Cancer Letters

Cancer Letters

Volume 340, Issue 1, 28 October 2013, Pages 22-29
Cancer Letters

P21-activated kinase 1 promotes colorectal cancer survival by up-regulation of hypoxia-inducible factor-1α

https://doi.org/10.1016/j.canlet.2013.06.024Get rights and content

Highlights

  • Up-regulation of PAK1 correlates with up-regulation of HIF-1α and β-catenin in CRC.

  • Activation of PAK1 correlates with increased expression of HIF-1α and β-catenin in CRC.

  • PAK1 stimulates CRC survival by up-regulation of HIF-1α.

Abstract

P21 activated kinase 1 (PAK1) enhances colorectal cancer (CRC) progression by stimulating Wnt/β-catenin and Ras oncogene, which promote CRC survival via stimulation of hypoxia-inducible factor 1α (HIF-1α). The aim of this study was to assess the mechanism involved in the stimulation by PAK1 of CRC survival. PAK1 promoted CRC cell survival by up-regulation of HIF-1α. PAK1 was over-expressed and hyper-activated in tumors of ApcΔ14/+ mice, which was correlated with over-expression of HIF-1α and β-catenin. Inhibition of PAK1 decreased tumor growth and the expression of HIF-1α and β-catenin in tumors of ApcΔ14/+ mice, and suppressed xenograft tumor survival in SCID mice. These findings indicate that PAK1 stimulates CRC survival by up-regulation of HIF-1α.

Introduction

Mutations of Ras and components of the Wnt/β-catenin pathway occur in 50% and 90%, respectively, of human colorectal cancers (CRC) [1], [2], [3]. Mutations of the adenomatous polyposis coli (Apc) gene, a component of the Wnt/β-catenin pathway, synergise with oncogenic Ras to enhance intestinal tumor formation and progression by stimulating the translocation of β-catenin to the nucleus [4], [5]. Ras activates the P21-activated kinase 1 (PAK1), which is over-expressed in human CRCs [6]. The expression of PAK1 increases with progression through the adenoma to carcinoma sequence, with the most dramatic increases in invasive and metastatic CRC [6]. We have reported previously that PAK1 knockdown inhibits growth, survival and migration of CRC cell lines by inactivation of ERK and AKT, the downstream targets of Ras [7], and abrogates growth and metastasis of CRC cell lines in xenograft and liver metastasis models in mice [8]. PAK1 associates with and phosphorylates β-catenin in CRC cells, and PAK1 knockdown inhibits β-catenin expression and suppresses β-catenin/TCF4 transcriptional activity [8], [9]. Together these findings indicate that PAK1 play a key role in mediating the cross-talk between Ras and Wnt/β-catenin signaling in CRC progression (for review see [10]).

Hypoxia-inducible factor 1 (HIF-1) regulates oxygen delivery (via angiogenesis) and metabolic adaptation to hypoxia (via glycolysis). HIF-1 consists of a constitutively expressed HIF-1β subunit and an oxygen- and growth-factor-regulated HIF-1α subunit. Intratumoral hypoxia induces and stabilizes the expression of HIF-1α, which in turn transactivates the gene products that mediate tumor angiogenesis and glycolytic metabolism [11]. Overexpression of HIF-1α has been found in primary and metastatic human cancers and the level of expression correlates with tumor angiogenesis and patient mortality [11]. The Wnt/β-catenin signaling pathway is also activated in response to the hypoxia associated with tumor expansion [12]. Under hypoxia, β-catenin switches from binding with TCF4 and promoting cell proliferation to binding with HIF-1α and enhancing HIF-1-mediated transcription, thereby promoting cell survival and adaptation to hypoxia [13]. In addition to hypoxia, HIF-1α can also be regulated by oncogenic pathways. For example Ras mutants have been shown to stimulate HIF-1α in CRC cells [14]. These findings suggest that HIF-1α plays an important role in the stimulation of CRC survival that results from activation of Ras and the Wnt/β-catenin pathway.

As mentioned above, PAK1 acts as a convergence point in CRC progression, by mediating the signals from Ras and the Wnt/β-catenin pathway [10]. In turn, activation of both Ras and the Wnt/β-catenin pathway stimulates CRC survival through regulation of HIF-1α. However, it is not known whether PAK1 promotes CRC survival by stimulation of HIF-1α. In order to test this hypothesis, the effect of PAK1 on HIF-1α expression, VEGF secretion and CRC cell survival in vitro and in vivo was assessed. The in vitro PAK1 expression was either decreased by transfection with plasmids encoding shRNAs or increased by over-expression of constitutively active PAK1. HIF-1α and β-catenin expression were measured by Western blotting and immunohistochemistry, VEGF secretion by ELISA, survival by cell counting in CRC cell lines. The in vivo studies used APCΔ14/+ C57BL/6 mice and human CRC cell xenografts in SCID mice. The in vivo PAK1 expression was reduced by treatment with siRNAs. The number and size of tumors were measured and the characteristics of the tumors determined by haematoxylin and eosin staining and by immunohistochemistry for PAK1, pPAK1, β-catenin and HIF-1α.

Section snippets

Cell culture and transfection

The human colorectal cancer (CRC) cell lines DLD1, HCT116, HT29 and SW480 were obtained from the ATCC and cultured in Dulbecco’s Modified Eagle’s medium (DMEM) containing 5% fetal bovine serum (FBS). The derivation of PAK1 knockdown (KD) clones of DLD1 cells by transfection with plasmid DNAs encoding either shRNA sequences (SABioscience, Frederick, MD, USA) to silence the PAK1 gene specifically or with a scrambled sequence as a negative control (NC) using Lipofectin Reagent (Invitrogen,

PAK1 knockdown inhibited HIF-1α expression and CRC cell survival

Both negative control (NC) and PAK1 knockdown (KD) DLD1 cells were cultured under normoxia or hypoxia (1% O2) for 48 h. The expressions of HIF-1α, total PAK1 and active phosphorylated PAK1 (pPAK1) were determined by Western blots, and VEGF secretion was measured by ELISA. The induction of HIF-1α expression by hypoxia was significantly lower in PAK1 KD cells than in NC cells (Fig. 1A). The reduced HIF-1α expression in PAK1 KD cells was associated with lower concentrations of total PAK1 and of

Discussion

In current study, we have demonstrated for the first time that PAK1 plays a role in the regulation of HIF-1α in CRC progression. In CRC cells in vitro, knocking down PAK1 inhibited HIF-1α expression and VEGF secretion in response to hypoxia. Conversely, constitutively active PAK1 stimulated HIF-1α expression and VEGF secretion. Likewise HIF-1α expression induced by CoCl2, a stimulus that mimics hypoxia, was inhibited in PAK1 knockdown cells, and the inhibition was associated with reduced cell

Conflict of Interest

The authors of this paper have no conflict of interest to declare.

Acknowledgements

This work was supported by Australian National Health of Medical Research Council (NHMRC) Grants 508908 (HH), 104183 (GB &AS), and 1020983 (GB), and by funding from the Austin Hospital Medical Research Foundation.

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