Varlitinib Downregulates HER/ERK Signaling and Induces Apoptosis in Triple Negative Breast Cancer Cells

Abstract

Triple-negative breast cancer (TNBC) is a complex disease associated with the aggressive phenotype and poor prognosis. TNBC harbors heterogeneous molecular subtypes with no approved specific targeted therapy. It has been reported that HER receptors are overexpressed in breast cancer including TNBC. In this study, we evaluated the efficacy of varlitinib, a reversible small molecule pan-HER inhibitor in TNBC. Our results showed that varlitinib reduced cell viability and induced cell apoptosis in most TNBC cell lines but not in MDA-MB-231 cells. MEK and ERK inhibition overcame resistance to varlitinib in MDA-MB-231 cells. Varlitinib inhibited HER signaling which led to inhibition of migration, invasion and mammosphere formation of TNBC cells as well as significant suppression of tumor growth of MDA-MB-468 xenograft mouse model. In summary, these results suggest that HER signaling plays an important role in TNBC progression and that pan-HER inhibition is potentially an effective treatment for TNBC patients.

Keywords: pan-HER inhibitor; triple-negative breast cancer.

Figure 1

Varlitinib exerts anti-proliferation ability and induces apoptosis in MDA-MB-453 and MDA-MB-468 cells but not MDA-MB-231 cells. (A) Whole-cell extracts were analyzed by western blot analysis using antibodies against anti-EGFR, anti-HER2, anti-HER3, anti-HER4 and anti-β-actin. (B) Cells were treated with various concentrations of varlitinib for 72 h for MTT assay. IC₅₀ values were determined and shown. (C) MDA-MB-231, MDA-MB-453 and MDA-MB-468 cells were treated with various concentrations of varlitinib for 72 h. The treated cells were analyzed using flow cytometry. The means ± SEM of three independent experiments performed in triplicate are shown. Student’s t-test, * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 2

Varlitinib inhibits MEK/ERK and Akt pathway in TNBC cells. (A,B) Whole-cell extracts of SK-BR-3 and MDA-MB-468 cells treated with indicated concentration of varlitinib for 48 h were prepared for western blot analysis using antibodies against anti-phospho-EGFR^Y1173, anti-phospho-EGFR^Y1068, anti-EGFR, anti-phospho-HER2^Y1221/1222, anti-HER2, anti-phospho-HER3^Y1289, anti-HER3, anti-phospho-HER4^Y1284, anti-HER4 and anti-β-actin. (C) The whole-cell extracts from MDA-MB-231, MDA-MB-453 and MDA-MB-468 cells treated with indicated concentration of varlitinib for 48 h were prepared for western blot analysis using antibodies against anti-phospho-EGFR, anti-EGFR, anti-phospho-MEK, anti-MEK, anti-phospho-ERK, anti-ERK, anti-phospho-Akt, anti-Akt, anti-PARP, anti-Caspase-3 and anti-β-actin.

Figure 3

ERK signaling mediates varlitinib-induced apoptosis in TNBC cells. (A) MDA-MB-231 cells were treated with MEK inhibitor GDC-0973, varlitinib or DMSO for 48 h. (B) MDA-MB-231 cells were treated with ERK inhibitor SCH772984, varlitinib or DMSO for 48 h. (C) MDA-MB-468 cells were transfected with ERK1-, ERK2-expressing or control plasmids (pCMV6) for 24 h, and the transfected cells were further treated with varlitinib or DMSO for 48 h. Apoptosis in the treated cells was analyzed by flow cytometry (left) and the whole-cell extracts of treated cells were analyzed by western blot analysis using antibodies against anti-phospho-ERK, anti-ERK, anti-PARP and anti-β-actin (right). The means ± SEM of three independent experiments performed in triplicate are shown. Student’s t-test, ** p < 0.01, *** p < 0.001 compared with cells treated with DMSO control. # p < 0.05 compared with cells transfected with pCMV6 vector and treated with varlitinib.

Figure 4

Varlitinib reduces the abilities of migration, invasion and mammosphere formation of TNBC cells. (A–C) MDA-MB-231 and MDA-MB-468 cells were treated with varlitinib or DMSO for subsequent migration (A), invasion (B) and mammosphere assays (C). The means ± SEM of three independent experiments performed in triplicate are shown (100× magnification times for A,B,C). Student’s t-test, * p < 0.05, ** p < 0.01.

Figure 5

Varlitinib inhibits tumor growth of TNBC. (A–E) MDA-MB-468-bearing mice were treated with varlitinib at 100 mg/kg BID or vehicle and the (A) xenografted tumors sizes, (B) weights and (C) the body weights of xenografted mice were measured. (D) The xenografted tumors were analyzed by western blotting using antibodies against anti-pEGFR, anti-EGFR, anti-pERK, anti-ERK, anti-PARP and anti-β-actin. (E) Immunohistochemical staining of pEGFR, EGFR, pERK, ERK and M30 in MDA-MB-468 xenografts (200× magnification times for E). Student’s t-test, * p < 0.05. Data are shown as mean ± SD.