In recent years, tumor necrosis factor receptor 2 (TNFR2) has attracted increasing attention for its important roles in promoting proliferation, migration and angiogenesis in several types of cancer. However, its role in drug resistance remain unclear. In the present study, TNFR2 expression levels in MDA-MB-231 and MCF-7 breast cancer cells were demonstrated to be associated with Adriamycin (ADM) resistance. Silencing of TNFR2 in MCF-7 cells significantly inhibited ADM resistance, while overexpression of TNFR2 in MDA-MB-231 cells significantly enhanced ADM resistance. ADM treatment induced phosphorylation of the histone family 2A variant X (pH2AX), an established marker of DNA damage. Silencing of TNFR2 in MCF-7 cells further induced pH2AX levels but inhibited the expression of the DNA damage repair protein, poly(ADP-ribose) polymerase (PARP). By contract, overexpression of TNFR2 in MDA-MB-231 cells decreased pH2AX levels and enhanced PARP expression. Of note, treatment with the PARP inhibitor ABT888 significantly abrogated the effects of TNFR2 on pH2AX expression. On a molecular mechanism level, TNFR2 significantly affected the phosphorylation of AKT serine/threonine kinase 1 (AKT) in both cell lines, and treatment with the AKT inhibitor LY294002 effectively abrogated TNFR2-induced PARP expression. A drug resistance assay demonstrated that treatment with either LY294002 or ABT888 inhibited ADM resistance in breast cancer cells, and combination treatment with both LY294002 and ABT888 exhibited a significantly stronger inhibition effect on ADM resistance. The present results indicated that TNFR2 promoted ADM resistance in breast cancer cells by regulating the DNA damage repair protein PARP via the AKT signaling pathway.
Breast cancer is one of the most common malignant cancers in women worldwide (
Tumor necrosis factor receptor (TNFR) 2 is a member of the TNFR family, and it is important in tumor progression and prognosis, by regulating the malignant behavior of tumor cells via stimulating AKT serine/threonine kinase 1 (AKT) or nuclear factor (NF)-κB signaling pathways (
In the present study, the role of TNFR2 in drug resistance was explored from the perspective of its effect on the DNA repair mechanism. The results demonstrated that TNFR2 induced ADM resistance in breast cancer cells, by enhancing DNA damage repair via regulating the DNA repair protein, poly(ADP-ribose) polymerase (PARP). Furthermore, the AKT signaling pathway was demonstrated to be required for TNFR2-induced PARP expression.
Human breast cancer cell lines MCF-7 and MDA-MB-231 were purchased from the American Type Culture Collection (Manassas, VA, USA). Both cell lines were cultured in minimum essential medium (MEM) (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS) (Invitrogen; Thermo Fisher Scientific, Inc.).
Cells were plated in a 6-well plate at 4×105 cells per well. After 24 h, plasmid pReceiver-M77-TNFR2 (410 ng/µl) (EX-A0254-M77; GeneCopoeia, Inc., Rockville, MD, USA) and control plasmid (320 ng/µl) were transfected into MDA-MB-231 cells to upregulate TNFR2 expression; plasmid psi-U6-GFP-TNFR2-sh (380 ng/µl) (RSH052309-CU6; GeneCopoeia, Inc.) and control plasmid (440 ng/µl) were transfected into MCF-7 cells to downregulate TNFR2 expression. All procedures were performed using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol and empty vectors were used as a control. After 48 h, cells were harvested and transfection efficiency was determined using western blot analysis. Sequence silencing TNFR2, 5′-TTGACACCCTACAAGCCAGAA-3′; sequence as control plasmid, 5′-GTTCTGCGAACGTGTCACGT-3′.
Cells were washed twice in PBS and lysed in radioimmunoprecipitation assay buffer (Beyotime Institute of Biotechnology, Haimen, China) containing 1% protease inhibitor. Protein concentration was measured by spectrophotometry (ND-1000; Nano Drop Technologies; Thermo Fisher Scientific, Inc., Waltham, MA, USA). Protein (200 µg) was separated by 10% SDS-PAGE and transferred to a polyvinylidene fluoride membrane. Following blocking in TBS/0.1% Tween-20 containing 5% non-fat dry milk for 1 h at room temperature, the membrane was incubated with primary antibodies (listed in
Cells were plated in 96-well plates in triplicate in MEM supplemented with 10% FBS at 8,000 cells per well. After 24 h, the medium was replaced with MEM containing 0.02, 0.08, 0.32, 1.28, 5.12, or 20.48 µmol/l ADM for 48 h and then MTT was dissolved in dimethyl sulfoxide (M1020-500T; Beijing Solarbio Science and Technology Co., Ltd., Beijing, China) and MTT assay was performed at 490 nm wavelength. The survival curves were constructed and the half maximal inhibitory concentration (IC50) was calculated. The experiment was repeated at least three times.
Data were expressed as mean ± standard deviation. SPSS 13.0 software (SPSS, Inc. Chicago, IL, USA) was used for statistical analysis. IC50 was calculated by regression analysis. Significance of differences between two groups was analyzed by Student two-tailed t-test. Significance of differences between multiple groups was analyzed by one-way analysis of variance followed by Student-Newman-Keuls test. P<0.05 was considered to indicate a statistically significant difference.
Firstly, the protein expression levels of TNFR2 were detected in the breast cancer cell lines MCF-7 and MDA-MB-231. As illustrated in
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To further study the potential molecular mechanism responsible for PARP expression, AKT and extracellular signal-regulated kinase (ERK) were examined as candidate signal targets. As illustrated in
Previous studies have reported two possible mechanisms responsible for the antitumor effects of ADM: Inhibition of DNA transcription and replication by intercalating between DNA base pairs, and induction of DNA double strand breakage by generating oxygen free radicals (
TNFR2, which differs from TNFR1 mainly due to the absence of death domain in its structure, promotes survival, proliferation, migration and invasion in multiple types of cancer. Tanimura
The mechanism of ADM resistance is complicated and TNFR2 effect on cell survival and proliferation may be partly responsible for this. In addition, integrity of DNA is crucial for cell survival (
MGMT and PARP are both important DNA repair proteases (
AKT and ERK are important signaling pathways for various cellular functions, including survival, proliferation, and migration in multiple types of tumors (
In conclusion, the present study demonstrated a role of TNFR2 in ADM resistance of breast cancer cells. This effect of TNFR2 was partly mediated by the induction of the DNA damage repair protease PARP via the AKT signaling pathway. The present results may enrich our understanding regarding the role of TNFR2 in breast cancer and in drug resistance and may provide novel therapy targets for breast cancer treatment.
ADM resistance assay in breast cancer cells. (A) Representative blots and (B) quantification of TNFR2 protein expression levels in MDA-MB-231 and MCF-7 cells, as assessed by western blotting. GAPDH was used as a loading control. (C) ADM resistance abilities of MDA-MB-231 and MCF-7 cells. (D) Quantification of IC50 values for MDA-MB-231 and MCF-7 cells following ADM treatment. (E) TNFR2 protein expression levels in MCF-7 cells treated with either control or TNFR2-specific shRNA (TNFR2-sh). (F) ADM resistance abilities and (G) IC50 values of control and TNFR2-sh MCF-7 cells. (H) TNFR2 protein expression levels in MDA-MB-231 cells treated with either control empty vector or a TNFR2-overexpressing vector. (I) ADM resistance abilities and (J) IC50 values of control and TNFR2-overexpressing MDA-MB-231 cells. *P<0.05. ADM, Adriamycin; TNFR2, tumor necrosis factor receptor 2; shRNA, short hairpin RNA; IC50, half maximal inhibitory concentration.
TNFR2 inhibits ADM-induced pH2AX expression. Protein expression levels of pH2AX and H2AX were assessed by western blotting in (A) control and TNFR2-knockdown (TNFR2-sh) MCF-7 cells, and in (B) control and TNFR2-overexpressing (TNFR2-vector) MDA-MB-231 cells, with or without ADM treatment. GAPDH was used as a loading control. *P<0.05. TNFR2, tumor necrosis factor receptor 2; ADM, Adriamycin; H2AX, histone family 2A variant X; p-, phosphorylated; sh, short hairpin RNA.
TNFR2 inhibits pH2AX expression through PARP. Protein expression levels of pH2AX and PARP were assessed in (A) control and TNFR2-overexpressing (TNFR2-vector) MDA-MB-231 cells, and in (B) control and TNFR2-knockdown (TNFR2-sh) MCF-7 cells, with or without ADM treatment. The effect of the PARP inhibitor ABT888 on the ADM-induced expression levels of pH2AX was assessed in (C) control and TNFR2-knockdown (TNFR2-sh) MCF-7 cells, and in (D) control and TNFR2-overexpressing (TNFR2-vector) MDA-MB-231 cells. GAPDH was used as a loading control. *P<0.05. TNFR2, tumor necrosis factor receptor 2; pH2AX, phosphorylated histone family 2A variant X; PARP, poly(ADP-ribose) polymerase; sh, short hairpin RNA; ADM, Adriamycin.
LY294002 and ABT888 inhibit ADM resistance induced by TNFR2. TNFR2-overexpressing (TNFR2-vector) MDA-MB-231 cells were challenged with increasing concentrations of ADM, in the absence or presence of LY294002 and/or ABT888 inhibitors. (A) Survival rate. (B) IC50 values. *P<0.05. ADM, Adriamycin; TNFR2, tumor necrosis factor receptor 2; IC50, half maximal inhibitory concentration.
TNFR2 promotes PARP expression via the AKT signaling pathway. Expression levels of total and phosphorylated ERK and AKT were assessed by western blotting in (A) control and TNFR2-overexpressing (TNFR2-vector) MDA-MB-231 cells, and in (B) control and TNFR2-knockdown (TNFR2-sh) MCF-7 cells. (C) Protein expression levels of PARP and pH2AX were assessed in TNFR2-overexpressing (TNFR2-vector) MDA-MB-231 cells in the absence or presence of the AKT inhibitor LY294002. GAPDH was used as a loading control. *P<0.05. TNFR2, tumor necrosis factor receptor 2; PARP, poly(ADP-ribose) polymerase; AKT, AKT serine/threonine kinase 1; ERK, extracellular signal-regulated kinase; pH2AX, phosphorylated histone family 2A variant X; p-, phosphorylated; sh, short hairpin RNA.
Primary antibodies used in western blot analyses.
Protein | Cat. no. | Final dilution | Supplier |
---|---|---|---|
TNFR2 | ab8161 | 1:1,000 | Abcam, Cambridge, MA, USA |
pH2AX | ab22551 | 1:1,000 | Abcam, Cambridge, MA, USA |
PARP | 13371-1-AP | 1:1,000 | Wuhan Sanying Biotechnology, Wuhan, China |
MGMT | 17195-1-AP | 1:2,000 | Wuhan Sanying Biotechnology, Wuhan, China |
p-ERK1/2 | ab214362 | 1:1,000 | Abcam, Cambridge, MA, USA |
ERK1/2 | 9102 | 1:1,000 | Cell Signaling Technology, Inc., Danvers, MA, USA |
p-AKT | 13038 | 1:1,000 | Cell Signaling Technology, Inc., Danvers, MA, USA |
AKT | 4685 | 1:1,000 | Cell Signaling Technology, Inc., Danvers, MA, USA |
GAPDH | Ab181602 | 1:2,000 | Abcam, Cambridge, MA, USA |
TNFR2, tumor necrosis factor receptor 2; pH2AX, phosphorylated histone family 2A variant X; PARP, poly(ADP-ribose) polymerase; MGMT, O6-methylguanine-DNA methyltransferase; p-, phosphorylated; ERK, extracellular signal-regulated kinase; AKT, AKT serine/threonine kinase 1.