Ganoderma lucidum Combined with the EGFR Tyrosine Kinase Inhibitor, Erlotinib Synergize to Reduce Inflammatory Breast Cancer Progression

Abstract

The high incidence of resistance to Tyrosine Kinase Inhibitors (TKIs) targeted against EGFR and downstream pathways has increased the necessity to identify agents that may be combined with these therapies to provide a sustained response for breast cancer patients. Here, we investigate the therapeutic potential of Ganoderma lucidum extract (GLE) in breast cancer, focusing on the regulation of the EGFR signaling cascade when treated with the EGFR TKI, Erlotinib. SUM-149, or intrinsic Erlotinib resistant MDA-MB-231 cells, and a successfully developed Erlotinib resistant cell line, rSUM-149 were treated with increasing concentrations of Erlotinib, GLE, or their combination (Erlotinib/GLE) for 72h. Treatment effects were tested on cell viability, cell proliferation, cell migration and invasion. To determine tumor progression, severe combined immunodeficient mice were injected with SUM-149 cells and then treated with Erlotinib/GLE or Erlotinib for 13 weeks. We assessed the protein expression of ERK1/2 and AKT in in vitro and in vivo models. Our results show that GLE synergizes with Erlotinib to sensitize SUM-149 cells to drug treatment, and overcomes intrinsic and developed Erlotinib resistance. Also, Erlotinib/GLE decreases SUM-149 cell viability, proliferation, migration and invasion. GLE increases Erlotinib sensitivity by inactivating AKT and ERK signaling pathways in our models. We conclude that a combinatorial therapeutic approach may be the best way to increase prognosis in breast cancer patients with EGFR overexpressing tumors.

Keywords: EGFR; Erlotinib; Ganoderma lucidum; drug resistance.; synergy.

Figure 1

Effect of Erl/GLE in EGFR-overexpressing cells. A, B. SUM-102 and SUM-149 cells were treated with GLE for 72h. Cell viability was calculated as in materials and methods. IC₅₀ was obtained from dose response curve fittings using non-linear regression. C. SUM-149 cells were treated with GLE for 72h, to measure BrdU incorporation. Significance against vehicle (*) (P≤0.05). SUM-149 cells were treated with Erlotinib, GLE or Erl/GLE for 72h. D, E. Cell viability and CIs were calculated. Significance against Erlotinib (*) or GLE (ᶲ) (P≤0.05). Columns represent means ± SEM. CIs were calculated based on the IC₅₀ at a constant Erl:GLE ratio (1:1000) and at NCR (Erlotinib dilutions+0.05 mg/mL-GLE). CIs and fraction affected (Fa) calculated from the Erlotinib (E) and GLE (G) combinations were obtained using CompuSyn®. Fa-CI plot shows the interaction between drugs in function of Fa (original software output). CI=1 (dashed line), CIs<1 (synergism). Experiments were repeated at least three times.

Figure 2

Effect of Erl/GLE in cell motility and cell aggregation. Quiescent SUM-149 cells were seeded on top chambers and treated with vehicle, 0.1 µM-Erlotinib, 0.05 mg/mL-GLE or Erl/GLE for 72h. A, B. Cells migrated or invaded towards 10%FBS medium. Cells were fixed and stained, micrographs were captured, and cells quantified (ImageJ). C. SUM-149 cells were seeded on MatTek dishes, labeled and overlaid with Matrigel. Next day, cells were treated as above. Micrographs were obtained with a 20x objective magnification at the same location. These represent the average of 10 photos taken by treatment. Scale bars = 100µm. Columns represent means ± SEM. Experiments were repeated at least three times. Significance against: vehicle (*), Erlotinib (ᶲ) and GLE (ᵟ). (P≤0.05).

Figure 3

Effect of Erl/GLE in BC cells with acquired and intrinsic Erlotinib resistance. A, B. rSUM-149 cells were treated with Erl, GLE or Erl/GLE for 72h and cell viability and CIs were calculated based on the IC₅₀ of each at a constant Erl:GLE (1:24) and at NCR (Erl+0.2 mg/mL-GLE). C. rSUM-149 cells were seeded in 3D (see Fig.2) and treated with vehicle, 2.0 µM-Erlotinib, 0.2 mg/mL-GLE or Erl/GLE for 72h. Micrographs were obtained with a 20x objective magnification at the same location. These represent the average of 10 photos taken by treatment. Scale bars = 100µm. D, E. MDA-MB-231 cells were treated with Erl, GLE or Erl/GLE for 72h and cell viability and CIs were calculated based on the IC₅₀ of each at a constant Erl:GLE (1:47) and at NCR (Erlotinib+0.2 mg/mL-GLE). Columns represent means ± SEM. Experiments were repeated at least three times. Significance against: Erlotinib (*) or GLE (ᶲ) (P≤0.05).

Figure 4

GLE downregulates AKT and ERK activity. A. SUM-149 cells were treated with vehicle, 0.1 and 0.5 µM-Erlotinib, 0.05 and 0.2 mg/mL-GLE or 0.1 µM-Erlotinib+0.05 mg/mL-GLE for 72h. B. Densitometric analysis of immunoblotting bands. C. SUM-149 cells were treated with vehicle, 0.1 or 0.2 mg/mL GLE for 72h. D. Densitometric analysis of immunoblotting bands. E. rSUM-149 and MDA-MB-231 cells were treated with vehicle, 2.0 μM-Erlotinib and 0.2 mg/mL-GLE for 72h. F, G. Densitometric analysis of immunoblotting bands. Immunoblots with the primary antibodies listed on the left side of the panel, β-actin is loading control. Densitometric analyses were done using Image J software. Data is expressed as phospho/total protein ratio. Columns represent means ± SEM. Experiments were repeated at least three times.

Figure 5

In vivo Erl/GLE effect. 1x10⁶ SUM-149 cells mixed with Matrigel (1:1) were injected into the mammary fat pad of SCID mice. Mice were gavaged daily with vehicles (open triangles and open diamonds) or experimental groups (closed circles and closed squares for 13wks. A. Weekly mouse weight. B. Weekly tumor volume. C. Average tumor volume. D. Final tumor weight. E, F. Immunoblot with the primary antibodies listed on the left side of the panel, β-actin is loading control. Densitometric analyses were done using Image J software. Data is expressed as phospho/total protein ratio. Each lane represents a different animal. Estimated marginal means ± SEM. Significance against: (*) vehicle Erl+GLE, (ᵟ) vehicle Erlotinib. P≤0.05.