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Int J Biol Sci 2024; 20(15):6181-6206. doi:10.7150/ijbs.103187 This issue Cite

Review

The Crosstalk between Autophagy and Nrf2 Signaling in Cancer: from Biology to Clinical Applications

Chan Shan^1,2 , Yuan Wang², Yin Wang^1,2

1. Key Laboratory of Maternal & Fetal Medicine of National Health Commission of China, Shandong Provincial Maternal and Child Health Care Hospital Affiliated to Qingdao University, Jinan, 250014, China.
2. Institute of Translational Medicine, College of Medicine, Qingdao University, Qingdao 266021, China.

✉ Corresponding authors: E-mail: shanchan2019com; wangyinedu.cn.

Abstract

Autophagy is a catabolic process that has been conserved throughout evolution, serving to degrade and recycle cellular components and damaged organelles. Autophagy is activated under various stress conditions, such as nutrient deprivation, viral infections, and genotoxic stress, and operates in conjunction with other stress response pathways to mitigate oxidative damage and maintain cellular homeostasis. One such pathway is the Nrf2-Keap1-ARE signaling axis, which functions as an intrinsic antioxidant defense mechanism and has been implicated in cancer chemoprevention, tumor progression, and drug resistance. Recent research has identified a link between impaired autophagy, mediated by the autophagy receptor protein p62, and the activation of the Nrf2 pathway. Specifically, p62 facilitates Keap1 degradation through selective autophagy, leading to the translocation of Nrf2 into the nucleus, where it transcriptionally activates downstream antioxidant enzyme expression, thus safeguarding cells from oxidative stress. Furthermore, Nrf2 regulates p62 transcription, so a positive feedback loop involving p62, Keap1, and Nrf2 is established, which amplifies the protective effects on cells. This paper aims to provide a comprehensive review of the roles of Nrf2 and autophagy in cancer progression, the regulatory interactions between the Nrf2 pathway and autophagy, and the potential applications of the Nrf2-autophagy signaling axis in cancer therapy.

Keywords: Autophagy, Nrf2 signaling, Crosstalk, Cancer biology, Cancer therapy

1. Introduction

Autophagy is a cellular process in which eukaryotic cells degrade cytoplasmic proteins and damaged organelles through the action of lysosomes under the regulation of autophagy-related genes (ATGs) [1]. This process serves to prevent cell damage, promote cell survival in the absence of nutrients, and respond to cytotoxic stimuli. Autophagy plays a crucial role in both normal physiological functions and pathological conditions within the body [2]. Under physiological conditions, autophagy functions to eliminate aging and damaged biological macromolecules and organelles, thereby preserving the normal biological functions of cells. During metabolic stress states, autophagy facilitates the degradation of aging and damaged biological components, providing cells with the necessary energy sources and materials for reconstruction in harsh environments. Importantly, dysregulation of autophagy has been implicated in the pathogenesis and progression of various diseases, including autoimmune disorders, neurological conditions, cardiovascular and cerebrovascular ailments, metabolic diabetes, and notably, cancer [3]. Current research indicates that autophagy plays a dual role in cancer, influenced by factors such as tumor stage, specific oncogenic mutations, and the surrounding cellular environment. Autophagy is widely recognized to be a suppressor of tumorigenesis; however, in established tumors, it facilitates the unregulated proliferation of cancer cells and their increased metabolic activity, resulting in dependence on autophagy for tumor maintenance [4, 5]. Moreover, autophagy plays a crucial role not only in cancer cells but also in adjacent stromal cells and the tumor microenvironment (TME), which are intricately linked to tumor growth and drug resistance [6, 7]. Therefore, comprehending the molecular mechanisms involved in autophagic control has significant potential for devising efficacious approaches for the prevention and treatment of cancer.

Nuclear factor (erythroid derived-2)-like 2 (Nrf2), a pivotal transcription factor encoded by NFE2L2 that is responsible for regulating antioxidant stress, assumes a critical function in triggering the body's antioxidant response [8]. In normal physiological conditions, the interaction between Nrf2 and Kelch-like ECH-associated protein 1 (Keap1) facilitates the ubiquitination of Nrf2, leading to its degradation by the 26S proteasome [9]. However, during oxidative stress, Keap1 undergoes conformational changes that disrupt the ubiquitination process of Nrf2. Consequently, Nrf2 translocates to the nucleus, where it binds to antioxidant response elements (AREs) and initiates the transcription of various cytoprotective genes, such as NAD(P)H quinone dehydrogenase 1 (NQO1), heme oxygenase 1, superoxide dismutase, glutamate cysteine ligase (GCL), and catalase.

Over the past decade, numerous studies have provided evidence of the crosstalk between autophagy and Nrf2 signaling, which is facilitated by the autophagy adaptor p62/sequestosome 1 [10-13]. In this scenario, various factors, such as oxidative stress, metabolic disorders, diseases, and autophagy inhibition, trigger the transcription of p62, leading to its accumulation in the cytoplasm. The aggregation of p62 then directly interacts with Keap1, resulting in the degradation of Keap1 through a selective autophagic pathway, which ultimately leads to the continuous activation of Nrf2 and the upregulation of genes encoding antioxidant enzymes. Nuclear Nrf2 also promotes the overexpression of the p62 gene, establishing a p62-Keap1-Nrf2-positive feedback loop. An increasing body of research has substantiated the pivotal significance of this feedback loop in the progression of cancer [12, 13]. In this review, we specifically focus on the regulatory functions of autophagy and Nrf2 signaling, as well as their crosstalk, in the progression of cancer. We also discuss their potential applications in cancer therapy.

2. The role of autophagy in cancer

2.1. Molecular mechanisms of autophagy

Autophagy serves as the fundamental process for the degradation and recycling of cellular components [1]. It involves the breakdown of impaired, aged, or dysfunctional organelles to promptly supply energy and essential resources for the maintenance of normal cellular activities. The autophagy process primarily consists of five distinct stages: initiation of autophagy, vesicle nucleation, vesicle elongation, fusion of autophagosomes with lysosomes, and ultimately the degradation of the enclosed contents (Figure 1).

Autophagy is triggered by both extracellular stimulation (e.g., nutrient shortage, hypoxia-ischemia, and growth factor induction) and intracellular factors (e.g., metabolic stress, organelle aging, protein misfolding, and DNA damage) [14]. The initiation of autophagy is regulated by the protein complex of the target of rapamycin (TOR) kinase. Specifically, the mammalian TOR (mTOR) is activated in response to cellular stress, which, in turn, activates downstream serine/threonine UNC-51-like kinases 1 and 2 (ULK1 and ULK2). ULK1 and ULK2 then form a complex with the 200 kDa family-interacting proteins ATG101 (also known as FIP200, a mammalian homolog of ATG17) and ATG13, thereby initiating the autophagy process.

Two distinct ubiquitin-like conjugation systems are involved in the formation and maturation of autophagosomes: the ATG5-ATG12 complex conjugated with ATG16L1 and microtubule-associated protein 1 light chain 3 (MAP1LC3, also known as LC3) conjugated with lipid phosphatidylethanolamine (PE) [1]. Upon activation by upstream signals, ATG12 and ATG5 undergo sequential conjugation facilitated by ATG7 and ATG10, ultimately leading to the formation of a complex with ATG16L, which plays a critical role in the assembly of autophagosomes. Following translation, pro-LC3 is immediately cleaved by ATG4 at the C-terminal peptide, exposing a glycine residue site and resulting in the formation of LC3 A, which is then distributed throughout the cytoplasm. Through the action of the E1-like enzyme ATG7, the E2-like enzyme ATG3, and the E3-like enzyme ATG12-ATG5-ATG16L, LC3 A covalently binds with PE to generate LC3-PE, which is also referred to as LC3 B. The lipophilicity of PE facilitates the binding of LC3 to the autophagosome membrane, thereby mediating membrane elongation and the selective degradation of cellular components. LC3 B has been widely used as a marker to monitor the autophagy process [15-17].

The fusion of autophagosomes and lysosomes is a critical process for cells to carry out autophagy, which involves the breakdown and recycling of cellular waste [18]. Specifically, autophagosomes initially fuse with endosomes to create late endosomes and then form autolysosomes through fusion with lysosomes. The process of autophagosome-lysosome fusion begins with the merging of the outer membrane of autophagosomes with lysosomes and concludes with the degradation of the inner membrane of autophagosomes and the release of their contents into the lysosomal lumen. Several functional proteins play a role in the fusion of autophagosomes and lysosomes, including small GTPases, RAB proteins (RAB5 and RAB7), the UV radiation resistance-associated gene protein (UVRAG), and SNARE proteins (VAMP8 and STX17) [1].

 Figure 1 

Autophagy is a cellular process that is activated in response to internal stressors, such as hypoxia or nutrient deprivation. Autophagy initiation involves activation of the class III phosphatidylinositol 3-kinase (PI3K) complex, which is composed of Beclin 1, AMBRA1, ATG14L, VPS15, and VPS34. This activation is facilitated by the ULK1 complex, which comprises FIP200, ATG13, and ATG101. Activation of the class III PI3K complex initiates vesicle nucleation, which serves as a scaffold for membrane expansion. During the membrane expansion phase, two ubiquitin-like conjugation systems play a role: the ATG5-ATG12 complex combined with ATG16L1 and LC3 combined with PE. These systems are responsible for elongating and forming autophagosomes. The final crucial step involves the fusion of autophagosomes with lysosomes to create autophagic lysosomes, where biological macromolecules are degraded and recycled.

Following fusion, the cellular material transported by autolysosomes undergoes degradation by hydrolases in the lysosomes, resulting in the production of substrates that can be circulated and metabolized [3]. Selective autophagy exhibits distinct characteristics compared to nonselective autophagy. For instance, cargo items must possess an ubiquitination label and attach to the inner surface of the phagophore via ATG8s, thereby forming a large aggregate that facilitates phagophore formation [19].

In addition, mitophagy, the process by which damaged mitochondria are cleared, is triggered in various situations such as hypoxia, mitochondrial DNA damage, oxidative stress, and mitochondrial depolarization [20]. In mammals, the classical pathways that mediate mitophagy can be categorized into two main pathways: ubiquitin dependent pathways and non-ubiquitin dependent. Among these, the PTEN-induced putative kinase 1 (PINK1)/PARKIN signaling pathway has been extensively studied [21]. Under normal conditions, PINK1 enters the inner mitochondrial membrane through the TOM/TIM complex and is then cleaved by proteases in the inner membrane and subsequently degraded by proteasomes. When mitochondria are damaged, the mitochondrial membrane potential decreases, preventing PINK1 from entering the inner membrane and causing its accumulation on the outer membrane of mitochondria. Subsequently, the receptor protein for autophagy accumulates on the outer membrane of mitochondria. Mitochondria are then transported to the autophagosome with the assistance of LC3 and ultimately degraded by lysosomes.

Together, elucidating the operational mechanisms of individual stages within the autophagy process and delineating the regulation and interplay of the pivotal receptor proteins involved in this process are essential. This is important for enhancing the understanding of the quality control mechanism in autophagy and cellular homeostasis.

2.2. Bipolar properties of autophagy in cancer progression

The intricate involvement of autophagy in the regulation of cancer development has been the subject of extensive research. The impact of autophagy on the fate of cancer cells varies depending on factors such as the type of cancer, its stage, and the genetic background of the individual [22]. On the one hand, autophagy functions as a quality control mechanism that preserves genomic stability by removing metabolic waste, damaged organelles, and other cellular components [23-25]. Based on this perspective, autophagy protects cells from damage induced by various microenvironmental stimuli, inflammatory responses, and nutritional imbalances, thereby acting as a tumor suppressor in the early stages of cancer [4, 26, 27]. On the other hand, as cancer progresses, cancer cells exploit autophagy as a protective and defensive mechanism, allowing cancer cells to sustain their metabolism, growth, and survival, ultimately facilitating cancer progression, metastasis, and the development of drug resistance [5, 28, 29]. The dual roles of autophagy in Table 1.

Evidence and suppressive functions of autophagy in tumorigenesis

As previously stated, autophagy serves as a mechanism employed by organisms to maintain metabolic, protein, and organelle integrity and to eliminate impaired proteins and organelles that have accumulated during periods of stress [1]. Consequently, autophagy plays a crucial role in preventing the development of tumors, especially in the early stages of tumorigenesis [4, 26, 27]. A growing body of research has revealed that defective autophagy promotes tumorigenesis, typically because of the deletion or impairment of specific ATGs within a particular tumor. An illustrative example is the tumor suppressor gene Beclin 1, which is implicated in the initiation of autophagy in the context of cancer. Monoallelic loss of Beclin 1 is sufficient to induce the development of breast cancer, ovarian cancer, B cell lymphoma, melanoma, and other malignancies [30-32]. However, the upregulation of Beclin 1 is associated with enhanced autophagic flux, a reduction in tumorigenesis, and the inhibition of malignant characteristics [33]. Additionally, dysfunctions in ATGs, such as ATG12, ATG14, and ATG5, can affect the autophagy process, thereby influencing the initiation and progression of cancer, as well as response to chemotherapy. For instance, in head and neck squamous cell carcinoma (HNSCC), the depletion of ATG12 leads to a diminished autophagic flux, which subsequently inhibits the proliferation of cancer cells and enhances their susceptibility to chemotherapeutic agents [34]. In the context of oral cancer, ATG5-dependent autophagy plays a crucial role in preserving tumor stemness, facilitating self-renewal, and conferring cisplatin resistance in oral CD44⁺ cells [35]. Overall, the downregulation and deletion of ATGs have been shown to increase the incidence of malignancies, indicating that intact autophagy plays a critical role in tumor suppression.

The PI3K/AKT/mTORC1 signaling pathway and its associated proteins also play a crucial role in the regulation of autophagy and tumorigenesis [36]. Autophagy can be inhibited by PI3K inhibitors, such as wortmannin and 3-Methylamphetamine, while rapamycin, an mTORC1 inhibitor, effectively induces autophagy. AKT also participates in the regulation of autophagy through its interaction with various proteins, including p62, Forkhead Box O3, ULK1, Phafin2, and Beclin 1. Research has demonstrated that the presence of chronic inflammation can elevate the likelihood of developing cancer, while autophagy serves as a fundamental mechanism within the inflammasome [37]. Impairments in autophagy have the potential to induce tissue harm, necrosis, persistent inflammation, and genetic instability, thereby augmenting the occurrence of cancer through alterations in the TME, heightened oxidative stress, and the generation of oncogenic mutations [38]. In conclusion, the a reduction in the expression of functional autophagy genes, defects in ATGs, and the inhibition of signaling pathways associated with autophagy all contribute to an elevated occurrence of cancer, suggesting that maintaining intact autophagy is crucial for its antitumor effects.

The role of autophagy in promoting cancer development

Preliminary evidence indicating the involvement of autophagy in cancer sustenance was observed through the detection of elevated levels of LC3 and lipidated LC3 (LC3 B) in certain tumor tissues, suggesting an accumulation of autophagosomes [39]. Further investigations have demonstrated that autophagy plays a crucial role in facilitating cancer progression by eliminating harmful oxygen free radicals and damaged proteins, preserving mitochondrial function, and meeting the metabolic and survival requirements of cancer cells in stressed conditions [22]. Tumor cells rely primarily on glycolysis because of metabolic alterations; however, mitochondrial function remains essential for specific anabolic processes. Autophagy plays a critical role in maintaining mitochondrial integrity, and deficiencies in autophagic processes can lead to the accumulation of dysfunctional mitochondria [40, 41].

In addition to the direct examination of cancer cells, evaluating the effects of autophagy on surrounding and distant stromal cells within the host organism is needed in the investigation of cancer autophagy. Research utilizing murine models has demonstrated that the systemic deletion of ATG7 results in widespread autophagy deficiencies across the host, which correlate with a significant regression of KRAS-driven tumors [42]. Likewise, a significant regression of KRAS-driven pancreatic cancer was observed in ATG4B-mutated mouse models of systemic autophagy inhibition [43]. These findings imply that autophagy, both in the host organism and within the cancer cells themselves, plays a critical role in facilitating cancer progression.

Moreover, tumors should not be regarded as isolated entities; rather, they are intricately linked and coordinated with the TME, which consists of stromal and immune cells, among others. Research has indicated that autophagic processes in stromal cells can enhance the anabolic activities of cancer cells, thereby facilitating tumor progression. For instance, pancreatic stellate cells can produce pyruvate through an autophagy-dependent mechanism, which is subsequently utilized by pancreatic cancer cells for oxidative metabolism [44]. Another important instance is cancer-associated fibroblasts (CAFs), which are significant cellular components within the tumor stroma. Metabolites generated through autophagy of CAFs, including glutamine, free fatty acids, and ketones, can be utilized by cancer cells as nutritional resources [45]. Autophagy in CAFs also facilitates the release of interleukin-6, interleukin-8, and a range of other cytokines, contributing to the malignant progression of HNSCC [46]. Collectively, these investigations highlight the significant function of stromal cell autophagy in advancing tumors, suggesting that modulation of this process may yield novel perspectives for cancer treatment.

Autophagic degradation may also affect stromal and immune cells, facilitating tumor progression. For instance, in the context of pancreatic ductal adenocarcinoma, hypoxia-induced autophagy in pancreatic stellate cells leads to lumica degradation within the extracellular matrix, which subsequently promotes cancer progression [47]. Additionally, autophagy contributes to the progression of cancer by influencing the functionality of immune cells. In liver cancer, autophagy is implicated in NF-κB p65 degradation, which drives the differentiation of bone marrow-derived macrophages into tumor-promoting M2 phenotypes via the TLR2 signaling pathway [48]. Another separate study indicated that inhibition of autophagy in myeloid-derived suppressor cells disrupts lysosomal degradation functions, resulting in an increased expression of MHC-II and activation of CD4⁺ T cells, thereby enhancing antitumor immunity and inhibiting melanoma progression [49]. Future investigations into the mechanisms by which autophagy influences the function of tumor stromal cells and immune cells within TME are essential, as these processes significantly affect cancer progression.

Pro-metastasis and anti-metastasis functions of autophagy in cancer

Metastasis is a significant contributor to cancer progression and mortality in affected individuals. The involvement of autophagy in cancer metastasis is currently a subject of debate within the scientific community. Autophagy has been implicated in facilitating cancer metastasis through various biological mechanisms, including the promotion of cancer cell migration and invasion [50], maintenance of cancer cell dormancy [51], preservation of cancer stem cells (CSCs) [52], suppression of senescence in cancer cells [53], regulation of epithelial-mesenchymal transition (EMT) [54], adaptation to nutrient deprivation and hypoxia conditions [5, 34], and cancer cell survival within the microenvironment outside the lesion, among other factors. Currently, the association between autophagy and cancer metastasis has been demonstrated across multiple cancer types. This understanding has facilitated the advancement of promising therapeutic approaches to managing cancer metastasis and recurrence through the inhibition of autophagy. For instance, fusobacterium nucleatum outer membrane vesicles (Fn OMVs) have been demonstrated to induce autophagy in oral cancer cells, decrease the expression of EMT-associated proteins, enhance migration and invasive capabilities, and facilitate lung metastasis in vivo [55]. These findings suggests that the inhibition of autophagy induced by Fn OMVs, whether through chemical or biological interventions, may hinder the metastatic progression of oral cancer, offering novel avenues for therapeutic strategies in the management of this malignancy. Moreover, the resistance exhibited by chemotherapy agents presents a significant challenge in clinical practice. Zamora et al. shed light on a novel mechanism underlying paclitaxel-induced metastasis in breast cancer [56]. Mechanistically, paclitaxel inhibits the migration and adhesion of lymphoendothelial cells via an autophagy-dependent pathway, simultaneously enhancing their permeability and thus facilitating metastasis and malignant progression of breast cancer to sentinel lymph nodes. Advances in innovative combination therapies may offer promising strategies to mitigate and potentially reverse paclitaxel resistance in breast cancer.

By contrast, recent research has underscored the critical role of autophagy in suppressing cancer metastasis, particularly highlighting its influence on cancer cell dormancy. In various malignancies, including breast cancer, prostate cancer, and small-cell lung cancer, cancer cells can spread from the primary tumor to secondary sites, entering a stage of growth arrest that may persist for more than a decade [51]. Under certain conditions, these dormant cells can reactivate, leading to metastatic recurrence. A growing body of evidence supports the notion that autophagy plays a pivotal role in inhibiting cancer cell dormancy, particularly in the context of metastatic colonization and proliferation. For instance, the ablation of ATG3 and ATG5 in dormant breast cancer cells prompts them to exit dormancy, resulting in the emergence of a malignant phenotype similar to CSCs, which is associated with aggressive growth and metastatic recurrence [57, 58]. Notably, cells that reactivated because of autophagy inhibition often exhibit genomic instability; however, the implications for cancer metastasis warrant further investigation. Additionally, as previously noted, autophagy that protects cellular integrity is implicated in metastasis primarily through the maintenance of cellular homeostasis, protecting cancer cells from death [1]. Conversely, in certain contexts, autophagy may hinder metastatic processes by preventing necrosis of cancer cells and infiltration of inflammatory cells [59]. Autophagic cell death can also contribute to the suppression of cancer cell metastasis by inducing the demise of these cells [60]. While an increasing body of evidence supports the significant role of autophagy in cancer metastasis, the precise functions and effects of autophagy in specific microenvironments require further investigation.

3. Nrf2 signaling in cancer

3.1. The structure of Nrf2

Throughout the course of evolution, cells have developed the capacity to withstand various internal and external pressures and stimuli. One particularly significant transcription factor, Nrf2, plays a crucial role in facilitating the regulation of the cellular antioxidant stress response [61]. Structurally, Nrf2 is a protein that consists of seven highly conserved ECH homology domains (Nehs). One of these domains, known as Neh1, contains a leucine CNC-bZIP region that facilitates the binding of Nrf2 to the ARE in DNA through interaction with the muscle tendon membrane fibrosarcoma (Maf) protein in the cell nucleus [62]. As a result, the transcription of various enzymes involved in antioxidant defense, ubiquitination, phase II detoxification, and proteasome activity is activated, thereby counteracting oxidative stress in cells. The Neh2 domain of Nrf2 contains ETGE and DLG domains, which exhibit high and low affinities for Keap1, respectively [63]. Neh2 is responsible for the degradation of Nrf2 and negatively regulates its transcriptional activity. Neh3 can interact with the transcription coactivator chromodomain-helicase-DNA-binding protein 6 and collectively modulate the activation of ARE-dependent gene transcription [64]. The Neh4 and Neh5 domains of Nrf2 can bind to the activating transcription factor cAMP response element-binding protein, facilitating the translocation of Nrf2 into the nucleus and its subsequent binding to ARE in the form of the Nrf2-Maf complex [65]. Neh6, which contains numerous serine residues, is involved in the ubiquitination degradation of Nrf2 mediated by the Skp1-Cul1-Rbx1/Roc1-β-TrCP ubiquitin ligase complex and does not rely on Keap1 [66]. Neh7 of Nrf2 primarily functions to recognize the retinoid X receptor alpha [67]. Nrf2 also possesses crucial phosphorylation sites that regulate the binding and dissociation of Nrf2 and Keap1. The structure of Nrf2 is shown in Figure 2A.

3.2. Nrf2 signaling

The activity of Nrf2 signaling is mainly regulated by the negative modulator Keap1. Keap1 can form homodimers and interact with Nrf2 through the ETGE and DLG sequences [68]. In normal physiological conditions, the Keap1-Nrf2 complex binds to the E3 ubiquitin ligase Cullin 3 (Cul3) via the Neh6 domain of Nrf2, leading to the ubiquitination and subsequent degradation of Nrf2 by the 26S proteasome [69]. However, when exposed to oxidative stress or chemical stimulation, Keap1 undergoes conformational changes and dissociates from Nrf2, interrupting the ubiquitination and degradation of Nrf2 [70]. Consequently, Nrf2 is released and translocated to the nucleus, where it functions as a transcription factor. Specifically, Nrf2 activates the transcription of a series of antioxidant enzymes by binding to Maf and ARE (Figure 2B).

It has been observed that Nrf2 can also be regulated independently of Keap1. Several studies have demonstrated that certain kinases, including protein kinase C, casein kinase II, protein kinase R-like endoplasmic reticulum kinase, c-Jun N-terminal kinase, and extracellular signal-regulated kinase, are capable of phosphorylating Nrf2 and facilitating its translocation into the nucleus [71-74]. Conversely, the phosphorylation of Nrf2 by glycogen synthase kinase-3β and mitogen-activated protein kinase can lead to its degradation [75].

Nrf2 signaling can also be modulated by genetic and epigenetic factors. Studies have demonstrated the presence of a potential ARE sequence within the promoter region of NFE2L2, suggesting the existence of a plausible feedback regulatory mechanism that can modulate the transcriptional activity of Nrf2 itself [63]. In addition, some transcription factors, such as AhR, have been demonstrated to modulate the transcriptional activity of Nrf2 by altering the TME and promoting redox homeostasis in breast cancer [76, 77]. The modulation of NFE2L2 by transcription factors plays a significant role in metabolic reprogramming and the malignant progression of cancer. In the context of HNSCC, C-MYC has been demonstrated to engage directly with the NFE2L2 promoter, leading to the upregulation of Nrf2 expression and subsequently facilitating the malignant characteristics of cancer by altering nucleotide biosynthesis pathways [78]. Notably, this regulatory mechanism may be of greater significance than Nrf2-mediated redox regulation. In recent years, numerous noncoding RNAs, such as microRNAs, circular RNAs, and long noncoding RNAs, have been extensively studied because of their involvement in modulating the Nrf2 pathway. Notable examples include miR-140-5p, miR-34a/b/c, circPIBF1, lncMALAT1, and lncMT1DP, which regulate Nrf2 expression or activity and thus play an important role in the development of various cancers and their responses to therapeutic interventions [79-83].

Nrf2 signaling is also influenced by epigenetic factors, including DNA methylation and chromatin modification. For example, in colon cancer, oxidative stress can induce DNA demethylation, leading to increased Nrf2 expression and resistance to 5-fluorouracil [84]. In another independent study, the reduction of methylation at the NFE2L2 promoter was found to enhance its expression, trigger apoptosis, and contribute to an anticancer effect [85]. The function of Nrf2 is influenced by a combination of genetic and epigenetic factors, resulting in a complex impact on cancer. However, the precise mechanisms underlying them require further investigation. In addition, the impact of protein posttranslational modifications, including acetylation, sumoylation, and glycosylation, on Nrf2 signaling has received significant attention. In colon cancer, ARD1 has been demonstrated to acetylate Nrf2, thereby inhibiting its degradation via the proteasome pathway, further facilitating Nrf2 nuclear translocation, enhancing the transactivation of downstream target genes, and ultimately contributing to colon cancer cell proliferation and malignant phenotype [86]. In the case of hepatocellular carcinoma (HCC), Nrf2 sumoylation was demonstrated to promote continuous HCC growth by enhancing the removal of intracellular reactive oxygen species (ROS) and regulating cellular metabolism, which plays a crucial role in the development of HCC and related metabolic stress [87]. A study by Sanghvi et al. also revealed that the glycosylation of Nrf2 destabilizes the protein and impairs the ability of cancer cells to withstand ROS stress in a Keap1-dependent or Keap1-independent manner [88]. Existing research has provided initial insights into the effects of various posttranslational modifications on the stability and activity of Nrf2. However, further investigation is warranted to elucidate the abundance, function, and regulatory mechanisms of each modification in different types of cancers.

3.3. Nrf2: tumor suppressor or oncogene?

In recent decades, a growing body of research has explored the diverse mechanisms involved in the regulation of Nrf2. These mechanisms include alternation in the canonical Nrf2-Keap1 complex, disruptions in noncanonical p62-dependent Nrf2-Keap1 interactions, and control of Nrf2 mRNA and protein expression through transcriptional and translational processes [89]. It is worth noting that Nrf2 plays a dual role in the initiation and progression of cancer, with its effects varying in different conditions.

 Figure 2 

The structure of Nrf2 and regulation of Nrf2 signaling. (A) Nrf2 is comprised of seven Nehs, which play a role in mediating its interaction with adaptors. (B) Under normal physiological conditions, the Keap1-Cul3-E3 ubiquitin ligase typically targets various lysine residues in the Nrf2 Neh2 domain, located between DLG and ETGE motifs, facilitating ubiquitination that subsequently leads to degradation by the 26S proteasome. Upon exposure to reactive oxygen species (ROS) and electrophilic stress, specific cysteine residues in Keap1 undergo modifications, resulting in structural alterations in the Keap1-Cul3-E3 ubiquitin ligase complex. This disrupts the ubiquitination of Nrf2, prompting its translocation to the nucleus where it binds to AREs in target genes through heterodimerization with the MAF protein. Consequently, this initiates a cascade of gene expression that serves to protect the cell.

Traditionally, Nrf2 has been recognized as a significant regulator of detoxification and redox homeostasis, playing a crucial cytoprotective role. In response to stress, Nrf2 activates cytoprotective mechanisms by inducing the expression of multiple genes involved in antioxidant signaling, exogenous biotransformation, autophagy, and proteostasis [9, 90, 91]. Research has demonstrated that when mice with mutated NFE2L2 are exposed to carcinogens, organ damage is increased, particularly in the liver, kidneys, and lungs [92]. Additionally, studies on mice lacking Nrf2 have shown inhibition of ARE-mediated genes, such as GST, γ-GCS, NQO1, HO-1, and GCL, which are involved in detoxification processes, leading to increased susceptibility to cancer [93]. It is important to note that recent investigations have uncovered additional functions of Nrf2 in cancer that go beyond its role in redox regulation, including response to ER stress, growth factor signaling, and nutritional status [94-96]. Therefore, Nrf2 is commonly acknowledged as a tumor suppressor, and the activation of Nrf2 signaling is employed in cancer chemoprevention to enhance cellular and systemic defenses against cancer [97]. The activation of Nrf2-related pathways via Nrf2 inducers has been demonstrated to impede cancer progression and enhance the sensitivity of cancer cells to chemotherapeutic agents. Among the plant-derived Nrf2 inducers are compounds such as sulforaphane, curcumin, epigallocatechin gallate, lycopene, and resveratrol [80, 98-101]. Chemically synthesized or derived substances, including indole and triterpenoid analogs, have also been identified as effective Nrf2 inducers [102, 103].

By contrast, in recent years, an increasing body of evidence has emerged suggesting that the activation of Nrf2 may not be advantageous for all types and stages of cancers. In fact, the activation of Nrf2 enhances the survival of not only normal cells but also cancer cells, supporting the notion that Nrf2 activation may play a role in the maintenance and advancement of cancer, as well as provide protection to tumor cells against oxidative damage that could potentially result in cell death [104-106]. Numerous studies have demonstrated that cancer patients exhibit elevated levels of Nrf2 compared to individuals without cancer [104]. The hyperactivation of Nrf2 in oncogenesis is frequently modulated by various factors. Apart from alterations in Nrf2/Keap1/p62/Cul3, the mutation and activation of oncogenes (e.g., KRAS^G12V/D, B-RAF^V619E, and MYC) or the inactivation of tumor suppressor genes (e.g., Trp53/p16) can also contribute to the hyperactivation of Nrf2 [107, 108].

Subsequent studies have further confirmed the facilitation of Nrf2 in the initiation and progression of cancer. For instance, in lung cancer, the loss of Keap1 heterozygosity and mutations result in decreased Keap1 expression, leading to the upregulation of Nrf2 expression and the activation of its downstream genes [109, 110]. Similarly, the Keap1^C23Y mutation identified in breast cancer reduces its ability to inhibit Nrf2 expression [111]. In the unstable oxidative microenvironment of hypoxia/reoxygenation, cancer cells also exhibit reduced Keap1 expression, which promotes nuclear translocation and increased expression of Nrf2, which, in turn, contributes to the removal of ROS and the progression of cancer [112]. In summary, the diminished expression and functional loss of Keap1 may result in the sustained activation of Nrf2, thereby providing growth support for cancer cells.

Chemotherapy resistance poses a significant obstacle to the clinical management of cancer. Recent research has indicated that Nrf2 may play a role in chemotherapy resistance. Specifically, there is a positive association between Nrf2 levels and the resistance of cancer cells to chemotherapy drugs, such as cisplatin, doxorubicin, and etoposide [69, 113]. Cancer cell resistance to drugs is heightened when Nrf2 expression is increased. For instance, Xu et al. established that in HNSCC, there is an obstruction to Nrf2 degradation mediated by the ubiquitin-proteasome pathway, leading to Nrf2 accumulation, which may contribute to the development of acquired cisplatin resistance in HNSCC [113]. Another independent study by Lee et al. found that Nrf2 is a key regulator of cell survival in ovarian cancer cells under conditions of GSH depletion [105]. Inhibiting Nrf2 using the GSH inhibitor L-buthionine-(S, R)-sulfoximine as a chemical sensitizer renders ovarian cancer cells more susceptible to doxorubicin. The Nrf2 pathway is also activated in doxorubicin and etoposide-resistant osteosarcoma cells [69]. DDRGK1 knockdown results in increased Nrf2 degradation and decreased Nrf2 stability, leading to an increase in ROS levels, which subsequently facilitates the apoptosis of cancer cells and heightens their sensitivity to chemotherapeutic agents, such as doxorubicin and etoposide. Combining DDRGK1 knockout with chemotherapy may yield enhanced therapeutic outcomes in the treatment of osteosarcoma. In conclusion, targeting Nrf2 and related pathways may offer novel strategies and avenues for reversing chemotherapy resistance in cancer.

4. Crosstalk between autophagy and Nrf2 signaling

A comprehensive understanding of the interplay between the Nrf2 signaling pathway and autophagy has been elucidated by several research groups [12, 35, 98, 114, 115]. These investigations simultaneously unraveled the mechanism underlying the connection between Nrf2 and various autophagy receptors, the functional implications of autophagy dysregulation, and the regulation of the persistent activation of the Nrf2 signal. Among these, p62, which functions as a scaffold protein and stress-inducing protein and plays a role in multiple biological processes, including autophagy, apoptosis, inflammation, cell survival, cell death, signal transduction, and tumorigenesis, has been extensively investigated [13, 114, 116]. Simply put, Nrf2 regulates the transcription of ATGs and, at the same time, stimulates autophagy directly through nontranscriptional pathways. The autophagy adaptor protein p62, in turn, is involved in the noncanonical activation of Nrf2. These findings suggest the existence of a positive feedback loop between Nrf2 signaling and autophagy (Figure 3).

4.1. The p62-Keap1-Nrf2 feedback loop

p62 is recognized as the first selective autophagy receptor and serves a significant regulatory function within the autophagy pathway. Through its interaction with LC3, p62 attaches to the autophagosome, facilitating the transport of ubiquitinated cargo to the lysosome for degradation [16]. It has been reported that p62 is implicated in the regulation of the Nrf2 pathway, leading to Nrf2 translocation to the nucleus and subsequent activation of the transcription of antioxidant enzyme genes [13]. This process involves several main steps. First, during the oxidative stress response, the accumulation of p62 protein resulting from autophagy dysfunction leads to the interaction between p62 and Keap1, which sequesters Keap1 within p62 aggregates [117]. Research has demonstrated that when cells are exposed to ROS, phosphorylation of the KIR domain of p62 significantly enhances binding affinity with the DGR domain of Keap1 [118]. Subsequently, p62 binds to LC3 through the LIR domain, forming the LC3-p62-Keap1 complex, which transports Keap1 to autophagosomes for degradation [119]. This binding reduces the formation of the Keap1-Cul3-E3 complex and reduces its degradation through the ubiquitin-proteasome pathway, resulting in the activation of Nrf2 signaling.

 Figure 3 

Crosstalk between the autophagy and Nrf2 pathway. P62 is a receptor protein involved in autophagy that binds to ubiquitin and serves as a key link between the Nrf2 pathway and autophagy. Through post-translational phosphorylation, p62 exhibits an increased affinity for Keap1, leading to the dissociation of Keap1 from Nrf2. This process results in the formation of p62-Keap1 heterodimers that recruit LC3, facilitating selective autophagic degradation of Keap1. As a result, Nrf2 accumulates in the cytoplasm, translocates to the nucleus, and activates transcription of various downstream genes. In addition, Nrf2 promotes p62 expression, establishing a positive feedback loop involving p62-Keap1-Nrf2. In particular, PINK1 localizes to the outer membrane of damaged mitochondria, recruiting PARKIN from the cytoplasm to damaged mitochondria, where PARKIN is phosphorylated. Phosphorylated PARKIN then ubiquitinates p62, facilitating its binding to LC3 B and promoting the recruitment of damaged mitochondria for degradation through mitophagy. p62, NDP52, and OPTN act as mitophagy-associated adaptors.

Interestingly, Nrf2 may participate in the autophagy pathway, either directly or indirectly. On the one hand, Nrf2 interacts with ARE located in the promoter region of the p62 gene, thereby facilitating p62 transcriptional activation [120]. That is, p62 is recognized as a target gene for Nrf2. On the other hand, overexpression of p62 may enhance Nrf2 activity by promoting Keap1 degradation and decreasing Nrf2 ubiquitination. This establishes an Nrf2-Keap1-p62 positive feedback loop that sustains Nrf2 activation [121]. This evidence indicates that p62, acting as a regulatory interactor of the Nrf2-Keap1 complex, is present within the cellular milieu alongside other interactors and regulators that participate in various mechanisms to modulate the unbound and active state of this transcription factor. Ultimately, these processes facilitate its degradation in the cytoplasm and nucleus, thereby regulating or potentially stopping Nrf2-dependent stress responses.

The interaction between p62 and Nrf2 in this feedback loop is influenced by various factors and holds significant importance in cellular antioxidant response and the development and treatment of cancer. For instance, Shi et al. demonstrated that inhibition of autophagy mitigates p62-mediated Keap1 sequestration, subsequently reducing the Nrf2-mediated transcriptional activation of antioxidant genes, which ultimately facilitates the initiation and progression of prostate cancer [13]. Similarly, autophagy activation in gastric cancer significantly enhances the interaction between p62 and Keap1 while simultaneously inhibiting the binding of Nrf2 to Keap1, which reduces the ubiquitination and degradation of Nrf2; this results in a sustained activation of the Nrf2 signaling pathway and inhibition of malignant progression [122]. In addition, investigations into the anticancer mechanisms of apatinib, a targeted angiogenesis inhibitor, have revealed that it can induce ROS production in lung cancer, suppress Nrf2 and p62 expression, and promote autophagy and apoptotic cell death in non-small cell lung cancer (NSCLC) [123]. This mechanism is considered one of the critical pathways through which apatinib exerts its antitumor effects, both in vitro and in vivo. In summary, the Nrf2 signaling pathway and autophagy exhibit mutual regulatory interaction through the p62-Keap1-Nrf2 positive feedback loop, and they perform distinct pathological and pharmacological functions across various cancer contexts.

4.2. Effect of the Nrf2-p62 loop on mitophagy

The association between Nrf2 and mitophagy was initially identified because of Nrf2's function as a transcriptional regulator. Murata et al. discovered that Nrf2 can bind to four AREs in the promoter region of the PINK1 gene, thereby influencing its transcription and subsequent expression [124]. Specifically, the overexpression of Nrf2 leads to an increase in PINK1 expression, whereas the suppression of Nrf2 has the opposite effect. These pieces of evidence support the idea that Nrf2 induces PINK1 to participate in mitophagy. Nrf2 has also been found to participate in the modulation of mitophagy independently of the PINK1/PARKIN pathway. Gumeni et al. discovered that in a fruit fly model lacking PINK1/PARKIN, the activation of Nrf2 can stimulate mitophagy and effectively mitigate and reverse neurodegenerative alterations resulting from PINK1/PARKIN suppression [125]. This suggests that alternative mechanisms could potentially exist through which Nrf2 regulates mitophagy, necessitating additional investigation.

The involvement of p62 in mitophagy is also linked to PINK1/PARKIN. PARKIN possesses E3 ubiquitin ligase activity, enabling it to ubiquitinate many proteins located on the outer membrane of mitochondria and subsequently triggering mitophagy [126]. PARKIN facilitates K63 polyubiquitination of mitochondrial substrates and recruits the ubiquitin and LC3 binding protein p62 to mitochondria [127]. p62 can interact directly with ubiquitinated molecules on autophagosomes, and p62 knockout completely blocks the clearance of damaged mitochondria. Therefore, activation of the PINK1/PARKIN/p62 axis plays a critical role in mitophagy, which is essential for maintaining mitochondrial quality control and homeostasis. However, a recent challenge to this conventional model has emerged, as PINK1 has been found to recruit two additional adaptors, OPTN and NDP52, instead of p62 [128]. This recruitment leads to the activation of the key autophagy serine/threonine kinase ULK1 and the induction of mitophagy. Notably, this process occurs independently of PARKIN. Comprehending the role of Nrf2 in mitophagy and autophagy is critical to gain insight into the mechanisms by which Nrf2/p62 regulates mitochondrial quality control and maintains mitochondrial homeostasis.

5. Implication of Nrf2-autophagy crosstalk in cancer progression and therapeutics

A growing body of research has indicated that p62 and several ATGs, including ATG5 and ATG7, are potential targets of Nrf2, indicating a close association between Nrf2 and autophagy [35, 129, 130]. Investigating the connection between Nrf2-related autophagy and cancer development warrants further investigation and may provide a scientific basis for the formulation of cancer treatment approaches that target Nrf2.

5.1 Effects of Nrf2-autophagy crosstalk on cancer progression and therapy response

As previously noted, the dark side of Nrf2 in cancer has gained significant interest since 2006 [131]. In adverse conditions, elevated levels of Nrf2 and activated autophagy within cancer cells foster a favorable cellular environment that facilitates proliferation and survival, thereby exacerbating cancer progression, recurrence, and metastasis. In addition, Nrf2-autophagy crosstalk contributes to the development of resistance to chemotherapy to some extent.

HCC is a widespread form of malignancy worldwide and ranks as one of the most common causes of cancer-related mortality. Recent research has revealed that the crosstalk between Nrf2 signaling and autophagy can affect the advancement of HCC. Komatsu et al. presented initial evidence of the involvement of the Nrf2-p62 axis in the development of HCC [132, 133]. Elevated p62 levels in livers with impaired autophagy sustain the progression of precancerous lesions and HCC. In particular, excessive p62 competes with the Nrf2 binding site on Keap1, leading to the stabilization of Nrf2 and the subsequent activation of Nrf2 target genes. In addition to p62 overexpression, p62 phosphorylation has been observed in HCC, both of which contribute to the activation of Nrf2 [134, 135].

Nrf2-autophagy crosstalk has been extensively investigated in various types of cancers. For instance, in prostate cancer, speckle-type POZ protein has been observed to bind to and trigger the nondegradable ubiquitination of p62, resulting in the inhibition of p62-dependent autophagy [13]. Consequently, this process leads to Keap1 isolation, ultimately reducing the transcriptional activation of Nrf2-mediated antioxidant genes, which in turn facilitates prostate cancer progression. Similarly, BDH2 facilitates the ubiquitination of Nrf2 by enhancing the interaction between Keap1 and Nrf2 in gastric cancer, leading to increased ROS accumulation, activation of cellular autophagy, and inhibition of gastric cancer progression [122]. Nrf2 has also been identified as an independent prognostic factor influencing overall survival in patients with NSCLC [136]. Notably, Nrf2 stimulates the formation of autophagosomes, thereby promoting the progression of NSCLC. Conversely, regulators inhibit the Nrf2-autophagy axis to suppress cancer progression. In osteosarcoma, TRIM22 has been observed to promote Keap1-independent degradation of Nrf2, thereby activating autophagy signaling pathways and ultimately impeding osteosarcoma progression [60]. This suggests that the TRIM22/Nrf2/autophagy signaling axis may represent a promising therapeutic target for osteosarcoma treatment. In general, these studies offer new perspectives on the comprehension of the Nrf2-autophagy signaling axis in carcinogenesis and development, and they have the potential to advance the creation of novel cancer treatment approaches.

5.2 Agents targeting the Nrf2-autophagy signaling axis in cancer therapy

The dynamic role of Nrf2-autophagy crosstalk in cancer raises the question of whether its regulation is beneficial to cancer patients. A growing body of research has demonstrated that both natural and synthetic ligands can modulate the Nrf2-autophagy signaling axis through direct or indirect means, thus affecting the progression and resistance to treatment of various types of cancers (Table 2). Recent investigations have also utilized a combination of antitumor drugs and regulators of the Nrf2-autophagy signaling axis to uncover the role of the Nrf2-autophagy signaling axis in the onset and progression of cancer.

Medicinal plants contain a wealth of potential treatments for various diseases. Researchers have discovered that a range of natural compounds and their derivatives can affect the Nrf2-autophagy signaling axis, leading to anticancer properties. For example, sulforaphane, an isothiocyanate compound obtained from cruciferous plants, has been shown to affect cancer progression by modulating various cellular processes, such as proliferation, metastasis, apoptosis, and angiogenesis [137]. Recent research has indicated a strong correlation between the pharmacological effects of sulforaphane and the Nrf2-autophagy signaling axis. In cervical cancer, sulforaphane has been found to induce p62 expression and p62 body formation through the transcriptional coactivator SPBP, thereby influencing the activation of cytoprotective autophagy signaling associated with Nrf2 [138]. In lung cancer, the triterpenoid glycoside oleifolioside B and sesquiterpene lactone compound isodeoxyelephantopin (ESI) have been found to stimulate cytoprotective autophagy [139, 140]. In particular, ESI prompts nuclear translocation of Nrf2 and triggers p62 transcription, which subsequently competes with Keap1 for binding, releasing Nrf2 and establishing a positive feedback loop for p62 activation. Regarding its anti-lung cancer properties, ESI demonstrates the selective inhibition of both the viability and clonogenic potential of lung cancer cell lines while exhibiting minimal toxicity to lung epithelial cells.

Targeting the Nrf2-p62-Keap1 regulatory axis combining ESI presents a potentially promising therapeutic approach for the fight against lung cancer. Moreover, research has demonstrated that lycopene can promote the activation of intracellular antioxidant enzymes and the nuclear translocation of Nrf2 [100]. Lycopene has also been found to increase p62 expression, leading to Keap1 degradation and the subsequent release of Nrf2. Subsequent research has demonstrated that lycopene exhibits a protective effect against the development of cutaneous papilloma in both cellular and animal models. These findings elucidate the mechanistic connection between lycopene-induced Nrf2-autophagy signaling and provide potential preclinical support for chemoprevention of skin cancer. The above research has provided promising prospects for identifying precursors targeting the Nrf2-autophagy signaling axis from natural products, which may serve as a foundation for further investigation and theoretical underpinning in the advancement of innovative cancer treatment approaches.

Chemically synthesized drugs play a crucial role in the treatment of cancer because of their rapid therapeutic action and evident efficacy. Certain small-molecule compounds and drugs have demonstrated the ability to affect the Nrf2-autophagy axis, resulting in promising anticancer properties. For instance, jaspine B derivative C-2 exhibits selective inhibition of gastric cancer cell proliferation compared to normal epithelial gastric cells [121]. Interestingly, C-2 induces autophagy and enhances p62 expression in gastric cancer cells by activating the JNK/ERK/Beclin 1 pathway. Consequently, p62 competitively binds to Keap1, leading to the release of Nrf2 and its translocation to the nucleus, thereby promoting the expression of downstream Nrf2 target genes and enhancing gastric cancer cell survival. These findings indicate that gastric cancer cells may activate protective autophagy via the p62/Keap1/Nrf2 pathway in the early stages to counteract C-2-induced cell death. Moreover, in NSCLC, apatinib has been observed to induce the generation of ROS, suppress the expression of Nrf2 and p62, trigger autophagy and apoptosis, and inhibit tumor proliferation, both in vitro and in vivo [141]. In a separate independent investigation, Yu et al. elucidated the cytotoxic properties of C-2 in the context of bladder cancer [142]. Mechanistically, C-2 activates the JNK pathway, which subsequently induces autophagy and increases p62 expression. C-2 also activates the Nrf2 signaling pathway, facilitates the transition from autophagy to apoptosis, and amplifies the apoptotic effect on cancer cells. Notably, the coadministration of C-2 with the JNK inhibitor SP600125 further enhances the tumor-suppressive effects of C-2, potentially because of the promotion of apoptosis and the attenuation of autophagy. These results provide a basis for further research and theoretical justification for the development of pharmacological agents that target the Nrf2-autophagy signaling axis.

The combination of small-molecule compounds or medications presents novel prospects for the prevention and management of cancer. Particularly, focusing on the Nrf2-autophagy axis has significant implications for overcoming resistance to cancer treatment. Colon cancer is a prevalent and deadly malignant tumor. mTOR inhibitors, including rapamycin, have been employed in the treatment of colon cancer, but the development of drug resistance frequently results in cancer evasion [143]. Li et al. investigated the potential of S-allylmercaptocysteine (SAMC) as an adjuvant to rapamycin in the treatment of colon cancer, with a particular focus on the interplay between Nrf2 and autophagy in the context of combination therapy [144]. The researchers found that the combined application of rapamycin and SAMC stimulated the transcription of Nrf2 and its downstream NQO1 gene while suppressing p62 expression. In terms of effectiveness, the combined treatment of SAMC and rapamycin has the potential to enhance the anticancer efficacy. These results provide novel insights into colon cancer treatment and suggest that the Nrf2-autophagy pathway could represent a promising new therapeutic target for colorectal cancer. The combination of sulforaphane with other pharmacological agents has also demonstrated significant anticancer efficacy across various malignancies, including prostate cancer, breast cancer, and esophageal cancer, among others. In particular, the combination of sulforaphane and vitamin D has been shown to diminish cell viability in prostate cancer by inducing oxidative stress, DNA damage, and autophagy, which is linked to the upregulation of several key proteins, including BAX, CASP8, CASP3, JNK, and Nrf2 [145]. Furthermore, in esophageal squamous cell carcinoma, sulforaphane promotes autophagy through the activation of the Nrf2 pathway, which subsequently inhibits the proliferation and clonogenic potential of cancer cells [115]. The use of the autophagy inhibitor chloroquine (CQ) has been found to inhibit the Nrf2 pathway while activating the caspase pathway, thereby enhancing the antitumor efficacy of sulforaphane in both in vitro and in vivo models. These findings provide a preclinical theoretical framework for the potential application of sulforaphane in future cancer treatment strategies.

The Nrf2-autophagy signaling pathway presents novel pharmacodynamic targets for certain anticancer drugs that are currently utilized in clinical practice. For instance, apatinib is a prominent antiangiogenic drug that demonstrates significant inhibitory effects on various solid tumors, including NSCLC [146]. Beyond its traditional mechanism of targeting the VEGFR2/STAT3 signaling pathway, apatinib has been found to enhance ROS generation, suppress Nrf2 and p62 expression, and subsequently induce autophagy and apoptotic cell death in NSCLC [141]. Similarly, in the context of breast cancer, apatinib has been observed to downregulate the Nrf2/HO-1 signaling pathway and suppress glutathione levels; this leads to ROS generation, which facilitates ROS-dependent autophagy and apoptosis, and ultimately results in the inhibition of both the proliferation and migration of breast cancer cells [147]. These findings enhance the pharmacodynamic investigation of apatinib and are expected to contribute to the broader clinical utilization of the drug. Moreover, temozolomide is a chemotherapeutic agent employed in the treatment of malignant glioma and malignant melanoma [148, 149]. Recent research indicates that downregulation of Nrf2 may exacerbate autophagy and the proliferation suppression of temozolomide-induced glioma cells [150]. Future investigation into the synergistic effects of Nrf2 inhibitors in combination with temozolomide may enhance therapeutic outcomes for glioma, requiring further pharmacological exploration. Furthermore, procaine is a widely utilized local anesthetic in clinical settings [151]. Recent studies have demonstrated that in oral squamous cell carcinoma (OSCC), procaine enhances the expression of the differentiation gene PAX9 and activates the Nrf2 signaling pathway, which subsequently leads to the inhibition of OSCC cell proliferation, differentiation, and stemness via an autophagy-dependent mechanism [152]. Elucidating the pharmacological effects and molecular mechanisms of procaine may offer a novel therapeutic approach for the treatment of OSCC. In conclusion, these findings indicate that the Nrf2-autophagy signaling axis holds promise as a target for anticancer therapy. Elucidating the mechanism of action of the Nrf2-autophagy signaling axis may offer a novel avenue for drug development.

5.3. Nrf2-p62 and other autophagy-related proteins in cancer monitoring

For a long time, the prompt detection of cancer and the evaluation of treatment effectiveness have been key components of clinical practice. Extensive efforts have been made to identify markers that can help predict cancer risk and establish preventive measures. These markers play a pivotal role in facilitating timely diagnosis and prognostic assessment, thus influencing the selection of clinical treatment options and secondary prevention strategies. Because of the involvement of the Nrf2-autophagy signaling axis in tumorigenesis and cancer resistance, significant efforts have been made to identify Nrf2-p62 and other autophagy-related markers for predicting cancer risk, establishing preventive measures, and facilitating timely diagnosis and prognosis assessment of cancer (Table 3).

Research has indicated that elevated levels of Nrf2 can facilitate cancer cell survival, enhance resistance to chemicals, and function as a prognostic indicator for various malignancies, including lung cancer, cervical cancer, pancreatic cancer, and breast cancer [153-156]. Additionally, p62 aggregation has been linked to an unfavorable prognosis in triple-negative breast cancer, lung adenocarcinoma, and glioma [157-159]. In this context, Nrf2, p62, and other autophagy-related proteins have been suggested and further investigated for their potential utility in clinical protocols for assessing cancer risk, establishing prognostic scores, and determining intervention criteria for cancer patients. In basic terms, prolonged elevation of Nrf2 expression and heightened activity increases the likelihood of developing precancerous lesions and cancers during the carcinogenesis stage. Conversely, in later stages of cancer, it is associated with a negative prognosis. Sustained activation of the Nrf2 pathway through increased autophagy signaling, particularly p62-mediated activation, may provide valuable clinical insights into the severity of precancerous lesions and associated cancer risk. Furthermore, the cancer-specific reprogramming of metabolic and stress response pathways prompted by p62-mediated activation of the Nrf2 pathway warrants further exploration as a potential prognostic and therapeutic indicator for cancer.

In addition, other proteins associated with autophagy may contribute to advancing and supporting the clinical utilization of the Nrf2-p62 pathway in cancer surveillance. For instance, LC3 can be valuable in distinguishing cancer progression and conducting prognostic assessment. LC3 A exhibits high expression in HCC tissues [160]. Significantly, LC3 A has a stone-like appearance in HCC, and its expression level correlates with elevated serum alpha-fetoprotein levels, a lower degree of tumor differentiation, and vascular invasion. In addition, elevated levels of stone-like LC3 A are strongly associated with unfavorable outcomes for HCC patients, thus serving as an independent prognostic factor. Conversely, LC3 B expression is closely related to a large tumor size, advanced tumor stages, and worse relapse-free and overall survival, making it a potential molecular marker for guiding the selection of cancer staging and surgical treatment options [161].

In mammals, alongside LC3, a group of autophagy-associated proteins that are homologous to yeast ATG8 belongs to the gamma-aminobutyric acid type A receptor-associated protein (GABARAP) family (e.g., GABARAP, GABARAPL1, and GABARAPL2). These proteins are reported to engage with molecules involved in vesicle transport, autophagy, and programmed cell death, serving a crucial function in regulating autophagy initiation and autophagosome closure [218]. Studies have shown a decrease in the expression of GABARAP and GABARAPL1 in different types of tumor tissues. For instance, in the context of breast cancer, decreased GABARAP levels have been observed to trigger EMT and facilitate tumor progression [214]. In addition, there exists an inverse correlation between GABARAP expression and advanced clinicopathological characteristics (e.g., tumor size and tumor node metastasis (TNM) stage) in clinical samples. Patients with reduced GABARAP levels tend to have an unfavorable prognosis. In summary, GABARAP has the potential to hinder the malignant progression of breast cancer and may serve as a promising target for diagnosis and therapeutic intervention. In a separate investigation, Gil et al. observed a significant decrease in GABARAP expression in colorectal cancer tissue [215]. GABARAP functions as a tumor suppressor in the development of colorectal cancer. A reduced GABARAP expression is associated with inferior differentiation of colorectal cancer and shorter overall survival, indicating its potential as a therapeutic target for colorectal cancer treatment. Moreover, Brigger et al. conducted clinical research demonstrating a notable decrease in the expression of GABARAP family members GABARAPL1 and GABARAPL2/GATE-16 in individuals diagnosed with primary acute myeloid leukemia [216]. Specifically, diminished levels of GABARAPL2 were linked to immature myeloid leukemia phenotypes. A reduced expression of these GABARAP family proteins is essential for early neutrophil differentiation in leukemia. However, the levels of GABARAP expression in various tumors are inconsistent. For example, GABARAP and GABR2 are notably increased in thyroid adenoma and early thyroid cancer tissues, and they contribute to tumor growth and spread [217]. This illustrates the intricate functional role of GABARAP in tumor development and highlights the complexity of the mechanisms underlying cancer initiation and progression. Further investigations are warranted to support these findings in the future.

UVRAG, a crucial regulator of macrophage/autophagy in mammals, interacts with Beclin 1, PIK3C3, and RUBCN. Shi et al. found that UVRAG has the potential to increase tumor migration and drug resistance, resulting in a negative prognosis for individuals with colorectal cancer [162]. These findings provide a basis for the development of treatment strategies for colorectal cancer patients. Moreover, research has indicated that UVRAG phosphorylation plays a negative role in autophagosome maturation. Specifically, phosphorylation levels at UVRAG S522 have been associated with a less favorable prognosis in patients with HCC, and further research is warranted to determine its clinical application value [163].

Moreover, ULK1 is a serine/threonine kinase that is recognized for its role in autophagy initiation. Wu et al. highlighted the significance of ULK1 as a crucial prognostic factor in patients with HCC [164]. The simultaneous incorporation of LC3 B in assessments can also enhance the accuracy of prognosis and survival predictions for these patients. In the context of breast cancer, ULK1 is strongly associated with disease progression and adverse prognostic outcomes, suggesting its potential as a novel therapeutic target and prognostic marker for breast cancer [165]. Furthermore, Yun et al. investigated ULK1 expression patterns and prognostic implications in nasopharyngeal carcinoma [166]. Their findings revealed a strong correlation between elevated ULK1 levels and aggressive clinical features, as well as poor survival rates in individuals with nasopharyngeal carcinoma.

Furthermore, Beclin 1 plays a crucial role in the assembly of a significant protein complex within the autophagy pathway, contributing to various stages including autophagosome initiation, elongation, and maturation [1]. A reduced Beclin 1 expression is associated with impaired autophagy in cancer, making it a significant biomarker associated with carcinogenesis and progression. Its reduced expression is believed to impact the prognosis and clinical outcomes of cancer patients. Du et al. found that NSCLC patients with reduced levels of Beclin 1 were diagnosed at a later stage, had a higher incidence of lymph node metastases, and exhibited lower tumor differentiation [167]. Consequently, Beclin 1 may offer more precise prognostic indicators for NSCLC patients, potentially benefiting a larger number of individuals with this condition. Similarly, a decreased expression of Beclin 1 in gastric cancer correlates with lymph node metastasis, TNM staging, dedifferentiation, and adverse prognosis [168]. In an independent study by Dong, it was observed that a decreased Beclin 1 expression was markedly linked to lymph node metastasis in intrahepatic cholangiocarcinoma, as well as to reduced overall survival and disease-free survival [169]. The expression of Beclin 1 is correlated with the advancement and spread of intrahepatic cholangiocarcinoma, suggesting its potential utility as a novel prognostic marker for patients. Together, Beclin 1 holds promise as a potential indicator for assessing the onset, invasion, metastasis, and prognosis of cancer, and may serve as a target for gene therapy in cancer.

Advances in technology and research have led to the discovery of an increasing number of autophagy-related proteins that significantly affect cancer prognosis and clinical medication monitoring. Utilizing autophagy-related proteins as biomarkers for cancer surveillance provides a thorough understanding of the interplay between autophagy and cancer, offering novel perspectives on cancer treatment strategies that focus on autophagy modulation.

5.4. Ongoing clinical trials of Nrf2 and autophagy signaling

Currently, the Clinical Trials Database (https://www.clinicaltrials.gov) contains approximately 130 records targeting Nrf2 and autophagy in cancer. The trials focused on advancing innovative anticancer drugs that specifically target Nrf2 and autophagy and on exploring the potential of combining established drugs. Given its significance as a regulatory element in cancer and a target for therapeutic measures, Nrf2 and autophagy signaling have attracted considerable interest in both preclinical and clinical investigations.

Clinical trials have been conducted to assess the safety, tolerability, pharmacological properties, and antitumor effects of Nrf2 inhibitors, including pyrimethamin (NCT05678348) and sulforaphane (NCT03182959), as well as the GLS1 inhibitor telaglenastat (NCT03872427, NCT04265534), TORC 1/2 inhibitor sapanisertib (NCT02417701), Keap1 activator VD-130037 (NCT05954312), and other compounds in Nrf2-mutated cancer types, such as HNSCC, NSCLC, and acute myeloid leukemia (Table 4). For instance, sapanisertib, a TORC1/2 inhibitor, has shown enhanced therapeutic efficacy and extended progression-free survival in NSCLC models and phase II clinical trial populations characterized by Nrf2 activation [170]. In combination with glutaminase inhibitors, it may also help reduce metabolic resistance to treatment in patients. These findings indicate that modulation of the Nrf2 pathway, in conjunction with metabolic targeting, could yield promising therapeutic strategies for NSCLC patients lacking genotype-directed therapies. A limited number of clinical trials have focused on Nrf2 as a potential target for anticancer treatment. As interest in Nrf2-targeted cancer therapies grows, there is a need for the continued development of improved Nrf2 modulators to enhance clinical efficacy.

Increased autophagy is believed to be the mechanism by which cancer cells survive and develop resistance to chemotherapy. It has been widely demonstrated that inhibiting autophagy can sensitize cancer cells to anticancer treatments. Small-molecule inhibitors of autophagy can be used independently or in combination with anticancer drugs. Currently, CQ/hydroxychloroquine (HCQ) is the only FDA-approved autophagy inhibitor for clinical trials [18]. CQ, an anti-malarial drug, inhibits autophagosome-lysosome fusion by affecting lysosomal acidification. Numerous studies have shown that CQ can enhance the sensitivity of cancer cells to chemotherapy drugs and even reverse resistance to chemotherapy [15, 171, 172]. Presently, the combination of CQ/HCQ with various chemotherapy drugs is undergoing clinical trials (Table 4). The development of drug resistance in cancer presents a significant challenge in cancer treatment. The potential use of autophagy inhibitors in combination with chemotherapy drugs holds promise for addressing this problem, particularly in recurrent and refractory cancer types.

6. Conclusion and perspective

The Nrf2-Keap1 pathway and autophagy are critical mechanisms that contribute to cellular resistance to various stimuli, primarily by increasing the expression of a range of antioxidant and cellular defense genes [109]. Normally, periodic activation of Nrf2 via the conventional pathway provides cellular security and operational efficiency. By contrast, continuous activation of Nrf2 promotes tumor growth and contributes to chemotherapy resistance because of genetic mutations in Nrf2-controlled genes [9, 69, 91, 173]. Autophagy is a cellular stress response essential for cellular homeostasis and organelle health [39]. Recent research has demonstrated a close relationship between Nrf2 and the autophagy adaptor protein p62, highlighting a mechanistic connection that supports the sustained activation of Nrf2 signaling [129]. In the context of oxidative stress, p62 facilitates Keap1 aggregation, leading to the stabilization of Nrf2 and the upregulation of ARE regulatory genes, including p62 itself. This interaction results in the establishment of an Nrf2-Keap1-p62 regulatory loop that plays a significant role in the regulation of cellular processes in cancer cells.

Targeting the Nrf2-autophagy signaling axis through either biological or pharmacological interventions may offer potential strategies for chemoprevention and the treatment of cancer. This paper provides a comprehensive overview of and recent advances in the functions and mechanisms of autophagy and the Nrf2 pathway, as well as their crosstalk in cancer progression. It also highlights the potential utility of targeting the Nrf2-autophagy signaling axis for cancer monitoring and therapeutic interventions. While further investigation is needed to fully understand this process, these findings suggest a promising avenue for the development of cancer therapies.

Despite advancements in understanding Nrf2, numerous questions remain to be addressed. For example, Nrf2 functions as a multifaceted transcription factor that regulates the expression of various genes implicated in cellular redox homeostasis, cell proliferation, apoptosis, inflammatory responses, and DNA repair, among others [104]. However, it is important to note that not all genes regulated by Nrf2 influence autophagy uniformly. The precise molecular mechanisms through which specific stimuli activate Nrf2 signaling to modulate autophagy have not yet been fully elucidated, and the protein interaction networks that mediate responses to diverse signals are still poorly understood. Numerous studies have also highlighted the involvement of Nrf2 in autophagy, and there remains a lack of clarity in distinguishing between the transcriptional and nontranscriptional roles of the Nrf2 signaling pathway in this context. Consequently, subsequent research endeavors may need to focus on elucidating these uncertainties to enhance the comprehension of Nrf2. Such insights could prove instrumental in establishing the targetability of Nrf2 in cancer and formulating intervention strategies that specifically target Nrf2. Furthermore, the limited number of ligands that specifically target Nrf2 is currently undergoing initial phases of clinical trials, and the overall quantity of pharmacological agents aimed at the Nrf2 signaling pathway remains constrained. Consequently, there is significant potential for further pharmacological investigation in this area.

Existing data suggest that the inhibition of autophagy through the use of ligands or drugs demonstrates promising clinical efficacy in cancer therapy. Recent studies have also highlighted the beneficial synergistic effects of ligands that target the autophagy pathway when used in conjunction with chemotherapeutic drugs. For instance, in preclinical models, autophagy inhibitors have proven to be effective adjuncts to various cytotoxic drugs and targeted therapies, which enhance the sensitivity of cancer cells to chemotherapy and mitigate resistance mechanisms [15, 171, 172]. Furthermore, clinical trials are increasingly exploring a range of combination therapies that incorporate both anticancer agents and autophagy inhibitors. Nonetheless, a comprehensive analysis and investigation into the selection of suitable autophagy inhibitors and chemotherapy agents are needed, as well as an understanding of their functions across various cancer treatment modalities, which is essential for identifying optimal combination therapies and their respective indications.

Abbreviations

3-MA: 3-methyladenine; AML: acute myelogenous leukemia; ARE: antioxidant response element; ATG: autophagy-related gene; CAFs: cancer-associated fibroblasts; CQ: chloroquine; Cul3: Cullin 3; CSCs: cancer stem cells; DSS: disease-specific survival; EMT: Epithelial-mesenchymal transition; ESI: isodeoxyelephantopin; Fn OMVs: fusobacterium nucleatum outer membrane vesicles; GABARAP: gamma-aminobutyric acid type A receptor-associated protein; GCL: glutamate cysteine ligase; HCC: hepatocellular carcinoma; HCQ: hydroxychloroquine; HNSCC: head and neck squamous cell carcinoma; ICC: intrahepatic cholangiocellular carcinoma; Keap1: kelch-like ECH-associated protein 1; LC3: microtubule-associated protein 1 light chain 3; Maf: muscle tendon membrane fibrosarcoma; mTOR: mammalian TOR; Neh: nrf2 ECH homology domain; NQO1: NAD(P)H quinone dehydrogenase 1; Nrf2: nuclear factor (erythroid derived-2)-like 2; NSCLC: non-small cell lung cancer; OSCC: oral squamous cell carcinoma; PE: phosphatidylethanolamine; PFS: progression free survival; PI3K: phosphatidylinositol 3-kinase; PINK1: PTEN induced putative kinase 1; RITA: reactivation of p53 and induction of tumor cell apoptosis; ROS: reactive oxygen species; SAMC: S-allylmercaptocysteine; SCLC: small cell lung cancer; TME: the tumor microenvironment; TNM: tumor node metastasis; TOR: target of rapamycin; ULK1/2: serine/threonine UNC-51-like kinases 1/2; UVRAG: UV radiation resistance-associated gene protein.

Acknowledgements

This work was supported by Natural Science Foundation of Shandong Province (Project No: ZR2022MH032), China Postdoctoral Science Foundation Funded Project (Project No.: 2018M632612), Open Foundation of the Key Laboratory of Molecular Pharmacology and Drug Evaluation of Yantai University (Project No.: P202202), and Qingdao Postdoctoral Applied Research Project.

Author contributions

Chan Shan and Yuan Wang drafted and wrote the manuscript. Yin Wang participated in the design of the review. All authors read and approved the final manuscript.

Competing Interests

The authors have declared that no competing interest exists.

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Corresponding authors: E-mail: shanchan2019com; wangyinedu.cn.

Received 2024-9-3
Accepted 2024-10-29
Published 2024-11-11

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