Cancer Res Treat.  2020 Jul;52(3):973-986. 10.4143/crt.2019.726.

RON and MET Co-overexpression Are Significant Pathological Characteristics of Poor Survival and Therapeutic Targets of Tyrosine Kinase Inhibitors in Triple-Negative Breast Cancer

• Weng TH
• Yao MY
• Xu XM
• Hu CY
• Yao SH
• Liu YZ
• Wu ZG
• Tang TM
• Fu PF
• Wang MH
• Yao HP

• KMID: 2504475
• DOI: http://doi.org/10.4143/crt.2019.726

Abstract

Purpose
Triple-negative breast cancer (TNBC) is highly malignant and has poor prognosis and a high mortality rate. The lack of effective therapy has spurred our investigation of new targets for treating this malignant cancer. Here, we identified RON (macrophage-stimulating 1 receptor) and MET (MET proto-oncogene, receptor tyrosine kinase) as a prognostic biomarker and therapeutic targets for potential TNBC treatment.
Materials and Methods
We analyzed RON and MET expression in 187 primary TNBC clinical samples with immunohistochemistry. We validated the targeted therapeutic effects of RON and MET in TNBC using three tyrosine kinase inhibitors (TKIs): BMS-777607, INCB28060, and tivantinib. The preclinical therapeutic efficacy of the TKIs was mainly estimated using a TNBC xenograft model.
Results
Patients with TNBC had widespread, abnormal expression of RON and MET. There was RON overexpression, MET overexpression, and RON and MET co-overexpression in 63 (33.7%), 63 (33.7%), and 43 cases (23.0%), respectively, which had poor prognosis and short survival. In vivo, the TKI targeting RON ant MET inhibited the activation of the downstream signaling molecules, inhibited TNBC cell migration and proliferation, and increased TNBC cell apoptosis; in the xenograft model, they significantly inhibited tumor growth and shrank tumor volumes. The TKI targeting RON and Met, such as BMS-777607 and tivantinib, yielded stronger anti-tumor effects than INCB28060.
Conclusion
RON and MET co-overexpression can be significant pathological characteristics in TNBC for poor prognosis. TKIs targeting RON and MET have stronger drug development potential for treating TNBC.

Keyword

Triple-negative breast cancer; RON receptor tyrosine kinase; MET receptor tyrosine kinase; Tyrosine kinase inhibitor; Targeting therapy

Figure

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  Fig. 1. RON and MET expression in primary triple-negative breast cancer (TNBC) samples. Semiquantitative immunohistochemical staining was performed on 187 primary TNBC samples. Representative images showing negative staining and different levels of RON immunoreactivity are shown. A combined score of 2-4 was deemed weakly positive, while a combined score ≥ 5.0 was considered high expression or overexpression. (A) TNBC samples using mouse anti-RON mAb Zt/f2 for RON staining. (B) TNBC samples using rabbit anti-MET mAb for MET staining.

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  Fig. 2. Kaplan-Meier plots with log-rank test of overall survival. Patients with RON high expression and MET high expression were considered to have high co-expression. +, high expression; –, low expression. Kaplan-Meier analysis showing that patients with triple-negative breast cancer with high RON (A), MET (B), and RON/MET co-expression (C, D) had shorter survival than patients with low expression (RON, p < 0.01; MET, p < 0.05; co-expression, p < 0.001).

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  Fig. 3. Cytotoxic and apoptosis effects of BMS-777607, INCB28060, and tivantinib on triple-negative breast cancer cell lines. HCC1806, HCC2185, SUM52PE, and MDA-MB-231 cells (8,000 cells per well in 96-well plates in triplicate) were treated with different concentrations of BMS-777607, INCB28060, or tivantinib for 96 hours. (A) Cell Counting Kit-8 measurement of cytotoxic effects after exposure to tyrosine kinase inhibitors (TKIs). (B) Caspase-Glo 3/7 assay measurement of apoptosis effects after exposure to TKIs.

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  Fig. 4. Tyrosine kinase inhibitors suppress MDA-MB-231 cell motility. MDA-MB-231 cells were serum starved overnight and scratched to generate artificial gaps. Cells were treated with different BMS-777607, INCB28060 or tivantinib in the serum-free medium. Cell motility was monitored and cells migrated into denuded area were scored. (A) Typical images of cell migration after tyrosine kinase inhibitor treatment (×50). Relative migration area as shown in the figure (%). (B) Open image area of cell migration. Data are from three independent experiments. ***The experimental group compared with the control group, p < 0.001.

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  Fig. 5. Expression of RON and MET signaling pathways in MDA-MB-231 cells. Western blot analysis of whole cell lysate following tyrosine kinase inhibitor treatment. MDA-MB-231 cells were serum starved overnight, pretreated with BMS-777607, INCB28060, or tivantinib for 30 or 60 minutes, and stimulated with macrophage-stimulating protein (MSP) and hepatocyte growth factor (HGF) for 15 minutes. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a loading control.

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  Fig. 6. RON and MET tyrosine kinase inhibitors inhibited triple-negative breast cancer xenograft growth in nude mice. (A) Average tumor volumes of HCC1806 and MDA-MB-231 xenografts. Cells were subcutaneously injected into the mouse left flank to establish xenograft models. All mice were observed, and the tumor volume were measured every 3 days. (B) Xenograft tumors obtained after the mice had been sacrificed.

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