β-Catenin accumulation and S33F mutation of CTNNB1 gene in colorectal cancer in Saudi Arabia

Introduction

The global occurrence of colorectal cancer (CRC) is third among cancer incidence in males and fourth among females [1, 2]. In Saudi Arabia CRC is less common as compared to its counterpart Gulf Cooperation Council States (GCCS) and the West. However, CRC was the second most common malignancy after breast cancer, and first most common malignancy among men between 1994 and 2004 as reported by Saudi Cancer Registry (SCR) [3]. The World Health Organization (WHO) reported in 2004 that the age-standardized death rate from CRC was 8.3% in KSA [4]. The incidence of CRC is increasing among the local population of Saudi Arabia as seen by a three-fold rise in incidence in males from 3.2% to 11.2% within around 7 years; a parallel trend in females was observed with an increase from 2.7% to 8.8% for the same period [3, 5]. Through established surveillance programs and subsequent early detection and removal of pre-cancerous colonic polyps, the incidence of CRC and its related deaths have decreased over the past 15 years in the United States, the high CRC incidence country [6, 7]. In contrast, due to the lack of surveillance programs and insufficient molecular investigations, the increasing CRC incidence in KSA suggests a potentially alarming situation developing in the coming decades [8].
It has been proposed that chromosomal abnormalities, genetic mutations, and epigenetic changes are the factors responsible for advancement of malignant carcinoma from benign colorectal adenoma [9]. These events lead to inactivation of DNA mismatch repair genes and tumor suppressor genes, or activation of oncogenes. The critical gene mutations in the development of colorectal cancer have been found in adenomatous polyposis coli (APC), Kirsten-ras (K-ras), p53 and β-catenin genes [10, 11, 12, 13]. Nonetheless, the initiating event for most of the colorectal cancers is observed to be due to the mutations in components of the Wntβ-catenin pathway [14].
The N-terminus of β-catenin harbors highly conserved residues, S33, S37, S45, and T41 encoded by exon 3 of the human CTNNB1 gene. These amino acids are not only putative GSK-3 phosphorylation sites but also constitute a part of a 6-amino acid stretch important for ubiquitination, similar to I-kB [15, 16]. Mutation of any one of these amino acid residues in exon 3 of the CTNNB1 gene generates a stabilized form of β-catenin which can no longer be phosphorylated and degraded and ultimately leading to constitutively active transactivation complexes, which appear to contribute to loss of cell growth control [17, 18]. Different types of tumors have been screened for mutations in exon 3 of the CTNNB1 gene and, indeed, mutations are found in these four residues [19, 20, 21, 22, 23]. These analyses indicate that a mutation in only one of these phosphorylation sites is sufficient to create a dominant positive form of β-catenin.
The available knowledge about the genetic factors that determine the development and clinical course of colorectal cancers in Eastern and Middle Eastern countries is limited. To date, the association between gene polymorphisms in exon 3 of β-catenin gene and the development of colorectal cancer in Saudi population has not yet been investigated. In the present study we tried to identify the mutations in exon 3 of β-catenin in primary colorectal carcinomas using PCR-based sequencing analysis.

Experimental methodology

Patient selection and tissue preservation

The study was performed based on approval by the Institutional Review Board of the Ethics Committee at King Khalid University Hospital in Riyadh, KSA. A total of 60 Saudi patients with CRC diagnosed with CRC with available formalin fixed and paraffin embedded tumor and normal tissues were selected from the Department of Pathology and Surgical Oncology King Khalid Hospital, Riyadh Saudi Arabia. Clinical and demographic features were recorded, including age at the time of diagnosis, gender, nationality, family history, smoking habits, disease behavior, disease location, and type of surgery.

DNA extraction and purification

DNA was extracted from fresh and formalin-fixed and paraffin-embedded (FFPE) tissue specimens by standard techniques as described previously [24]. From all CRC tissue samples 5 micron sections were cut from each FFPE block and mounted onto glass slides. Subsequently, tissue sections were deparaffinized in xylene followed by rehydration in a series of graded ethanol solutions and lastly with ultra-pure water. Using standard protocols one section from each block was stained with haematoxylin and eosin. An expert histopathologist examined and confirmed the presence of tumor tissue in FFPE blocks. Corresponding FFPE tumor tissue blocks were used for DNA extraction using QIAamp DNA FFPE Tissue Kit (Qiagen).

Mutation analysis of exon 3 of CTNNB1 gene

DNA was extracted from two 20-µm-thick formalin-fixed, paraffin-embedded tissue rolls cut from blocks with at least 70% tumor using the QIAamp DNA mini kit DNA isolation kit (Qiagen Valencia, CA). The genomic DNA isolated from tumor and normal control tissue (n = 60) from the same patient was used as substrate for amplification of exon 3 of the CTNNB1 gene. The polymerase chain reaction (PCR) used primers BCAT-F:5’-ATTTGATGGAGTTGGACATGGC-3’ and BCAT-R:5’-CCAGCTACTTGTTCTTGAGTGAAGG-3’, which yielded an amplicon measuring 224 bp in size with thermocycling conditions previously reported to amplify exon 3 of CTNNB1 with appropriate controls. PCR products were detected by gel electrophoresis in 2% agarose, and amplicon bands were purified using the QIAquick gel extraction kit (Qiagen). Sequencing of these products were performed with forward and reverse primers by Macrogen Inc. Obtained sequences were analyzed and aligned with CTNNB1 reference sequence from the Gen Bank database accession number, NG_013302.
All PCR and sequencing experiments were repeated at least two times. DNA mutations were confirmed by re-sequencing from an independent PCR reaction using same primers in the same sample.

Immunohistochemistry

Standard immunohistochemistry was performed on formalin fixed, paraffin embedded tissue using an anti-β-catenin monoclonal antibody (Sigma-Aldrich) and performed with ultraView DAB Detection Kit (Ventana, Arizona, USA) on a BenchMark XT automated staining system (Ventana, Arizona, USA). Tissue sections 3 micron thickness were cut into a coated slides by using Leica RM2235 Rotary Microtome (Leica Biosystems, Wetzlar, Germany), incubated for 15 minutes in a hot air oven at 60°C. The tissue sections were deparaffinized with EZ Prep (Ventana, Arizona, USA) at 75°C, heat pretreated in Cell Conditioning 1 (CC1; Ventana, Arizona, USA) using “standard cell conditioning” for antigen retrieval at 100°C, and then incubated with the anti-β-catenin primary antibody (diluted 1 : 50) for 32 min at 37°C after inactivation of the endogenous peroxidase with hydrogen peroxide for 4 min. Ultraview universal HRP multimer was then added as the secondary antibody. The immunolocalized β-catenin protein was visualized using a copper-enhanced DAB reaction. The slides were counterstained by Hematoxylin II and Bluing Reagent (Ventana, Arizona, USA) for 4 min and then liquid coverslip (LCS) was applied atop aqueous reagents by the NexES Special Stains automated slide stainer to prevent reagent evaporation and ensure complete slide coverage. Following that, samples for mounting in DPX were dehydrated by passing through graded alcohols: 70% ethanol, 96% ethanol and absolute ethanol. Then a small drop of DPX was added to the specimen. After each step Reaction Buffer was used to wash slides to provide a stable aqueous environment. Immunostained sections were reviewed by Olympus BX51 light Microscope and DP72 Olympus Digital Camera (magnification 100× and 200×) (Olympus America Inc, Center Valley, PA, USA).

Results

All the 60 CRC samples were screened for mutations in exon 3 of the β-catenin gene. The demographic and clinic data of the 60 patients enrolled in this study were reported in Table I.
The average age of patients was 55.9 and the number of male and female was the same. The types of tumors are adenocarcinoma for 91.7% located in the colon and 8.3% are mucinous carcinoma located in the rectum. The majority of patients (88.3%) were in grade II of CRC.
Sequencing of exon 3 of the CTNNB1 gene revealed an activating mutation (S33F) in one of the tumor samples (Fig. 1). The tumor and adjacent normal tissue was obtained from a 63 years old female patient with cecal cancer and retroperitoneal mass. Our data thus indicate for the first time that β-catenin may play a role in the development of colorectal carcinoma and that activating mutations of the β-catenin gene may substitute biallelic APC inactivation in this tumor type in KSA.
The mutation at codon 33 changed serine, a hydrophyllic neutral amino acid to phenyalanine, a hydrophobic amino acid [TCT (Ser)  TTT (Phe)]. It is noteworthy that this mutation was heterozygous as evidenced by the presence of double peak pattern in the chromatogram of the tumor DNA in codon 33. Sequencing analysis of the corresponding non-tumorous tissue did not reveal a mutation (Fig. 1).
To determine the β-catenin protein expression level in tumor and the adjacent normal tissue, we performed immunohistochemical analysis on formalin-fixed, paraffin-embedded tissue using anti β-catenin monoclonal antibody. The tumor cells with the S33F mutation displayed an increased protein expression versus that in normal adjacent cells with wild type β-catenin protein (Fig. 2). The enhanced protein expression in S33F mutated tumor cells was localized to both cytoplasm and nuclei.

Discussion

β-catenin plays a key role in the Wnt signaling cascade.β-catenin is mutated in up to 10% of all sporadic colorectal carcinoma by point mutations or inframe deletions of the serine and threonine residues that are phosphorylated by GSK-3β [14]. These mutations result in stabilization of β-catenin and activation of WNT-signaling. Mutations in β-catenin occur in exclusivity to APC aberrations as both molecules are components of the same pathway [25]. It has been found that the cells containing a mutant APC have elevated β-catenin levels and 80% of colon cancers possess APC mutations [26]. β-catenin is assumed to transactivate mostly unknown target genes, which may stimulate cell proliferation (acts as an oncogene) or inhibit apoptosis. The β-catenin level in the cell is regulated by its association with the adenomatous polyposis coli (APC) tumor-suppressor protein, axin, and GSK-3β. Phosphorylation of β-catenin by the APC-axin-GSK-3β complex leads to its degradation by the ubiquitin-proteasome system [27, 28, 29]. Thus far three serines (S33, 37, 45) and one threonine (T41) are considered to be the substrates for GSK-3βphosphorylation. Indeed, these amino acids are regularly mutated in tumors, resulting in accumulation of β-catenin molecules with enhanced transcriptional activity [30].
Our data indicate for the first time in Saudi Arabia that mutations are detected at codon 33 of β-catenin/CTNNB1 gene in colorectal cancer tissue. In this study, we have analyzed the status of β-catenin protein and of exon 3 of the CTNNB1 gene in colorectal cancer for the first time in KSA. Compared to normal adjacent tissue, we found a clear increase of cytoplasmatic and nuclearβ-catenin concentration in the malignant cells of one of the 60 investigated cases. Thus, although the number of investigated cases is small and incidence of S33F mutation is very low; our results suggest a possible role ofβ-catenin accumulation as a result of S33F mutation in the pathogenesis of colorectal cancer in some of the patients in. Our observations need to be confirmed on a larger number of tumors in Saudi Arabia.
An association of β-catenin accumulation and malignancy has been shown for several tumor types, including colorectal carcinomas [21, 31, 32], hepatocellular carcinomas [12, 33], endometroid ovarian carcinomas [34], melanomas [35], and desmoid tumors [36, 37, 38]. Although, the increased oncogenic β-catenin activity in these heterogeneous tumor types may be as a result of different mechanisms but colorectal carcinomas, desmoid tumors, and hepatoblastomas have in common that they more frequently affect patients with FAP (familial adenomatous polyposis). However, the most probable mechanism of β-catenin accumulation in FAP-associated tumors is its decreased degradation due to APC dysfunction. The same mechanism is likely to be true for sporadic cases of the above-mentioned tumor types, in which biallelic somatic inactivation of APC have been shown [39, 40, 41, 42]. It has been found that the alterations of the β-catenin itself could also be responsible for elevated protein levels. Mutations in CTNNB1 gene, which substitute APC inactivation on a molecular level, have recently been described in colorectal carcinoma cell lines [21, 43] as well as in colorectal cancer patients [31]. The mutations were found in exon 3 of the CTNNB1 gene and resulted in an amino acid change at GSK-3β phosphorylation sites at codons 33, 37, 41, and 45. Lack of phosphorylation at any of these sites decreases sequestration of β-catenin by APC [44]. Our results show for the first time in KSA that CTNNB1 mutations that may substitute for biallelic APC inactivation also occur in colorectal cancer. We found a missense mutation in codon 33 of the CTNNB1 gene in one of the investigated cases. The mutation (TCT to TTT) results in a substitution of phenylalanine for a serine phosphorylation site. Mutations of the same codon (S33) has earlier been described for hepatocellular carcinoma [45], glomangiopericytoma [46], endometrial hyperplasia [47], brain tumours [48] and colon cancer cells [43] and is considered oncogenic.
In conclusion, our results indicate that colorectal cancer is a tumor in which β-catenin accumulation is implicated in tumor development. As a genetic reason for protein accumulation, we have identified an activating mutation in the CTNNB1 gene in one of the analyzed cases. Furthermore, the cause of this genetic mutation needs to be investigated with relation to toxicogenetic effects of changed lifestyle and environment. Larger numbers of colorectal cancers should be screened for β-catenin alterations in order to evaluate the validity of our findings.

The authors declare no conflict of interest.
The authors acknowledge the Distinguished Scientist Fellowship Program at King Saud University for financial support for this work.

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Address for correspondence

Lamjed Mansour

Department of Zoology, College of Science
King Saud University
PO. Box: 2455, Riyadh, 11451, Saudi Arabia
tel. +966550145746
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e-mail: lamjed.mansour@gmail.com