Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis
Tumors from rats given 1,2-dimethylhydrazine plus chlorophyllin or indole-3-carbinol contain transcriptional changes in β-catenin that are independent of β-catenin mutation status
Introduction
There is growing interest in the β-catenin/T-cell factor (TCF)/lymphoid enhancer factor (LEF) signaling pathway and its role in human cancer development [1]. β-Catenin is a cadherin-binding protein that also functions as a transcriptional activator when complexed in the nucleus with members of the TCF/LEF family [2]. Cytosolic β-catenin interacts with APC, Axin, glycogen synthase kinase-3β (GSK-3β) and other protein partners, leading to phosphorylation of Ser33, Ser37, Thr41 and Ser45 residues in the N-terminal region of β-catenin, followed by ubiquitination and proteosomal degradation [1], [2], [3]. In primary human colon tumors and colorectal cancer cell lines, mutations in CTNNB1 substitute one of the four critical Ser/Thr residues and stabilize β-catenin, leading to accumulation of β-catenin/TCF complexes in the nucleus, and activation of downstream target genes [4], [5], [6].
Mutations in β-catenin also have been detected in colon tumors of animals treated with chemical carcinogens, such as 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), azoxymethane, 1,2-dimethylhydrazine (DMH), and methylazoxymethanol acetate plus 1-hydroxyanthraquinone [7], [8], [9], [10], [11]. In fact, there are a number of similarities between human and rat colon tumors with respect to the β-catenin pathway. First, as in the human situation, colon tumors in the rat contain mutations in Apc or Ctnnb1, but not in both of these genes [7]. Second, β-catenin/Tcf downstream targets frequently are over-expressed, including c-Myc, c-Jun and cyclin D1 [9], [11]. Third, genetic changes in human CTNNB1 or murine Ctnnb1 substitute amino acids within the GSK-3β regulatory domain of β-catenin. However, whereas the vast majority of β-catenin mutations in human colon cancers substitute critical Ser/Thr residues directly, in rat colon tumors the mutations often localize to two CTGGA ‘hotspot’ sequences and substitute amino acids adjacent to Ser33 [7], [8], [9], [10], [11].
To complicate matters, the spectrum of β-catenin mutations can be influenced by exposure to dietary phytochemicals, such as chlorophyllin (CHL) and indole-3-carbinol (I3C). The latter compound is found in cruciferous vegetables [12], [13], [14], whereas CHL is a water soluble derivative of chlorophyll, the ubiquitous pigment in green, leafy vegetables [15], [16], [17]. In the tumors from rats given DMH or IQ alone, virtually all of the β-catenin mutations substituted amino acid residues adjacent to Ser33, whereas in animals given carcinogen followed by I3C or CHL, β-catenin mutations more often substituted one of the critical Ser/Thr residues [9]. Subsequent work [18] showed that amino acid substitutions adjacent to Ser33 retard, rather than completely block, the proteasome degradation pathway, leading to a range of β-catenin protein expression levels in colon tumors. High levels of β-catenin and c-Jun were detected in tumors that contained mutations affecting Ser45 or Thr41 of β-catenin, tumors with genetic changes substituting Gly34 and Asp32 had intermediate levels of β-catenin and c-Jun, and the lowest levels of β-catenin and c-Jun were observed in tumors with wild type β-catenin [11].
In the latter experiments, it was assumed that each specific mutation in β-catenin affected its relative stability and turnover at the protein level, the degree of nuclear trafficking and interaction with Tcf/Lef transcription factors, and thus the extent to which downstream target genes became activated. However, in the present investigation of more than 50 DMH-induced tumors in the rat, we observed that β-catenin frequently was over-expressed at the mRNA level. This suggested transcriptional dysregulation of β-catenin, as distinct from the β-catenin protein stabilization reported before [11]. Expression of β-catenin mRNA was correlated with cyclin D1, c-myc and c-jun mRNA levels, but not with the mutation status of β-catenin, or treatment with I3C or CHL.
Section snippets
Source of tumors
Tumors were from a study in which male F344 rats were initiated during the first 5 weeks with DMH (20 mg/kg body weight, by subcutaneous injection), and treated post-initiation with 0.1, 0.01 or 0.001% CHL in the drinking water, or with 0.1, 0.01 or 0.001% I3C in the diet. Full details were provided in the original report [19].
Mutation screening
A subset of 57 DMH-induced small intestine and colon tumors, from each of the treatment groups in the original study, was screened for mutations in β-catenin. The
Confirmation of a mutational ‘hotspot’ involving codons 32 and 34
In the present investigation, 16 tumors from the DMH control group, 25 tumors from the DMH + CHL group, and 22 tumors from the DMH + I3C group were screened using PCR-SSCP analysis (Fig. 1). One of the samples lacked any of the bands for the wild type, indicating that the tumor was likely to be homozygous for the corresponding β-catenin mutation. Sequencing of this tumor identified a mutation in codon 32 of β-catenin (GAT → AAT, D32N). Table 2 shows the complete list of β-catenin mutations, confirmed
Discussion
The present investigation builds upon prior work showing that phytochemicals such as CHL and I3C can alter the spectrum of β-catenin mutations in DMH- and IQ-induced tumors [9], [11]. Under conditions of tumor promotion, there was an increase in mutations affecting Ser37, Thr41 and Ser45 of β-catenin [11]. The latter mutations have been reported in human cancers, but they occur less frequently in the colon tumors from rats exposed to chemical carcinogens in the absence of a tumor promoter [7],
Acknowledgments
Thanks are extended to Meirong Xu, who was responsible for overseeing the original carcinogenicity bioassay. We also thank Qingjie Li for help with primer design, and members of the Tanguay and Hagen laboratories for access to qPCR equipment. Sequencing was performed in the Center for Gene Research and Biotechnology at Oregon State University. This work was supported in part by NIH grants CA65525, CA80176, and CA90890.
References (25)
Indole-3-carbinol: anticarcinogen or tumor promoter in brassica vegetables?
Chem. Biol. Interact.
(1998)- et al.
Dietary cancer and prevention using antimutagens
Toxicology
(2004) - et al.
Activator protein 2α associates with adenomatous polyposis coli/β-catenin and inhibits β-catenin/T-cell factor transcriptional activity in colorectal cancer cells
J. Biol. Chem.
(2004) - et al.
Characterization of dysplastic aberrant crypt foci in the rat colon induced by 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine
Am. J. Pathol.
(2003) - et al.
Analysis of differential gene expression in colon cancer and cancer stroma using microdissected tissues
Gastroenterology
(2005) - et al.
Cloning of the rat β-catenin gene (Ctnnb1) promoter and its functional analysis compared with the Catnb and CTNNB1 promoters
Genomics
(2004) WNT/PCP signaling pathway and human cancer
Oncol. Rep.
(2005)- et al.
Oncogenic β-catenin signaling networks in colorectal cancer
Cell Cycle
(2005) - et al.
β-Catenin is a target for the ubiquitin-proteosome pathway
EMBO J.
(1997) - et al.
Identification of c-MYC as a target of the APC pathway
Science
(1998)
Target genes of β-catenin-T-cell factor/lymphoid-enhancer-factor signaling in human colorectal carcinomas
Proc. Natl. Acad. Sci. U.S.A.
β-Catenin regulates expression of cyclin D1 in colon carcinoma cells
Nature
Cited by (13)
Memories of a friend and colleague – Takashi Sugimura
2020, Mutation Research - Reviews in Mutation ResearchCitation Excerpt :Subsequently, at the 27th International Symposium of the Princess Takamatsu Cancer Research Fund, in 1996, a discussion during lunch break with Dr. Minako Nagao led to a sabbatical at the National Cancer Center in Tokyo, where friendships were renewed. In addition to weekly interactions during journal clubs and seminars, personal and scientific interests often were aired over green tea in Dr. Sugimura’s office as the President Emeritus, including topics such as cabbage butterflies [27–29], heterocyclic amine mutagens [16–23], and Wnt/β-catenin signaling [31–35]. As the one-year sabbatical was ending, Dr. Sugimura took time from his busy schedule to escort me around his alma mater at The University of Tokyo Faculty of Medicine.
3,3'-Diindolylmethane, but not indole-3-carbinol, inhibits histone deacetylase activity in prostate cancer cells
2012, Toxicology and Applied PharmacologyCitation Excerpt :DIM has been shown to be well tolerated in humans and inhibits the growth of prostate cancer both in vitro and in mouse models (Cho et al., 2011). Despite the fact that I3C did not strongly affect HDAC activity, I3C has been shown to be effective in limiting cancer growth in several other models in vitro and in vivo, and still may be an important chemopreventive agent through alternate mechanisms (Lubet et al., 2011; Wang et al., 2006, 2012; Yu et al., 2006). The chemopreventive properties of DIM are likely attributed to targeting multiple mechanisms.
Prevention and therapy of 1,2-dimethyl hydrazine induced colon carcinogenesis by Ferula assafoetida hydroalcoholic extract
2015, Turkish Journal of BiochemistryChemopreventive potential of chlorophyllin: A review of the mechanisms of action and molecular targets
2015, Nutrition and CancerChemopreventive effects of caraway powder and oils to suppress 1, 2- dimethylhydrazine-induced colon carcinogenesis
2014, Turkish Journal of Biochemistry