Volume 129, Issue 1, Pages 156-169 (July 2005) The Ras Effector RASSF2 Is a Novel Tumor-Suppressor Gene in Human Colorectal Cancer  Kimishige Akino, Minoru Toyota, Hiromu Suzuki, Hiroaki Mita, Yasushi Sasaki, Mutsumi Ohe-Toyota, Jean-Pierre J. Issa, Yuji Hinoda, Kohzoh Imai, Takashi Tokino  Gastroenterology  Volume 129, Issue 1, Pages 156-169 (July 2005) DOI: 10.1053/j.gastro.2005.03.051 Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 1 Expression profiles of RASSF1-6 in CRC cell lines. (A) Structure of RASSF family genes: The RA domain is shown as a solid box, the protein kinase C conserved domain as a hatched box. (B) RT-PCR analysis of RASSF1-6 carried out using cDNA prepared from 10 CRC cell lines (shown on the top); the genes analyzed are shown on the left. Expression of GAPDH was examined to monitor the integrity of cDNA. Gastroenterology 2005 129, 156-169DOI: (10.1053/j.gastro.2005.03.051) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 2 Analysis of RASSF gene methylation. (A) CpG islands of RASSF1-6: Vertical bars indicate the CpG sites; horizontal bars labeled GM1 indicate regions analyzed by COBRA; and arrows indicate the transcription start sites. (B) Bisulfite-PCR analysis of RASSF1-6. The methylation status of RASSF1-6 was examined by COBRA. The cell lines examined are shown on the top. M, methylated alleles. Gastroenterology 2005 129, 156-169DOI: (10.1053/j.gastro.2005.03.051) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 3 Methylation mapping of RASSF2. (A) Schematic diagram of the sequence around exon 1 of RASSF2: Vertical bars indicate the CpG sites; solid bars (A–F) indicate the regions analyzed. (B) Analysis of RASSF2 methylation. COBRA was carried out using bisulfite-treated DNA from 10 CRC cell lines. The region analyzed and the restriction enzymes used are shown on the left. M, methylated alleles. (C) Bisulfite sequencing of RASSF2. The entire CpG island of RASSF2 was analyzed using 4 sets of primers. The PCR products were cloned into pCR4.0 vector using a TOPO-TA-cloning kit. At least 10 clones are sequenced: Black columns indicate methylated CpGs; white columns indicate unmethylated CpGs. The cell lines examined are shown on the left. Gastroenterology 2005 129, 156-169DOI: (10.1053/j.gastro.2005.03.051) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 4 Role of DNA methylation and histone deacetylation in the silencing of RASSF2. (A) Restoration of RASSF2 in CRC cell lines treated with 5-Aza-dC. Three methylated and 3 unmethylated cell lines were examined for RASSF2 expression using real-time PCR. The RASSF2 signal was then normalized to that of GAPDH. (B) RT-PCR and real-time PCR showing that restoration of RASSF2 was enhanced by treatment with 5-Aza-dC + TSA. As indicated, cells were treated with mock, 2.0 μmol/L 5-aza-dC for 72 hours (Aza), 300 nmol/L TSA for 24 hours (TSA), 0.2 μmol/L 5-aza-dC for 72 hours (Aza 0.2 μmol/L), or 0.2 μmol/L 5-aza-dC for 72 hours, followed by 300 nmol/L TSA for 24 hours (A+T). For real-time PCR, the RASSF2 signal was normalized to that of GAPDH. (C) Luciferase assays carried out to determine the extent of the RASSF2 promoter. A series of reporter constructs containing the 5′-flanking region of the human RASSF2 gene were transiently transfected into HEK293, RKO, and HCT116 cells. Solid boxes represent the CACCC box, SP1 site, and exon 1. Relative luciferase activity was defined as the luciferase activity in cells transfected with the indicated reporter construct divided by activity in cells transfected with control vector. Cell lines used are shown on the left. (D) Acetylation status of histone H3 in CRC cell lines with or without RASSF2 methylation determined by chromatin immunoprecipitation using antiacetylated histone H3 antibody. Methylation levels and expression of RASSF2 are shown below. Gastroenterology 2005 129, 156-169DOI: (10.1053/j.gastro.2005.03.051) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 5 Morphological changes induced by transfection of RASSF2 into DLD-1 and RKO cells. Phalloidin staining (red) of DLD-1 (A) and RKO (B and C) cells transfected with empty vector reveals an abundance of actin stress fibers. These fibers are absent in cells transfected with RASSF2, and there is a concomitant increase of cortical actin. RASSF2 is stained with FITC-conjugated anti-Flag monoclonal antibody (green). (D) Morphologic changes of cells transfected with RASSF2 are not due to apoptosis. pCMV-TAG2-RASSF2 or pCMV-TAG2-p53 was transfected into DLD-1 cells by electroporation. After 12 hours, apoptotic cells were detected in TUNEL assays. Apoptotic cells were detected among cells transfected with p53 but not among those transfected with RASSF2. (E) Suppression of Rho by RASSF2. DLD-1 cells were transfected with either pCMV-Tag2B or pCMV-Tag2B-RASSF2, after which levels of the activated form of RhoA were assessed using pull-down assays. Gastroenterology 2005 129, 156-169DOI: (10.1053/j.gastro.2005.03.051) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 6 Tumor-suppressor function of RASSF2. (A) Schematic representation of the RASSF2 deletion constructs used in this study; the RA domain is shown by a solid box. (B) Assay showing the effect of RASSF2 on colony formation. DLD-1 and RKO cells were transfected with RASSF2, after which the cells were selected for 2 weeks in medium containing G418. Colonies were then stained with Giemsa. (C) Quantitative analysis of growth suppression by RASSF2. Gastroenterology 2005 129, 156-169DOI: (10.1053/j.gastro.2005.03.051) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 7 Induction of apoptosis by exogenous expression RASSF2 in CRC cell lines that otherwise lack RASSF2. (A) FACS analysis carried out to assess apoptosis after transfecting RKO cells with RASSF1 or RASSF2. (B) TUNEL assays. RKO cells were transfected with either empty vector or RASSF2 and subjected to TUNEL staining after 48 hours. (C) Quantitative analysis of apoptosis induced by transfecting cells with RASSF2. Gastroenterology 2005 129, 156-169DOI: (10.1053/j.gastro.2005.03.051) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 8 The role of RASSF2 in K-ras-induced cell transformation. (A) Comparison of the amino acid sequences of human, mouse, and rat RASSF2: Conserved amino acids are shown in red. The region used for siRNA is underlined. (B) Knockdown of RASSF2 in KNRK cells. After transfection with RASSF2 siRNA, the cells were harvested, and RASSF2 mRNA levels were determined by real-time PCR and normalized to those of GAPDH. (C) Enhancement of K-ras-mediated cell growth mediated by knockdown of RASSF2. Shown are representative colonies formed in soft agar (top) or on plates (bottom) after treatment with control or RASSF2 siRNA. (D) Quantitative analysis of colony formation under the indicated conditions; values are normalized with the number obtained with Mock. (E) Western blot analysis of phosphorylated p44/42 in extracts prepared from KNRK cells transfected with RASSF2 siRNA or control siRNA (top). Loading control: p44/42 (bottom). Gastroenterology 2005 129, 156-169DOI: (10.1053/j.gastro.2005.03.051) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 9 Aberrant methylation and silencing of RASSF2 in primary colorectal tumors. (A) COBRA of RASSF2 methylation (top) and bisulfite sequencing (bottom): N, normal colon tissues; C, CRC. The primer set RASSF2C and the restriction enzyme HhaI were used for COBRA analysis. M, methylated alleles; m, methylated CpG sites; u, unmethylated CpG sites. (B) Real-time PCR analysis of RASSF2 in methylated or unmethylated CRC xenografts. (C) Analysis of methylation RASSF family genes in primary CRCs: The genes analyzed are shown on the left; the CRC cases examined are shown on the top. M, methylated alleles. Gastroenterology 2005 129, 156-169DOI: (10.1053/j.gastro.2005.03.051) Copyright © 2005 American Gastroenterological Association Terms and Conditions