P-21 activated kinase 1 knockdown inhibits β-catenin signalling and blocks colorectal cancer growth

Abstract

The p21-activated kinase 1 (PAK1) plays important roles in cell growth, motility, and transformation. The aims of this study were to delineate the signalling mechanisms downstream of PAK1, and to investigate the importance of PAK1 for colorectal cancer (CRC) growth and metastasis in vivo. PAK1 knockdown in human CRC cell lines inhibited β-catenin expression, β-catenin/TCF4 transcriptional activity, and the expression of c-Myc. In mouse models PAK1 knockdown suppressed the growth and metastasis of human CRC cells by decreasing proliferation and increasing apoptosis. Our findings demonstrate for the first time the crucial role of PAK1 in CRC progression in vivo.

Introduction

Mutations in Wnt signalling components play key roles in the initiation and progression of colorectal cancer (CRC). Aberrant activation of β-catenin, which is a central molecule of the Wnt pathway, promotes cell proliferation and survival, and initiates colorectal tumorigenesis [1], [2]. Accumulation of nuclear β-catenin, and the consequent formation of a constitutively active complex with the transcription factor T-cell factor 4 (TCF4) [3], [4], promotes cell growth and drives tumor progression through up-regulation of target genes such as c-Myc [1], [5].

Oncogenic KRas synergistically enhances Wnt/β-catenin signalling in intestinal tumors. Ras mutations are found in 40% of CRC and occur early during the adenoma–carcinoma sequence [6], [7], [8]. Synchronous detection of activated KRas and β-catenin nuclear accumulation, which is the hallmark of activated Wnt signalling, identifies a group of patients with poor prognosis and resistance to chemotherapy [9]. In a mouse model carrying mutations in both the adenomatous polyposis coli (Apc) and KRas genes, Wnt/β-catenin signalling is enhanced, with resultant increases in tumor initiation and progression toward malignancy [10]. The increased expression of some Wnt-pathway target genes is consistent with synergistic co-operation between mutant KRas and Apc in this model [10], and combined inhibition of β-catenin and KRas in CRC cells further reduces proliferation and promotes apoptosis [11].

The observations that PAK1 is essential for malignant transformation induced by Ras, Rac and Cdc42 [12], [13], [14] identify PAK1 as a convergence point in transformation induced by small GTPases. Activated PAK1 then phosphorylates Raf and thus enhances Ras/Raf/MAPK signalling [6], [15], [16]. We have previously reported that PAK1 also interacts with β-catenin and promotes β-catenin/TCF4 activation in gastric epithelial cells [17]. Phosphorylation of β-catenin at Ser⁶⁷⁵ by PAK1 increases the stability and transcriptional activity of β-catenin in CRC cells [18]. Thus PAK1, acting downstream of small GTPases, may coordinate Ras/Raf/MAPK and Wnt/β-catenin signalling in CRC initiation and progression.

Alterations in PAK1 expression and activation have been detected in human tumors. In CRC, PAK1 expression was found to increase with progression through the adenoma to carcinoma sequence, with the most dramatic increases in invasive and metastatic CRC [19]. Higher expression and activity of PAK1 were also associated with a lower survival rate in CRC patients [20]. Recently we have shown that PAK1 is required for CRC cell proliferation, migration/invasion and survival in vitro [21]. Here we have further investigated the effect of PAK1 on CRC growth and metastasis in vivo, and the mechanisms involved. In particular the connections between PAK1 and oncogenic Ras and Wnt/β-catenin signalling were examined more closely since, as described above, both pathways play important parts in CRC development. Short hairpin RNA (shRNA) was used to knockdown PAK1 in two CRC cell lines, and the resultant changes in cell signalling pathways and in the growth and metastasis of CRC in vivo were observed.