HER2-mediated GLI2 stabilization promotes anoikis resistance and metastasis of breast cancer cells

Abstract

Breast cancer metastasis is a multi-step process and requires cells to overcome anoikis. Anoikis is defined as cell-death that occurs due to loss of cell adhesion. During the course of cancer progression, tumor cells acquire resistance to anoikis. However, mechanisms of anoikis resistance are not clear. Human epidermal growth receptor 2 (HER2) overexpressing breast tumors are known to be highly aggressive and metastatic. The mechanisms correlating HER2 with metastasis are poorly understood. We observed increased anoikis resistance in HER2 overexpressing breast cancer cells. In addition, we identified that HER2 overexpression was also associated with increased sonic hedgehog (SHH) signaling especially GLI2, and that inhibition of SHH pathway suppressed anoikis resistance. GSK3β is known to facilitate proteasome-mediated degradation of GLI2. Moreover, we observed that silencing of GLI2 resulted in reduced migration and invasion of HER2 overexpressing cells. Anoikis resistant HER2 overexpressing cells also showed increased rate and extent of metastasis in vivo, as compared to wild type anoikis resistant cells. Taken together, this study indicates a novel role of HER2/GSK3β/GLI2 axis in anoikis resistance and metastasis, and that GLI2 could be a potential target for anti-cancer therapies.

Keywords: Anoikis resistance; Breast cancer metastasis; GLI2; GSK3β; HER2; Sonic hedgehog (SHH).

Figure 1–. HER2 promotes anoikis resistance.

Cell survival was analyzed in cells after 48h of anchorage independent conditions, using anoikis assay. A) Comparison of anoikis resistance in MDA-MB-231 and HH cells and in HH cells after silencing HER2 using shRNA transfection. B) Analysis of Anoikis resistance in SKBR3 and HER2 shRNA transfected SKBR3 cells. C) Bright field images of sulforhodamine B stained MDA-MB-231, HH and HH with HER2shRNA cells, re-attached after 48h of anchorage independent conditions. Values were plotted as mean ± SD (n=3).

Figure 2–. Proliferation of anoikis resistant cells.

Proliferation rate of anoikis resistant MDA-MB-231, HH and SKBR3 cells was analyzed by comparing cell densities after 24h incubation. Proliferation rates of anoikis resistant MDA-MB-231, HH and HH with HER2shRNA cells. Proliferation rates of anoikis resistant SKBR3 cells and SKBR3 transfected with shRNA HER2. C) Western blot analysis for proliferation marker PCNA under adherent vs anchorage independent condition in MDA-MB-231 (i) cells and SKBR3 (ii) cells. D) Mammosphere formation assay in MDA-MB-231, HH and HH with HER2shRNA in agar gel for 30 days; Multiphoton image of mammosphere formed in HH cell culture, Green fluorescence indicates HER2 staining. Values were plotted as mean ± SD (n=3).

Figure 3–. SHH signaling promote anoikis resistance.

A) Western blot analysis for protein modulations in adherent vs anchorage independent MDA-MB-231 cells. B) Anoikis assay for MDA-MB-231 cells treated with SHH ligand and respective control cells. C) Anoikis assay for MDA-MB-231 cells transfected with empty vector or GLI2 overexpression plasmid. D) Anoikis assay (i) and western blot analysis (ii) for HH cells treated with cyclopamine, a pharmacological inhibitor of SHH signaling. E) Anoikis assay (i) and western blot analysis (ii) for HH cells transfected with scrambled shRNA or GLI2 shRNA to compare anoikis resistance. F) Anoikis assay in SKBR3 cells transfected with GLI2 shRNA. Values were plotted as mean ± SD (n=3).

Figure 4–. HER2 regulates SHH signaling.

A) Western blot analysis for comparison of constitutive protein expressions of SHH signaling in MDA-MB-231 vs HH cells (i) and in SKBR3 parent cells vs SKBR3 cells transfected with siRNA HER2 (ii). B) PCR assay for analysis of transcript levels of GLI2 and SHH in MDA-MB-231 and HH cells. C) Immunofluorescence for GLI2 (indicated by red color) in MDA-MB-231 and HH cells. D) Western blot analysis for GLI2 in cytosolic and nuclear extracts of MDA-MB-231 and HH cells. E) Western blot analysis for GLI2 in MDA-MB-231 and HH cells treated with cycloheximide at different time points (i). The GLI2 expression was quantitated and values from three independent experiments were plotted against time (ii). F) Western blot analysis for GLI2 and SHH in MDA-MB-231 cells treated with proteasome inhibitor MG-132 (i) and GLI2 overexpression plasmid transfection (ii). Values were plotted as mean ± SD (n=3).

Figure 5–. GSK3β mediates GLI2 stability in HH cells.

A) Constitutive expression of GSK3β in MDA-MB-231 and HH cells. B) Immunoprecipitation of ubiquitin in MDA-MB-231 and HH cells and immunoblotting for GLI2. C) Western blot analysis for GLI2 signaling in MDA-MB-231 cells after 24h treatment with GSK3β inhibitor, XIX, IM-12. D) Effect of HER2 silencing on GLI2 expression in HH cells. HH cells were transfected with scrambled or GLI2 shRNA. The control and GLI2 silenced cells were plated and analyzed for cell migration using wound healing and cell invasion using Boyden’s chamber assay in HH cells (E&F) and in SKBR3 cells (G&H). Values were plotted as mean ± SD (n=3).

Figure 6–. Increased metastasis of HH cells in vivo and increased expression of HER2 and GLI2 in tumors from anoikis resistant MDA-MB-231 and HH cells.

About 0.5 × 10⁶ MDA-MB-231 or HH anoikis resistant cells were injected by tail vein route in athymic nude female mice (n=6). The mice were imaged periodically for bioluminescence after luciferin injections. A&B) Time dependent luminescence imaging data from athymic nude mice for MDA-MB-231 and HH cells. C) Luminescence imaging data from isolated lungs and livers of from MDA-MB-231 and HH injected mice at the end of experiment. D) Time dependent luminescence signal from mice injected with HH and HH with GLI2shRNA cells. E) Average luminescence of the lungs from HH and HH-GLI2 shRNA group, which were imaged ex vivo at the end of the experiment. Values were plotted as means ± SEM (n=6). The lungs from mice of both the groups were fixed in formalin and processed for microscopic evaluation of tumors. F) H&E staining for tumors from different mouse lung samples of MDA-MB-231 and HH injected mice. G) immunohistochemical staining for HER2 and GLI2 in tumors from MDA-MB-231 and HH injected mice lungs. The inset in all panel shows enlarged view of tumor cells. Values were plotted as mean ± SEM (n=6).

Figure 7–

Schematic molecular mechanism of SHH pathway regulation by HER2 in breast cancer cells