Human Cancer Biology Fatty Acid Binding Protein 6 Is Overexpressed in Colorectal Cancer Ta k a h i r o Oh m a c hi , 1, 2 , 3 Hiroshi Inoue,1, 2 Koshi Mimori,1, 2 Fumiaki Tanaka,1, 2 Atsushi Sasaki,1, 2 Ta t s u o K a n d a , 4 Hiroshi Fujii,4 Katsuhiko Yanaga,3 and Masaki Mori1, 2 Abstract Purpose: Fattyacid binding protein 6 (FABP6) is a cancer-related protein that acts as an intra- cellular transporter of bile acid in the ileal epithelium. Because bile acids are implicated in the carcinogenesis of colorectal cancer, we evaluated FABP6 expression in colorectal cancer. Experimental Design:The expression of FABP6 mRNA was evaluated in 78 paired samples of cancer/normal tissue representing colorectal cancer cases, plus 16 adenomas, and 16 metastatic lymph nodes. An immunohistochemical study was conducted with paraffin sections. In vitro transfection was done to determine FABP6’s biological roles. Results: The expression of FABP6 mRNA was significantlyhigher in cancer (75 of 78, 96.2%) thaninnormaltissue(P < 0.001). The expression of mRNA was increased in cancer compared with adenoma, but was dramaticallydecreased in node metastases. Tumors with high FABP6 expression were smaller in size (P < 0.01), more often in the left colon (P < 0.05), and had shal- lower invasion into the bowel wall (P < 0.05) compared with those with low expression. There was no significant difference between high- and low-expression tumors regarding clinicopatho- logic variables such as histologic type, lymph node, or liver metastasis, Dukes’ classification, and prognosis. Immunohistochemical studyrevealed that FABP6 expression was primarily observed in cancer cells. In vitro transfection revealed that transfectants showed weakerinvasive- ness (P < 0.05), more dominant proliferation (P < 0.001), and less apoptosis than mock cells. Conclusions: The expression of FABP6 was higher in primarycolorectal cancers and adenomas than in normal epithelium, but was dramaticallydecreased in lymph node metastases, suggesting that FABP6 mayplayan important role in earlycarcinogenesis. To further our understanding of cancer biology, it is important intracellular transporter of bile acids in ileal epithelial cells, to discern cancer-related genes. To identify such genes, we helping to catalyze and metabolize cholesterol. It is also known evaluated differences in the gene expression profiles between that bile acids induce colon carcinogenesis and cancer colon cancer cells and normal colonic epithelial cells using development in experimental models (1–3). Bile acids induce a combination of laser microdissection and cDNA microarray apoptosis and inflammation in colonic epithelial cells (4) and techniques. Consequently, we identified 84 cancer-related indirectly cause oxidative DNA damage that leads to genetic genes that were overexpressed in cancer cells.5 One example modulation during epithelial restoration (5). However, the is fatty acid binding protein 6 (FABP6), which acts as an significance of the expression of FABP6 in colorectal cancer has not been studied to date. In this study, we clarified, through clinical and experimental studies, that the relative expression level of FABP6 is associated with colorectal cancer. Authors’ Affiliations: 1Department of Surgeryand Molecular Oncology,Medical Institute of Bioregulation, Kyushu University, Beppu, Japan; 2Core Research for Evolutional Science and Technology, Japan Science and Technology Agency, Saitama, Japan; 3Department of Surgery, Jikei University School of Medicine, Materials and Methods Minato-ku, Tokyo, Japan; and 4Department of Biochemistry, Niigata University School of Medicine, Niigata, Japan Tissue sampling. A total of 186 tissue samples obtained from 104 Received 9/17/05; revised 5/6/06; accepted 5/11/06. patients were used in this study. Tumors and adjacent normal tissues Grant support: Core Research for Evolutional Science and Technology, Japan were obtained surgically from 78patients in Kyushu University Science and Technology, Uehara Memorial Foundation, Japan Society for the Hospital in Beppu, Japan, and immediately embedded in Tissue Tek Promotion of Science Grant-in-Aid for Scientific Research (grant nos. 17015032, 17109013, 17591411, 17591413, and 16390381), Health and Labour Sciences optimum cutting temperature compound medium (Sakura, Tokyo, À j Research Grants, Third Term Comprehensive Control Research for Cancer Japan), samples were kept frozen at 80 C until used. Sixteen (16271201). (4 samples from the aforementioned 78patients, and an additional The costs of publication of this article were defrayed in part by the payment of page 12 samples) metastatic lymph nodes were also obtained and prepared charges. This article must therefore be herebymarked advertisement in accordance for analysis. Finally, an adenoma obtained by endoscopic mucosal with 18 U.S.C. Section 1734 solelyto indicate this fact. resection from an additional 16 patients was also used. Requests for reprints: Masaki Mori, Department of Surgeryand Molecular Oncology, Medical Institute of Bioregulation, Kyushu University, 4546 Tsurumihara, Beppu 874-0838, Japan. Phone: 81-977-1645; Fax: 81-977-1651; E-mail: [email protected]. F 2006 American Association for Cancer Research. 5 Ohmachi T, Tananka F, Mimori K, Inoue H, Yanaga K, Mori M. Clinical significance doi:10.1158/1078-0432.CCR-05-2045 ofTROP2 expression in colorectal cancer. Clin Cancer Res 2006;12:3057 ^63. Clin Cancer Res 2006;12(17) September 1, 2006 5090 www.aacrjournals.org Downloaded from clincancerres.aacrjournals.org on October 1, 2021. © 2006 American Association for Cancer Research. FABP6 Is Overexpressed in Colorectal Cancer Written informed consent was obtained from all patients. The study mRNA were used for examination to verify the results of in vitro assays. design was approved by the Institutional Review Board in Kyushu The clones from mock cells showing no difference from the parent cell University prior to implementation. line, DLD-1, were used for controls. Reverse transcriptase-PCR and semiquantitative real-time reverse Invasion assays were done using the BD BioCoat Tumor Invasion transcriptase-PCR. Total RNA was extracted from each sample and System (BD Biosciences, Franklin Lakes, NJ) to evaluate invasive cells as cDNA was synthesized from 8.0 Ag total RNA as described previously described previously (10). Briefly, cells (5.0 Â 104 cells/well) were (6). The purity and concentration of total RNA were determined using placed in the upper chamber, and the lower chamber was filled with an Agilent 2100 bioanalyzer (Agilent Technologies, Palo Alto, CA). The 750 AL of DMEM with 10% FCS as a chemoattractant. After 48hours of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene served as incubation at 37jC, membranes were labeled with Calcein-AM an internal control. Semiquantitative real-time reverse transcriptase- solutions. Invasive cells that had migrated through the membrane to PCR assay was done using 78surgically resected paired cancer and the lower surface were evaluated in a fluorescence plate reader at normal tissue samples, 16 adenomas, and 16 metastatic lymph nodes. excitation/emission wavelengths of 485/530 nm. Invasiveness was The following primers were used to amplify genes: FABP6 (sense evaluated as the percentage of fluorescence of invasive cells of HT-1080, primer, 5¶-ACTACTCCGGGGGCCACACCAT-3¶ and antisense primer, the fibrosarcoma cell line that served as a control. 5¶-GTCTCTTGCTCACGCGCTCATAGG-3¶) and GAPDH (sense primer, Proliferation assays were done by cell counting for three clones each 5¶-TTGGTATCGTGGAAGGACTCA-3¶ and antisense primer, 5¶-TGTCAT- of the transfectants and mock cells. Each clone was harvested in RPMI j CATATTTGGCAGGTTT-3¶). The reaction was done in a LightCycler 1640 with 10% fetal bovine serum at 37 C in a 5% humidified CO2 system (Roche Applied Science, Indianapolis, IN) using the LightCycler- atmosphere. They were plated at a density of 106 cells per well in three FastStart DNA Master SYBR Green I kit (Roche Applied Science) as 10-cm plates and were harvested and counted on days 3, 5, and 7. The described previously (7). In brief, thermal cycling for all genes was medium was changed every 72 hours. This experiment was done in initiated with a denaturation step of 95jC for 10 minutes, followed triplicate. by 40 cycles at 95jC for 10 seconds, 64jC (60jC for GAPDH) for Apoptosis assays were done using FITC-conjugated monoclonal 10 seconds, and 72jC for each optimal length (s/25 bp). At the end of rabbit anti-active caspase-3 antibody apoptosis kit 1 (BD Biosciences) the PCR cycles, melting curve analysis was done to reconfirm the and flow cytometer as described previously (11). Briefly, cells (2.0 Â 6 expected PCR product. We determined the levels of FABP6 and GAPDH 10 ) were incubated for 72 hours in serum-free medium at 37jC and mRNA expression by comparisons with cDNA obtained from Human fixed in 70% ethanol at À20jC. Next, the cells were washed and Universal Reference RNA (Clontech, Palo Alto, CA). All calculated resuspended in the buffer with FITC-conjugated monoclonal rabbit concentrations of target genes were divided by the amount of anti-active caspase-3 antibody. The FITC fluorescence was evaluated endogenous reference (GAPDH) to obtain normalized FABP6 expres- using an EPICS XL flow cytometer (Beckman Coulter Corp., Tokyo, Â 4 sion values. Each assay was done in triplicate to verify the results. Japan) until the total cell count reached 3 10 . The experiment was Immunohistochemistry. Immunohistochemical studies