Pathophysiological Potentials of NRF3-Regulated Transcriptional Axes in Protein and Lipid Homeostasis

NRF3 (NFE2L3) belongs to the CNC-basic leucine zipper transcription factor family. An NRF3 homolog, NRF1 (NFE2L1), induces the expression of proteasome-related genes in response to proteasome inhibition. Another homolog, NRF2 (NFE2L2), induces the expression of genes related to antioxidant responses and encodes metabolic enzymes in response to oxidative stress. Dysfunction of each homolog causes several diseases, such as neurodegenerative diseases and cancer development. However, NRF3 target genes and their biological roles remain unknown. This review summarizes our recent reports that showed NRF3-regulated transcriptional axes for protein and lipid homeostasis. NRF3 induces the gene expression of POMP for 20S proteasome assembly and CPEB3 for NRF1 translational repression, inhibiting tumor suppression responses, including cell-cycle arrest and apoptosis, with resistance to a proteasome inhibitor anticancer agent bortezomib. NRF3 also promotes mevalonate biosynthesis by inducing SREBP2 and HMGCR gene expression, and reduces the intracellular levels of neural fatty acids by inducing GGPS1 gene expression. In parallel, NRF3 induces macropinocytosis for cholesterol uptake by inducing RAB5 gene expression. Finally, this review mentions not only the pathophysiological aspects of these NRF3-regulated axes for cancer cell growth and anti-obesity potential but also their possible role in obesity-induced cancer development.


POMP, a 20S Proteasome Assembly Factor
The 26S proteasome is essential for ubiquitin-dependent protein degradation and consists of two subcomplexes: a 20S proteasome and a 19S-regulatory particle (RP) [34]. Several chaperones strictly coordinate the assembly of 20S proteasome and 19S-RP [35,36]. NRF1 induces the expression of almost all proteasome-related genes required for 26S proteasome [7]. Meanwhile, NRF3 does not affect the expression of almost all proteasomerelated genes, but induces the gene expression of POMP [31], a chaperone of the 20S proteasome assembly [37]. ChIP experiments showed an ARE-like sequence (TGAGCG-GCG) near the transcription start site of the POMP gene as the NRF3 binding region [31] ( Figure 1A). Furthermore, POMP-ARE mutations using CRISPR/Cas9-based genome editing reduced not only NRF3 recruitment on POMP-ARE but also POMP gene expression induced by NRF3 [31]. Proteasome activity assays using a fluorogenic substrate showed that NRF3 increases the amount and activity of 20S proteasome [31] ( Figure 1B). These results provided direct evidence that POMP is an NRF3 target gene for enhancing the 20S proteasome activity.

NRF3-POMP-20S Proteasome Assembly Axis for Cancer Development
The 20S proteasome, a homodimer of a half-mer proteasome composed of an outer αring and an inner β-ring, contains proteolytic sites with different specificities: chymotrypsin-, caspase-, and trypsin-like activities. Meanwhile, the 20S proteasome lacks the 19S-RP that selects and unfolds ubiquitin substrates. Previous studies suggested that the 20S proteasome contributes to the ubiquitin-independent degradation of several tumor suppressor proteins, such as p53 and retinoblastoma (Rb) [38]. Surprisingly, NRF3 decreases p53 and Rb proteins without alteration of their mRNA levels under treatment with a ubiquitin-activating enzyme E1 inhibitor TAK-243, which inhibits 26S proteasome-mediated protein degradation by covalently binding with ubiquitin proteins [39] ( Figure 1B). Furthermore, POMP-ARE mutation impairs the NRF3-mediated reduction in p53 and Rb protein, irrespective of TAK-243 treatment. More importantly, p53 and Rb inhibit cancer cell proliferation by inducing cell-cycle arrest or apoptosis in response to DNA damage [40]. NRF3 suppresses the expression of p53 target genes, including the cell-cycle inhibitory effector gene p21 [41] and the proapoptotic gene PUMA (p53 upregulated modulator of apoptosis) [42]. NRF3 further inhibits p53-dependent cell-cycle arrest and apoptosis induction, leading to continuous cancer cell growth [31] ( Figure 1B). These results indicated that the NRF3-POMP-20S proteasome assembly axis affects the ubiquitin-independent degradation of endogenous p53 and Rb proteins. and metastasis, whereas POMP-ARE mutation inhibits this tumor burde portantly, clinical analyses indicated a negative correlation between POM levels and the survival rates of patients with colorectal adenocarcinoma highly expressed [31]. These insights shed light on the crucial functi POMP-20S proteasome axis on cancer development, by inhibiting tumor nals of p53 and Rb through ubiquitin-independent degradation. The up axis also confers resistance to a BTZ-type proteasome inhibitor [44].

CPEB3, a Translational Repressor of NRF1
NRF3 increases 20S proteasome activity through POMP expressi NRF1 maintains 26S proteasome activity by inducing the expression o teasome-related genes under proteasome inhibition [7], implying the bio of NRF1 and NRF3 for proteasome activity. In fact, the double knockdo NRF3 impairs basal proteasome activity in living cells [32]. Compared to down of NRF1 or NRF3, double knockdown of NRF1 and NRF3 redu teasome-related genes, including PSMB3, PSMB7, PSMC2, PSMD3, PSM POMP [32]. ChIP experiments showed an ARE sequence near the trans of each gene. These results indicated that NRF1 and NRF3 complemen expression of several proteasome-related genes to maintain the proteaso  The proteasome is a target for cancer chemotherapy, and several proteasome inhibitors have been developed as anticancer agents. Among proteasome inhibitor anticancer agents, bortezomib (BTZ) inhibits both 20S and 26S proteolytic activities by binding to catalytic sites within the 20S proteasome [43]. Expectedly, upregulation of the NRF3-POMP-20S proteasome axis confers resistance to BTZ [31] ( Figure 1B). Furthermore, xenograft and hepatic metastatic mouse models showed that NRF3 increases tumorigenesis and metastasis, whereas POMP-ARE mutation inhibits this tumor burden [31]. More importantly, clinical analyses indicated a negative correlation between POMP/NRF3 mRNA levels and the survival rates of patients with colorectal adenocarcinoma, where NRF3 is highly expressed [31]. These insights shed light on the crucial function of the NRF3-POMP-20S proteasome axis on cancer development, by inhibiting tumor suppression signals of p53 and Rb through ubiquitin-independent degradation. The upregulation of the axis also confers resistance to a BTZ-type proteasome inhibitor [44].

CPEB3, a Translational Repressor of NRF1
NRF3 increases 20S proteasome activity through POMP expression [31], whereas NRF1 maintains 26S proteasome activity by inducing the expression of almost all proteasomerelated genes under proteasome inhibition [7], implying the biological relevance of NRF1 and NRF3 for proteasome activity. In fact, the double knockdown of NRF1 and NRF3 impairs basal proteasome activity in living cells [32]. Compared to the single knockdown of NRF1 or NRF3, double knockdown of NRF1 and NRF3 reduces several proteasome-related genes, including PSMB3, PSMB7, PSMC2, PSMD3, PSMG2, PSMG3, and POMP [32]. ChIP experiments showed an ARE sequence near the transcription start site of each gene. These results indicated that NRF1 and NRF3 complementarily induce the expression of several proteasome-related genes to maintain the proteasome activity [32].
Interestingly, NRF3 represses the translation of NRF1 proteins by decreasing the amount of NRF1 mRNA in polysomes, although NRF3 does not only affect the levels of NRF1 mRNA but also the degradation of NRF1 proteins [32]. Gene expression analysis identified CPEB3 as the candidate NRF3 target gene for this NRF1 translation repression [32] ( Figure 2A). CPEB family proteins recognize a CPEB recognition motif (5 -UUUUA-3 , CPE) in the 3 -untranslated region (UTR) of a target gene for translation regulation [45]. CPEB3 interacts with the NRF1-3 -UTR which contains five CPEs, decreasing NRF1 protein levels and the amount of NRF1 mRNA in polysomes [32] (Figure 2A). Meanwhile, NRF3 deficiency or CPE mutation of the NRF1-3 -UTR increases NRF1 translation [32] ( Figure 2B). These results indicate that NRF3 directly induces CPEB3 gene expression, and then CPEB3 inhibits ribosome recruitment to NRF1 mRNA, resulting in the repression of NRF1 translation. Interestingly, NRF3 represses the translation of NRF1 proteins by decreasing the amount of NRF1 mRNA in polysomes, although NRF3 does not only affect the levels of NRF1 mRNA but also the degradation of NRF1 proteins [32]. Gene expression analysis identified CPEB3 as the candidate NRF3 target gene for this NRF1 translation repression [32] (Figure 2A). CPEB family proteins recognize a CPEB recognition motif (5′-UUUUA-3′, CPE) in the 3′-untranslated region (UTR) of a target gene for translation regulation [45]. CPEB3 interacts with the NRF1-3′-UTR which contains five CPEs, decreasing NRF1 protein levels and the amount of NRF1 mRNA in polysomes [32] (Figure 2A). Meanwhile, NRF3 deficiency or CPE mutation of the NRF1-3′-UTR increases NRF1 translation [32] ( Figure 2B). These results indicate that NRF3 directly induces CPEB3 gene expression, and then CPEB3 inhibits ribosome recruitment to NRF1 mRNA, resulting in the repression of NRF1 translation.

Clinical Significance of the NRF3-CPEB3-NRF1 Translational Repression Axis
In NRF3-deficienct cells, CPEB3 represses NRF1 translation and then reduces the expression levels of PSMB3, PSMB7, PSMC2, PSMG2, and POMP genes, resulting in the suppression of 26S proteasome activity [32]. CPEB3 also confers resistance to BTZ in NRF3-deficient cells [32]. Furthermore, colorectal cancer patients with higher CPEB3/NRF3-expressing tumors exhibited shorter overall survival rates, but higher CPEB3/NRF1 expression was not associated with poor prognosis [32]. These results suggested that the NRF3-CPEB3-NRF1 translational repression axis is involved in cancer development by shunting ubiquitin-dependent protein degradation through the NRF1-26S proteasome regulatory axis to ubiquitin-independent protein degradation through the POMP-20S proteasome axis (Figure 2).

NRF3-SREBP2-HMGCR Axis for Mevalonate Biosynthesis
Lipids, such as cholesterol and fatty acids, influence cell signaling, energy storage, and membrane formation. SREBPs are membrane-bound transcription factors crucial for lipid metabolism [46]. In response to cholesterol depletion, SREBP1 and SREBP2 proteins are cleaved in the Golgi apparatus, resulting in the translocation to the nucleus. SREBP1 induces the gene expression of enzymes required for fatty acid biosynthesis and adipocyte differentiation, whereas SREBP2 induces the gene expression of enzymes required for mevalonate/cholesterol biosynthesis.

Clinical Significance of the NRF3-CPEB3-NRF1 Translational Repression Axis
In NRF3-deficienct cells, CPEB3 represses NRF1 translation and then reduces the expression levels of PSMB3, PSMB7, PSMC2, PSMG2, and POMP genes, resulting in the suppression of 26S proteasome activity [32]. CPEB3 also confers resistance to BTZ in NRF3-deficient cells [32]. Furthermore, colorectal cancer patients with higher CPEB3/NRF3expressing tumors exhibited shorter overall survival rates, but higher CPEB3/NRF1 expression was not associated with poor prognosis [32]. These results suggested that the NRF3-CPEB3-NRF1 translational repression axis is involved in cancer development by shunting ubiquitin-dependent protein degradation through the NRF1-26S proteasome regulatory axis to ubiquitin-independent protein degradation through the POMP-20S proteasome axis (Figure 2).

NRF3-SREBP2-HMGCR Axis for Mevalonate Biosynthesis
Lipids, such as cholesterol and fatty acids, influence cell signaling, energy storage, and membrane formation. SREBPs are membrane-bound transcription factors crucial for lipid metabolism [46]. In response to cholesterol depletion, SREBP1 and SREBP2 proteins are cleaved in the Golgi apparatus, resulting in the translocation to the nucleus. SREBP1 induces the gene expression of enzymes required for fatty acid biosynthesis and adipocyte differentiation, whereas SREBP2 induces the gene expression of enzymes required for mevalonate/cholesterol biosynthesis.

NRF3-GGPS1-GGPP Production Axis for Lipogenesis Inhibition
Interestingly, NRF3 does not affect the intracellular levels of chole increases the expression levels and enzymatic activity of HMGCR. M duces that of lanosterol [33]. Lanosterol is not only a precursor of ch downstream metabolite of farnesyl pyrophosphate, which is also meta a reaction catalyzed by GGPS1 (Figure 3, (2)). Furthermore, NRF3 dire expression [33], implying that NRF3 reprograms cholesterol biogenes of GGPP rather than lanosterol. GGPP suppresses SREBP1-dependen thesis and intracellular lipid accumulation [49,50]. In fact, DNA m showed a negative correlation between the expression levels of NRF3 to fatty acid metabolism [33]. More directly, intracellular levels of ne creased by NRF3 knockdown and reduced by GGPP treatment [33] ( sistently, a few body mass index-associated genomic loci near the NR identified previously [51,52]. These results indicated the potential

NRF3-GGPS1-GGPP Production Axis for Lipogenesis Inhibition
Interestingly, NRF3 does not affect the intracellular levels of cholesterol, even if NRF3 increases the expression levels and enzymatic activity of HMGCR. Meanwhile, NRF3 reduces that of lanosterol [33]. Lanosterol is not only a precursor of cholesterol but also a downstream metabolite of farnesyl pyrophosphate, which is also metabolized to GGPP in a reaction catalyzed by GGPS1 (Figure 3(2)). Furthermore, NRF3 directly induces GGPS1 expression [33], implying that NRF3 reprograms cholesterol biogenesis to the production of GGPP rather than lanosterol. GGPP suppresses SREBP1-dependent fatty acid biosynthesis and intracellular lipid accumulation [49,50]. In fact, DNA microarray analysis showed a negative correlation between the expression levels of NRF3 and genes related to fatty acid metabolism [33]. More directly, intracellular levels of neutral lipids are increased by NRF3 knockdown and reduced by GGPP treatment [33] (Figure 3(2)). Consistently, a few body mass index-associated genomic loci near the NRF3 gene have been identified previously [51,52]. These results indicated the potential role of the NRF3-GGPS1-GGPP production axis (Figure 3(2)).

NRF3-RAB5-Macropincytosis Induction Axis for Cholesterol Uptake
Intracellular cholesterol is derived from not only de novo biosynthesis but also endocytic uptake [53]. NRF3 decreases lanosterol levels, but it does not change cholesterol levels in cells [33] (Figure 3(3)), implying that NRF3 enhances endocytosis for cholesterol uptake to compensate for the potential depletion in cholesterol levels following lanosterol reduction. Low-density lipoprotein receptor (LDLR) is a key endocytosis regulator of LDL [54]. However, NRF3 does not induce LDLR gene expression. Meanwhile, NRF3 induces the gene expression of three isoforms of RAB5A, RAB5B, and RAB5C [33] (Figure 3(3)). These RAB5 proteins act as early endocytosis regulators [55] and are involved in macropinocytosis, a bulk and fluid-phase endocytosis process [56]. NRF3 further increases posttranslational prenylation RAB5 proteins [33] essential for proper localization and function in membranes [57]. The previous section (Figure 3(2)) described that NRF3 induces the production of GGPP, which functions as a required substrate for protein prenylation [58]. Altogether, NRF3 enhances RAB5-mediated endocytosis rather than LDLR-mediated endocytosis for cholesterol uptake through GGPP production ( Figure 3, (3)). NRF3 enhances the uptake of fluorogenic LDL in a RAB5-dependent manner. Moreover, NRF3-enhanced uptake of other fluorogenic macropinocytosis indicators based on 70 kDa dextran and bovine serum albumin is abolished by treatment with 5-(N-ethyl-N-isopropyl)amiloride [33], also known as an inhibitor of macropinocytosis and a selective blocker of Na + /H + exchanger [59]. Similarly, NRF3 enhances the uptake of two fluorogenic cholesterols through macropinocytosis [33]. These results indicated the crucial function of the NRF3-RAB5-macropinocysosis induction (NRF3-RAB5-macropinocysosis) axis on cholesterol uptake (Figure 3(3)). The next section discusses the pathophysiological potential of this axis.

Concluding Remarks
Increased ubiquitin-independent proteasomal activity causes tumor growth, metastasis, and resistance to the proteasome inhibitor BTZ. NRF3 induces POMP expression, leading to ubiquitin-independent protein degradation of tumor suppressors Rb and p53 ( Figure 1). Upregulation of the POMP-20S proteasome axis further results in poor prognosis of colorectal cancer patients [31]. NRF3 also induces the expression of proteasome-related genes in parallel with NRF1 translational repression by inducing CPEB3 expression [32] ( Figure 2A). If the NRF3 gene is deficient, NRF1 escapes from CPEB3-mediated translational repression, and complementarily plays a transcriptional role for the robust maintenance of basal proteasome activity in cancer cells ( Figure 2B). Although NRF3 shares several target genes with NRF1 and NRF2 on ARE [30,60], this review showed the translation-mediated crosstalk between NRF3 and NRF1.
Nrf1 is ubiquitously expressed in normal tissues, and Nrf1 knockout mice suffer from embryonic lethality [8]. Meanwhile, Nrf3 expression levels are low, except in several mouse tissues, such as the placenta [5,6], and Nrf3 knockout mice do not exhibit any obvious abnormalities under normal physiological conditions [18][19][20]. However, NRF3 is highly expressed in many cancer cells [31], implying that the proteasome in cancer or normal cells is maintained through the CPEB3-NRF1 axis or the negative feedback regulation of NRF1. Higher CPEB3/NRF3 expression, but not higher CPEB3/NRF1 expression, is associated with poor prognosis of cancer patients [32]. Therefore, NRF1 maintains a 26S proteasome activity for normal development, whereas NRF3 alternatively maintains 20S proteasome activity for cancer development through both POMP-20S proteasome and CPEB3-NRF1 axes.
Furthermore, NRF3 is involved in lipid metabolism through three regulatory axes [33] (Figure 3): (1) NRF3 induces the gene expression of SREBP2 required for cholesterol biosynthesis through the mevalonate pathway. NRF3 also leads to SREBP2 activation through direct induction of gene expression. NRF3 and SREBP2 synergistically induce HMGCR expression and the following mevalonate biosynthesis. (2) NRF3 then upregulates GGPS1-mediated GGPP production for lipogenesis inhibition. (3) In parallel, NRF3 confers RAB5-mediated induction of macropinocytosis for cholesterol uptake. This gene expression is induced in colon and/or rectal tissue of newly generated NRF3-transgenic mice [33]. Dietary cholesterol in the blood is absorbed in the intestine [61], and its dysregulation is associated with obesity, resulting in an increased risk of cardiovascular diseases (CVD) and colorectal cancer [62,63]. The gut microbiota has been identified as a CVD risk factor and regulates host cholesterol homeostasis [64][65][66], suggesting the pathophysiological potential of the NRF3-regulated host lipid metabolism to the gut-heart connection through the gut microbiota.
Epidemiological studies have associated obesity with a range of cancers [67,68]. Furthermore, many efforts have been made to identify the key factor for obesity-induced cancer, including insulin resistance, increased steroid hormones and adipokine, and aberrant inflammation [69]. However, these findings implied possible opposite roles for NRF3 in obesity-induced cancer development ( Figure 4): NRF3-SREBP2-HMGCR and the following NRF3-GGPS1-GGPS axes regulate lipid homeostasis and confer resistance to obesity through lipogenesis inhibition, while the NRF3-POMP-20S proteasome and NRF3-CPEB3-NRF1 axes regulate protein homeostasis and confer ubiquitin-independent protein degradation for continuous cancer cell growth. This review further showed that NRF3 maintains cholesterol homeostasis through the RAB5-macropinocytosis axis ( Figure 3, (3)). Interestingly, macropinocytosis is associated with obesity-related disorders, such as increased diabetic mouse macrophages and chronic inflammation [70,71]. Furthermore, NRF2 induces macropinocytosis and contributes to the escape of autophagy-deficient cancer cells from metabolic decline and anticancer drugs, such as gemcitabine and doxorubicin, which target the anabolic dependencies of cancer cells [72,73]. These insights implied the possibility that the NRF3-RAB5-macropinocytosis axis paradoxically interferes with obesity-induced cancer development through attenuation of obesity-induced inflammation and resistance to therapy targeting cancer anabolism (Figure 4). A big issue of the NRF3 study is identifying the endogenous cue of NRF3 activation, although NRF3 is experimentally activated by treatment with a proteasome inhibitor. Recently, NRF1 senses cholesterol levels in the ER membrane through the cholesterol recognition amino acid consensus motif domain (CRAC), and it is activated in response to cholesterol depletion [74]. Because the CRAC domain is conserved in NRF3 proteins [27], NRF3 acts as a cholesterol sensor in the ER membrane similarly to NRF1.