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Molecular and Cellular Pathobiology

Metastatic Consequences of Immune Escape from NK CellCytotoxicity by Human Breast Cancer Stem Cells

Bin Wang1, Qiang Wang1, Zhe Wang1, Jun Jiang2, Shi-Cang Yu1, Yi-Fang Ping1, Jing Yang1, Sen-Lin Xu1,Xian-Zong Ye1, Chuan Xu1, Lang Yang1, Cheng Qian1, Ji Ming Wang3, You-Hong Cui1, Xia Zhang1, andXiu-Wu Bian1

AbstractBreast cancer stem-like cells (BCSC) are crucial for metastasis but the underlying mechanisms remain elusive.

Here, we report that tumor-infiltrating natural killer (NK) cells failed to limit metastasis and were not associatedwith improved therapeutic outcome of BCSC-rich breast cancer. Primary BCSCs were resistant to cytotoxicitymediated by autologous/allogeneic NK cells due to reduced expression of MICA and MICB, two ligands for thestimulatoryNKcell receptorNKG2D. Furthermore, the downregulation ofMICA/MICB inBCSCswasmediated byaberrantly expressed oncogenic miR20a, which promoted the resistance of BCSC to NK cell cytotoxicity andresultant lung metastasis. The breast cancer cell differentiation–inducing agent, all-trans retinoic acid, restoredthe miR20a–MICA/MICB axis and sensitized BCSC to NK cell–mediated killing, thereby reducing immuneescape–associated BCSC metastasis. Together, our findings reveal a novel mechanism for immune escape ofhuman BCSC and identify the miR20a–MICA/MICB signaling axis as a therapeutic target to limit metastaticbreast cancer. Cancer Res; 74(20); 5746–57. �2014 AACR.

IntroductionThe escape of cancer cells from immune surveillance has

been considered as a prerequisite for metastasis (1), the majorcause of death in breast cancer. Cancer cells are highly het-erogeneous and a small fraction of cancer cells with stem cellproperties, known as tumor-initiating cells, or cancer stemcells (CSC), may be responsible for tumorigenesis (2), angio-genesis (3) and metastasis (4). Recently, selection by adaptiveimmunity has been shown to generate or enrich cancer cellswith stemness (5–9), suggesting the presence of intimateinteraction between CSCs and the skewed adaptive immunityin patients with cancer. However, the role of innate immunityin affecting CSCs and their metastasis remains unclear.

Cancer immunoediting, defined by phases of elimination,equilibrium, and escape, consists of both tumor-limiting and-promoting effects (10). Selection by adaptive and innate

immunity components, together with clonal evolution ofmalignant cells, contributes to the generation of cancer cellswith better survival advantages and functional heterogeneity(10). Recent studies using mice deficient in adaptive immunitydemonstrate that cancer immunoediting may occur in theabsence of adaptive immune cells through IFNg-producingnatural killer (NK) cells (11). Thus, we hypothesize thatimproper immunoediting by NK cells may result in the failureof cancer cells with stem cell property to respond to anticancerimmunity and, thus, promote cancer metastasis.

As innate immune effectors, NK cells are one of the majorinfiltrating immune cells in breast cancer and are endowedwith the capability of recognizing nascent transformed cells toprevent tumorigenesis and subsequent metastasis (12–15).Breast cancer stem-like cells (BCSC) are resistant to therapiesand are highly metastatic (16–18). However, it remains unclearwhether BCSCs succumb to NK cell cytotoxicity. In this study,we attempt to address this issue and report that BCSCs escapefrom NK cell cytotoxicity and metastasize through miR20a-mediated downregulation of MICA and MICB, two ligands forthe NK cell–activating receptor NKG2D.

Materials and MethodsBreast cancer specimens and immunohistochemistry

Breast cancer tissues were obtained from 591 consecutiveconsented patients who underwent surgical resection at theBreast Surgery Center at SouthwestHospital from2006 to 2007,with approval from the Institutional Ethics Committee. Allspecimens were formalin-fixed and paraffin-embedded in theInstitute of Pathology at Southwest Hospital. The median ageof patients at diagnosis was 45 years (range, 23–87 years). Thepatients received radical mastectomy, or modified radical

1Institute of Pathology and Southwest Cancer Center, Southwest Hospital,Third Military Medical University, and Key Laboratory of Tumor Immuno-pathology, Ministry of Education of China, Chongqing, China. 2BreastSurgery Center, Southwest Hospital, Third Military Medical University,Chongqing, China. 3Laboratory of Molecular Immunoregulation, Cancerand Inflammation Program, Center for Cancer Research, National CancerInstitute at Frederick, Frederick, Maryland.

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

B. Wang, Q. Wang, and Z. Wang contributed equally to this work.

Corresponding Author: Xiu-wu Bian, Institute of Pathology and South-west Cancer Center, Southwest Hospital, ThirdMilitaryMedical University,Gaotanyan 30, Chongqing 400038, China. Phone: 86-23-6875-4431; Fax:86-23-6539-7004; E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-13-2563

�2014 American Association for Cancer Research.

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mastectomy, or breast-conserving surgery. The axillary lymphnodes were routinely examined for metastasis. Tumor size,location, distal metastasis, and final tumor–node–metastasisstage were determined. The histologic diagnosis was madeaccording to the WHO standard. The expression of the breastcancermarker aldehyde dehydrogenase 1 (ALDH1) and the NKcell marker CD56 was examined by immunohistochemistry(IHC; Supplementary Materials and Methods).

Cell sorting by FACSPrimary breast cancer cells at 90% confluence were sus-

pended in ALDEFLUOR assay buffer containing an ALDHsubstrate and incubated for 45 minutes at 37�C (ALDEFLUORassay kit; StemCell Technologies; refs. 18, 19). Negative controlcells were incubated with an ALDH inhibitor, diethylamino-benzaldehyde (DEAB). The cells were then suspended in assaybuffer for sorting ALDHþ and ALDH� cells on a BDAria IIsorter (BD Biosciences). ALDHþ cells were maintained asmammospheres for further analysis.

Culture of peripheral blood NK cells and NK cellcytotoxicity assayPeripheral blood NK cells (pNK cells) from patients with

breast cancer and healthy donors were isolated for directanalysis or for further expansion (Supplementary Materialsand Methods). NK cell cytotoxicity was measured by a 51chro-mium (51Cr) release assay. The target cells (1 � 106) werelabeled with 250 mCi Na251CrO4 for 2 hours at 37�C, seeded into96-well round bottom plates at 5,000 per well, and coculturedwith pNK cells (effector cells) at indicated effector:target (E/T)ratios. K562 cells were served as a positive control for killing.After incubation for 4 hours at 37�C, cell supernatants werecollected, and spontaneous and maximal releases of 51Cr weremeasured. The percentage of 51Cr release (cytotoxicity) wascalculated as 100 � [(experimental release � spontaneousrelease)/(maximum release� spontaneous release)]. In block-ing experiments, pNK cells were pretreated with 20 mg/mLanti–NKG2D-neutralizing mAb (R&D Systems) for 20 minutesat room temperature before the assay.Cell death of targets induced by NK cell cytotoxicity was also

examined by flow cytometry (Supplementary Materials andMethods).

IFNg quantification by ELISABriefly, target cells were seeded in laminin-coated 96-well

plates and cultured for 24 hours. pNK cells (5 � 105/mL) wereincubated with the targets (2.5 � 105/mL) in a final volume of200 mL of fresh NK cell medium (Supplementary Materials andMethods) for 24 hours (E/T¼ 2:1). Blockade ofNKG2D receptorbefore incubation was performed using an anti–NKG2D-neu-tralizing mAb (R&D Systems). To test whether cancer cellsreleased IFNg , we included target cells alone as control. Aftercentrifugation, the supernatant was collected to determine thelevel of IFNg using a human IFNg ELISA Kit (Invitrogen).Because the levels of IFNg secretion varied among donors, weshowed the representative data usingNK cells derived fromonedonor for presentation, andall of the assayswere repeatedusingNK cells from at least three different donors.

Clearance by NK cells and lung metastasisThe elimination of target cells by NK cells in vivo and

immunoevasion-associated metastasis were assessed in ashort-term lung clearance assay and an experimental lungmetastasis model in NK cell (mNK)–depleted NOD/SCID micetransferred with human pNK cells (Supplementary Materialsand Methods).

Statistical analysisAll experiments were performed at least in triplicates and

representative data were shown. The correlation between thenumber of CD56þ NK cells or ALDH1 positivity and clinico-pathologic parameters was assessed by the nonparametricWilcoxon rank-sum test, the c2 test, or the Fisher exact testas appropriate. Overall survival (OS) and metastasis-free sur-vival (MFS) rates were plotted as Kaplan–Meier curves andanalyzed using the log-rank test. The Cox proportional hazardregression model was used to determine the influence ofvarious parameters on patient survival in univariate andmultivariable analysis. The Student t test or ANOVA was usedwhen appropriate. All data were analyzed using SPSS 13.0statistical software. Data were shown as the means � SD. AP value of <0.05 was considered statistically significant.

ResultsFailure of tumor-infiltrating NK cells to prevent themetastasis of BCSC-rich human breast cancers

We first examined the presence of CD56þ NK cells andBCSCs in breast cancer specimens from 591 patients usingALDH1 as a stem cell marker (18–20). On the basis of the IHCstaining, 212 cases of breast cancers were ALDH1high and 379cases were ALDH1low (Supplementary Table S1). High level ofinfiltrating CD56þ cells was detected in 33.0% of cancers. Ofnote, we frequently detected codistribution of CD56þ cells inthe areas with either non-BCSCs (ALDH1low) or BCSCs(ALDH1high) cancer cells (Fig. 1A). In ALDH1low cancers, thelevel of infiltratingCD56þ cells was lower in cancer tissueswith�4 lymph node metastasis (LNM) than those with <4 LNM(Fig. 1B). Although close localization of infiltrating CD56þ cellswith BCSCs was observed (Fig. 1C), there was no significantdifference in the number of tumor-infiltrating CD56þ cellsbetween cases with �4 LNM and those with <4 LNM inALDH1high tumors (Fig. 1D).

We then analyzed the correlation of infiltrating CD56þ cellswith patient survival in ALDH1low and ALDH1high subgroups.Significantly longer OS and MFS were found in the patientswith higher level of infiltrating CD56þ cells than those withlower level of infiltrating CD56þ cells in ALDH1low, but notALDH1high cancers (Fig. 1E and F). Moreover, increased level ofinfiltrating CD56þ cells in tumors was a favorable prognosticfactor for OS and MFS in ALDH1low, but not the ALDH1high

patient subgroup (Supplementary Table S2). Furthermore,increased level of infiltrating CD56þ cells was able to inde-pendently predict fewer distal metastases in the ALDH1low

tumors (Supplementary Table S3). However, in the ALDH1high

subgroup, no significant difference in the relative risk todevelop metastasis was observed in patients with CD56high

tumors as compared with those with CD56low tumors. These

Immune Escape of Cancer Stem Cells from NK Cells

www.aacrjournals.org Cancer Res; 74(20) October 15, 2014 5747




results indicate that high level of infiltrating NK cells in breastcancer fails to prevent ALDH1high BCSCs from metastasis inpatients.

BCSCs are resistant toNKcell cytotoxicity and contributeto lung metastasis

We isolated ALDHþ cells from primary breast cancer cellsand found that they exhibited potent self-renewal capacitywith multiple differentiation potentials and markedlyenhanced tumorigenicity in vivo (Supplementary Fig. S1),confirming the BCSC property of ALDHþ cells (18, 19). We

next examined activation and degranulation of NK cellscocultured with either BCSCs or non-BCSCs (ALDH� cells).Flow cytometric analysis showed reduced expression ofCD107a, a surfacemarker for NK cell degranulation, on humanpNK cells cocultured with BCSCs (Fig. 2A and SupplementaryFig. S2A). Because IFNg released by activated NK cells isimportant for immunoregulation, we determined the level ofIFNg and found significantly reduced production of IFNg bypNK cells cocultured with BCSCs (Fig. 2B and SupplementaryFig. S2B). Thus, primary BCSCs are unable to activate normalNK cells.

Figure 1. Infiltrating NK cells fail tolimit metastasis of human breastcancers with high level of BCSCs. A,in ALDH1low cancers (top), lowor high levels of infiltrating CD56þ

cells (bottom) were detected by IHCstaining on serial sections,demonstrating codistribution of NKcells with non-BCSCs. B, averagenumbers of infiltrating CD56þ cellsin ALDH1low cancers with �4 or <4LNM. C, in ALDH1high cancers (top),low or high levels of infiltratingCD56þ cells (bottom) were alsorevealed, showing codistribution ofNK cells with BCSCs. D, averagenumbers of infiltrating CD56þ cellsin ALDH1high cancers with �4 or<4 LNM. LN, lymph node. E and F,Kaplan–Meier plot ofOS andMFSofthe patients with ALDH1low (E) orALDH1high cancers (F), stratified bythe levels of infiltrating CD56þ cells.Mean � SD (B and D); bar, 100 mm.

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To examine the capacity of NK cells to destroy BCSCs, weused pNK cells from both patients with breast cancer andhealthy donors. Activated pNK cells showed a weaker lyticactivity on autologous BCSCs than non-BCSCs (Fig. 2C). BCSCswere also resistant to killing by either long-term expanded orfresh pNKs from healthy donors (Fig. 2D and Supplementary

Fig. S2C and S2D). Pretreatment of NK cells with an anti-CD16mAb inhibited both degranulation and cytotoxicity by NK cellsthat were cocultured with BCSCs or non-BCSCs (Fig. 2A and D,Supplementary Fig. S2A, S2C, and S2D), indicating the spec-ificity of the function of NK cells. Moreover, the levels of celldeath in BCSCs incubated with either long-term expanded or

Figure 2. Resistance of BCSCs to the cytotoxicity of autologous or allogeneic pNK cells and the contribution of BCSCs to lung metastasis. A, detection ofCD107aonpNKcells by FACS in pNKcells coculturedwith primary BCSCsor non-BCSCs (E:T¼5:1). K562 cells served aspositive control. Alternatively, pNKcells were pretreated with anti-CD16 mAb (3 mg/mL) to demonstrate the specificity for the function of NK cells. pNK represents human peripheral blood NKcells that were expanded for 2 to 3 weeks. Fresh pNK represents freshly isolated NK cells. B, IFNg released by pNK cells in the coculture supernatant(E:T¼ 2:1). C, lysis of primary BCSCs and non-BCSCs by autologous pNK cells asmeasured by 51Cr release. Data from six patients are shown (triple repeats).D, lysis of BCSCs and non-BCSCs by allogeneic pNKs from healthy donors. Anti-CD16 mAb was applied to specifically inhibit NK cell activity. E, flowcytometric analysis of target cell death cultured with pNK. The middle panel shows representative flow cytometric graph and total dead cells (%).F, the percentages of ALDHþ cells (left) or efficacy of sphere formation (right) of cancer cells that survivedNKcell–mediated lysis. G, the relative survival ratio ofBCSCs or non-BCSCs (DiD-labeled) in relation to HeLa cells (GFP-labeled, internal control) in a short-term lung clearance assay in NOD/SCIDmice (n¼ 3). H,the numbers of pulmonary metastasis established by BCSCs and non-BCSCs (n ¼ 3); mean � SD (A–I); �, P < 0.05; ��, P < 0.01; ns, no significance.






freshly isolated pNK cells were reduced (Fig. 2E and Supple-mentary Fig. S2E). In addition, short-term exposure to pNKcells did not significantly affect cell-cycle distribution andproliferation of BCSCs (Supplementary Fig. S2F and S2G).These results suggest that the resistance of BCSCs to NK cellcytotoxicity is due to reduced cell death. To test possibleimmunoselection of BSCSs versus non-BCSCs by NK cells, wecultured primary cancer cells with allogeneic pNK cells andfound that the residual viable cancer cells were enriched withBCSCs, as indicated by increased number of ALDH1þ cells andhigh efficacy of tumorsphere formation (Fig. 2F and Supple-mentary Fig. S2H). Thus, BCSCs are more resistant than non-BCSCs to lysis by NK cells.

To test the consequences of BCSC resistance to NK cellcytotoxicity, we used an in vivo short-term lung clearance assayof cancer cells in mice (Supplementary Fig. S3A; refs. 21, 22).Although no significant difference of cell survival rates wasfound between BCSCs and non-BCSCs i.v. injected into NOD/SCID mice depleted of NK (mNK) cells (mNK cell–depletedmice), less BCSCs were eliminated in themice transferred withhuman pNK cells (Fig. 2G and Supplementary Fig. S3B). Theseresults suggest that circulating BCSCs are resistant to elimi-nation by NK cells in vivo. In a lung metastasis model (Sup-plementary Fig. S3C), i.v. injected BCSCs formed higher num-bers of metastases in the lungs of normal mice as comparedwith non-BCSCs (Fig. 2H and Supplementary Fig. S3D). Incontrast, there was no difference in the number of metastaticfoci formed by either BCSCs or non-BCSCs in mNK cell–depleted mice. However, i.v. injected BCSCs established moremetastatic foci in the lungs of mice adoptively transferred withhuman pNK cells. The results indicate that BCSCs are resistantto NK cell cytotoxicity in vivo, thus are prone to form lungmetastasis.

Reduced expression of MICA and MICB by BCSCscontributes to the resistance to NK cell cytotoxicity andthe resultant metastasis

Tumor-infiltrating immune cells release cytokines such asIFNg to regulate leukemic stemcells (8). However,we found thatthe expressionof IFNg receptora chain, an essential componentof IFNg receptor for ligand binding and trafficking, was com-parable between BCSCs and non-BCSCs (Supplementary Fig.S4A). Because NK cell receptor ligands expressed by target cellsdetermine the sensitivity to NK cell cytotoxicity, we comparedthe expression of inhibitory receptor ligands HLA-A/B/C, HLA-E, HLA-G by BCSCs and non-BCSCs and also found no signifi-cant difference (Supplementary Fig. S4B). We then comparedthe levels of activating receptor ligands (Supplementary Fig.S4C–S4E) on BCSCs and non-BCSCs, and found that the expres-sion of both MICA and MICB, two ligands for the activatingreceptorNKG2D,was significantly lower onBCSCs as comparedwith non-BCSCs (Fig. 3A and Supplementary Fig. S4F).

To examine whether the lower expression of MICA andMICB was responsible for the resistance of BCSCs to NK cellcytotoxicity, we transfected BCSCs with MICA and MICB(Supplementary Fig. S4G). Coculture of transfected BCSCswith pNK cells significantly increased the expression ofCD107a by pNK cells, which was inhibited by pretreatment

of pNK cells with an NKG2D-blocking mAb (Fig. 3B andSupplementary Fig. S4H). In addition, these cocultured pNKcells showed increased IFNg secretion (Fig. 3C). BCSCs withincreased MICA or MICB, but not nontransfected parentalBCSCs or vector control, were significantly more sensitive toallogeneic pNK-mediated lysis (Fig. 3D and SupplementaryFig. S4I). Increases in IFNg secretion and NK cell–mediatedcytotoxicity were inhibited by blocking NKG2D but not NKp30receptor on NK cells (Fig. 3C and D, Supplementary Fig. S4I).Short-term lung clearance assay showed a reduced survival ofMICA- or MICB-transfected BCSCs in the lungs of mNK cell–depleted mice transferred with human pNK cells. In contrast,blocking NKG2D on NK cells promoted the survival of BCSCs(Fig. 3E and Supplementary Fig. S4J). Moreover, BCSCs trans-fected with NKG2D ligands formed fewer metastatic foci in thelungs of these mice. The NKG2D-blocking mAb restored thelung metastasis of MICA- or MICB-transfected BCSCs (Fig. 3Fand Supplementary Fig. S4K). These results indicate that lowerlevels of MICA and MICB contribute to BCSC resistance to NKcell lysis, thus increasing their lung metastasis.

To further examine the relevance of MICA and MICB totumor cell sensitivity to NK cell cytotoxicity, we used siRNA todownregulate the ligands originally expressed at higher levelson non-BCSCs (siRNA–non-BCSCs; Supplementary Fig. S5A).Coculture of pNK cells with siRNA–non-BCSCs reduced theirCD107a expression (Fig. 4A and Supplementary Fig. S5B),accompanied by reduced IFNg secretion (Fig. 4B), suggestingthat cancer cells with lower levels of MICA or MICB are unableto trigger NK cell killing. These nonactivated NK cells, there-fore, were less effective in lyzing siRNA–non-BCSCs, but notnontransfected non-BCSCs or scramble siRNA control (Fig.4C). NKG2DmAb, but not isotype IgG or NKp30 mAb, cast theeffect similar toMICA/MICBknockdownon the IFNg secretionand cytotoxicity (Fig. 4B and C), suggesting an important roleof MICA/MICB–NKG2D interaction in the immune surveil-lance of non-BCSCs by NK cells. Moreover, siRNA–non-BCSCswith downregulatedNKG2D ligands showed improved survival(Fig. 4D and Supplementary Fig. S5C), and, thus, metastasizedmore frequently to the lungs of mNK cell–depleted micetransferred with human pNK cells (Fig. 4E and SupplementaryFig. S5D). These in vivo results demonstrate that loss of MICAor MICB results in enhanced survival and metastasis of breastcancer cells.

High level of miR20a in BCSCs downregulates MICA/Band promotes metastasis

The mechanisms underlying the loss of MICA and MICB inBCSCs were explored. We failed to detect differences in themRNA levels of eitherMICA orMICB between BCSCs and non-BCSCs (Supplementary Fig. S6A). However, the level of onemicroRNA, miR20a, was found significantly higher in BCSCsthan non-BCSCs (Fig. 5A). We, therefore, hypothesized thatmiR20a might be involved in the downregulation of MICAand/or MICB on BCSCs (Supplementary Fig. S6B). Dual lucif-erase reporter assays showed that integration of MICA andMICB (Fig. 5B) 30 untranslated region (UTR) markedly inhib-ited the luciferase activities. However, the inhibition wasreversed whenmiR20a-binding sites were mutated, suggesting

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Figure 3. The expressions of NGK2D ligands MICA and MICB on BCSCs and their contribution to NK cell cytotoxicity and tumor metastasis.A, representative data of MICA and MICB expression by primary BCSCs and non-BCSCs (left). Right, quantitative data from six cases ofprimary cancers. B, detection of CD107a on pNK cells cocultured with BCSCs overexpressing MICA or MICB (E:T ¼ 5:1). To abrogateMICA/B–NKG2D interaction, pNK cells were pretreated with an NKG2D-blocking mAb (20 mg/mL). C, IFNg levels in the coculture supernatantof pNK cells with NKG2D ligands transfected with BCSCs (E:T ¼ 2:1). Experimental controls included pNK cells alone, or nontransfectedBCSCs alone, or NKp30 mAb (2 mg/mL)- or NKG2D mAb-pretreated pNK cells with BCSCs. D, the cytotoxicity of pNK cells to BCSCs(mean � SEM). Controls included K562 cells, nontransfected or vector-transfected tumor cells, or pNK cells pretreated with NKG2D or NKp30mAb. E, the survival of BCSCs overexpressing MICA or MICB in vivo (n ¼ 3). F, numbers of lung metastatic nodules formed by BCSCs (n ¼ 3).Mean � SD (A–C, E, and F); �, P < 0.05.






that endogenousmiR20a binds to 30-UTR ofMICA andMICB toregulate their expression. These results were confirmed by thefindings that the miR20a inhibitor (Supplementary Fig. S6C)markedly upregulated the expression of MICA and MICB onBCSCs (Fig. 5C). Thus, miR20a appears to interfere with theposttranscriptional modification of MICA and MICB.

To study the contribution of increased miR20a in BCSCs totheir escape fromNKcell killing, the cells were transfectedwithan miR20a inhibitor (miRi-BCSCs). After coculture with miRi-BCSCs, pNK cells expressed a significantly higher level ofCD107a (Fig. 5D and Supplementary Fig. S6D) and secretedincreased amounts of IFNg (Fig. 5E). miRi-BCSCs were alsomore sensitive to the cytotoxicity (Fig. 5F and SupplementaryFig. S6E) and the clearance by NK cells in vitro and in vivo(Fig. 5G and Supplementary Fig. S6F). Masking the NKG2D

receptor onNK cells reversed the effect of themiR20a inhibitor.Moreover, inhibition of miR20a reduced the metastasis ofBCSCs in the lungs of the mice, which was also reversed byblockade of the NKG2D receptor on pNK cells (Fig. 5H andSupplementary Fig. S6G). Although the miR20a inhibitor sligh-tly reduced sphere formation, ALDH expression, and prolifer-ation by BCSCs (Supplementary Fig. S6H and S6I), inhibition ofmiR20a alone did not significantly increase the apoptosis of thecells without the presence of NK cells (Supplementary Fig. S6J).Inhibition of miR20a alone did not significantly attenuate cellsurvival andmetastatic capacity of BCSCs in the absence of NKcells in vivo (Fig. 5G and H and Supplementary Fig. S6F andS6G). Therefore, miR20a downregulates MICA and MICB andincreases the capacity of BCSCs to metastasize by evading NKcell clearance.

Figure 4. The effect ofMICA orMICB knockdown on the sensitivity of non-BCSCs toNK cell–mediated lysis and their metastatic potential. A, FACS analysis ofCD107a expression in pNK cells that were incubated with non-BCSCs treated with indicated siRNAs (E:T ¼ 5:1). B, IFNg levels in the supernatant weredetermined by ELISA (E:T ¼ 2:1). Controls included NK cells alone, nontransfected non-BCSCs, and siRNA–non-BCSCs in the presence of effectorcells or pNK cells pretreated with NKG2D mAb, NKp30 mAb, or isotype mAb. C, killing efficacy of non-BCSCs by activated pNK cells (mean � SEM).Experiment controls were identical to B. D, the survival of non-BCSCs transfected with indicated siRNAs in vivo (n¼ 3). E, the numbers of metastatic nodulesformed by non-BCSCs (n ¼ 3) mean � SD (A, B, D, and E); �, P < 0.05.

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Targeting the miR20a–MICA/B axis by all-trans retinoicacid sensitizes BCSCs to NK killing and preventsmetastasisBecause differentiation therapy targeting CSC represents a

promising therapeutic approach (2, 23, 24), we tested the

capacity of all-trans retinoic acid (ATRA), a well-establisheddifferentiation-inducing agent for CSCs from leukemia andglioblastoma (25, 26), to affect immune evasion by BCSCs.ATRA markedly enhanced the expression of both MICA andMICB proteins (Fig. 6A) but not their mRNA levels

Figure 5. Regulation of MICA/MICB expression, the sensitivity to NK cell–mediated lysis, and metastatic potential by miR20a in BCSCs. A, the expression ofmiR20a in BCSCs and non-BCSCs by cells from six primary cancers was examined by qRT-PCR (U6B as a loading control; horizontal bars, means).B, luciferase activity was detected in BCSCs transfected with Mock or p-MICA 30UTR (or mutant) or p-MICB 30UTR (or mutant) reporters in the presenceof control miRNA (ctrl-miRNA) or anmiR20a inhibitor (anti-miR20a). C, FACS analysis ofMICA orMICB expression by BCSCs transfectedwith anti-miR20a orctrl-miRNA for 48 hours. D and E, pNK cells, with or without preincubation using NKG2D-blocking mAb, were cocultured with BCSCs transfectedwith anti-miR20a. CD107a expression on pNK cells was detected (D) and IFNg levels in the supernatant were analyzed (E). F, cytotoxicity of pNK cells toCSCs (mean � SEM). G, the short-term lung survival of BCSCs (n ¼ 3). H, numbers of lung metastasis formed by BCSCs (n ¼ 3); mean � SD (B, D, E, G,and H); �, P < 0.05.






(Supplementary Fig. S7A) by BCSCs. Moreover, ATRA treat-ment downregulated miR20a (Fig. 6B), whereas its restorationwith miR20a mimics (miR20a M) in ATRA-treated BCSCs(Supplementary Fig. S7B) reduced MICA and MICB on BCSCs(Fig. 6C). These results suggest that ATRA regulates MICA andMICB expression by repressing aberrantly expressed miR20a.

The enhancing effect of ATRA on MICA and MICB expres-sion prompted us to investigate the potential of induceddifferentiation to sensitize BCSCs to NK cell cytotoxicity,thus preventing metastasis. BCSCs treated with ATRAshowed reduction of miR20a and were more sensitive toNK cell–mediated lysis. Restoration of miR20a in ATRA-treated BCSCs or blocking NKG2D on pNK cells attenuatedthe sensitivity of ATRA-treated BCSCs to the lysis by NK cells(Fig. 6D). Furthermore, pretreatment of BCSCs with ATRAreduced their survival (Fig. 6E and Supplementary Fig. S7C)and metastatic capacity (Fig. 6F and Supplementary Fig.S7D) in mice transferred with human pNK cells. The effect ofATRA was reversed by introduction of exogenous miR20a inBCSCs or blockade of NKG2D on NK cells. In addition, ATRAtreatment resulted in measurable reduction in the propa-gation and stem-like properties of BCSCs (Supplementary

Fig. S7E and S7F). However, ATRA alone neither inducedsignificant apoptotic death of BCSCs (SupplementaryFig. S7G), nor attenuated their survival/metastatic capacityin vivo (Fig. 6E and F), indicating that ATRA inhibits BCSCmetastasis mainly through facilitating elimination of BCSCsby NK cells. Moreover, ATRA showed less effect on theexpression of miR20a and MICA/MICB as well as the sen-sitivity to NK cell cytotoxicity in non-BCSCs (SupplementaryFig. S7H–S7J), suggesting that ATRA preferentially targetsBCSCs. These observations indicate that targeting themiR20a–MICA/B axis by induction of differentiation sensi-tizes BCSCs to NK cell killing and prevents their metastasis.

DiscussionCSCs are the cellular origin of multiple malignant properties

in cancer, including unrestrained self-renewal (2), neovascu-larization (3), resistance to chemo- or radiotherapy (2, 27),dissemination (4), and evasion of antitumor immunity (5–9).The metastasis of BCSCs is a result of epithelial-to-mesenchy-mal transition (EMT) induced by genetic/epigenetic altera-tions or microenvironmental cues (4). In this study, we dem-onstrate that BCSCs evade NK cell surveillance to establish

Figure 6. The effect of ATRA on the sensitivity to NK cell–mediated lysis and themetastatic potential of BCSCs. A and B, BCSCswere treatedwith ATRA for 48hours. The expression of MICA, MICB (A), and miR20a (B) was examined. C, BCSCs with or without transfection of miR20a mimics (miR20aM) were treatedwith ATRA for 48 hours, andMICA orMICBexpressionwas detected. D, lysis of BCSCs by pNK cells (mean�SEM). E, the survival of BCSCs in vivo; (n¼ 3). F,numbers of lung metastatic foci formed by BCSCs in vivo (n ¼ 3); mean � SD (B, E, and F); �, P < 0.05.

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tumor metastasis, representing a revelation of novel mechan-isms by which BCSCs establish distant metastasis.Tumor heterogeneity has been recognized as the conse-

quence of genetic diversity within cancer and the presence ofCSCs (2). Immunoediting may also promote cancer progres-sion and select tumor cells with enhanced malignancy (10).The potential for CSCs to avoid destruction by adaptiveimmunity has been documented in the context of CTL-medi-ated cytotoxicity (5–7, 9, 28). In addition, IFNg released byCTL promotes the proliferation of CSCs and contributes toleukemia progression (8). These observations demonstratethat tumor metastasis is associated with dynamic regulationof CSC evasiveness from adaptive immunity.The impact of innate immunity on cancer progression and

enrichment of CSCs has largely been unclear. It has beenreported that several types of normal stem cells were efficientlylyzed by fresh allogeneic NK cells (29–31). Similarly, stem-likecells in oral squamous carcinoma (31), colorectal carcinoma(32), and glioblastoma (33) were susceptible to NK cell–medi-ated cytotoxicity. However, leukemic stem cells were resistantto allogeneic NK cell cytotoxicity in vitro (34). Evasion of BCSCsfrom NK cell surveillance was implicated in trastuzumabresistance, in which treatment of MCF-7 cells with trastuzu-mab resulted in enrichment of CD44þCD24� cells by NK cells(35), although BCSCs and remaining cells from other cell lineswere equally sensitive to allogeneic NK cell–mediated lysisin vitro (13). Our findings, using primary breast cancer cells,demonstrate that ALDHþ BCSCs were resistant to both autol-ogous and allogeneic NK cell cytotoxicity, and that cancer cellssurviving NK cell killing were enriched with ALDHþ BCSCs.Therefore, CSCs from neoplastic tissues of different originexhibit diverse sensitivity to lysis by NK cells. Understandingthis diversity has important implication for the development ofrational immune therapy against CSCs.NK cell anergy is detected in a great number of patients

with cancer, especially those with late-stage cancer (15, 36).By using in vitro expanded anergic NK cells, we demonstratethat immunoevasion of BCSCs from NK cell cytotoxicity isessential for establishment of breast cancer metastasis.Therefore, resistance to NK cell lysis may be an intrinsicproperty of BCSCs to initiate metastasis in the patients withdysfunctional NK cells. In cancer tissues, we observed codis-tribution of CD56þ cells with ALDH1þ BCSCs, indicatingthat these cells may play wider roles in tumor microenvi-ronment. IFNg released by immune cells regulates thebiology of CSCs in chronic myeloid leukemia (8). Neverthe-less, no significant difference of IFNg receptor a chainexpression between BCSCs and non-BCSCs indicates thatthe immune evasion of BCSCs is not likely due to thedifferential activation of IFNg signaling by infiltrating NKcells.We found that the lack of stimulatory signals in BCSCs for

NK cells was associated with reduced expression of MICA andMICB, two ligands for the NK-activating receptor NKG2D (37),in BCSCs. MICA and MICB are stress-inducible antigens rarelyexpressed by healthy cells but are frequently upregulated ontransformed or virus-infected cells (38). Reduced expression ofMICA/B facilitates the evasion of epithelial tumors from

surveillance by NK cells and subsets of T cells (39). Wedemonstrated that reduced MICA/B expression by BCSCscontributed to their resistance to NK cell cytotoxicity andenhanced metastatic capacity in vivo. Therefore, it is plausiblethat reduction in MICA/B expression promotes BCSC metas-tasis by evading NK cell killing.

Despite the lower expression of MICA/B proteins by BCSCs,no substantial difference inMICA/BmRNA levels was detectedbetween BCSCs and non-BCSCs, suggesting posttranscription-al regulation of MICA/B in tumor cells. Small noncoding RNAs,miRNAs, are important posttranscriptional regulators in CSCs(40, 41). miRNAs targeting MICA or MICB have been identifiedin studies of viral infection and IFNg treatment (42–45). As amember of the oncogenic miR17-92 cluster, the expression ofmiR20a is elevated in several malignancies (46–48). We dem-onstrated that overexpression of miR20a reduced the levels ofboth MICA and MICB in BCSCs, resulting in their immuneevasion with increased metastasis. Thus, miR20a is a pivotalregulator of immune evasion by BCSCs, and represents apotential target for strengthening immunosurveillance toinhibit tumor metastasis.

Promoting differentiation is a promising therapeuticapproach against CSCs. Induced differentiation of CSCs resultsin their loss of chemoresistance and tumorigenicity (24). Onesuch clinically applied differentiation agent is ATRA, which iswell-known for its potent differentiation-inducing effect onCSCs from leukemia and glioblastoma (25, 26). In the presentstudy, BCSC differentiated by ATRA showed diminishedimmune evasiveness with reduced expression of miR20a butenhancedMICA/B expression. Thus, the resistance of BCSCs toNK cell surveillance is reversible by differentiation and increas-ing the levels of ligands for NK cell stimulating receptorNKG2D.

In conclusion, our present study reveals that an aberrantmiR20a–MICA/B axis is responsible for the escape of BCSCsfrom innate immunity, which is critical for increased meta-static capacity of BCSCs. Thus, targeting the miR20a–MICA/Baxis may represent a promising immune therapeutic strategyto prevent breast cancer metastasis.

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

Authors' ContributionsConception and design: B. Wang, Z. Wang, X.-W. BianDevelopment of methodology: B. Wang, Z. Wang, J. Jiang, S.-C. Yu, Y.-H. CuiAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): B. Wang, Q. Wang, Z. Wang, J. Jiang, S.-C. Yu, Y.-F.Ping, J. Yang, S.-L. Xu, X.-Z. Ye, C. Xu, L. Yang, C. Qian, Y.-H. Cui, X.-W. BianAnalysis and interpretation of data (e.g., statistical analysis, biostati-stics, computational analysis): B. Wang, Z. Wang, S.-C. Yu, L. Yang, Y.-H. Cui,X.-W. BianWriting, review, and/or revision of the manuscript: B. Wang, J.-M. Wang,Y.-H. Cui, X. Zhang, X.-W. BianAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): Z. Wang, S.-C. Yu, Y.-H. Cui, X.-W. BianStudy supervision: Y.-H. Cui, X.-W. Bian

AcknowledgmentsThe authors thank Dr. Peng Tang for collecting fresh specimens from patients

with breast cancer (Breast SurgeryCenter, Southwest Hospital, theThirdMilitaryMedical University) and Qing-hua Ma for assistance in FACS analyses (Institute






of Pathology and Southwest Cancer Center, Southwest Hospital, Third MilitaryMedical University).

Grant SupportThis work was supported by grants from the National Basic Research

Program of China (973 Program, no. 2010CB529403; X.-W. Bian), the NationalNatural Science Foundation of China [nos. 81071771, 30930103, and 30725035(X-W. Bian); 81101631 (B. Wang); and 81101606 (Z. Wang)], the NationalScience and Technology Major Program (no. 2011ZX09102-010-02; X.-W.

Bian), and the Natural Science Foundation Project of CQ CSTC (no.CSTC2012JJA10124; B. Wang). J.-M. Wang was supported by the IntramuralResearch Program of the National Cancer Institute, NIH.

The costs of publication of this article were defrayed in part by the paymentof page charges. This article must therefore be hereby marked advertisementin accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received September 6, 2013; revised June 30, 2014; accepted August 16, 2014;published OnlineFirst August 27, 2014.

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2014;74:5746-5757. Published OnlineFirst August 27, 2014.Cancer Res Bin Wang, Qiang Wang, Zhe Wang, et al. Cytotoxicity by Human Breast Cancer Stem CellsMetastatic Consequences of Immune Escape from NK Cell

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