Response in Ovarian Cancer

The RASSF1A tumor suppressor gene is frequently inactivated by promoter methylation in human tumors. The RASSF1A protein forms an endogenous complex with tubulin and promotes the stabilization of microtubules. Loss of RASSF1A expression sensitizes cells to microtubule destabilizing stimuli. We have observed a strong correlation between the loss of RASSF1A expression and the development of Taxol resistance in primary ovarian cancer samples. Thus, we sought to determine if RASSF1A levels could dictate the response to Taxol and whether an epigenetic therapy approach might be able to reverse the Taxol resistant phenotype of RASSF1A negative ovarian tumor cells. We found that knocking down RASSF1A expression in an ovarian cancer cell line inhibited Taxol-mediated apoptosis and promoted cell survival during Taxol treatment. Moreover, using a combination of small molecule inhibitors of DNA Methyl Transferase enzymes, we were able restore RASSF1A expression and Taxol sensitivity. This identifies a role for RASSF1A in modulating the tumor response to Taxol and provides proof of principal for the use of epigenetic therapy to overcome Taxol resistance.

Abstract 2186: Characterization of a novel ubiquitin E3 ligase that targets tumor suppressor RASSF1A for degradation during mitosis

Abstract Ras association domain family 1 isoform A (RASSF1A) is an important tumor suppressor. Loss or altered expression of RASSF1A has been associated with pathogenesis of a variety of cancers. RASSF1A is known to play an important role in regulation of mitosis, however, little is known about how RASSF1A is regulated during mitotic-phase of the cell cycle. In the present study, we have identified Cul4A as a RASSF1A E3 ligase and DDB1 serves as an adaptor protein to bring RASSF1A into the Cul4A-DDB1 complex. We have found that depletion of Cul4A E3 ligase results in increased levels of RASSF1A protein due to increased half-life; whereas overexpression of the Cul4A ligase markedly enhances RASSF1A protein ubiquitination resulting in reduced RASSF1A levels. We have further shown that Cul4A-mediated RASSF1A degradation occurs during mitosis and depletion of the E3 ligase markedly reverses mitotic-phase-stimulated RASSF1A degradation. Thus, our present study demonstrates that Cul4A is important for regulation of RASSF1A during mitosis and it may contribute to inactivation of RASSF1A thereby promoting cell cycle progression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2186. doi:10.1158/1538-7445.AM2011-2186

Quantification and application of the placental epigenetic signature of the RASSF1A gene in maternal plasma

The aims of the detection of hypermethylated RASSF1A gene in maternal plasma from all three trimesters of pregnancy were to show its feature of cell-free fetal DNA and to make up deficients of genetic markers. This study also aimed at investigating of its application value in pre-eclampsia compared with third trimester. Eighty pregnant women (7-41 gestational weeks) including normal pregnant women (60 cases) and pre-eclamptic pregnant women (20 cases) were selected as study groups and 20 normal non-pregnant women were selected as control group. Free DNA of plasma samples was extracted, and RASSF1A levels before and after double-methylation-sensitive restriction enzyme digestion of HinP1I and HhaI were determined to measure total and fetal cell-free DNA, respectively. beta-Actin gene was detected as a control to confirm complete enzyme digestion. The median concentrations of hypermethylated RASSF1A gene were 46 copies/mL in first trimester, 96 copies/mL in second trimester, 527 copies/mL in third trimester and 1947 copies/mL in pre-eclampsia. There was positive correlation between fetal-derived hypermethylated RASSF1A levels and the severity of pre-eclampsia. Hypermethylated RASSF1A gene may be considered as an epigenetic marker to detect the fetal DNA in maternal plasma and expands the clinical application of non-invasive prenatal diagnosis.