Hypoxia upregulates the core pluripotency factors NANOG, SOX2, and OCT4, associated with tumor aggressiveness and resistance to conventional anticancer treatments. We have previously reported that hypoxia-induced NANOG contributed in vitro to tumor cell resistance to autologous-specific CTL and in vivo to the in situ recruitment of immune-suppressive cells. In this study, we investigated the mechanisms underlying NANOG-mediated tumor cell resistance to specific lysis under hypoxia. We demonstrated the tumor-promoting effect of hypoxia on tumor initiation into immunodeficient mice using human non–small lung carcinoma cells. We next showed a link between NANOG and autophagy activation under hypoxia because inhibition of NANOG decreased autophagy in tumor cells. Chromatin immunoprecipitation and luciferase reporter assays revealed a direct binding of NANOG to a transcriptionally active site in a BNIP3L enhancer sequence. These data establish a new link between the pluripotency factor NANOG and autophagy involved in resistance to CTL under hypoxia.
Microenvironmental hypoxia is a prominent feature of most solid tumors and is involved in fostering the neoplastic process by, at least, inducing a cancer stem cell (CSC)–like phenotype (1). NANOG, a homeodomain transcription factor, is involved in the maintenance of pluripotency of embryonic stem cells (2) as well as tumor progression (3, 4) and escape from immune killing (4–6). However, the mechanisms of NANOG-dependent intrinsic resistance of hypoxic tumor cells to T cell–mediated lysis remain not well understood.
Autophagy is a homeostatic process responsible for the sequestration and lysosomal degradation of cytoplasmic materials (7). We and others have shown that hypoxia activates autophagy, which confers tumor resistance to cell-mediated cytotoxicity (8–10). The role of hypoxia in inducing a CSC phenotype is also well documented (1); however, little is known about whether there is interplay between CSC features and autophagy, with both processes being regulated by hypoxia.
In this study, we investigated the involvement of the pluripotency factor NANOG in the activation of autophagy under hypoxia. We show that NANOG directly activates the expression of BNIP3L and contributes to autophagy under hypoxia. Our data reveal a new link between hypoxia-induced NANOG and the induction of autophagy required for maintenance of tumor resistance to killer cells.
Materials and Methods
The IGR-Heu human lung carcinoma cell line and Heu171 autologous T cell clone were grown under hypoxia (1% pO2) in a hypoxia workstation (Invivo2 400; Ruskinn) (5).
Real-time PCR and Western blot
Real-time PCR and Western blotting were performed as described (5).
Autophagosomes were quantified after stable transfection of IGR-Heu cells with a Tomato-LC3 vector as previously described (8).
Chromatin immunoprecipitation assay
Chromatin immunoprecipitation (ChIP) was performed using anti-NANOG Ab (Cell Signaling Technology) as previously described (11). Primer sequences are available upon request.
Luciferase reporter assay
Luciferase assay was performed as previously described (12).
Tumor growth monitoring
NOD–SCID–IL-2Rγ−/− mice were raised and housed at Gustave Roussy animal facility following institutional animal guidelines.
Data were analyzed with GraphPad Prism. A Student t test was used for single comparisons. A p value <0.05 was considered significant.
Results and Discussion
Hypoxia increases CSC-like features in IGR-Heu cells
In this study, we show that hypoxic preconditioning of IGR-Heu cells significantly facilitated tumor initiation after engraftment into immune-deficient mice (Fig. 1A). This is in agreement with our previous findings using B16 melanoma tumors (4) and in line with other reports using HIF targeting in tumors (13, 14). We also observed that targeting NANOG in hypoxic IGR-Heu cells reduced tumor initiation (Supplemental Fig. 1A–C). NANOG, SOX2, and OCT4 mRNAs and proteins were consistently increased under hypoxic stress (Supplemental Fig. 1D) as previously reported (5). To investigate the relative contribution of these proteins in the modulation of immune reactivity, we selectively inhibited their expression by RNA interference in hypoxic IGR-Heu cells. We showed a prominent role of NANOG in hypoxia-induced IGR-Heu resistance to autologous CTL (Fig. 1B). These data clearly show that hypoxic preconditioning of IGR-Heu cells increases their tumorigenic potential, which may correlate with an enrichment of CSC-like populations. We cannot exclude that several other mechanisms triggered under hypoxia also contribute to this increased tumorigenic potential. Fig. 1B also shows that NANOG emerges as a prominent factor in hypoxia-induced tumor cell resistance to specific lysis.
NANOG targeting impairs hypoxia-induced autophagy in IGR-Heu cells
We next sought to determine the existence of a link between hypoxia-induced NANOG and autophagy. We investigated autophagy levels in NANOG defective hypoxic IGR-Heu cells by assessing the level of p62 and LC3-II with or without the autophagy inhibitors E64d/pepstatin A (E/P), which inhibit lysosomal proteases. Western blot analysis showed that hypoxia induced a decrease in p62 and an increase in LC3-II, as previously reported (Fig. 2A) (8), which were inhibited when NANOG was knocked down (Fig. 2A). A decrease in LC3-II can be either related to an inhibition of autophagosome formation or an increase in autophagic flux due to enhanced lysosomal activity, faster fusion between autophagosome and lysosome compartments, or accelerated trafficking of autophagosomes to lysosomes. To evaluate the autophagic flux, hypoxic IGR-Heu cells were treated with E/P. E/P increased hypoxia-induced LC3-II levels in control cells, highlighting the autophagic flux. The accumulation of LC3-II was decreased in hypoxic IGR-Heu cells knocked down for NANOG expression, indicating a decreased autophagic flux (Fig. 2A). This suggests that NANOG targeting inhibited autophagosome formation. We next assessed autophagosome formation by transfecting IGR-Heu cells with a Tomato-LC3 plasmid (red dots). Microscope analysis clearly showed that NANOG targeting in hypoxic Tomato-LC3 IGR-Heu cells suppresses the hypoxia-dependent autophagosome formation (Fig. 2B).
To further delineate the role of NANOG in autophagy activation, we evaluated autophagy in normoxia after NANOG overexpression in IGR-Heu cells. It resulted in a decrease of p62 and an enhancement of LC3-II levels (Fig. 3A). Microscopic analyses also showed a consistently higher number of autophagosomes per cell when NANOG was overexpressed under normoxia (Fig. 3B, 3C). These results clearly demonstrate that NANOG positively regulates autophagy under hypoxic stress.
Hypoxia-induced NANOG contributes to autophagy activation by directly activating BNIP3L expression
We have previously shown that hypoxia induced autophagy in IGR-Heu cells by upregulating BNIP3 and BNIP3L proteins, which then released BECLIN from its inhibitory interaction with Bcl-2 (8, 10). Therefore, we first focused on a putative interaction of hypoxia-induced NANOG with the promoter regions of BNIP3 and BNIP3L. Under our experimental conditions, hypoxia-induced NANOG and BNIP3L were detected in the same cells (Supplemental Fig. 2A). Knockdown of NANOG under hypoxic conditions decreased both BNIP3 and BNIP3L transcripts and protein levels (Supplemental Fig. 2B, 2C). The regulatory effect of NANOG on BNIP3L was also found in melanoma and breast tumor cells (Supplemental Fig. 2D). The presence of NANOG binding consensus sequences TAAT(G/T)(G/T) (15) and (C/G)(G/A)(C/G)C(G/C)ATTAN(G/C) (2) in the promoter region (−2000 to +2000 bp) of BNIP3 and BNIP3L was analyzed using fuzznuc (EMBOSS explorer) software. Three putative NANOG binding sites (NBS) containing the consensus sequence TAAT(G/T)(G/T) were found for each gene. ChIP assay demonstrated hypoxia-inducible binding of NANOG to NBS3 of BNIP3L (+1303 to +1308 bp) (Fig. 4A, 4B) but not to BNIP3 promoter (data not shown). This demonstrates that NANOG regulated BNIP3L expression by directly interacting with a NBS downstream and distal to BNIP3L transcription start site, suggesting an enhancer function for NANOG. It also suggests an indirect regulating effect on BNIP3 expression. To determine whether NBS3 is a transcriptionally active site, IGR-Heu cells were cotransfected with pGL4-hRluc/SV40 vector and pGL3-NBS3 (Fig. 4C). Measurement of firefly and Renilla activities after 48 h showed a significant increase in luciferase activity for the pGL3-NBS3 reporter (≥2-fold) under hypoxia as compared with normoxia, indicating NBS3 enhancer activity (Fig. 4D). Furthermore, siRNA-mediated knockdown of NANOG consistently reduced luciferase signal under hypoxia, as well as base substitution mutation in NBS3 (pGL3-NBS3-Mut vector) (Fig. 4D). These results demonstrate that, under hypoxic stress, NANOG contributes to autophagy induction at least by activating BNIP3L expression in IGR-Heu cells after binding to the distal enhancer site NBS3. Whether NANOG induction and binding to NBS3 influence BNIP3L promoter occupancy by other transcription factors under hypoxia remains to be determined. Bioinformatics analysis of the promoter regions of the autophagy genes ATG5, BECN1, and LC3 also revealed the presence of putative NBS, suggesting that additional autophagy genes could be target genes for NANOG. Recently, the core pluripotency factor SOX2 has been shown to regulate autophagy in embryonic cells by repressing mTOR (16). These findings and ours support the idea that pluripotency factors can play a direct role in autophagy activation. Interestingly, Cho et al. (17) demonstrated that, in embryonic cells, autophagy is involved in degradation of pluripotency factors for homeostasis purposes. This suggests interactions between pluripotency factors and autophagy at different levels.
Targeting BNIP3L in hypoxic IGR-Heu cells decreases their resistance to specific lysis
The link between hypoxia-induced BNIP3L and IGR-Heu resistance to CTL was investigated by knocking down BNIP3L expression in hypoxic IGR-Heu cells using siRNA. BNIP3L knockdown reversed hypoxia-induced inhibition of CTL-mediated killing at all E:T ratios tested (Fig. 5). Because we demonstrated that BNIP3L is required for autophagosome formation in hypoxic IGR-Heu cells (8) and the ability of autophagosomes to degrade granzyme B in hypoxic tumor cells (9), it is likely that BNIP3L targeting in hypoxic IGR-Heu cells suppresses this protective mechanism. A report argued for a death mediator function for hypoxia-induced BNIP3L (18). Indeed, hypoxia is reported to induce p53-dependent increase in BNIP3L, causing cell death, but whether autophagy was involved in BNIP3L-mediated death was not established.
In conclusion, our studies highlight a new hypoxia-induced pathway in which NANOG activates BNIP3L expression, contributing to autophagy induction in hypoxic tumor cells and their resistance to killing by CTL. These results suggest that targeting hypoxia-induced autophagy-related stemness may be an important approach to reverse immune resistance.
This work was supported by Ligue contre le Cancer Grant EL2015.LNCC/SaC.
The online version of this article contains supplemental material.
Abbreviations used in this article:
cancer stem cell
NANOG binding site
small interfering RNA.
The authors have no financial conflicts of interest.