The effect of a germline mutation in the APC gene on ?-catenin in human embryonic stem cells


Colorectal cancer (CRC) is one of the leading causes of cancer-related mortality [1]. About 50 % of all CRC patients will develop metastases and ultimately die from the disease. Most CRC cases arise from two somatic unrelated events, however, approximately 5 % of CRCs are initiated by an inherited genetic mutation which inevitably leads to the acquisition of a second somatic mutation. In all cases, progression to carcinoma occurs through the accumulation of multiple somatic mutations, leading to malignant transformation and development of an invasive cancer [13].

One of the most critical genes mutated in CRC is the adenomatous polyposis coli (APC) tumor suppressor gene [1, 2]. APC encodes a large multi-functional protein [4], and its main role in tumorigenesis lies in its ability to negatively regulate Wnt signaling by controlling cellular levels of ?-catenin [1]. Wnt signalling is a key developmental pathway involved in embryonic development, cell differentiation, cell proliferation and tissue maintenance in adults [5, 6]. However, the aberrant constitutive activation of the Wnt pathway that is caused by APC mutations in many cases leads to uncontrolled cell proliferation and tumorigenic transformation, CRC being the most notable among them [6].

Since APC mutations are detected very early in the adenoma-carcinoma sequence, the APC protein has been suggested to act as a “gatekeeper” of colorectal carcinogenesis, which means that functional loss of APC is a prerequisite for the progression towards malignancy. Around 85 % of all sporadic and hereditary colorectal tumors show loss of APC function [1]. Individuals affected by familial adenomatous polyposis (FAP) carry a germline mutation in the APC gene (‘first hit’), and show autosomal dominant inheritance with essentially 100 % penetrance (i.e., all will develop cancer [3, 7, 8]). Young FAP patients start to acquire additional mutations (somatic mutations or the ‘second hit’) in the second allele of the APC gene, leading to its functional loss and to the development of adenomatous colon polyps, which invariably progress to colon cancer if not removed.

The APC gene includes a mutation cluster region (MCR) which is prone to mutations. The cell will have a selective advantage for tumor formation when at least one of the mutations (germline or somatic) is located within the MCR region that includes multiple ?-catenin binding sites. Indeed, APC mutations in colorectal tumors are distributed non-randomly within the gene [9], with the position and type of the somatic APC mutation depending on the germline mutation [916].

Most of our knowledge about the initiation and development of CRC came from studies performed in cancer cells derived from CRC-affected patients [17]. In addition to the APC mutation, these already differentiated cells reportedly carry some other mutations that are only partly characterized and thus have limitations in providing necessary data on the initial molecular steps leading to cancer formation. Another research model for CRC is genetically manipulated mice with different mutations in the APC gene. Although most of these APC mutations in mice are embryonically lethal, the severity of the cancer predisposition is variable [18]. Numerous APC genetically altered mice have been generated and serve as models for colon adenoma and cancer, but their phenotypes are different from the human disease [19]. For example, several genetic mouse models generate tumors predominantly in the small intestine, in contrast to human CRC, in which tumors are found in the colonic epithelium [20]. Carcinogen treatment of mice generates colonic neoplasia, but these mice show specific gene expression patterns that do not represent the entire development of human CRC [20]. Therefore, although these models are very important for studying colon carcinogenesis, they are inadequate for the study of the earliest molecular mechanisms underlying malignant transformation in humans.

Human embryonic stem cells (hESCs) have already been proven to be a valuable tool for studying human genetic disorders [2125]. Pre-implantation genetic diagnosis (PGD), a procedure used to obviate the inheritance of mutations in affected families, has recently been established for FAP families as well. In the current study, we use two FAP hESC lines derived from embryos that inherited the APC germline mutation following PGD for FAP carriers (Lis25_FAP1 published in [26]). These cell lines, to the best of our knowledge, are available solely in our lab, and they comprise a valuable model for unraveling the very early mechanisms leading to malignant transformation in the colon.