Research ArticleImmunotherapy

Immunological Visibility: Posttranscriptional Regulation of Human NKG2D Ligands by the EGF Receptor Pathway

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Science Translational Medicine  09 Apr 2014:
Vol. 6, Issue 231, pp. 231ra49
DOI: 10.1126/scitranslmed.3007579

Abstract

Human cytolytic T lymphocytes and natural killer cells can limit tumor growth and are being increasingly harnessed for tumor immunotherapy. One way cytolytic lymphocytes recognize tumor cells is by engagement of their activating receptor, NKG2D, by stress antigens of the MICA/B and ULBP families. This study shows that surface up-regulation of NKG2D ligands by human epithelial cells in response to ultraviolet irradiation, osmotic shock, oxidative stress, and growth factor provision is attributable to activation of the epidermal growth factor receptor (EGFR). EGFR activation causes intracellular relocalization of AUF1 proteins that ordinarily destabilize NKG2D ligand mRNAs by targeting an AU-rich element conserved within the 3′ ends of most human, but not murine, NKG2D ligand genes. Consistent with these findings, NKG2D ligand expression by primary human carcinomas positively correlated with EGFR expression, which is commonly hyperactivated in such tumors, and was reduced by clinical EGFR inhibitors. Therefore, stress-induced activation of EGFR not only regulates cell growth but also concomitantly regulates the cells’ immunological visibility. Thus, therapeutics designed to limit cancer cell growth should also be considered in terms of their impact on immunosurveillance.

INTRODUCTION

Although variation in human health is genetically determined, it is increasingly acknowledged to be massively affected by the “exposome,” which refers to the totality of environmental challenges to which genomes equip individuals to respond (1). Although the challenge of microbial exposure has received considerable attention, inanimate components of the exposome, collectively termed “stress,” are also important. In this regard, lymphoid stress surveillance describes how lymphocytes, as opposed to myeloid cells, respond rapidly and polyclonally to endogenous molecules whose expression is substantially altered by cell and/or tissue dysregulation (2). One molecular manifestation of this is provided by major histocompatibility complex (MHC) class I–related antigens of the MICA/MICB and ULBP families in humans, and by the murine Rae1, H60, and Mult1 genes. By engaging NKG2D, an activating receptor on natural killer (NK) cells and subsets of T cells, these ligands provoke immune effector responses including cytolysis and cytokine production, either by primary activation of responding lymphocytes or by costimulation of cells receiving signals through the T cell antigen receptor (TCR) (35). By either means, NKG2D ligand up-regulation can be a major source of immunogenicity of dysregulated cells, complementing the activities of microbe-associated molecules such as Toll-like receptor ligands. It therefore becomes important to understand the mechanisms by which NKG2D ligands are regulated because their expression can be a key factor in determining whether or not cells become visible to the immune system. Were NKG2D ligand expression not to be regulated appropriately, normal cells might become targets of immune attack, potentially provoking autoinflammatory diseases such as psoriasis, to which NKG2D ligands are conspicuously linked by genome-wide association studies (6, 7).

NKG2D regulation and lymphoid stress surveillance are of particular relevance to cancer immunotherapy. Many human tumors express very high levels of MICA, MICB, and one or more ULBPs (8), the significance of which is implied by the many immuno-evasive mechanisms adopted by tumors as well as by viruses to suppress NKG2D-mediated lymphocyte activation (911). The common association of NKG2D ligand expression with cancer cells is consistent with evidence that murine Rae1 is up-regulated in response to DNA damage (12). Nonetheless, human NKG2D ligands showed much lower levels of responsiveness to DNA damage (12). Moreover, MICA up-regulation is frequently associated with scenarios such as osmotic and oxidative stress, virus infection, and increased cellular proliferation that cannot collectively be explained by DNA damage (1315). Hence, the regulation of human NKG2D ligand expression merited more thorough investigation.

Another fascinating and unresolved aspect of NKG2D-dependent lymphoid stress surveillance is the multiplicity of ligands (such as human MICA, MICB, and ULBP1 to ULBP6) (16), which appear functionally nonredundant despite their all engaging the same receptor (17). One hypothesis is that multiple ligands permit multiple means of regulation, enabling the host to respond to myriad components of the exposome and collectively confounding immuno-evasive strategies (18). Consistent with this, murine Rae1 genes, but not Mult1 or H60 genes, were expressed by primary fibroblasts upon explant to culture (19). In sum, there may be forms of regulation that apply to all NKG2D ligands and those that apply to only one or few.

The stress responsiveness of MICA was first implied by the identification of a heat shock response element in the MICA promoter (20). Thus, many studies have focused on NKG2D ligand gene transcription, including recent work showing regulation of murine Rae1 by the E2F transcription factor (19, 21). This notwithstanding, the profound impact of NKG2D ligands on cellular recognition by T lymphocytes and NK cells makes it probable that they are regulated at multiple levels. Consistent with this, some human NKG2D ligand mRNAs could be regulated under specific circumstances via microRNA (miRNA) docking sites in their 3′ untranslated regions (UTRs) (22, 23). Furthermore, disease-associated allelic variation in MICA was shown to profoundly affect the posttranscriptional stability of MICA mRNA with directly proportionate effects on surface protein expression (5).

Considering these various perspectives, this study reassessed human NKG2D ligand regulation in epithelial cells. The surprising result was that the regulation of MICA by a broad spectrum of challenges could be more readily attributed to activation of the epidermal growth factor receptor (EGFR) than to DNA damage. This mode of regulation applied generally to human NKG2D ligand genes, albeit with slight variation in mechanism, but did not apply to most murine NKG2D ligand genes. Because the EGFR pathway is one of the most frequently dysregulated and commonly targeted pathways in human cancer (24), this study directly links tumor cell surveillance by T cells and NK cells to a major cell-autonomous component of cancer progression that is also a prime target of cancer therapy.

RESULTS

Ultraviolet B induces human NKG2D ligand up-regulation

Because the murine NKG2D ligand Rae1 is regulated by DNA damage, MICA expression was measured after ultraviolet B (UVB) treatment of human keratinocytes, the cell type from which MICA complementary DNA (cDNA) was originally cloned (25, 26). Although immortalized, the human HaCat keratinocyte cell line maintains marked contact-dependent inhibition of growth at confluence and expresses little MICA mRNA, thus permitting the gene’s up-regulation by environmental agents to be studied. However, because MICA expression can be perturbed by changes in cell growth conditions, all experiments were meticulously controlled (see Materials and Methods).

Acute exposure to UVB (60 mJ/cm2) (equivalent to twice the minimal erythematous dose), taking about 1 to 2 min, reproducibly up-regulated total MICA protein expression to levels comparable to those induced by acute heat shock, the prototypic means of MICA up-regulation (Fig. 1A). Quantitative reverse transcription polymerase chain reaction (RT-PCR) was used to measure MICA RNA relative to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) RNA, which was previously demonstrated to be extremely stable in response to UVB, including in HaCat cells (2730). In each of nine independent experiments, MICA RNA was significantly up-regulated, in several cases by >10-fold (comparable to the levels of protein induction shown in Fig. 1A), and in other cases by 3- to 5-fold (Fig. 1B). This response was not limited to HaCat cells, because MICA RNA was also significantly up-regulated by UVB in four independent experiments using Int407, a fetal human intestinal epithelial cell line that also shows strict growth control (Fig. 1B). It might be argued that UVB irradiation provoked MICA mRNA up-regulation via transient heat shock. However, this was not compatible with the observation that whereas MICA mRNA levels induced by heat shock peaked within 2 hours and returned almost to normal by 24 hours, those induced by UVB did not peak until after 24 hours (fig. S1, A and B). UVB-induced NKG2D ligand up-regulation extended to cultured primary human keratinocytes where increases in cell surface protein were detected using either an antibody specific for both MICA and MICB or one specific for ULBP2 (Fig. 1C).

Fig. 1. UVB up-regulates NKG2D ligand expression via EGFR.

(A) Confluent HaCat cells were untreated (control) or exposed to UVB (60 mJ/cm2) or heat shock (HS; 90 min at 42°C), labeled with [35S]methionine/cysteine immediately after heat shock and 24 hours after UVB, and lysed. MICA immunoprecipitates were analyzed relative to known size markers (kD) by SDS–polyacrylamide gel electrophoresis (SDS-PAGE) and autoradiography. (B) Confluent HaCat (top) or Int407 (bottom) cells were untreated or exposed to UVB. RNA was extracted 24 hours later, and MICA was quantified relative to GAPDH by real-time quantitative PCR (qPCR). Data were compiled from nine (HaCat) and four (Int407) independent experiments. Numbers in italic indicate P values (paired t test). (C) Primary human keratinocytes were untreated (Mock) or UVB-irradiated (60 mJ/cm2), and MICA/B and ULBP2 expression was monitored by flow cytometry 24 hours after treatment. Numbers in italic indicate respective MFI values. (D) Confluent HaCat cells were exposed to UVB or treated with doxorubicin (Dox; 1 μg/ml), hydroxyurea (HUrea; 1 mM), or 4NQO (1 μg/ml). MICA expression was monitored as in (B). Each dot represents the analysis of an individual sample. Numbers in italic indicate P values relative to control (unpaired t test). (E) Confluent HaCat cells were untreated (control) or treated with the EGFR inhibitor AG1478 (AG) alone or 30 min before UVB irradiation (left) or serum-deprived overnight and incubated ± EGF (500 ng/ml; right), and mRNA levels were quantified as in (B). Results from five independent experiments are shown. Numbers in italic indicate P values (paired t test). (F) Confluent primary murine keratinocytes in SFM were untreated or stimulated with EGF (100 ng/ml) for 24 hours (left). As a positive control for Rae1b and H60a up-regulation, cells were UVB-irradiated (right). Expression was monitored by real-time qPCR. Data were normalized to cyclophilin and expressed as the means of three treatments ± SD. Numbers in italic indicate P values (unpaired t test).

To examine the impact of other genotoxic stresses on MICA mRNA, we treated HaCat cells with doxorubicin, which intercalates into DNA (31); hydroxyurea, which stalls or causes collapse of DNA replication forks after inhibition of ribonucleotide reductase (32); and 4-nitroquinoline-1-oxide (4NQO), a carcinogen that—akin to UV light—provokes the formation of bulky purine adducts, which are targeted by the nucleotide excision repair pathway, and which additionally induces DNA single-strand breaks and alkali-labile sites (33). Most unexpectedly, none of these significantly up-regulated MICA mRNA levels (Fig. 1D), although the agents were effective as judged by the up-regulation of p21 RNA (fig. S1C) that is induced by the DNA damage–dependent activation of p53 (34). Thus, MICA mRNA expression was up-regulated in keratinocytes and intestinal epithelial cells by UVB but not obviously by a mechanism relating to DNA damage.

UVB up-regulates NKG2D ligand RNAs via the EGFR

UVB irradiation can induce signaling through the EGFR, the major growth regulatory pathway for epithelial cells (35). To assess the potential contribution of this pathway to the regulation of MICA mRNA, we exposed cells to UVB but with or without AG1478, which inhibits several receptors with tyrosine kinase activity including EGFR. In each of five independent experiments, AG1478 significantly inhibited UVB-induced up-regulation of MICA mRNA (Fig. 1E, left panel). Moreover, in five independent experiments in which confluent HaCat cells were rested in serum-free medium (SFM) and then treated with EGF, MICA mRNA was sharply up-regulated (Fig. 1E, right panel), with kinetics and dose dependence very similar to an established EGF-regulated gene, the urokinase-like plasminogen activator receptor (UPAR) (36, 37) (fig. S1D). Cell surface expression of MICA/B and ULBP2 proteins was likewise induced by EGF with similar kinetics, with the most overt increase evident between 12 and 24 hours after treatment (fig. S1E).

Consistent with the cells’ strict contact inhibition, neither UVB nor EGF alone induced cell cycling in confluent HaCat cells (fig. S1F), thus distinguishing this regulation of MICA mRNA from the up-regulated transcription of the MICA and ULBP2 genes induced by increased cell division (19). The experiments were then repeated in primary murine keratinocytes. Predictably, NKG2D ligand RNAs were up-regulated by UVB irradiation: Rae1b and H60a by more than four- to fivefold, and Mult1 by about twofold. However, Mult1 was the only NKG2D ligand gene up-regulated by EGF (again by about twofold) (Fig. 1F). Thus, the mechanism of NKG2D ligand regulation by EGF/UVB most likely reflects a property that is shared by the Mult1, MICA/B, and ULBP2 genes, but not by the Rae1b and H60a genes (see below).

EGFR promotes NKG2D-dependent cell killing

EGF treatment of confluent HaCat cells increased surface expression of ULBP2 and of MICA/B detected either with a widely used antibody that detects both proteins or with monospecific antibodies for MICA and MICB, respectively (Fig. 2A and fig. S2A), but it did not induce expression of ULBP1 and ULBP3, which were not expressed before treatment (Fig. 2A). Either EGF does not up-regulate NKG2D ligands de novo or it specifically does not regulate ULBP1 and ULBP3. This was clarified by showing that EGF up-regulated ULBP1, ULBP2, and ULBP3 and, to a lesser extent, MICA/B proteins on the surface of Caco-2 cells, an immortalized human intestinal epithelial cell line that, like HaCat, shows strong contact-dependent growth inhibition at confluence (Fig. 2A). Thus, over the course of 12 to 24 hours, EGF up-regulated the surface display of several human NKG2D ligands in two distinct epithelial cell types but was seemingly more effective at increasing the level of expressed ligands rather than inducing ligands de novo. No EGF-mediated NKG2D ligand up-regulation was shown by HCT116 cells that carry a KRAS mutation, preventing surface EGFR expression (Fig. 2A) (38), whereas the ligands were up-regulated on both HaCat and HCT116 cells by phorbol 12-myristate 13-acetate that activates signaling components downstream of EGFR (fig. S2B).

Fig. 2. Cell surface expression of NKG2D ligands is up-regulated by EGF and cellular stresses.

(A) Confluent HaCat, Caco-2, and HCT116 cells were serum-deprived overnight and stimulated with EGF (500 ng/ml) (dark blue and dark red histograms) or left in SFM (light blue and light red histograms) for 24 hours, and cell surface expression of the indicated NKG2D ligands was monitored by flow cytometry. Data are representative of at least three independent experiments. Iso, isotype control staining. (B) HaCat and HCT116 cells were treated as in (A) (see fig. S2D for assessment of NKG2D ligand expression) and then co-incubated for 5 hours at 37°C with PBMCs from three healthy donors (HD1 to HD3) in the presence of phycoerythrin (PE)–conjugated anti-CD107a antibody and a blocking anti-NKG2D or isotype control antibody (10 μg/ml). Cells were washed and stained with allophycocyanin (APC) anti-CD3 and fluorescein isothiocyanate (FITC) anti–pan-γδTCR or with APC anti-CD3 and FITC anti-CD56 to analyze responses of γδ T and NK cells, respectively (see fig. S3 for gating controls). Data are means of triplicate stimulations ± SD. Numbers in italic indicate P values (unpaired t test). (C) HaCat cells serum-deprived overnight were treated with EGF (500 ng/ml), sorbitol (Sorb; 0.5 M), or hydrogen peroxide (H2O2; 0.6 mM) with or without a 1-hour pretreatment with AG1478 (AG, 10 μM). Surface MICA, MICB, and ULBP2 expression levels were monitored 24 hours later by fluorescence-activated cell sorting (FACS). Data were normalized to untreated cells (Ctrl) and expressed as means of three treatments ± SD. Numbers in italics are P values (unpaired t test).

The implication of EGFR in the response of human NKG2D ligands to UVB and EGF raised the question of whether this major tyrosine kinase receptor pathway that regulates cell growth also contributed to the levels of ligand expression in actively growing cells. Supporting this, dividing, subconfluent HaCat cells treated with AG1478 showed dose-dependent reductions in cell surface MICA/B and ULBP2 expression (fig. S2C), whereas no such reductions were seen in HCT116 cells in which the EGFR is impaired and does not regulate cell growth (fig. S2C).

Although the increases in NKG2D ligand surface expression were modest, they were well within the range reported for biologically relevant miRNA-mediated regulation of MICA and MICB (22) and typical of the levels of miRNA-mediated gene regulation more generally. Moreover, NKG2D+ γδ T cells and NK cells can be sensitive to only small changes in MICA expression, albeit with donor-to-donor variation (5). Therefore, to examine whether EGF regulates this critical function of NKG2D ligands, we treated HaCat and HCT116 cells with increasing concentrations of EGF; measured the surface expression of MICA/B and ULBP2 on aliquots of cells (fig. S2D); cocultured parallel aliquots of cells with primary peripheral blood mononuclear cells (PBMCs) from three independent healthy donors; and measured the induced killing potential of NK cells and γδ T cells within the PBMCs by flow cytometry of CD107a that comes to the cell surface as cells degranulate cytolytic lysosomes (5). Notwithstanding predictable donor-specific response variation, EGF-treated HaCat cells consistently provoked greater degranulation-dependent CD107a expression by NK cells and γδ T cells than did control cultures (Fig. 2B and fig. S3). Indeed, in some cases, ~20% of primary NK or γδ T cells were functionally activated. These effects were NKG2D-dependent (Fig. 2B) and not attributable to relief of NK inhibition by potential EGF-induced reductions in human leukocyte antigen (HLA) expression, because levels of HLA-A/B/C and HLA-E were unaltered by EGF (fig. S2E). Indeed, the lack of any impact on HLA refutes the hypothesis that EGF treatment provokes generalized immune dysregulation. Notably, the lack of an EGF response pathway in HCT116 cells fully correlated with a failure to stimulate NK and γδ T cell degranulation (Fig. 2B and fig. S3). In sum, EGFR-mediated regulation markedly increased cells’ NKG2D-dependent susceptibility to immune surveillance.

To investigate the generality of this regulatory pathway in relation to cell stress, we measured cell surface MICA, MICB, and ULBP2 expression on HaCat cells treated with EGF, with sorbitol (which induces osmotic shock), and with hydrogen peroxide (which induces oxidative stress). Notably, the EGFR can be activated by sorbitol and by oxidative stress, as well as by UV irradiation (39, 40). MICA, MICB, and ULBP2 were each up-regulated through an AG1478-sensitive mechanism (Fig. 2C and fig. S4). Thus, activation of EGFR and/or a closely related AG1478-sensitive receptor tyrosine kinase (RTK) mediates the up-regulated surface expression of human NKG2D ligands in response to multiple components of the exposome.

EGF stabilizes MICA/B RNAs via the 3′UTR

The sustained induction of MICA mRNA by UVB and EGF, rather than its induction de novo, might reflect accumulation of stabilized mRNA. To test this, we treated confluent HaCat cells with actinomycin D to arrest transcriptional initiation 24 hours after treatment with either EGF or control medium. Northern analysis of RNAs sampled at hourly time points thereafter permitted quantification relative to GAPDH. This experiment showed that MICA and MICB mRNAs had very low half-lives (t1/2) in confluent cells (MICA, ≤30 min; MICB, ~1.5 hours), consistent with the biological importance of limiting NKG2D ligand expression in growth-controlled cells. However, both mRNA species were substantially stabilized by EGF treatment (t1/2: MICA, >2 hours; MICB, ≥4 hours) (Fig. 3A and fig. S5A).

Fig. 3. MICA mRNA stability is regulated by its 3′UTR.

(A) HaCat cells were serum-deprived and incubated overnight with or without EGF (500 ng/ml). Actinomycin D was added, and RNA was extracted at the indicated time points [t (h)] for Northern analysis. The blot was probed for MICA and MICB, and reprobed for GAPDH. Representative of three experiments. Densitometry analysis was performed with ImageJ, and raw values are indicated in italics for each sample. ND, not detectable. See fig. S5A for the evaluation of mRNA half-lives. (B) The sequence of the full 3′UTR of MICA was deduced from cloning and sequencing and from the genomic sequence (accession number NC_000006.11) to include poly(A) signals (underlined), relative to which the poly(A) processing site identified by resequencing (blue) shows a different location than the previously identified site (yellow) (see also fig. S6A). Also shown are miRNA seeding sites (green) and a canonical ARE (bold). (C) HaCat cells were transfected with the pmaxFP-Green-N plasmid (GFP) or a modified version including the 3′UTR of MICA (GFP-M3U). GFP expression was monitored by flow cytometry 24 hours after transfection. Plots are representative of three independent transfections. Numbers in italic indicate MFI. (D) HaCat and HCT116 cells were transfected as in (B). Medium was supplemented 24 hours after transfection with G418 (0.5 mg/ml). After 14 days, confluent cells were serum-deprived overnight and further treated (or not) with EGF for 24 hours before GFP expression was analyzed by flow cytometry. Data were normalized to untreated cells for each transfectant and are means of triplicate EGF treatments ± SD. Numbers in italic indicate P values (unpaired t test).

RNA instability is commonly regulated via the 3′UTR, in relation to which seeding sites for miRNAs mapped within the 3′UTRs of MICA/MICB mRNAs (green annotation in Fig. 3B) were shown to reduce MICA/MICB expression (22, 23). However, the 3′ end also harbors a conspicuous AU-rich element (ARE) of the kind that can destabilize >5% of all cellular transcripts (41, 42). By resequencing and reannotation of the 3′UTR of the MICA gene relative to published and archived sequences, the ARE could be oriented 6 nucleotides downstream of a canonical AATAAA polyadenylate [poly(A)] signal (bold, Fig. 3B) relative to which a new poly(A) tail addition site was identified (blue, Fig. 3B) 26 nucleotides downstream, consistent with the reported mammalian mRNA range of 5 to 30 nucleotides (43, 44). This contrasts with the previously reported poly(A) addition site (yellow, Fig. 3B), which would have been at a highly atypical distance of 71 nucleotides downstream of the AATAAA signal (Fig. 3B).

To test the potential impact of the MICA 3′UTR region on gene expression, we cloned the MICA 3′UTR region (starting immediately after the translational stop codon) downstream of the green fluorescent protein (GFP) coding region, creating a chimeric allele termed GFP-M3U. HaCat cells transiently transfected with GFP-M3U displayed reproducibly lower GFP expression than those transfected with unmodified GFP, consistent with GFP being down-regulated by the MICA 3′UTR (Fig. 3C). Moreover, essentially the same result was shown by other human cell lines, in each case there being fewer cells expressing high GFP levels (fig. S5B).

HaCat and HCT116 cells transfected with GFP and GFP-M3U constructs were then established as stable lines by drug selection over a 2-week period during which the expression of GFP in GFP-M3U transfectants continued to decline significantly, relative to unmodified GFP transfectants, as judged by both mean fluorescence intensity (MFI) and percent cells positive (fig. S5C). However, the MFI of GFP expression was significantly up-regulated in EGF-treated GFP-M3U HaCat transfectants relative to mock-treated cells, whereas it was not up-regulated in EGF-treated GFP HaCat transfectants. GFP was also not up-regulated in HCT116 cells transfected with either the GFP or GFP-M3U constructs (Fig. 3D), consistent with the refractoriness of HCT116 cells to EGF. Collectively, these data establish that the MICA 3′UTR destabilizes gene expression, but that this can be relieved by EGF activation. Notably, AREs are conserved in all human NKG2D ligand genes (including several MICA alleles) for which reliable 3′UTR sequences are available, and in Mult1, the only murine NKG2D ligand tested that was regulated by EGF (fig. S6), but are not conserved in Rae1b or H60a genes.

An ARE regulates NKG2D ligands

To confirm that MICA mRNA instability was attributable to the ARE, we subjected the chimeric reporter constructs to several mutations that did not change the length of the constructs. Individual doublet mutations (M3U-mut1, M3U-mut2, and M3U-mut3) within the ARE significantly rescued the low expression levels of GFP-M3U expression (purple, blue, and cyan columns, fig. S7A), whereas mutation of the overlapping miRNA seeding site (M3U-mut0) had a lesser effect (red column). However, as an independent measure of mRNA destabilization, further chimeras were generated by fusing wild-type and mutant versions of the MICA 3′UTR to Renilla luciferase (Rluc) that encodes a less stable protein than GFP, thereby providing a more direct readout of the destabilizing effect of the 3′UTR. In these studies, the activity of a co-introduced, unmanipulated Firefly luciferase served to normalize Rluc expression across different transfectants. Again, relative to nonchimeric expression, luciferase levels were greatly reduced by the 3′UTR (green column) (Fig. 4A). These levels were marginally rescued by mutation of the overlapping miRNA seeding site (M3U-mut0, red column) but were almost completely restored to normal by independent mutations of the ARE site (purple, blue, and cyan columns) (Fig. 4A). Collectively, the results obtained with chimeric GFP and luciferase constructs demonstrated the capacity of the MICA 3′UTR ARE to destabilize different RNAs at steady state, with relief of this regulation being provided by EGFR activation.

Fig. 4. The ARE and AUF1 destabilize MICA mRNA.

(A) HaCat cells were transfected with plasmids encoding Rluc (pGL4.74, Ctrl) or modified versions including the wild-type (wt) or mutated (M3Umut0 to M3Umut3) 3′UTR of MICA and co-transfected with a plasmid encoding Firefly luciferase (Fluc) as an internal control. At 24 hours after transfection, luciferase activity was measured using a Dual-Luciferase Reporter Assay System, and luminescence values for Rluc were normalized to the internal Fluc values and to the wt Rluc control. Data are the means of three independent transfections ± SD. Numbers in italic indicate P values (unpaired t test). Relevant sequences are indicated, including the miRNA seeding site (italic), ARE (bold), and mutations generated (red). (B) HaCat cells were mock-transfected or transfected with pCR3.1 plasmids either empty or encoding each AUF1 isoform as indicated. Cell surface expression of MICA, MICB, and ULBP2 was analyzed by flow cytometry at 36 hours after transfection. Data are normalized to mock-transfected cells expressed as the means of three transfections ± SD. Numbers in italic indicate P values (unpaired t test). (C) HaCat cells grown on coverslips were treated as indicated, fixed and permeabilized, stained for endogenous AUF1, and analyzed by confocal microscopy. Images are representative of three independent experiments and show overlays of AUF1 (green) and 4′,6-diamidino-2-phenylindole staining (blue) (see fig. S9B for individual channels).

In considering how EGFR might promote mRNA stabilization, it was reported that several regulators of mRNA stability that bind to AREs are regulated by mitogen-activated protein kinases (MAPKs) (45). Therefore, confluent HaCat cells were stimulated with EGF with or without increasing doses of inhibitors of MAPK kinase (MEK), p38, and c-Jun N-terminal kinase (JNK). MEK inhibition by PD184352 recapitulated the inhibitory effects of AG1478 in a dose-dependent fashion; conversely, JNK inhibition (by SP600125) had no effect, and p38 inhibition (by SB203580) impaired EGF-responsive up-regulation of ULBP2 but not of MICA/B (fig. S7B). Thus, the EGFR-regulated cell surface display of NKG2D ligands shows selective dependence on MAPKs.

AUF1 is one of the most widely implicated factors that destabilize mRNAs by binding to AREs (46). However, the four main isoforms vary in their effects on different genes (47), and it was therefore important to investigate whether any or all AUF1 isoforms might regulate NKG2D ligands at steady state. Thus, cell surface expression was measured on HaCat cells transfected with constructs encoding each of the four AUF1 isoforms (p37, p40, p42, and p45), respectively. Relative to mock-transfected cells, or cells transfected with the empty vector (pCR3.1), cells transfected with each of the four isoforms showed significant reductions in cell surface MICA and MICB and ULBP2 (Fig. 4B and fig. S8).

To investigate whether EGFR-mediated rescue of NKG2D ligand expression might reflect regulation of AUF1 by EGFR, we treated serum-starved cells with EGF with or without signaling inhibitors, and AUF1 protein was assessed. There was no measurable impact on total or phosphorylated protein levels (fig. S9A). However, cell fractionation experiments had previously shown that generalized MAPK activation could provoke nuclear AUF1 relocalization to the cytoplasm (48), which is noteworthy considering that deletion of the AUF1-p37 nuclear localization signal showed that nuclear import is required for AUF1-mediated regulation of RNA turnover (49). EGF treatment provoked near-complete exclusion of AUF1 from the nucleus (Fig. 4C and fig. S9B). Notably, this was prevented by AG1478 and PD184352, which both inhibited surface up-regulation of MICA/B and ULBP2, as described above, but not by either SP600125 or SB203580, neither of which inhibited EGFR regulation of MICA/B (fig. S9B).

EGFR and NKG2D ligand expression correlate in human cancer

Were EGFR-mediated regulation of human NKG2D ligand mRNAs in vitro to reflect events in vivo, overt changes in EGFR activity should be reflected in NKG2D ligand expression. In this regard, many human carcinomas display specific dysregulation of the EGFR (24, 5052). Thus, gene expression profiles of a collection of 172 fresh-frozen primary breast carcinomas (53) were examined for six NKG2D ligand RNAs (note: probes for MICA were not present on the array platform used) in relation to EGFR, the estrogen receptor (ESR1), and HER2, which respectively represent the major growth regulatory pathways for human breast carcinomas (5456). Tumors graded in the lowest quartile (B25) for their expression of EGFR showed significantly lower expression of four of six NKG2D ligand RNAs assayed (ULBP1, ULBP2, ULBP3, and ULBP5) (Fig. 5A), none of which showed any correlation with ESR1 or HER2 expression (fig. S10A). Independently corroborating the correlation of NKG2D ligand expression and the EGFR pathway, the expression of all NKG2D ligand mRNAs except ULBP4 negatively correlated with LRIG1 RNA, which encodes a negative regulator of the EGFR (57) (Fig. 5A). Furthermore, all NKG2D ligand mRNAs showed negative correlations with HNRNPD RNA encoding AUF1, albeit the correlations with MICB and ULBP1 failed to reach statistical significance (Fig. 5A).

Fig. 5. The EGFR-mediated up-regulation of NKG2D ligand up-regulation has clinical implications.

(A) Expression of NKG2D ligands in primary breast cancers with the bottom 25% (B25) EGFR or top 25% (T25) LRIG1 and HNRNPD gene expression compared to the remaining samples. Numbers in italic indicate P values (Wilcoxon rank sum test). (B) HaCat cells were grown to confluence, serum-deprived for 24 hours, treated or not with EGF (500 ng/ml) for 24 hours with or without a 1-hour pretreatment with erlotinib (10 μM), and stained for MICA, MICB, and ULBP2. Data were normalized to untreated cells (control) and expressed as the means of three treatments ± SD. Numbers in italic indicate P values (unpaired t test). (C) Actively growing subconfluent HaCat cells grown in complete medium were treated or not with increasing doses of erlotinib for 24 hours and stained for MICA, MICB, and ULBP2. Data were normalized to untreated cells and are means of three treatments ± SD. (D) Differentiated Caco-2 monolayers grown on collagen-coated Transwell inserts until establishing electrically resistant epithelial sheets were serum-deprived overnight, treated (or not) with EGF (500 ng/ml) for 24 hours with or without a 1-hour pretreatment with erlotinib (10 μM), and stained for MICA/B and ULBP2. Numbers in italic indicate MFI.

Because MICA was missing from the array, its expression, together with that of MICB, was investigated in a data set of 26 triple-negative breast cancer cell lines (58): both genes showed positive correlation to EGFR expression (fig. S10B). In sum, although primary human tumors and cell lines are inevitably complex and heterogeneous, this complexity was superimposed upon by an expression pattern fully consistent with the regulation of most NKG2D ligand RNAs by the opposing effects of EGFR activation and AUF1.

The EGFR pathway is also a major therapeutic target in cancer. Illustrating the potential impact of such modalities on NKG2D ligand expression, erlotinib (a clinically used EGFR inhibitor) prevented the EGF-mediated up-regulation of different NKG2D ligands on confluent HaCat cells (Fig. 5B and fig. S10C). Likewise, erlotinib reduced NKG2D ligand expression on actively growing HaCat cells (Fig. 5C and fig. S10D) and phenocopied the effects of AG1478 and PD184352 on both subconfluent and confluent Caco-2 cells (fig. S11). Analogous dose-dependent effects in subconfluent and confluent HaCat cells were also achieved by cetuximab, another clinical inhibitor of EGFR (fig. S12).

Finally, the regulation of NKG2D ligand by EGFR activation and its response to erlotinib were examined in differentiated, nonproliferative human intestinal epithelial cells. To achieve this, we cultured Caco-2 cells (which are commonly used to model a fully differentiated human intestinal epithelium) in collagen-coated Transwells in which they formed electrically resistant monolayers (59). Over a 3-week period, electrical resistance was regularly measured across a Caco-2 cell sheet seeded in this way, and when it had reached a stable plateau of 500 ohms, the cells were transferred to SFM, with or without EGF. MICA/B, ULBP1, ULBP2, and ULBP3 surface expression were up-regulated by EGF (Fig. 5D), demonstrating that this form of regulation can occur in a fully confluent, functional monolayer, as might be the case at the earliest stages of human epithelial cell transformation in vivo. These EGF-mediated increases were substantially inhibited by erlotinib (Fig. 5D).

DISCUSSION

The NKG2D axis is increasingly viewed as a key component of host interactions with the environment. In response to microbial and nonmicrobial components of the exposome, NKG2D ligands can promote immunogenicity in the innate phase of immunity and can activate effector/memory cells in the adaptive phase. Considering these profound potentials, it was predicted that NKG2D ligand expression would be tightly regulated. In that regard, this study has identified a pathway by which cell surface ligand expression is restricted via posttranscriptional regulation, in large part mediated by a consensus ARE that can be destabilized by AUF1 proteins (Fig. 6A). In cells experiencing environmental perturbation by at least four components of the exposome—UVB irradiation, growth factors, osmotic stress, and oxidative stress—this restriction was relieved by signaling via the EGFR that, among other things, induced cellular relocalization of AUF1 (Fig. 6B).

Fig. 6. The EGFR pathway induces NKG2D ligand up-regulation via the abrogation of AUF1-mediated mRNA destabilization.

(A) Under normal conditions, NKG2D ligand mRNAs are constitutively targeted by AUF1 for degradation via their AREs. (B) Activation of the EGFR, either by high levels of its ligand, by any of several physicochemical stresses, or by genetic dysregulation in cancer, activates the MEK pathway and the exclusion of AUF1 from the nucleus, allowing NKG2D ligand mRNAs to be stabilized and translated and the proteins to be expressed at the cell surface, where they may engage NKG2D on cytolytic lymphocytes.

These findings are consistent with evidence that diverse environmental stress provokes the oxidative inactivation of active sites of phosphatases that constitutively suppress EGFR activity (35). Some quantitative variation in the levels of induction across experiments (for example, see Fig. 1, B, D, and E) most likely reflects the fine balance in any cell preparation between the activity of those phosphatases, the factors mediating RNA degradation including AUF1, and the levels of MAPK activity induced by various components of the exposome. This variation notwithstanding, the increases in surface ligand expression consistently induced by UVB and other EGFR activators were sufficient to render cells targets for NK and γδ T cell–mediated immune surveillance.

All human NKG2D ligand transcripts for which relevant sequence data are available contain AREs, and considering that surface expression of several NKG2D ligands was regulated in parallel with that of MICA and MICB, this mode of NKG2D ligand regulation is probably a general one in humans. Indeed, no differential responses of surface MICA and MICB protein were noted to any of the agents tested, although this does not preclude additional and differential means of regulation under different circumstances. The convergence of many different forms of stress on a common node of NKG2D ligand activation should provoke efficient and robust immune surveillance of many potentially threatening components of the exposome.

At the same time, the fine details of the regulation of different ligands can vary (21). Thus, p38 inhibition shows a differential impact on ULBP2 expression compared to MICA/B, with our ongoing studies implicating HuR in the regulation of ULBP2 but not MICA/B RNAs. Such differential regulation is most evident in the mouse where regulation via EGFR applied only to Mult1, the one mouse gene clearly harboring canonical 3′ ARE sequences. It is evident from the comparison of heat shock and UVB regulation of MICA (Fig. 1A and fig. S1, A and B) that NKG2D ligands are regulated in multiple ways. Therefore, it is possible that the DNA damage pathway and primary transcriptional control play a greater role in the regulation of murine Rae1 and H60 than they do in the regulation of most human NKG2D ligands. A species-specific dichotomy in the major pathways of regulation need not be surprising because the main ligands for NKG2D in mice and humans are not strictly orthologs, as is also the case for NK inhibitory receptors. Nonetheless, it is clearly appropriate to seek further clarification over the mechanisms by which human NKG2D ligands may be regulated by DNA damage. In the meantime, this study strengthens the implication of human NKG2D ligands in human cancer biology because the EGFR pathway is one of the most frequently dysregulated in tumors, particularly during the transformation of epithelial cells that are among the most established targets of lymphoid stress surveillance (24).

This study has begun to describe a pathway of human NKG2D ligand regulation, but there are as yet many gaps to fill. For example, how does AUF1 ordinarily destabilize NKG2D ligand RNAs and how does MAPK signaling promote its relocalization? Moreover, future studies should examine whether disease-associated allelic variation in NKG2D ligands might affect the sensitivity to this form of gene regulation. The dissection of gene regulation described here is very difficult to undertake in primary cells, and considering the lack of conservation of this form of regulation of NKG2D ligands in the mouse, it is equally challenging to design appropriate animal models. Here, differentiated Caco-2 cells and primary human keratinocytes were used to validate key observations, but this does not detract from the need to cross-reference to other independent data sets as much as is possible. This was achieved here by the gene expression analysis of more than 170 primary cancers that supported the hypothesis that EGFR regulates human NKG2D ligands in vivo.

Additionally, this study has not as yet integrated different means of NKG2D ligand regulation. For example, do conditions that up-regulate MICA/B or ULBP promoter activity also stabilize the respective RNAs or do these steps require separate regulation, perhaps as a checkpoint averting the precocious expression of NKG2D ligands by normal cells? Related to this, this study has not begun to ask which mechanism of regulation might be most relevant in particular circumstances. Nonetheless, the gene expression profiling of primary human breast cancers showed a positive correlation of most NKG2D ligands with EGFR expression and a negative correlation with both LRIG1, an EGFR inhibitor, and HNRNPD (AUF1), which was shown here to destabilize NKG2D ligand RNAs. These correlations are particularly striking considering the complexity that will inevitably underlie the composition of cancer transcriptomes (60). Indeed, the correlation might argue that the impact of the EGFR pathway may be a dominant means of NKG2D ligand regulation in vivo. Only ULBP4 showed no significant correlations with either EGFR or LRIG1, perhaps reflecting a ligand-specific difference because ULBP4, by contrast to other ligands, showed a significant positive correlation with HER2, which is related to but distinct from EGFR (fig. S6A). Thus, the possibility that ULBP4 is regulated independently should be investigated. Likewise, this study has been limited to the regulation of NKG2D ligands by the EGFR pathway, whereas the likelihood exists that some or all NKG2D ligand genes are also regulated by MAPK-dependent RNA stabilization downstream of other RTKs, such as the platelet-derived growth factor receptor, which is likewise implicated in pathologic cellular dysregulation.

The evidence provided here that EGFR activation can modulate NKG2D ligand expression in confluent cells independent of cell proliferation offers a mechanism of immune surveillance that may be relevant to the early stages of cancer when the EGFR pathway and/or EGF levels become dysregulated within a small focus of transformed cells amid a differentiated epithelium. Conversely, this study does not suggest that normal epithelial cells responding to physiologic levels of EGF as part of their normal growth cycle will necessarily up-regulate NKG2D ligands and thereby activate immune responses unnecessarily. A similar consideration was applied to the E2F-mediated induction of NKG2D ligands in proliferating cells (19). Rather, it is more likely that acquired “immunological visibility” is a general property of epithelial cells that are either responding to supraphysiologic EGFR activation provoked by high levels of various “stressors” or experiencing abortive cell proliferation cues, unable to balance their differentiated state with chronic EGFR/MAPK activation. Indeed, this may reflect the status of differentiated cells after infection with a number of viruses, several of which express molecules that suppress NKG2D-mediated immune surveillance (6164).

Posttranscriptional regulation is increasingly acknowledged to play a major role in the regulation of gene expression, particularly in response to environmental change. This has been brought into focus by the identification of many noncoding RNAs that regulate coding mRNAs in trans. However, ARE-mediated regulation remains a potent force, particularly in immune responses where it also limits the expression of cytokines. A survey recently identified 17 categories of p37 AUF1-regulated genes, among which immunity and defense genes were the ones with the highest statistical significance (65). Notably, in being co-regulated by both EGF and AUF1, MICA segregates with a subset of profoundly important biological functions, including CXCL5 and IL-11 (6568)—two genes that, like NKG2D ligands, are expressed by epithelial cells and have immunostimulatory potential.

Thus, one can view the data as further evidence that EGFR activation is de facto an immunological regulator. Consistent with this, EGFR can be transactivated after lipopolysaccharide treatment (69), whereas two recently published papers described a large number of immunological mediators acutely regulated, up or down, by clinical inhibitors of the EGFR pathway, including erlotinib (70, 71). This impact on immune effectors could be related to skin pathologies that commonly accompany therapeutic use of EGFR inhibitors and which are a major problem in that they deter patient compliance. More specifically, the common association of EGFR inhibitor usage with susceptibility to Staphylococcus aureus and virus infection (72) would be consistent with defects in lymphoid stress surveillance caused by EGFR inhibition and the consequent impairment of NKG2D ligand up-regulation, as predicted by this study. Indeed, there is widespread implication of the NKG2D pathway in antiviral defenses and EGFR is reportedly triggered by several S. aureus toxins (7375) and by cytomegalovirus infection (76), highlighting the potential host benefit of the EGFR being directly linked to lymphoid stress surveillance.

Consistent with the mechanism of ligand regulation elucidated in this study, and on the basis of the demonstrated lack of response of MHC class I expression to EGFR activation, one may conclude that the EGFR-mediated regulation of NKG2D ligands is somewhat specific. Nonetheless, it must be noted that other authors have claimed that NKG2D ligand expression can be up-regulated by application of erlotinib to human cells including Caco-2 cells (77, 78). Because this seemingly contrasts with this study, the experiments described here were repeated in multiple variations, but always with the same result, namely, the suppression of any of several NKG2D ligands by erlotinib and by other EGFR inhibitors, consistent with the detailed molecular mechanism deduced. Possibly cells in other studies had become so stressed by inhibitor treatment that secondary mechanisms began to affect NKG2D ligand expression.

However, this study adds to several others that highlight that the capacity of clinical inhibitors of the EGFR pathway to regulate immune surveillance may militate the effects of those therapeutics, particularly considering the capacity of a derepressed immune system to be of therapeutic benefit to patients with solid and hematological tumors. This fuels the view that EGFR, RTK, and MAPK pathway inhibitors that are used in cancer should be stratified according to their effects on immune surveillance. Moreover, the identification of specific factors and mechanisms regulating human NKG2D ligands, as has been achieved here, might facilitate the targeting of EGFR by strategies that do not impair NK cell and T cell recognition of tumors. The current study also argues for a clinical trial to test whether therapeutics targeting RTKs might best be supplemented with immune enhancement, rather than anti-inflammatories, as a means of promoting cancer therapy and reducing adverse events such as defects in skin barrier protection.

MATERIALS AND METHODS

Study design

The core of the investigative approach was the comparison of NKG2D ligand RNA and protein expression under defined circumstances. Because NKG2D ligand expression can be perturbed by changes in cell growth conditions, all experiments were meticulously controlled. Thus, the cell lines primarily used (HaCat and Caco-2) cease growth at confluence owing to contact inhibition and were subjected to stress or growth factor addition only after reaching confluence 48 hours beforehand. All control and experimental cell aliquots were treated comparably, for example, the cells’ temporary removal from incubators for experimental or mock treatment, and their subsequent reincubation in medium in which they had been previously growing and that had been stored pro tem at 37°C. All experiments were repeated independently, sometimes by different investigators (P.V., C.W., and A. Turner) over a period of more than 5 years, and were internally controlled, such that all conclusions reached could be based on the analysis of single experiments repeated several times, rather than relying on comparisons across experiments. The sample sizes and experimental repetitions were sufficient to permit rigorous statistical analysis as described in the figure legends and text, including correction for multiple parameter analysis for the comparison of gene expression profiles. The specificity of reagents, particularly antibodies, was verified.

Cells and culture conditions

Caco-2, HCT116, Int407, and HeLa cells were from the American Type Culture Collection. Polarized Caco-2 monolayers were established as described (59). Primary human keratinocytes were isolated as described (79) from human skin samples obtained as discarded material after cutaneous surgery (St Thomas’ Hospital, London) under ethical approval of the Guy’s and St Thomas’ NHS Foundation Trust Research Ethics Committee (06/Q0704/18), adhering to the principles of the Declaration of Helsinki. All cells were cultured in Dulbecco’s modified Eagle’s medium + 10% fetal calf serum (FCS), penicillin (50 U/ml), streptomycin (50 μg/ml), and glutamine (2 mM).

Primary murine keratinocytes were obtained from shaved mouse skin digested in a trypsin-GNK solution for 2 hours to dissociate epidermis from dermis. The former was further digested for 15 min in trypsin-GNK and deoxyribonuclease, filtered through a 70-μm cell strainer, and washed twice with RPMI. Cells were plated at 106 cells/ml in keratinocyte-SFM supplemented with EGF in wells precoated with rat tail collagen type I (50 μg/ml) (BD). PBMCs were isolated by Ficoll gradient centrifugation from blood obtained from healthy volunteers after informed consent, according to the Declaration of Helsinki.

Antibodies and reagents

Alexa Fluor 647 anti-MICA/B, APC anti-CD3, FITC anti-CD56, APC anti–HLA-E, PE anti–HLA-A/B/C, blocking anti-NKG2D, and all isotype controls were from BioLegend. PE anti-MICA, anti-MICB, anti-ULBP1, anti-ULBP2, and anti-ULBP3 were from R&D Systems. FITC anti–pan-γδTCR was from Beckman Coulter. PE anti-CD107a was from BD. Cetuximab (Erbitux) was from Merck. Recombinant human EGF was from Calbiochem. All cell culture media and reagents, Alexa 488 goat anti-rabbit immunoglobulin G, and ProLong Gold were from Invitrogen. Chemical inhibitors and rabbit polyclonal anti-AUF1 and anti–phospho-AUF1 were from Sigma-Aldrich. The Dual-Luciferase Reporter Assay System was from Promega.

Metabolic labeling and immunoprecipitation

Medium was replaced with methionine- and cysteine-free RPMI and 5% dialyzed FCS for 1 hour. 35S-labeled methionine/cysteine (150 μCi) (Amersham) was added, and cells were cultured for 6 hours at 37°C and 5% CO2. Cells were washed three times with phosphate-buffered saline (PBS) and lysed with 1% NP-40 and 1 mM phenylmethylsulfonyl fluoride buffer containing protease inhibitor cocktail (50 μl/ml) (lysis buffer). Lysates were precleared for 2 hours at 4°C with protein G–Sepharose beads. Immunoprecipitation was carried out overnight at 4°C with 2 μg of MICA monoclonal antibody coupled to protein G–Sepharose beads. Beads were then washed twice with lysis buffer and once with PBS. SDS-PAGE sample buffer was added; the samples were boiled for 5 min and run on a 10% polyacrylamide gel. The gels were dried in a Bio-Rad gel drier at 80°C for 1 hour and exposed to film.

UV irradiation

Cells were irradiated under a single Westinghouse FS20 tube (8 cm from the source). The dose was calibrated with a phototherapy radiometer (IL442A) and head SEE 1240. Medium was removed from cells and stored at 37°C, monolayers were rinsed with prewarmed PBS, and irradiation was carried out on cells covered with a meniscus of PBS. After irradiation, PBS was removed and the cells’ original medium that had been maintained pro tem at 37°C was added back to the cells. Control cells were treated in identical fashion but not irradiated.

RNA isolation and first-strand cDNA synthesis

RNA was isolated with TRIzol reagent (Invitrogen) from cell monolayers lysed directly in their flasks, followed by extraction with chloroform and precipitation with isopropanol. RNA (5 μg) was reverse-transcribed for 1 hour in the presence of 500 μM deoxynucleotide triphosphates, 25 nM oligo(dT)12–18 primer, 10 μM dithiothreitol, and 200 U of SuperScript II Reverse Transcriptase (Invitrogen).

Quantitative PCR

Quantitative real-time PCR was carried out with the TaqMan system for cDNAs generated from human cell lines. Primers and probes for MICA and MICB were designed with the Primer-Express software (Perkin-Elmer). GAPDH primers and probe have been reported (80). Amplicons were designed to cover an exon-exon boundary. Reporter and quencher dyes were 6-carboxyfluorescein (FAM) and 6-carboxytetramethylrhodamine (TAMRA), respectively. For monitoring murine NKG2D ligand expression, the QuantiTect SYBR Green PCR Kit was used (Qiagen). All qPCR primers are listed in table S1A.

Plasmids, RT-PCR, cloning, and generation of MICA-3′UTR mutants

pGL4.74 (encoding Rluc) was from Promega; pmaxFP-Green-N was from Lonza. All molecular biology reagents were from New England Biolabs. PCR was performed in 50 μl with Phusion Taq DNA polymerase according to the manufacturer’s instructions. All primers are listed in table S1B. AUF1 cDNA was cloned from HaCat cells. The amplicon product (generated in the presence of 10% dimethyl sulfoxide because the first exon contains >80% GC) was directly digested with Eco RI and Xho I restriction enzymes, followed by ligation into the pCR3.1 plasmid (Invitrogen). All four isoforms (p37, p40, p42, and p45) were obtained, and their sequence was verified. GFP reporter constructs were generated by overlapping PCR. GFP was cloned from the pmaxFP-Green-N plasmid (primers GFP forward and reverse), and the 3′UTR of MICA including all three poly(A) signals was amplified from genomic DNA extracted from HeLa cells (primers GFP-M3U forward and reverse). Both PCR products (2 μl) were used as a template for the overlapping PCR with primers GFP forward and GFP-M3U reverse. The resulting PCR product was cloned into pmaxFP-Green-N with Hind III and Alf II restriction enzymes, and the sequence was verified. Mutants were generated from this template by overlapping PCR with primers GFP forward and GFP-M3U reverse and the M3U-mut0 to M3U-mut3 primer pairs. Rluc reporter constructs were generated in the same way with the pGL4.74 plasmid, primers RLuc, Rluc-M3U forward, and Rluc-M3U reverse, and Hind III and Bam HI restriction enzymes. All transfections were carried out with Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions.

Flow cytometry

Cells were collected with PBS–10 mM EDTA and stained in 50 μl of PBS–5% FCS (FACS buffer) with the indicated antibodies (all used at a 1:100 final dilution) for 30 min at 4°C. Cells were washed twice with FACS buffer, and data were acquired on a FACSCalibur flow cytometer (Becton Dickinson) and analyzed with FlowJo (Tree Star).

CD107a assay

Activation of effector cells by measuring the cell surface translocation of the CD107a marker was performed as described previously (81), except that brefeldin A was omitted. Briefly, target cells and PBMCs were mixed 1:1 in the presence of the PE anti-CD107a antibody (1:30 final dilution) and incubated for 5 hours at 37°C and 5% CO2. Cells were washed with FACS buffer and stained with combinations of APC anti-CD3 and FITC anti-CD56 (NK cells) or APC anti-CD3 and FITC anti–pan-γδTCR (γδ T cells), acquired, and analyzed as described above.

mRNA stability experiments

Confluent HaCat cells were treated (or not) with EGF (500 ng/ml). Actinomycin D (5 μg/ml) (Sigma) was then added to replicate flasks, which were subsequently harvested for mRNA extraction at defined time points. mRNA was quantified by spectrophotometry, and 10 μg of each sample was separated on a 1.2% formalin-agarose gel and transferred overnight onto nylon membranes (Hybond N+, Amersham) by capillary blotting in 10× SSC. Blots were UV-crosslinked before hybridization. MICA, MICB, and GAPDH probes were prepared by PCR and labeled with [α-32P]deoxycytidine triphosphate with Megaprime labeling kit (Amersham) following the manufacturer’s instructions. Probes were denatured at 95°C for 5 min, and hybridization was carried out in ExpressHyb solution (Clontech) following the manufacturer’s instructions. Blots were incubated with probe in roller bottles for 1 hour at 68°C, washed with 2× SSC, 0.5% SDS at room temperature, then with 0.1× SSC, 0.1% SDS at 50°C, exposed to a storage phosphor screen, and acquired with the Storm PhosphorImager (Amersham). Bands were quantitated with ImageJ software.

Confocal microscopy

HaCat cells were grown to confluence on 13-mm coverslips placed in 24-well plates and treated as described. Cells were then washed twice with PBS, fixed for 20 min at room temperature with BD CellFix buffer, washed twice with PBS, and permeabilized with PBS–0.1% Triton X-100 for 30 min. Cells were then incubated with primary and secondary antibodies in PBS–0.02% Triton X-100 for 1 hour each, washed twice with PBS and once in ultrapure water, and then mounted on glass slides with 10 μl of ProLong Gold. Samples were analyzed with a TCS SP2 AOBS confocal microscope [equipped with an HCX PL APO CS (confocal scanning), 63.0×/1.4 oil objective; Leica].

Analysis of gene expression in breast cancer samples and cell lines

The Human Exon 1.0 ST Affymetrix Array was used to generate the gene expression data of 172 primary breast carcinomas as described (53). Briefly, gene expression data were normalized with the quantile normalization method of Aroma Affymetrix package (82) of the R software project (http://www.r-project.org/), followed by combat normalization to adjust for batch (83). The difference in gene expression of several NKG2D ligands was determined between tumors with the lowest 25% EGFR, ESR1, or HER2 or highest 25% LRIG1 and HNRNPD expression versus the remaining samples. Publicly available gene expression data of 26 triple-negative breast cancer cell lines were obtained from and preprocessed as described (58). Probe sets with the highest variability across the data set were used as a representative for each gene (MICA, MICB, and EGFR). The association between their gene expression levels was determined with Pearson’s correlation.

Statistical analysis

Statistical significance for all experimental data was determined by paired or unpaired (as indicated for each experiment) two-tailed t tests with the Excel software (Microsoft). Correlations between NKG2D ligands and EGFR, ESR2, HER2, LRIG1, and HNRNPD expression in primary breast cancer samples and triple-negative breast cancer cell lines were analyzed by Wilcoxon rank sum test and Pearson’s correlation, respectively. The appropriateness and correct implementation of the statistical tests were independently verified.

SUPPLEMENTARY MATERIALS

www.sciencetranslationalmedicine.org/cgi/content/full/6/231/231ra49/DC1

Fig. S1. Induction of MICA by UVB and EGF is not due to heat shock, DNA damage, or cell proliferation.

Fig. S2. NKG2D ligands are up-regulated at the cell surface after EGF treatment.

Fig. S3. Cell surface increase in NKG2D ligand expression by EGF is detected by cytotoxic lymphocytes.

Fig. S4. NKG2D ligand induction by various stress components of the exposome is EGFR-dependent.

Fig. S5. NKG2D ligand expression is regulated posttranscriptionally.

Fig. S6. NKG2D ligand mRNAs contain ARE sequences in their 3′UTRs.

Fig. S7. The ARE sequence and the MEK pathway regulate NKG2D ligand expression.

Fig. S8. AUF1 regulates NKG2D ligand expression.

Fig. S9. The EGFR/MEK pathway regulates AUF1 localization.

Fig. S10. NKG2D ligand expression correlates with EGFR expression levels and is abrogated by erlotinib.

Fig. S11. EGF-induced up-regulation of cell surface NKG2D ligand expression by confluent differentiated Caco-2 cells is abrogated by EGFR and MEK inhibitors.

Fig. S12. Cetuximab inhibits NKG2D ligand expression.

Table S1. Primers used in this study.

REFERENCES AND NOTES

  1. Acknowledgments: We are grateful to A. Young [Department of Photobiology, St John’s Institute for Dermatology (SJID), London, UK] for help during the early phases of this research and to many colleagues for discussions. HaCat cells were a gift from M. Allen (SJID). The monoclonal antibody to MICA (hu MIC-A-M673) for immunoprecipitation was a gift of D. Cosman (Amgen Corp.). Primary human materials were provided through the National Institute for Health Research (NIHR) Biomedical Research Centre of Guy’s and St Thomas’ Hospital and King’s College London. Statistical tests were independently verified by L. Abeler-Dörner. Funding: Cancer Research UK (A.H. and P.V.); a Wellcome Trust Programme grant (A.H.); the European Community’s Seventh Framework Programme FP7/2007-2013 under grant agreement #PIEF-GA-2009-255285 (P.V.); the UK Medical Research Council (C.M.S.); and NIHR Biomedical Research Centre at Guy’s and St Thomas’ NHS Foundation Trust and King’s College London. (A.H., A.G., and A. Tutt). Author contributions: P.V. designed and performed the experiments, analyzed the data, and co-wrote and revised the manuscript. C.W. and A. Turner designed and performed experiments, analyzed the data, and revised the manuscript. C.M.S. provided key reagents, designed the experiments relating to mRNA stability, analyzed the data, and revised the manuscript. Y.H. and O.S. performed experiments and analyzed data. A.G. and A. Tutt provided access to and analyzed the array data generated from primary breast cancer samples and revised the manuscript. A.H. designed the study, analyzed and interpreted the data, and wrote and edited the manuscript. Competing interests: The authors declare that they have no competing interests.

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