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. Author manuscript; available in PMC: 2010 Mar 23.
Published in final edited form as: Oncogene. 2009 Sep 7;28(47):4189–4200. doi: 10.1038/onc.2009.266

LARG at chromosome 11q23 has functional characteristics of a tumor suppressor in human breast and colorectal cancer

DCT Ong 1, YM Ho 1, C Rudduck 2,8, K Chin 3, W-L Kuo 3,4, DKH Lie 1, CLM Chua 1, PH Tan 2, KW Eu 5, F Seow-Choen 5, CY Wong 6, GS Hong 6,9, JW Gray 3,4, ASG Lee 1,7
PMCID: PMC2844776  NIHMSID: NIHMS185870  PMID: 19734946

Abstract

Deletion of 11q23–q24 is frequent in a diverse variety of malignancies, including breast and colorectal carcinoma, implicating the presence of a tumor suppressor gene at that chromosomal region. We examined a 6-Mb region on 11q23 by high-resolution deletion mapping, using both loss of heterozygosity analysis and customized microarray comparative genomic hybridization. LARG (leukemia-associated Rho guanine-nucleotide exchange factor) (also called ARHGEF12), identified from the analysed region, is frequently underexpressed in breast and colorectal carcinomas with a reduced expression observed in all breast cancer cell lines (n=11), in 12 of 38 (32%) primary breast cancers, 5 of 10 (50%) colorectal cell lines and in 20 of 37 (54%) primary colorectal cancers. Underexpression of the LARG transcript was significantly associated with genomic loss (P=0.00334). Hypermethylation of the LARG promoter was not detected in either breast or colorectal cancer, and treatment of four breast and four colorectal cancer cell lines with 5-aza-2′-deoxycytidine and/or trichostatin A did not result in a reactivation of LARG. Enforced expression of LARG in breast and colorectal cancer cells by stable transfection resulted in reduced cell proliferation and colony formation, as well as in a markedly slower cell migration rate in colorectal cancer cells, providing functional evidence for LARG as a candidate tumor suppressor gene.

Keywords: LARG, tumor suppressor, breast cancer, colorectal cancer

Introduction

Chromosome 11q23–q24 deletion is frequent in a variety of tumor types, including tumors of the breast, colorectum, ovary, stomach, lung, cervix, nasopharynx and malignant melanoma, implicating that this region is important in the tumorigenesis of diverse tumor types (Rasio et al., 1995; Tomlinson et al., 1995; Baffa et al., 1996; Gabra et al., 1996; Hui et al., 1996; Robertson et al., 1996; Lee et al., 2000; Pulido et al., 2000; Martin et al., 2003).

Functional evidence suggesting the involvement of 11q23–q24 in tumorigenesis has been demonstrated by microcell-mediated chromosome transfer (Negrini et al., 1994). The MCF-7 cell line transferred with the entire chromosome 11 was non-tumorigenic, whereas the MCF-7 line, which had the transfer of chromosome 11 lacking the distal portion of 11q, maintained the tumorigenic phenotype, suggesting the presence of one or more tumor suppressor gene(s) in the distal region of 11q (Negrini et al., 1994). Furthermore, significant tumor suppression has been demonstrated in fibrosarcoma cells and in lung carcinoma cell lines transfected with yeast artificial chromosomes mapping to the 11q23 region (Murakami et al., 1998; Koreth et al., 1999).

Two independent regions of loss of heterozygosity (LOH) at 11q23 have been identified previously in breast cancer (Negrini et al., 1995). The BCSC-1 candidate tumor suppressor gene is located in the second, more distal region (LOH11CR2), and is implicated as the target of deletion in breast cancer on the basis of LOH analysis, northern analysis on cell lines (but not primary tumors), suppression of colony formation in vitro and tumorigenicity in vivo (Martin et al., 2003). To our knowledge, no tumor suppressor gene has been identified from the first region.

We, along with others, have identified a third region of LOH in breast and colorectal cancers, which lies between these two regions and from which a candidate tumor suppressor gene is yet to be identified (Tomlinson et al., 1995, 1996; Koreth et al., 1997; Lee et al., 2000). In this study, this third region of LOH was analysed by high-resolution deletion mapping, and a candidate tumor suppressor gene, LARG (leukemia-associated Rho guanine-nucleotide exchange factor), was identified. We show here that expression of LARG is frequently silenced in primary breast and colorectal cancers and cell lines. Furthermore, the tumor suppressive function of LARG was demonstrated in breast and colorectal cancer cell lines by reduced colony formation and cell proliferation, as well as by inhibition of cell migration.

Results

High-resolution deletion mapping

The frequency of LOH and the heterozygosity rate of seven microsatellite markers on chromosome 11q23 in 58 primary breast carcinoma specimens are shown in Figure 1. The demographic and clinical details of these patients are summarized in Supplementary Table S1. The frequency of LOH was high for all markers, ranging from 45% at D11S4104 to 66% at D11S29, and heterozygosity rates ranged from 0.586 to 0.877 (Figure 1a). Overall, 41 of 58 (71%) tumors showed LOH for, at least, one of the seven microsatellite markers (Figure 1b). Notably, 16 cases had either LOH and/or homozygosity at all seven microsatellite markers, suggesting that chromosomal nondisjunction may have occurred with loss of the entire chromosomal region (Figure 1b). Representative examples of LOH are shown in Figure 1c.

Figure 1.

Figure 1

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Loss of heterozygosity (LOH) analysis in primary breast cancers. (a) The frequency of LOH and the heterozygosity rate at seven microsatellite markers on chromosome 11q22–q23 in breast carcinoma. (b) Results of microsatellite analyses on 58 breast carcinoma samples. L, LOH; Ho, homozygous or noninformative; He, heterozygosity retained; MI, microsatellite instability; nd, not determined. (c) Examples of LOH on chromosome 11 in representative breast carcinoma samples. Top, case numbers. Left, microsatellite markers. N, normal; T, tumor. Arrowheads indicate the allele lost in tumor DNA.

A customized comparative genomic hybridization (CGH) microarray was constructed to further define the region of deletion. The microarray included 41 bacterial artificial chromosome (BAC) clones within an ~6Mb region from 11q23.3 to 11q24.1, and spanned the microsatellite markers D11S29 to D11S1345 (Figure 2). All BAC clones were tested by fluorescence in situ hybridization (FISH) on normal metaphase chromosome spreads to verify that the clones were indeed from this chromosomal region. Our FISH analysis revealed that 11 clones either hybridized to other chromosomes (RP11-8K10, RP11-158K18, RP11-271P14) or gave nonspecific signals on FISH (RP11-712L22, RP11-630O14, RP11-778017, RP11-640N11, RP11-812L16, RP11-166D19, RP11-811I7, RP11-93E4). These clones were subsequently excluded from array CGH analysis.

Figure 2.

Figure 2

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Location of microsatellite markers, bacterial artificial chromosome (BAC) clones and cancer-related genes across the chromosome 11q23–q24 region on the basis of Ensembl (release 43).

The frequency plot of copy number alterations for the remaining 30 BAC clones is shown in Figure 3a. A heat map representing the array CGH copy number alterations for the primary breast cancer tumors analysed showed a high frequency of copy number loss with RP11-15I6 (Figure 3b). RP11-15I6 was selected for further characterization, as several tumors (B4, B12, B16, B20, B28, B45 and B46) had copy number losses at RP11-15I6, but not at adjacent BAC clones, suggesting that a tumor suppressor gene may lie within the genomic region encompassed by RP11-15I6.

Figure 3.

Figure 3

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Array comparative genomic hybridization (CGH) analysis of 40 primary breast tumors and two breast cancer cell lines. (a) Frequency plot of copy number gains or losses for 30 BAC clones on chromosome 11q23. (b) Heat map of DNA copy number ratios for 30 BAC clones on chromosome 11q23. Color codes are indicated below the heat map.

Dual color FISH using BAC clone RP11-15I6 and a chromosome 11 centromeric probe was conducted on frozen sections from six available primary breast tumors to confirm the copy number losses observed from array CGH analysis. Copy number losses were detected in all primary breast tumors and representative examples are shown in Supplementary Figure S1.

The gene LARG maps within BAC RP11-15I6, and was selected for further characterization as a candidate tumor suppressor gene. LARG spans 152.7 kb and comprises 40 exons with a transcript length of 9453 bp (www.ensemble.org, release 44).

Frequent underexpression of LARG in breast and colorectal cancer

To determine whether LARG is underexpressed in breast and colorectal cancer, real-time RT–PCR analysis was performed on 38 breast tumor samples, on 11 breast cancer cell lines, 37 colorectal cancers and on 10 colorectal cancer cell lines. A reduced mRNA expression of LARG of less than 50% relative to the calibrator (normal tissue control) was observed in all breast cancer cell lines, in 12 of 38 (32%) primary breast cancers, 5 of 10 (50%) colorectal cell lines and in 20 of 37 (54%) primary colorectal cancers (Figure 4).

Figure 4.

Figure 4

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Leukemia-associated Rho guanine-nucleotide exchange factor (LARG) expression in breast and colorectal primary tumors and cell lines. The relative mRNA levels of LARG were measured by quantitative RT–PCR (mean±s.d.), using GAPDH as the endogenous control gene in (a) primary breast tumor samples (n=38) and breast cancer cell lines (n=11). The level of LARG mRNA was expressed relative to normal human breast RNA (Ambion1), the calibrator, shown in black. Three additional normal breast RNA samples were included, shown in gray (Stratagene, Ambion2 and B7N). (b) Primary colorectal tumor samples (n=37) and colorectal cancer cell lines (n=10). The level of LARG mRNA was expressed relative to normal human colon RNA (Stratagene), the calibrator. Two additional normal colon RNA samples were included, shown in gray (Ambion, C24N).

Importantly, underexpression of the LARG transcript significantly correlates with genomic loss detected by either LOH analysis or array CGH in breast carcinoma (P=0.00334, Fisher’s exact test) (Supplementary Tables S3 and S4).

Mutation analysis of LARG

As inactivating mutations are a known mechanism for gene silencing, the entire coding sequence and the intron–exon boundaries of LARG were screened for mutations in the same panel of primary breast cancers, with available genomic DNA (n=40). A missense mutation was detected in one case, resulting in the aminoacid substitution, Q1219P (Supplementary Table S5). Several polymorphisms were also detected, which included previously documented single-nucleotide polymorphisms (www.genecards.org) and new alterations present in normal controls. All cases with intronic splice site alterations were subjected to RNA analysis and none showed aberrant splicing.

Lack of methylation of the LARG promoter

To explore the possibility that silencing of LARG expression may be a result of methylation of CpGs within the CpG island upstream of the transcription start site of LARG, bisulfite sequencing of genomic DNA from 10 breast cancer cell lines was carried out. CpG island methylation was not detected in any of the cell lines.

A lack of CpG island methylation in breast and colorectal cancer was observed in primary breast cancer samples (n=24), in breast cancer cell lines (n=6), in primary colorectal cancers (n=26) and in the SW620 colorectal cancer cell line through qualitative high-throughput analysis of DNA methylation by base-specific cleavage and mass spectrometry using the SEQUENOM MassARRAY System (Sequenom, San Diego, CA, USA), with the exception of the BT20 breast cancer cell line (Supplementary Figures S2a and S2b).

Effect of a demethylating agent and/or a histone deacetylase inhibitor

Treatment of four breast cancer and four colorectal cancer cell lines that underexpress LARG with the demethylating agent 5-aza-2′-deoxycytidine (5Aza-dC) did not lead to the reactivation of LARG, further suggesting that the silencing of LARG was not because of the methylation of CpGs (Figure 5). To determine whether inactivation of LARG may alternatively have occurred through epigenetic silencing by histone modification, the same cell lines were treated with the histone deacetylase inhibitor, trichostatin A (TSA) (Figure 5). Reactivation of LARG was not observed in breast cancer or colorectal cancer cell lines.

Figure 5.

Figure 5

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Effect of 5-aza-2′deoxycytidine and trichostatin A (TSA) on the expression of leukemia-associated Rho guanine-nucleotide exchange factor (LARG) in breast and colorectal cancer cell lines. Cells were treated with 5-aza-2′deoxycytidine, TSA or a combination of both. Expression was quantified by quantitative RT–PCR (mean±s.d.) and normalized using GAPDH.

Tumor suppressive function of LARG

To investigate the tumor suppressive function of LARG in vitro, constructs containing LARG or empty vector were transfected into MCF-7 and SW620 cell lines (Figure 6). Growth suppressive activity was assessed using colony formation, cell proliferation and wound healing assays for three stable transfectants. For both MCF-7 breast cancer and SW620 colorectal cancer cell lines, colony numbers of cells transfected with LARG were significantly decreased compared with those of empty vector-transfected cells (P=0.0243, P=0.0102 and P=0.0067, paired t-test) (Figure 6b). Growth inhibition was also observed in 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) cell proliferation assays of MCF-7 and SW620 cells transfected with LARG (Figure 6c). The wound healing assay showed that the cell migration rate of SW620 cells expressing LARG was markedly slower than that of cells transfected with empty vector, over a 24-h period (Figure 6d). In addition, siRNA knockdown of LARG expression in SW620 cells stably transfected with LARG showed an increased cell proliferation rate compared with control cells (Figure 7). Thus, LARG has the functional characteristics of a tumor suppressor gene in vitro.

Figure 6.

Figure 6

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Tumour suppressive function of leukemia-associated Rho guanine-nucleotide exchange factor (LARG) in breast and colorectal carcinoma. (a) Expression of LARG in stable cell lines of MCF-7 and SW620, transfected with pcDNA3.1 (empty vector) or a pcDNA3.1 construct with LARG, was verified by RT–PCR (left) and western blot analysis (right). (b) Colony formation assay was carried out for cells transfected with pcDNA3.1 (empty vector) or a pcDNA3.1 construct with LARG, grown for 14 days and visualized by crystal violet staining (left). Cell colonies with more than 50 cells were counted in triplicate experiments (right). *P=0.0243; **P=0.0102; ***P=0.0067, paired t-test). (c) Cell proliferation assay of MCF-7 and SW620 cells transfected with pcDNA3.1 (empty vector) or LARG showed slower growth of cells transfected with LARG. (d) Wound healing assay of SW620 cells revealed that expression of LARG inhibited cell migration during a 24-h period.

Figure 7.

Figure 7

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siRNA knockdown of leukemia-associated Rho guanine-nucleotide exchange factor (LARG) in two SW620 clones, pLARG-1 and pLARG-6, which stably express LARG. (a) Real-time RT–PCR (left) and western blot analysis (right) showing reduced expression of LARG in LARG siRNA-transfected cells as compared with non-targeting siRNA (negative control)-transfected cells. (b) Cell proliferation assay showing an increased growth rate in LARG siRNA-transfected cells as compared with non-targeting siRNA (negative control)-transfected cells.

Discussion

We have identified a candidate tumor suppressor gene, LARG, from a region on 11q23, which is frequently deleted in breast and colorectal carcinoma and other cancers. To our knowledge, this is the first candidate tumor suppressor gene identified from this LOH region, spanning from markers D11S924 to D11S4107 (Figure 2) (Lee et al., 2000). Interestingly, two other candidate tumor suppressor genes, TSLC1 and BCSC-1, have been previously identified from 11q23 but are >4Mb centromeric and >3Mb telomeric from LARG, respectively (Kuramochi et al., 2001; Martin et al., 2003).

Although LARG has previously been implicated in the pathogenesis of acute myeloid leukemia (Kourlas et al., 2000), it has not been studied in any solid tumors. We observed a high frequency of underexpression of LARG in primary tumors and cell lines of breast and colorectal cancer. These frequencies were based on the selection of the control sample (normal tissue) with the lowest expression for LARG, and thus the most stringent control, to be used as the calibrator. If, however, we use the least stringent control (the normal control sample with the highest expression), an even higher frequency of underexpression of LARG will be obtained, that is, 53% for primary breast cancer and 68% for primary colorectal cancer.

A reduced expression of a tumor suppressor gene is frequently due to epigenetic modifications, mutations or allelic loss (Plass, 2002; Ting et al., 2006). Epigenetic changes, such as promoter hypermethylation and histone modifications (for example, acetylation, methylation, phosphorylation, SUMOylation and ubiquitylation) alter the structure of chromatin into a closed configuration resulting in transcriptional silencing (Shilatifard, 2006; Esteller, 2007; Rice et al., 2007). Our investigations revealed a lack of methylation in the LARG promoter in both primary breast and colorectal carcinomas, and the absence of histone deacetylation in breast and colorectal cell lines treated with TSA, a histone deacetylase inhibitor, which did not induce re-expression of LARG. Thus, other regulatory mechanisms may control transcript expression of LARG. Importantly, underexpression of LARG transcript significantly correlated with genomic loss (P=0.00334).

A further confirmation of the possible function of LARG as a tumor suppressor was obtained through multiple independent in vitro assays using breast and colorectal cell lines transfected with LARG. LARG was shown to reduce colony formation and cell proliferation in both breast and colorectal cancer, as well as inhibit cell migration in colorectal cancer, as demonstrated by wound healing assay.

LARG was initially identified as a new gene, found to be fused with the mixed-lineage leukemia (MLL) gene in a patient with primary acute myeloid leukemia (Kourlas et al., 2000). The in-frame MLL–LARG fusion is thought to have occurred as a result of an interstitial deletion rather than a balanced translocation, with the break point in LARG at its 5′ end after nucleotide 931, resulting in the deletion of the amino-terminal end and the region encoding the PDZ domain (Kourlas et al., 2000). It is possible that tumorigenesis in acute myeloid leukemia resulting from the MLL–LARG fusion may be due to the loss of N-terminal and PDZ domains.

The predicted protein of LARG is a member of the Dbl family of proteins, which functions as guanine nucleotide exchange factors (GEFs), usually for the Rho family of GTPases (Kourlas et al., 2000). GEFs mediate the activation of Rho proteins, which function as molecular switches by cycling between an active (GTP bound) and an inactive (GDP bound) state. Rho GTPases regulate numerous actin-dependent processes, including cell migration and adhesion; microtubule cytoskeleton; gene expression and cell-cycle progression (Rossman et al., 2005).

LARG contains a PDZ domain, which mediates protein–protein interactions, usually through binding of the C-terminal of the target protein (Harris and Lim, 2001). We speculate that the tumor suppressive activity of LARG could be mediated through such protein–protein interactions, particularly as the PDZ domain of LARG has been shown to have a broad recognition profile, interacting with diverse binding partners, such as the insulin-like growth factor-1 receptor, lysophosphatidic acid-1, plexin-B1 receptor and CD44 (Taya et al., 2001; Swiercz et al., 2002; Yamada et al., 2005; Bourguignon et al., 2006; Smietana et al., 2008). Other domains in LARG are the RGS-like domain, which functions primarily as a GTPase activating protein (to accelerate GTPase activity) for Gα proteins (Siderovski et al., 1999; Fukuhara et al., 2000); the Dbl homology domain, for GEF activity (Whitehead et al., 1997); and a pleckstrin homology domain. The pleckstrin homology domain has been thought to be involved in the subcellular localization of the Rho GEF protein and to directly regulate the Dbl homology domain (Whitehead et al., 1997; Liu et al., 1998).

Several members of the Rho GEF family function as oncogenes (Van Aelst and D’Souza-Schorey, 1997). SIRT1 and Notch are two notable examples of proteins that can function as oncogenes or tumor suppressors. The divergent functions of SIRT1 in metabolism, aging and cancer are due to complex regulation by several factors during transcription, translation and posttranslational modification. Although SIRT1 exerts an oncogenic function by downregulating p53 activity, it functions as a tumor suppressor in a mutated p53 background (Brooks and Gu, 2009). In human T cell leukemia, Notch activation promotes tumorigenesis, whereas in the skin/keratinocyte system, Notch functions as a tumor suppressor in both mice and humans. These opposing roles are context dependent in different cell types and are regulated by a complex signaling network (Radtke and Raj, 2003; Dotto, 2008). An understanding of the precise regulatory mechanisms governing the diverse functions of LARG, a Rho GEF, remains to be elucidated.

In summary, we report here the identification of LARG as a candidate tumor suppressor gene and show evidence that LARG has tumor suppressor function in both breast and colorectal carcinoma. Further investigations with a larger series of clinical specimens of breast and colorectal cancer are warranted to determine whether loss of LARG expression is associated with clinical parameters.

Materials and methods

Samples

Breast and colorectal tumor tissues were collected from the Singapore General Hospital, snap-frozen in liquid nitrogen on resection and stored at −70 °C or under liquid nitrogen. Peripheral blood samples were collected in EDTA tubes from each of the patients, and frozen at −70 °C. Informed consent from all patients was obtained. Demographic and clinical information on the cases is summarized in Supplementary Tables S1 and S2. The study protocol was approved by the Institutional Review Boards of the Singapore General Hospital and the National Cancer Centre of Singapore.

DNA and RNA extraction

DNA and RNA were isolated from microdissected tumor tissues as described previously (Lee et al., 2000). Only samples that had, at least, 70% tumor cells were processed for DNA extraction using DNAzol (GIBCO/BRL, Carlsbad, CA, USA) according to the manufacturer’s instructions, or for RNA extraction using a column-based method (RNeasy; Qiagen, Hilden, Germany) or TRIzol (Invitrogen, Carlsbad, CA, USA). DNA was extracted from blood samples using sucrose lysis buffer and proteinase K digestion.

Microsatellite analysis

Loss of heterozygosity at the 11q22–23 chromosomal region was assessed using seven microsatellite markers (Figure 2), as previously described (Lee et al., 2000). PCRs were carried out using 100–400 ng of tumor or normal (blood) DNA. The sense primer was end labeled with 33P. PCR reaction was carried out for 1 min at 94 °C, for 1 min between 60 and 67 °C, and for 1 min at 72 °C for 24 cycles. The PCR products were separated on an 8% polyacrylamide gel and exposed to X-ray film overnight and also exposed to CS phosphor screens (Bio-Rad, Hercules, CA, USA) for 4–6 h.

Assessment of LOH

Loss of Heterozygosity was assessed by densitometry by scanning the CS screens with a Molecular Imager (Bio-Rad, USA). LOH was determined by quantification of the signal intensity of each allele, and comparing the ratios of the intensity of alleles from the tumor DNA with that of constitutional (blood) DNA, using the formula T1:T2/N1:N2. Samples with ratios of less than 0.5 or more than 1.5, indicating a reduction of more than 50% of one allele, were deemed to have LOH. All samples with LOH were re-analysed by repeating the microsatellite analysis to confirm the results.

Array CGH

The BAC arrays were constructed at the University of California San Francisco Cancer Centre, and included 41 BAC clones from 11q23 (Figure 2). Array CGH analysis was carried out as described by Massion et al. (2002). In brief, 1 μg of DNA from primary breast cancer tumors (test) and reference DNAs were digested with DpnII and labeled with Cy3-dUTP or Cy5-dUTP, respectively (Amersham Biosciences, Piscataway, NJ, USA), by random priming (Invitrogen, Carlsbad, CA, USA), and were co-hybridized with tRNA and human Cot-1 DNA (Roche, CA, USA) onto array slides for 48 h. Slides were then washed and TIF (tagged image file) images were captured using a GenePix scanner (Axon, Molecular Devices, Sunnyvale, CA, USA). The images were analysed using University of California San Francisco Spot and Sproc software (Jain et al., 2002) and an Excel macro developed at University of California San Francisco. After normalization, mean log2 ratios were plotted and fluorescent Cy3 (test)/Cy5 (reference) log2 ratios were classified as genomic gain, if greater than 0.3, or genomic loss, if lower than −0.3.

Fluorescence in situ hybridization

DNA from the BAC clone, RP11-15I6, from chromosome 11q23 was labeled by nick translation with SpectrumGreen (Vysis, Downers Grove, IL, USA) as described by Lee et al. (2004). A centromeric chromosome 11 probe to D11Z1 labeled with SpectrumOrange (Vysis) was used as an internal control. Interphase FISH was performed by hybridizing the probes to frozen tumor sections and counterstaining with 4′,6-diamidino-2-phenylindole. At least 100 cells were examined using a fluorescent imaging workstation by two independent investigators (Applied Imaging, Santa Clara, CA, USA). Samples with signal ratios of test (RP11-15I6) to control (chromosome 11 centromeric probe) probes of <0.8 were deemed as having deletion.

Real-time RT–PCR

cDNA was generated from 500 ng of total RNA from each sample by reverse transcription using the iScript cDNA Synthesis kit (Bio-Rad). The expression level of the LARG gene was determined using the Assay-on Demand Gene Expression product (Applied Biosystems, Foster City, CA, USA), with the GAPDH housekeeping gene as endogenous control. Total RNA from normal breast and colonic tissue were included as controls (Ambion, Austin, TX, USA and Stratagene, La Jolla, CA, USA; B7N, normal breast; C24N, normal colon). For normal breast, two different lots of total RNA were purchased from Ambion, Ambion1 and Ambion2. A control sample (normal tissue) with the lowest expression for LARG (and thus the most stringent control) was selected to be used as the calibrator and designated the relative value of 1.0. Reactions were performed in triplicate on the ABI 7500 Fast Real-Time PCR System (Applied Biosystems).

Sequencing

The entire coding region and flanking exon–intron boundaries of the LARG gene were screened for mutations by direct sequencing on a CEQ 2000 sequencer (Beckman Coulter, Fullerton, CA, USA) as described by Lee et al. (2003).

DNA methylation analysis

Bisulfite sequencing

Genomic DNA of 10 breast cancer cell lines (MCF-7, BT20, BT549, HCC2218, HCC1937, HCC1500, ZR75.1, T47D, BT474 and MBA MD231) was modified by bisulfite treatment using the MethylSEQr Bisulfite Conversion Kit (Applied Biosystems). The bisulfite-treated genomic DNA was PCR amplified (six overlapping fragments of 356, 476, 397, 420, 382 and 362 bp) and directly sequenced (BigDye Terminator v3.1 cycle sequencing kit, Applied Biosystems). A region encompassing 1334 bp before the transcription start site and 474 bp after the first exon (42 bp) was analszed.

Base-specific cleavage and mass spectrometry

In addition, DNA methylation analyses for 24 primary breast cancers, 7 breast cancer cell lines, 1 nontumorigenic breast cell line (MCF10A), 1 normal breast tissue sample (B13N), 26 primary colorectal cancers, 1 colorectal cell line and 1 normal colonic sample (C6N) were performed by Sequenom. using the MassARRAY system, which uses matrix-assisted laser desorption/ionizationtime of flight mass spectrometry analysis of base-specifically cleaved amplification products (Ehrich et al., 2005). Two CpG-rich regions upstream of exon 1 of LARG (−12240 to −11474 and −1585 to −197 relative to the transcription start site) were screened for methylation.

Treatment of Cells with 5-aza-2′deoxycytidine (5Aza-dC) and TSA

Cells (2×105) were seeded in 60-mm dishes and treated 24 h later with 5 μm 5Aza-dC (dissolved in dimethyl sulfoxide) (Calbiochem, La Jolla, CA, USA) for 72 h or with 0.5 μm TSA (dissolved in ethanol) (Sigma, St Louis, MO, USA) for 24 h. For co-treatment of cells with 5Aza-dC and TSA, cells were treated initially with 5Aza-dC (5 μm) for 48 h, followed by the addition of TSA (0.5 μm) and treatment for an additional 24 h. After treatment, expression of LARG was evaluated by RT–PCR.

Plasmid construction and gene transfection

Full-length LARG cDNA in pCMV6-XL4 was obtained commercially (Origene Technologies, Rockville, MD, USA). As pCMV6-XL4 lacks an antibiotic-resistant gene for selection of stable transfectants, the full-length LARG cDNA was subcloned into the Not1 restriction site of pcDNA3.1 (Invitrogen). The pcDNA3.1–LARG construct was transfected into the breast cancer cell line, MCF-7, and into the colorectal cancer cell line, SW620, using Lipofectamine 2000 (Invitrogen), and stable transfectants were selected with 400 or 1600 μg/ml geneticin (Invitrogen), respectively. MCF-7 and SW620 cell lines were also transfected with the pcDNA3.1 vector with no insert (empty vector), as controls. Cell lines were purchased from the American Type Culture Collection (Manassas, VA, USA) and were maintained in Roswell Park Memorial Institute 1640 medium or Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum. To confirm that selected clones were successfully transfected, clones were screened by PCR, real-time RT–PCR and western blotting analysis both before and after performing cell proliferation and colony formation assays.

Screening of transfectants by PCR

The PCR primers to screen empty vector clones were 5′-CCA CTGCTTACTGGCTTATC-3′ (forward) and 5′-TAGAAGG CACAGTCGAGG-3′ (reverse) generating a PCR product of 202 bp. The primers for screening the pcDNA3.1–LARG construct were 5′-CAGAATACTCACTCCGATGG-3′ (forward, exon 38) and 5′-TAGAAGGCACAGTCGAGG-3′ (reverse, pcDNA3.1), generating an amplicon of 743 bp.

Western blot analysis

Whole-cell lysates (30μg) were electrophoresed on 6% SDS–polyacrylamide gels and transferred to polyvinylidene difluoride membranes (Bio-Rad). Membranes were incubated with either a customized rabbit polyclonal anti-LARG primary antibody (Zymed, San Francisco, CA, USA) or the rabbit polyclonal antibody, LARG (H-70) (Santa Cruz Biotechnology, Santa Cruz, CA, USA), followed by a horseradish peroxidase-conjugated antirabbit secondary antibody (Amersham Biosciences). Signals were visualized by enhanced chemiluminescence, using enhanced chemiluminescence western blotting reagents (Amersham Biosciences).

Colony formation assay

A total of 900 cells were seeded onto 10-cm Petri dishes in triplicate and maintained for 14 days in selection medium. Cells were fixed with methanol–acetic acid (3:1; vol/vol) for 15 min and stained with 1% (wt/vol) crystal violet for 1 h. Colonies with more than 50 cells were counted.

Cell proliferation assay

The cell proliferation rate of cells transfected with LARG was compared with that of cells transfected with empty vector using the CellTitre 96 Aqueous One Solution Cell Proliferation Assay (Promega Corporation, Madison, WI, USA), according to the manufacturer’s instructions. Cells were seeded at a density of 5000 cells per well in 100 μl of medium in triplicate onto 96-well plates. The estimated number of cells was determined every 48 h from day 1 for 7 days (MCF-7) or every 24 h from day 1 for 5 days (SW620). In brief, cells were incubated for 2 h at 37 °C in a humidified, 5% CO2 environment with 20 μl of MTS solution. The absorbance of a formazan product, bioreduced from MTS tetrazolium by dehydrogenase enzymes in metabolically active cells, was recorded at 450 nm using a microplate reader.

The cell proliferation rate of cells treated with 5Aza-dC and TSA was determined as described above, but using 24-well rather than 96-well plates to enable more cells to be counted. Each well was seeded with 3×104 cells in 300 μl of medium, and using 60 μl of MTS solution. After incubation, 100 μl of the medium was then transferred to a 96-well plate for reading on the microplate reader.

Wound healing assay

SW620 cells transfected with either empty vector or with LARG were grown to confluency on six-well plates. A wound was introduced onto the cell layer using a sterile pipette tip, and cell migration was monitored over a period of 24 h, with the cells photographed under a phase contrast microscope. The experiment was carried out in triplicate for two stable cell lines transfected with LARG, and a control stable cell line transfected with empty vector.

RNA interference

SW620 cells stably expressing LARG were transfected with 20 nm siGenome siRNA against LARG (Dharmacon, Lafayette, CO, USA) using Lipofectamine RNAiMax (Invitrogen) according to the manufacturer’s instructions. Nontargeting siGenome siRNA pool (Dharmacon) (20 nm) was transfected in parallel as a control. After 48 h of transfection, cells were collected and cell proliferation assay was carried out as described above. Real-time RT–PCR and western blotting were performed 48 h after transfection to confirm the knockdown of LARG.

Supplementary Material

Supplementary Information
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Acknowledgments

We thank Dr Glenn Koh for assistance with review of case notes; YC Seo, Angela Chang, S Tohari, Irene HK Lim and Gan Yar Chze for excellent technical assistance; and Dr Eric Yap for helpful discussions. This study was supported by Grants from the National Medical Research Council (NMRC) of Singapore (NMRC/0076/1995, NMRC/0440/2000, NMRC/0570/2001, NMRC/0843/2004); SingHealth Foundation (SHF/FG235P/2005); the Singapore Cancer Society, SGH Research Fund, Cancer Research Education Fund, NCC and Department of Clinical Research, SGH, to AL. We gratefully acknowledge the grant support from the US Department of Energy under Contract No. DE-AC02-05CH11231, USAMRMC BC 061995; National Institutes of Health, National Cancer Institute (P50 CA 58207, P50 CA 83639, P30 CA 82103, U54 CA 112970, U24 CA 126477 P01 CA 64602); National Human Genome Research Institute (U24 CA 126551) and SmithKline Beecham Corporation, to JWG.

Footnotes

Conflict of interest

The authors declare no conflict of interest.

Supplementary Information accompanies the paper on the Oncogene website (http://www.nature.com/onc)

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Associated Data

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Supplementary Materials

Supplementary Information
NIHMS185870-supplement-Supplementary_Information.doc (27.5KB, doc)
fig 1
NIHMS185870-supplement-fig_1.jpg (53KB, jpg)
fig 2
NIHMS185870-supplement-fig_2.jpg (278.5KB, jpg)
fig 2b
NIHMS185870-supplement-fig_2b.jpg (247.6KB, jpg)
tbl 1
NIHMS185870-supplement-tbl_1.doc (46KB, doc)
tbl 2
NIHMS185870-supplement-tbl_2.doc (22.5KB, doc)
tbl 3
NIHMS185870-supplement-tbl_3.doc (61.5KB, doc)
tbl 4
NIHMS185870-supplement-tbl_4.doc (23KB, doc)
tbl 5
NIHMS185870-supplement-tbl_5.doc (28KB, doc)

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