The sonic hedgehog signaling pathway stimulates anaplastic thyroid cancer cell motility and invasiveness by activating Akt and c-Met

Abstract

The sonic hedgehog (Shh) pathway is highly activated in thyroid neoplasms and promotes thyroid cancer stem-like cell phenotype, but whether the Shh pathway regulates thyroid tumor cell motility and invasiveness remains unknown. Here, we report that the motility and invasiveness of two anaplastic thyroid tumor cell lines, KAT-18 and SW1736, were inhibited by two inhibitors of the Shh pathway (cyclopamine and GANT61). Consistently, the cell motility and invasiveness was decreased by Shh and Gli1 knockdown, and was increased by Gli1 overexpression in KAT-18 cells. Mechanistic studies revealed that Akt and c-Met phosphorylation was decreased by a Gli1 inhibitor and by Shh and Gli1 knockdown, but was increased by Gli1 overexpression. LY294002, a PI-3 kinase inhibitor, and a c-Met inhibitor inhibited the motility and invasiveness of Gli1-transfected KAT-18 cells more effectively than the vector-transfected cells. Knockdown of Snail, a transcription factor regulated by the Shh pathway, led to decreased cell motility and invasiveness in KAT-18 and SW1736 cells. However, key epithelial-to-mesenchymal transition (EMT) markers including E-cadherin and vimentin as well as Slug were not affected by cyclopamine and GANT61 in either SW1736 or WRO82, a well differentiated follicular thyroid carcinoma cell line. Our data suggest that the Shh pathway-stimulated thyroid tumor cell motility and invasiveness is largely mediated by AKT and c-Met activation with little involvement of EMT.

Keywords: PI-3 kinase; Snail; c-Met; cell motility and invasion; sonic hedgehog signaling pathway.

Figure 1. Inhibition of the Shh pathway leads to reduced cell motility and invasive potential

KAT-18 A. and SW1736 B. seeded in uncoated or Matrigel-coated Boyden chambers in duplicate were incubated for 24 hr in the presence of 0.5% DMSO, cyclopamine (5 μM) or GANT61 (10 μM). Cells migrating through the pored PET membrane were stained in Diff-Quik solutions. Data represent the mean ± SD of the numbers of the cells in five random fields (10X) in duplicate. The experiment was repeated at least twice with similar results. *p < 0.05; **p < 0.01.

Figure 2. Effect of the Shh pathway knockdown or Gli1 overexpression on cell motility and invasiveness

(A & B) KAT-18 cells stably transfected with an expression vector encoding a control, Shh or Gli1 miRNA A. or transfected with pcDNA3.1 or pcDNA/Gli1 B. were analyzed for Shh and Gli1 expression by Western blot with their specific antibodies. Actin was included as a loading control. The density of the bands was analyzed by using an NIH Image-J software and normalized by the arbitrary units of the density of actin. The results were the mean ± standard deviation from three independent experiments. C & D. Effect of the Shh pathway on cell motility and invasiveness. KAT-18 cells stably transfected with control miRNA, Shh-miRNA 658 or Gli-miRNA 1519 C. or KAT-18 cells transfected with pcDNA3.1 or pcDNA/Gli1 D. were seeded in uncoated or Matrigel-coated Boyden chambers and for their migratory and invasive potential after incubation for 24 hr. Data represent the mean ± SD of the numbers of the cells in five random fields (10X) in duplicate. Data represent the results of one of two experiments with similar results. *p < 0.05; **p < 0.01.

Figure 3. Effect of the Shh pathway on c-Met and AKT phosphorylation

KAT-18 A. and SW1736 B. cells were treated with 0.5% DMSO, cyclopamine (5 μM) or GANT61 (10 μM) for 72 hr. The cells were harvested and analyzed for AKT (S473) phosphorylation, c-Met tyrosine phosphorylation at Y1230/1234/1235m and Gli1 expression by Western blot with their specific antibodies. The density of the bands from three independent experiments was analyzed and plotted in a bar graph. *p < 0.05; **p < 0.01. C. KAT-18 cells stably transfected with encoding a control, Shh or Gli1 miRNA or transfected with pcDNA3.1 or pcDNA/Gli1 were analyzed for AKT and c-Met phosphorylation by Western blot with their specific antibodies. Actin was included as a loading control.

Figure 4. AKT and c-Met activation is required for the Shh pathway-induced cell motility and invasiveness

pcDNA3.1 and pcDNA/Gli1-transfected KAT-18 cells seeded in 6-well plates were treated with a c-Met inhibitor III A. or an AKT inhibitor LY294002 B. with indicated concentrations for 24 hr. Cells were harvested and analyzed for c-Met tyrosine phosphorylation and AKT S473 phosphorylation by Western blot. The density of the bands was quantified by using an Image-J software and normalized by the arbitrary units of the density of actin. The numbers beneath the blots are a representative of two independent experiments with similar results. C.-H. Effect of c-Met inhibitor III and LY294002 on cell migration and invasiveness. pcDNA3.1 (C & D) and pcDNA/Gli1-transfected (E & F) KAT-18 cells seeded in Boyden chambers were incubated in the presence of DMSO (0.5%), c-Met inhibitor III (5 μM) (C & E) or LY294002 (10 μM) (D & F) for 24 hr. The cells migrating through the pored PET membrane were stained using Diff-Quik solution. The data represent the mean ± SD of the numbers of the cells in five random fields (10X) in duplicate (G & H). Experiments were repeated at least twice with similar results. The experiment was repeated twice with similar results. *p < 0.05; **p < 0.01.

Figure 5. Snail knockdown inhibits cell motility and invasiveness

KAT-18 and SW1736 cells seeded in 35-mm dishes were transfected with a scrambled control siRNA or Snail siRNA (2.5 nmole each). After incubation for 48 hr, the cells were harvested and analyzed for Snail and actin expression by Western blot A. or for cell motility and invasiveness of KAT-18 B. and SW1736 cells C. in uncoated or Matrigel-coated Boyden chamber. Data represent the results of one of two independent experiments with similar results. *p < 0.05; **p < 0.01.

Figure 6. Effect of the Shh pathway on EMT

A. & B. Cell lysates of KAT-18, SW1736, WRO82, TPC1, and BCPAP cell lines were analyzed for the expression of E-cadherin, Snail, and actin A. or pAKT, AKT, pMet, Met, and actin B. by Western blot. KAT-18 cells stably transfected with an expression vector encoding a control, Shh or Gli1 miRNA C. or transfected with pcDNA3.1 or pcDNA/Gli1 C. were analyzed for E-cadherin expression. (D-F) The effect of the Shh pathway inhibitors on E-cadherin expression. KAT-18 cells D. were treated with 0.5% DMSO as a vehicle control, cyclopamine (5 μM) or GANT61 (10 μM) for 72 hr. The cells were harvested and analyzed for E-cadherin and actin expression by Western blot with their specific antibodies. SW1736 E. and WRO82 F. cells were incubated in the presence of vehicle (0.5% DMSO) or the indicated concentrations of cyclopamine or GANT61 for 72 hr. The cells were harvested and analyzed for the expression of several genes involved in EMT.

Figure 7. Mechanisms of the Shh pathway-induced cell motility and invasiveness

The binding of Shh to the 12-pass transmembrane receptor Ptch leads to the translocation and activation of Smo, a G protein-coupled receptor. Smo activates Gli1 through inhibiting SUFU, an inhibitors of Gli1. In addition, Smo activation also leads to the formation of the G_β and G_γ heterodimer, which activates the PI-3-kinase pathway. AKT activation leads to cytoskeleton remodeling and cell motility. Snail expression can be transcriptionally induced by Gli1. The Shh pathway may also contribute to increased invasiveness by converting cells to cancer stem-like cell phenotype and c-Met activation.