Expression, Signaling Proficiency, and Stimulatory Function of the NKG2D

Expression, signaling proﬁciency, and stimulatory function of the NKG2D lymphocyte receptor in human cancer cells

Andrea Caballero Benitez1, Zhenpeng Dai1, Henning H. Mann, Rebecca S. Reeves, Daciana H. Margineantu, Ted A. Gooley, Veronika Groh, and Thomas Spies2

Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, WA 98109

Edited* by Robert N. Eisenman, Fred Hutchinson Cancer Research Center, Seattle, WA, and approved January 27, 2011 (received for review December 10, 2010)

The stimulatory natural killer group 2 member D (NKG2D) lympho- MICA (sMICA) in many cancer patients, NKG2D function be- cyte receptor and its tumor-associated ligands are important comes systemically impaired (13). In addition, tumor-associated mediators in the immune surveillance of cancer. With advanced MICA and sMICA drive proliferative expansions of normally rare + human tumors, however, persistent NKG2D ligand expression may NKG2D CD4 T cells that have immunosuppressive functions favor tumor progression. We have found that cancer cells them- (14). Taken together, these negative imprints on the immune selves express NKG2D in complex with the DNAX-activating protein system may account, at least in part, for poor clinical outcomes that 10 (DAP10) signaling adaptor. Triggering of NKG2D on ex vivo have been associated with the presence of NKG2D ligands in can- – cancer cells or on tumor lines which express only few receptor cer patients (1, 15 17). complexes activates the oncogenic PI3K–protein kinase B (PKB/ As tumor immune surveillance fails in advanced cancer patients, AKT)–mammalian target of rapamycin (mTOR) signaling axis and we suspected that persistent NKG2D ligand expression, in addi- downstream effectors, the ribosomal protein S6 kinase 1 (S6K1) tion to promoting immune evasion, might constitute another, perhaps more fundamental, tumor survival asset. We report here and the translation initiation factor 4E-binding protein 1 (4E-BP1). that breast, ovarian, prostate, and colon cancer cells express In addition, as in lymphocytes, NKG2D ligand engagement sti- NKG2D–DAP10 receptor complexes. In vitro experimental and mulates phosphorylation of JNK and ERK in MAP kinase cascades. correlative clinical data support the idea that this immunoreceptor Consistent with these signaling activities, above-threshold expres- complements the presence of its ligands on cancer cells for stim- – sion of NKG2D DAP10 in a ligand-bearing tumor line increases its ulation of tumor growth, thus suggesting functional similarity to bioenergetic metabolism and proliferation, thus suggesting func- oncogenic growth factor receptors. tional similarity between this immunoreceptor and tumor growth factor receptors. This relationship is supported by significant corre- Results lations between percentages of cancer cells that are positive for Expression of NKG2D–DAP10 on Cancer Cells. In the course of ex- surface NKG2D and criteria of tumor progression. Hence, in a con- amining primary breast and epithelial ovarian cancer specimens ceptual twist, these results suggest that tumor co-option of NKG2D for infiltrating lymphocytes by immunohistochemistry, we observed immunoreceptor expression may complement the presence of its unambiguous cancer tissue staining for the NKG2D receptor using ligands for stimulation of tumor growth. the specific 1D11 mAb and HRP-conjugated secondary reagent (Fig. 1A) (3). The staining patterns were similar to those recorded growth stimulation | signal transduction for the MIC ligands of NKG2D that were detected with the bispecific 6D4 mAb (7). In comparison, tumor-infiltrating CD3+ he stimulatory natural killer group 2 member D (NKG2D) lymphocytes, among which most CD8 T cells are positive for Tlymphocyte receptor and its ligands are important mediators NKG2D, were infrequent and scattered. Staining for NKG2D of of tumor immunity but also are instrumental in promoting tumor normal breast, ovary, and prostate tissue sections including well- B immune evasion and immune suppression (1, 2). Human NKG2D recognizable epithelial areas gave negative results (Fig. 1 ). in complex with the DNAX-activating protein 10 (DAP10) sig- Cell-surface expression and composition of NKG2D receptor complexes were examined with cancer-cell suspensions sorted for naling adaptor is expressed on most natural killer (NK) cells and + − CD8 T cells (3, 4). DAP10 signals upon Tyr phosphorylation of an epithelial cell adhesion molecule (EpCAM) CD45 pheno- type to ensure analysis of epithelial tumor cells and exclusion of YXXM motifs in its cytoplasmic tail by binding either the p85 + − subunit of PI3K or the growth factor receptor-bound protein 2 hematopoietic cells. The epithelial nature of EpCAM CD45

fi IMMUNOLOGY (Grb2), thus activating protein kinase B (PKB/AKT) and MAP cells was con rmed separately by staining for pan-cytokeratin. kinase cascades (4–6). NKG2D ligands include the MHC class I- Flow cytometry analysis showed that 12/12 breast, 14/14 colon, related chains A and B (MIC, A and B), which are absent from 29/30 epithelial ovarian, and four of four prostate cancer specimens included cancer-cell populations that were positive for the surface of most normal cells but are induced by generic – responses to cellular stress in diseased cells such as epithelial surface NKG2D, with proportional ranges of 4 83% (mean 23%, SD ± 22.3), 4–39% (mean 16%, SD ± 10.6), 1–65% (mean 18%, tumor cells (1, 7). Additional NKG2D ligands comprise six ± – ± members of the UL16-binding protein (ULBP) family, which can SD 17.3), and 3 33% (mean 11%, SD 14.7), respectively be tumor associated as well (8). MIC frequently are expressed in cancers and stimulate NK- cell and CD8 T-cell responses against tumor cells in vitro (3, 7). Author contributions: D.H.M., V.G., and T.S. designed research; A.C.B., Z.D., H.H.M., R.S.R., Evidence for NK-cell– and T-cell–mediated protective functions and D.H.M. performed research; D.H.M., T.A.G., V.G., and T.S. analyzed data; and V.G. and of NKG2D in tumor immunity has been derived from mouse T.S. wrote the paper. model experiments and a study of NKG2D-deficient mice (9–12). The authors declare no conflict of interest. At least in humans, however, tumor progression coupled with *This Direct Submission article had a prearranged editor. persistent surface expression and shedding of soluble MICA point 1A.C.B. and Z.D. contributed equally to this work. to changes in dynamics that may signify host tumor susceptibility 2To whom correspondence should be addressed. E-mail: [email protected]. instead of resistance (13). NKG2D is subject to ligand-induced This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. down modulation. Because of the ubiquitous presence of soluble 1073/pnas.1018603108/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1018603108 PNAS | March 8, 2011 | vol. 108 | no. 10 | 4081–4086 Downloaded by guest on September 25, 2021 (Fig. 1C). Sizeable proportions of all cancer-cell suspensions expressed the MIC ligands of NKG2D. We next tested whether cancer cells also express the DAP10 signaling adaptor (4–6). By standard 30-cycle RT-PCR, NKG2D and DAP10 cDNA amplicons were readily detected with six − of seven breast, ovarian, colon, and prostate EpCAM+CD45 cancer-cell suspensions. Upon sequencing, we found no changes in the canonical amino acid coding regions. Only faint signals were recorded with mRNAs from non-lymphocyte-depleted normal breast, ovarian, and skin tissues (Fig. 1D). Cancer-cell expression of NKG2D–DAP10 complexes was conﬁrmed by immunoprecipitation using bead-coupled anti-NKG2D 5C6 mAb (3) fol- lowed by SDS/PAGE and sequential immunoblot probing for NKG2D and DAP10. Direct comparison of a breast cancer sample with matched nonaffected tissue control further illustrated the malignancy-associated expression of NKG2D–DAP10 (Fig. 1E).

Triggering of NKG2D Stimulates PI3K-Dependent Phosphorylation of AKT. Our ﬁndings gave rise to the idea that cancer cells might co- opt expression of NKG2D to exploit the presence of its ligands for self-stimulation of tumor growth. In NK cells and T cells, phosphorylation of DAP10 activates branched signaling cascades that include the PI3K–AKT axis (4, 5, 18). We tested the sig- − naling capacity of NKG2D–DAP10 in sorted EpCAM+CD45 breast, ovarian, and prostate cancer cells after serum deprivation by 1D11 mAb-mediated receptor cross-linking and subsequent Ab probing of total cell lysate immunoblots for phosphorylation of AKT (19). Phospho-AKT (P-AKT) was induced in all four cancer samples tested but not in matched ovarian cancer cells sorted for absence of surface NKG2D. Its appearance was sensitive to wortmannin and thus was dependent on PI3K (Fig. 1F) (4, 19). Exposure to insulin provided positive control activation except for unresponsive prostate cancer cells. Crosslinked F(ab′)2 fragments of the anti-NKG2D 5C6 mAb also were effective in P-AKT induction, thus precluding Ab Fc region/Fc receptor-mediated or unspeciﬁc stimulatory events (Fig. 1G).

Minimal NKG2D–DAP10 Expression Is Sufficient for AKT Activation in Tumor Lines. A thorough investigation of NKG2D–DAP10 signaling and its physiological effects required serial analyses and approaches for which ex vivo cancer cells were unsuitable. We thus switched to studying tumor lines. However, unlike cancer cells, 15 breast, colon, gastric, ovarian, and prostate tumor lines tested by flow cytometry were either negative for surface NKG2D or displayed only minor shifts in the fluorescence intensity profile. No increased expression was apparent after treatment of cells with inhibitors of proteasomal or lysosomal degradation or after exposure to IL-15, which induces T-cell NKG2D (13, 20, 21). By real- time quantitative PCR (qPCR), 12 randomly selected tumor lines contained an average of no more than one copy of NKG2D and DAP10 mRNA per cell. In comparison, five freshly prepared − NKG2D+EpCAM+CD45 cancer cell suspensions contained averages of 15 and 100 copies of NKG2D and DAP10 mRNAs,

Fig. 1. Cancer-cell expression of NKG2D–DAP10 and stimulation of PI3K- dependent AKT phosphorylation. (A) Micrographs of Ab staining for NKG2D NKG2D and DAP10 using cell lysates of control NKL cells (1 × 106 cells) and and MIC of breast and ovarian cancer tissue cryosections. Staining for CD3 EpCAM+CD45− cancer cells (3–5 × 106 cells) corresponding to those shown in identifies tumor-infiltrating T cells. (B) Staining of normal breast, ovary, and D. Panels at right show protein data from an additional breast cancer and prostate tissue sections for NKG2D. (C) Flow cytometry of freshly isolated matched nonaffected tissue (NAT) control. Numbers at left indicate molec- breast (BT), ovarian (OT), prostate (PT), and colon (CT) cancer cells (gated for ular masses (in kDa) of NKG2D and DAP10. (F) Stimulation of purified − − EpCAM+CD45 ; see upper left dot plot for a representative example) for EpCAM+CD45 breast, ovarian, and prostate cancer cells by cross-linked anti- surface NKG2D and MIC. Numbers in dot plots indicate percentages of cells NKG2D mAb 1D11 results in detection of P-AKT (S473) on total cell lysate in quadrants. (D) Detection of mRNA for NKG2D, DAP10, and control GAPDH immunoblots. P-AKT was not detected in ovarian cancer cells (OT25−) sorted by RT-PCR in NKL NK cells and in breast (BT), colon (CT), ovarian (OT), and for absence of NKG2D. Exposure to insulin provides positive control activa- prostate (PT) cancer cells. Note that sample OT91 is essentially negative for tion except with the nonresponsive prostate cancer cells. The PI3K inhibitor − NKG2D–DAP10. RNAs were prepared from sorted EpCAM+CD45 cancer-cell wortmannin (Wort) inhibits AKT phosphorylation. DMSO is added as solvent suspensions. Panels at right show minimal NKG2D–DAP10 expression in control. (G) Detection of P-AKT (S473) after stimulation of purified breast + control breast, ovary, and skin tissue specimens nondepleted for CD45 cells. and ovarian cancer cells with cross-linked anti-NKG2D mAb 5C6 F(ab′)2 Numbers at left indicate cDNA amplicon sizes (in bp). (E) NKG2D immuno- fragments. Ig lanes in F and G represent cells exposed to primary mouse

precipitations (IP) with bead-coupled mAb 5C6 and immunoblotting (IB) for control IgG and secondary goat anti-mouse F(ab′)2.

4082 | www.pnas.org/cgi/doi/10.1073/pnas.1018603108 Benitez et al. Downloaded by guest on September 25, 2021 respectively. Nonetheless, with a subset of tumor lines (breast 4 regulator of T-cell activation, flow cytometry can be insuffi- MCF-7, BT-20, and MDA-MB-453; ovarian HTB-78; colon ciently sensitive to detect minimal expression of functionally active DLD-1; and gastric AGS) chosen because of low constitutive AKT cell-surface receptors (22). phosphorylation, PI3K-dependent induction of P-AKT was read- Complementary evidence was obtained with the breast MDA- ily observed after desensitization of cells and 1D11 mAb- or 5C6 MB-231, prostate PC3, and melanoma A375 tumor lines that – F(ab′) -mediated NKG2D cross-linking (Fig. 2A). lacked detectable NKG2D DAP10 complexes and showed no 2 A B These results implying functional NKG2D–DAP10 expression inducible AKT phosphorylation (Fig. 2 and ). Taken to- were difficult to reconcile with the minimal expression of the gether, these experiments with tumor lines replicated the results obtained with freshly isolated cancer cells, except for the much corresponding mRNAs. However, the mRNAs may be distrib- – uted unevenly among tumor cells and template multiple trans- lower expression of NKG2D DAP10. We also tested for expression of NKG2D receptors in mouse lation cycles. In fact, protein expression of NKG2D–DAP10 was cancer specimens including 7,12-dimethylbenz(a)anthracene/ detected when lysates of large numbers of cells (∼5 × 107 per ∼ – × 6 12-O-tetradecanoyl-phorbol-13-acetate (DMBA/TPA) carcinogen- SDS/PAGE lane; with cancers, we used 3 5 10 cells) were induced squamous cell carcinoma, transgenic adenocarci- used in immunoprecipitation and immunoblot experiments using noma of the mouse prostate (TRAMP) model aggressive and B a highly sensitive chemiluminescent reagent (Fig. 2 ). Alto- late-arising autochthonous prostate cancers, and human epi- gether, these results indicate that few receptor complexes are dermal growth factor receptor 2 (HER-2)/neu–transgenic mam- sufficient for signal transduction in tumor lines, possibly because mary carcinoma (23–25). By RT-PCR, all T-cell– and NK-cell– of their sensitized activation status. As exemplified by the CTLA- depleted cancer-cell samples were devoid of both the long and short variants of murine NKG2D and its DAP10 and DAP12 signaling adaptors (26) (Fig. S1A). No NKG2D receptor protein was detected in any lysates (5 × 106 cancer cells) by immunoprecipitation with bead-coupled Ab and immunoblot (Fig. S1B). It thus appears that NKG2D receptor expression does not occur in mouse models of cancer.

Genetic Confirmation of NKG2D–DAP10 Signaling in Tumor Cells. Because tumor cell expression and signaling proficiency of NKG2D–DAP10 may have profound implications, we sought definitive experimental proof. Ectopic expression of NKG2D– DAP10 in stable transfectants of the A375 melanoma line (A375- TF cells) restored PI3K-dependent AKT phosphorylation after Ab-mediated receptor cross-linking (Fig. 1 A and B). In a complementary approach, lentiviral transduction of siRNAs in breast tumor MCF-7 cells linked NKG2D and DAP10 depletion to loss of inducible AKT phosphorylation (Fig. S2A). Demonstration of protein depletion, as done for NKG2D (Fig. S2B), was not fea- sible for DAP10 because the DAP10 siRNA-transduced MCF-7 cells proliferated poorly and could not be expanded to the ∼5 × 107 cells required. Hence, we used NKG2D–DAP10 MCF-7 cell transfectants (MCF-7–TF cells) and a CD8 T-cell line to dem- onstrate fully the efficacy of RNAi targeting of NKG2D and DAP10 (Fig. S2 B–D). Taken together, these results confirmed the signaling capacity of NKG2D–DAP10 in tumor cells.

Activation of Mammalian Target of Rapamycin and Downstream Effectors and Evidence for Self-Stimulation. The growth factor- responsive PI3K–AKT signal transduction pathway regulates intersecting cellular processes including cell-cycle progression, metabolic activity, and survival and commonly is hyperactive in cancer (27). To evaluate further the proﬁciency of NKG2D– DAP10 signaling in tumor cells, we examined activation of the mammalian target of rapamycin (mTOR) kinase downstream of AKT and its catalytic activity on effectors controlling protein IMMUNOLOGY synthesis and cell growth, the ribosomal protein S6 kinase 1 (S6K1) and the translation initiation factor 4E-binding protein 1 (4E-BP1) (28, 29). Along this axis, mTOR is part of the rapamycin-sensitive mTOR complex 1 (mTORC1), which is ac- Fig. 2. PI3K-dependent AKT phosphorylation correlates with minimal tivated separately by nutrient supply. The detection of target NKG2D–DAP10 expression in tumor lines. (A) Immunoblot detection of P- phosphorylation events following NKG2D cross-linking thus ne- AKT (S473) in lysates of desensitized MCF-7, BT-20, MDA-MB-453, HTB-78, cessitated extensive prior cell starvation in serum-free medium DLD-1, and AGS cells but not of MDA-MB-231, PC3, and A375 cells after mAb ′ – and HBSS for 24 h and a minimum of 4 h, respectively, to reduce 1D11 or mAb 5C6 F(ab )2 cross-linking. Transfection of NKG2D DAP10 ﬁ restores AKT phosphorylation in A375-TF cells. Insulin provides positive constitutive activation suf ciently (30). Under these conditions, control activation. Ig control lanes represent cells exposed to mouse IgG and freshly isolated cancer cells and most tumor lines became unresponsive to NKG2D triggering because of impaired viability secondary goat anti-mouse F(ab′)2. DMSO is added as solvent control. As with the cancer-cell suspensions, AKT phosphorylation is sensitive to wort- and probable loss of the scarcely expressed receptor proteins, mannin (Wort). (B) AKT phosphorylation correlates with the detection of respectively. However, with MCF-7–TF cells, which were com- small amounts of NKG2D–DAP10 protein complexes by immunoprecipitation parable to ex vivo cancer cells in surface NKG2D expression with bead-coupled anti-NKG2D mAb 5C6 from lysates of typically 5 × 107 (Fig. 1C and Fig. S2C), and the HTB-78 ovarian tumor line we cells (2 × 107 BT-20 and 5 × 106 A375-TF cells) and sequential immunoblot recorded robust induction of phosphorylation of mTOR, S6K1, probing for NKG2D and DAP10. and 4E-BP1 (Fig. 3A). The appearance of the phosphoproteins

Benitez et al. PNAS | March 8, 2011 | vol. 108 | no. 10 | 4083 Downloaded by guest on September 25, 2021 was both PI3K dependent and rapamycin sensitive. Thus, these to Grb2 results in phosphorylation of JNK (5, 6, 31). In cancer results provided evidence for the capacity of NKG2D–DAP10 to cells, these MAP kinases are activation targets of the EGF re- stimulate the oncogenic PI3K–AKT–mTOR–S6K1/4E-BP1 sig- ceptor (EGFR), among other receptor Tyr kinases, which, be- naling axis in tumor cells. By inference from this example, other cause of mutation or aberrant expression, frequently cause effectors coupled to AKT signal transduction that promote cell- excessive tumor cell proliferation and increased motility and cycle progression, differentiation, and survival are likely to be af- survival (32). To test the relevance of these pathways for NKG2D– fected by NKG2D–DAP10 as well. DAP10 signaling in tumor cells, we used freshly isolated − For the signaling experiments throughout this study, cells EpCAM+CD45 ovarian cancer cells and HTB-78 and MCF-7– typically were grown at low density to minimize NKG2D ligand- TF cells that were desensitized and stimulated with recombinant mediated self-stimulation. However, this functional activity was sMICA cross-linked by anti–His-tag Ab. As with the EGF control unproven, although it was central to our model of the biological activation, stimulation of NKG2D–DAP10 resulted in phos- significance of tumor expression of NKG2D. To obtain evidence phorylation of the ERK1/2 and JNK1/2 isoforms as determined by for self-stimulation, we examined P-AKT induction in a time- immunoblot using phosphoprotein-specific Abs (Fig. 3C). Tumor course experiment comparing desensitized A375 mock-transfected cell exposure to sMICA or anti–His-tag Ab alone had no effect. − − control cells (NKG2D DAP10 ) and A375-TF cells (NKG2D+ The appearance of phosphorylated ERK1/2 (P-ERK1/2) and DAP10+) that were spun into pellets to enforce cell contacts phosphorylated JNK1/2 (P-JNK1/2) was diminished by inhibitors mimicking solid cancer-cell compaction. Like the other tumor lines, of the MAP kinase kinase (MEK) upstream of ERK (UO126) and A375 melanoma cells are positive for several NKG2D ligands (Fig. JNK itself (SP600125), respectively, but not vice versa (Fig. 3C). P- S3). Although no signal increase occurred in the negative control ERK1/2 but not P-JNK1/2 was sensitive to wortmannin and thus, cells, P-AKT was induced in A375-TF cells after 10 min of in- as in lymphocytes, dependent on PI3K (6). Consequently, P- cubation. This activity was blocked in the presence of a mixture of JNK1/2 was in all likelihood downstream of Grb2. This arrange- anti-MIC and anti-ULBP mAb (14), thus reflecting productive ment was supported by immunoblot detection of Grb2 in transient NKG2D receptor–ligand interactions (Fig. 3B). association with DAP10, which was immunoprecipitated from lysates of MCF-7–TF cells after brief Ab-mediated NKG2D Activation of ERK and JNK MAP Kinases. As determined thus far, cross-linking (Fig. 3D). As with tumor cell ligand-induced AKT signaling pathway activation by NKG2D–DAP10 in tumor cells phosphorylation (Fig. 3B), P-ERK1/2 and P-JNK1/2 were detec- was similar to that in lymphocytes. In activated human NK cells ted in time-course experiments with compacted A375-TF cells in and T cells, signal transduction initiated by PI3K also leads to the absence but not in the presence of a ligand-masking Ab mix- phosphorylation of ERK, whereas alternative coupling of DAP10 ture (Fig. 3 E and F).

Fig. 3. Activation of mTOR–S6K1/4E-BP1 and MAP kinase cascades. (A) Immunoblot detection of P-mTOR (S2448), phosphorylated S6K1 (P-S6K1) (T389), and phosphorylated 4E-BP1 (P-4E-BP1) (T37/46) in lysates of HTB-78 and MCF-7–TF cells after starvation and mAb 1D11 cross-linking. Phosphorylation events are sensitive to both wortmannin (Wort) and rapamycin (Rapa). Insulin provides positive control activation. Ig control lanes are as in Fig. 2. DMSO is added as solvent control. (B) Induction of P-AKT after 10 min in pellets of A375-TF but not in mock-transfected A375 cells and inhibition by anti-MIC/ULBP mAb mixture. (C) Immunoblot detection of P-ERK1/2 (T202/Y204) and P-JNK1/2 (T183/Y185) in lysates of desensitized EpCAM+CD45− ovarian cancer (OT63), and HTB-78 and MCF-7–TF cells after exposure to recombinant sMICA and cross-linking anti–His-tag Ab. EGF provides positive control activation. UO126 and SP600125 are inhibitors of MEK/ERK and JNK, respectively. Phosphorylation of ERK but not JNK is sensitive to wortmannin. (D) Immunoprecipitation of DAP10 and immunoblot detection of transiently associated Grb2 in lysates of MCF-7–TF cells (5 × 107 cells per SDS/PAGE lane) after 2 or 5 min of Ab-mediated NKG2D cross- linking. (E) Induction of P-ERK1/2 in pellets of A375-TF cells and inhibition by anti-NKG2D ligand Ab mixture as in B.(F) Induction of P-JNK1/2 in pellets of A375-TF cells and inhibition by anti-NKG2D ligand Ab mixture.

4084 | www.pnas.org/cgi/doi/10.1073/pnas.1018603108 Benitez et al. Downloaded by guest on September 25, 2021 Stimulation of Cellular Proliferation and Bioenergetic Metabolism. Joint Committee on Cancer (AJCC) staging (Table S1). To allow According to our analysis of representative components of sig- a combined analysis of all four cancer types studied, we applied naling pathways associated with tumorigenesis, NKG2D–DAP10 tumor size/spread, node involvement, and metastasis (TNM) displayed activities similar to growth factor receptors such as status rather than cancer type-specific staging. Survival/outcome EGFR and insulin-like growth factor-1 receptor (IGF-1R) (32, information was not available. Linear regression analysis revealed 33). We thus explored effects of NKG2D stimulation on cellular significant correlations between mean percentages of NKG2D+ functions using the MCF-7 mock-transfected control and MCF- cancer cells (%2D) with tumor stage [P < 0.0001; stage I (n = 14), 7–TF model cells. Cells were plated at near confluence in the %2D 3.7; stage II (n = 15), %2D 10.9; stage III (n = 30), %2D absence of growth factors with or without the relevant anti-MIC 28.3; stage IV (n = 1), %2D 45.7] and tumor size/spread [P < and anti-ULBP1/3/4 Abs or control Ig 24 h before assay time 0.0001; T1 (n = 12), %2D 4.5; T2 (n = 13), %2D 6.3; T3 (n = 34), (Fig. S3). Cell-cycle analysis by propidium iodide (PI) staining %2D 27.7; T4 (n = 1), %2D 34.0]. By t-test assessment, a statis- identified significantly enlarged proportions of MCF-7–TF cells tically significant association also was observed with lymph node P < n n with DNA content corresponding to S- and G2-phase transitions. status [ 0.009; negative ( = 33), %2D 12.3; positive ( = 18), This effect was reversed in the presence of the anti-NKG2D li- %2D 24.6]. There were no statistically significant associations gand Ab mixture or by RNAi targeting of NKG2D (Fig. 4A). with tumor grade (P = 0.025), presence or absence of lymphatic These results confirmed ligand-mediated NKG2D stimulation and/or vascular invasion (P = 0.10), or patient age (P = 0.59). and were corroborated by determinations of cellular ATP as an Together, these results provide ex vivo correlative evidence as- independent parameter of cellular proliferation (Fig. 4B). sociating NKG2D expression with criteria of tumor progression, Changes in bioenergetic cell metabolism were measured using thereby lending support to its tumor growth factor receptor-like an extracellular flux analyzer that allows real-time determinations stimulatory functions. of oxygen consumption rates (OCR) and of extracellular acidification rates (ECAR) as a measure of glycolysis-derived lactic acid Discussion (34). Both energy-producing pathways, oxidative phosphoryla- This study has uncovered the surface expression and signaling tion and glycolysis, were markedly stimulated in MCF-7–TF cells proficiency of the human NKG2D–DAP10 immunoreceptor in – (Fig. 4B). We conclude that, based on key criteria tested, in vitro cancer cells. Because NKG2D DAP10 activates oncogenic sig- stimulation of cellular proliferation, and bioenergetic metabo- naling cascades, it may complement the presence of its ligands lism, NKG2D–DAP10 is functionally similar to tumor growth for stimulation of tumor growth. This functional relationship is factor receptors. supported by in vitro proliferation and bioenergetic metabolism data and by the positive correlation of NKG2D with criteria of Correlation Between NKG2D Expression and Tumor Progression. To tumor progression. Tentatively, NKG2D thus may resemble tu- obtain evidence for the pathophysiological significance of mor growth factor receptors such as the EGFR, IGF-1R, and β β NKG2D expression in cancers, we tested for clinical correlations. TGF- receptor type 1 (TGF 1R). Although these receptors also have roles in tissue development and differentiation, NKG2D Pathology reports for all 60 primary cancer specimens examined fi were abstracted for clinical and histopathological information, normally is con ned to regulating lymphocyte functions alone. and data on tumor size/spread, lymph node involvement, and, Irrespective of their tissue origin, nearly all cancer specimens where available, tumor metastasis used to generate American heterogeneously expressed NKG2D within wide proportional ranges. The identity of cancer cells was ascertained by staining for EpCAM and confirmation of absence of CD45. Because loss of surface EpCAM is common (35), proportions of NKG2D+ cancer cells may be underestimated. At this point, there is no hint at mechanisms of gene regulation that may underlie cancer-cell expression of NKG2D and DAP10, which might be induced by cytokines or growth factors in permissive cancer-cell subsets. It may be relevant here that the promoter region of the DAP10 gene includes an activator protein 1 (Ap-1) transcription factor- binding site (36). Conversely, constitutive expression of NKG2D– DAP10 opposed by inducible down-regulation [e.g, by TGF-β, macrophage migration inhibitory factor (MIF), or IL-21] is a formal although unlikely possibility (37–39). In contrast to ex vivo cancer cells, most tumor lines expressed only scarce amounts of NKG2D–DAP10, which nonetheless were signaling proficient as substantiated by RNAi targeting and transfection experiments. Tumor lines may down-modulate NKG2D–DAP10 as an escape from detrimental effects of ligand-mediated self-stimulation IMMUNOLOGY under in vitro culture conditions. We have not explored ectopic NKG2D receptor expression in hematopoietic malignancies, which has been reported for acute myeloid leukemia. However, the significance here is less clear and presumably is unrelated to our observations in cancers, because NKG2D appears to confer cytolytic ability (40). The question arises as to whether NKG2D imparts cellular Fig. 4. Stimulation of proliferation and bioenergetic metabolism. (A) Cell- – – – effects in cancer environments that are distinct from or merely cycle analysis of MCF-7 mock-transfected, MCF-7 TF, and MCF-7 TF NKG2D synergistic to those conferred by prototypic growth factor recep- RNAi cells, plated for 24 h in the presence or absence of anti-MIC/ULBP1/3/4 Ab mixture, by PI staining and quantitative evaluation of flow cytometry tors. Distinct from T cells, we observed no stimulatory effect of data based on Dean–Jett–Fox curve fitting. Data shown are representative noncross-linked recombinant sMICA in suspended cancer cells fi and tumor lines. However, sMICA could be stimulatory, given of three experiments. P values indicate statistical signi cance of data pair fi comparisons. (B) NKG2D–DAP10 signaling is associated with increased met- permissive NKG2D licensing in speci c cancer environments (20). abolic activity. MCF-7 mock-transfected (control) and MCF-7–TF cells were In conclusion, our results suggest that the NKG2D immu- compared for total cellular ATP (Left), for real-time OCR (Center), and for noreceptor expressed by human cancer cells may promote tumor ECAR (Right). P values indicate statistical significance. Data shown are rep- progression and thus represent a determining factor underlying resentative of three experiments. the typically poor clinical outcomes that have been correlated

Benitez et al. PNAS | March 8, 2011 | vol. 108 | no. 10 | 4085 Downloaded by guest on September 25, 2021 with tumor-associated expression of its ligands (1, 15–17). This Santa Cruz Biotechnology). Immunoblots were probed with polyclonal tentative role of NKG2D will require scrutiny by in vivo experi- antibodies to NKG2D, DAP10 (N-20 and N-17; Santa Cruz Biotechnology), or mental models of tumor growth. If confirmed, previous mouse Grb2 (Cell Signaling Technology) and developed using secondary reagents models of NKG2D-mediated tumor immune surveillance may not and Supersignal West Dura Extended Duration Substrate (Pierce). Further account adequately for human cancer conditions, because our details are given in SI Materials and Methods. data suggest the absence of NKG2D expression in mouse cancer cells. We propose that in humans NKG2D-mediated promotion RT-PCR, NKG2D-DAP10 Transfection, and siRNA Transduction. Details of PCR of tumor growth becomes effective concurrent with the failure of primers and conditions, qPCR, transfections, and siRNA sequences are given in immune defense at advanced tumor stages. However, dynamics SI Materials and Methods. Annealed siRNA oligonucleotides were ligated resulting from the contributions of NKG2D to both tumor im- into lentiviral pRRLsin-cPPT-PGK-GFPwpre vector modified by insertion of mune surveillance and growth cannot be considered, because a U6 gene promoter cassette (41). there is no knowledge of its expression during earlier tumorigenesis stages. Cell-Cycle Analysis and Metabolic Activity Assays. Details of cell-cycle analysis are given in SI Materials and Methods. Cellular ATP was determined using Materials and Methods the ATPlite luminescence assay system (PerkinElmer). Real-time oxygen Cancer Specimens and Cell Lines, Immunohistochemistry, and Flow Cytometry. consumption and extracellular acidification rates were measured using the Primary cancer, nonaffected tissue specimens, and pathology reports were Seahorse Bioscience Extracellular Flux Analyzer XF24 (34). provided by the Cooperative Human Tissue Network (CHTN)–Western Di- vision. This activity was approved by the Institutional Review Board of the Statistical Analysis. Details of statistical analysis are given in SI Materials Fred Hutchinson Cancer Research Center. If two tumor grades were found within one cancer specimen, the higher grade was used for classification. and Methods. Tumor lines were from the American Type Culture Collection. Details of cell culture conditions, immunohistochemistry, flow cytometry, antibodies, and ACKNOWLEDGMENTS. We thank H.-W. Chung for pathology report abstrac- conjugates are given in SI Materials and Methods. tions; D. Hockenbery and V. Vasioukhin for advice; J. Fry for technical help; and R. Chmelar, C. Kemp, and H. Lu for mouse tumor specimens. This work was supported by the Deutsche Forschungsgemeinschaft (H.H.M.), by grants Immunoprecipitations and Immunoblots. NKG2D was immunoprecipitated from the Safeway Breast Cancer Pilot Project Fund, the Marsha Rivkin Center using mAb 5C6 immobilized on AminoLink Plus Coupling Resin (Pierce). for Ovarian Cancer Research (to T.S.), and by Grant AI30581 from the Na- DAP10 was immunoprecipitated using rabbit polyclonal antibodies (FL-93; tional Institutes of Health (to T.S).

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