The aE-catenin gene (CTNNA1) acts as an invasion-suppressor gene in human colon cancer cells Stefan J Vermeulen 1 , Friedel Nollet 2 , Erik Teugels 3 , Krist'l M Vennekens 1 , Fransiska Malfait 1 , Jan Philippe 4 , Frank Speleman 5 , Marc E Bracke 1 , Frans M van Roy 2 and Marc M Mareel 1 1 Laboratory of Experimental Cancerology, Department of Radiotherapy and Nuclear Medicine, University Hospital Gent, De Pintelaan 185, B-9000 Gent; 2 Department of Molecular Biology, Laboratory of Molecular Cell Biology, University of Gent and VIB, Ledeganckstraat 35, B-9000 Gent; 3 Laboratory of Medical Oncology, University Hospital Brussels, Laarbeeklaan 101, B-1010 Jette; 4 Department of Clinical Chemistry, Microbiology and Immunology, University Hospital Gent, De Pintelaan 185, B-9000 Gent; 5 Department of Medical Genetics, University Hospital Gent, De Pintelaan 185, B-9000 Gent, Belgium The acquisition of invasiveness is a crucial step in the malignant progression of cancer. In cancers of the colon and of other organs the E-cadherin/catenin complex, which is implicated in homotypic cell-cell adhesion as well as in signal transduction, serves as a powerful inhibitor of invasion. We show here that one allele of the aE-catenin (CTNNA1) gene is mutated in the human colon cancer cell family HCT-8, which is identical to HCT-15, DLD-1 and HRT-18. Genetic instability, due to mutations in the HMSH6 (also called GTBP) mismatch repair gene, results in the spontaneous occurrence of invasive variants, all carrying either a mutation or exon skipping in the second aE-catenin allele. The aE-catenin gene is therefore, an invasion- suppressor gene in accordance with the two-hit model of Knudsen for tumour-suppressor genes. Keywords: colon cancer; invasion; aE-catenin; genetic instability Introduction Colon cancer progression is characterized by histo- pathological changes starting from the normal mucosa over hyperplasia and sometimes polyp formation, towards carcinoma in situ and ®nally invasive cancer. This histopathological evolution is associated with the accumulation of a number of distinct genetic altera- tions (Kinzler and Vogelstein, 1996). Activation of several oncogenes and inactivation of several tumour- suppressor genes has been associated with growth disturbance preceeding invasive lesions. Fewer genetic alterations could be associated so far with the acquisition of the invasive and potentially metastatic phenotype, which is crucial for cancer malignancy and responsible for therapeutic failure (Mareel et al., 1991, 1996). Experimental as well as clinical observations have demonstrated that the E-cadherin/catenin complex is a powerful inhibitor of invasion (Behrens et al., 1989; Vleminckx et al., 1991; Takeichi, 1991; Bracke et al., 1996; Mareel et al., 1997). Disturbance of one of the elements of this complex, as evidenced by loss or truncation of the protein, has been associated with the transition from the noninvasive towards the invasive phenotype and with a worse prognosis in many human cancers. We have only started to understand how activation or inactivation of cadherin and catenin genes are implicated in tumour progression. The E-cadherin gene acts as an invasion-suppressor gene in lobular breast cancer since mutation of one allele in combination with loss of the other allele leads to invasive tumours (Berx et al., 1995). b-Catenin acts as an oncogene in colon cancer and in melanoma since heterozygous mutation results in protein stabilization and correlates with tumour progression (Morin et al., 1997; Rubinfeld et al., 1997). For aE-catenin the eect on tumour progression of heterozygous as compared to homozygous mutations has not been examined. There are indications from immunohistochemical studies that aE-catenin is reduced in 80% of invasive colon cancers (Shiozaki et al., 1994, 1995). In some colon cancer cell lines, de®ciencies at the mRNA and protein level were described for E-cadherin and aE-catenin but not for b-catenin (Vermeulen et al., 1996). Cultivating the human colon cancer cell line HCT-8, we found that invasive variants regularly emerged from noninvasive clones (Vermeulen et al., 1995a). Such invasive variants were recognized through their round (R) morphotype against a background of epithelioid (E) cells. Similar R variants were observed in the cell lines HCT-15, HRT-18 and DLD-1 which all have the same genetic background as HCT-8 (Vermeulen et al., 1998, in press). R variants were de®cient in aE-catenin protein and this explained their invasiveness in vitro. We have used this tumour model to address the question whether or not the aE-catenin gene (CTNNA1) meets the criteria of an invasion- suppressor gene in accordance with the two-hit hypothesis of Knudson (1985) for tumour-suppressor genes. Results Regular transition from the epithelioid (E) to the round (R) morphotype HCT-8 subclones that were either positive or negative for aE-catenin protein could be selected by their epithelioid (E) or round (R) morphotype respectively (Vermeulen et al., 1995a). Using phase contrast microscopy we estimated that R variants reproducibly Correspondence: MM Mareel Received 16 March 1998; revised 5 August 1998; accepted 6 August 1998 Oncogene (1999) 18, 905 ± 915 ã 1999 Stockton Press All rights reserved 0950 ± 9232/99 $12.00 http://www.stockton-press.co.uk/onc