NQO1 promotes an aggressive phenotype in hepatocellular carcinoma via amplifying ERK‐NRF2 signaling

Abstract Patients with hepatocellular carcinoma (HCC) are usually diagnosed at the later stages and have poor survival outcomes. New molecules are urgently needed for the prognostic predication and individual treatment. Our study showed that high levels of NQO1 expression frequently exist in HCC with an obvious cancer‐specific pattern. Patients with NQO1‐high tumors are significantly associated with poor survival outcomes and serve as independent predictors. Functional experiments showed that NQO1 promotes the growth and aggressiveness of HCC in both in vitro and in vivo models, and the underlying mechanism involved NQO1‐derived amplification of ERK/p38‐NRF2 signaling. Combined block of ERK and NRF2 signaling generated stronger growth inhibition compared with any single block, especially for HCC with high‐NQO1. Therefore, NQO1 is a potential biomarker for HCC early diagnosis and prognosis prediction, and also attractive for cancer‐specific targets for HCC treatment.


| BACKG ROU N D
Liver cancer is one the most commonly diagnosed cancers, an estimated 782 500 new cases and 745 500 deaths occurred in 2012 worldwide. 1 More than 75% of the cases occur in the Asia-Pacific region and China alone accounted for 50% of cases and deaths. 1 Most (70% to 90%) primary liver cancers are hepatocellular carcinoma (HCC). The prognosis of patients with HCC is significantly different among patients with local, regional, and distance lesions, with 5-y survival rates of 30%, 11%, and 3%, respectively. 1,2 However, only 40% of patients with HCC are discovered with local lesions. 2 Moreover, the heterogeneity and evolution of HCC have forced the recognition of the profound importance of stratifying patients for clinical management at an individual level. [3][4][5] Therefore, it is important to search for more effective biomarkers for early diagnosis and individual treatment. NAD(P)H quinone oxidoreductase 1 (NQO1) is a flavoenzyme, capable of catalyzing obligatory two-electron reductions of a wide range of quinones to hydroquinones in an NAD(P)H-dependent manner. [6][7][8][9] It is also a key player in the cellular defense mechanism against oxidative stress, and substantially increases in response to various stimuli. [6][7][8][9] The generation of stable hydroquinones by NQO1 is believed to be a detoxification mechanism because this process can avoid one-electron reductions. [6][7][8][9] In some patients, rather than detoxifying, NQO1-related reduction can bioactivate certain antitumor quinines into potent cytotoxic compounds. [6][7][8][9][10][11][12] Beyond its catalytic function, NQO1 also has a critical role in protecting various regulatory proteins containing intrinsically unstructured domains from degradation by the 20S proteasome, 7,13-17 such as p53, p73a, p33, p63g, c-Fos, HIF1a, and the translation initiation factor (eIF) 4GI, many of which are very important for cell survival and cancer progression.
Interest in NQO1 has largely been sparked by its overexpression in many tumors, [6][7][8][9][18][19][20][21] at levels 5-200-folds above that in normal tissues. The elevated activity of NQO1 is closely associated with tumor progression, resistance to chemotherapy, and poor survival. 6,7,[18][19][20][21] Although some effort has been made to explore the small molecule inhibitor of NQO1 as an anticancer strategy, little success has been achieved. Recently, it was found that some specific substrates for NQO1 generate superoxide substances that may be used for cancer early diagnosis and treatment. 7 Drugs may be more effective when combined with PARP inhibitors for cancers with high levels of NQO1. 10,12 However, the significance of NOQ1 changes in HCC are poorly understood and controversial. [22][23][24][25][26][27] In the present study, we found that NQO1 expression was elevated significantly in HCC, significantly associated with hypomethylation of NQO1 and/ or mutations of NRF2 (a key transcription factor of NQO1), and high levels of NQO1 were associated with poor prognosis. Furthermore, NQO1 can significantly affect the growth and/or aggressiveness of HCC cells both in vitro and in vivo. An alternative mechanism for NQO1 in HCC is its action on amplifying ERK/p38-NRF2 signaling, supported by the evidence that combined block of NRF2 and ERK has a stronger inhibitory effect on HCC growth than any single block. Therefore, our results indicated that NQO1 plays a crucial role in HCC and may be a promising prognostic biomarker and therapeutic target.

| Bioinformatics analysis
Raw data from 3 microarray data sets (GSE14520, GSE36376 and GSE62944) were downloaded from the Gene Expression Omnibus (GEO) (https://www.ncbi.nlm.nih.gov/geo/). The expression profiles of the first 2 datasets were extracted using the fRMA package 28 and expression profiles of GSE62944, containing all RNA sequencing data from The Cancer Genome Atlas (TCGA), were processed from feature count data to obtain the log2-based values. Then, all 3 datasets were used to investigate the differential expression of NQO1 between cancerous and normal tissues, and the datasets of GSE14520 and TCGA-LIHC, annotated with survival and clinical information, were used to perform survival analysis. Associations between mRNA expression and copy number or DNA methylation of NQO1 were assessed using the processed data of TCGA-LIHC from the UCSC Xena online database (http://xena.ucsc.edu/). The associations between NQO1 mRNA expression and the mutation sites of NRF2 or KEAP1 were also examined using the processed data of TCGA-LIHC from cBioportal database (http://www.cbiop ortal.org/), and the annotation information of the constructed domains of NRF2 and KEAP1 were retrieved from previous reports. 29,30 We also examined the association between gene dependence and NQO1 expression status (high or low) across more than 17 000 genes among 325 cell lines (containing 14 HCC cells) using information from the CERES database. 31  infection (HBsAg and HBeAg), was also documented, as shown in Table S1. Follow-up information on patients with HCC was collected following a standard procedure as previously described. 32 In this study, the outcomes of interest were disease-free survival (DFS) and overall survival (OS). DFS was defined as the months from the date of surgery to the date of HCC relapse, and OS was defined as the months from the date of surgery to the date of death due to any disease. All patients provided written informed consent, and the study had also been approved by the hospital (Research ID: EHBHKY2019-01-001).

| Immunohistochemistry (IHC)
Tissue microarrays (TMAs) from the FFPE specimens of patients were commercially developed (Outdo Biotech). Rabbit polyclonal antibody against NQO1 (1:200, HPA007308, Sigma) was used according to the manufacturer's protocol. Immune staining was performed simultaneously on all arrays to eliminate interassay variation.
Levels of NQO1 staining were evaluated using an H-score method, as previously described, 33,34 and results were recorded as staining intensity (0, negative; 1, weakly positive; 2, moderately positive; 3, strongly positive) multiplied by the percent of tumor-positive area (0%-100%). Two observers (YY and JZ) blinded to the clinical status of the donor independently assessed the H-score of each tissue dot, and the scores of the 2 observers were averaged for analysis. Any controversial cases (defined as a difference in IHC scores more than 10% of the average score) were jointly re-evaluated until a consensus was reached.

| Cell lines, quantitative RT-PCR and western blot analysis
Human HCC cells (Huh7, Hep3B and HepG2) were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China).
All cell lines were authenticated using short tandem repeat (STR) analysis. Details regarding cell culture, quantitative RT-PCR and western blot analysis are included in Supplementary Material 2.

| Knockdown or overexpression of NQO1 in HCC cells
The control siRNAs and siRNAs targeting NQO1 transcripts, as shown in Table S2, were synthesized by Shanghai Invitrogen. Using  (Table S2) was designed and synthesized as previously described. 35 The template was subjected to PCR amplification with primers containing XhoI or EcoRI restriction sites. PCR products were purified and cloned into the pInducer 10 vector. For the process of overexpression of NQO1, the complete cDNA sequence of NQO1 (NCBI reference sequence: NM_000903) was first synthesized and confirmed commercially (Obio Technology). cDNA products were subcloned into the pENTR™ 3C entry vector, and the NQO1 sequence was then Gateway-recombined into the pInducer 20 vector.
Then, lentiviral particles were produced in HEK293T cells using the Lenti-X ™ HTX Packaging System (Clontech Laboratories, Inc) and titrated with HCC cells in medium to achieve optimal knockdown or overexpression of the target protein with minimal viral load. Finally, NQO1 expression in cells with NQO1 knocked down or overexpressed was verified by qPCR (primers shown in Table S2) and western blot analysis, respectively.

| In vivo tumor growth
Male BALB/c nude mice (5-6 wk old) were purchased from SLAC Laboratory Animal Co. Prior to being injected with HCC cells, the mice were acclimated in a pathogen-free facility. Then, constructed Hep3B cells with stable shNQO1 or shCont expression and constructed HepG2 with stable overexpression of NQO1 or controls were injected subcutaneously into the flanks of nude mice (5 × 10 6 cells/ mouse), respectively. These mice constituted the treatment and control groups (5 mice per group). Inhibitor of NRF2 signaling (ML385, 30 mg/kg) and/or the inhibitor of ERK signaling (SCH772984, 25 mg/ kg) were also used to treat the tumors. At 10 d after cell injection, tumor size was determined by external measurements every 3 or 4 d and tumor volume was calculated using the following formula: tumor volume = 1/2(length (mm) × width (mm)) 2 . The mice were sacrificed after 24-28 d, and tumor xenografts were harvested. All procedures were performed according to the National Research Council's Guide for the Care and Use of Laboratory Animals.

| Statistical analysis
To compare NQO1 expression between HCC and adjacent normal tissues, we used independent-sample t tests for non-paired samples and paired t tests for paired samples. Categorical data, such as sex and tumor differentiation grade were compared and analyzed using a χ 2 test or Mann-Whitney U test. In the survival analysis, patient subgroups, which were divided using maxstat software 36 according to NQO1 mRNA or protein levels, were evaluated using Kaplan-Meier curves and Cox proportional hazards models. A log-rank test was used to test the statistical significance of the Kaplan-Meier curves. Boolean analysis 37 was also used to explore the association between the status of NQO1 expression and the dependence scores of each of more than 17 000 genes across 325 cells based on the CERES database. 31 All statistical tests were

| Elevated NQO1 expression in HCC
The expression pattern of NQO1 in HCC was first explored with a bioinformatics method among 3 data sets (GSE14520, GSE36376, and TCGA-LIHC) with transcriptional profiling. As shown in Figure

| High levels of NQO1 expression in HCC predict poor survival
NQO1 protein immunostaining and survival information from the Shanghai cohort were used to investigate the prognostic value of NQO1. With the maxstat algorithm focused on DFS, we identified an IHC score of 75 as the optimal cut-value to define patient subgroups with high (n = 101) or low NQO1 protein expression levels (n = 195), which have the most significant discriminatory ability for determining survival. Using this cut-off value, NQO1 expression was found to be significantly associated with multiple nodes (P = .015), satellite lesions (P = .022) and microvessel invasion (P = .020), as shown in supplementary

| Elevated NQO1 expression is significantly associated with DNA hypomethylation of NQO1 and NRF2 mutations
Elevated NQO1 expression in HCC may be caused by genetic and/ or epigenetic variations of NQO1 and the aberrant activities of its upstream genes such as those in the KEAP1-NRF2 pathway. 29,30,38 Based on TCGA-LIHC data from the UCSC Xena website, we found

| NQO1 promotes the growth and aggressiveness of HCC cells
Considering the increased expression of NQO1 in HCC, we first investigated the effects of NQO1 knockdown on HCC cells. The NQO1 siRNA duplexes (siRNA) were tested in Huh7, Hep3B, and HepG2 cells, as shown in Figure 3A,B, and the result showed that NQO1 expression (mRNA and protein) in all cells was markedly inhibited by the siRNAs. Next, we assessed whether the proliferation,   Figure 3C-E. The inhibitory effects on HCC proliferation were also observed when we used the drug dicoumarin, which is an inhibitor of NQO1 ( Figure S3). Furthermore, we overexpressed NQO1 in 2 HCC cell lines (Huh7, Hep3B) ( Figure S4A,B) to verify the function of NQO1. As shown in Figure S4C-F, overexpression of NQO1 significantly promoted the growth and aggressiveness phenotypes of HCC cells. In addition, the knockdown or overexpression of NQO1 also resulted in delayed or accelerated wound healing of targeted HCC cells, respectively, as shown in Figure S5A,B. Therefore, our data consistently suggested that a high level of NQO1 expression promotes the growth and aggressiveness of HCC cells.

| NQO1 promotes the growth of HCC in an animal model
To investigate the effect of NQO1 on tumorigenicity in vivo, we first

Western Blot
to grow more slowly starting at approximately the 9th d ( Figure 4B) post-injection compared with the tumors in the control group.
Tumor size ( Figure 4B) was significantly smaller in NQO1 shRNAexpressing mice compared with the control animals from 9 d to 27 d post-injection, and the final weights obtained for the isolated tumors confirmed the significant reduction caused by NQO1 knockdown ( Figure 4C). Therefore, low NQO1 expression reduced tumor growth in vivo. We also constructed cells stably overexpressing NOQ1 in HepG2 and the corresponding control cells ( Figure 4D). Consistent with above results, higher NQO1 expression in HCC cells resulted in the rapid growth of the tumors in mice, which was sustained to the end of the experiment ( Figure 4E,F). Therefore, both knockdown and overexpression of NQO1 in HCC cells consistently supported that conclusion that NQO1 serves as an oncogene during HCC growth in vivo.

| NQO1 accelerates HCC growth by promoting ERK/p38-NRF2 signaling
To explore the pathways effected by NQO1 expression, we compared the differences in gene dependence scores of more 17 000 genes across 325 cell lines based on the CERES database considering the expression status of NQO1 (high or low expression).
Interestingly, we found that the cells most sensitive to NRF2 knockdown were almost all those with high levels of NQO1 expression, as shown in Figure 5A, and the pattern was reiterated in the remaining 13 HCC cells ( Figure 5B). This pattern suggested that the effect of NRF2 knockdown may depend on expression status of NQO1. Furthermore, we found that knockdown of NQO1 in HCC cells significantly reduced the levels of p-NRF2 ( Figure 5C) and vice versa ( Figure 5D), indicating that NQO1 may activate NRF2 via a positive feedback mechanism. Therefore, we explored the effect of NQO1 on the upstream molecules of NRF2 signaling, such as ERK and p38. Interestingly, the results showed that NQO1 knockdown in HCC cells significantly reduced the activation of p-ERK and p-p38 ( Figure 5C), and its overexpression increased the levels of these proteins ( Figure 5D). As we expected, the xenograft tumors with NQO1 overexpression had higher expression of p-ERK and p-NRF2 compared with that in those tumors with the control NQO1, as shown in Figure S6. The results above indicated that elevated NQO1 amplifies tNRF2 signaling with a positive feedback through activated ERK signaling.

| ERK and NRF2 inhibitors significantly inhibited HCC especially with NQO1-high
Furthermore, we compared the role of the block of NRF2 and ERK signaling with their inhibitors on the HCC with overexpressed NQO1 (NQO1-high) or control NQO1 (NQO1-low). As expected, we found that the xenografts tumors with NQO1-high had the faster growth compared with those tumors with NQO1-low ( Figure 6A). However, inhibition with SCH772984, ML385, or both generated relative more small tumor sizes in the groups with NQO1-high compared with that in the groups with NQO1-low ( Figure 6A), indicating more significant effects of these treatments in HCC with NQO1-high expression compared with HCC with NQO1-low. Combined block consistently generated the strongest inhibition among all treatment subgroups with either NQO1-high tumors or NQO1-low tumors ( Figure 6A).
Moreover, the result above was confirmed using the isolated final tumors from all subgroups, as shown in Figure 6B. However, the combined effect of the 2 inhibitors on HCC was more obvious in NQO1-high tumors compared with in the NQO1-low tumors ( Figure 6B). Next, we examined the expression status of NQO1, p-ERK and p-NRF2 in the isolated tumor specimens from the groups with NQO1-high or NQO1-low tumors, and the result showed that the combined inhibition resulted in more significantly reduced signaling of NQO1, p-ERK and p-NRF2 compared with that in the other subgroups ( Figure 6C). Furthermore, our colony assessed with HepG2 and Huh7 cells consistently showed that the combination of ML385 and SCH772984 generated the strongest inhibition effects among all tested subgroups ( Figure 6D), which supported a combined block of NRF2 and ERK signaling in HCC treatments. In addition, we also combined ML385 with SB202190 (an inhibitor for p38 signaling) for the joint effect on the colony formation of HCC, and found that the combination resulted in the strongest inhibition effects among all tested subgroups ( Figure S7), supporting that a block of p38 signaling sensitizes the role of NRF2 inhibition on HCC growth suppression.  tumor progression. In this study, we first explored the differences in NQO1 mRNA expression between HCC tissues and adjacent normal tissues using 3 publicly available data sets (GSE14520, GSE36376

| D ISCUSS I ON
and TCGA-LIHC), and consistently found that NQO1 expression was significantly elevated in cancerous lesions. This pattern of NQO1 expression was further confirmed at the protein level by IHC in samples from the Shanghai cohort. All these results were consistent with those from recent studies. 23,26 More importantly, we noticed that approximately 36%-45% of HCC specimens expressed high levels of NQO1, which was much higher than the 97.5% quartile of expression levels in the normal specimen groups, suggesting a strong HCC-specific expression pattern that will provide new opportunities for HCC drugs that are highly efficient and relatively nontoxic. 7,8 Recently, certain quinone compounds (such as mitomycin C, E09, RH1 and β-lapachone) have been shown to be cancer-specific drugs in tumors with high-NQO1, [6][7][8][9] and have provided greater efficiency of treatment when combined with other drugs, such as PARP1 inhibitors. 10,12 In addition, some quinone compounds can be marked with chemical fluorescence probes and used in imaging to diagnose cancers with high-NQO1 expression. 7 The prognostic value of NQO1 expression was first evaluated in the Shanghai cohort. With an optimal IHC score of NQO1 identified by the maxstat algorithm as a cut-off value, we classified the patients into 2 subgroups, namely, NQO1-high and NQO1-low. The  expression level in HCC. The accumulation of p62 in cancer has been verified to stabilize NRF2 and activate its target genes by disrupting the NRF2-KEAP1 interaction. 40 Mutations located in NRF2 and KEAP1 frequently exist in HCC, and will activate NRF2 irrespective of the P62 status. Therefore, the regulation mechanism of NRF2 by P62 in HCC will be more complex and needs further study.
The biological function of NQO1 in HCC was further investigated.
Knockdown of the gene significantly reduced the proliferation, colony formation, migration, and invasion abilities of 3 HCC cell lines.
These results were consistent with those in a recent report. 22, 26 We also constructed HCC cells with stable overexpression of NQO1 and evaluated their biological function. As expected, the overexpression of NQO1 in HCC cells promoted the proliferation and aggressiveness of HCC cells. Moreover, we also verified the suppression of tumor growth by NQO1 knockdown using mouse xenograft models.
These results in a functional assessment were consistent with clinical analysis, confirming the oncogenic function of NQO1 in HCC.
NQO1 is an important transcriptional target of NRF2 29,30,38 ; however, no study has investigated the effect of NQO1 on the activation of NRF2 transcription. Based on a publicly available database that contains the gene dependence scores of more than 17 000 genes across nearly 400 cells, we found that the sensitivities of cancer cells to the knockdown of NRF2 depended on the status of NQO1 expression and that the same pattern was observed in HCC cells.
The results strongly suggested that high levels of NQO1 expression are necessary for the cells that are sensitive to NRF2 knockdown.
Interestingly, we further found that NQO1 could elevate the level of p-NRF2 in HCC cells, and the results supported the conclusion that NQO1 plays a role in NRF2 activation via a positive feedback mechanism. Furthermore, in our study, the upstream activators of NRF2, such as ERK and p38, 38 were also found to be activated by NQO1, suggesting that ERK/p38 serves as an important mediator for the feedback activation of NRF2 due to NQO1. Interestingly, a recent study also reported that high NQO1 in HCC may activate ERK and AKT signaling, 26 partly consistent with our results. At the same time, enhanced ERK/p38 signaling also promotes cancer development and progression in combination with other signaling pathways.
The results above suggested that the combination of the inhibitors of NRF2 and ERK signaling perhaps more effectively blocked the growth of HCC with NQO1-high tumors. Therefore, we tested the hypothesis with NQO1-high or NQO1-low expressing tumors and found that the combined inhibition with ML385 and SCH772984 generated more obviously inhibited effects on the NQO1-high tumors, although the effects from subgroups with NQO1-low tumors were also significantly. Effects also were observed in the assays of colony formation of 2 cell lines. This evidence further supported the idea that NQO1 may be a key mediator for amplification of NRF2-ERK signaling in HCC.
In summary, we systematically investigated the clinical significance and biological role of NQO1 and provided some clues regarding its elevated expression and function in HCC. NQO1 may be a potential prognostic biomarker for the risk stratification of HCC patients. Significantly higher expression of NQO1 in HCC tissues compared with normal tissues provides many opportunities for early diagnosis and HCC-specific treatment with NQO1-substrate-related chemical probes or drugs. 7 Targeting NQO1 expression reduces the growth and aggressiveness of HCC, suggesting that an oncogenic role is played by NQO1 in HCC tumorigenesis. Notably, NQO1 may amplify ERK/p38-NRF2 signaling, supported by the strongest inhibition effects derived from combined block of NRF2 and ERK signaling among all tested subgroups. We conclude that NQO1 may be a promising prognostic and predictive biomarker with potential use as a therapeutic target in HCC.

E TH N I C A L A PPROVA L
The ethics approval was already obtained by authors.