Luke B. Hesson

The Role of RASSF1A Methylation in Cancer

Tumour suppressor gene inactivation is critical to the pathogenesis of cancers; such loss of function may be mediated by irreversible processes such as gene deletion or mutation. Alternatively tumour suppressor genes may be inactivated via epigenetic processes a reversible mechanism that promises to be more amenable to treatment by therapeutic agents. The CpG dinucleotide is under-represented in the genome, but it is found in clusters within the promoters of some genes, and methylation of these CpG islands play a critical role in the control of gene expression. Inhibitors of the DNA methyltransferases DNMT1 and DNMT3b have been used in a clinical setting, these nucleotide analogues lack specificity but the side effects of low dose treatments were minimal and in 2004 Vidaza (5-azacitidine) was licensed for use in myelodysplastic syndrome. Methylation inhibitors are also entering trials in conjunction with another class of epigenetic modifiers, the histone deacetylase inhibitors and this epigenetic double bullet offers hope of improved treatment regimes. Recently there has been a plethora of reports demonstrating epigenetic inactivation of genes that play important roles in development of cancer, including Ras-association domain family of genes. Epigenetic inactivation of RASSF1A (Ras-association domain family 1, isoform A) is one of the most common molecular changes in cancer. Hypermethylation of the RASSF1A promoter CpG island silences expression of the gene in many cancers including lung, breast, prostate, glioma, neuroblastoma and kidney cancer. Several recent studies have illustrated the diagnostic and prognostic potential of RASSF1A methylation. This presents RASSF1A methylation as an attractive biomarker for early cancer detection which, for most cancers, results in improved clinical outcome. DNA methylation analysis is applicable to a range of body fluids including serum, urine, bronchioalveolar lavage and sputum. The ease with which these body fluids can be acquired negates the need for invasive procedures to obtain biopsy material. This review will discuss the feasibility of using RASSF1A methylation as a diagnostic and prognostic marker in cancer management.

Enhanced binding of TBK1 by an optineurin mutant that causes a familial form of primary open angle glaucoma

TANK‐binding kinase 1 (TBK1) was identified as a binding partner for Optineurin (OPTN) in two‐hybrid screens, an interaction confirmed by overexpression/immunoprecipitation experiments in HEK293 cells and by coimmunoprecipitation of endogenous OPTN and TBK1 from cell extracts. A TBK1 binding site was located between residues 1‐127 of OPTN, residues 78‐121 displaying striking homology to the TBK1‐binding domain of TANK. The OPTN‐binding domain was localised to residues 601‐729 of TBK1, while TBK1[1‐688] which cannot bind to TANK, did not interact with OPTN. The OPTN[E50K] mutant associated with Primary Open Angle Glaucoma (POAG) displayed strikingly enhanced binding to TBK1, suggesting that this interaction may contribute to familial POAG caused by this mutation.

RASSF1A Interacts with Microtubule-Associated Proteins and Modulates Microtubule Dynamics

The candidate tumor suppressor gene RASSF1A is inactivated in many types of adult and childhood cancers. However, the mechanisms by which RASSF1A exerts its tumor suppressive functions have yet to be elucidated. To this end, we performed a yeast two-hybrid screen to identify novel RASSF1A-interacting proteins in a human brain cDNA library. Seventy percent of interacting clones had homology to microtubule-associated proteins, including MAP1B and VCY2IP1/C19ORF5. RASSF1A association with MAP1B and VCY2IP1/C19ORF5 was subsequently confirmed in mammalian cell lines. This suggested that RASSF1A may exert its tumor-suppressive functions through interaction with the microtubules. We demonstrate that RASSF1A associates with the microtubules, causing them to exist as hyperstabilized circular bundles. We found that two naturally occurring tumor-associated missense substitutions in the RASSF1A coding region, C65R and R257Q, perturb the association of RASSF1A with the microtubules. The C65R and R257Q in addition to VCY2IP1/C19ORF5 showed reduced ability to induce microtubule acetylation and were unable to protect the microtubules against the depolymerizing action of nocodazole. In addition, wild-type RASSF1A but not the C65R or the R257Q is able to block DNA synthesis. Our data identify a role for RASSF1A in the regulation of microtubules and cell cycle dynamics that could be part of the mechanism(s) by which RASSF1A exerts its growth inhibition on cancer cells.

Frequent epigenetic inactivation of RASSF1A and BLU genes located within the critical 3p21.3 region in gliomas.

RASSF1A is a major tumor suppressor gene located at 3p21.3. We investigated the role of aberrant promoter region hypermethylation of RASSF1A in a large series of adult gliomas. RASSF1A was frequently methylated in both primary tumors (36/63; 57%) and tumor cell lines (7/7; 100%). Hypermethylation of RASSF1A in glioma cell lines correlated with loss of expression and treatment with a demethylating agent-reactivated RASSF1A gene expression. Furthermore, re-expression of RASSF1A suppressed the growth of glioma cell line H4 in vitro. Next, we investigated whether other members of the RASSF gene family were also inactivated by methylation. NORE1B and RASSF3 were not methylated in gliomas, while NORE1A and RASSF5/AD037 demonstrated methylation in glioma cell lines but not in primary tumors. We then investigated the methylation status of three other candidate 3p21.3 tumor suppressor genes. CACNA2D2 and SEMA3B were not frequently methylated, but the BLU gene located just centromeric to RASSF1 was frequently methylated in glioma cell lines (7/7) and in 80% (35/44) of glioma tumors. In these tumor cell lines, BLU expression was restored after treatment with a demethylating agent. Loss of BLU gene expression in glioma tumors correlated with BLU methylation. There was no association between RASSF1A and BLU methylation. RASSF1A methylation increased with tumor grade, while BLU methylation was seen at similar frequencies in all grades. Our data implicate RASSF1A and BLU promoter methylation in the pathogenesis of adult gliomas, while other RASSF family members and CACNA2D2 and SEMA3B appear to have only minor roles. In addition, RASSF1A and BLU methylation appear to be independent and specific events and not due to region-wide changes in DNA methylation.

NORE1A, a homologue of RASSF1A tumour suppressor gene is inactivated in human cancers.

We recently demonstrated that RASSF1A, a new tumour-suppressor gene located at 3p21.3 is frequently inactivated by promoter region hypermethylation in a variety of human cancers including lung, breast, kidney and neuroblastoma. We have identified another member of the RASSF1 gene family by in silico sequence analysis using BLAST searches. NORE1 located at 1q32.1 exists in three isoforms (NORE1Aα, NORE1Aβ and NORE1B). Both NORE1A and NORE1B isoforms have separate CpG islands spanning their first exons. NORE1Aα Produces a 418 aa protein containing a Ras-association (RA) domain and a diacylglycerol (DAG) binding domain. NORE1Aβ produces a C-terminal truncation of the RA domain. NORE1B also contains the RA domain but not the DAG domain. NORE1 is the human homologue of the mouse Ras effector Nore1. No inactivating somatic mutations were found in lung tumour lines; however, NORE1A promoter region CpG island was hypermethylated in primary tumours and tumour cell lines. NORE1A promoter was methylated in 10/25 breast, 4/40 SCLC, 3/17 NSCLC, 1/6 colorectal and 3/9 kidney tumour cell lines, while NORE1B promoter was unmethylated in the same tumour cell lines. While 24% (6/25) of primary NSCLC underwent NORE1A methylation, methylation in SCLC was a rare event (0/22); (P=0.0234). NORE1A expression in tumour cell lines was reactivated after treatment with a demethylating agent. There was no correlation between NORE1A and RASSF1A methylation status in NSCLC. Our results demonstrate that NORE1A is inactivated in a subset of human cancers by CpG island promoter hypermethylation, and in lung cancer this hypermethylation may be histological type specific.

Evaluation of the 3p21.3 tumour-suppressor gene cluster

Deletions of the 3p21.3 region are a frequent and early event in the formation of lung, breast, kidney and other cancers. Intense investigation of allelic losses and the discovery of overlapping homozygous deletions in lung and breast tumour-cell lines have defined a minimal critical 120 kb deletion region containing eight genes and likely to harbor one or more tumour-suppressor genes (TSGs). The candidate genes are HYAL2, FUS1, Ras-associated factor 1 (RASSF1), BLU/ZMYND10, NPR2L, 101F6, PL6 and CACNA2D2. Recent research indicates that several of these genes can suppress the growth of lung and other tumour cells. Furthermore, some genes (RASSF1A and BLU/ZMYND10) are very frequently inactivated by non-classical mechanisms such as promoter hypermethylation resulting in loss of expression. These data indicate that the 120 kb critical deletion region at 3p21.3 may represent a TSG cluster with preferential inactivation of particular genes depending on tumour type. The eight genes within this region and their potential role in cancer will be the focus of this review.

Frequent epigenetic inactivation of the SLIT2 gene in gliomas.

The SLIT family of genes consists of large extracellular matrix-secreted and membrane-associated glycoproteins. The Slits (Slit1–3) are ligands for the repulsive guidance receptors, the robo gene family. The Slit–Robo interactions mediate the repulsive cues on axons and growth cones during neural development. In a recent report, we demonstrated that promoter region CpG island of human SLIT2 was frequently hypermethylated in lung, breast and colorectal tumours and the silenced gene transcript suppressed the malignant phenotype in in vitro assays. In this report we undertook epigenetic, genetic and expression analysis of SLIT2 gene in a large series of gliomas and glioma cell lines. Promoter region CpG island of SLIT2 was found to be methylated in 71% (5/7) of glioma cell lines and was unmethylated in five DNA samples from normal brain tissues. The hypermethylation of the SLIT2 promoter region in glioma cell lines correlated with loss of expression and treatment with the demethylating agent 5-aza-2’-deoxycytidine reactivated SLIT2 gene expression. In primary gliomas, SLIT2 was methylated in 59% (37/63) of tumours analysed. In addition, SLIT2 expression was downregulated in methylated gliomas relative to unmethylated tumour samples, as demonstrated by quantitative real-time RT–PCR. Loss of heterozygosity analysis revealed that SLIT2 methylated gliomas retained both alleles of a microsatellite marker within 100 kb of the SLIT2 gene at 4p15.2. Exogenous expression of SLIT2 in a glioma cell line that was heavily methylated for SLIT2 decreased in vitro colony formation. Our data indicate that SLIT2 is frequently inactivated by promoter region CpG island hypermethylation in gliomas and may be a good candidate for a glioma tumour suppressor gene (TSG) located at 4p15.2. Furthermore, our data suggest that a detailed analysis of both the cancer genome and epigenome will be required to identify key TSGs involved in glioma development.

The RASSF1A tumor suppressor activates Bax via MOAP-1.

The novel tumor suppressor RASSF1A is frequently inactivated during human tumorigenesis by promoter methylation. RASSF1A may serve as a node in the integration of signaling pathways controlling a range of critical cellular functions including cell cycle, genomic instability, and apoptosis. The mechanism of action of RASSF1A remains under investigation. We now identify a novel pathway connecting RASSF1A to Bax via the Bax binding protein MOAP-1. RASSF1A and MOAP-1 interact directly, and this interaction is enhanced by the presence of activated K-Ras. RASSF1A can activate Bax via MOAP-1. Moreover, activated K-Ras, RASSF1A, and MOAP-1 synergize to induce Bax activation and cell death. Analysis of a tumor-derived point mutant of RASSF1A showed that the mutant was defective for the MOAP-1 interaction and for Bax activation. Moreover, inhibition of RASSF1A by shRNA impaired the ability of K-Ras to activate Bax. Thus, we identify a novel pro-apoptotic pathway linking K-Ras, RASSF1A and Bax that is specifically impaired in some human tumors.

CpG island promoter hypermethylation of a novel Ras-effector gene RASSF2A is an early event in colon carcinogenesis and correlates inversely with K-ras mutations

We report in silico identification and characterisation of a novel member of the ras association domain family 1 (RASSF1)/NORE1 family, namely, RASSF2, located at chromosomal region 20p13. It has three isoforms, all contain a ras association domain in the C-terminus. The longest isoform RASSF2A contains a 5′ CpG island. RASSF2A was cloned from a brain cDNA library and directly sequenced, confirming the genomic gene structure. In previous reports, we and others have demonstrated that RASSF1A is epigenetically inactivated in a variety of cancers, including sporadic colorectal cancer (CRC). In the present report, we analysed the methylation status of RASSF2A promoter region CpG island in sporadic CRC and compared it to K-ras mutation status. RASSF2A promoter region CpG island was hypermethylated in a majority of colorectal tumour cell lines (89%) and in primary colorectal tumours (70%), while DNA from matched normal mucosa was found to be unmethylated (tumour-specific methylation). RASSF2A expression was reactivated in methylated tumour cell lines after treatment with 5-aza 2-deoxycytidine. RASSF2A methylation is an early event, detectable in 7/8 colon adenomas. Furthermore, 75% of colorectal tumours with RASSF2A methylation had no K-ras mutations (codons, 12 and 13) (P=0.048), Fishers exact test). Our data demonstrate that RASSF2A is frequently inactivated in CRCs by CpG island promoter hypermethylation, and that epigenetic (RASSF2A) and genetic (K-ras) changes are mutually exclusive and provide alternative pathways for affecting Ras signalling.

RASSF6 is a novel member of the RASSF family of tumor suppressors

RASSF family proteins are tumor suppressors that are frequently downregulated during the development of human cancer. The best-characterized member of the family is RASSF1A, which is downregulated by promoter methylation in 40–90% of primary human tumors. We now identify and characterize a novel member of the RASSF family, RASSF6. Like the other family members, RASSF6 possesses a Ras Association domain and binds activated Ras. Exogenous expression of RASSF6 promoted apoptosis, synergized with activated K-Ras to induce cell death and inhibited the survival of specific tumor cell lines. Suppression of RASSF6 enhanced the tumorigenic phenotype of a human lung tumor cell line. Furthermore, RASSF6 is often downregulated in primary human tumors. RASSF6 shares some similar overall properties as other RASSF proteins. However, there are significant differences in biological activity between RASSF6 and other family members including a discrete tissue expression profile, cell killing specificity and impact on signaling pathways. Moreover, RASSF6 may play a role in dictating the degree of inflammatory response to the respiratory syncytial virus. Thus, RASSF6 is a novel RASSF family member that demonstrates the properties of a Ras effector and tumor suppressor but exhibits biological properties that are unique and distinct from those of other family members.