Epigenetic Regulation of RIP3 Suppresses Necroptosis and Increases Resistance to Chemotherapy in NonSmall Cell Lung Cancer

Abstract

Introduction: The efficacy of chemotherapeutic agents in killing cancer cells is mainly attributed to the induction of apoptosis. However, the tremendous efforts on enhancing apoptosis-related mechanisms have only moderately improved lung cancer chemotherapy, suggesting that other cell death mechanisms such as necroptosis could be involved. In this study, we investigated the role of the necroptosis pathway in the responsiveness of nonsmall cell lung cancer (NSCLC) to chemotherapy.

Methods: In vitro cell culture and in vivo xenograft tumor therapy models and clinical sample studies are combined in studying the role of necroptosis in chemotherapy and mechanism of necroptosis suppression involving RIP3 expression regulation.

Results: While chemotherapeutic drugs were able to induce necroptotic cell death, this pathway was suppressed in lung cancer cells at least partly through downregulation of RIP3 expression. Ectopic RIP3 expression significantly sensitized lung cancer cells to the cytotoxicity of anticancer drugs such as cisplatin, etoposide, vincristine, and adriamycin. In addition, RIP3 suppression was associated with RIP3 promoter methylation, and demethylation partly restored RIP3 expression and increased chemotherapeutic-induced necroptotic cell death. In a xenograft tumor therapy model, ectopic RIP3 expression significantly sensitized anticancer activity of cisplatin in vivo. Furthermore, lower RIP3 expression was associated with worse chemotherapy response in NSCLC patients.

Conclusion: Our results indicate that the necroptosis pathway is suppressed in lung cancer through RIP3 promoter methylation, and reactivating this pathway should be exploited for improving lung cancer chemotherapy.

Figure 1

Cisplatin induces necroptosis in NSCLC cells. A. The cells were treated with Necrostatin-1 (Nec-1, 10 μM) or NSA (5 μM) for 30 min or remained untreated, and then with cisplatin (cDDP, 10 μM) for 48 hours and cell death was detected by LDH release assay. B. A549 cells were treated with Nec-1 (10 μM) or NSA (5 μM) plus cDDP (5 μM) for 2 days, then cultured in fresh medium for 10 days and stained with methylene blue. C. H460 cells were treated with cDDP (5 μM) for the indicated times. Top, the indicated proteins in cell lysates were detected by Western blot. GADPH was detected as an input control. Bottom, Cyclophilin A (CypA) in culture media was detected by Western blot. The major band detected in the gel by coomassie blue staining representing the major media protein was used as an input control. D. A549 cells were stably transfected with MLKL shRNA or negative control (NC) shRNA, MLKL knockdown was confirmed by Western blot (top). The cells were treated with cisplatin for 48 hours. Cell death was detected by LDH releasing assay. Data shown are the mean ± SD. *P < 0.05, **P < 0.01.

Figure 2

RIP3 expression is reduced in NSCLC. A. Human lung tumors with paired adjacent normal lung tissues in tissue arrays were stained for RIP3. Representative images (200×) are shown. Bar size: 100 μM. The airway and alveolar epithelial cells are strongly positive RIP3 in the adjacent tissues, whereas the tumor cells are weakly positive in the tumor tissues. B. Quantitative results derived from IHC of the tissue array (top) and surgical dissected samples (bottom). LUAD: Lung adenocarcinomas; LUSC: Lung squamous carcinomas. C. RIP3 mRNA expression quantified by qRT-PCR. Lung cancers are compared with paired normal lung (n = 12). Average of RIP3 expression in lung cancer cell lines are shown (n = 14). The RIP3 expression in distant normal lung tissue (DNLT) was used as a normal expression level for comparison. D. Data from MethHC was analyzed. The related RIP3 expression in tumor–normal pairs was shown. The tumor/normal ratios <1 means decreased RIP3 expression in tumors.

Figure 3

Reduced RIP3 expression associated with RIP3 promoter methylation in human NSCLC. A. RIP3 was detected in nontransformed (HBEC-2, -13) and lung cancer cell lines by Western blot. β-actin was detected as an input control. Relative RIP3 expression fold (HBEC-2 was set as 1) is shown at the bottom. B. Upper, A diagram of RIP3 promoter structure with the location of the primer and restriction sites analyzed by COBRA is shown. B, BSTUI, T, Taq I. Lower, NSCLC lines and primary human bronchial epithelial cells (PHBC1 and 2) were analyzed by COBRA using the restriction enzymes BSTUI (B) or Taq I (T) for digesting the RIP3 promoter PCR products. C. Quantitative analysis of RIP3 methylation. Genome-wide methylation of PHBC derived from a cancer free individual, lung tumor–normal pairs from 16 NSCLC patients, and the Calu6 NSCLC cells were evaluated with the Illumina HM450 Beadchip. Methylation level measured in β-values ranges from 0 to 1 and indicate 0–100% methylation, respectively. Five probes located within the RIP3 promoter CpG island were analyzed and β ≥ 0.3 were considered methylated. Out of 16, 4 (25%) tumors, but not PHBC and normal lung tissues (0/16, 0%), show methylation of all five probes. N: normal tissue, UT: unmethylated tumor, MT: methylated tumor. D. The cell lines were treated with 1 μM 5-aza-2′-deoxycytidine (DAC) every 24 hours for 96 hours and/or 300 nM Trichostatin A (TSA) for 18 hours, and RIP3 mRNA expression was detected by qRT-PCR. *P < 0.05; **P < 0.01. E. The cells were treated as described in D, and RIP3 protein expression was detected by Western blot. β-actin was detected as an input control.

Figure 4

Ectopic RIP3 expression sensitizes cisplatin-induced cytotoxicity. A. Stable transfection of RIP3-EGFP or negative control EGFP vector in A549 cells. The expression of ectopic RIP3-EGFP was confirmed by Western blot. B. The cells were treated with cisplatin (cDDP) for 48 hours and cell death was detected by LDH release assay. *P < 0.05. C. The cells were treated with indicated concentrations of cDDP for 2 days, then cultured in fresh medium for 10 days and stained with methylene blue. D. The cells were treated with Nec-1 (10 μM) or NSA (5 μM) plus cisplatin for 48 hours, and cell death was detected by LDH release assay. E. The cells were transfected with RIP3- or negative control (NC)-siRNA. RIP3 knockdown was confirmed by Western blot. F. The cells were treated with for 30 min or remained untreated, and then with cisplatin (cDDP, 30 μM) or etooside (Ept, 20 μM) for 48 hours and cell death was detected by LDH release assay. *P < 0.05, **P < 0.01.

Figure 5

Restoration of RIP3 expression sensitizes cisplatin response in vivo. A. Tumor growth was measured every three days and the mean tumor volume of each group was shown. Comparison: NC (cDDP) vs RIP3 (cDDP); *, p < 0.05, **, p < 0.01. B. Resected xenograft tumors. C. Tumor weights and, D. Tumor inhibition rates were compared between treatment groups. E. Detection of apoptosis and necroptosis in tumor tissues by TUNEL assay combined with immunofluorescent staining of active caspase-3. Nucleus was stained with DAPI. Apoptosis (active caspase-3 and TUNEL positive) and necroptosis (TUNEL-positive but active caspase-3-negative) cells were quantified by counting in a total of 300 cells in five fields (200 × ). Representative images were shown. White arrows, active caspase-3 positive cells (yellow). Green arrows, TUNEL-positive cells (red). F. Average apoptotic cell numbers and necroptotic cell numbers were shown as mean ± SD. Columns represent mean in each group, bars represent SD. **, P < 0.01, ***, P < 0.001.