Methylation-dependent loss of RIP3 expression in cancer represses programmed necrosis in response to chemotherapeutics - PubMed (original) (raw)

doi: 10.1038/cr.2015.56. Epub 2015 May 8.

Michael J Morgan 2, Da-Gyum Lee 3, Woo-Jung Kim 1, Jung-Ho Yoon 1, Ja Seung Koo 4, Seung Il Kim 5, Soo Jung Kim 6, Mi Kwon Son 6, Soon Sun Hong 6, Jean M Mulcahy Levy 7, Daniel A Pollyea 8, Craig T Jordan 8, Pearlly Yan 9, David Frankhouser 9, Deedra Nicolet 10, Kati Maharry 10, Guido Marcucci 9, Kyeong Sook Choi 1, Hyeseong Cho 1, Andrew Thorburn 2, You-Sun Kim 1

Affiliations

Methylation-dependent loss of RIP3 expression in cancer represses programmed necrosis in response to chemotherapeutics

Gi-Bang Koo et al. Cell Res. 2015 Jun.

Abstract

Receptor-interacting protein kinase-3 (RIP3 or RIPK3) is an essential part of the cellular machinery that executes "programmed" or "regulated" necrosis. Here we show that programmed necrosis is activated in response to many chemotherapeutic agents and contributes to chemotherapy-induced cell death. However, we show that RIP3 expression is often silenced in cancer cells due to genomic methylation near its transcriptional start site, thus RIP3-dependent activation of MLKL and downstream programmed necrosis during chemotherapeutic death is largely repressed. Nevertheless, treatment with hypomethylating agents restores RIP3 expression, and thereby promotes sensitivity to chemotherapeutics in a RIP3-dependent manner. RIP3 expression is reduced in tumors compared to normal tissue in 85% of breast cancer patients, suggesting that RIP3 deficiency is positively selected during tumor growth/development. Since hypomethylating agents are reasonably well-tolerated in patients, we propose that RIP3-deficient cancer patients may benefit from receiving hypomethylating agents to induce RIP3 expression prior to treatment with conventional chemotherapeutics.

PubMed Disclaimer

Figures

Figure 1

Figure 1

Expression of RIP3 contributes to sensitivity to DNA-damaging agents. (A) HeLa, MDA-MB231, and Huh7 cells ectopically expressing RIP3 were treated with the indicated concentration of doxorubicin or etoposide for 2 days and cell viability was analyzed by MTT assay (right). Results are presented as means ± SEM. (B) HT-29 cells with stable shRNA expression were analyzed by western blotting (left). Cells were treated with doxorubicin or etoposide for 48 h and cell viability was analyzed by MTT assay (right). Results are presented as means ± SEM. (C) Huh7 cells from A were treated with doxorubicin (Doxo, μM), etoposide (Etopo, μM), taxol (nM), 5-fluorouracil (5-FU, μM), camptothecin (CPT, μg/ml), or cisplatin (μM), as indicated, and after 48 h, cell viability was analyzed by MTT assay. Results are presented as means ± SEM. (D) HT-29 cells were pretreated with the RIP3 inhibitor dabrafenib (10 μM) for 1 h and then treated with TCZ/TSZ, doxorubicin, or etoposide for 48 h. Cell viability was analyzed by MTT assay. Results are presented as means ± SEM. (E) Inhibition of downstream event of RIP3 kinase activation affects cell death in response to DNA-damaging agents. HeLa and RIP3-expressing HeLa cells were pretreated with dabrafenib (10 μM) or NSA (1 μM) for 1 h and then treated with TCZ/TSZ or doxorubicin (1 μM) for 24 h. Cell viability was analyzed by MTT assay. Results are presented as means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 2

Figure 2

MLKL phosphorylation induced by DNA-damaging agents is dependent on RIP3 kinase activity. (A) HeLa and RIP3-expressing HeLa cells were treated with different concentrations of doxorubicin for 24 h and cell lysates were analyzed by western blotting. (B) RIP3-expressing HeLa cells were treated with doxorubicin (0.5 μM) or etoposide (12.5 μM) for the indicated times and cell lysates were analyzed by western blotting. (C) HeLa cells were transiently transfected with GFP-hRIP3 and cells were treated with TCZ, doxorubicin (0.5 μM), and etoposide (12.5 μM) for the indicated times. Cells were stained with phospho-MLKL antibody and analyzed by confocal fluorescence microscopy. Scale bar, 10 μm. (D) HeLa and RIP3-expressing HeLa cells were treated with doxorubicin (1 μM) for 12 h and cell lysates were immunoprecipitated with anti-MLKL antibody. (E) HT-29 cells were treated with doxorubicin (1.25 μM) for 24 h. Cell lysates were immunoprecipitated with anti-MLKL antibody and analyzed by western blotting for RIP3. The asterisk (*) indicates the IgG band. §The upper band in this blot is thought to be phosphorylated RIP3, and appears only in the doxorubicin-treated cells. (F, G) RIP3-expressing HeLa (F) or HT-29 cells expressing MLKL shRNAs or non-silencing control (G) were analyzed by western blotting (insets). Cells were treated with TSZ, doxorubicin, or etoposide for 48 h and cell viability was analyzed by MTT assay. Results are presented as means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 3

Figure 3

RIP3 is silenced by methylation in multiple cancer cell lines. (A) Western blotting analysis of lysates from multiple cancer cell lines showing RIP3 expression. (B) Western blotting analysis of RIP3 expression is shown in melanocyte, keratinocyte and fibroblasts from human normal skin. Other lysates from human mammary epithelial cells (HMLE: immortalized, hTERT + T antigens; HME: primary), primary prostate epithelial cells (two specimens: PPE7, PPE30), normal hepatocyte cell line HL7702 and primary brain tissue (two specimens: E3, E37) were also probed for RIP3 expression. (C) HeLa, A549, and HT-29 cells were either lysed in M2 buffer (left) or directly dissolved and boiled in Laemmli SDS buffer (right) and lysates were analyzed by western blot. Blotting for Vimentin is included as a positive control for detection of insoluble protein. (D) Immunoblotting showing NIK protein expression in HeLa and DLD-1 cells upon proteosome inhibition with MG132 (10 μM). (E) Western blotting analysis of HeLa cells treated with sodium butyrate (2.5 mM) or Trichostatin A (0.25 μg/ml) for 1 day in the presence or absence of 5-AD. (F) Western blotting analysis of RIP3 expression in HeLa, MDA-MB231, and DLD-1 cells treated with 5-AD for the indicated times (2 μM) and with different concentrations (4 days).

Figure 4

Figure 4

Methylation of the RIP3K TSS is negatively associated with RIP3 expression in cancer cell lines. (A) Western blotting showing the response of various cancer cells to 5-AD (2 μM, 4 days). (B) Reverse transcription-PCR products from cells following treatment with 5-AD (2 μM, 4 days). A sFRP2 product is shown as a positive control for methylation. (C) Western blotting and reverse transcription-PCR results after 5-AZA treatment of HeLa (0.625 μM, 8 days) or DLD-1 (1.25 μM, 4 days) cells. (D) Western blotting and reverse transcription-PCR results after treatment of HeLa cells with 5-AD (2 μM, 4 days) or RG108 (100 μM, 8 days). (E) Methylation-specific PCR (MSP) of genomic DNA from HeLa, MDA-MB231, K562, and HT-29 cells. M and U indicate methylated-specific and unmethylated-specific primers, respectively, to the RIPK3 sequence. (F) MSP of genomic DNA from HeLa, DLD-1, HCT-116, MDA-MB231, and K562 cells treated with 5-AD (2 μM, 4 days). MSP was repeated on genomic DNA from HeLa and DLD-1 cells with a second set of methylated- and unmethylated-specific primers. (G, H) HeLa and DLD-1 cells were infected with a lentivirus encoding DNMT1 shRNAs, or non-silencing control. After selection with puromycin, cells with stable shRNA expression were analyzed by reverse transcription-PCR (G) or western blotting (H) after 5-AD (2 μM) treatment of HeLa or DLD-1 cells, showing reverse correlation between DNMT1 and RIP3 expression.

Figure 5

Figure 5

RIP3 is silenced in breast cancer. (A) Western blotting for RIP3 expression is shown for breast cancer tumors compared to normal tissue from the same patients (N, normal tissue; T, tumor tissue). In 3 cases where the actin immunoblots had problems, ERK protein is shown as a protein loading control. (B, C) Immunohistochemical staining of RIP3 in paraffin-embedded tissue specimens from tumor and non-tumor samples in breast cancer patients. Arrows indicate entrapped normal ducts. Paired tumor and normal tissue from each patient was quantitated (B, right**)**. Intensity of histological staining grade was quantitated according to American Society of Clinical Oncology (ASCO)/College of American Pathologists (CAP) guidelines using the following categories: 0, no immunostaining; 1+, weak incomplete membranous staining, < 10% of tumor cells; 2+, complete membranous staining, either uniform or weak in at least 10% of tumor cells; and 3+, uniform intense membranous staining in at least 30% of tumor cells. Staining grade is shown for various subtypes of breast cancer (C). Scale bar, 100 μm. (D) Methylation-specific PCR of genomic DNA from tumor and non-tumor tissue of three triple-negative breast cancer patients (patients A-C). M and U indicate methylated-specific and unmethylated-specific primers (set 1), respectively, to the RIPK3 genomic sequence. (E) Kaplan-Meier curve showing metastasis relapse (MR)-free survival of breast cancer patients having above or below median expression of RIP3 (RIPK3). Hazard ratio (HR) based on relative risk of event occurrence between the group of patients with RIP3 expression above the median value in the selected population and the group with RIP3 expression below the median value.

Figure 6

Figure 6

Restoration of RIP3 by hypomethylating agents enhances sensitivity to DNA-damaging agents. (A, B) HeLa and MDA-MB231 cells were treated with 5-AD (2 μM, 4 days), then with doxorubicin or etoposide for 48 h and cell viability was analyzed by MTT assay (A) or ATP depletion (B). Results are presented as means ± SEM. (C-E) HT-29 cells, which endogenously express RIP3, were treated with 5-AD (2 μM, 4 days) and then with doxorubicin or etoposide for 48 h, and cell viability was analyzed by MTT assay (C). Human dermal fibroblasts (HDF), which are untransformed and have RIP3 expression, were treated with 5-AD (2 μM, 4 days), then with doxorubicin (2 μM), etoposide (25 μM), 5-FU (50 μM), or CPT (1 μg/ml) for 48 h and cell viability was analyzed by MTT assay (D). A549-N cells, which do not upregulate RIP3 expression in response to hypomethylating agents, were treated with 5-AD (2 μM) for 4 days and cell viability was analyzed by MTT assay (E). Results are presented as means ± SEM. (F) HeLa cells with stable shRNA expression were pretreated with 5-AD (2 μM, 4 days) and then treated with doxorubicin or etoposide for 2 days and cell viability was analyzed by MTT assay. Results are presented as means ± SEM. (G) MDA-MB231 cells with stable shRNA expression were pretreated with 5-AD (2 μM) for 4 days and then treated with doxorubicin (μM), etoposide (μM), CPT (μg/ml), and cisplatin (μM) for 48 h and cell viability was analyzed by relative ATP level analysis. Results are presented as means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 7

Figure 7

The combination of 5-AD plus doxorubicin inhibits breast cancer growth in a mouse xenograft model. (A) Effect of 5-AD plus doxorubicin on MDA-MB231 breast cancer cell xenograft in athymic BALB/c nude mice. MDA-MB231 tumors were established orthotopically and treated with 5-AD for the first week and then doxorubicin for 33 days. Tumor volume was monitored and is shown in the graph. Data are presented as means ± SD (n = 7). (B) At the end of treatment mice were killed and tumors are shown (left). Tumor mass was measured and is summarized (right). (C) H&E staining for liver section of mice receiving the indicated treatment. (D) Top panel shows detection of cell death by TUNEL assay in tumor sections of mice receiving the indicated treatment. Sections were also stained for p-H2AX and H&E. (E) PCR of tumor tissues to detect RIP3 restoration upon 5-AD injection in mice. (F) Immunohistochemical staining of RIP3 in paraffin-embedded tissue specimens from tumor of mouse xenograft.

References

    1. Hanahan D, Weinberg RA. Hallmarks of cancer: the next generation. Cell. 2011;144:646–674. - PubMed
    1. Vandenabeele P, Galluzzi L, Vanden Berghe T, Kroemer G. Molecular mechanisms of necroptosis: an ordered cellular explosion. Nat Rev Mol Cell Biol. 2010;11:700–714. - PubMed
    1. Vanlangenakker N, Vanden Berghe T, Vandenabeele P. Many stimuli pull the necrotic trigger, an overview. Cell Death Differ. 2012;19:75–86. - PMC - PubMed
    1. Morgan M, Liu Z. Programmed cell death with a necrotic-like phenotype. BioMol Concepts. 2013;4:259–275. - PubMed
    1. Kaiser WJ, Upton JW, Long AB, et al. RIP3 mediates the embryonic lethality of caspase-8-deficient mice. Nature. 2011;471:368–372. - PMC - PubMed

Publication types

MeSH terms

Substances

LinkOut - more resources