Transmembrane TNF-alpha promotes chemoresistance in breast cancer cells

Abstract

Chemoresistance remains a major obstacle to successful treatment of breast cancer. Although soluble tumor necrosis factor-α (sTNF-α) has been implicated in mediating drug-resistance in human cancers, whether transmembrane tumor necrosis factor-α (tmTNF-α) plays a role in chemoresistance remains unclear. Here we found that over 50% of studied patients expressed tmTNF-α at high levels in breast cancer tissues and tmTNF-α expression positively correlated with resistance to anthracycline chemotherapy. Alteration of tmTNF-α expression changed the sensitivity of primary human breast cancer cells and breast cancer cell lines to doxorubicin (DOX). Overexpression of N-terminal fragment (NTF) of tmTNF-α, a mutant form with intact intracellular domain of tmTNF-α to transmit reverse signals, induced DOX-resistance. Mechanistically, the tmTNF-α/NTF-ERK-GST-π axis and tmTNF-α/NTF-NF-κB-mediated anti-apoptotic functions were required for tmTNF-α-induced DOX-resistance. In a xenograft mouse model, the combination of tmTNF-α suppression with chemotherapy significantly enhanced the efficacy of DOX. Our data indicate that tmTNF-α mediates DOX-resistance through reverse signaling and targeting tmTNF-α may be beneficial for the treatment of DOX-resistant breast cancer.

Fig. 1

tmTNF-α promotes proliferation, clonogenicity, migration and invasion of MDA-MB-231. MDA-MB-231 breast cancer cells were stably transfected with control or tmTNF-α shRNA. a tmTNF-α expression determined by Western blot in parental or shRNA-transfected MDA-MB-231 cells. b Cell proliferation was detected after incubation for indicated time points by a CCK8 assay. c Quantification of DNA content by flow cytometry for analysis of cell cycle following propodium iodide staining. d Clonogenicity of parental or shRNA-transfected MDA-MB-231 cells was detected by colony formation assay after a 24-h culture. e Cell migration was evaluated using a wound-healing assay after 0 or 24-h culture. f Number of invasive cells cross through matrigel and membrane porous over 24 h. c–f Representative images on the left, and the quantitative data on the right are presented as mean ± SEM of three independent experiments. **p < 0.01, ***p < 0.001

Fig. 2

tmTNF-α is required for DOX-resistance of breast cancer. a Primary human breast cancer cells were isolated from patients with low (n = 5) or high (n = 15) expression of tmTNF-α and incubated with DOX for 72 h. IC50 values of DOX-mediated cytotoxicity was determined by ATP-TCA. b, c Primary breast cancer cells from three patients were transfected with control or tmTNF-α shRNA for 48 h. DOX was added in indicated concentrations and incubated for 72 h. b Dose dependent DOX-induced cytotoxicity determined by ATP-TCA. c IC50 values of DOX to primary cancer cells or those transfected with shRNA. d FACS analysis of tmTNF-α expression in parental or shRNA stably transfected MDA-MB-231 cells. e, f Cytotoxicity and DNA fragmentation of parental or shRNA-transfected MDA-MB-231 cells treated with 3 μM DOX for 24 h detected by MTT assay and ELISA, respectively. g Scheme of NTF-tmTNF-α-mediated reverse signaling without delivering forward signaling. h FACS analysis of NTF expression in parental, or empty vector or NTF-transfected MCF-7 cells. i and j Cytotoxicity and DNA fragmentation of parental or NTF-transfected MCF-7 cells treated with 3 μM DOX for 24 h. Data are represented as mean ± SEM of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 3

ERK-GST-π is involved in tmTNF-α-mediated DOX-resistance. a Representative IHC images of GST-π and BCRP expression in paratumor and breast cancer tumor sections. b, c Histogram showing co-expression rate of GST-π or BCRP with tmTNF-α in tumor tissues of 105 patients. d, e Immunoblotting analysis of phosphorylation levels of ERK1/2 in parental or shRNA-transfected MDA-MB-231 cells (d) and in parental or NTF-transfected MCF-7 cells (e) after treatment with or without 3 μM DOX for 24 h. f, g Immunoblotting analysis of GST-π expression in parental or shRNAs-transfected MDA-MB-231 cells and in parental or NTF-transfected MCF-7 cells treated with 10 μM BAY117082 (BAY), 10 μM PD98059 (PD) or 1 μM SCH772984 (SCH) for 24 h. h, i A 24-h DOX (3 μM)-induced apoptosis in parental or shRNA-transfected MDA-MB-231 cells and in parental or NTF-transfected MCF-7 cells pretreated with 10 μM PD98059 for 30 min using Annexin V/PI staining. Data are represented as mean ± SEM of three independent experiments. ***p < 0.001

Fig. 4

Knockdown of GST-π expression blocked tmTNF-α-mediated DOX-resistance. Parental or shRNAs-transfected MDA-MB-231 cells and parental or NTF-transfected MCF-7 cells were transfected with 50 μM control or GST-π siRNA for 48 h and then treated with DOX (3 μM) for another 24 h. a Immunoblotting analysis of GST-π expression in MDA-MB-231 cells. b, c DOX fluorescence intensity was determined by FACS. d, e Immunoblotting analysis of phosphorylation levels of ERK1/2. f, g The apoptosis was determined by PI/Annexin V staining. Representative cytogram on the right and quantitative data on the left. Data are represented as mean ± SEM of three independent experiments. ***p < 0.001

Fig. 5

NF-κB is required for tmTNF-α-mediated DOX-resistance. a Representative IHC images of phosphorylation of NF-κB p65 in paratumor and breast cancer tumors, and histogram showing co-expression rate of phosphorylated NF-κB p65 and tmTNF-α in tumors of 105 patients. (b-i) Parental or shRNA-transfected MDA-MB-231 cells and parental or NTF-transfected MCF-7 cells were treated with or without 3 μM DOX for 24 h. (b and d) Representative immunoblotting of levels of IκB-α, cIAP1, cIAP2, and XIAP in total protein and translocation of NF-κB p65 from cytoplasmic fraction to nuclear fraction. LamB1 or β-actin served as a nuclear protein or a cytoplasmic/total protein loading control. c, e NF-κB activity was detected by ELISA. f–i Real-time PCR analysis of Bcl-X_L (f and g) and BAX (h and i). j, k A 24 h-DOX (3 μM)-induced apoptosis was determined in parental or shRNA-transfected MDA-MB-231 cells (j) and in parental or NTF-transfected MCF-7 cells (k) pretreated with 10 μM BAY117082 (BAY) for 30 min using Annexin V/PI staining. Data are represented as mean ± SEM of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 6

Knockdown of tmTNF-α expression enhances the therapeutic efficacy of DOX in vivo. 1 × 10⁶ MDA-MB-231 cells transfected with control or tmTNF-α shRNA were injected into mammary fat pad of nude mice. When tumors reached 100 mm³, the mice were treated intraperitoneally with DOX (4 mg/kg) or PBS, once a week for three weeks (n = 6 each group). a Representative images of tmTNF-α expression in tumor tissue section detected by IHC. b Growth curves of tumors after tumor cell inoculation. c Tumor sizes at the end point (day 21) of experiments. d Inhibition rate of tumor growth. e Caspase 3 activity in tumor tissues at day 21. f Immunoblotting of levels of IκB-а, cIAP1, cIAP2, XIAP, BAX, and Bcl-X_L in total protein and translocation of NF-κB p65 from cytoplasmic fraction to nuclear fraction of tumor tissues. g Immunoblotting of levels of phosphorylation of ERK and GST-π in tumor tissues. h Kaplan–Meier survival curves of mice after tumor cell inoculation (n = 6). Data are represented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 7

tmTNF-α induces DOX-resistance of breast cancer cells via its reverse signaling. tmTNF-α can be overexpressed in breast cancer cells and tends to induce chemoresistance. tmTNF-α constitutively activates both ERK and NF-κB pathways through reverse signaling. tmTNF-α promotes GST-π expression via ERK pathway, leading to degradation and detoxification of DOX in tumor cells, and tmTNF-α-induced GST-π in turn further activates ERK pathway, forming a positive feedback to promote DOX-resistance. tmTNF-α can also upregulate antiapoptotic molecules via NF-κB pathway and downregulate proapoptotic molecule, resulting in survival and anti-apoptosis of tumor cells. Targeting tmTNF-α may sensitize tumor cells to chemotherapy