c-MYC-directed NRF2 drives malignant progression of head and neck cancer via glucose-6-phosphate dehydrogenase and transketolase activation.

Rationale: NRF2, a redox sensitive transcription factor, is up-regulated in head and neck squamous cell carcinoma (HNSCC), however, the associated impact and regulatory mechanisms remain unclear.
Methods: The protein expression of NRF2 in HNSCC specimens was examined by IHC. The regulatory effect of c-MYC on NRF2 was validated by ChIP-qPCR, RT-qPCR and western blot. The impacts of NRF2 on malignant progression of HNSCC were determined through genetic manipulation and pharmacological inhibition in vitro and in vivo. The gene-set enrichment analysis (GSEA) on expression data of cDNA microarray combined with ChIP-qPCR, RT-qPCR, western blot, transwell migration/ invasion, cell proliferation and soft agar colony formation assays were used to investigate the regulatory mechanisms of NRF2.
Results: NRF2 expression is positively correlated with malignant features of HNSCC. In addition, carcinogens, such as nicotine and arecoline, trigger c-MYC-directed NRF2 activation in HNSCC cells. NRF2 reprograms a wide range of cancer metabolic pathways and the most notable is the pentose phosphate pathway (PPP). Furthermore, glucose-6-phosphate dehydrogenase (G6PD) and transketolase (TKT) are critical downstream effectors of NRF2 that drive malignant progression of HNSCC; the coherently expressed signature NRF2/G6PD/TKT gene set is a potential prognostic biomarker for prediction of patient overall survival. Notably, G6PD- and TKT-regulated nucleotide biosynthesis is more important than redox regulation in determining malignant progression of HNSCC. Conclusions: Carcinogens trigger c-MYC-directed NRF2 activation. Over-activation of NRF2 promotes malignant progression of HNSCC through reprogramming G6PD- and TKT-mediated nucleotide biosynthesis. Targeting NRF2-directed cellular metabolism is an effective strategy for development of novel treatments for head and neck cancer. © The author(s).

Introduction

Nuclear factor erythroid 2-related factor 2 (NRF2) is a member of Cap'n'Collar family of transcription factors that share a highly conserved basic region‐leucine zipper structure. NRF2 is tightly regulated by Kelch‐like ECH associating protein 1 (KEAP1), a substrate adaptor protein for a Cullin3‐based E3 ubiquitin ligase, resulting in expression of NRF2 at low transactivating levels in all human organs 1, 2. When cells are exposed to electrophiles or oxidative stress, NRF2 is released from KEAP1 and transported into the cell nucleus to activate the transcription of specific cytoprotective genes by binding to the antioxidant response element (ARE) as a heterodimer with the small Maf protein 1, 2. Abundant evidence has shown that activation of NRF2 can suppress carcinogenesis 3, 4. However, recent evidence has revealed that aberrant activation of NRF2 is associated with cancer hallmarks 4-10, including promoted tumorigenesis 11, 12, sustained proliferative signaling and survival advantage 7, 13, 14, resistance to treatment 15-18, deregulated redox homeostasis 19-21, enhanced tissue invasion and metastasis 14, 22, 23, and altered cellular metabolism 13, 24-26.

Head and neck squamous cell carcinoma (HNSCC) is the fifth most common malignancy worldwide and comprises a diverse set of cancers arising from the squamous epithelium of the oral cavity, larynx, and pharynx, including the nasopharynx, oropharynx, hypopharynx, etc. 27. The primary risk factors for HNSCC include alcohol consumption, betel nut chewing, cigarette smoking, human papillomavirus (HPV) infection (for oropharyngeal cancer), and Epstein-Barr virus (EBV) infection (for nasopharyngeal cancer) 28. Even though the curability of early-stage HNSCC is high, the survival rate of patients with recurrent or metastatic HNSCC diminishes drastically 29. Exploring robust biomarkers that contribute to the malignant progression of HNSCC may aid the development of useful therapeutic strategies. For example, identification of aberrant signaling pathways and targeting these molecules (e.g., EGFR) has been an effective approach to HNSCC treatment 30.

Genomic studies using The Cancer Genome Atlas Network revealed that the activation of the NRF2/KEAP1/CUL3 pathway is a common feature of HPV-negative HNSCC 29. In addition, several groups demonstrated that increased NRF2 expression was observed in HNSCC and positively correlated with poor clinical outcome 19, 31-33. There is limited research on the effects of NRF2 on the malignant progression of HNSCC, which includes NRF2-mediated radiation tolerance and drug resistance 16, 34, 35, activated oncogenic Notch 14 or mTOR signaling 36, and NRF2 activity of as a central node in the maintenance of low ROS levels and stemness properties 19. Thus, the biological consequences of NRF2 in head and neck cancer remain unclear, and the critical NRF2 downstream effectors that participate in NRF2-promoted malignant progression have yet to be conclusively identified in HNSCC. In this study, we provide evidence that activation of NRF2 induces tumorigenesis and promotes tumor growth and metastasis. We also report our discovery of potential upstream modulators and downstream effectors of NRF2 in head and neck cancer. Among them, we identified c-MYC, which is associated with chemical induced carcinogenesis and a modulator of NRF2 activation. In addition, glucose-6-phosphate dehydrogenase (G6PD) and transketolase (TKT) exhibit exclusive roles in determining how NRF2 drives malignant progression of HNSCC.

Materials and Methods

HNSCC patient samples

Human HNSCC clinical specimens at different clinicopathological stages were obtained from National Cheng Kung University Hospital, Tainan, Taiwan in accordance with the associated Institutional Review Board (A-ER-107-147) and National Health Research Institutes, Zhunan, Miaoli, Taiwan (EC1070105).

Cell lines

Normal human oral keratinocytes (NHOK), human dysplastic oral keratinocytes (DOK), and human HNSCC cells (Ca9-22, HSC-3, OC3, HONE-1 and OEC-M1) were maintained as described previously 37-40. The Ca9-22-D1 cell line and the carcinogen-transformed DOK cells were established in Dr. Ching-Chuan Kuo's laboratory (, ). See for details. All cell lines were authenticated using Short-tandem-repeat (STR) profiling (DNA fingerprinting).

Genetic manipulation

For transient gene silencing, the siRNAs specific to NRF2, G6PD, and TKT and siRNA negative controls were purchased from Invitrogen and delivered into cells using Lipofectamine RNAiMAX reagent (Thermo Fisher Scientific, Waltham, MA., USA). For establishment of stable NRF2-knockdown HNSCC cells, a set of pGFP-C-shLenti vectors containing four independent 29-mer shRNAs against human NRF2 and a non-targeting shRNA were purchased from OriGene Technologies Inc. (Rockville, MD., USA). After transfection, the cells were selected by puromycin treatment. For generation of stable NRF2-overexpressing DOK cells, human NRF2 was amplified from Gene PoolTM normal human prostate cDNA (Life Technologies, Carlsbad, CA., USA) and cloned into the pIRES2-EGFP vector between the Nhe1 and Xho1 cloning sites. The specific primers used for plasmid cloning are listed in . After transfection, the cells were selected by geneticin (G418) treatment.

Evaluation of mRNA and protein levels

The real-time polymerase chain reaction (PCR) primer pairs used for amplification of target genes are listed in . Gene expression was determined by quantitative real-time PCR. Western blot analysis and immunohistochemistry (IHC) staining were used to analyze protein levels of cell lysates and tissue sections, respectively. The antibodies used for recognizing specific proteins are listed in .

Assessment of malignant features

(1) In vitro: Cell proliferation was assessed by methylene blue staining. Anchorage-independent growth was examined using the soft agar colony formation assay. Trans-well migration and invasion assays were performed to evaluate cell motility and invasiveness, respectively. (2) In vivo: Animals used in this study were purchased from BioLASCO Taiwan Co., Ltd (Taipei, Taiwan) and maintained at the Laboratory Animal Center of National Health Research Institutes (NHRI), Taiwan. The in vivo tumorigenic assay and the experimental lung metastasis assay were performed. See for details.

Chromatin immunoprecipitation-quantitative polymerase chain reaction (ChIP-qPCR)

Chromatin immunoprecipitation (ChIP) was performed using the Magna ChIP™ A/G Chromatin Immunoprecipitation Kit (Merck Millipore, Burlington, MA, USA) with an antibody specific for NRF2 (Abcam, Cambridge, MA., USA), c-MYC (Abcam, Cambridge, MA., USA) or normal rabbit IgG (Santa Cruz Biotechnology, Santa Cruz, CA., USA). Following ChIP, quantitative PCR was utilized to amplify and quantify the immunoprecipitated DNA using primers specific for the NRF2-targeted antioxidant response element (ARE) within G6PD or TKT, as well as primers for a c-MYC binding site within the NRF2 promoter region (). The c-MYC binding site within the NRF2 promoter region was obtained from the ENCODE Consortium. The ChIP-qPCR values were normalized to that of input control and represented as fold enrichment relative to the anti-normal rabbit IgG control.

Statistical analysis

The experimental results were analyzed and expressed as the mean ± standard deviation (S.D.) or mean ± standard error (S.E.) for the in vitro and in vivo experiments, respectively. One-way analysis of variance (ANOVA) and Student's t-test were used to determine statistical significance.

Results

Expression of NRF2 is positively correlated with the malignant characteristics of HNSCC

The clinicopathological significance of NRF2 was validated in an in-house cohort of clinical specimens of HNSCC (n=77) by IHC staining. In tumor-adjacent normal epithelium, few cells with positive staining for NRF2 were detected and most of the NRF2-positive cells were found in the active para-basal and basal layers. Conversely, in the oral epithelial dysplastic lesions and oral squamous cell carcinoma, NRF2-positive cells spread into the intermediate layer and the staining intensity was dramatically higher than in cancer-adjacent normal epithelia (Figure ). Based on immuno-reactive score (IRS) analysis, we noted that NRF2 was localized predominantly in cell nuclei, and both nuclear and cytosolic NRF2 levels increased significantly with malignant progression, i.e., progression from tumor-adjacent normal epithelia to epithelial dysplasia to squamous cell carcinoma (Figure ). We also compared differential expression of NFE2L2, the gene encoding NRF2, in HNSCC and normal tissue from clinical cohorts using the online cancer microarray database Oncomine 41. As shown in , increased NRF2 expression in tumors compared to normal tissue appeared in many HNSCC datasets. While analyzing NRF2 expression with histological differentiation, we demonstrated that NRF2 level was positively correlated with the degree of differentiation (Figure ). Intriguingly, IHC analysis demonstrated higher staining intensity of NRF2 in the invasive oral squamous cell carcinoma and invasive nests than in primary carcinoma (Figure ). Similar to clinical findings, the expression of NRF2 protein was markedly higher in the HNSCC cell lines than in dysplasia oral keratinocytes (DOK; Figure ), and lower levels of NRF2 protein were observed in normal human oral keratinocytes (NHOK) compared to DOK and HNSCC cells (). Increased nuclear localization of NRF2 led to upregulation of NRF2 downstream targets, such as AKR1C1, NQO1 (Figure ), and TrxR (. Notably, overexpression of NRF2 was positively related to motility of HNSCC cells (Figure ). These results indicate that expression of NRF2 is positively correlated with malignant features of HNSCC.

NRF2 is upregulated by c-MYC and acts as an important determinant for malignant progression of HNSCC

To confirm that NRF2 drives the malignant progression of HNSCC, we knocked down the NRF2 gene using RNA interference in three HNSCC cell lines (HONE-1, Ca9-22-D1, and OEC-M1; ) and found that both transient (Figure ) and stable () knockdown of NRF2 markedly reduced cell migratory, invasive (Figure ), and growth (Figure , ) capacity compared to control. In contrast, stably overexpressed NRF2 facilitates cellular motility and growth; however, when NRF2 is re-suppressed, the migration and growth abilities were decreased in NRF2 overexpressing DOK cells (Figure ). Furthermore, by performing the soft agar colony formation assay, we found that knockdown of NRF2 significantly suppressed the ability of anchorage-independent growth in HNSCC cells () and vice versa in NRF2 overexpressing cells (). In addition, the commercially available NRF2 inhibitor ML-385 produced results similar to genetic manipulation (). These in vitro findings suggest that NRF2 is important for maintaining the malignant characteristics of HNSCC.

Next, we questioned whether carcinogenic substances modulate NRF2 during head and neck carcinogenesis. Cigarette smoking, alcohol consumption, and betel quid chewing are the leading risk factors for HNSCC 42-44. IHC staining showed that HNSCC patients who smoke, drink, and chew betel nut have higher nuclear NRF2 levels than those who do not have those habits (). Therefore, we used nicotine and arecoline to further investigate the role of NRF2 in HNSCC, as long-term nicotine (e.g., cigarette smoking) and arecoline (e.g., betel nut chewing) exposure have been reported to promote head and neck carcinogenesis and progression 28, 45, 46. We found that long-term exposure to nicotine or arecoline increased NRF2 expression, which was accompanied by an increased proliferation index in carcinogen-transformed DOK cells (Figure ). We previously established a 4-NQO (simulation of smoking) plus arecoline (simulation of betel nut chewing) induced mouse model of oral carcinogenesis 47, which mimics the pathogenesis of squamous cell carcinoma (SCC) in patients from Southeast Asia and Taiwan 48-50. For in vivo validation, we analyzed normal and SCC samples from tongue tissue from the 4NQO/arecoline co-induction oral SCC mouse model. The results demonstrated that expression and nuclear localization of NRF2 protein was increased in SCC compared to normal tongue tissue ().

It is known that NRF2 is modulated by several oncogenes 20. By performing micro-western arrays, which enable quantitative, sensitive, and high-throughput assessment of protein abundance and modifications, we identified that BRAF, phospho-EGFR and c-MYC are significantly increased in both nicotine (NIC)- and arecoline (AC)-transformed DOK cells (). Further validation revealed that increased expression levels of BRAF and c-MYC were observed in most carcinogen-transformed DOK cells (Figure ). Thus, we investigated whether BRAF or c-MYC were upstream modulators to upregulate NRF2 in HNSCC cells. As shown in , we found silencing BRAF did not suppress NRF2 level in HNSCC cells. On the other hand, knockdown of MYC gene significantly inhibits NRF2 expression (Figure ). Similar results were observed in both nicotine (NIC)- and arecoline (AC)-transformed DOK cells (Figure ).

It is noteworthy that increased and differential MYC expression was observed in tumors of relevant clinical cancer datasets (). Therefore, we further explored the relationship between NRF2 and c-MYC. In addition to DOK cells, we confirmed the positive correlation between the expression of NFE2L2 (NRF2) and MYC in long-term arecoline-transformed normal human oral keratinocytes (NHOK) (). Next, we determined whether NRF2 could regulate the expression of c-MYC and found that knockdown of NRF2 does not affect the expression of c-MYC protein in HNSCC cells (). To investigate how c-MYC regulates NRF2 expression, we performed RT-qPCR analysis, and found that knockdown of c-MYC significantly decreased NRF2 mRNA levels in HNSCC cells (Figure ). Furthermore, we confirmed that c-MYC binds to the NRF2 promoter, and knockdown of c-MYC significantly reduced the binding strength of c-MYC at the NRF2-promoter in HNSCC cells (Figure ). These results indicate that c-MYC is involved in directing NRF2 expression in head and neck cancer.

NRF2 drives tumor progression of HNSCC in vivo

We next performed in vivo experiments to substantiate whether aberrant activation of NRF2 enhanced malignant progression of HNSCC. Firstly, we generated DOK cells with stable overexpression of NRF2 to examine the role of NRF2 in promoting malignant transformation in vivo. The pre-malignant epithelial cell line DOK is derived from human dorsal tongue tissue and is non-tumorigenic in mice 40. There was no tumor formation after implantation of mock control DOK cells in vivo (Figure , upper panel). In contrast, implantation of DOK cells with stable overexpression of NRF2 resulted in tumor development in vivo (Figure , lower panel). The tumor incidences of the NRF2-overexpressing and mock control groups were 100% and 0%, respectively (Figure ). In our next experiment, we investigated how NRF2 expression could affect tumor growth using the Ca9-22-D1 (human origin) xenograft tumor model. As shown in Figure , tumor growth was inhibited in mice bearing stable NRF2-knockdown Ca9-22-D1 xenograft tumors. In addition, we found that the expression levels of NRF2 and AKR1C1 (the classical NRF2 downstream target) proteins were significantly reduced in NRF2-knockdown tumors compared to controls (Figure ). To evaluate whether the depletion of NRF2 suppresses metastasis of HNSCC in vivo, we used an experimental metastasis model by injecting mice with HONE-1 or Ca9-22-D1 cells, either with stably expressing NRF2-shRNA or empty expression vector. Compared with the mock control group, a decrease in lung-specific metastatic foci formation was observed in NRF2-knockdown groups (Figure ), suggesting that genetic ablation of NRF2 remarkably hindered pulmonary metastasis. The hematoxylin and eosin (H&E) and IHC staining of lung tissue sections demonstrated that mice bearing NRF2-knockdown Ca9-22-D1 cells significantly inhibited lung-specific metastatic tumor foci formation compared with mice bearing mock control cells, and NRF2 expression level is positively correlated with the number of tumor foci (Figure ). These results suggest that augmentation of NRF2 expression drastically enhances the potential for malignant transformation, tumor growth, and metastasis of HNSCC in vivo.

NRF2-mediated antioxidant capacity is not the primary cause of malignant progression of HNSCC

Oxidative stress is known to play a pivotal role in the development of HNSCC 51. The primary function of NRF2 is to maintain cellular redox homeostasis through enhancing antioxidant gene expression. Therefore, we investigated whether the change in redox balance is the primary cause of NRF2-mediated malignant progression in HNSCC. Gene Set Enrichment Analysis (GSEA) revealed that the HALLMARK REACTIVE OXYGEN SPECIES pathway genes were highly enriched in NRF2-silenced HNSCC cells and that there was a concomitant decrease in the expression of a panel of NRF2-mediated antioxidant genes (Figure ). Profoundly increased intracellular ROS levels were also found in both Ca9-22-D1 and HONE-1 cells (Figure ). In addition, DOK cells with stable overexpression of NRF2 exhibited lower ROS levels than mock controls (). When cells were challenged with oxidative stress, the reduced form of glutathione (GSH) was oxidized to form glutathione-disulfide (GSSG), which brought on a decrease in intracellular GSH levels. Knockdown of NRF2 significantly reduced intracellular GSH levels in HNSCC cells (Figure ). Although treatment of cells with N-acetyl cysteine (NAC), a widely used pharmacological antioxidant, effectively reduced ROS production (Figure , ), addition of NAC did not restore cell growth (Figure , ) and migration ability (Figure , ) in NRF2-knockdown HNSCC cells. Similarly, treatment with another antioxidant, Trolox, had no effect in the rescue of cell motility in NRF2-knockdown HNSCC cells (Figure ). These results suggested that NRF2-mediated antioxidant capacity was not a primary cause of malignant progression of HNSCC.

Pentose phosphate pathway (PPP) is the most highly enriched NRF2-mediated metabolic pathway in HNSCC cells

After a close examination of GSEA data, we noticed that 33% (10/30) of the top 30 most highly enriched down-regulated gene sets in NRF2-knockdown cells were related to cell metabolism (). Among them, the highest-ranking metabolic pathway is the KEGG PENTOSE PHOSPHATE PATHWAY (PPP; ). The enrichment plot and heat map of differentially expressed genes displayed obvious suppression of PPP in NRF2-knockdown HONE-1 and Ca9-22-D1 cells (Figure ). Since the PPP is a major source of reduced nicotinamide adenine dinucleotide phosphate (NADPH), we checked and observed that NADPH was also significantly suppressed upon NRF2 inhibition ().

To explore which biological processes or molecular pathways could be regulated by NRF2 signaling in the milieu of tumors, we again performed GSEA analysis on head and neck tumor samples annotated in The Cancer Genome Atlas Head-Neck Squamous Cell Carcinoma (TCGA-HNSC) dataset. We queried significant enrichment of gene sets that were co-expressed with NRF2. At nominal (NOM) p-value < 0.05 and FDR < 0.25, we found 511 gene sets were highly co-expressed with NRF2. As expected, PPP pathway also showed significant positive enrichment (NES = 1.895) among these gene sets, indicating a positive correlation between PPP genes and NRF2 level (Figure ). We conducted another independent GSEA assay based on Cox regression model to query whether any enrichment of genes was associated with clinical outcomes of HNSCC. Notably, the PPP pathway was found to be significantly associated with patient survival (NES = 2.458; Figure ). These results indicate that increased PPP activity may predict poor overall survival of the patients with HNSCC.

We next identified the specific PPP genes enriched in HNSCC by performing qPCR analysis, and the results demonstrated that G6PD, 6-phosphogluconate dehydrogenase (PGD), and TKT were significantly suppressed in NRF2-knockdown HONE-1 and Ca9-22-D1 cells (Figure ). Remarkably, knockdown of NRF2 drastically suppressed the protein levels and enzymatic activity of G6PD and TKT in HNSCC cells (Figure , ). Consistently, the protein levels or enzyme activity of G6PD and TKT were increased in stable NRF2-overexpressing cells (Figure ,) and could be re-suppressed by NRF2-targeted siRNA treatment (Figure ). Furthermore, the in vivo study revealed that knockdown of NRF2 markedly decreased the protein levels () and enzymatic activities (Figure ) of G6PD and TKT in the excised tumors from Ca9-22-D1 xenograft tumor-bearing mice.

We further dissected the mechanisms by which NRF2 regulates G6PD and TKT in HNSCC cells. ChIP-qPCR assays demonstrated direct binding of NRF2 to the ARE consensus sequence in the promoter region of G6PD and TKT in both HONE-1 and Ca9-22-D1 cells (Figure ). Analysis of data from the TCGA-HNSC cohort demonstrated that the level of G6PD or TKT gene expression was positively correlated with NRF2 (G6PD vs. NRF2: Pearson correlation = 0.49, p < 4.7e-33; TKT vs. NRF2: Pearson correlation = 0.36, p < 2.7e-17; ). These results indicated that NRF2 repurposes PPP metabolism by direct modulation of G6PD and TKT in head and neck cancer.

G6PD and TKT are key NRF2-downstream effectors to drive malignant progression of HNSCC

We next investigated whether G6PD and TKT were critical downstream effectors in driving NRF2-mediated malignant progression of HNSCC. First, we found that the levels of G6PD and TKT expression (Figure ) were positively correlated with hallmarks of cell malignancy (i.e., motility as shown in Figure ). As expected, knockdown of G6PD or TKT lead to suppression of cell migration, invasion, and anchorage-independent growth of HNSCC cells (Figure ). We also observed that increase of NRF2 drives cell motility, invasiveness, and proliferation in stably NRF2-overexpressing DOK cells, and these effects could be re-suppressed after blocking the function of G6PD and TKT (Figure ). These results indicated that G6PD and TKT are key NRF2-downstream effectors to drive malignant features of HNSCC.

The combination of pathway targeted therapies with standard-of-care (S.O.C.) treatment is a promising therapeutic strategy that may generate synthetic lethality 52. Cisplatin concurrent chemoradiotherapy (CCRT) is a S.O.C. therapy for treatment of HNSCC, therefore, we questioned whether targeted G6PD or TKT could elicit a synergistic interaction with cisplatin. We treated HNSCC cells with either low dose cisplatin alone, or in combination with G6PD or TKT inhibitors. Strikingly, three different G6PD inhibitors, including dehydroepiandrosterone (DHEA) 53, trans-polydatin 54, and 6-aminonicotinamide (6-AN) 55 were found to cause synthetic lethality in HNSCC cells when combined with cisplatin (Figure , ), while co-treatment with the TKT inhibitor (oxythiamine chloride hydrochloride) and cisplatin showed no synergistic cytotoxicity (data not shown). These results indicate that combined treatment with cisplatin plus a G6PD inhibitor may be particularly effective as a synthetic lethal therapeutic regimen for HNSCC, and clinical application of this strategy merits further investigation. Although the combination of a TKT inhibitor and cisplatin did not show synergy in HNSCC cells, targeting TKT may have a synergistic interaction with other approved drugs; further research is needed to explore additional combinations.

To expand the level of evidence, the clinical outcome of NRF2/G6PD/TKT-gene signature in HNSCC patients was analyzed in TCGA-HNSC cohort. A total of 215 patient samples were divided into the NRF2/G6PD/TKT-high group (red; n = 102) and the NRF2/G6PD/TKT-low group (blue; n = 113) based on gene expression patterns. The survival probability estimates for the two groups were visualized as Kaplan-Meier plots. Notably, overall survival analysis revealed that the patients in the NRF2/G6PD/TKT-high (red) group had worse survival outcomes than the NRF2/G6PD/TKT-low (blue) group (p = 0.05; Figure ). These results suggested that the NRF2/G6PD/TKT gene set could be a potential prognostic biomarker to predict the clinical outcome of HNSCC.

Ribose-5-phosphate generation is more important than NADPH production in determining malignant features of head and neck cancer

NADPH and ribose 5-phosphate (R5P) are the main products or intermediates of G6PD and TKT. NADPH in cells maintains glutathione in the reduced state (GSH) and is important for reductive biosynthesis (e.g., fatty acid synthesis). R5P is required for de novo nucleotide biosynthesis 56. The metabolite analysis showed that intracellular GSH, NADPH, NADP+ and nucleotide metabolism-related metabolites, especially R5P, were down-regulated in NRF2-knockdown HNSCC cells (). We found that replenishment of metabolites can effectively rescue the levels of NADPH, NADP+, GSH and R5P in NRF2-knockdown cells (Figure ). However, addition of NADPH and GSH does not significantly restore cell motility in NRF2-knockdown HNSCC cells (Figure ). Notably, unlike NADPH and GSH, R5P significantly restored cell motility and invasiveness in NRF2-knockdown HNSCC cells (Figure ;).

Nucleotide biosynthesis begins with the conversion of R5P to 5-phosphoribosyl-1-pyrophosphate (PRPP), which is then catalyzed to become phosphoribosylamine, a nucleotide precursor. Therefore, we performed a rescue experiment by adding PRPP to the growth media of NRF2-knockdown HNSCC cells. The result demonstrated that, similar to R5P, PRPP could rescue the cell migration ability under NRF2 knockdown condition (). These results suggested that the regulatory function of G6PD/TKT on nucleotide biosynthesis appears to be more important than redox regulation for malignant progression of head and neck cancer.

Discussion

In HNSCC, activation of NRF2 occurs through gain-of-function mutations in NFE2L2 (gene encoding NRF2) 57-59, loss-of-function mutations in the KEAP1 gene 57, disruption of the KEAP1/CUL3/RBX1 E3-ubiquitin ligase complex 31, modulation by IL-6 18 or GRP78/PERK signaling 19, and increased protein stability of NRF2 via O-GlcNAcylation 23. In this study, we are the first to provide evidence that aberrant activation of NRF2 is modulated by c-MYC in chemical-induced head and neck tumorigenesis. Major chemical-associated risk factors for development of HNSCC, including alcohol consumption, betel nut chewing, and cigarette smoking, all generate oxidative stress 60. NRF2 is a key determinant for cells coping with oxidative stress and a tobacco exposure-related signature in HNSCC 57, 61. Long-term exposure of dysplastic DOK cells to nicotine (simulating smoking) or arecoline (simulating betel nut chewing) was used to mimic the malignant transformation in vitro, and the result demonstrated that increased cell proliferation was accompanied by increased NRF2, B-RAF, and c-MYC expression in carcinogen-transformed cells (Figure , ). Through additional experiments we excluded B-RAF and confirmed that c-MYC positively modulates NRF2 through binding to the NRF2 promoter region in HNSCC cells (Figure ). ChIP-seq data from the ENCODE consortium demonstrated that c-MYC binds directly to the NFE2L2 locus and increases NRF2 transcription 20. The MYC gene has been shown to be amplified in HNSCC, particularly in HPV(-) HNSCC 57. Our study demonstrated that long-term exposure of DOK and NHOK cells with carcinogens induces c-MYC expression (Figure , and S10), suggesting that chemical factors may be more important to initiate dysregulation of c-MYC oncogene than viral factors during head and neck carcinogenesis.

Through systematic investigation by combining RNAi, microarrays, and GSEA approach, we noted that 33% (10/30) of the 30 most highly enriched down-regulated gene sets in NRF2-knockdown HNSCC cells were related to cellular metabolism (). Metabolic reprogramming is firmly established as a hallmark of cancer 62. During malignant development, cancer cells are obliged to rewire their metabolism to support aberrant cell growth, enhance their metastatic capacity, and adapt to the stress of survival in the austere tumor microenvironment 63. A characteristic feature of head and neck cancer is enhanced glycolysis, providing the basis for clinical use of FDG-PET as a diagnostic imaging agent 64. In addition, recent studies on NRF2-mediated cellular metabolism in HNSCC are focused on glycolysis (the Warburg effect). Chang et al. demonstrated that the Warburg effect and the stemness of cancer-initiating cells of the head and neck were mediated by GRP78/p-PERK/NRF2 signaling 19. Fu et al. found that hyperactive NRF2 causes metabolic reprogramming and up-regulation of glycolysis genes, such as pyruvate kinase M2 (PKM2), in the mouse esophagus 26. In this study, we observed that KEGG GLYCOLYSIS GLUCONEOGENESIS pathway is the fourth most highly enriched down-regulated gene set in NRF2-knockdown HNSCC cells (), which mirrored previous reports 19, 26.

In addition to highlighting glycolysis, we have now identified the PPP pathway as the most highly enriched down-regulated metabolism gene set in NRF2-knockdown HNSCC cells (). More importantly, as supported by clinical evidence, the expression of PPP genes is positively correlated with NRF2 (Figure ), and increased PPP predicted poor overall survival of patients with HNSCC (Figure ). PPP branches off from glycolysis and comprises irreversible oxidative and reversible non-oxidative phases. The enzymes involved in the oxidative phase are G6PD, 6-phosphogluconolactonase (PGLS), and PGD. Ribose-5-phosphate isomerase (RPI), ribulose 5-phosphate 3-epimerase (RPE), transaldolase (TALDO1), and TKT function in the non-oxidative phase of the PPP. To date, very little is known regarding these PPP enzymes in head and neck cancer. NRF2-regulated PPP genes, including G6PD, PGD, TKT, and TALDO1 were first identified in lung adenocarcinoma A549 cells 13. In this study, we confirmed that G6PD and TKT mRNA level, protein level, and enzyme activity were consistently regulated by NRF2 in HNSCC in vitro and in vivo (Figure , ).

G6PD is overexpressed in a number of cancer types 65, including HNSCC (). Several oncogenic pathways associated with PPP flux regulation are linked with G6PD 65. G6PD can be transcriptionally activated by TAp73 66, YY1 67, and NRF2 13. However, only one prior study revealed that G6PD was modulated by FOXM1 for participating radio-resistance in head and neck cancer 68. TKT had been reported to be involved in the malignant progression of breast cancer 69 and liver cancer 70, however, there is very limited information on TKT in HNSCC. Based on Oncomine database analysis, increased TKT expression was observed in tumors in several clinical HNSCC datasets (). It has been shown that transketolase like 1 (TKTL1), a TKT isoform, is activated by promoter hypomethylation and contributes to HNSCC carcinogenesis 71. TKT, TKTL1, and TKTL2 encoded enzymes all have transketolase activity. Our study showed that transketolase activity was significantly reduced in NRF2-knockdown HNSCC cells and xenograft tumors (Figure , ), and NRF2 did not affect the expression of TKTL1 or TKTL2 gene in HNSCC (data not shown). These results suggested that knockdown of NRF2-mediated TKT suppression did not cause compensatory effects by increasing TKTL1 and TKTL2. Knockdown of TKT has been reported to inhibit the proliferation of oral cancer cells and be accompanied by an increased uptake of glucose and glutamine, as well as increased production of lactate 72, which suggested that cancer cells may overcome single defective pathways through secondary metabolic network adaptations. Therefore, inhibition of multiple metabolic pathways or their common upstream regulator may be required to impede tumor progression. Accordingly, inhibition of NRF2 may be more advantageous compared to targeting a particular downstream metabolic pathway. Although there have yet to be any FDA-approved drugs targeting NRF2 activity in cancer, there have been increasing efforts toward the development of novel NRF2 inhibitors 73 and continued research in this area has great potential for clinical application.

NRF2 has been shown to activate PPP genes through direct binding to the ARE in the gene promoters 13, 21, producing effects such as attenuation of miR-1 and miR-206 12 or driving telomerase reverse transcriptase 74. We demonstrated that NRF2 bound directly to the ARE consensus sequence in the promoters of G6PD and TKT in HNSCC cells (Figure ), and G6PD and TKT acted as the key NRF2-downstrem effectors of malignant progression of HNSCC (Figure ). G6PD is the irreversible rate limiting enzyme in the oxidative phases of the PPP that ensures sufficient NADPH and R5P levels. The reversible enzyme TKT bridges the PPP with glycolysis, and is therefore a key enzyme controlling the direction of PPP and R5P levels 56. Although the requirement for NADPH is higher than that for R5P in cell physiological functions, the addition of R5P can restore cell motility in NRF2-knockdown HNSCC cells, whereas the addition of NADPH does not rescue motility (Figure ). These results suggest that G6PD- and TKT-regulated nucleotide biosynthesis may be more important than redox regulation in driving malignant progression of head and neck cancer. NADPH could be generated by both PPP and the folate-mediated one-carbon metabolism and malic enzymes in cancer cells and other proliferating cells 75, 76, therefore, we speculate that inhibition of the PPP in HNSCC cells may reprogram other NADPH homeostasis systems. Additional research is needed to explore this possibility.

In fact, the mechanism by which NRF2 regulates nucleotide biosynthesis varies among cancer types. Saigusa et al. found that TALDO1 may involve in NRF2-mediated de novo purine nucleotide synthesis in non-small-cell lung carcinoma 77. However, we noted that in all test HNSCC cells, the expression level of the TALDO1 gene did not consistently change after genetic ablation of NRF2 (Figure ). In addition, Mitsuishi et al. has reported that MTHFD2, the metabolism enzyme for nucleotide biosynthesis, was decreased in lung cancer cell lines with NRF2 knockdown 13. DeNicola et al. demonstrated that NRF2 controls the expression of PHGDH, PSAT1, PSPH, and SHMT2 via ATF4 to support nucleotide production in non-small cell lung cancer 25. However, according to our real-time PCR validation, the expression levels of these genes (PHGDH, PSAT1, PSPH, SHMT1/2, MTHFD1/2) did not change upon NRF2 silencing in HNSCC cells (), suggesting that NRF2-mediated regulatory mechanisms may vary substantially in head and neck versus lung cancer, further supporting the unique role of NRF2-mediated G6PD and TKT expression in nucleotide biosynthesis of head and neck cancer. More importantly, we have identified the NRF2/G6PD/TKT gene signature as a potential prognostic biomarker for prediction of HNSCC survival outcomes (Figure ). Even though it was known that NRF2 can regulate PPP 12, 13, 21, no study has demonstrated that NRF2 promotes cancer progression directly through PPP components, particularly in head and cancer. In this study, we provided the first evidence to confirm that G6PD and TKT are critical downstream effectors of NRF2-driven malignant progression in HNSCC. We believe this is an important breakthrough for clarifying the role of NRF2 in cancer development.

In addition to PPP and glycolysis, the ten most highly enriched down-regulated metabolism pathways in NRF2 knockdown HNSCC cells also included amino acid, nucleotide, xenobiotics, heme, and fatty acid metabolism as well as oxidative phosphorylation (), and many of them have been known to modulate head and neck oncogenesis 78. Interestingly, we noted that the KEGG VALINE, LEUCINE AND ISOLUCINE DEGRADATION pathway was the second most highly enriched downregulated gene set in response to NRF2 silencing in HNSCC cells. Valine, leucine, and isoleucine are branched-chain amino acids (BCAAs). BCAA metabolism can influence diverse cellular processes, ranging from protein synthesis to epigenetic regulation, and dysregulation of BCAA metabolism contributes to cancer progression by diverse mechanisms 79. To date, no literature has been published on the regulation between NRF2 and BCAA metabolism. In addition, the role of valine, leucine, and isoleucine degradation in the progression of head and neck cancer remains to be clarified. Further exploration of the link between BCAA metabolism and NRF2 in HNSCC is warranted.

This is the first study providing data from systematic analysis of the regulatory mechanisms and function of NRF2 in HNSCC. We discovered that aberrant activation of NRF2 occurred via c-MYC-mediated upregulation and is associated with chemical-induced carcinogenesis. NRF2 is involves in reprogramming the broad metabolic milieu in HNSCC, and PPP is the most highly enriched NRF2-mediated metabolic pathway. Notably, G6PD and TKT are critical NRF2-downstream effectors that drive malignant progression of HNSCC. Furthermore, G6PD- and TKT-regulated nucleotide biosynthesis is more important than redox regulation for maintaining the malignant features of head and neck cancer. Taken together, these data support the potential for use of the NRF2/G6PD/TKT gene signature as a prognostic biomarker and for the development of NRF2-targeted therapies that alter cellular metabolism as novel and promising treatment options for head and neck cancer.