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Colorectal cancer is the second most common cause of cancer-related deaths in the United States and the third most common cancer in both genders, with an estimated 135,400 new cases and 56,700 deaths occurring in the year 2001 (American Cancer Society, 2001). Therefore, it is crucial to discover and better understand molecular pathways underlying this type of cancer, including the identification of new tumor suppressor genes as well as mechanisms involved in their inactivation.

The activin type II receptor gene (ACTRII) was identified by genome-wide frameshift mutational screening as a novel candidate tumor suppressor gene mutated very frequently (in 58.1% of MSI-H colorectal cancers, ie, tumors with high microsatellite instability) at an (A)8 mononucleotide repeat within its coding region (Mori et al, 2001). ACTRII is a member of the transforming growth factor β (TGF-β) receptor family and is associated with the activin-SMAD signaling pathway, which is in turn involved in the induction of differentiation, growth suppression, and apoptosis in a variety of cell types, including those arising from the intestinal epithelium (Attisano et al, 1996; Sonoyama et al, 2000). Interference with the ACTRII signaling pathway in vitro inhibits cell differentiation (Li et al, 1998; Liu et al, 2000) and apoptosis (Chen et al, 2000; Choi et al, 2001). The TGF-β–SMAD signaling pathway, which shares the downstream effectors SMAD2 and SMAD4 with the activin-SMAD pathway, is reported to be responsible for human tumor suppression (Attisano et al, 1996; Liu et al, 2000; Sonoyama et al, 2000; Su et al, 2001). ACTRII forms an active heterodimeric receptor complex with ACTRI (Attisano et al, 1993). Of interest, somatic mutation of ACTRI was recently described in pancreatic carcinoma (Su et al, 2001).

To investigate whether ACTRII was involved in all colorectal tumors, rather than just those with frequent microsatellite instability, we studied inactivation of ACTRII in non–MSI-H tumors. We performed detailed loss of heterozygosity (LOH) studies of 2q22.1-q23.3, the region containing the ACTRII gene, in 51 non–MSI-H colorectal tumors. LOH-positive tumors were further tested for somatic point mutations within ACTRII.

Results

Allelotyping revealed that 17 (33%) of 51 colorectal tumors showed LOH at one or more of four loci on chromosome 2q23.1. These results are summarized in Table 1. Locus D2S141 showed the highest rate of LOH (12 of 27 informative cases or 44.4%). Nine (17.6%) of the 51 tumors manifested LOH flanking the ACTRII locus: these comprised 7 adenocarcinomas and 2 adenomas, one of which contained focal high-grade dysplasia.

Table 1 LOH Mapping Results at ACTRII Locus
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Quantitative RT-PCR revealed one tumor totally lacking ACTRII mRNA expression (Fig. 1). This finding was confirmed with multiple repeat assays. DNA sequencing of the remaining six tumors from which RNA was available revealed one containing a single-nucleotide change (TTT to ATT, amino acid substitution Ile to Phe) at codon 117 in the extracellular domain of ACTRII. This sequence alteration also appeared in the corresponding normal sample (Fig. 2). In addition, a silent polymorphism at codon 118 (CCG to CCA) was discovered in 3 (50%) of the 6 primary tumor samples; this polymorphism was then found to have been previously described (D’Abronzo et al, 1999). This polymorphism was also observed in five (62.5%) of the eight colorectal cancer cell lines we studied (D’Abronzo et al, 1999). Finally, among these eight cell lines, we detected a one-base deletion within an (A)8 repeat at codon 437 in SW48 cells, which are known to be MSI-H (Branch et al, 1995).

Figure 1
figure 1

ACTRII gene expression in tumors with LOH. RT-PCR analysis of the loss of heterozygosity (LOH)+ samples. This figure shows the gel picture of the RT-PCR product for seven LOH-positive tumors. The results for β-actin RT-PCR are presented as an internal control. The RT-PCR was performed according to the Invitrogen/Life Technologies protocol. PCR conditions were described previously (Mori et al, 2001). Top, The 1100-bp fragment of ACTRII gene; bottom, the 436-bp RT-PCR product of the β-actin gene. Lane 6 presents total lack of mRNA expression of the ACTRII gene.

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Figure 2
figure 2

Germline sequence alteration in ACTRII. Nucleotide change at codon 117. Nucleotide change from normal T (left) to A (right) at codon 117, resulting in amino acid change from Ile to Phe in the extracellular domain of the receptor. The figures show the sense sequence, and the codon 117 is marked in both panels. An arrow indicates the mutated nucleotide.

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Discussion

ACTRII is frequently mutated in MSI-H colorectal cancers (Mori et al, 2001). Moreover, activin is known to play a role in intestinal cell growth and differentiation (Attisano et al, 1996; Sonoyama et al, 2000). Finally, there are strong structural and functional similarities between the activin and TGF-β receptors (Attisano et al, 1993), the latter of which is known to be involved in both MSI-H and non–MSI-H colorectal tumorigenesis (Calin et al, 2000; Grady et al, 1998, 1999; Orimo et al, 1998). For these reasons, we studied somatic alterations of the ACTRII gene in non–MSI-H colorectal cancers. The occurrence of allelic deletion at 2q23.1 in two adenomas (one with focal-grade dysplasia), as well as in seven adenocarcinomas, suggests that LOH at this locus may occur early in colorectal neoplasia.

To evaluate whether deletion of one allele of ACTRII was associated with inactivation of the remaining allele, we performed DNA sequencing of the entire coding region of ACTRII in seven primary LOH-positive tumors from which RNA was available, as well as in eight colorectal cancer cell lines. This mutational analysis revealed, in addition to a silent polymorphism at codon 118 in five of eight cell lines and three of six tumors showing ACTRII expression, a single patient with a novel alteration at codon 117 in both tumor and corresponding normal samples.

Notably, the codon 117 alteration (I117F) found in one primary tumor has not been previously described. To determine whether I117F represented a germline mutation or a polymorphism, we sequenced normal genomic DNA from 23 additional individuals, focusing on the second exon of the ACTRII gene (containing codon 117). None of these individuals possessed this alteration, suggesting that I117F is not a frequent polymorphism in the general population.

To explore the involvement of ACTRII codon 117 in human tumorigenesis in general, we performed a literature search. Three amino acids within the ACTRII extracellular domain, 42F, 60W, and 83F, are known to be involved in the binding of activin and inhibin to ACTRII: mutations of these residues in the full-length receptor cause disruption of activin and inhibin binding (Gray et al, 2000).

Complete lack of ACTRII mRNA expression was discovered in one of seven LOH-positive primary non–MSI-H tumors but in none of eight colorectal cancer cell lines. It is possible that promoter hypermethylation caused this lack of expression; however, a promoter region for ACTRII could not be identified in public DNA sequence databases, nor were upstream CpG islands found at the ACTRII genomic locus. Finally, a mutation at codon 437, resulting in a predicted truncated protein, was observed in SW48, the only MSI-H colorectal cancer cell line studied.

These data imply that genetic alterations of the ACTRII gene (ie, missense mutation, total loss of mRNA expression, and LOH) occur in non–MSI-H colorectal cancers, but at a lower frequency than in MSI-H cancers. Thus, events predicted to result in ACTRII inactivation occur in both MSI-H and non–MSI-H colorectal tumors, further supporting the candidacy of ACTRII as a colorectal tumor suppressor gene.

Materials and Methods

Tissue Preparation, DNA, and RNA Extraction

Fifty-one non–MSI-H colorectal cancers showing MSI at no more than one of five consensus loci (BAT 25, BAT 26, D2S123, D5S346, and D17S250) were obtained with matching normal control tissues. All specimens were harvested at the time of surgical procedures or endoscopies and immediately frozen in liquid nitrogen. DNA and RNA were extracted from frozen tissue samples using a DNeasy Tissue Kit and RNeasy Mini Kit from Qiagen (Valencia, California), following the manufacturer’s protocol.

Alellotyping Analysis

For the LOH study, four microsatellite markers localizing within a 17-Mb region at 2q22.1-q23.3 including the ACTRII gene (2q23.1) were used: D2S127, D2S151, D2S2299, and D2S141 (Table 2). The ACTRII gene is located between D2S151 and D2S2299. PCR reactions were performed in a total volume of 10 μl containing 20 ng of genomic DNA, 0.5 μm each primer, 1 × Taq polymerase buffer (Life Technologies, Gaithersburg, Maryland), 0.1 mm each dNTP, 1.5 mm MgCl, and 0.1 IU of TaqDNA polymerase (Life Technologies). PCR conditions were described previously (Mori et al, 2001). Ten microliters of each PCR product were analyzed on an automated DNA sequencer (MegaBace 1000; Amersham LifeSciences/Molecular Dynamics, Sunnyvale, California) using the software program Genetic Profiler, Version 1.0 (Amersham Life Sciences/Molecular Dynamics). We classified a tumor-specific alteration as LOH when it resulted in a change of more than 50% in tumor allele peak height relative to the corresponding normal.

Table 2 Details of the Four Microsatellite Markers Used in LOH Mapping at 2q22.1-q23.3
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Mutational Analyses

RT-PCR for Expression Levels and cDNA Sequencing.

RNA was available from seven of the LOH-positive tumors, as well as from eight colorectal cancer cell lines (SW480, SW48, Colo320HSR, WiDr, SW620, SW948, Colo201, and Colo32DM). Reverse transcription used 10 μg of total mRNA in a total reaction volume of 20 μl, containing 0.5 μg of Oligo dT (Invitrogen, Carlsbad, California), 0.1 mm dNTP mix (Invitrogen), 4 μl of 5 × First-Strand Buffer (ibid), 2 μl 0.1 m dithiothreitol (Invitrogen), and 1 μl of RNase OUT Recombinant Ribonuclease Inhibitor 40 U/μl (Invitrogen). PCR protocol was performed in a total volume of 20 μl, containing 1.75 μl of primer (Invitrogen), 0.1 mm dA/T/C/GTP, 0.1 μl of TaqDNA polymerase (Qiagen), and 10 × Taq polymerase buffer (Qiagen) under the same conditions as described in allelotyping analysis. Internal control RT-PCR for the β-actin gene consisted of the same protocol, with the primers 5′-CCA GAG CAA GAG AGG TAT CC-3′ and 5′-CTG TGG TGG TGA AGC TGT AG-3′. PCR product electrophoresis was performed on a 1.5% agarose gel. Sequencing analysis was performed on both sense and antisense strands of cDNA obtained by RT-PCR.

cDNA Sequence Analysis.

Sequencing reactions were performed using the DYEnamic ET terminator kit (Amersham Pharmacia Biotech) following the manufacturer’s protocol. Electrophoresis was performed on a MegaBace 1000 (Molecular Dynamics) and analyzed using Sequence Analyzer software, Version 2.0 (Amersham Pharmacia Biotech). DNA sequences from tumors or cell lines were compared with the wild-type ACTRII sequence retrieved from the National Center for Biotechnology Information (NCBI) database (www.ncbi.nlm.nih.gov). Genomic DNA from 24 colorectal cancers was sequenced at the second exon of the ACTRII gene in accordance with the protocol described above, using primers 5′-TTC TCT GCT TAT TTA TAG GAC TG-3′and 5′-GTT TCC AAT CTA CAG TTG AGC-3′.