Hypercholesterolemia Increases Colorectal Cancer Incidence ......Molecular and Cellular Pathobiology...

Molecular and Cellular Pathobiology

Hypercholesterolemia Increases ColorectalCancer Incidence by Reducing Production of NKTand gd T Cells from Hematopoietic Stem CellsGuodong Tie1, Jinglian Yan1, Lyne Khair1, Julia A. Messina2, April Deng3, Joonsoo Kang3,Thomas Fazzio4, and Louis M. Messina1

Abstract

Obesity will soon surpass smoking as the most preventablecause of cancer. Hypercholesterolemia, a common comorbidity ofobesity, has been shown to increase cancer risk, especially colo-rectal cancer. However, the mechanism by which hypercholester-olemia or any metabolic disorder increases cancer risk remainsunknown. In this study, we show that hypercholesterolemiaincreases the incidence and pathologic severity of colorectal neo-plasia in two independent mouse models. Hypocholesterolemiainduced an oxidant stress–dependent increase in miR101c, which

downregulated Tet1 in hematopoietic stem cells (HSC), resultingin reduced expression of genes critical to natural killer T cell (NKT)and gd T-cell differentiation. These effects reduced the number andfunction of terminally differentiated NKT and gd T cells in thethymus, the colon submucosa, and during early tumorigenesis.These results suggest a novel mechanism by which a metabolicdisorder induces epigenetic changes to reduce lineage priming ofHSC toward immune cells, thereby compromising immunosur-veillance against cancer. Cancer Res; 77(9); 2351–62. �2017 AACR.

IntroductionObesity will soon surpass smoking as the most preventable

cause of cancer (1, 2). Studies to determine which metabolicdisorder or combination of disorders in obese people increasestheir cancer risk have been inconclusive (3). Hypercholesterol-emia, a common metabolic disorder in obese people, has beenshown to increase cancer risk and substantial epidemiologicevidence links hypercholesterolemia to an increased risk of colo-rectal cancer (4). It was originally proposed that hypercholester-olemia exerts a systemic, conditional influence that affects immu-nosurveillance against colorectal cancer. In support of thishypothesis, hypercholesterolemia has been shown to reduce thefrequency and function of the cellular components of tumorimmunosurveillance (5, 8). The mechanism by which hypercho-lesterolemia reduces the number and function of innate immunecells is unknown, nor is it clear if such an effect compromisesimmunosurveillance against colorectal cancer.

Emerging evidence suggests that, although hematopoieticstem cells (HSC) maintain an undifferentiated state, activatingepigenetic marks and low-level expression of lineage-associatedgenes, a process known as lineage priming, keep HSCs respon-sive to physiologic and pathologic demands of immune cells(9–12). Recently, we have shown that hypercholesterolemiainduces oxidant stress in HSCs that accelerates their aging andimpairs their repopulation capacity (13). With these findings,we hypothesize that hypercholesterolemia-induced oxidantstress reduces HSC lineage priming toward innate immunecells and thereby impairs immunosurveillance against colorec-tal cancer.

Here, we show that hypercholesterolemia-induced oxidantstress downregulates Ten Eleven Translocation 1 (Tet1) in HSCs,resulting in increased DNA hypermethylation and histone mod-ifications in the genes critical to natural killer T cell (NKT) and gdT-cell differentiation. These effects reduced the number andfunction of terminally differentiated NKT and gdT cells in thethymus, the colon submucosa and at the early stages of tumor-igenesis and thereby impaired immunosurveillance against colo-rectal neoplasia.

Materials and MethodsMice

All mice were purchased from Jackson Laboratories. Care ofmice was in accordance with NIH guidelines. ApoE�/� and wild-type (WT) mice were fed standard mouse chow (5.4 g fat/100 gdiet, 0%cholesterol).HCDmicewere fed adietwith 10 g fat/100gdiet, 11.25 g cholesterol/100 g diet (Research Diets). NAC wasgiven for 8 weeks (150 mg/kg/day via drinking water).

Cell lines293T (CRL-3216) cell lines were obtained from the ATCC

repository. Cell line characterization by ATCC is conducted by

1Diabetes Center of Excellence and Division of Vascular and EndovascularSurgery, University ofMassachusettsMedical School,Worcester,Massachusetts.2Division of Infectious Diseases, Duke University School of Medicine, Durham,North Carolina. 3Department of Pathology, University of Massachusetts MedicalSchool, Worcester, Massachusetts. 4Department of Molecular, Cell, and CancerBiology, University ofMassachusetts Medical School,Worcester, Massachusetts.

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

G. Tie and J. Yan contributed equally to this article.

Corresponding Author: Louis M. Messina, University of Massachusetts MedicalSchool, 55 Lake Avenue North, Worcester, MA 01655. Phone: 508-856-5599;Fax: 508-856-8329; E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-16-1916

�2017 American Association for Cancer Research.

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short tandem repeat (STR) analysis. OP9-DL1 cells were kindlyprovided by Dr. Juan Carlos Z�u~niga-Pfl€ucker (University of Tor-onto, Toronto, Canada). Upon receiving the cell lines, frozenstocks were prepared within one to five passages and new stockswere thawed frequently to keep the original condition. The celllines were passaged for less than 3 months after receipt orresuscitation. Cells were authenticated by morphology, pheno-type, and growth, routinely screened for mycoplasma.

Tumor induction and analysisThe colorectal neoplasia experiments were performed as

described in previous publications (14). Three-month-old micewere subcutaneously injected with a solution of AOM at a doserate of 15 mg/kg body weight, once weekly for 3 successiveweeks. Two percent of DSS was given in the drinking water over5 days in the last week. Mice were sacrificed 10 weeks after thelast injection of AOM. Tumor counts and histopathologicstaging of tumors were performed by a cancer pathologist ina blind fashion.

Flow cytometry and HSC isolationCells were stained with mAbs conjugated to various fluorop-

robes. These antibodies included: cKit (2B8), Sca-1 (E13-161.7),CD4 (L3T4), CD8 (53-6.72), CD90.1, CD25, CD44, TCRb,NK1.1, gdTCR, CD45.1, CD45.2. The lineage cocktail consistedof CD4, CD8, B220 (RA3-6B2), TER-119, Mac-1 (MI/70), andGr-1 (RB6-8C5). All antibodies were purchased from BD Bio-science. CD1d-GalCer tetramer was obtained from the NIHTetramer facility. FACS analysis was carried out on a FACS Divaor MoFlow. HSCs were isolated from the bone marrow anddefined as cKitþ sca-1þ CD90.1lo/�Lin� (KTLS).

Lentiviral particle preparation and transductionThe Tet1 specific and control shRNA plasmids were purchased

from Santa Cruz Biotechnology. The plasmid with Tet1 catalyticdomain (pTYF-U6-shCONT-EF1-Puro-2A-CD1) was a gift fromDr. Yi Zhang (Boston Children's Hospital, Boston, MA). Theenvelope and helper plasmids were purchased from ABM. Thelentiviral particles were prepared according to the kit instructions.Fresh isolated KSL cells were transduced with lentivirus for 24hours and then selected with puromycin (2 mg/mL; Santa CruzBiotechnology) for 72 hours.

Preparation of oxLDLNative LDL (nLDL) was purchased from Sigma. OxLDL was

prepared by incubating nLDL with 10 mmol/L CuSO4 at 37�Cfor 24 hours. The material was dialyzed against a sterile solution(150 mmol/L NaCl, 1 mmol/L EDTA, 100 mg/mL polymyxin B,pH 7.4) and then sterilized by filtration. The extent of LDLoxidation was estimated by agarose gel electrophoresis and bymeasuring the amount of thiobarbituric acid reactive substancesgenerated (Supplementary Fig S1A and S1B).

HSCs and OP9 cell cocultureThe coculture was performed as described (15, 16). KTLS cells

were seeded at 4� 103 cells/well into 12-well tissue culture platescontaining a confluent monolayer of OP9-DL1 cells. All cultureswereperformed in thepresence of 5ng/mL IL2, 10ng/mLGM-CSF(Stem cell Technology), 5 ng/mL IL7, and 5 ng/mL mFLT3(PeproTech).

IHCWe used a standard protocol to detect NKT and gdT cells in

colon and tumor tissues. The antibodies were purchased from BDBiosciences. For indirect IHC, we used rabbit-specific IgG conju-gated with FITC or PE (Chemicon) as a secondary antibody.Fluorescent images were obtained using a confocal laser scanningmicroscope (Carl Zeiss LSM 510 system; Carl Zeiss).

Chromatin immunoprecipitationChromatin immunoprecipitation (ChIP) was performed fol-

lowing a standard protocol. A detailed description canbe found inSupplementary Methods.

qRT-PCRWe reverse transcribed cDNAs from total RNA isolated from

each cell fraction using RNAqueous-Micro Kit (Ambion). Tran-scription to cDNA was performed using SuperScript III (Invitro-gen). All PCRs were carried out in triplicate using an EppendorfMastercycler (Eppendorf).

DNA extraction, bisulfite conversion, and pyrosequencingGenomic DNA was extracted with a standard protocol. PCRs

were performed using a converted DNA by 2xHiFi HotstartUracilþReady Mix RCR Kit (Kapa Biosystems). Please see Sup-plementary Methods for detailed description.

miRNA microarray expression profiling, miRNA targetprediction, and mRNA 30-UTR cloning and luciferase reporterassay

Total RNAwas isolated using themirVanamiRNA Isolation Kitaccording to manufacturer's instructions (Applied Biosystems).The predicted target genes of differentially expressed miRNAswere obtained using the following tools: TargetScan v6.2 andmiRDB. Please see Supplementary Methods for detaileddescription.

Statistical analysisAll data were shown as means � SD. Statistical analyses

were carried out with either GraphPad Prism (GraphPadSoftware) or SPSS v19 (IBM) software. Statistical significancewas evaluated by using a one- or two-way ANOVA or anunpaired t test. Significance was established for P values of atleast <0.05.

ResultsHypercholesterolemia increases the incidence andhistopathologic severity of colorectal neoplasia by an HSC-autonomous mechanism

We first induced colorectal neoplasia with azoxymethane(AOM) in two common mouse models of hypercholesterolemia,the ApoE�/� mouse, and the C57BL/6 mouse fed a high choles-terol diet (HCD). The average tumor numberwas almost two-foldhigher in hypercholesterolemic mice than in WT mice (Fig. 1A),indicating that hypercholesterolemia increases the incidence ofcolorectal neoplasia. The tumors at late stages of tumorigenesis,including adenomaþþþ and carcinomas, were not found inWT control mice, but constituted more than 10% of the tumorsfound in hypercholesterolemic mice. Meanwhile the, tumors atthe early stages of tumorigenesis, including hyperplasia andadenomaþ, were dramatically reduced in hypercholesterolemic

Tie et al.

Cancer Res; 77(9) May 1, 2017 Cancer Research2352




mice (Fig. 1B), indicating that hypercholesterolemia significantlyincreases the incidence and the pathological severity of colorectalneoplasia.

To determine whether hypercholesterolemia-induced oxidantstress causes an HSC-autonomous defect that increases the inci-dence of colorectal neoplasia by impairing HSC lineage specifi-cation, we induced colorectal neoplasia in a chimeric modelwhereby hematopoiesis was reconstituted in lethally irradiatedWT recipient mice (CD45.1) with HSCs from either ApoE�/� orWT mice. In the recipient WT mice, the thymus was repopulatedwith cells that were derived from the donor HSCs (CD45.2;Supplementary Fig. S1C). As expected, the serum cholesterol andwhite blood cell counts of the recipient mice were normal andcomparable to those of WT mice (Supplementary Fig. S1D andS1E). Despite normal serum cholesterol levels, the average tumornumber and their histopathologic severity was significantly great-er in recipients that received HSCs from ApoE�/� mice than inthose that received HSCs from WT mice (Fig. 1C and D). Thus,both chimeric models in which WT mice received HSCs fromApoE�/� mice recapitulated the increased tumor incidence andgreater histopathologic severity seen in hypercholesterolemicmice. These results show that the increased incidence of colorectalneoplasia in hypercholesterolemic mice is due to a HSC-auton-omous mechanism.

Hypercholesterolemia specifically reduces the differentiationof HSCs toward NKT and gdT cells

Upon TCR activation, NKT and gdT cells rapidly secrete avariety of cytokines that are critical for the antitumor functionsof cytotoxic T cells. In addition to the cytokines producedby antigen-presenting cells with which NKT and gdT cellsinteract, these cytokines recruit and stimulate the antitumorfunctions of cytotoxic T cells, boosting innate and adaptiveantitumor responses. Activated NKT and gdT cells both havestrong cytotoxic effector activity (17–19). In this context,NKT and gdT cells function as major participants in tumorimmunosurveillance.

The populations of gdT cells and NKT cells in the thymus weresignificantly lower in hypercholesterolemic mice than inWTmice(Fig. 2A and B; Supplementary Fig. S2A and S2B). IntrathymicNKT-cell development in ApoE�/� mice was identical to WT atphases 1 (CD44�NK1.1�) and 2 (CD44þNK1.1�; SupplementaryFig. S2C) as well as the CD4 subsets of NKT cells (SupplementaryFig. S2D). T-cell developmental intermediates were similar in allgroups (Supplementary Fig. S2E and S2F). Except for a slightdecrease in B cells and a slight increase in NK cells, FACS analysisdid not show any significant change in CD3eþ, CD4þ, and CD8þ

T-cell populations in peripheral blood of hypercholesterolemicmice (Supplementary Fig. S2G). In the thymus of WT recipient

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Figure 1.

Hypercholesterolemia increasesaverage tumor number andhistopathologic stage of colorectalneoplasia through a hematopoieticstem cell autonomous manner.A, Average tumor number per mousefrom WT, ApoE�/�, and HCD mice.B, Histopathologic stages of the tumorsfrom WT, ApoE�/�, and HCD mice.n ¼ 12. C, Average tumor number fromWT recipient mice reconstituted withHSCs from WT or ApoE�/� mice.D, Histopathologic stages of the tumorsfrom WT recipient mice reconstitutedwith HSCs from WT or ApoE�/� mice.n ¼ 12. See also Supplementary Fig. S3.

Hypercholesterolemia Impairs Colon Cancer Immunosurveillance

www.aacrjournals.org Cancer Res; 77(9) May 1, 2017 2353




mice reconstituted with HSCs from ApoE�/�mice, we observed anearly identical decrease in differentiation toward NKT and gd Tcells as that seen in ApoE�/� mice (Fig. 2C and D). These results

indicate that hypercholesterolemia specifically induces a cell-autonomous reduction in lineage specification of HSCs towardNKT and gd T cells.

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Hypercholesterolemia significantly impairs the differentiation of HSCs toward NKT and gdT cells, which are critical components of innate immunity againstcolorectal neoplasia. A, Frequency and total number of NKT cells in thymus of WT and ApoE�/� mice. n ¼ 8. B, Frequency and total number of gdT cells inthymus of WT and ApoE�/� mice. n ¼ 8. C, Frequency and total number of NKT cells in thymus of lethally irradiated WT recipients reconstituted with HSCsfrom WT or ApoE�/� mice. n ¼ 8. D, Frequency and total number of gdT cells in thymus of lethally irradiated WT recipients reconstituted with HSCs fromWT or ApoE�/� mice. n ¼ 8. E, Frequency of submucosal NKT cells in colon of WT and ApoE�/� mice. n ¼ 8. F, Frequency of submucosal gdT cells in colon of WTand ApoE�/� mice. n ¼ 8. G, Average tumor number and histopathologic stage of AOM-induced colorectal neoplasia in CD1d�/� mice. n ¼ 10. H, Averagetumor number and histopathologic stages of AOM-induced colorectal neoplasia in Tcrd�/� mice. n ¼ 10. I, Frequency of NKT cells in the tumors from WT andApoE�/� mice. n ¼ 8. J, Frequency of gdT cells in the tumors from WT and ApoE�/� mice. n ¼ 8. See also Supplementary Fig. S2.

Tie et al.





Consistent with the reduction of NKT and gd T-cell maturationin the thymus, we also found a robust reduction of NKT cells(up to 6-fold) and gd T cells (3-fold) in the colon submucosaof ApoE�/� mice (Fig. 2E and F). Meanwhile, the other majorcellular components of cancer immunosurveillance, includingNKcells, CD11b�dendritic cells, CD11bþdendritic cells, CD11c�

macrophages, and CD11cþ macrophages in the colon of hyper-cholesterolemic mice did not show significant changes (Supple-mentary Fig. S2H).

NKT and gdT cells are critical components ofimmunosurveillance against colorectal neoplasiainduced by AOM

To confirm if this substantial reduction of NKT and gdT cellsimpairs tumor immunosurveillance,wedetermined the incidenceof AOM-induced colorectal neoplasia in Tcrd�/�mice, which lackgdT cells, and CD1d�/� mice, which lack NKT cells. Both micestrains showed significantly higher incidence and greater histo-pathologic severity of colorectal neoplasia than their controlstrains (Fig. 2G and H). In addition, we also found significantlyfewer NKT and gdT cells infiltrated into the early but not laterstages of tumor progression fromhypercholesterolemicmice thanin those from WT mice (Fig. 2I and J). Together, these resultsindicate that hypercholesterolemia reduces the differentiation ofHSCs toward NKT and gdT cells, resulting in impaired tumorimmunosurveillance against colorectal neoplasia.

The incidence of colorectal neoplasia is a linear function ofhypercholesterolemia-induced HSC oxidant stress

We previously showed that hypercholesterolemia induces anoxLDL-dependent increase of oxidant stress in HSCs that accel-erated their ageing and impaired their repopulation capacity, bothof which were reversed by the antioxidant N-acetylcysteine (NAC;ref. 13). Interestingly, NAC administration rescued the otherwiseimpaired differentiation toward NKT and gd T cells in hyper-cholesterolemic mice (Supplementary Fig. S3A and S3B). NACalso significantly decreased the average tumor number inApoE�/�

and HCD mice. Although NAC reduced the histopathologicseverity of tumors in HCD mice, the reduction in the histopath-ologic severity of tumors in ApoE�/�mice did not reach statisticalsignificance (Supplementary Fig. S3C and S3D). Finally, NACincreased significantly the infiltration of NKT and gd T cells inearly stages of tumor development in both ApoE�/� and HCDmice (Supplementary Fig. S3E and S3F). Regression analysisbetween the degree of HSC oxidant stress and the number oftumors per mouse revealed a remarkable linear correlationbetween these variables (R2 ¼ 0.87; Supplementary Fig. S3G).Together, these findings show that hypercholesterolemia-inducedHSC oxidant stress directly mediates the reduction of HSC dif-ferentiation toward NKT and gd T cells and the consequentincrease in tumor number and histopathologic severity.

Hypercholesterolemia-induced downregulation of Tet1 inHSCs impairs their differentiation toward NKT and gdT cells

The cell autonomous defect in HSC differentiation caused byhypercholesterolemia-induced oxidant stress raised the possibil-ity that oxidant stress disrupted an epigenetic regulatory pathwaynecessary for proper HSC differentiation to NKT and gdT cells. Insupport of the possibility, we observed an oxidant stress-depen-dent reduction in the expression of Tet1 in HSCs from hyper-cholesterolemic mice (Fig. 3A and B).

Within the Tet family, Tet2 has been shown to have a criticalrole in regulating the self-renewal, proliferation, and differenti-ation of HSCs (20–23), whereas the role of Tet1 in HSC differ-entiation is as yet unknown. To determine if this reduction of Tet1expression is directly responsible for the decrease in NKT and gdTcells and the impairment of tumor immunosurveillance in hyper-cholesterolemic mice, we measured the percentage and totalnumber of NKT and gdT cells in the thymus of WT and Tet1�/

� mice. Consistent with our findings in hypercholesterolemicmice, the percentage and total number of NKT and gdT cells in thethymuswas significantly lower in Tet1�/�mice thanWTmice (Fig.3C and D). The percentage and number of NKT and gdT cells wasalso decreased in the colon submucosa of Tet1�/� mice (Fig. 3Eand F).Meanwhile, Tet1�/�mice didnot show significant changesin T-cell intermediate populations in the thymus (SupplementaryFig. S4A and S4B). With the exception of a slight increase in NKcells and a slight decrease in B cells, we did not find significantchanges in CD3eþ, CD4þ, and CD8þ cells in peripheral blood ofTet1�/� mice (Supplementary Fig. S4C). The other major cellularcomponents of cancer immunosurveillance, including NK cells,CD11b� dendritic cells, CD11bþ dendritic cells, CD11c� macro-phages, and CD11cþ macrophages did not show significantchanges in the colon of Tet1�/� mice (Supplementary Fig.S4D). In an in vitroHSC differentiation assay, knockdown of Tet1expression in HSCs from either WT or ApoE�/� mice greatlyreduced their differentiation toward NKT and gdT cells (Supple-mentary Fig. S5A–S5C).

The overexpression of Tet1 in HSCs fromWT or ApoE�/� micecaused a seven-fold increase inWT and almost 20-fold increase inApoE�/� mice in HSC differentiation toward NKT cells both invitro and in vivo. The overexpression of Tet1 in HSCs also caused10-fold increase in WT and 20-fold increase in ApoE�/� mice inHSC differentiation toward gdT cells (Fig. 3G–I; SupplementaryFig. S5A, S5D, and S5E). The overexpression of Tet1 in HSCs didnot affect the daughter T-cell intermediate populations in thymusas well as B cells, NK cells, CD3eþ, CD4þ, and CD8þ cells inperipheral blood of recipient mice (Supplementary Fig. S5F–S5H). These results further support the novel and specific role ofTet1 in HSCs lineage specification toward NKT and gdT cells.

IL17 is a critical cytokine in both innate and adaptive immu-nity. CCR6 regulates the migration and recruitment of T cellsduring inflammatory and immunological responses (24–27). gdTcells from ApoE�/� mice also showed significant decreases in theproduction of IL17 (Supplementary Fig. S5I and S5J). Interest-ingly, gdT cells derived from Tet1-overexpressing HSCs also dis-played greater expression of CCR6 and IL17 than WT mice(Supplementary Fig. S5K and S5L). These results also indicatethat Tet1 expression in HSCs is a pivotal determinant not only ofthe lineage specification of HSCs toward NKT and gdT cells butalso of the function of terminally differentiatedNKT and gdT cells.

We next sought to determine in vivowhether the overexpressionof Tet1 in HSCs from hypercholesterolemic mice could restoretheir normal lineage specification toward NKT and gdT cells andthereby immunosurveillance against colorectal neoplasia. Wereconstituted hematopoiesis of WT recipient mice withWTHSCs,Tet1-overexpressing WT HSCs or Tet1-overexpressing ApoE�/�

HSCs. When we tried to reconstitute WT and ApoE�/� mice withHSCs that overexpress Tet1, all the mice died. We assumed thesedeaths were secondary to failed reconstitution of the bone mar-row of the irradiated mice. To address this problem, the trans-plantation of Tet1-overexpressing WT HSCs was supported with






normal, nontransduced WT HSCs and similarly the transplanta-tion of Tet1 overexpressing ApoE�/� HSCs was supported withApoE�/� HSCs, both at the ratio of 3:1 (Fig. 4A). Under these

conditions, all mice survived and overexpression of Tet1 inApoE�/� HSCs restored the number of NKT and gdT cells in thethymus of recipient WT mice to that of recipient WT mice

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Hypercholesterolemia-induced oxidant stressdownregulates the expression of Tet1 in HSCsthat impair their differentiation toward NKTand gdT cells. A, Expression of Tet1, Tet2, andTet3 in HSCs from WT and ApoE�/� mice; n ¼6, �� , P <0.01, versusWT.B,Downregulation ofTet1 expression in HSCs from ApoE�/� isoxidant stress dependent in mice; n ¼ 6,�P < 0.05; �� , P < 0.01, versus ApoE�/�.C, Frequency and number of NKT cells inthymus of WT and Tet1�/� mice; n ¼ 5.� , P < 0.05, versus WT. D, Frequency andnumber of gdT cells in thymus of WT andTet1�/� mice; n ¼ 5. �, P < 0.05, versus WT.E, Frequency of submucosal NKT cells in colonof WT and Tet1�/� mice; n ¼ 5, � , P < 0.05,versus WT. F, Frequency of submucosal gdTcells in colon of WT and Tet1�/� mice.n ¼ 5; � , P < 0.05, versus WT. G, Tet1-relativeexpression following its overexpression in WTand Apoe�/� HSCs. n ¼ 6; � , P < 0.05,versus WT; ##, P < 0.01, versus ApoE�/�.H, Frequency of NKT cells in thymus ofrecipient mice transplanted with WT HSCs,ApoE�/�HSCs, Tet1-overexpressingWTHSCs,or Tet1-overexpressing ApoE�/� HSCs.n ¼ 6; � , P < 0.05; �� , P < 0.01, versusWTþMock; ##, P < 0.01, versusApoE�/�þMock. I, Frequency of gdT cells inthymus of recipientmice transplantedwithWTHSCs, ApoE�/�HSCs, Tet1-overexpressingWTHSCs, or Tet1-overexpressing ApoE�/� HSCs.n ¼ 6; � , P < 0.05; �� , P < 0.01, versusWTþMock; ##, P < 0.01, versusApoE�/�þMock. See also SupplementaryFig. S4.

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Figure 4.

Reconstitution of lethally irradiated WT mice with ApoE�/- HSCs that overexpress Tet1 restores immunosurveillance against colorectal neoplasia. A, Frequency ofcells derived from Tet1-overexpressing HSCs. The transplantation of Tet1-overexpressing WT HSCs was supported with WT HSCs and the transplantation of Tet1-overexpressing ApoE�/� HSCs was supported with ApoE�/� HSCs, both at the ratio of 3:1. n ¼ 8; � , P < 0.05; �� , P < 0.01, versus WTþMock; ##, P < 0.01,versus ApoE�/�þMock. B, Frequency and total number of NKT cells in thymus of the recipients after transplantation with WT HSCs, ApoE�/� HSCs, Tet1-overexpressing WT HSCsþWT HSCs, or Tet1-overexpressing ApoE�/� HSCsþApoE�/� HSCs. n ¼ 8; � , P < 0.05, versus WTþMock!WT; #, P < 0.05, versusApoE�/�þMock!WT. C. Frequency and total number of gdT cells in thymus of the recipients. n ¼ 8; � , P < 0.05, versus WTþMock!WT; #, P < 0.05, versusApoE�/�þMock!WT. D, Frequency of NKT cells in colon submucosa of the recipients. n ¼ 8; ��, P < 0.01, versus WTþMock!WT; #, P < 0.05, versusApoE�/�þMock!WT. E, Frequency of gdT cells in colon submucosa of the recipients. n ¼ 8; �� , P < 0.01, versus WTþMock!WT; #, P < 0.05, versusApoE�/�þMock!WT. F, Average tumor number per mouse in the recipients. n¼ 12; � , P < 0.05, versus WTþMock!WT; #, P < 0.05, versus ApoE�/�þMock!WT.G, Histopathologic stages of tumors. n ¼ 12; � , P < 0.05; �� , P < 0.01 versus WTþMock!WT; #, P < 0.05; ##, P < 0.01, versus ApoE�/�þMock!WT. See alsoSupplementary Figure S5.






reconstituted with WT HSCs (Fig. 4B and C). Overexpression ofTet1 in ApoE�/� HSCs restored the number of submucosal NKTand gdT cells in recipient WT mice (Fig. 4D and E). Most signif-icantly, overexpression of Tet1 in ApoE�/� HSCs reduced theaverage tumor number of colorectal neoplasia in recipient irra-diatedWT to a level similar to that of overexpression of Tet1 inWTmice (Fig. 4F and G). Moreover, overexpression of Tet1 in bothApoE�/� and WT HSCs had a profound effect on the histopath-ologic severity of the tumors. Indeed, lethally irradiated WTrecipient mice reconstituted with either Tet1-overexpressing WTHSCs or Tet1-overexpressing ApoE�/� HSCs eliminated the pro-gression of any tumors to the carcinoma stage in both groups (Fig.4G). These results indicate that restoration of Tet1 expression inApoE�/�HSCs rescues both their reduced lineage specificationtoward NKT and gdT cell populations as well as their effectiveimmunosurveillance against colorectal neoplasia. The increase inNKT and gdT cell populations in the mucosa and submucosa andthe consequent reduction in the histopathologic severity of theAOM-induced tumors in WT mice transplanted with Tet1 over-expressing HSCs was an unexpected finding that might havepotential immunotherapeutic implications.

MiR101cmediates the downregulation of Tet1 inHSCs isolatedfrom hypercholesterolemic mice

We performed miRNA microarray analysis in HSCs isolatedfrom WT and hypercholesterolemic ApoE�/- mice (Supplemen-tary Fig. S6A). MiR101c, which is predicted to directly target Tet1,showed a significant upregulation in HSCs from ApoE�/� mice.This increased level of miR101c was further validated by RT-PCR(Fig. 5A; Supplementary Fig. S6B). The administration of NACeffectively reduced the overexpression of miR101c in HSCs fromhypercholesterolemic mice (Fig. 5A; Supplementary Fig. S6B).Transfection of miR101c mimics in HSCs from WT mice signif-

icantly repressed Tet1 expression (Fig. 5B and C), whereas trans-fection of miR101c inhibitors in HSCs from ApoE�/� micesignificantly increased Tet1 expression (Fig. 5D and E). miR101csignificantly repressed luciferase activity in cells transfected withthe constructs containing the predicted miR101c binding sites inthe Tet1 30-UTR regions. In contrast, miR101c failed to alterluciferase activity when we mutated the Tet1-binding sites, there-by confirming that Tet1 is the direct binding target of miR101c(Fig. 5F). These findings represent the first known effects ofmiR101c on gene expression in vivo.

Tet1 directly induces the expression of genes critical for HSCdifferentiation toward NKT and gdT cells

Although the molecular mechanism underlying the differenti-ation of NKT and gd T cells is still incompletely characterized, agroup of genes that have been shown to mediate the differenti-ation and maturation of NKT and gd T cells have been identified(28, 29). To determine the mechanism by which hypercholester-olemia-induced downregulation of Tet1 impairs differentiationof HSCs toward NKT and gd T cells, we examined the expressionand epigenetic regulation of genes necessary for NKT and gd T-cellspecification (Supplementary Table S1). Only five genes, Fyn,Sox13, IL15R, ITK, and SH2D1a, had lower expression inApoE�/� HSCs than in WT HSCs (Fig. 6A). Moreover, the expres-sion of these genes increased when Tet1 was overexpressed inHSCs from WT and hypercholesterolemic mice (Fig. 6A; Supple-mentary Fig. S7A), thereby indicating a repression of the genesessential for NKT and gd T differentiation in HSCs from hyper-cholesterolemic mice.

Because Tet-dependent DNA demethylation typicallyincreases the transcription of target genes (23, 30), we nextsought to characterize the changes in DNA methylation at theregulatory regions of the five genes whose expression was

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miR101c mediates the downregulationof Tet1 in HSCs isolated fromhypercholesterolemic mice.A, Oxidant stress–dependentupregulation of miR101c in HSCsfrom ApoE�/�mice. n¼ 6; � , P < 0.05,versus WT. #, P < 0.05, versusApoE�/�. B, Expression of miR101cmimics in WT HSCs. C, Expression ofTet1 in WT HSCs after transfectionof miR101c mimics. n ¼ 6; � , P < 0.05;��, P < 0.01 versus WT control. D,Expression of miR101c in HSCs fromApoE�/� mice after transfection ofmiR101c inhibitor. E, Expression of Tet1in HSCs from ApoE�/� mice aftertransfection of miR101c inhibitor.n ¼ 6; � , P < 0.05, versus ApoE�/�

control. F, Detection of direct bindingbetween miR101c and Tet1 30 UTRs byluciferase reporter assay. C1, construct1; C2, construct 2; M1, mutant 1; M2,mutant 2. *, P < 0.05, vs. control. Seealso Supplementary Fig. S6.

Tie et al.





reduced in ApoE�/�mice. Pyrosequencing analysis showed thatFyn, Sox13, IL15R, ITK, and SH2D1a were more hypermethy-lated in ApoE�/� HSCs than in WT HSCs (Fig. 6B). In contrast,Tet1 overexpression in HSCs from WT and hypercholesterol-emic mice significantly decreased the methylation and corre-spondingly increased the expression of these genes in both WTand ApoE�/� HSCs (Fig. 6B and Supplementary Fig. S7B).Interestingly, several genes (ETV5, EGR2, SLAMF1, ZBTB16,and RELb) whose expression did not change significantly inApoE�/� HSCs were also increased after overexpression of Tet1in HSCs of both WT and ApoE�/� mice (Supplementary Fig.S7A), suggesting that Tet1 overexpression to levels higher thanthose found in WT HSCs can increase the expression of genesrequired for NKT and gd T specification. Consistent with thispossibility, methylation of ETV5, EGR2, and NFKB1c wassignificantly higher in cells derived from ApoE�/� HSCs thanthose from WT HSCs, and this hypermethylation was reducedto levels at or below WT upon overexpression of Tet1 (Sup-

plementary Fig. S7B). Taken together, these results show thatTet1 directly activates genes required for NKT and gd T spec-ification, and this activation is impaired upon Tet1 downregu-lation in hypercholesterolemic HSCs.

Tet proteins also participate in the regulation of histone mod-ifications via distinct pathways. The O-linked N-acetylglucosa-mine (O-GlaNAc) transferase,OGT is an evolutionarily conservedenzyme that catalyzesO-linkedprotein glycosylation. Tet proteinswere identified as stable partners of OGT in the nucleus (31–33).The interaction of Tet2 and Tet3 with OGT leads to the GlcNA-cylation of Host Cell Factor 1 and contributes to the integrity ofthe H3K4 methyltransferase SET1/COMPASS complex, revealingthat Tet proteins increase the level of H3K4me3, a modificationthat functions in transcriptional activation (34). Although anearly observation showed that the interaction between Tet1 andOGT was limited to embryonic stem cells, our immunoprecipi-tation studies show that OGT also interacts with Tet1 in HSCs(Supplementary Fig. S8A). In accordancewith the decrease in Tet1

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In HSCs, Tet1 regulates the expression of the key regulatory genes in their differentiation into NKT and gdT cells. A,Gene expression in cells fromWTHSCs, ApoE�/�

HSCs, Tet1-overexpressing WT HSCs, or Tet1-overexpressing ApoE�/� HSCs. n ¼ 4; � , P < 0.05, �� , P < 0.01, versus WTþMock; #, P < 0.05, ##, P < 0.01,versus ApoE�/�þMock. B, DNA methylation status of the genes analyzed in A. n ¼ 4; � , P < 0.05, ��, P < 0.01, versus WTþMock; #, P < 0.05, ##, P < 0.01, versusApoE�/�þMock. See also Supplementary Fig. S7.






expression, the Tet1–OGT interactionwas significantly reduced inHSCs isolated from hypercholesterolemic mice. This overexpres-sion of Tet1 significantly increased the interaction of Tet1 andOGT, but did not influence the expression or interaction of Tet3and OGT in the cells (Supplementary Fig. S8A and S8B). Con-sistent with these findings, overexpression of Tet1 caused anincrease ofH3K4me3methylation near the promoters of all genesinvestigated, except RELb andNFKB1. The results suggest that, byinteractingwithOGT, Tet1 also increases histoneH3K4me3 levelsand maintains active chromatin structure near many of the genescritical for the differentiation of HSCs toward NKT and gdT cells(Supplementary Fig. S8C). Consequently, Tet1 promotes theexpression of genes drivingNKT and gdT specification bymultiplemechanisms.

Hypercholesterolemia also downregulates Tet1 expression inhuman HSCs and impairs their differentiation towardNKT and gd T cells

To test whether these findings in mouse models of hypercho-lesteremia were applicable to human HSCs, human HSCs wereexposed to oxLDL, the primary source of oxidant stress in theHSCs of hypercholesterolemic mice (13) and their differentiationcapacities towardNKT and gd T cells were examined.We observedan oxLDL concentration–dependent decrease in the differentia-tion of human HSCs toward NKT and gd T cells (SupplementaryFig. S9A and S9B). Congruent with our mouse studies, thetreatment with oxLDL inhibited Tet1 expression in human HSCsin a dose dependent manner (Supplementary Fig. S9C). Thus,these results in human HSCs are parallel to those in hypercholes-terolemic mice, suggesting that these findings may be generaliz-able to humans.

DiscussionHypercholesterolemia has been shown to increase the risk of

all-cause mortality, including an increased risk of death fromcancer (12, 24). Here, we show that hypercholesterolemiaincreases the incidence and pathologic severity of experimentalcolorectal neoplasia by inducing an oxidant stress dependentdownregulation of Tet1 in HSCs that impairs their differentiationtoward NKT and gdT cells. This reduction of Tet1 expressiondownregulates genes critical to the differentiation ofHSCs towardNKT and gd T cells in large part by loss of activating histoneH3K4me3modifications and gain of repressiveDNAmethylationmarks near the promoters of the key differentiation genes. TheseTet1-induced effects onHSC differentiation reduce the number ofterminally differentiated NKT and gd T cells in the circulation, thesubmucosa of the gut, and finally within the early stages of tumordevelopment. These findings reveal a novel mechanism by whichinnate immunity canbemodulated by ametabolic abnormality atthe level of HSC rather than at terminally differentiated immunecells. Thus, this pathologic change in gene expression was "pre-programmed" in HSCs and carried through bone marrow pro-genitor cells, thymic intermediate cells, and terminally differen-tiated NKT and gd T cells.

Although genome-wide ChIP studies established the centralrole of epigeneticmodifications inHSC fate decision (9–12), littleis known of how the epigenetic regulators of lineage priming arelinked to a given lineage specification. In this study, we show thatthe inhibition of Tet1 inHSCs imposed repressivemodification ofthe genes that controls their differentiation toward gdT cells and

NKT cells. This overexpressionof Tet1 led to activemodificationofthe genes and greatly increased the differentiation ofHSCs towardNKT and gdT cells both in vivo and in vitro. These finding indicatethat Tet1 is a master epigenetic regulator of HSC differentiationtoward NKT and gdT cells.

Emerging evidence suggests that Tet1 functions as a criticaltumor suppressor in multiple human cancers, including colorec-tal cancer (35). The analysis of tumor methylomes showed thatTet1, as a methylated target, is frequently methylated and down-regulated in cell lines and primary tumors ofmultiple carcinomasand lymphomas, including gastric and colorectal carcinomas(36–38). The expression of the Tet1 catalytic domain is able toinhibit the CpGmethylation of tumor suppressor gene promotersand reactivate their expression (37). The downregulation of Tet1leads to repression of the DKK gene and constitutive activation ofthe WNT pathway, resulting in the initiation of tumorigenesis incolon. In addition, the reexpression of Tet1 in colon cancer cellsinhibits their proliferation and the growth of tumor xenograftseven at late stages (38). These studies provide convincing evidencethat Tet1 is a critical regulator in preventing themalformation andtransformation of colon cells. We found that Tet1, by supportingthe differentiation from HSCs toward NKT and gd T cells, alsofunctions as a critical regulator of innate immunity against colo-rectal neoplasia. Our findings provide new evidence for under-standing how tumors escape immunosurveillance in the contextof hypercholesterolemia.

In general, NKT and gd T cells are critical components of innateimmunity against cancer and initiate the cascade of immunereactions to recognize and eliminate transformed cells in the earlystage of immunosurveillance (1, 39, 40). Suppression of the T-cellresponses to tumors has been observed frequently in cancerpatients, specifically NKT and gd T cells have been shown to bedecreased or functionally hyporeactive in both cancer bearingmice and humans (41–43). In agreement with these studies, wealso found that both NKT and gd T cells of hypercholesterolemicmice were decreased in the colon submucosa and at early stages oftumorigenesis. The overexpression of Tet1 significantly increasedthe differentiation of HSCs toward NKT and gd T cells both in vivoand in vitro. Despite reconstituting less than 10% of the T cells inthe thymus of the transplanted recipient mice, gd T cell and NKTcell numbers in the thymus and most importantly in the submu-cosa of the colon were normal. The clinical implications of thesefindings could be important.

The immunoscore is a more accurate predictor of tumor-freesurvival in patients who have colorectal neoplasia than is theclassical Dukes Classification or the TMN score (44). Although,weak immune infiltrates have been postulated to be due to adefect in the host immune response, no etiology for this impairedhost immune response has heretofore been identified (45). Thus,the identification of a new level of regulation of innate immunityby the epigenetic regulation of HSC differentiationmay provide acontext to understand why patients are found to have impairedImmunoscores in response to tumors. Creation of chimeric micethat received either ApoE�/� or WT HSCs that overexpressed Tet1had a substantial effect on the pathologic stage of the tumorswhere not only were all carcinoma tumors eliminated in themicereceiving ApoE�/� transfected cells that overexpress Tet1 but sodid the chimeric mice that received WT HSCs that overexpressedTet1. Thus, a miR101c-Tet1 based HSC immunotherapy mightnot only be effective in patients found to have an impairedimmunoscore but also in those who have relatively normal

Tie et al.





Immunoscores. However, much remains to be done to determinehow these changes inHSC gene expression are carried through thecomplex process of their differentiation into terminally differen-tiated NKT and gdT cells.

Finally, the relationship between cardiovascular risk factors andcancer incidence is being actively investigated and sometimesreferred to as CardioOncology (1). Cardiovascular disease andcancer share many similar risk factors. The relationship of hyper-cholesterolemia and colorectal cancer has been studied for dec-ades. Some studies found an inverse relationship between serumhypercholesterolemia and colorectal cancer that raised questionsabout the relationship. However, it was learned subsequently thatcolonic adenocarcinoma cells can aggressively metabolize cho-lesterol and so studies that look for the relationship between thesevariables in patients with established cancers can lead to anerroneous conclusion. An additional issue is the findings thatserum levels of 27-hydroxycholesterol (1), a metabolite of cho-lesterol, is associated with many cancers, especially breast cancer.We assume this is the result of a direct effect of 27-hydroxycho-lesterol on tumor cells. It will be important to see if it also affectslineage priming of HSCs.

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

Authors' ContributionsConception and design: G. Tie, L.M. MessinaDevelopment of methodology: G. Tie, J. Yan, J. KangAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): G. Tie, J. Yan, L. KhairAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): G. Tie, J. Yan, J.A. Messina, A. Deng, L.M. MessinaWriting, review, and/or revision of the manuscript: G. Tie, J. Yan, L. Khair,J.A. Messina, T. Fazzio, L.M. MessinaAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): G. Tie, J. Yan, L.M. MessinaStudy supervision: L.M. MessinaOther (advice, consultation, and technical support): T. Fazzio

AcknowledgmentsWe thank Dr. Oliver Rando and Katelyn Sylvia (University of Massachusetts

Medical School) for their great support. We thank Dr. Juan Carlos Zuniga-Pflucker (University of Toronto) for providing the OP9-DL1 cells. We thankDr. Yi Zhang (Mass General Hospital, Boston, MA) for providing pTYF-U6-shCONT-EF1-Puro-2A-CD1. We thank Dr. Tin-Lap Lee (The Chinese Universityof Hong Kong) for providing the pmirGLO vector. We thank the NIH TetramerFacility for the CD1d tetramer.

The costs of publication of this articlewere defrayed inpart by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received July 24, 2016; revisedOctober 31, 2016; accepted February 24, 2017;published OnlineFirst March 1, 2017.

References1. Koene RJ, Prizment AE, Blaes A, Konety SH. Shared risk factors in cardio-

vascular disease and cancer. Circulation 2016;133:1104–14.2. Hennekens CH, Andreotti F. Leading avoidable cause of premature deaths

worldwide: case for obesity. Am J Med 2013;126:97–8.3. Font-Burgada J, Sun B, Karin M. Obesity and cancer: the oil that feeds the

flame. Cell Metab 2016;23:48–62.4. Notarnicola M, Altomare DF, Correale M, Ruggieri E, D'Attoma B, Mas-

trosimini A, et al. Serum lipid profile in colorectal cancer patients with andwithout synchronous distant metastases. Oncology 2005;68:371–374.

5. Drechsler M, Megens RT, van Zandvoort M, Weber C, Soehnlein O.Hyperlipidemia-triggered neutrophilia promotes early atherosclerosis. Cir-culation 2010;122:1837–1845.

6. Klingenberg R, Gerdes N, Badeau RM, Gistera�A, Strodthoff D, Ketelhuth

DF, et al. Depletion of FOXP3þ regulatory T cells promotes hypercholes-terolemia and atherosclerosis. J Clin Invest 2013;123:1323–1334.

7. Sag D, Wingender G, Nowyhed H, Wu R, Gebre AK, Parks JS, et al. ATP-binding cassette transporter G1 intrinsically regulates invariant NKT celldevelopment. J Immunol 2012;189:5129–5138.

8. Sag D, Cekic C, Wu R, Linden J, Hedrick CC. The cholesterol transporterABCG1 links cholesterol homeostasis and tumour immunity. Nat Com-mun 2015;6:6354.

9. vanGalen P, Kreso A,Wienholds E, Laurenti E, Eppert K, Lechman ER, et al.Reduced lymphoid lineage priming promotes human hematopoietic stemcell expansion. Cell Stem Cell 2014;14:94–106.

10. GuoG, Luc S,Marco E, Lin TW, Peng C, Kerenyi MA, et al. Mapping cellularhierarchy by single-cell analysis of the cell surface repertoire. Cell StemCell2013;13:492–505.

11. Mercer EM, Lin YC, Benner C, Jhunjhunwala S, Dutkowski J, FloresM, et al.Multilineage priming of enhancer repertoires precedes commitment to theB and myeloid cell lineages in hematopoietic progenitors. Immunity2011;35:413–425.

12. Orkin SH. Priming the hematopoietic pump. Immunity 2003;19:633–4.13. Tie G, Messina KE, Yan J, Messina JA, Messina LM. Hypercholesterolemia

induces oxidant stress that accelerates the ageing of hematopoietic stemcells. J Am Heart Assoc 2014;3:e000241.

14. Greten FR, Eckmann L, Greten TF, Park JM, Li ZW, Egan LJ, et al. IKKbetalinks inflammation and tumorigenesis in a mouse model of colitis-asso-ciated cancer. Cell 2004;118:285–96.

15. Schmitt TM, de Pooter RF, Gronski MA, Cho SK, Ohashi PS, Z�u~niga-Pfl€ucker JC. Induction of T cell development and establishment of T cellcompetence from embryonic stem cells differentiated in vitro. Nat Immu-nol 2004;5:410–7.

16. Nunez-Cruz S, Yeo WC, Rothman J, Ojha P, Bassiri H, JuntillaM, et al. Differential requirement for the SAP-Fyn interactionduring NK T cell development and function. J Immunol 2008;181:2311–20.

17. Chien YH, Meyer C, Bonneville M. gd T cells: first line of defense andbeyond. Annu Rev Immunol 2014;32:121–55.

18. Taniguchi M, Seino K, Nakayama T. The NKT cell system: bridging innateand acquired immunity. Nat Immunol 2003;4:1164–5.

19. Todaro M, D'Asaro M, CaccamoN, Iovino F, FrancipaneMG, Meraviglia S,et al. Efficient killing of human colon cancer stem cells by gammadelta Tlymphocytes. J Immunol 2009;182:7287–96.

20. Ito S,D'AlessionAC, TaranovaOV,HongK, Sowers LC, ZhangY. Role of Tetproteins in 5mC to 5hmC conversion, ES-cell self-renewal and inner cellmass specification. Nature 2010;466:1129–33.

21. Ko M, Huang Y, Jankowska AM, Pape UJ, Tahiliani M, Bandukwala HS,et al. Impaired hydroxylation of 5-methylcytosine in myeloid cancers withmutant TET2. Nature 2010;468:839–43.

22. Ito S, Shen L, Dai Q,Wu SC, Collins LB, Swenberg JA, et al. Tet proteins canconvert 5-methylcytosine to 5-formylcytosine and 5-carboxylcytosine.Science 2011;333:1300–3.

23. Ko M, Bandukwala HS, An J, Lamperti ED, Thompson EC, Hastie R, et al.Ten-Eleven-Translocation 2 (TET2) negatively regulates homeostasis anddifferentiation of hematopoietic stem cells inmice. ProcNatl Acad SciUSA2011;108:14566–71.

24. Corpuz TM, Stolp J, Kim HO, Pinget GV, Gray DH, Cho JH,et al. Differential responsiveness of innate-like IL-17- and IFN-g-producing gd T cells to homeostatic cytokines. J Immunol 2016;196:645–54.

25. Shibata S, Tada Y, Hau CS,Mitsui A, KamataM, Asano Y, et al. Adiponectinregulates psoriasiform skin inflammation by suppressing IL-17 productionfrom gd-T cells. Nat Commun 2015;6:7687.

26. Wilson RP, Ives ML, Rao G, Lau A, Payne K, Kobayashi M, et al. STAT3 is acritical cell-intrinsic regulator of human unconventional T cell numbersand function. J Exp Med 2015;212:855–64.






27. Zarin P, Chen EL, In TS, AndersonMK, Z�u~niga-Pfl€ucker JC. Gammadelta T-cell differentiation and effector function programming, TCR signalstrength, when and how much? Cell Immunol 2015;296:70–5.

28. Matsuda JL, Gapin L. Developmental program of mouse Valpha14i NKTcells. Curr Opin Immunol 2005;17:122–30.

29. Garbe A, von Boehmer H. TCR and Notch synergize in alphabeta versusgammadelta lineage choice. Trends Immunol 2007;28:124–31.

30. Wu H, Zhang Y. Mechanisms and functions of Tet protein-mediated 5-methylcytosine oxidation. Genes Dev 2011;25:2436–52.

31. Vella P, Scelfo A, Jammula S, Chiacchiera F,Williams K, CuomoA, et al. Tetproteins connect the O-linked N-acetylglucosamine transferase Ogt tochromatin in embryonic stem cells. Mol Cell 2013;49:645–56.

32. Chen Q, Chen Y, Bian C, Fujiki R, Yu X. TET2 promotes histone O-GlcNAcylation during gene transcription. Nature 2013;493:561–4.

33. Shi FT, Kim H, Lu W, He Q, Liu D, Goodell MA, et al. Ten-eleventranslocation 1 (Tet1) is regulated by O-linked N-acetylglucosamine trans-ferase (Ogt) for target gene repression inmouse embryonic stem cells. J BiolChem 2013;288:20776–84.

34. Deplus R, Delatte B, Schwinn MK, Defrance M, M�endez J, Murphy N, et al.TET2 and TET3 regulate GlcNAcylation and H3K4 methylation throughOGT and SET1/COMPASS. EMBO J 2013;32:645–55.

35. Cimmino L, Dawlaty MM, Ndiaye-Lobry D, Yap YS, Bakogianni S, Yu Y,et al. TET1 is a tumor suppressor of hematopoietic malignancy. NatImmunol 2015;16:653–62.

36. Chapman CG, Mariani CJ, Wu F, Meckel K, Butun F, Chuang A, et al. TET-catalyzed 5-hydroxymethylcytosine regulates gene expression in differen-tiating colonocytes and colon cancer. Sci Rep 2015;5:17568.

37. Li L, Li C,MaoH,DuZ, ChanWY,Murray P, et al. Epigenetic inactivation ofthe CpGdemethylase TET1 as aDNAmethylation feedback loop in humancancers. Sci Rep 2016;6:26591.

38. Neri F, Dettori D, Incarnato D, Krepelova A, Rapelli S, Maldotti M, et al.TET1 is a tumour suppressor that inhibits colon cancer growth by dere-pressing inhibitors of the WNT pathway. Oncogene 2015;34:4168–76.

39. DunnGP, Bruce AT, IkedaH,Old LJ, Schreiber RD.Cancer immunoediting:from immunosurveillance to tumor escape. Nat Immunol 2002;3:991–998.

40. Vantourout P, Hayday A. Six-of-the-best: unique contributions of gd T cellsto immunology. Nat Rev Immunol 2013;13:88–100.

41. Strid J, Roberts SJ, Filler RB, Lewis JM, Kwong BY, Schpero W, et al. Acuteupregulation of anNKG2D ligand promotes rapid reorganization of a localimmune compartment with pleiotropic effects on carcinogenesis. NatImmunol 2008;9:146–154.

42. Bendelac A, Savage PB, Teyton L. The biology of NKT cells. Annu RevImmunol 2007;25:297–336.

43. DhodapkarMV,GellerMD,ChangDH, ShimizuK, Fujii S,Dhodapkar KM,et al. A reversible defect in natural killer T cell function characterizes theprogression of premalignant to malignant multiple myeloma. J Exp Med2003;197:1667–76.

44. Themeli M, Kloss CC, Ciriello G, Fedorov VD, Perna F, Gonen M, et al.Generation of tumor-targeted human T lymphocytes from induced plu-ripotent stem cells for cancer therapy. Nat Biotechnol 2013;31:928–33.

45. Galon J, Pages F, Marincola FM, Angell HK, ThurinM, Lugli A, et al. Cancerclassification using the Immunoscore: a worldwide task force. J Transl Med2012;10:205–214.


Tie et al.




2017;77:2351-2362. Published OnlineFirst March 1, 2017.Cancer Res Guodong Tie, Jinglian Yan, Lyne Khair, et al. Stem Cells

T Cells from HematopoieticδγReducing Production of NKT and Hypercholesterolemia Increases Colorectal Cancer Incidence by

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