Hypoxia-Dependent Inhibition of Tumor Cell Susceptibility to CTL-Mediated Lysis Involves NANOG Induction in Target Cells

The tumor microenvironment is an integral part of tumor physiology, structure, and function, playing a critical role in tumor cell survival and growth and contributing to cell transformation and tumor development. Indeed, it is well established that a disrupted relationship between tumoral and stromal cells is essential for tumor cell growth, progression, and development (1), suggesting that an improved understanding of this interaction may provide new and valuable clinical targets for controlling tumor progression.

Tissue oxygenation is an important component of the microenvironment and can acutely alter cell behavior through the direct regulation of genes involved in cell survival, apoptosis, glucose metabolism, and angiogenesis (2). Evidence indicates that hypoxia, defined as oxygen deprivation, is an important feature of the microenvironment of a wide range of solid tumors. Its critical role in radio- and chemoresistance and its significance as an adverse prognosis factor have been well established during the last decades (2). Hypoxia has been shown to induce the loss of differentiation markers of several tumor types while increasing the expression of embryonic markers such as the transcription factors NANOG, octamer-binding transcription factor (OCT) 4, SOX2, and the Notch ligand (3, 4). This reprogramming of non-stem cancer cells toward a cancer stem phenotype is associated with increased tumorigenic capacity (4).

At a cellular level, hypoxia evokes a complex molecular response mainly characterized by changes in gene expression that are mediated by hypoxia-inducible factors (HIFs) (2). The HIFs are heterodimers that consist of α and β subunits. Three isoforms of the α subunit are described: HIF-1α, HIF-2α, and HIF-3α (2). Under hypoxic conditions, the α subunit is stabilized and activated and binds the HIFβ isoform, which is not regulated by oxygen levels and is constitutively present in the nucleus (2). A broad range of tumors respond to hypoxia by stabilizing the HIF-1α subunit (5).

In a previous study, we reported that tumor hypoxia decreases tumor susceptibility to CTL-mediated lysis via HIF-1α stabilization and STAT3 phosphorylation (6). Although it has been reported that tumor stem cells were intrinsically susceptible to immune effector cell cytotoxicity (7), the effects of hypoxia-driven tumor dedifferentiation on the response to CTL-mediated lysis have not been reported.

NANOG, a homeodomain-containing protein expressed in embryonic cells, germ cells, and pluripotent stem cells, has the ability when overexpressed to inhibit in vitro embryonic stem cell differentiation and maintain their self-renewal capability independently of the LIF/gp130/STAT3 pathway (8). In human cancers, NANOG expression has been found in germ cell tumors where NANOG functions are required, and it has been reported to increase in several tumoral tissues (breast, prostate, kidney) compared with matched benign tissues (9). Although accumulating evidence suggests that the protumoral functions of NANOG rely on promoting tumor cell proliferation (9), stimulation of STAT3 transcriptional activity has also been reported, leading to tumor resistance to chemotherapy and improved cell survival (10). In line with these reports, NANOG expression in head and neck cancers has recently been associated with unfavorable clinical outcome (11). However, whether NANOG interferes with tumor cell susceptibility to CTL-mediated cytotoxicity is not yet known.

The present study shows that hypoxia increases NANOG expression in non-small cell lung carcinoma cells and that this induction contributes to hypoxia-induced tumor target resistance to CTL-mediated lysis. Such induction is also associated with the regulation of cell replication and of nuclear translocation of STAT3. Our findings provide new insights into understanding the role of hypoxia-induced NANOG in regulating the cellular response to hypoxia and tumor target susceptibility to CTL-dependent killing.

The IGR-Heu lung carcinoma cell line was derived and maintained in culture as described previously (12). Briefly, the cells were grown in DMEM/F12 medium supplemented with 10% heat-inactivated FCS, 1% Ultroser G, 1% penicillin-streptomycin, and 1 mM sodium pyruvate (Life Technologies, Cergy Pontoise, France). The Heu161 cytotoxic T cell clone was derived from autologous tumor-infiltrating lymphocytes (13).

Protease inhibitors and sodium orthovanadate were purchased from Roche Molecular Biochemicals. Abs against CD133 and OCT4 were from Abcam. Abs against NANOG were purchased from Santa Cruz Biotechnology (for Western blotting) and R&D Systems (for confocal microscopy). Ab against HIF-1α was from BD Transduction Laboratories. Abs against total STAT3, phospho-STAT3 (STAT3Y705), total Src, and phosphor-Src (Y416) were from Cell Signaling Technology. Abs against β-actin and GAPDH were from Sigma-Aldrich.

For hypoxia treatment, cell cultures were incubated in a hypoxia chamber (Invivo₂ 400 hypoxia workstation; Ruskinn) with a humidified atmosphere containing 5% CO₂, 01% O₂, and 94% N₂ at 37°C (14). Cells were exposed to these hypoxic conditions for 24, 48, and 72 h. Cells for protein analysis were harvested while inside the hypoxia workstation and were not reoxygenated before harvesting.

Flow cytometry analysis was performed using a FACSCalibur flow cytometer. Data were processed using CellQuest software (BD Biosciences). Anti-HLA class I (W6/32) Ab was used as reported previously (15).

Total RNA was extracted using TRIzol (Sigma-Aldrich) following the manufacturer’s instructions. For quantification of OCT4, NANOG, and hairy and enhancer of split (HES) 1 expression, cDNA was synthesized from 1 μg mRNA using an Applied Biosystems kit (N808-0234). Real-time PCR analysis was performed using a LightCycler (Roche Applied Science, Meylan, France) and the LightCycler-FastStart DNA Master SYBR Green I mix (Roche Applied Science) according to the manufacturer’s instructions with an initial denaturation step at 95°C for 10 min, followed by 45 cycles at 95°C for 10 s and annealing at specific temperature for 10 s and at 72°C for 10s. Primers used for NANOG, OCT4, and HES1 amplification were: NANOG forward, 5′-CAGCTGTGTGTACTCAATGATAGA-3′, reverse, 5′-ACACCATTGCTATTCTTCGGCCAG-3′; OCT4 forward, 5′-ACATCAAAGCTCTGCAGAAAGAACT-3′, reverse, 5′-CTGAATACCTTCCCAAATAGAACCC-3′; HES1 forward, 5′-AGGCGGCACTTCTGGAAATG-3′, reverse, 5′-CGGTACTTCCCCAGCACACTT-3′ (at a 63°C annealing temperature). Specificity of PCR amplicons was confirmed by melting curve analysis. Expressions of target genes were normalized to that of RPL13. Relative expression of tested genes was calculated using the target threshold cycle value (Ct) and the 2^−ΔCt method. For each gene, values were averaged over three independent measurements and the relative transcript level was calculated.

For quantification of apoptosis-related genes, reverse transcription was performed using a high-capacity cDNA reverse transcription kit (Ambion). Real-time PCR was done using Platinum SYBR Green qPCR SuperMix-UDG w/ROX (Invitrogen) and an Applied Biosystems 7900HT Fast real-time PCR system. Apoptosis-dedicated arrays were elaborated by the PETC platform of INSERM Unité 576.

Adherent tumor cells were washed in 1× PBS and lysed in plates with lysis buffer (62.5 mM Tris-HCl [pH 6.8], 2% w/v SDS, 10% glycerol, 1 mM sodium orthovanadate, 2 mM PMSF, 25 μM leupeptin, 5 mM benzamidine, 1 μM pepstatin, 25 μM aprotinin). Lysates were sonicated on ice, resolved by SDS-PAGE electrophoresis (60 μg/lane), and transferred onto nitrocellulose membranes. After incubation in blocking buffer, the membranes were probed overnight at 4°C with the indicated Abs. The labeling was visualized using peroxidase-conjugated secondary Abs and with an ECL kit (Amersham International). Blots were scanned and processed by Adobe Photoshop 7.0 software.

Four-hour chromium release assays were performed as described previously (6). Briefly, different E:T ratios were used on 1000 target cells per well (round-bottom 96-well plates). After 4 h coculture, the supernatants were transferred to LumaPlate-96 wells (PerkinElmer), dried down, and counted on a Packard Instrument TopCount NXT. Percentage-specific cytotoxicity was calculated conventionally as described earlier (6). All cytotoxicity experiments with [⁵¹Cr] were performed under normoxic conditions.

TNF-β release was measured as described previously (16). Briefly, TNF-β was detected by measuring the cytotoxicity of the culture medium on the TNF-sensitive WEHI-164c13 cells with an MTT colorimetric assay.

Cells were plated in 8 well μ-slide (Ibidi) and incubated for 24, 48, and 72 h at 37°C and 01% partial pressure of O₂ (pO₂). Afterwards, cells were washed twice in 1× PBS, fixed with 4% paraformaldehyde for 10 min, permeabilized with 0.1% Triton X-100 for 10 min, and blocked for 45 min with 0.1% Triton X-100, 10% FCS, and 1% BSA. After blocking, cells were incubated overnight with diluted anti-STAT3Y705 and anti-NANOG primary Abs, followed by incubation with Alexa 488 and Alexa 546 secondary Abs (Molecular Probes), respectively, in the dark for 1 h at room temperature. Wells were covered with Fluoromount-G (SouthernBiotech, Birmingham, AL) and analyzed the following day by a Zeiss laser scanning confocal microscope (LSM-510; Zeiss, Jena, Germany). Z-projection of slices was performed using LSM Image Examiner software (Zeiss).

Gene silencing of NANOG was performed using sequence-specific small interfering RNA (siRNA; Santa Cruz Biotechnology). Briefly, 8 × 10⁶ cells were electroporated twice in 48 h in serum-free medium with 20 μM siRNA in an EasyJect Plus electroporation system (EquiBio; 260 V, 450 μF) and then allowed to grow for 24, 48, and 72 h in hypoxia. siRNA targeting luciferase was used as a negative control (5′-GCAAGCUGACCCUGAAGUUCAU-3′). Gene-specific targeting was evaluated by quantitative real-time PCR (RT-qPCR) or Western blot.

For isolation of cytoplasmic and nuclear proteins, the ProteoJET cytoplasmic and nuclear protein extraction kit (Fermentas) was used following the manufacturer’s instructions. Briefly, hypoxic or normoxic cells were harvested after trypsinization and solubilized in lysis buffer with protease inhibitors and DTT. After centrifugation, the cytoplasmic fraction was isolated. Nuclear pellets were washed twice and then lysed in nuclear lysis buffer added with protease inhibitors and DTT. Nuclear lysates were obtained after centrifugation.

Electroporated IGR-Heu cells were incubated in normoxia or hypoxia (01% pO₂) for 72 h. In the last 10 min, BrdU was added to the culture medium. Then, cells were harvested and fixed in 80% ice-cold ethanol. For BrdU detection, DNA was first denaturated in a pepsine buffer (0.5 mg/ml pepsine, 2 N 30 mM HCl) for 20 min at 37°C. Cell pellets were then resuspended in 2 N HCl for 20 min at room temperature. After washing in ice-cold 1× PBS, detection of replicating cells was performed by BrdU staining. Cells were washed in BrdU detection buffer (0.5% FCS, 0.5% Tween 20, 20 mM HEPES in 1× PBS) and BrdU was detected in the dark using a specific primary Ab for 45 min followed by a FITC-conjugated secondary Ab. To detect total DNA, cell pellets were resuspended in a propidium iodide solution (25 μg/ml propidium iodide, 50 μg/ml RNase A in 1× PBS) and incubated in the dark at room temperature for 30 min. Cells were then analyzed by flow cytometry.

Data were analyzed with GraphPad Prism. A Student t test was used for single comparisons. A p value of <0.05 was considered statistically significant.

As previously reported, data depicted in Fig. 1A show that exposure of the lung tumor cell line IGR-Heu to hypoxic treatment resulted in an inhibition of its susceptibility to autologous CTL-mediated killing. We next examined the relationship between hypoxia-induced resistance to specific lysis and the adoption of a stem cell-like phenotype. Indeed, cancer stem cells have been shown to resist cell death induced by apoptotic and chemo- or radiotherapeutic agents (17). For this purpose, we first investigated the influence of hypoxic stress on the expression of the cell surface protein CD133 and the cytosolic enzyme aldehyde dehydrogenase (ALDH) 1. These two markers are respectively associated with lung cancer stem cell proliferation and chemoresistance (18–20). Flow cytometry analysis indicated that hypoxia did not affect the cell surface expression of CD133 (Fig. 1B,a). Most of the IGR-Heu cells express CD133 constitutively, suggesting that CD133 may not be an exhaustive marker for detection of stem cell-like tumor cells. Total cellular extracts were analyzed by Western blot, and expression levels of CD133 (Fig. 1B,b) and ALDH1 (Fig. 1B,c) were not influenced by exposure to hypoxic stress. Because epithelial to mesenchymal transition (EMT) has been reported to be associated with both acquisition of an immature phenotype (21) and hypoxic stress (22), we investigated the induction of EMT in hypoxic IGR-Heu cells. The expression of the epithelial marker E-cadherin and of the EMT activator Snail, which is also an important regulator of E-cadherin expression (23), was analyzed. Fig. 1C,a shows that the total expression of E-cadherin, as well as surface E-cadherin expression (data not shown), remained unchanged after 72 h exposure to hypoxia. In contrast, Snail expression was significantly decreased after 24 h hypoxia (Fig. 1C,a). Because EMT is associated with morphological changes resulting in a distinct elongated phenotype, cell morphology of hypoxic IGR-Heu cells was therefore visualized by phalloidin staining. Data shown in Fig. 1C b indicate that hypoxia had no effect on IGR-Heu morphology for up to 72 h. Phalloidin staining also showed the absence of cell rounding, which is a morphological feature associated with the acquisition of immature phenotypes (24). These results indicate that hypoxia-induced tumor resistance was not associated with the acquisition of a stem cell-like phenotype in IGR-Heu cells.

Although hypoxic stress did not result in phenotypic changes in IGR-Heu cells, hypoxia-induced dedifferentiation may be associated with altered expression of specific genes. Thus, to evaluate the potential role of dedifferentiation genes in hypoxic tumor cells, we selected OCT4 and NANOG genes, as well as HES1, a Notch target gene, based on their increased expression under hypoxia (4, 25, 26) as well as their reported role in the control of stem cell self-renewal and multipotency (27–29). Using RT-qPCR, we measured their respective mRNA levels under hypoxic conditions and found that OCT4 and NANOG mRNAs but not HES1 mRNA markedly increased following exposure to hypoxia for 24, 48, and 72 h (Fig. 2A). Whereas Western blot analysis did not reveal OCT4 expression (data not shown), fitting with its marginal mRNA levels under hypoxia, NANOG protein was induced up to 72 h hypoxia (Fig. 2B). This was confirmed by confocal microscopy analysis, which revealed increased staining of NANOG along with the time of exposure to hypoxia (data not shown). Fig. 2B also showed that the induction of NANOG under hypoxia correlated with HIF-1α and HIF-2α stabilization. To delineate the potential link between hypoxia-induced NANOG and HIF-1α or HIF-2α, NANOG protein expression was analyzed following HIF-1α or HIF-2α knockdown using specific siRNAs. As shown in Fig. 2C, specific silencing of HIF-1α resulted in the inhibition of hypoxia-induced NANOG after 24, 48, and 72 h of hypoxic stress whereas HIF-2α silencing had no effect (Fig. 2D). These results indicate that hypoxia-induced NANOG was dependent on HIF-1α and raise the possibility that NANOG may play a role in the acquisition of tumor cell resistance to CTL lysis under hypoxic conditions.

To delineate the role of NANOG induction in hypoxia-mediated tumor cell resistance to specific lysis, we investigated the susceptibility of NANOG-targeted IGR-Heu cells using siRNA under hypoxic conditions. The inhibition of NANOG in hypoxic tumor cells was performed using specific siRNA and confirmed by RT-qPCR, showing an efficient and specific inhibition of NANOG mRNA expression after 72 h hypoxia, whereas luciferase siRNA used as a control had no effect on NANOG mRNA level (Fig. 3A). As shown in Fig. 3B, NANOG knockdown in hypoxic IGR-Heu cells was associated with sensitization of IGR-Heu cells to CTL-mediated lysis following 48 h exposure to hypoxia as compared with control hypoxic cells. We also show that the targeting of NANOG in hypoxic IGR-Heu cells was not associated with an alteration in autologous CTL reactivity, as TNF-β secretion by CTL was not affected (Fig. 3C). Moreover, such targeting did not alter MHC class I expression in IGR-Heu cells (Fig. 3D). This is consistent with the fact that attenuation of hypoxia-induced tumor cell resistance following NANOG targeting was not due to an alteration in CTL reactivity and target cell recognition.

To investigate the molecular mechanisms associated with the attenuation of target cell resistance to CTL following NANOG targeting under hypoxic conditions, we analyzed the expression of a series of antiapoptotic and proapoptotic genes using an apoptosis-related array of 94 genes. NANOG knockdown in hypoxic tumor cells revealed an increase in the expression of proapoptotic and antiapoptotic genes (Fig. 4A). However, NANOG depletion in hypoxic cells resulted in a limited but consistent increase in both apoptotic (sub-G₁) and nonreplicating (S-BrdU-negative) cells, suggesting that one of the roles of NANOG in hypoxia is to sustain cell viability (Fig. 4B). Additionally, flow cytometry analysis of BrdU-labeled cells revealed that hypoxia modified the overall level of nucleotides incorporation (Fig. 4B b). Indeed, we noticed the appearance of a cell subpopulation characterized by lower BrdU incorporation. This subpopulation represented the majority of S-phase cells in hypoxic conditions. Importantly, NANOG depletion restored a cell cycle profile similar to that observed in normoxic conditions. These results indicate that NANOG knockdown in hypoxic tumor cells correlates with increased replication levels, an event associated to and responsible for DNA damage and apoptosis (30).

We have recently shown that hypoxia-induced resistance of IGR-Heu cells to specific lysis was critically dependent on STAT3 phosphorylation (6). Additionally, a functional cooperation between STAT3 and NANOG for gene transcription has been reported in cancer drug resistance (10). Therefore, we asked whether a functional interaction exists between NANOG and STAT3 upon hypoxic stress. Fig. 5A indicates that silencing of hypoxia-induced NANOG resulted in an inhibition of STAT3 phosphorylation on Tyr⁷⁰⁵, a crucial event for STAT3 dimerization and translocation to the nucleus (31). We next examined in hypoxic NANOG-downregulated cells the phosphorylation state of JAK2 and Src, two kinases involved in STAT3 phosphorylation (32) (33). Results indicate that NANOG downregulation under 48 h hypoxia resulted in selective inhibition of Src (Fig. 5A) but had no effect on JAK phosphorylation (data not shown).

Phosphorylation of STAT3-Y705 is crucial for STAT3 translocation to the nucleus (31). However, unphosphorylated nuclear forms of STAT3 have already been described (34). Therefore we investigated the role of NANOG on STAT3 nuclear translocation in hypoxic IGR-Heu cells using siRNA-targeting NANOG. Fig. 5B indicates that knocking down NANOG in hypoxic NANOG-targeted IGR-Heu cells suppressed STAT3 phosphorylation and its subsequent translocation into the nucleus after 24, 48, and 72 h of hypoxic stress. In contrast, in control hypoxic cells, phosphorylated STAT3 was induced and located in nuclear fractions. It also shows that in NANOG-targeted and control cells, hypoxia-induced NANOG did not translocate to the nucleus but remained in the cytoplasm (Fig. 5B). Confocal microscopy analysis indicated a constant decrease of pSTAT3-Y705 nuclear staining in siRNA-NANOG–treated IGR-Heu cells under 24, 48, and 72 h hypoxia compared with control cells (Fig. 5C). This further points to the role of NANOG in STAT3 phosphorylation on Y705.

It is well established that microenvironmental factors, in particular hypoxia, act to shift the normal balance toward malignancy. Accumulating evidence indicates that cancer cells that remain viable in hypoxic conditions often possess an increased survival potential and tend to grow particularly aggressively. Hypoxia induces tumor progression through multiple mechanisms, including several transcriptional programs (2). In this study we show, in human non-small cell lung carcinoma cells, that hypoxic stress selectively induces the stem cell marker NANOG, which is involved in the hypoxia-mediated resistance to specific lysis. Hypoxia-induced NANOG has the ability to control the expression of a series of proapoptotic genes, to regulate cell replication, and to contribute to STAT3 and Src phosphorylation.

The selective increase in NANOG expression under hypoxia was not associated with alteration of CD133 or ALDH1 expression, two reported markers for lung cancer stem cells (20). Additionally, IGR-Heu cells did not lose their epithelial features under hypoxia, as Snail expression was decreased and E-cadherin level remained unchanged in IGR-Heu hypoxic cells. Whether Snail decrease is due to transcriptional repression or increased degradation following hypoxic stress is currently under investigation. Nonetheless, this suggests that in our experimental system, tumor cells did not undergo a phenotypic dedifferentiation but indeed displayed increased expression of NANOG, a transcription factor associated with stemness. NANOG induction in hypoxia has been recently reported (4, 35, 36), suggesting its putative involvement in cell adaptation to hypoxic stress. In this regard, it has been shown that NANOG possesses proangiogenic activities by controlling vascular endothelial growth receptor-2 expression in endothelial cells (37). Although induction of this transcription factor under hypoxia is usually described to be dependent on HIF-2 in cancer stem cells (38, 39) and embryonic stem cells (35), our results clearly indicate that only HIF-1α selectively regulates NANOG induction in our experimental model.

To determine the functional relevance of NANOG in hypoxic tumor resistance to CTL-mediated lysis, we targeted NANOG and found that it resulted in the attenuation of target resistance to CTL-mediated killing. How this transcription factor regulates tumor susceptibility to lysis is not yet established. Nevertheless, our data considering the relevance of NANOG induction following hypoxic stress support the idea that NANOG is a significant mediator of the adaptive cellular response to hypoxia. NANOG has been reported to have antiapoptotic functions by inhibiting apoptosis in transformed human stem cells (40) and choriocarcinoma cells (41). NANOG targeting significantly increased the percentage of sub-G₁ dead cells, suggesting that hypoxia-induced NANOG has a protective role on tumor cells. By analyzing cell cycle transition, we show a decrease in BrdU incorporation under hypoxia. This could be related to the activation of DNA repair processes subsequent to DNA damage known to be induced by exposure to hypoxia (42). Additionally, a major event in shifting cells form normoxia to hypoxia is cell cycle arrest (43) (44). Interestingly, our data show that NANOG knockdown restored BrdU incorporation in hypoxic cells, suggesting that NANOG, under conditions of hypoxic stress, could be involved in the activation of checkpoint signaling resulting in slowing down S phase. This is supported by our finding that hypoxia induced p53 protein accumulation and that this event was inhibited following NANOG knocking down (data not shown). To further delineate the role of NANOG in hypoxia, we explored the effect of NANOG on hypoxia-induced STAT3 activation. Bourguignon et al. (10) demonstrated functional cooperation of NANOG with activated STAT3 in inducing expression of the chemoresistance gene MDR1. Previously, we provided evidence indicating that STAT3 phosphorylation was a major event in the acquisition of hypoxic tumor cell resistance to CTL-mediated killing (6). In the present report, our data point to the potential role of NANOG in regulating STAT3 phosphorylation and its subsequent nuclear translocation. To our knowledge, this is the first demonstration involving a functional interaction between NANOG and STAT3 in the regulation of tumor cell susceptibility to CTL-dependent cytotoxicity. More importantly, we showed that NANOG targeting resulted in the inhibition of hypoxia-induced Src phosphorylation. Because Src is known to be required for STAT3 phosphorylation (32, 33), it is tempting to speculate that NANOG controls STAT3 phosphorylation by a mechanism involving Src phosphorylation under hypoxia. This is in agreement with our recent observations showing that chemical inhibition of Src phosphorylation in hypoxic IGR-Heu cells was able to block hypoxia-induced STAT3 phosphorylation (45).

Along with inhibition of hypoxia-induced delay on cell replication levels, NANOG knockdown seems to allow hypoxic tumor cells to gain a “normoxia-like” state that may shift CTL-resistant hypoxic tumor cells to sensitive ones.

Collectively, these results point to a novel function of NANOG and suggest the existence of a new relationship between stem cell markers, hypoxia, and resistance to CTL-mediated lysis.

The authors have no financial conflicts of interest.