La notificación

The Wnt signalling pathway can cause tumour formation when abnormally activated. Colorectal cancer (CRC) and gastrointestinal tumours have been associated with mutations in the tumour suppressor gene APC which can mimic Wnt stimulation (Giles et al., 2003b). Up to 80% of sporadic CRC and 76% of gastric adenomas can be linked to a mutation of APC which results in loss of the binding domain for !-catenin and therefore accumulation of !-catenin in the nucleus. !-catenin itself acts on transcription factors and activates transcription (van Es and Clevers, 2005). Activation of the Wnt pathway due to !-catenin mutations has also been found in hair matrix cell tumours, hepatocellular carcinoma in Hepatitis C patients, ovarian cancer and to a low but consistent level in prostate cancer. Among childhood cancers, !-catenin mutations were detected in over 92% of hepatoblastomas (Giles et al., 2003b). In addition to that, the inhibition of Wnt using Dickkopf-1, a potent antagonist, resulted in blocking of proliferation and loss of proliferative crypts in adult mice (Kuhnert et al., 2004).

Another reason for studying !-catenin in CaP is that many !-catenin – TCF/LEF target genes have been associated with cancer, e.g. c-Myc (He et al., 1998, Yochum et al., 2010, Yochum et al., 2007) and Cyclin D1 (Tetsu and McCormick, 1999, Yochum et al., 2007). c-Myc has recently been shown to be sufficient, on its own, to induce an embryonic stem cell like programme in normal human keratinocytes, increasing the tumour initiating cells by 150-fold (Wong et al., 2008).

In colon cancer active !-catenin was associated with cancer cells at the invasive front of the tumour, at the site of potential cancer stem cells (Fodde and Brabletz, 2007).

1.3.1 A connection between !-catenin and the androgen receptor

The majority of androgen-independent prostate tumours still express androgen receptor (AR). An aberrantly activated AR pathway, in the absence of normal levels of androgen, is believed to play a role in tumour progression (Zhu et al., 2004).

One of the proteins which has been found to interact with the AR and potentiate its transcriptional activity is !-catenin, one of the major players of canonical Wnt signalling (Chesire and Isaacs, 2002, Heinlein and Chang, 2002, Pawlowski et al., 2002, Truica et al., 2000, Yang et al., 2002).

Even though activating !-catenin mutations and APC mutations are rarely found in clinical prostate tumour samples (Chesire et al., 2000, Voeller et al., 1998, Watanabe et al., 1996), they have been reported in castration resistant CaP and a link between an aberrantly activated canonical Wnt pathway and androgen independence in prostate cancer is believed to occur (Zhu et al., 2004).

High levels of both the AR protein and mRNA have been found to be associated with the transition of hormone sensitive to androgen-independent CaP (Chen et al., 2004, Koivisto et al., 1997, Linja et al., 2001, Schweizer et al., 2008). A recent study showed that while physiological levels of androgen inhibit tumour cell growth, the interaction between AR and Wnt signalling provides a growth advantage for prostate cancer cells at castration levels of androgen (Schweizer et al., 2008).

1.3.2 !-catenin and E-cadherin

Apart from its apparent role in Wnt signalling, !-catenin is also recognised as a key molecule in the E-cadherin adherens junctions and the link between Wnt signalling and adhesion through !-catenin has been investigated for some time (Moon et al., 1997). Under normal physiological condition, E-cadherin mediates cell adhesion of epithelial cells by connecting to the actin cytoskeleton via catenins in the cytoplasm (Giehl and Menke, 2008, Heasley and Petersen, 2004, Jamora and Fuchs, 2002, Morita et al., 1999, Götz, 2008) (Figure 14). Loss of this cell adhesion has been found to lead to tumour progression and formation of metastasis (Beavon, 2000).

In prostate cancer it is thought that differences in E-cadherin expression are important for the development from a non-invasive into an invasive tumour (Isaacs et al., 1994, Isaacs et al., 1995, Verras and Sun, 2006). Loss of E-cadherin or reduced expression has been

associated with more poorly differentiated tumours and therefore later stages of prostate cancer (Bussemakers et al., 1992, Luo et al., 1999, Richmond et al., 1997, Umbas et al., 1992).

The significance of this was exemplified in a study by Ewing and colleagues. By expressing #-catenin in PC-3 prostate cells, which contained a dysfunctional adhesion complex due to loss of #-catenin, they were able to re-establish a normal cell-cell adhesion pathway. This also lead to decreased tumourgenicity when the PC-3 variant was injected into nude mice, therefore establishing that normal functioning of the E-cadherin complex is required to suppress tumour growth (Ewing et al., 1995). It was also previously investigated whether loss of E-cadherin in advanced prostate cancer was resulting in increased !-catenin in the cytoplasm and nucleus (Sasaki et al., 2000).

In our study, we wanted to elucidate the importance of !-catenin in prostate cancer. Taking the relationship between !-catenin and E-cadherin into account our research was based on the following assumptions: Reduced E-cadherin due to the advanced stage of a tumour will lead to less !-catenin being required at cell junctions, which therefore will result in an increase of !-catenin availability in the cytoplasm. This in turn will increase the chances of !-catenin being transferred into the nucleus which mimics increased canonical Wnt signalling. The lentivirally delivered shRNAs against !-catenin would then target this aberrantly active signalling cascade.

Figure 14: Basic model of an adherens junction

E-cadherin forms a complex with !-catenin and "-catenin to connect to the actin cytoskeleton within the cell (Modified from Giehl and Menke, 2008, Heasley and Petersen, 2004 and Götz, 2008).