Bisphenol A activates EGFR and ERK promoting proliferation, tumor spheroid formation and resistance to EGFR pathway inhibition in estrogen receptor-negative inflammatory breast cancer cells

Abstract

Emerging evidence from epidemiological studies suggests a link between environmental chemical exposure and progression of aggressive breast cancer subtypes. Of all clinically distinct types of breast cancers, the most lethal phenotypic variant is inflammatory breast cancer (IBC). Overexpression of epidermal growth factor receptors (EGFR/HER2) along with estrogen receptor (ER) negativity is common in IBC tumor cells, which instead of a solid mass present as rapidly proliferating diffuse tumor cell clusters. Our previous studies have demonstrated a role of an adaptive response of increased antioxidants in acquired resistance to EGFR-targeting drugs in IBC. Environmental chemicals are known to induce oxidative stress resulting in perturbations in signal transduction pathways. It is therefore of interest to identify chemicals that can potentiate EGFR mitogenic effects in IBC. Herein, we assessed in ER-negative IBC cells a subset of chemicals from the EPA ToxCast set for their effect on EGFR activation and in multiple cancer phenotypic assays. We demonstrated that endocrine-disrupting chemicals such as bisphenol A (BPA) and 2,2-bis(p-hydroxyphenyl)-1,1,1-trichloroethane can increase EGFR/ERK signaling. BPA also caused a corresponding increase in expression of SOD1 and anti-apoptotic Bcl-2, key markers of antioxidant and anti-apoptotic processes. BPA potentiated clonogenic growth and tumor spheroid formation in vitro, reflecting IBC-specific pathological characteristics. Furthermore, we identified that BPA was able to attenuate the inhibitory effect of an EGFR targeted drug in a longer-term anchorage-independent growth assay. These findings provide a potential mechanistic basis for environmental chemicals such as BPA in potentiating a hyperproliferative and death-resistant phenotype in cancer cells by activating mitogenic pathways to which the tumor cells are addicted for survival.

Figure 1.

Endocrine-disrupting chemicals increase mitogenic signaling. Western immunoblot analysis of SUM149 cells treated for 30 min with indicated doses of BPA, HPTE, methoxychlor, esfenvalerate, chlorothalonil and niclosamide. Blots probed for pEGFR, EGFR, pERK and ERK expression, with GAPDH used as a loading control. Numbers represent fold change of phospho to total protein (EGFR or ERK) normalized to untreated.

Figure 2.

Select ToxCast environmental compounds exhibit differential growth and health effects in SUM149 cells. SUM149 cells were treated for 24 h with the indicated ToxCast compounds and bars represent mean ± SD normalized to untreated for three independent assays carried out in triplicate for (A) % nuclear count, (B) % MTT proliferation and (C) % YOYO-1 cell viability. Statistical significance was calculated by the Student’s t-test. *P < 0.05, **P < 0.01, ^# P < 0.005. Inset: representative high-content imaging fields (×10; overlay of Hoechst = blue, YOYO-1 = green) for vehicle, cell kill control (1 μM staurosporine), 1 nM BPA, esfenvalerate, HPTE and methoxychlor, and 5 μM niclosamide and chlorothalonil.

Figure 3.

EGFR-ERK signaling is necessary for BPA’s proliferative effect. (A) SUM149 cells treated for 24 h with either vehicle, BPA (40 nM), EGFR inhibitor GW583340 (2.5 μM), or EGFRi + BPA co-treatment and assessed for proliferation by MTT. Inset: western immunoblot analysis of SUM149 cells treated for 24 h with BPA (40 nM), EGFRi (2.5 μM) and probed for pEGFR and EGFR, with GAPDH used as a loading control. (B) rSUM149 were treated for 24 h with BPA at the indicated concentrations and assessed for proliferation by MTT. Inset: western immunoblot analysis of rSUM149 cells treated for 24 h with BPA (10 μM) and probed for pEGFR and EGFR, with GAPDH used as a loading control. (C) SUM190 cells were treated for 24 h with BPA at the indicated concentrations and assessed for proliferation by MTT. Inset: western immunoblot analysis of SUM190 cells treated for 24 h with EGFR/HER2i (GW583340; 7.5 μM), BPA (1 nM) or the combination and probed for pHER2 and HER2, with GAPDH used as a loading control. For each cell line study, proliferation data represents mean ± SD percent normalized to untreated control for a minimum of three independent assays, each carried out in triplicate. Statistical significance was calculated by the Student’s t-test. *P < 0.05, ^# P < 0.005, ns = not significant. For all western blots, numbers represent fold change of phospho proteins to total proteins and total proteins to GAPDH, normalized to untreated.

Figure 4.

BPA promotes clonogenic and tumor spheroid growth and attenuates EGFR inhibitory effect on anchorage-independent growth. (A) Clonogenic growth: SUM149 cells were treated for 24 h with vehicle (V), 5 μM niclosamide (N), 5 μM chlorothalonil (C), 1 μM methoxychlor (M), 100 nM esfenvalerate (Es), 1 nM HPTE (H) and 10 μM BPA (B), followed by 5 days of additional growth. Data represent mean ± SD percentage of colonies relative to vehicle/untreated for a minimum of three independent assays. Right: representative photomicrographs (×1) of vehicle and BPA (10 μM) treated SUM149 colonies. (B) Tumor spheroid: SUM149 cells were seeded in spheroid promoting media and treated with either vehicle control (0.1% dimethyl sulfoxide), low-dose (1 nM) or high-dose BPA (10 μM) or EGFR inhibitor GW583340 (7.5 μM) at the time of seeding. Cells were grown for 5–6 days and mature spheroids (>1000 pixels) counted and assessed using ImageJ. Spheroid count data represent mean ± SD percent normalized to untreated control for at least two independent experiments comprising a minimum of six replicate wells. Right: representative photomicrographs (×5) are shown for each treatment. (C) SUM149 cells were treated for 24 h with BPA (40 nM), EGFRi (2.5 μM) or BPA + EGFRi co-treatment, followed by an additional 2 weeks of anchorage independent growth. Bars represent mean ± SD percentage of anchorage-independent colony number formed relative to untreated for at least two independent experiments comprising a minimum of six replicate wells. Right: representative photomicrographs (×10) of anchorage-independent colonies are shown for each treatment.

Figure 5.

BPA increases downstream survival proteins and GPER while attenuating the growth inhibitory effects of EGFR inhibition. (A) SUM149 cells were treated for 30 min with BPA at the indicated concentrations and western blotting carried out for Bcl-2, SOD1 and GPER expression with GAPDH used as a loading control. Numbers represent fold change of total protein relative to GAPDH normalized to untreated. (B) Schema: the effect of BPA in ER-negative, GPER-positive (29) SUM149 IBC cells. BPA activates EGFR and ERK signaling leading to an increased proliferative phenotype, increased alternate estrogen receptor GPER levels and upregulation of survival and antioxidant proteins Bcl-2 and SOD1. Antagonism of EGFR activation by GW583340 (13,15,17), respectively, reverses the proliferative effect of BPA. However, BPA can overcome the growth inhibitory effect of GW583340 in a longer-term assay of cancer growth and progression. The numbers on the schema represent references as described above.