Interaction between p21-activated kinase 1 and beta-catenin

© 2012 Dr. He LiuColorectal cancer (CRC) was the second most frequently occurring cancer and the second leading cause of cancer death in Australia in 2007 (AIHW2010). Hyper-activation of the Wnt/β-catenin signaling pathway is a hallmark of colorectal cancer. The Wnt signaling pathway plays a critical role in embryonic development and homeostasitic maintenance in mature tissues, particularly in regeneration of intestinal epithelium (Lynch and Lynch 2005). In studies of human colon cancer over the last two decades, mutations have been identified in genes coding for Wnt/β-catenin pathway components, such as axin, adenomatous polyposis coli (APC) and β-catenin, which are known to contribute to tumor progression. Tumor genetic studies have revealed that mutations in these members of the Wnt/β-catenin pathway occur in approximately 90% of colorectal cancers (Bienz and Clevers, 2000; Cottrell et al., 1992; Morin et al., 1997; Polakis, 2000; Powell et al., 1992; Vogelstein and Kinzler, 2004). Under normoxic conditions (having a normal atmospheric oxygen concentration of 20~21%), the transcription factor 4 (TCF4) stably binds to β-catenin in the nuclei of colon carcinoma cells and is constitutively activated. This activation stimulates cell migration and proliferation, and contributes to the development of colorectal tumors (Munemitsu et al., 1995). Under hypoxic conditions β-catenin interacts with the heterodimeric transcription factor hypoxia inducible factor-1α (HIF-1α), enhances HIF-1-mediated transcription, and further promotes cell survival and adaptation to hypoxia (Kaidi et al., 2007). In a mouse model carrying a mutation in the APC gene, the gastrin gene has been identified as a downstream target of the β-catenin/TCF4 signaling pathway (Koh et al., 2000). Similarly the expression of a constitutively active β-catenin causes a threefold increase in gastrin promoter activity (Koh et al., 2000). In previous studies from this laboratory, p21-activated kinase 1 (PAK1) was found to interact with β-catenin and to be required for the regulation of the β-catenin signaling pathway by gastrins (He et al., 2008). PAK1 kinase activity has been implicated in various cellular processes such as gene regulation, cytoskeletal reorganization, cell growth, motility, and morphogenesis (Kumar et al., 2006). PAK1 also functions as a key node in various signaling pathways leading to cell growth, migration and survival. PAK1 has oncogenic functions in a broad range of cancers including CRC and its hyper-activation has been well documented in breast cancer (Dummler et al., 2009; Kumar et al., 2006). PAK1 expression has also been reported to increase in the progression of colorectal carcinomas to metastasis (Carter et al., 2004). However, the specific role of PAK1 in β-catenin signaling and the mechanism by which PAK1 interacts with β-catenin in CRC have not been investigated in detail. The studies in Chapter 3 demonstrate that PAK1 is required for maximal expression of β-catenin and its downstream targets and is important for Wnt signaling pathways in CRC, that β-catenin/TCF4 transcriptional activity is also significantly reduced in PAK1 knockdown cells, and that knocking down PAK1 decreases cell proliferation, migration, HIF-1α expression and cell survival. The mechanism by which PAK1 interacts with β-catenin was further investigated by studying the signaling networks of both proteins in Chapter 4. Cellular β-catenin expression is regulated at the protein level through phosphorylation by glycogen synthase kinase 3-beta (GSK-3β). In the cytoplasm, β-catenin forms a complex with APC, axin, GSK-3β and casein kinase 1 (CK1) (Giles et al., 2003; Kikuchi et al., 2006), and GSK-3β then induces serine-threonine phosphorylation at the amino-terminal of β-catenin, and the phosphorylated β-catenin binds to βTrCP, an E3 ubiquitin ligase that promotes the degradation of β-catenin (Wu et al., 2003). Without phosphorylation by GSK-3β, the stabilized β-catenin accumulates and is translocated to the nucleus, where it interacts with transcription factors of the TCF/LEF-1 family (mainly TCF4), leading to the increased expression of genes which stimulate cell proliferation and migration, and contribute to the development of tumors (He et al., 1998; Shtutman et al., 1999; Tetsu and McCormick, 1999). According to the results of the studies of Chapter 4, there is no change in either expression of GSK-3β protein or in kinase activity of GSK-3β as measured by phosphorylation at Serine 9 in PAK1 knockdown cells. These results indicate that PAK1 may not regulate β-catenin through GSK-3β signaling in CRC cell lines. Other effectors of β-catenin activity have also been studied in Chapter 4. The integrin-linked kinase (ILK) has been reported to be involved in β-catenin/TCF4 signaling through multiple mechanisms (Novak et al., 1998; Tan et al., 2001). The Inhibitor of β-catenin and TCF4 (ICAT) is reported to inhibit β-catenin nuclear signaling by competing with TCF4 for binding with β-catenin (Tago et al., 2000). ICAT is also located downstream of ILK in progastrin-mediated signaling in CRC tumors (Pannequin et al., 2007). Interestingly, PAK1 has been shown to be responsible for phosphorylation-dependent translocation and gene regulation of ILK (Acconcia et al., 2007). The studies of Chapter 4 demonstrate that PAK1 is required for ILK activity, that knocking down PAK1 increases ICAT expression and that PAK1 regulates β-catenin through the ILK/ICAT signaling pathway. After the investigation of the PAK1 and β-catenin interaction in CRC cells in vitro, the role of PAK1 in β-catenin signaling is further explored in vivo using animal models in Chapter 5. In this study, the in vivo role of the PAK1 protein and its interaction with β-catenin was examined using Severe Combined Immunodeficiency (SCID) mice and genetically modified APCΔ14/+ mice, and siRNAs were used as a treatment to inhibit expression of the proteins of interest. The growth of CRC cell lines as xenografts in SCID mice was studied and tumor histology in the genetically modified CRC mouse model APCΔ14/+ mice was analysed. In xenograft studies with human CRC cells in Chapter 5, PAK1 knockdown suppressed tumor growth by inhibition of proliferation and stimulation of apoptosis. In addition, PAK1 siRNA treatment delayed the growth of wildtype human CRC cells at an early stage of tumor development and contributed to tumor necrosis at later stages. In studies with APCΔ14/+ mice, expression of both PAK1 and β-catenin protein was reduced in tumors from APCΔ14/+ mice treated with PAK1 siRNA compared with mice treated with control siRNA. PAK1 siRNA treatment decreased tumor numbers significantly and slowed the bodyweight drop caused by tumor development. This thesis provides detailed information on the Wnt/β-catenin and PAK1 signaling pathways, contributes to understanding of the mechanism of human CRC development, and provides a novel direction for effective CRC treatment. The results presented here indicate that PAK1 could be a suitable target for CRC therapy. Future studies focusing on PAK1 as a drug development target may have promising outcomes in clinical trials as well as in cancer therapy