Locking Src/Abl Tyrosine Kinase Activities Regulate Cell Differentiation and Invasion of Human Cervical Cancer Cells Expressing E6/E7 Oncoproteins of High-Risk HPV

Abstract

In this study, we compared the effects of SKI-606 with Iressa, Src/Abl and EGF-R kinase inhibitors, respectively, on selected parameters in HeLa and SiHa cervical cancer cell lines, which express E6/E7 oncoproteins of high-risk HPV types 18 and 16, respectively. Our results show that SKI-606 and Iressa inhibit cell proliferation and provoke G(0)-G(1) cell cycle arrest and reduction of S and G(2)-M phase using 2 and 5 μM concentrations of these inhibitors. In contrast, SKI-606 induces differentiation to an epithelial phenotype "mesenchymal-epithelial transition"; thus SKI-606 causes a dramatic decrease in cell motility and invasion abilities of HeLa and SiHa cancer cells, in comparison to untreated cells and Iressa-treated cells in which these parameters are only slightly affected. These changes are accompanied by a regulation of the expression patterns of E-cadherin and catenins. The molecular pathway analysis of Src/Abl inhibitor revealed that SKI-606 blocks the phosphorylation of β-catenin and consequently converts its role from a transcriptional regulator to a cell-cell adhesion molecule. Our findings indicate that SKI-606 inhibits signaling pathways involved in regulating tumor cell migration and invasion genes via β-catenin alteration, suggesting that Src inhibitor, in comparison to EGF-R, is a promising therapeutic agent for human cervical cancer.

Figure 1

Effect of SKI-606 and Iressa on cell growth of HeLa human cervical cancer cells. We note that cell proliferation slightly relies on Src/Abl and EGF-R inhibitor concentrations at 2 and 5 μM each; in contrast, Src/Abl inhibitor, but not EGF-R, drastically inhibits cell proliferation of HeLa and SiHa cells (data not shown) at 10 μM. Bars, SDs of each point.

Figure 2

SKI-606 and Iressa (to a lesser extent) provoke G₀-G₁ cell cycle arrest and reduction of S and G₂-M phase of HeLa and SiHa cells. A, propidium iodide staining shows a significant increase (P < .0001 and P < .001) in the proportion of HeLa and SiHa cells, respectively, in the G₀-G₁ phase of the cell cycle following 48 hours of SKI-606 and Iressa treatments (5 μM). B, SKI-606, but not Iressa, treatment for 48 hours results in changes in key G₀-G₁ cell cycle phase regulators in HeLa cells.

Figure 3

SKI-606 and Iressa (to a lesser extent) induce morphological changes in HeLa and SiHa cell lines. Untreated (control) cells possess a fibroblast-like (mesenchymal) cell phenotype, whereas 48 hours treatment with SKI-606 (5 μM) significantly induces a mesenchymal-epithelial transition of these cells in comparison with Iressa-treated cells.

Figure 4

Effect of SKI-606 and Iressa on colony formation ability of HeLa cell line. HeLa cells are able to form large colonies in soft agar. In contrast, SKI-606 inhibits the colony formation ability of HeLa (P < .0001) and SiHa cells (data not shown) in soft agar, whereas, Iressa slightly reduce the colony formation following treatment with 5 μM as described in the Materials and Methods section. Magnification is 100x. We have obtained the same results using SiHa cell line.

Figure 5

Comparison of SKI-606 and Iressa activity against cell migration and invasion ability of the HeLa cell line. SKI-606 and, to a lesser extent, Iressa block cell migration at the same concentrations, as indicated in Materials and Methods, (arrows) within 48 hours in comparison with untreated (control), (P < .001) using a cell wounding assay. In parallel, SKI-606 dramatically inhibits cell invasion ability of HeLa and SiHa cells (data not shown) in comparison with Iressa-treated and untreated (control) cells (arrows indicate invasive cells), (P < .0001) using Boyden chambers. Experiments conducted on SiHa cells showed the same data regarding cell migration and invasion ability.

Figure 6

Immunofluorescence analysis of E-cadherin, α-, β-, and γ-catenin expression in HeLa-untreated (control) and SKI-606 as well as Iressa-treated cells. We note that SKI-606-treatment restores the expression patterns of E-cadherin and α-, β-, as well as γ-catenin, on cell surfaces and undercoat membrane, at 5 μM concentration, in HeLa cells when compared with Iressa-treated and untreated cells at the same concentrations.

Figure 7

Comparison between the influence of SKI-606 and Iressa activity on P-cadherin, fascin, Id-1, IGF-R1, and EGF-R expression as well as β-catenin phosphorylation in HeLa cells. (a) Western blot analysis of P-cadherin, fascin, Id-1, IGF-R1, and EGF-R expression in HeLa-untreated and treated cells. We found that SKI-606 decreases the expression of P-cadherin, fascin, Id-1 and IGF-R1 in HeLa cells in comparison with Iressa-treated (as indicated in the Materials and Methods) and untreated cells; in contrast, Iressa largely reduces the expression of EGF-R in comparison with SKI-606. (b) Tyrosine phosphorylation analysis of β-catenin in HeLa cells and SKI-606 as well as Iressa-treated cells. Src/Abl inhibitor (SKI-606) blocks the constitutive phosphorylation of Src and consequently β-catenin phosphorylation in HeLa cancer cells. Cells were grown for two days in both the absence (control) and presence of 2 and 5 μM of SKI-606 and 5 μM of Iressa. Cell lysates were immunoprecipitated with anti-β-catenin antibody and analyzed by immunoblotting with antiphosphotyrosine antibody as described in Section 2.