Antitumor effects of novel mAbs against cationic amino acid transporter 1 (CAT1) on human CRC with amplified CAT1 gene

Abstract

Copy number alterations detected by comparative genomic hybridization (CGH) can lead to the identification of novel cancer-related genes. We analyzed chromosomal aberrations in a set of 100 human primary colorectal cancers (CRCs) using CGH and found a solute carrier (SLC) 7A1 gene, which encodes cationic amino acid transporter 1 (CAT1) with 14 putative transmembrane domains, in a chromosome region (13q12.3) with a high frequency of gene amplifications. SLC7A1/CAT1 is a transporter responsible for the uptake of cationic amino acids (arginine, lysine, and ornithine) essential for cellular growth. Microarray and PCR analyses have revealed that mRNA transcribed from CAT1 is overexpressed in more than 70% of human CRC samples, and RNA interference-mediated knockdown of CAT1 inhibited the cell growth of CRCs. Rats were immunized with rat hepatoma cells expressing CAT1 tagged with green fluorescent protein (GFP), and rat splenocytes were fused with mouse myeloma cells. Five rat monoclonal antibodies (mAbs) (CA1 ~ CA5) reacting with HEK293 cells expressing CAT1-GFP in a GFP expression-dependent manner were selected from established hybridoma clones. Novel anti-CAT1 mAbs selectively reacted with human CRC tumor tissues compared with adjacent normal tissues according to immuno-histochemical staining and bound strongly to numerous human cancer cell lines by flow cytometry. Anti-CAT1 mAbs exhibited internalization activity, antibody-dependent cellular cytotoxicity, and migration inhibition activity against CRC cell lines. Furthermore, CA2 inhibited the in vivo growth of human HT29 and SW-C4 CRC tumors in nude mice. This study suggested CAT1 to be a promising target for mAb therapy against CRCs.

Keywords: CAT1; CRC; SLC7A1; mAb; oncogene addiction.

Figure 1

Amplifications and homozygous deletions in colorectal cancer (CRC) and correlation between SLC7A1 gene amplification and prognosis and metastasis. A, Green‐colored area indicates an increased genome copy number (G/R > 1.2), and red‐colored area indicates a reduced genome copy number (G/R < 0.8). One hundred samples were analyzed. B, High‐level amplification and homozygous deletion in CRCs. C, Frequent gene amplification of SLC7A1 located on 13q12. D and E, Correlation between progression‐free survival (D), and lymph node and distant metastasis (liver, etc.) (E) of CRC patients and amplification of SLC7A1/CAT1 gene. Data were obtained from The Cancer Genome Atlas (Data set ID: TCGA. COADREAD. SampleMap/Gistic2 _CopyNumber_Gistic2_all_data_by_genes)

Figure 2

Correlation between gene amplification and mRNA expression of SLC7A1/CAT1 and CAT1 small interfering RNA (siRNA)–mediated growth inhibition of colorectal cancer (CRC) cells. A, Relative mRNA expression (ratio from a mean value of normal tissues) in normal and cancer tissues. B, Correlation between gene amplification and mRNA expression of SLC7A1. C, CAT1 mRNA levels in HCT116 and LS1034 cells treated with CAT1 siRNA for 24 hours. D, CRC cell growth 48 hours after treatment with CAT1 siRNA

Figure 3

Specificity of anti‐human CAT1 monoclonal antibodies (mAbs) demonstrated by flow cytometry (FCM). A, Representative FCM dot plot of HEK293F cells expressing CAT1‐green fluorescent protein (GFP) stained with anti‐CAT1 mAbs. B, Representative FCM histograms of anti‐CAT1 mAb–stained HT29 cells, which were treated with CAT1 small interfering RNA (siRNA) (orange lines) or control siRNA (red lines) for 72 h

Figure 4

Immunostaining of human colorectal tissues by anti‐CAT1 monoclonal antibody (mAb) and patient information. A, Representative staining of human colorectal cancer (CRC) tissues (sample 3 was paired with normal colon) by CA2. Immunoscores (1‐3) and comparative genomic hybridization (CGH) values are shown. B, Clinical information of CRC patients is summarized. Patient sex: M, male; F, female. Tumor location: A, ascending colon; S, sigmoid colon; Rs, rectosigmoid; Ra, upper rectum; Rb, lower rectum; C, cecum. Tumor histology: mod, moderately differentiated adenocarcinoma; well, well‐differentiated adenocarcinoma; muc, mucinous adenocarcinoma

Figure 5

Specificity of anti‐CAT1 monoclonal antibodies (mAbs) against human cancer cell lines. A, Representative flow cytometry (FCM) histograms of human colorectal cancer (CRC) cell lines stained with anti‐CAT1 mAbs. B and C, Different human CRC cell lines (B), and pancreatic, breast, and lung cancer cell lines (C) were reacted with CA2 or isotype‐matched control mAb, followed by incubation with phycoerythrin (PE)‐labeled anti‐rat IgG polyclonal antibody (pAb). The fluorescence intensity of individual cells was measured by FCM

Figure 6

Internalization, antibody‐dependent cellular cytotoxicity (ADCC), and migration inhibition assays by anti‐CAT1 monoclonal antibodies (mAbs). A, Representative images of anti‐CAT1 mAb–treated HEK293F cells expressing CAT1‐green fluorescent protein (GFP) proteins. Nuclei were stained with DAPI. B and C, Quantitative analysis of internalization by flow cytometry (FCM). Subtracted mean fluorescence intensity (ΔMFI) was calculated based on the MFI with or without primary mAbs, and indicated as mAb binding. D‐F, ADCC activity was measured by lactate dehydrogenase release assay. D, ADCC assay at an effector/targe (E/T) ratio of 50 and 20 μg/mL with CA1 ~ CA5 mAbs. E, E/T ratio–dependent assay at 20 μg/mL with CA2 and CA3 mAbs. F, mAb concentration–dependent assay at an E/T ratio of 50 with CA2 and CA3 mAbs. G, Abrogation of NRG‐1–induced vitronectin (VN)‐mediated migration of highly liver‐metastatic LS‐LM4 cells by anti‐CAT1 mAb. VN‐mediated migration of LS‐LM4 cells, enhanced by NRG‐1 (10 ng/mL) is abolished by 10 μg/mL of CA2 anti‐CAT1 mAb (**P < .0035, two‐way ANOVA)

Figure 7

Comparative analysis with monoclonal antibodies (mAbs) against amino acid (AA) transporters and in vivo antitumor effects of anti‐CAT1 mAb. A and B, Reactivity of mAbs against CAT1, LAT1, or xCT AA transporter was compared. Maximum binding and K_A of mAbs against HT29 and SW‐C4 cells was measured by Scatchard plot analysis. C and D, Tumor growth of grafted HT29 or SW‐C4 cells in nude mice. On days 0 and 7, CA2 mAb or isotype control IgG (γ2a/κ) (100 μg/mouse) was administered intraperitoneally. Data are presented as the mean ± SEM (n = 4). ΔMFI, subtracted mean fluorescence intensity