ripk1 binds mcu to mediate induction of mitochondrial ca ...essential signaling molecule in pathways...

Molecular Cell Biology

RIPK1 Binds MCU to Mediate Induction ofMitochondrial Ca2þ Uptake and PromotesColorectal OncogenesisFanxin Zeng1,2, Xiao Chen3,Weiyi Cui1,2, Wei Wen1,2, Fujian Lu1, Xueting Sun1,2,DongweiMa1,2,YeYuan1,2, Zezhong Li1,2, NingHou1,2, HongZhao3, XinyuBi3, Jianjun Zhao3,Jianguo Zhou3, Yan Zhang1,2,4, Rui-Ping Xiao1,2,4, Jianqiang Cai3, and Xiuqin Zhang1,2

Abstract

The receptor-interacting protein kinase 1 (RIPK1) is anessential signaling molecule in pathways for cell survival,apoptosis, and necroptosis. We report here that RIPK1 isupregulated in human colorectal cancer and promotes cellproliferation when overexpressed in a colon cancer cell line.RIPK1 interacts with mitochondrial Ca2þ uniporter (MCU) topromote proliferation by increasing mitochondrial Ca2þ

uptake and energy metabolism. The ubiquitination site of

RIPK1 (RIPK1-K377) was critical for this interaction with MCUand function in promoting cell proliferation. These findingsidentify the RIPK1-MCU pathway as a promising target to treatcolorectal cancer.

Significance: RIPK1-mediated cell proliferation through MCUis a central mechanism underlying colorectal cancer progressionandmay prove to be an important therapeutic target for colorectalcancer treatment. Cancer Res; 78(11); 2876–85. �2018 AACR.

IntroductionColorectal cancer is one of the most common human cancers

worldwide. It is the second leading cause of cancer deaths in theUnited States (1). Colorectal cancer has taken a significant upwardage-standardized incidence rate from 2000 to 2011 in China (2).Colorectal cancer is a multifactorial disease, with an etiologyrelated to environmental exposure, genetic factors, and inflam-matory conditions of the digestive tract (3).

Traditionally, colorectal carcinogenesis is believed to involvethe gatekeeper and caretaker pathways (4). The gatekeeper path-way is characterized by mutations in genes that regulate tumorgrowth, such as the tumor-suppressor genes APC, p53, and nm32and the oncogenes K-ras and c-myc (5). The caretakers are genesthatmaintain the integrity of the genome, such asmismatch repairgenes (6). Now, carcinogenesis has been correlated directly withmitochondrial dysfunction, and reprogramming energy metabo-

lism is considered to be an emerging hallmark of this process (7).Still, the mechanisms underlying colorectal cancer carcinogenesisremain elusive.

Receptor-interacting protein kinase 1 (RIPK1) is a key com-ponent at the crossroads of stress-induced signaling pathwaysand determines whether a cell lives or dies (8, 9). When cells aretreated with TNFa, RIPK1 suppresses adenine nucleotide trans-locase activity and inhibits ADP transport into mitochondria,reducing ATP and causing necrotic cell death (10). Previousstudies have focused on cell death induced by RIPK1, especiallyin regulating apoptosis or necroptosis (11–14). Mice that lackRIPK1 die perinatally while exhibiting apoptosis in multipletissues (15). RIPK1 maintains epithelial homeostasis by inhi-biting apoptosis and necroptosis and is a master regulator ofepithelial cell survival (16). A recent study has shown thatRIPK1 is highly expressed in pancreatic ductal adenocarcinomaand that blocking RIPK1 protects against pancreatic oncogen-esis in vivo (17). Moreover, RIPK1 acts as an oncogenic driver,and is upregulated commonly in human melanoma in amanner that depends on NFkB (18). Nonetheless, the role ofRIPK1 in colorectal cancer remains unclear.

Ca2þ regulates various fundamental cellular processes, includ-ing those relevant to tumor progression and metastasis, suchas proliferation, gene transcription, differentiation, and cell death.A wide variety of studies have demonstrated that specific Ca2þ

channels, pumps, or exchangers are upregulated or downregu-lated in some cancers (19–21). Mitochondria modulate theoverall intracellular Ca2þ signaling and are responsible for rapidCa2þ uptake, which is mainly mediated by the mitochondrialCa2þ uniporter (MCU).MCU is an evolutionarily conserved Ca2þ

channel, which is expressed widely and localized to the innermitochondrial membrane (22–24). MCU-dependent mitochon-drialCa2þhomeostasis is essential to regulate aerobicmetabolismand cell survival.

1Institute of Molecular Medicine, Peking University, Beijing, China. 2Beijing KeyLaboratory of Cardiometabolic Molecular Medicine, Peking University, Beijing,China. 3Department of Hepatobiliary Surgery, National Cancer Center/CancerHospital, Chinese Academy of Medical Sciences and Peking Union MedicalCollege, Beijing, China. 4State Key Laboratory of Biomembrane and MembraneBiotechnology, Peking-Tsinghua Center for Life Sciences, Beijing, China.

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

F. Zeng, X. Chen, and W. Cui contributed equally to this article.

Corresponding Authors: Xiuqin Zhang, Institute of Molecular Medicine, PekingUniversity, Beijing 100871, China. Phone: 8610-6275-3420; E-mail:[email protected]; and Jianqiang Cai, Department of Hepatobiliary Surgery,National Cancer Center/Cancer Hospital, Chinese Academy of Medical Sciencesand Peking Union Medical College, Beijing 100021, China. E-mail:[email protected]

doi: 10.1158/0008-5472.CAN-17-3082

�2018 American Association for Cancer Research.

CancerResearch

Cancer Res; 78(11) June 1, 20182876

on December 21, 2020. © 2018 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

Published OnlineFirst March 12, 2018; DOI: 10.1158/0008-5472.CAN-17-3082

http://crossmark.crossref.org/dialog/?doi=10.1158/0008-5472.CAN-17-3082&domain=pdf&date_stamp=2018-5-22

http://cancerres.aacrjournals.org/

The role of MCU in inducing mitochondrial Ca2þ uptake incarcinogenesis is still controversial. Previous studies have revealeda correlation betweenMCUandbreast cancer. Patientswith breastcancer with high levels of expressed MCU have a poor prognosis(25). In the MDA-MB-231 cell line, a triple-negative breast cancermodel, caspase-independent cell death is potentiated by silencingMCU,which suggests that overexpression ofMCUoffers a survivaladvantage by acting against an apoptotic pathway (26). In addi-tion, the role of MCU in controlling breast cancer cell migrationhas been ascribed to a store-operated Ca2þ entry-dependentmechanism (27). Although the physiology of MCU has beeninvestigated for decades, its molecular properties in colorectalcancer remain unclear.

Given the above uncertainties, we designed experiments toinvestigate the role of RIPK1 and MCU in cell proliferation andcolorectal cancer oncogenesis.

Materials and MethodsReagents and materials

Unless indicated otherwise, all chemicals were from Sigma-Aldrich. Rabbit anti-MCU antibody was from Sigma-Aldrich(1:1,000); mouse anti-RIPK1 antibody was from BD Biosciences(1:1,000); and mouse anti–b-catenin, rabbit anti–caspase-8, rab-bit anti–NFkB, rabbit anti-IkB, and rabbit anti-Ub antibodieswere from Cell Signaling Technology (1:1,000). Rabbit anti–p-MLKL and mouse anti-MICU1 antibodies were from Abcam(1:1,000), and rabbit anti-RIPK3 antibody was from Imgenex(1:1,000). Rhod-2/AM and Fluo-4/AM were from Invitrogen.

Human sample collectionPatient samples were from the Cancer Hospital, Chinese Acad-

emy of Medical Sciences (CAMS), Beijing China. Informed,written consent was given by all patients. The study protocolconformed to the ethical guidelines of the 1975 Declarationof Helsinki and was approved by the Human Ethics Committeeof the Cancer Hospital, Chinese Academy of Medical Sciences.

Cell culture, adenoviral infection, and plasmid transfectionHT29 cells (Cell Resource Center, Institute of Basic Medical

Sciences, CAMS/Peking Union Medical College, Beijing, China)were cultured at 37�C under 5% CO2 in DMEM supplementedwith 10% FBS (Gibco), and 1% penicillin–streptomycin. Approx-imately, 80% confluent HT29 cells were transfected with vector orplasmid DNA at 2 mg/mL using Lipofectamine 2000 (Invitrogen)following the manufacturer's instructions and then cultured for afurther 48 hours. Cells were infected with adenovirus DNAexpressing RIPK1 or MCU at a multiplicity of infection (MOI)of 25 and grown for a further 48 hours. For siRNA transfection,HT29 cells at approximately 80% confluence were transfectedwith 200 pmol of control siRNA and specific siRNA using Lipo-fectamine RNAiMAX (Invitrogen) following the manufacturer'sinstructions, and incubated for a further 48 hours. The siRNAsequences for transfection were as follows: RIPK1-targetingsequences: 50-GCCAGCUGCUAAGUACCAATT-30 (siRNA1-1),50-UUGGUACUUAGCAGCUGGCTT-30 (siRNA1-2); 50-GCAAA-GACCUUACGAGAAUTT-30 (siRNA2-1), 50-AUUCUCGUAAGG-UCUUUGCTT-30 (siRNA2-2); and MCU-targeting sequences: 50-GCGCCAGGAAUAUGUUUAUTT-30 (siRNA1-1), 50-AUAAACA-UAUUCCUGGCGCTT-30 (siRNA1-2); 50-UGACUUAACAUACC-ACGUATT-30 (siRNA2-1), 50-UACGUGGUAUGUUAAGUCATT-

30 (siRNA2-2); scrambled siRNA, 50-UUCUCCGAACGUGUCAC-GUTT-30, 50-ACGUGACACGUUCGGAGAATT-30.

Plasmids for RIPK1 point mutationPoint mutation plasmids of RIPK1 were constructed using the

HiFi HotStart DNA Polymerase (KAPA Biosystems; Roche) alongwith custom mutagenic primers to create substitutions in theH-RIPK1-HA plasmid. After PCR amplification, the PCR productwas digested with Dpn1 restriction enzyme (NEB) and trans-formed into TransT1 cells (TransGen Biotech). Then the colonieswere cultured, plasmid DNA were extracted and sequenced toverify the desired mutations.

Western blot analysisTissues or cells were lysed in lysis buffer (30mmol/LHEPES pH

7.6, 100 mmol/L NaCl, 0.5% Nonidet P-40, and protease inhib-itor mixture) on ice for 30 minutes, and the lysates were centri-fuged at 13,000 rpm for 10minutes. Protein samples (50–100 mg)were separated by 10% SDS-PAGE. Blots were incubated at 4�Covernight with the primary antibodies, followed by anti-rabbit oranti-mouse horseradish peroxidase–labeled secondary antibodiesfor 1 hour at room temperature. An electrochemiluminescencedetection system was used to reveal the peroxidase label, and therelative abundance was quantified by densitometry using Quan-tity One 4.6.7 software (both from Bio-Rad).

ImmunoprecipitationFor RIPK1 or MCU immunoprecipitation, the cells or human

tissues were lysed in lysis buffer (30 mmol/L HEPES pH 7.5,100mmol/LNaCl, 1mmol/L EDTA, and 0.5%NP40). The lysateswere precleaned with Protein G agarose beads (Thermo FisherScientific) at 4�C for 4hours. Control IgG, anti-RIPK1 antibody, orMCU antibody was coupled to Protein G agarose beads in lysisbuffer for 4 hours at 4�C. Then the cleaned lysates were incubatedwith beads coupledwith RIPK1 andMCUantibody or control IgGovernight at 4�C. After that, the beads were washed three times inice-cold lysis buffer andboiledwith loading buffer for 10minutes.The bead mixture was vortexed, centrifuged for 1 minute, and thesupernatant was collected for Western blot analysis.

Mass spectrometryFor liquid chromatography/tandem mass spectrometry

(LC/MS-MS) analysis, peptides were separated by a 40-minutegradient elution at a flow rate 0.300mL/minute with a ThermoFisher Scientific Dionex UltiMate 3000 HPLC System, which wasdirectly interfacedwith a ThermoFisher ScientificOrbitrap FusionLumos mass spectrometer. MS-MS spectra from each LC/MS-MSrun were searched against the RIPK1 andMCUdatabase using theProteome Discoverer (version PD1.4; Thermo Fisher Scientific)searching algorithm. Relative protein quantification was per-formed using ProteomeDiscoverer software (version 1.4) accord-ing to manufacturer's instructions on the six reporter ion inten-sities per peptide. Quantitation was carried out only for proteinswith two or more unique peptide matches. Protein ratios werecalculated as themedian of all peptide hits belonging to a protein.Quantitative precision was expressed as protein ratio variability.

Real-time PCRTotal RNA was isolated with TRIzol reagent (Invitrogen) and

2 mg was reverse transcribed into cDNA using RT-MLV reversetranscriptase (Promega). The RT reaction mixture was used as a

RIPK1 and MCU Promote Colorectal Cancer Progression

www.aacrjournals.org Cancer Res; 78(11) June 1, 2018 2877




template to perform the real-time PCR (StepOnePlus Real-TimePCR System; Applied Biosystems). The relative mRNA levelswere determined by normalizing to the actin mRNA level. Theprimers for human ripk1 were 50-CCAAgACgAAgCCAACTACC-30

(forward) and 50-CTCgTAAggTCTTTgCTgTg-30(reverse). The pri-mers for human mcu were 50-TCCAgAAgCCAgAgACAgAC-30

(forward) and 50-TgTCggAgAggCAgATgTAC -30 (reverse).

Histologic analysisThe human colorectal cancer tissues for histologic analysis were

fixed in 4% paraformaldehyde (pH 7.4) overnight, embedded inparaffin, serially sectioned at 5 mm, and stained with hematoxylinand eosin.

Immunofluorescence for confocal microscopyHT29 cells cultured in Nunc glass bottom dishes (Thermo

Fisher Scientific) were cotransfected with plasmids expressingRIPK1-GFP and Mito-DsRed (Clontech) or MCU-GFP andMito-DsRed. Twenty-four hours posttransfection, cells werewashed three times with PBS and imaged using a Zeiss LSM710 confocal microscope.

Oxygen consumptionThe oxygen consumption rate (OCR) was measured at 37�C

using an XF24 extracellular analyzer (Seahorse Bioscience). Thecells were seeded in 24-well plates. After 24 hours, cells wereloaded into the analyzer for O2 concentration determinations.Cells were exposed sequentially to oligomycin (1 mmol/L),carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP;1 mmol/L), and rotenone (1 mmol/L) plus antimycin (1 mmol/L).OCRwasmeasured after each exposure. BasalOCRwas calculatedas the difference between OCR before and after oligomycinexposure. Maximum OCR was calculated as the differencebetween OCR after FCCP and that after exposure to rotenoneplus antimycin.

Simultaneous measurements of cytoplasmic andmitochondrial Ca2þ concentrations ([Ca2þ]c and [Ca2þ]m)

HT29 cells were grown on 25-mm glass coverslips for 48 hoursand loadedwith2mmol/LRhod-2/AM(20minutes) and2mmol/LFluo-4/AM (10 minutes) in the extracellular medium. The cover-slips were mounted in an open perfusion microincubator(PDMI-2;Harvard Apparatus) at 37�Cand imaged. After 1minuteof baseline recording, the agonist histamine (100 mmol/L) wasadded and confocal imageswere recorded every 2 s (LSM710;CarlZeiss) at 488- and 561-nm excitation using a 40� oil objective.Images were analyzed and quantified using ImageJ (NIH).

Mouse tumor xenograft modelThe mouse study was approved by the Institutional Animal

Care and Use Committee of Peking University and was in accor-dancewith theprinciples of laboratory animal care of theNationalAcademies of Sciences/National Research Council. For colorectalcancer tumor xenograft experiments, HT29 (1 � 107), RIPK1-knockdown HT29 (RIPK1-KD, 1 � 107), or MCU-knockdownHT29 (MCU-KD, 1 � 107) cells were subcutaneously injected inaxilla of 4-week-old male BALB/c nude mice. The mice weresacrificed 16 days after tumor cell transplantation, the tumorswere weighed, and the volumes were measured. The length (A)and width (B) of the tumors were measured by a caliper. Tumorvolume was calculated using the formula (A � B2)/2.

Statistical analysisData are expressed as mean � SEM. Statistical analyses were

performed with GraphPad PRISM version 5.01 (GraphPad Soft-ware) and the SPSS 18.0 software package (SPSS). Student t testwas used to evaluate differences between control and experimen-tal groups. Comparisons between multiple groups were assessedby one-way ANOVA.

ResultsRIPK1 is upregulated in colorectal cancer

To investigate the function of RIPK1 in the oncogenesis ofcolorectal cancer, we collected colorectal cancer samples frompatients diagnosed by a pathologist as having adenocarcinoma(Supplementary Table S1) and graded them on their glandularformations (Fig. 1A). RIPK1, the key molecule in cell death andsurvival pathways, was upregulated significantly at both theprotein and mRNA levels in colorectal cancer tissue comparedwith the normal paracarcinoma tissue (Fig. 1B–E). Moreover,RIPK1 protein expression levels showed an increasing trend thatwas correlated with the stage of cancer progression (Supplemen-tary Fig. S1A), and 3 yearsmortality was positively correlated withRIPK1 protein expression levels (Supplementary Fig. S1B). As amarker of colorectal adenocarcinoma, b-catenin was also signif-icantly higher in colorectal cancer than in normal paracarcinomatissue (Fig. 1B and F). The transcription factor NFkB, which alsoserves as a downstreammolecule of RIPK1-mediated cell survivalsignaling, was upregulated (Fig. 1B andG), and its expressionwashigher in both cytoplasm and nuclear in most colorectal cancertissue than in normal control (Supplementary Fig. S2A), whereasits inhibitor, IkB, was downregulated (Fig. 1B and H). These datasuggested that RIPK1may be involved in cell proliferation duringcolorectal cancer development.

RIPK3 and p-MLKL, the other well-known cell death signalingmolecules regulated by RIPK1 and used as markers and executorsof necroptotic cell death (28), were decreased in colorectal cancer.However, the level of apoptotic executionmolecule caspase-8washigher in colorectal cancer than in normal paracarcinoma tissue(Supplementary Fig. S3A–S3D). These data suggested that incolorectal cancer, necroptosis might decrease, whereas apoptosisincreased, but these changes did not correlate well with RIPK1upregulation in the context of cell fate during cancer develop-ment. Therefore, RIPK1 may regulate colorectal cancer develop-ment by means other than traditional pathways.

MCU, a protein that interacts with RIPK1, is upregulated incolorectal cancer

Although NFkB is involved in the cell survival pathway regu-lated by RIPK1, it does not interact directly with RIPK1 (29–31).Next, we analyzed the profiles of proteins that interacted withRIPK1 in colorectal cancer samples using coimmunoprecipitationand a mass spectrum approach. Among the proteins that inter-acted with RIPK1, MCU scored highly molecules, and was evenmore desirable as a component of the RIPK1 complex (Supple-mentary Fig. S4A and S4B). Further analysis showed that theMCUmRNAandprotein levels were increased significantly in colorectalcancer tissues compared with normal paracarcinoma tissues(Fig. 2A–D). The protein expression level of MCU was signifi-cantly correlated with the RIPK1 protein expression in colorectalcancer (Fig. 2E and F). Previous studies have shown that mito-chondrial Ca2þ uptake 1 (MICU1) is a negative regulator of MCU

Zeng et al.

Cancer Res; 78(11) June 1, 2018 Cancer Research2878




(32). We found less MICU1 in colorectal cancer than in normalpara-carcinoma tissue (Supplementary Fig. S5A and S5B), sug-gesting thatMCUmay have played an important role in colorectalcancer development regulated by RIPK1.

RIPK1 interacts with MCU in mitochondriaWe performed coimmunoprecipitation assays to further inves-

tigate the relevance of RIPK1 and MCU to colorectal cancer, andrevealed a physical interaction between RIPK1 and MCU, whichwas enhanced markedly in colorectal cancer (Fig. 3A and B).

Because MCU is expressed on the inner membranes of mitochon-dria (23), we fractionated the total protein into mitochondrialand cytoplasmic proteins to identify the locations of RIPK1 andMCU. RIPK1was expressed in bothmitochondria and cytoplasm,whereas MCU was exclusively expressed in mitochondria. Theirinteractions occurred primarily in mitochondria and not in cyto-plasm (Fig. 3C). Moreover, using immunofluorescence confocalmicroscopy, we observed substantial colocalization of mitochon-dria (indicated by Mito-DsRed) and MCU (infected with MCU-GFP virus) in HT29 cells (Fig. 3D), a human colon

Figure 1.

RIPK1 is upregulated in colorectal cancer. A, Representative hematoxylin and eosin staining of normal and colorectal cancer (CRC) tissues. Scale bar, 100 mm. B,RepresentativeWestern blots of RIPK1, b-catenin, NFkB, and IkB from normal and colorectal cancer tissues. C, Statistical analysis ofWestern blots for RIPK1. Resultsare expressed as mean� SEM. �� , P < 0.001, normal vs. colorectal cancer (n¼ 40). D,mRNA expression of RIPK1 from normal and colorectal cancer tissues. Resultsare expressed as mean � SEM. �� , P < 0.01, normal vs. colorectal cancer (n ¼ 6). E, Representative photomicrographs of IHC for RIPK1 in normal and colorectalcancer tissues. Scale bar, 100 mm. F, Statistical analysis of b-catenin. Results are expressed as mean � SEM. � , P < 0.05, normal vs. colorectal cancer (n ¼ 40).G, Statistical analysis of NFkB. Results are expressed as mean � SEM. � , P < 0.05, normal vs. colorectal cancer (n ¼ 40). H, Statistical analysis of IkB.Results are expressed as mean � SEM. �� , P < 0.001, normal vs. colorectal cancer (n ¼ 40).

Figure 2.

MCU is upregulated and correlates with RIPK1 expression in colorectal cancer. A, Representative Western blots of MCU from normal and colorectal cancer(CRC) tissues. B, Statistical analysis of MCU. Results are expressed as mean � SEM. �� , P < 0.001, normal vs. colorectal cancer (n ¼ 40). C, mRNA expressionanalysis of MCU from normal and colorectal cancer tissues. Results are expressed as mean � SEM. �� , P < 0.01, normal vs. colorectal cancer (n ¼ 6). D,Representative photomicrographs of IHC for MCU in normal and colorectal cancer tissues. Scale bar, 100 mm. E, Relative expression of RIPK1 and MCU levelsin colorectal cancer (n ¼ 40). F, Regression analysis of the relationship between RIPK1 and MCU levels in colorectal cancer (n ¼ 40).






adenocarcinoma cell line. Meanwhile, mitochondria (indicatedbyMito-DsRed) and RIPK1 (infected with RIPK1-GFP virus) werealso colocalized in HT29 cells (Fig. 3D). Consistent with our IHCand coimmunoprecipitation results, the interaction score ofRIPK1 and MCU in the mass spectrum was also higher in colo-rectal cancer than in normal tissue (Fig. 3E–H). Taken together,the above results indicated that RIPK1 interacted with MCUin mitochondria and played a critical role in colorectal cancercarcinogenesis.

RIPK1 increases MCU expression in HT29 cellsTo evaluate the relationship between RIPK1 and MCU, we

silenced RIPK1 using siRNA. This, in turn, downregulated MCU(Fig. 4A and B), whereas overexpression of RIPK1 by adenovirus(Ad-RIPK1) led toupregulationofMCU inHT29 cells (Fig. 4CandD). However, knocking down (Fig. 4E and F) or overexpressingMCU (Fig. 4G and H) did not change the expression level ofRIPK1. These data indicated that RIPK1 triggered expression ofMCU upstream, and that MCU acted downstream on RIPK1.

Figure 3.

RIPK1 interacts with MCU in mitochondria. A, Coimmunoprecipitation of RIPK1 from normal and colorectal cancer (CRC) tissues (n ¼ 3). B,Coimmunoprecipitation ofMCU fromnormal and colorectal cancer tissues (n¼ 3).C,Coimmunoprecipitation of RIPK1 fromcytosolic (Cyto) andmitochondrial (Mito)fractions from normal and colorectal cancer tissues (n ¼ 3). D, Representative photomicrographs of HT29 cells transfected with Mito-DsRed, MCU-GFP, andRIPK1-GFP. Scale bar, 5 mm. E–H, Mass spectrum scores from normal and colorectal cancer tissues. MS, mass spectrum; IP, immunoprecipitation.

Figure 4.

RIPK1 increases expression of MCU. A, Representative Western blots of RIPK1 and MCU in HT29 cells transfected with si-RIPK1 (200 pmol/L). B, Statisticalanalysis of RIPK1 andMCU inHT29 cells transfectedwith si-RIPK1 (200pmol/L). Results are expressed asmean�SEM. �� ,P<0.01; �� ,P<0.001, control vs. treatment(n ¼ 6). C, Representative Western blots of RIPK1 and MCU in HT29 cells infected with Ad-RIPK1 (MOI 25). D, Statistical analysis of RIPK1 and MCU in HT29cells infectedwith Ad-RIPK1 (MOI 25). Results are expressed asmean� SEM. � , P <0.05; �� , P <0.001, control vs. treatment (n¼ 6). E, RepresentativeWestern blotsof RIPK1 and MCU in HT29 cells transfected with si-MCU (200 pmol/L). F, Statistical analysis of RIPK1 and MCU in HT29 cells transfected with si-MCU (200 pmol/L).Results are expressed as mean � SEM. �� , P < 0.01; N.S., not significant, control vs. treatment (n ¼ 6). G, Representative Western blots of RIPK1 and MCU inHT29 cells infected with Ad-MCU (MOI 25). H, Statistical analysis of RIPK1 and MCU in HT29 cells infected with Ad-MCU (MOI 25). Results are expressedas mean � SEM. � , P < 0.05; N.S., not significant, control vs. treatment (n ¼ 6).

Zeng et al.





RIPK1 and MCU promote cell proliferation by upregulatingmitochondrial Ca2þ uptake and energy metabolism

To investigate whether RIPK1 and MCU contributed to prolif-eration of tumor cells, we overexpressed or knocked down RIPK1or MCU in HT29 cells. Overexpression of RIPK1 increased cellproliferation, whereas knocking down RIPK1 decreased prolifer-ation (Fig. 5A). Cell proliferation also increased when MCU wasoverexpressed inHT29 cells but did not change significantly whenMCU was knocked down (Fig. 5B). Moreover, we found thatknockdown of RIPK1 or MCUwith shRNA significantly inhibitedtumor growth in a nude mouse tumor inoculation model whencomparedwith control cells (Fig. 5C–E). These data suggested thatboth RIPK1 and MCU promoted cellular proliferation.

MCU is critical for Ca2þ homeostasis and controls cell deathand survival (22, 33, 34). To test whether RIPK1 and MCU

mediate cell proliferation through mitochondrial Ca2þ uptake,we used Ca2þ imaging to measure mitochondrial and cytosolicCa2þ in the presence or absence of RIPK1orMCU.Overexpressionof RIPK1 profoundly augmented the level of mitochondrial Ca2þ

in HT29 cells treated with histamine (Fig. 5F–H). Notably, mito-chondrial Ca2þ uptake induced by RIPK1 was blocked by knock-ing down MCU (Fig. 5F and I). These results underscored theimportance of RIPK1 in regulating uptake of mitochondrial Ca2þ

through MCU.We used OCR to evaluate cellular energy state to reveal any

causal relationship between RIPK1 or MCU and energy metabo-lism. O2 consumption increased with overexpression of RIPK1 orMCU (Fig. 6A–D). Basal and maximal respirations were signifi-cantly higher inHT29 cells that overexpressedRIPK1orMCU thanin controls (Fig. 6A–F). Moreover, RIPK1 or MCU silencing

Figure 5.

RIPK1 and MCU promote cell proliferation by upregulating mitochondrial Ca2þ uptake. A, Statistical analysis of cell number in HT29 cells infected withAd-RIPK1 (MOI 25) or si-RIPK1 (200 pmol/L). Results are expressed as mean � SEM. �� , P < 0.001, control vs. Ad-RIPK1; ##, P < 0.01, control vs. si-RIPK1 (n ¼ 5).B, Statistical analysis of cell number in HT29 cells infected with Ad-MCU (MOI 25) or si-MCU (200 pmol/L). Results are expressed as mean � SEM. �� , P < 0.001,control vs. Ad-MCU (n ¼ 5). C, Representative photograph of tumors in HT29, RIPK1-KD, and MCU-KD cell–transplanted nude mice. D and E, Statisticalanalysis of mouse tumor volumes (D) andweights (E) in different groups (n¼ 4 in each group). � , P < 0.05; �� , P < 0.01, shNC vs. shMCU; #, P < 0.01, shNC vs. shRIPK1.F, Statistical analysis of mitochondrial Ca2þ levels in HT29 cells infected with Ad-RIPK1 (MOI 25) or Ad-RIPK1 þ si-MCU (200 pmol/L), as measured by Fluo-4 andRhod-2 fluorescence following histamine stimulation (100 mmol/L). Results are expressed as mean � SEM. �� , P < 0.001, control vs. Ad-RIPK1 (n¼ 13); ##, P < 0.01,Ad-RIPK1 vs. Ad-RIPK1 þ si-MCU (n ¼ 6). G, Representative cytosolic and mitochondrial Ca2þ levels in HT29 cells infected with Ad-b-gal. H, Representativecytosolic and mitochondrial Ca2þ levels in HT29 cells infected with Ad-RIPK1. I, Representative cytosolic and mitochondrial Ca2þ levels in HT29 cells infectedwith Ad-RIPK1 þ si-MCU. Black arrow, histamine stimulation.






mediated by siRNA inhibited OCR (Supplementary Fig. S6A andS6B), and OCR upregulation induced by RIPK1 was blocked byMCU knockdown (Fig. 6G–I). Both indicated that MCU wasimportant for RIPK1 to modulate energy metabolism.

The RIPK1-K377 site is critical for its interaction with MCUTo identify which site in RIPK1 was most critically interacting

with MCU, we made seven point mutation constructs of RIPK1(Fig. 7A). RIPK1 with the K377R point mutant, which is aubiquitination site, reduced RIPK1 interaction with MCU(Fig. 7B and C), and this mutation did not change the mitochon-drial location of RIPK1 (Supplementary Fig. S7A). A previousstudy showed that ubiquitination controls the switch between thepro-life and pro-death activity of RIPK1 (35). We found thatRIPK1 ubiquitination was upregulated in colorectal cancer com-paredwith normal paracarcinoma tissue (Fig. 7D).MitochondrialCa2þ uptake and the OCR were inhibited when HT29 cells weretransfected with RIPK1-K377R (Fig. 7E and F). Lactate dehydro-genase release increased and cell number decreased in HT29 cellstransfected with RIPK1-K377R (Fig. 7G and H), indicating thatcell death increased in HT29 cells transfected with RIPK1-K377R.Taken together, these data showed that the RIPK1-K377 ubiqui-tination site was pivotal, because it interacted with MCU tomediate cellular proliferation.

DiscussionRIPK1 is very important in determining cell death or survival

(31, 36–38). However, its possible roles in carcinogenesis remainlargely unknown. In this study, we demonstrated that the RIPK1

expression was upregulated in colorectal cancer samples, andoverexpression of RIPK1 in the HT29 human colon adenocarci-noma cell line was associated positively with cell proliferation,whereas knockdown of RIPK1 inhibited HT29 cell proliferation,together suggesting that RIPK1 was involved in colorectal cancerprogression by promoting cell proliferation. We also demonstrat-ed that MCU was a target of RIPK1 and mediated RIPK1-depen-dent cell proliferation by upregulating mitochondrial Ca2þ

uptake and energy metabolism, thereby revealing that RIPK1mediates a new cell proliferation pathway. These results not onlydemonstrated the role of RIPK1 in promoting colorectal cancerdevelopment and progression but also showed that RIPK1 helpsMCU mediate proliferation in colorectal cancer. Notably, anRIPK1-K377 mutant (RIPK1-K377R) was unable to recruit theessential modulator NFkB to the TNFR1 complex and failed toundergo ubiquitination; this process is important for RIPK1 toregulate cell survival (39, 40). Here, we found that RIPK1-K377was also critical for the interaction of RIPK1 with MCU inpromoting proliferation. A mutation of RIPK1 (K377R) reducedproliferation by inhibiting mitochondrial Ca2þ uptake by MCUand reducing metabolism.

There are considerable lines of evidence that altering expressionlevels or activating Ca2þ signaling via Ca2þ channels, Ca2þ trans-porters, or Ca2þ-ATPases are associated with carcinogenesis(21, 41, 42). In this study, we found that overexpression of MCUwas positively associated with colorectal cancer carcinogenesis;however, contradictory results have been reported. Maichi andcolleagues reported that overexpression of cancer-related miR25in HeLa cells significantly downregulates MCU expressionand mitochondrial Ca2þ uptake, and protects HeLa cells from

Figure 6.

RIPK1 regulates energy metabolism via MCU. A, OCRs of HT29 cells transfected with Ad-RIPK1 (MOI 25). B, Statistical analysis of basal respiration of HT29cells transfected with Ad-RIPK1 (MOI 25). Every point represents average of three different wells. Results are expressed as mean � SEM. �� , P < 0.001,control vs. treatment (n ¼ 5). C, Statistical analysis of maximal respiration of HT29 cells transfected with Ad-RIPK1 (MOI 25). Every point representsaverage of three different wells. Results are expressed as mean � SEM. �� , P < 0.001, control vs. treatment (n ¼ 5). D, OCRs of HT29 cells transfected withAd-MCU (MOI 25). E, Statistical analysis of basal respiration of HT29 cells transfected with Ad-MCU (MOI 25). Every point represents average of three different wells.Results are expressed as mean � SEM. �� , P < 0.001, control vs. treatment (n ¼ 4). F, Statistical analysis of maximal respiration of HT29 cells transfectedwith Ad-MCU (MOI 25). Every point represents average of three different wells. Results are expressed as mean� SEM. �� , P < 0.001, control vs. treatment (n¼ 4).G, OCRs of HT29 cells transfected with Ad-RIPK1 (MOI 25) or Ad-RIPK1 (MOI 25) þ si-MCU (200 pmol/L). H, Statistical analysis of basal respiration of HT29cells transfected with Ad-RIPK1 (MOI 25) or Ad-RIPK1 (MOI 25) þ si-MCU (200 pmol/L). I, Statistical analysis of maximal respiration of HT29 cellstransfected with Ad-RIPK1 (MOI 25) or Ad-RIPK1 (MOI 25) þ si-MCU (200 pmol/L). Every point represents average of three different wells. Results areexpressed as mean � SEM. �� , P < 0.001, Ad-RIPK1 vs. Ad-RIPK1 (MOI 25) þ si-MCU (200 pmol/L; n ¼ 5).

Zeng et al.





apoptosis (43). They also found thatmiR25 is increased andMCUis reduced in human colon cancer tissue and in many cancer-derived cell lines, and they hypothesize that decreased mitochon-drial Ca2þ may be related to cancer cell survival (43). However,there is no direct evidence associating MCU downregulation withcarcinogenesis. On the contrary, studies have shown that expres-sion of MCU correlates positively with tumor size and lymphnode infiltration in triple-negative breast cancer, demonstratingthat mitochondrial Ca2þ uptake is important for tumor growthand metastasis (44). Other experiments demonstrated that MCUis important for survival and migration of breast carcinoma cells(25, 27). Therefore, the role of MCU in previous studies in celldeath and tumorigenesis remains controversial. To our knowl-edge, no data have been reported on the functional interdepen-dence of MCU and RIPK1 in the progression of human colorectalcancer. In this study, we provide evidence that in colon cancercells, RIPK1 primarily regulates a critical survival signal via MCUin the context of cell proliferation evoked by increasingmitochondrial Ca2þ uptake and metabolism. Mechanistically,RIPK1 constitutes an important upstream molecule for MCU,which regulates mitochondrial Ca2þ and metabolism. We pin-pointedMCUas an essential downstream component of RIPK1 ininducing cell proliferation and energy metabolism. However, thepossibility that a cell survival function of traditional RIPK1-NFkBmight be relevant to human colorectal cancer could notbe excluded.

With regard to the mechanism by which RIPK1 is upregu-lated in colorectal cancer, we found that RIPK1 mRNA wassignificantly increased. In addition, the ubiquitination ofRIPK1, a modification that is generally thought to stabilizethe RIPK1 protein (45), also was higher in colorectal cancerthan in normal paracarcinoma tissues, suggesting that both

transcriptional and posttranslational mechanisms wereinvolved in upregulating RIPK1. High levels of RIPK1 in coloncancer may lead to severe disturbance of calcium balance andabnormal energy metabolism, leading to tumor progressionand poorer prognosis.

Our findings, together with previous reports, confirmed thatubiquitination of RIPK1 plays a role in cell proliferation andidentify a distinct RIPK1-mediated molecular signaling path-way (31, 39). RIPK1 may be a better target for developingtreatments because it is known to regulate cancer cell deathinduced by various stimuli (14, 46). Our results not onlydefined RIPK1 as a regulator of mitochondrial Ca2þ uptakebut also established RIPK1-mediated proliferation throughMCU as a central mechanism underlying colorectal cancerprogression. RIPK1 and MCU were marked as potentiallyimportant therapeutic targets to treat colorectal cancer.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: F. Zeng, Y. Yuan, R.-P. Xiao, J. Cai, X. ZhangDevelopment of methodology: F. Zeng, W. Cui, F. Lu, X. Sun, D. Ma, Y. Zhang,X. ZhangAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): F. Zeng, X. Chen, W. Cui, W. Wen, F. Lu, Z. Li,H. Zhao, X. Bi, J. Zhao, J. Zhou, Y. Zhang, X. ZhangAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): F. Zeng, X. Chen, W. Cui, H. Zhao, X. ZhangWriting, review, and/or revision of themanuscript: F. Zeng,W. Cui, R.-P. Xiao,X. ZhangAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): F. Zeng, X. Sun, D. Ma, N. Hou, X. ZhangStudy supervision: F. Zeng, J. Cai, X. Zhang

Figure 7.

The K377 site of RIPK1 is critical for its interaction with MCU. A, Schema of RIPK1 structure. B and C, Coimmunoprecipitation of RIPK1 (B) or HA (C) in HT29 cellstransfected with RIPK1, RIPK1-K45A, RIPK1-S161A, RIPK1-D324K, RIPK1-K377R, RIPK1-K530A, or RIPK1-I539A (2 mg/mL) for 48 hours. D, Ubiquitination (Ub) ofRIPK1 from normal and colorectal cancer (CRC) tissues (n ¼ 3). E, Statistical analysis of mitochondrial and cytosolic Ca2þ levels in HT29 cells infected withRIPK1-K377R (2 mg/mL), as measured by Fluo-4 and Rhod-2 fluorescence following stimulation with histamine (100 mmol/L). �� , P < 0.001, RIPK1 vs. RIPK1-K377R(n ¼ 12). F, Oxygen consumption of HT29 cells infected with RIPK1-K377R. �� , P < 0.001, RIPK1 vs. RIPK1-K377R (n ¼ 5). G, Statistical analysis of lactatedehydrogenase (LDH) release inHT29 cells infectedwithRIPK1-K377R. �� ,P<0.001, RIPK1 vs. RIPK1-K377R (n¼ 12).H,Statistical analysis of cell number inHT29 cellsinfected with RIPK1-K377R. �� , P < 0.001, RIPK1 vs. RIPK1-K377R (n ¼ 5). Results are expressed as mean � SEM.






AcknowledgmentsThe authors thank Drs. Heping Cheng, I.C. Bruce, and Jon K. Moon for

constructive comments on the manuscript and help with English editing. Theauthors also thankYanruWang for Seahorse technique assistance. Thisworkwassupported by grants from the National Natural Science Foundation of China(81471063 to X. Zhang; 81270883 to X. Zhang; 81672461 to H. Zhao), theNational Science and Technology Major Projects for "Major New Drug Inno-vation and Development" (2013ZX09501014 to X. Zhang), the National KeyBasic ResearchProgramofChina (2013CB531200 toH.Cheng; 2012CB518000to R.-P. Xiao), the CAMS Innovation Fund for Medical Sciences (CIFMS;

2016-I2M-1-001 to J. Cai; 2017-12M-4-002 to H. Zhao), and the NationalHigh-tech R&D (863) Program of China (2015AA020408 to J. Cai).

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received October 10, 2017; revised February 11, 2018; accepted March 6,2018; published first March 12, 2018.

References1. Siegel RL, Miller KD, Jemal A. Cancer statistics, 2016. CA Cancer J Clin

2016;66:7–30.2. ChenW, Zheng R, Baade PD, Zhang S, ZengH, Bray F, et al. Cancer statistics

in China, 2015. CA Cancer J Clin 2016;66:115–32.3. CunninghamD, AtkinW, LenzHJ, LynchHT,Minsky B, Nordlinger B, et al.

Colorectal cancer. Lancet 2010;375:1030–47.4. Kinzler KW, Vogelstein B. Cancer-susceptibility genes. Gatekeepers and

caretakers. Nature 1997;386:761.5. Calvert PM, Frucht H. The genetics of colorectal cancer. Ann Intern Med

2002;137:603–12.6. Lynch HT, de la Chapelle A. Hereditary colorectal cancer. N Engl J Med

2003;348:919–32.7. Hanahan D, Weinberg RA. Hallmarks of cancer: the next generation. Cell

2011;144:646–74.8. Festjens N, Vanden Berghe T, Cornelis S, Vandenabeele P. RIP1, a kinase on

the crossroads of a cell's decision to live or die. Cell Death Differ2007;14:400–10.

9. Meylan E, Tschopp J. The RIP kinases: crucial integrators of cellular stress.Trends Biochem Sci 2005;30:151–9.

10. Temkin V, Huang Q, Liu H, Osada H, Pope RM. Inhibition of ADP/ATPexchange in receptor-interacting protein-mediated necrosis. Mol Cell Biol2006;26:2215–25.

11. Sun X, Yin J, Starovasnik MA, Fairbrother WJ, Dixit VM. Identification ofa novel homotypic interaction motif required for the phosphorylationof receptor-interacting protein (RIP) by RIP3. J Biol Chem 2002;277:9505–11.

12. Pasparakis M, Vandenabeele P. Necroptosis and its role in inflammation.Nature 2015;517:311–20.

13. Wu XN, Yang ZH,Wang XK, Zhang Y,WanH, Song Y, et al. Distinct roles ofRIP1-RIP3 hetero- and RIP3-RIP3 homo-interaction in mediating necrop-tosis. Cell Death Differ 2014;21:1709–20.

14. Wang L,Du F,WangX. TNF-alpha induces two distinct caspase-8 activationpathways. Cell 2008;133:693–703.

15. Kelliher MA, Grimm S, Ishida Y, Kuo F, Stanger BZ, Leder P. The deathdomain kinase RIP mediates the TNF-induced NF-kappaB signal. Immu-nity 1998;8:297–303.

16. DannappelM, Vlantis K, Kumari S, Polykratis A, KimC,Wachsmuth L, et al.RIPK1 maintains epithelial homeostasis by inhibiting apoptosis andnecroptosis. Nature 2014;513:90–4.

17. Seifert L, Werba G, Tiwari S, Giao Ly NN, Alothman S, Alqunaibit D,et al. The necrosome promotes pancreatic oncogenesis via CXCL1and Mincle-induced immune suppression. Nature 2016;532:245–9.

18. Liu XY, Lai F, Yan XG, Jiang CC, Guo ST, Wang CY, et al. RIP1 kinase is anoncogenic driver in melanoma. Cancer Res 2015;75:1736–48.

19. MonteithGR,McAndrewD, FaddyHM, Roberts-Thomson SJ. Calcium andcancer: targeting Ca2þ transport. Nat Rev Cancer 2007;7:519–30.

20. Prevarskaya N, Skryma R, Shuba Y. Calcium in tumour metastasis: newroles for known actors. Nat Rev Cancer 2011;11:609–18.

21. Cui CC, Merritt R, Fu LW, Pan Z. Targeting calcium signaling in cancertherapy. Acta Pharm Sin B 2017;7:3–17.

22. Baughman JM, Perocchi F, Girgis HS, Plovanich M, Belcher-Timme CA,Sancak Y, et al. Integrative genomics identifies MCU as an essentialcomponent of the mitochondrial calcium uniporter. Nature 2011;476:341–5.

23. De Stefani D, Raffaello A, Teardo E, Szabo I, Rizzuto R. A forty-kilodaltonprotein of the inner membrane is the mitochondrial calcium uniporter.Nature 2011;476:336–40.

24. Kamer KJ, Mootha VK. The molecular era of the mitochondrial calciumuniporter. Nat Rev Mol Cell Biol 2015;16:545–53.

25. Hall DD, Wu Y, Domann FE, Spitz DR, Anderson ME. Mitochondrialcalcium uniporter activity is dispensable for MDA-MB-231 breast carcino-ma cell survival. PLoS One 2014;9:e96866.

26. Curry MC, Peters AA, Kenny PA, Roberts-Thomson SJ, Monteith GR.Mitochondrial calcium uniporter silencing potentiates caspase-indepen-dent cell death in MDA-MB-231 breast cancer cells. Biochem Biophys ResCommun 2013;434:695–700.

27. Tang S, Wang X, Shen Q, Yang X, Yu C, Cai C, et al. Mitochondrial Ca(2)(þ) uniporter is critical for store-operated Ca(2)(þ) entry-dependentbreast cancer cell migration. Biochem Biophys Res Commun 2015;458:186–93.

28. Sun L, Wang H, Wang Z, He S, Chen S, Liao D, et al. Mixed lineage kinasedomain-like protein mediates necrosis signaling downstream of RIP3kinase. Cell 2012;148:213–27.

29. Ting AT, Pimentel-Muinos FX, Seed B. RIP mediates tumor necrosis factorreceptor 1 activation of NF-kappaB but not Fas/APO-1-initiated apoptosis.EMBO J 1996;15:6189–96.

30. MicheauO, Tschopp J. Induction of TNF receptor I-mediated apoptosis viatwo sequential signaling complexes. Cell 2003;114:181–90.

31. Christofferson DE, Li Y, Yuan J. Control of life-or-death decisions by RIP1kinase. Annu Rev Physiol 2014;76:129–50.

32. Perocchi F, Gohil VM, Girgis HS, Bao XR, McCombs JE, Palmer AE, et al.MICU1 encodes a mitochondrial EF hand protein required for Ca(2þ)uptake. Nature 2010;467:291–6.

33. Clapham DE. Calcium signaling. Cell 2007;131:1047–58.34. Rizzuto R, De Stefani D, Raffaello A, Mammucari C. Mitochondria as

sensors and regulators of calcium signalling. Nat Rev Mol Cell Biol2012;13:566–78.

35. O'Donnell MA, Ting AT. RIP1 comes back to life as a cell death regulator inTNFR1 signaling. FEBS J 2011;278:877–87.

36. Jin L, Chen J, Liu XY, Jiang CC, Zhang XD. The double life of RIPK1. MolCell Oncol 2016;3:e1035690.

37. Han J, Zhong CQ, Zhang DW. Programmed necrosis: backup to andcompetitor with apoptosis in the immune system. Nat Immunol2011;12:1143–9.

38. Mandal P, Berger SB, Pillay S, Moriwaki K, Huang C, Guo H, et al. RIP3induces apoptosis independent of pronecrotic kinase activity. Mol Cell2014;56:481–95.

39. Ea CK, Deng L, Xia ZP, Pineda G, Chen ZJ. Activation of IKK by TNFalpharequires site-specific ubiquitination of RIP1 and polyubiquitin binding byNEMO. Mol Cell 2006;22:245–57.

40. Li H, Kobayashi M, Blonska M, You Y, Lin X. Ubiquitination of RIP isrequired for tumor necrosis factor alpha-induced NF-kappaB activation.J Biol Chem 2006;281:13636–43.

41. Roderick HL, Cook SJ. Ca2þ signalling checkpoints in cancer: remodellingCa2þ for cancer cell proliferation and survival. Nat Rev Cancer2008;8:361–75.

42. Monteith GR, Davis FM, Roberts-Thomson SJ. Calcium channels andpumps in cancer: changes and consequences. J Biol Chem 2012;287:31666–73.

Zeng et al.





43. Marchi S, Lupini L, Patergnani S, Rimessi A, Missiroli S, Bonora M, et al.Downregulation of the mitochondrial calcium uniporter by cancer-relatedmiR-25. Curr Biol 2013;23:58–63.

44. Tosatto A, Sommaggio R, KummerowC, Bentham RB, Blacker TS, Berecz T,et al. The mitochondrial calcium uniporter regulates breast cancer pro-gression via HIF-1alpha. EMBO Mol Med 2016;8:569–85.

45. Bertrand MJ, Milutinovic S, Dickson KM, Ho WC, Boudreault A,Durkin J, et al. cIAP1 and cIAP2 facilitate cancer cell survival byfunctioning as E3 ligases that promote RIP1 ubiquitination. Mol Cell2008;30:689–700.

46. Christofferson DE, Yuan J. Necroptosis as an alternative form of pro-grammed cell death. Curr Opin Cell Biol 2010;22:263–8.






2018;78:2876-2885. Published OnlineFirst March 12, 2018.Cancer Res Fanxin Zeng, Xiao Chen, Weiyi Cui, et al. Uptake and Promotes Colorectal Oncogenesis

2+RIPK1 Binds MCU to Mediate Induction of Mitochondrial Ca

Updated version

10.1158/0008-5472.CAN-17-3082doi:

Access the most recent version of this article at:

Material

Supplementary

http://cancerres.aacrjournals.org/content/suppl/2018/03/10/0008-5472.CAN-17-3082.DC1

Access the most recent supplemental material at:

Cited articles

http://cancerres.aacrjournals.org/content/78/11/2876.full#ref-list-1

This article cites 46 articles, 6 of which you can access for free at:

Citing articles

http://cancerres.aacrjournals.org/content/78/11/2876.full#related-urls

This article has been cited by 4 HighWire-hosted articles. Access the articles at:

E-mail alerts related to this article or journal.Sign up to receive free email-alerts

Subscriptions

Reprints and

[email protected]

To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at

Permissions

Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.http://cancerres.aacrjournals.org/content/78/11/2876To request permission to re-use all or part of this article, use this link



http://cancerres.aacrjournals.org/lookup/doi/10.1158/0008-5472.CAN-17-3082

http://cancerres.aacrjournals.org/content/suppl/2018/03/10/0008-5472.CAN-17-3082.DC1

http://cancerres.aacrjournals.org/content/78/11/2876.full#ref-list-1

http://cancerres.aacrjournals.org/content/78/11/2876.full#related-urls

http://cancerres.aacrjournals.org/cgi/alerts

mailto:[email protected]

http://cancerres.aacrjournals.org/content/78/11/2876


ripk1 binds mcu to mediate induction of mitochondrial ca ...essential signaling molecule in pathways...

Documents