Cancer Letters

Cancer Letters

Volume 317, Issue 1, 1 April 2012, Pages 65-71
Cancer Letters

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

https://doi.org/10.1016/j.canlet.2011.11.014Get rights and content

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.

Section snippets

Cell culture and shRNA transfection

The human colon cancer cell lines DLD1, HCT116, HT29 and SW1222 were obtained from the ATCC, and cultured in Dulbecco’s Modified Eagle’s medium (DMEM) containing 5% fetal bovine serum (FBS). PAK1 knockdown (KD) clones of DLD1 and HCT116 cells were derived by transfection with plasmid DNAs encoding either shRNA sequences (SABioscience, Frederick, MD, USA) to silence the PAK1 gene specifically or a scrambled sequence as a negative control (NC) using Lipofectin Reagent (Invitrogen, Melbourne,

Results

PAK1 associated with β-catenin in four human CRC cell lines (Fig. 1A), as previously observed in the murine gastric epithelial cell line IMGE [17].

Discussion

The results presented here reveal that PAK1 is crucially important for growth and metastasis of CRC cell lines in vivo. The importance of PAK1 in CRC development had previously been suspected from the observation that PAK1 expression in CRC increased with progression through the adenoma to carcinoma sequence, with the most dramatic increases in invasive and metastatic CRC [19], and that higher expression and activation of PAK1 were associated with poorer survival of CRC patients [20]. We now

Acknowledgments

We thank Ms. Linh Nguyen for her assistance with the mouse liver metastasis model, and Dr. Suzana Kovac and Dr. Kathryn Marshall for helpful discussions. This work was supported by National Health and Medical Research Council of Australia Grant 508908 to HH.

References (31)

  • P.J. Morin et al.

    Activation of beta-catenin-Tcf signaling in colon cancer by mutations in beta-catenin or APC

    Science

    (1997)
  • S.T. Yuen et al.

    Similarity of the phenotypic patterns associated with BRAF and KRAS mutations in colorectal neoplasia

    Cancer Res.

    (2002)
  • B. Vogelstein et al.

    Genetic alterations during colorectal-tumor development

    New Engl. J. Med.

    (1988)
  • H.J. Andreyev, A.R. Norman, D. Cunningham, J. Oates, B.R. Dix, B.J. Iacopetta, J. Young, T. Walsh, R. Ward, N. Hawkins,...
  • B. Zhang et al.

    Beta-Catenin and ras oncogenes detect most human colorectal cancer

    Clin. Cancer Res.

    (2003)
  • Cited by (48)

    • Role of the Wnt and GTPase pathways in breast cancer tumorigenesis and treatment

      2022, Cytokine and Growth Factor Reviews
      Citation Excerpt :

      PAK1 interacts with and phosphorylates β-catenin on S663 and S675 in BC and CRC cell lines [150,151], promoting the nuclear translocation and transcriptional upregulation of T-cell factor-responsive genes, such as myc and cyclin D1, major drivers of cell cycle progression. PAK1 binds to β-catenin in CRC cells, and PAK1 knockdown inhibited β-catenin expression and β-catenin/TCF4 transcriptional activity, according to recent findings [152]. PAK1 is involved in cross-talk between the Ras effector pathway and the Wnt signaling pathway, both of which are crucial in carcinogenesis, and could be targets in cancer treatment [148].

    • Interplay of Wnt β-catenin pathway and miRNAs in HBV pathogenesis leading to HCC

      2019, Clinics and Research in Hepatology and Gastroenterology
      Citation Excerpt :

      Paks have been engaged in activation of components of cell progression pathways such as Wnt, Akt and Erk signaling pathways [192]. Recently, interaction of Pak with Wnt/β-catenin has been unveiled in numerous studies [193–195]. Pak1 has been found in β-catenin phosphorylation at S-663 and S-675 sites, and resulted in promotion and its re-localization to nucleus to up-regulate Cyclin D1 and MYC expression.

    View all citing articles on Scopus
    View full text