Frequent epigenetic inactivation of RASSF2 in thyroid cancer and functional consequences

In our study we aimed to reveal the role of the classical RASSF members which harbor a C-terminal RA domain and a SARAH domain and their effectors MST1, MST2 and WW45 in thyroid carcinogenesis. Since RASSF1A function and silencing was already studied in detail, we focused our work on RASSF2, RASSF3, RASSF4, RASSF5A, RASSF5C and RASSF6 which all encode a C-terminal SARAH domain [7, 10, 13]. First we analyzed the promoter methylation of these genes in thyroid cancer cell lines (Fig. 1). RASSF6 contains the only promoter, which is not located within a CpG island [13] and was not included in the methylation analysis. RASSF2 promoter hypermethyation was observed in seven out of eight (88%) thyroid cancer cell lines (Fig. 1 and Tab. 1). RASSF5A hypermethylation was found in all analyzed cancer cell lines, however RASSF5C was only partially methylated in 8305C and Hth74 (Fig. 1). RASSF5A and RASSF5C are transcribed from different CpG island promoters [13]. CpG island hypermethylation of RASSF2 and RASSF5A was confirmed by bisulfite sequencing (additional file 1). CpG island promoter hypermethylation of RASSF3, RASSF4, MST1, MST2 and WW45 were not found in thyroid cancer cell lines (Fig. 1). RASSF1A methylation was analyzed previously and is summarized in Table 1[7, 10]. RASSF1A was hypermethylated in all analyzed thyroid cancer cell lines.

Next we analyzed the expression of RASSF2, RASSF5A and RASSF5C in all thyroid cancer cell lines and human fibroblasts (HF53) (Fig. 2). In human fibroblasts, which were unmethylated for all three promoters, high expression of RASSF2 and RASSF5A was detected, however RASSF5C expression was low (Fig. 2A). Expression of RASSF2 was reduced in several thyroid cancer cell lines (e.g. FTC 236 and 1736). RASSF5A expression was also diminished in different thyroid cancer cell lines (Fig. 2A) compared to HF53. Additionally, two thyroid cancer cell lines (FTC 236 and 1736) were treated with 5-Aza-2'-deoxycytidine (Aza) and RASSF2 and RASSF5A expression was analyzed by qRT-PCR (Fig. 2B and 2C). Inhibition of DNA methyltransferases by Aza induced an upregulation of RASSF2 and RASSF5A expression. In summary, we observed frequent epigenetic silencing of RASSF2 and RASSF5A in thyroid cancer cell lines.

Subsequently, we analyzed the methylation of RASSF2 and RASSF5A promoter in 31 primary thyroid carcinomas (3 MTC, 10 FTC, 12 PTC and 6 UTC), 10 follicular adenomas, 12 goiters and 12 normal thyroid controls (Fig. 3 and Tab. 1). In 19 out of 30 (63%) thyroid carcinomas, the promoter of RASSF2 was partially methylated (Tab. 1). In contrast, none of 12 normal thyroid tissues and 10 follicular adenomas exhibited a methylation of RASSF2 (Fig. 3A). Only two out of 12 (17%) goiters were methylated (Tab. 1). Statistical analysis revealed a significant increase in RASSF2 methylation frequency in thyroid cancer compared to normal thyroid controls, follicular adenomas or goiters (p < 0.001, p = 0.001 or p = 0.015; Fisher's exact test, respectively). Furthermore, we examined the methylation frequency of RASSF2 in different types of thyroid carcinoma (Tab. 1). Medullary thyroid carcinoma showed the lowest frequency (0/3 = 0%) in comparison to PTC (6/11 = 54%), to FTC (8/10 = 80%) and to UTC (5/6 = 83%). Methylation of RASSF2 was significantly higher in thyroid tumors obtained from older patients (90%; >60 year) compared to those from younger patients (38%; <40 years; p = 0.043) or patients from 41 to 59 years (58%). Other correlations with histopathological parameters were not revealed. We also analyzed the RASSF5A methylation in primary thyroid cancers and other thyroid tissues (Fig. 3B). RASSF5A methylation was frequently found in cancer cell lines (9/9 = 100%) and primary thyroid cancer but was also detected in all analyzed control tissues, goiters and follicular adenomas (Tab. 1). Since RASSF5A methylation was not tumor specific, we further focused on RASSF2 where methylation was preferentially found in cancers.

Next, we analyzed the distinct tumor suppressive features associated with RASSF2. First, the localization of RASSF2 and the other classical RASSFs was investigated in FTC133 cells (Fig. 4). RASSF1A and RASSF1C are both found in the cytoplasm together with microtubule. The other RASSFs (RASSF2, Rassf3, RASSF4, RASSF5 and RASSF6) were not associated with microtubule but were observed in the cytoplasm and nucleus (Fig. 4 and data not shown). For Rassf3, RASSF4, RASSF5 and RASSF6 cluster formation was detected (Fig. 4 and data not shown). Deletion of the SARAH domain did not alter the localization of the fluorescent RASSF2, Rassf3 and RASSF6. However RASSF1A and RASSF4 appear to localize to nucleus from cytoplasm. It is interesting to note that the utilized RASSF5A isoform (variant 4) contains a partially truncated SARAH domain. The SARAH domain is a protein-protein interaction domain, which is found in all classical RASSFs and their proapoptotic effector MST1 and MST2 and the adapter protein WW45. Therefore, we analyzed the interaction of these proteins in yeast two hybrid and pull down assays (Table 2 and Fig. 5). We found an interaction of all RASSFs with MST1 and MST2 in the yeast two hybrid assay (additional file 2). However, truncation of the SARAH of RASSF5A (variant 4) and RASSF2 reduced the interaction in the yeast two hybrid or abolished interaction in the pulldown assay (Fig. 5, additional file 2 and additional file 3). Interaction of WW45 with RASSF1A, RASSF3, RASSF4 and MST1 and MST2 in the yeast two hybrid was found. Precipitation of MST1 with WW45, RASSF1A, RASSF2, RASSF3, RASSF5C and RASSF6 was also confirmed by pull down (Tab. 2 and Fig. 5). Interaction of RASSF2 with RASSF1A, RASSF3 and RASSF5C was only found in yeast two hybrid assay (Tab. 2).

The interaction of RASSF2 with kinases MST1 and MST2 indicate that RASSF2 is involved in the proapoptotic RASSF-pathway. Therefore, we analyzed RASSF2-induced growth suppression and apoptosis in thyroid cancer cell lines (Fig. 6). The thyroid cancer cell line 1736 was transfected with a construct expressing RASSF2 under control of the CMV promoter or an empty vector (Fig. 4). After selection for 4 weeks a 65% decrease of colony formation in cancer cells transfected with RASSF2 (mean = 4.6) compared to cells transfected with the empty vector (mean = 13.6; p = 0.019, one-way ANOVA test) was observed (Fig. 6A and 6B). Additionally, we analyzed the RASSF2 induced apoptosis in 1736 and FTC133 cells (Fig. 6C and 6D; additional file 4). Transfection of fluorescent RASSF2 caused a significantly increased apoptosis indicated by nuclei condensation/fragmentation compared to the fluorescent vector in 1736 and FTC133 (p < 0.001 and p = 0.07, respectively). Apoptotic cells were exemplarily verified by TUNEL assay (Fig. S4). In FTC1736 the ratio of intact vs. apoptotic cells was for the vector: 96% vs. 4%; for RASSF2wt: 88% vs. 12% and for RASSF2ΔSARAH: 94% vs. 6%, respectively. In FTC133 a similar trend was observed (Fig. 6D). For FTC133 the ratio of intact vs. apoptotic cells was the following for the vector: 75% vs. 25%; for RASSF2wt: 62% vs. 38% and for RASSF2ΔSARAH: 78% vs. 22%, respectively. Thus, RASSF2ΔSARAH exhibited a significantly reduced rate of apoptotic cells in 1736 and FTC133 cells compared to wt RASSF2 (p < 0.05). In summary, our data suggest that RASSF2 tumor suppression function involves apoptotic regulation through binding of the proapoptotic kinases MST.

65 thyroid tissues, including 31 primary thyroid carcinomas (6 UTC, 3 MTC, 10 FTC, and 12 PTC), 10 follicular adenomas, 12 goiters and 12 normal thyroid samples were analyzed [7, 10]. All patients signed informed consent at initial clinical investigation. The study was approved by local ethic committees. The mean age of the study population at the time of surgical resection was 51 ± 15 years and 40% of the patients were male. Nine human thyroid cancer cell lines: BcPaP (PTC), FTC238, FTC236, FTC133, C643 (UTC), 8505C (UTC), 1736 (UTC), 8305C (UTC) and HTh74 (UTC) were cultured in the recommended medium and included in this study [7, 10]. Genomic DNA was extracted from frozen tissues and cultured cells by a standard phenol/chloroform procedure.

Methylation of promoter regions (RASSF2, RASSF3, RASSF4, RASSF5A, RASSF5C, WW45, MST1 and MST2) was determined by COBRA and bisulfite sequencing [38, 39]. Briefly, 100 ng of bisulfite-treated DNA were amplified with 10 pmol of primers in a reaction buffer containing 0.2 mM dNTP mix, 1.5 mM MgCl₂, 10 pmol of each primer and 1.5 U Taq polymerase (InViTek GmBH, Berlin, Germany). For PCR primers and condition see additional file 5. 20 to 50 ng of PCR products were digested with 10 U of TaqI or HpyCH4IV (NEB, Frankfurt, Germany) and analyzed on a 2% Tris-borate EDTA agarose gel. For bisulfite sequencing PCR products were cloned in the pGEM-T vector (Promega, Heidelberg, Germany) and sequenced (Genterprise, Mainz, Germany).

Two thyroid cancer cell lines (1736 and FTC236) were treated for 4 days with 5 μM or 10 μM of 5-aza-dC (Sigma, Taufkirchen, Germany). RNA was isolated using TRIzol-Reagent (Invitrogen, Karlsruhe, Germany). To eliminate DNA contamination 1 μg RNA was incubated with 1U DNase I (Fermentas GmbH, St. Leon-Rot, Germany), 1 μl 10X DNase I buffer and DEPC-treated water in 10 μl. After 15 minutes incubation at room temperature DNase was inactivated by adding 1 μl of 25 mM EDTA and incubation at 65°C for 15 min and reversely transcribed using poly-dT primers and random hexamers in 20 μl of RT-mix with MMLV reverse transcriptase (Promega, Heidelberg, Germany) for 1 h at 42°C. Subsequently, 2 μl of cDNA was amplified. For PCR primers and condition see additional file 6.

Statistical analysis was carried out using SPSS17 (SPSS, München, Germany). Categorical variables were plotted into contingency tables and evaluated using Fisher's exact test or one way ANOVA test. All reported p-values are two-sided and considered significant for p < 0.05.

The cDNAs of RASSF2, RASSF3, RASSF4, RASSF5A (variant 4), RASSF5C (variant 3), RASSF6, MST1, MST2 and WW45 were cloned after amplification of fragments from EST-clones DKFZp781O1747Q, IMAGp998A0813502Q, IRAKp961I1269Q, IRALp962N0113Q (BC004270), IRAKp961L2026Q; DKFZp686K23225Q, IRAKp961C0282Q, IRAKp961I0613Q and IRAKp961L0427Q (RZPD, Berlin, Germany), respectively. RASSF1A, RASSF1C and Rassf3 were described previously [3, 40]. Mutant forms of constructs were generated with the QuickChange^® XL Site-Directed Mutagenesis Kit (Stratagene, La Jolla, USA) and appropriate primers. All constructs were confirmed by sequencing.

cDNAs were cloned into the fluorescence vector pEYFP (Clontech, Mountain View, USA). After transient transfection into FTC133 cells with HEKfectin (BioRad, München, Germany), the localization of fluorescent RASSF variants and the vector control were investigated with a fluorescence microscope (Axio Observer Z1 Zeiss, Jena, Germany). Nuclei of the cells were visualized by staining with DAPI (0,1 μg/ml in PBS).

The Matchmaker Two-hybrid system (Clontech, Mountain View, USA) was utilized. The yeast strain PJ69-4A was co-transformed with 0.5 μg of each plasmid using the PEG/LiAc method. The interaction analysis was carried out on SD minimal medium plates without adenine and histidine and the transformation efficiency was determined on SD plates with adenine and histidine. The strength of interaction was investigated by quantification of the expression of the β-galactosidase reporter gene with o-nitrophenyl-β-D-galactopyranoside (ONPG) as substrate at OD 420 nm.

cDNAs were cloned into the vector pCMV-Tag1 (Stratagene, La Jolla, USA) and/or in the modified vector pEBG (GST and FLAG tag). To investigate the interaction of specific RASSF forms, MST1, MST2 and WW45, co-transfections with HEKfectin (BioRad, München, Germany), were performed in HEK293T cells. Two days after transfection, total protein was extracted in FLAG-lysis buffer. The GST-fused proteins were precipitated with glutathione-sepharose (Amersham Biosciences, Freiburg, Germany). FLAG-tagged proteins were precipitated with anti-Flag-agarose (Sigma, Steinheim, Germany). Samples were separated on a 10% PAGE gel and blotted. The interaction was determined with anti-FLAG-antibodies (Sigma, Steinheim, Germany).

RASSF2 was cloned into the vector pCMV-Tag1 (Stratagene, La Jolla, USA). The thyroid cancer cell line 1736 was transfected using HEKfectin (BioRad, München, Germany). Colonies were selected under 1 mg/ml Geneticin (Gibco, Karlsruhe, Germany) in DMEM for 4 weeks. Expression of RASSF2 was confirmed by RT-PCR (data not shown).

Thyroid cancer cells FTC133 and 1736 were seeded on glass slides and transfected with different RASSF2 constructs using HEKfectin (BioRad, München, Germany) and analyzed after two days of incubation. Therefore cells were fixed with paraformaldehyde, permeabilized with TritonX, DAPI stained and embedded in MOWIOL for fluorescent analysis. Transfected cells (n > 100 for FTC133 and n > 200 for 1736) were analyzed for intact, condensed, fragmented or missing nucleus.