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. 2013 Mar 28:4:35.
doi: 10.3389/fphar.2013.00035. eCollection 2013.

Association of the epithelial-to-mesenchymal transition phenotype with responsiveness to the p21-activated kinase inhibitor, PF-3758309, in colon cancer models

Todd M Pitts  1 , Gillian N Kulikowski, Aik-Choon Tan, Brion W Murray, John J Arcaroli, John J Tentler, Anna Spreafico, Heather M Selby, Maria I Kachaeva, Kelly L McPhillips, Blair C Britt, Erica L Bradshaw-Pierce, Wells A Messersmith, Marileila Varella-Garcia, S Gail Eckhardt
Affiliations

Association of the epithelial-to-mesenchymal transition phenotype with responsiveness to the p21-activated kinase inhibitor, PF-3758309, in colon cancer models

Todd M Pitts et al. Front Pharmacol. .

Abstract

The p21-activated kinase (PAK) family of serine/threonine kinases, which are overexpressed in several cancer types, are critical mediators of cell survival, motility, mitosis, transcription, and translation. In the study presented here, we utilized a panel of colorectal cancer (CRC) cell lines to identify potential biomarkers of sensitivity or resistance that may be used to individualize therapy to the PAK inhibitor PF-03758309. We observed a wide range of proliferative responses in the CRC cell lines exposed to PF-03758309, this response was recapitulated in other phenotypic assays such as anchorage-independent growth, three-dimensional (3D) tumor spheroid formation, and migration. Interestingly, we observed that cells most sensitive to PF-03758309 exhibited up-regulation of genes associated with a mesenchymal phenotype (CALD1, VIM, ZEB1) and cells more resistant had an up-regulation of genes associated with an epithelial phenotype (CLDN2, CDH1, CLDN3, CDH17) allowing us to derive an epithelial-to-mesenchymal transition (EMT) gene signature for this agent. We assessed the functional role of EMT-associated genes in mediating responsiveness to PF-3758309, by targeting known genes and transcriptional regulators of EMT. We observed that suppression of genes associated with the mesenchymal phenotype conferred resistance to PF-3758309, in vitro and in vivo. These results indicate that PAK inhibition is associated with a unique response phenotype in CRC and that further studies should be conducted to facilitate both patient selection and rational combination strategies with these agents.

Keywords: EMT; PAK; PF-3758309; colorectal cancer; intrinsic resistance.

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Figures

FIGURE 1
FIGURE 1
Effect of PF-3758309 on proliferation after 72 h in a panel of 27 colorectal cancer cell lines. Absolute IC50 values were calculated and graphed (green, sensitive; red, resistant). KRAS mutational status was obtained by direct sequencing and PAK4 gene amplification was obtained by fluorescence in situ hybridization.
FIGURE 2
FIGURE 2
Induction of apoptosis of CRC cell lines following 72-h treatment with PF-3758309 at concentration 1.0 μmol/L.
FIGURE 3
FIGURE 3
Proteome profiler analysis of two sensitive and two resistant CRC cell lines to PF-3758309. (A) Total protein lysates from control (top) and PF-3758309 treated (bottom) were analyzed by a human phospho-kinase array. (B) Semi-quantitative analysis of the spots was measured by densitometry and the mean (n = 2 spots) is presented in the graphs as a fold increase over control. (C) HCT116 and GEO were treated with 1 μmol/L of PF-3758309 for 8 h and levels of B-catenin were assessed.
FIGURE 4
FIGURE 4
Heat map showing differential expression of selected genes involved in EMT compared to sensitivity to PF-3758309.
FIGURE 5
FIGURE 5
Effects of PF-3758309 on anchorage-independent growth, spheroid formation and migration. (A) HCT116 and GEO were plated in a soft agar assay untreated or treated with 15 nmol/L of PF-3758309 for 14 days. (B) HCT116 and GEO were plated in 3D culture in matrigel to observe spheroid formation. (C) Quantification of spheroid formation of two sensitive and two resistant cell lines. The average spheroids from three separate experiments are displayed. Data is presented as mean ± SEM (t-test: *p <0.001). (D) RKO and SW480 were evaluated for their migratory phenotype in the presence of PF-3758309. Cells grown to 80% confluence were cleared with a scratch and migration was measured by the ability of cells to migrate into scratch after 48 h.
FIGURE 6
FIGURE 6
Antiproliferative effects of PF-3758309 in cell line xenografts. (A) Two sensitive and two resistant cell lines in vitro were implanted into athymic nude mice to confirm sensitivity in vivo. (B) Overall the EMT phenotype was recapitulated in vivo. HCT116_A and SW948_A are cell line gene expression. HCT116_X and SW948_X are gene expression from xenografted tumors.
FIGURE 7
FIGURE 7
Proliferation effects of knockdown of EMT-related genes. (A–E) Transfected cell lines with EMT-related genes knocked down exposed to varying concentrations of PF-3758309. Proliferation by SRB (at 0.03 nmol/L of PF-3758309). (F) RKO cell line transfected with an miRNA 200c expression vector. Proliferation by SRB (at 0.03 nmol/L of PF-3758309).
FIGURE 8
FIGURE 8
Three-dimensional (3D) culture of RKO vimentin knockdown. (A) The transfected cell lines were plated in 3D culture in matrigel to observe spheroid formation and treated with 30 nmol/L of PF-3758309 for 21 days. (B) The number of spheroids was counted from three independent experiments. Data presented as mean +/- SEM (t-test: *p <0.05, when compared to scramble control).
FIGURE 9
FIGURE 9
RT-PCR data of shRNA knockdown and expression. (A–D, F) RT-PCR data of CRC cell lines following transfection with shRNA to EMT-related genes. (E) RT-PCR of miRNA 200c following transfection with an expression vector containing miRNA 200c.
FIGURE 10
FIGURE 10
Antiproliferative effects of an miRNA 200c transfected RKO cell line in a xenograft model.
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