Volume 136, Issue 1, Pages 206-216 (January 2009) Decreased Expression of the RAS-GTPase Activating Protein RASAL1 Is Associated With Colorectal Tumor Progression  Miki Ohta, Motoko Seto, Hideaki Ijichi, Koji Miyabayashi, Yotaro Kudo, Dai Mohri, Yoshinari Asaoka, Motohisa Tada, Yasuo Tanaka, Tsuneo Ikenoue, Fumihiko Kanai, Takao Kawabe, Masao Omata  Gastroenterology  Volume 136, Issue 1, Pages 206-216 (January 2009) DOI: 10.1053/j.gastro.2008.09.063 Copyright © 2009 AGA Institute Terms and Conditions

Figure 1 RASGAP gene expression in CRC cells. (A) The expression of 12 genes containing a RASGAP domain was analyzed in 18 CRC cell lines using qRT-PCR, and mutations in KRAS and BRAF were identified using DNA sequencing. The tumors were categorized into 3 groups: those with mutant KRAS (Mut-KRAS, n = 7), those with mutant BRAF (Mut-BRAF, n = 4), and those with wild-type KRAS/BRAF (Wt, n = 7). The expression of each RASGAP gene was normalized relative to the GAPDH gene. (B) Quantification of RASAL1 gene expression using qRT-PCR in each cell line. The expression of RASAL1 gene was normalized relative to the GAPDH gene. (C) Immunoblot analysis using anti-RASAL1 antibody was performed for the 18 CRC cell lines. *Nonspecific bands. (D) The correlation of the RASAL1 mRNA and protein levels in each of the 18 CRC cell lines. The RASAL1 mRNA and protein levels were normalized using the GAPDH mRNA and ACTB protein expression levels, respectively. The RASAL1 and ACTB protein levels were measured using densitometry. (E) The RASAL1 expression levels were compared among the Mut-KRAS (n = 7), Mut-BRAF (n = 4), and wild-type KRAS/BRAF tumors (n = 7). (F) The cellular localization of endogenous RASAL1 was examined using immunofluorescence microscopy. NCIH630 and SKCO1 CRC cells were immunostained with anti-RASAL1 antibody and the secondary antibody Alexa Fluor 488 (green); the nuclei were stained with propidium iodide (PI) (red). SKCO1 cells treated without anti-RASAL1 antibody were included as a control. Scale bar, 40 μm. Gastroenterology 2009 136, 206-216DOI: (10.1053/j.gastro.2008.09.063) Copyright © 2009 AGA Institute Terms and Conditions

Figure 2 The RASGAP activity of RASAL1. (A) The RASAL1 expression plasmids or pcDNA3.1 control plasmids were transfected into HeLa cells. Total lysate was obtained 72 hours later, and small GTPase pull-down assays were performed. (B) The RASAL1 expression plasmids or Myc-KRAS expression plasmids were transfected into HeLa cells, and the RAS-GTP pull-down assay was performed 72 hours later. (C) The RASAL1 expression plasmids or empty control plasmids were transfected into HeLa cells. Epidermal growth factor (EGF) was added (50 ng/mL) 72 hours later, and the total lysate was obtained at 0, 10, 20, 30, 45, and 90 minutes. Immunoblotting was performed using anti-phosoho-ERK1/2 (p-ERK), total ERK1/2, phospho-MEK1/2 (p-MEK), and total MEK1/2. (D) The MTT cell growth assay was performed using the cell clones overexpressing RASAL1 and control cells. The RASAL1 expression plasmids were transfected into CaCo2 cells, and geneticin-resistant clones overexpressing RASAL1 were selected. Empty pcDNA3.1 plasmids were also transfected, and a geneticin-resistant cell pool was obtained as a control. The overexpression of RASAL1 was confirmed using immunoblotting. (E) The activity of transfected KRAS was examined using a RAS-GTP pull-down assay in the 7 CRC cell lines with wild-type KRAS and 2 with mutant KRAS. CRC with RASAL1 (+) or (−) means the cell lines with or without RASAL1 expression in immunoblotting assays, respectively (see also Figure 1C). The optical density of RAS-GTP was normalized to total RAS and described as the normalized RAS-GTP level. (F) Immunoblotting was performed using anti-p-ERK1/2 and anti-total ERK1/2 antibodies in 18 CRC cell lines. The optical density of p-ERK was normalized to total ERK and described as the normalized p-ERK level. Gastroenterology 2009 136, 206-216DOI: (10.1053/j.gastro.2008.09.063) Copyright © 2009 AGA Institute Terms and Conditions

Figure 3 RASAL1 knockdown in CRC cells. (A) Two RASAL1-knockdown clones were established using RNA interference with PMFko14 cells (shRasal1-1, -2). Two lines of GFP-knockdown cells were also established (shGFP-1, -2) as controls. The knockdown was confirmed by immunoblotting. The amount of activated Ras was assessed using affinity precipitation analysis. Phosphorylated ERK1/2 and MEK1/2 were also assessed by immunoblotting. (B) Changes in the xenograft volumes of each clone of RASAL1 knockdown and the control cells in nude mice (n = 6 each). Gastroenterology 2009 136, 206-216DOI: (10.1053/j.gastro.2008.09.063) Copyright © 2009 AGA Institute Terms and Conditions

Figure 4 RASAL1 expression in colorectal neoplasms. (A–F) Representative immunohistochemical results using anti-RASAL1 antibody with colon tumors. Human normal colon epithelial (A and B) and tumor tissues (C–E). Each tumor was categorized based on the number of tumor cells that expressed RASAL1: more than 50% (C, retained), 10%–50% (D, moderately reduced), or less than 10% (E, severely reduced). (D) Some tumor cells had reduced RASAL1 expression (white arrowheads). (F) An example of a “severely reduced” RASAL1 tumor. T, tumor cells; N, nontumor epithelial cells. (G) RASAL1 expression in clinical colorectal neoplasms was assessed using immunohistochemistry and compared according to tumor progression in small adenomas (diameter <10 mm), large adenomas (diameter >10 mm), and adenocarcinomas. Original magnification: A and F, ×40; B–E, ×100. Gastroenterology 2009 136, 206-216DOI: (10.1053/j.gastro.2008.09.063) Copyright © 2009 AGA Institute Terms and Conditions

Figure 5 KRAS, RASAL1, and colorectal tumor progression. (A) The frequency of mutant KRAS and reduced RASAL1 expression in the CRCs (n = 64). (B and C) The samples were divided into 4 groups based on the presence of KRAS mutations and RASAL1 expression in the tumor cells and were then compared using the clinicopathologic/molecular features of the patients. The groups included mutant KRAS (Mut-KRAS; n = 27), wild-type KRAS with reduced RASAL1 expression (Wt-KRAS/reduced-RASAL1; n = 25), and wild-type KRAS with normal RASAL1 expression (Wt-KRAS/retained-RASAL1; n = 12). (B) The dots indicate the age of each patient; the bars indicate the mean age of each group. (C) Abbreviations: dist, distal site within the colon or rectum; por, poorly differentiated adenocarcinoma; sig, signet ring cell adenocarcinoma; CTNNB1(+), nuclear localization of CTNNB1; TP53(+), abnormally strong TP53 expression. (D) Progression of colorectal neoplasms in relation to RASAL1. KRAS mutation/activation and reduced RASAL1 expression were frequent and tended to be mutually exclusive in CRCs. Reduced RASAL1 expression was observed more frequently in advanced lesions than in early adenomas. On comparing CRCs with wild-type KRAS and retained RASAL1 expression, CRCs with mutant KRAS or reduced RASAL1 tended to occur distally within the colon or rectum in younger patients. MSI, microsatellite instability. Gastroenterology 2009 136, 206-216DOI: (10.1053/j.gastro.2008.09.063) Copyright © 2009 AGA Institute Terms and Conditions

Figure 6 Methylation analysis of RASAL1. (A) Bisulfite sequence analysis of the 5′ CpG sites of RASAL1. The PCR products were cloned into pCR2.1-TOPO using a TOPO TA cloning kit. Six clones were sequenced: black circles indicate methylated CpG sites; white circles indicate unmethylated CpG sites. (B and C) The qRT-PCR of RASAL1 and PITX1 genes (B) and immunoblot analysis of RASAL1 (C) were performed in the CRC cells. Cells were cultured in 1 μmol/L 5-Aza-2′-deoxycytidine (5-aza) or DMSO for 96 hours, and then total RNA and protein extracts were prepared for analysis. (D) Bisulfite sequencing of the 5′ CpG island of RASAL1 in cells cultured as described in C. Gastroenterology 2009 136, 206-216DOI: (10.1053/j.gastro.2008.09.063) Copyright © 2009 AGA Institute Terms and Conditions