Regulation and function of autophagy in pancreatic cancer

ABSTRACT

Oncogenic KRAS mutation-driven pancreatic ductal adenocarcinoma is currently the fourth-leading cause of cancer-related deaths in the United States. Macroautophagy (hereafter “autophagy”) is one of the lysosome-dependent degradation systems that can remove abnormal proteins, damaged organelles, or invading pathogens by activating dynamic membrane structures (e.g., phagophores, autophagosomes, and autolysosomes). Impaired autophagy (including excessive activation and defects) is a pathological feature of human diseases, including pancreatic cancer. However, dysfunctional autophagy has many types and plays a complex role in pancreatic tumor biology, depending on various factors, such as tumor stage, microenvironment, immunometabolic state, and death signals. As a modulator connecting various cellular events, pharmacological targeting of nonselective autophagy may lead to both good and bad therapeutic effects. In contrast, targeting selective autophagy could reduce potential side effects of the drugs used. In this review, we describe the advances and challenges of autophagy in the development and therapy of pancreatic cancer.

Abbreviations: AMPK: AMP-activated protein kinase; CQ: chloroquine; csc: cancer stem cells; DAMP: danger/damage-associated molecular pattern; EMT: epithelial-mesenchymal transition; lncRNA: long noncoding RNA; MIR: microRNA; PanIN: pancreatic intraepithelial neoplasia; PDAC: pancreatic ductal adenocarcinoma; PtdIns3K: phosphatidylinositol 3-kinase; SNARE: soluble NSF attachment protein receptor; UPS: ubiquitin-proteasome system.

KEYWORDS:

• Disease
• lysosome
• macroautophagy
• pancreatic ductal adenocarcinoma
• pdac
• tumor

Introduction

Pancreatic ductal adenocarcinoma (PDAC) is the main type of pancreatic cancer, accounting for approximately 3% of all cancers, ranks fourth in cancer-related deaths in the United States, and has a 5-year overall survival rate of 9% [1]. Epidemiologically, risk factors for developing PDAC include smoking, heavy drinking, obesity, low physical activity, low intake of vegetables and fruits, and high intake of fat [2]. These risk factors may jointly cause chronic inflammation of the pancreas, an important extrinsic cause of tumorigenesis. The intrinsic cause of pancreatic tumorigenesis is related to gene mutations, especially mutations in KRAS (KRAS proto-oncogene, GTPase), TP53 (tumor protein p53), SMAD4 (SMAD family member 4), and BRCA2 (BRCA2 DNA repair associated) [2]. As a main driver of pancreatic cancer, KRAS mutations are observed in 99% of patients. The forced expression of KRAS^G12D mutations in the pancreas is sufficient to induce the development and progression of pancreatic intraepithelial neoplasia (PanIN) to PDAC with significant stromal responses in mice [3]. Despite unremitting efforts, the interaction between extrinsic and intrinsic factors during tumor transformation is still the basis for understanding the molecular pathogenesis of PDAC.

As one of the most aggressive solid malignant tumors, the diagnosis of PDAC usually occurs at an advanced stage, and patients may lack the opportunity for surgical resection. Although radiation therapy and immunotherapy are also potential options, chemotherapy is currently the main method of treating PDAC patients who are not eligible for resection [4]. For PDAC patients with metastatic disease, FOLFIRINOX (a combination of cancer drugs including folinic acid, fluorouracil, irinotecan, and oxaliplatin) and nab-paclitaxel plus gemcitabine, are currently two standard combination therapies, showing a mild survival advantage compared to gemcitabine-mediated monotherapy [5,6]. However, primary or secondary drug resistance is a common phenomenon that eventually leads to chemotherapy failure. Therefore, understanding the signals and regulation of cell survival and cell death of PDAC is essential to overcoming drug resistance.

Macroautophagy (hereinafter referred to as autophagy) is a conserved cellular homeostasis mechanism that degrades various cargoes (e.g., proteins, organelles, and pathogens) [7,8]. It mainly plays a role in promoting survival under environmental stress, but also causes cell death in some cases [9]. In addition to regulating noncancer diseases (e.g., neurodegenerative and infectious diseases), the pathological role of autophagy in cancer also depends on the context. Autophagy may play a role in limiting the earliest stages of tumor development while promoting survival in established tumors [10]. In this review, we first introduce the process of autophagy, and then focus on the function of autophagy in the development and treatment of PDAC in both preclinical and clinical research. Research in autophagy is rapidly developing, and the understanding we gain not only helps us understand the basic molecular machinery of homeostasis, but also can guide the clinical decision-making for targeted therapy that is autophagy related.

The basic process of autophagy

Cells need integrated degradation mechanisms, such as autophagy and the ubiquitin-proteasome system (UPS), to remove intracellular waste generated during metabolism or aging [11]. Unlike the UPS, the process of autophagy is mediated by the formation and maturation of multiple membrane vesicles, especially phagophores, autophagosomes, and autolysosomes Figure 1. Phagophores are sac-like structures whose membranes are produced from a variety of resources, such as endoplasmic reticulum (ER), mitochondria contact sites, the Golgi apparatus, and plasma membrane. The nucleation and expansion of phagophores result in the production of autophagosomes (a spherical structure with double-layer membranes), which engulf various types of cargoes, such as endogenous proteins or organelles and exogenous microorganisms. Later, autophagosomes deliver cytoplasmic components to lysosomes by forming autolysosomes, which ultimately lead to lysosomal enzyme-mediated degradation. Some components of degraded cargo can be recycled for protein synthesis and energy production, thereby promoting cell survival.

Figure 1. The process and modulation of autophagy. Autophagy is an intracellular lysosomal-dependent degradation pathway that occurs with the dynamic generation of specific membrane structures (e.g., phagophores, autophagosomes, and autolysosomes). This process is regulated by the ATG family and other proteins (particularly kinases) that form different protein complexes. Abbreviations: AMPK, AMP-activated protein kinase; ATG, autophagy related; BECN1, beclin 1; MTOR, mechanistic target of rapamycin kinase; PIK3C3, phosphatidylinositol 3-kinase catalytic subunit type 3; PIK3R4, phosphoinositide 3-kinase regulatory subunit 4; RB1CC1, RB1 inducible coiled-coil 1; Ub-like, ubiquitin-like; ULK1, unc-51 like autophagy activating kinase 1

The autophagy process is fine-tuned by multiple signals or regulators at different levels. A series of ATG (autophagy-related) genes are responsible for the induction and execution of autophagy. To date, the ATG family includes more than 40 members; the first Atg protein was discovered in S. cerevisiae in the 1990s [12–14], and half of the known Atg proteins have homologs in mammalian cells [15]. The proteins encoded by ATG genes form the distinct complexes required for the dynamic regulation of autophagy membrane structures, and this process is further regulated by kinase-mediated phosphorylation modifications [16].

AMP-activated protein kinase (AMPK) and MTOR (mechanistic target of rapamycin kinase) play opposite roles in autophagy by controlling the activation of ULK1 (unc-51 like autophagy activating kinase 1; a homolog of yeast Atg1) through phosphorylation. Unlike MTOR, which is a repressor of autophagy, the energy sensor AMPK is an autophagy promoter that controls the balance or metabolism between the production and consumption of adenosine triphosphate (ATP). In mammalian cells, MTOR is a component of MTOR complex 1 (MTORC1) and MTORC2, complexes with different functions. Active AMPK binds and inhibits MTORC1, and then stimulates the activating phosphorylation of ULK1, leading to the subsequent formation of the ULK1 complex (containing ULK1, ATG13 [autophagy related 13], ATG101 and RB1CC1 [RB1 inducible coiled-coil 1]). MTORC2 may indirectly suppress autophagy through the activation of MTORC1 [17]. In addition to the ULK1 complex, the activation of the class III phosphatidylinositol 3-kinase (PtdIns3K) complex (containing PIK3C3 [phosphatidylinositol 3-kinase catalytic subunit type 3], PIK3R4 [phosphoinositide 3-kinase regulatory subunit 4], BECN1 [beclin 1], and ATG14), as well as ATG9 (a multispanning membrane protein), work together to control phagophore formation and maturation during autophagy induction.

After phagophore formation, cells further rely on two ubiquitin-like conjugation systems, namely ATG12 and MAP1LC3 (microtubule associated protein 1 light chain 3; also known as Atg8 in yeast), to drive the biogenesis of the autophagosomal membrane. The conjugation reactions and expansion of the phagophore are catalyzed and achieved by a series of ATGs (e.g., the E1 enzyme ATG7 and the E2 enzyme ATG3 or ATG10). Because MAP1LC3 is converted from MAP1LC3-I to MAP1LC3-II, recruited to phagophore membranes, and remains associated with completed autophagosomes, MAP1LC3-II is a widely-used marker for monitoring the numbers of autophagosomes [18]. In addition to the MAP1LC3 subfamily (including MAP1LC3A, MAP1LC3B, MAP1LC3B2 and MAP1LC3C), the GABARAP (GABA type A receptor-associated protein) subfamily (including GABARAP, GABARAPL1, and GABARAPL2) also play a similar role in mediating autophagosome formation and/or maturation [19]. The MAP1LC3 and GABARAP families are collectively referred to as Atg8-family proteins. Although they have different tissue or cell expression specificities, it is still unclear how cells selectively use different members of the MAP1LC3 or GABARAP subfamilies to induce autophagy.

The process of forming autolysosomes is mainly regulated by some non-ATG proteins, such as LAMP2 (lysosomal associated membrane protein 2), soluble NSF attachment protein receptors (SNAREs), and the homotypic fusion and protein sorting/vacuolar protein sorting complex. Finally, the degradation of autophagic substrates, including MAP1LC3-II itself, inside the lysosome requires proteases, acid phosphatases, lipases, and nucleases. Because lysosomes are the intersection of many transport pathways, there may be the possibility of other intracellular transport proteins regulating the formation of the autolysosome.

Overall, the term “autophagic flux” is used to describe the entire dynamic process of autophagy. One of the most common methods for monitoring autophagic flux is to measure the protein level of MAP1LC3-II in the absence or presence of early (e.g., 3-methyladenine and spautin-1) or late (e.g., bafilomycin A₁ and chloroquine [CQ]) autophagy inhibitors [20]. Several fluorescent biosensors, such as mCherry-GFP-LC3, mRFP-GFP-LC3, and GFP-LC3-RFP-LC3∆G, are also widely used in vitro to measure autophagic flux [21]. However, testing autophagy flux in real-time in vivo remains a challenge.

Depending on whether the degradation of autophagy substrates requires a specific autophagy receptor and a ligand to mark the cargo, autophagy is divided into nonselective and selective types [22,23]. The autophagy receptors (e.g., SQSTM1/p62 [sequestosome 1], CALCOCO2/NDP52 [calcium binding and coiled-coil domain 2], and OPTN [optineurin]) act by interacting simultaneously with cargoes and Atg8-family proteins. Generally, the autophagy receptors interact with lipidated MAP1LC3 or GABARAP subfamily proteins by LC3-interacting region (LIR) motifs that bind to the LIR docking site of Atg8-family proteins in a cargo-dependent manner [24]. The increasing identification of different types of selective autophagy indicates that cells may use special pathways to degrade different substrates without affecting other functions. This selectivity may be important for targeting autophagy to treat diseases.

The mechanism of autophagy upregulation in PDAC

PDAC tissues or cells have a higher level of ATG expression or autophagy activity [25] through transcription or post-translational modification. On the one hand, PDAC cells maintain high levels of autophagy and lysosomal activity by decoupling the regulation of the microphthalmia/transcription factor E (MiT/TFE) family of transcription factors from MTOR signaling [26]. The MiT/TFE family can control the expression of several genes involved in autophagy, including genes related to autophagy initiation (e.g., BECN1, UVRAG, and ATG9B), extension (e.g., GABARAP, MAP1LC3B, and ATG5), substrate capture (e.g., SQSTM1), and autophagosome transport and fusion with lysosomes (e.g., RAB7) [27]. On the other hand, PDAC cells effectively dephosphorylate ULK1 through PTPA/PP2A (protein phosphatase 2 phosphatase activator), thereby maintaining a high degree of basal autophagy [28].

Autophagy in pancreatic cancer initiation

The etiology of PDAC is multifaceted, involving chemical exposure, inflammation induction, and gene mutation. A dysfunction in autophagy plays a dual role in inhibiting or promoting pancreatic tumorigenesis mediated by these factors, as described below.

Pancreatic acinar cells are functional units of the exocrine pancreas. Although the origin of PDAC is controversial, acinar cells are the most likely candidate cells for pancreatic tumorigenesis. Since the 1980s, electron microscopy and autoradiography studies have shown that after exposure to carcinogenic nitrosamines, such as methylnitrosourea/MNU, N-methyl-N’-nitro-N-nitrosoguanidine/MNNG, and N-nitrosobis(2-hydroxypropyl)amine, increased autophagy is related to cell damage, dysplastic morphology, and metaplasia in pancreatic acinar cells [29,30]. In a chemical carcinogenic model in which Wistar rats were fed with azamatrine, the autophagy capacity is also increased in pancreatic premalignant atypical acinar nodule cells [31]. Later, short-term exposure to cigarette smoke extract or smoking compounds, such as 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone/NNK, were shown to induce autophagy by the activation of AMPK to protect pancreatic ductal cells from damaged organelles or gene alterations during tumorigenesis [32]. However, long-term exposure to smoking compounds limits apoptosis and autophagy, therefore promoting tumor formation in mice [32]. Similarly, MAGEA6 (MAGE family member A6; a bioenergy sensor) suppresses autophagy and then accelerates the initiation of PDAC in mice [33]. In contrast, when nutrients are depleted, the UPS-mediated degradation of MEGA6 may restore autophagy, leading to the subsequent development of PDAC in vitro and in vivo [33]. The restriction of autophagy can be eliminated upon nutrient depletion by degrading MAGEA6 through the ubiquitin-proteasome system, thereby further supporting tumor progression [33]. These studies highlight the effect of autophagy on the development of pancreatic tumors caused by different chemical risk factors or mutant protein expression that can promote cancer depending on the exposure time or tumor stage.

Inflammation is considered an extrinsic factor and plays an indispensable role in the development of pancreatic tumors. Autophagy has a complex role in regulating inflammation during pancreatic cancer initiation. During the activation of oncogenic signaling (e.g., gene mutation and inflammation), a pathological change called acinar-ductal metaplasia (ADM) is the precursor of PanIN. The conditional knockout of Atg7 in pancreatic epithelial cells (Pdx1-Cre;atg7^fl/fl) leads to severe degeneration of acinar cells [34], accompanied by the formation of ADM through ER stress, mitochondrial dysfunction, and impaired protein synthesis [34]. Atg7 depletion in the pancreas (Pdx1-Cre;atg7^fl/fl) also leads to inflammation and fibrosis in mice by regulating cell death signals [35], indicating the potential role of ATG7-mediated inflammation regulation in the initiation of pancreatic cancer.

Autophagy plays a carcinogenic role in KRAS-driven pancreatic tumorigenesis by increasing the TBK1 (TANK binding kinase 1)-mediated dysplasia [36,37]. However, autophagy inhibitors or Atg5 deletion (Pdx1-Cre;atg5^fl/fl) exacerbate pancreatitis and dysplasia because increased autophagy reduces TBK1-mediated inflammation in mice [37]. Therefore, autophagy plays a dual role in the process of inflammation-related pancreatic tumorigenesis. HMGB1 (high mobility group box 1) is a DNA-binding protein and damage-associated molecular pattern (DAMP) molecule that promotes autophagy through a variety of mechanisms [38]. Similar to the conditional knockout of Atg7 in the pancreas, the conditional knockout of Hmgb1 in the pancreas (Pdx1-Cre;hmgb1^fl/fl) also accelerates inflammatory injury and inflammation-mediated KRAS activation and pancreatic tumors in mice [39].

Some proteins control autophagic flux through a protein-protein interaction to regulate the occurrence of pancreatic cancer. For example, the expression of VMP1 (vacuolar membrane protein 1), a KRAS-mediated ER-related membrane protein, promotes the initiation of PDAC, which is related to enhanced autophagy through the formation of the VMP1-BECN1 complex in cells [40]. In contrast, the administration of CQ not only inhibits autophagy flux, but also reverses PanIN formation induced by VMP1 overexpression in Pdx1-Cre;Kras^G12D;vmp1 mice [41], although CQ may have other functions independent of autophagy inhibition [42]. Similarly, accumulation of the autophagy receptor SQSTM1 accelerates PanIN to PDAC progression and results in shorter survival in mice [43]. The activation of NFE2L2/NRF2 (nuclear factor, erythroid 2 like 2) is regulated by SQSTM1-mediated KEAP1 (kelch like ECH associated protein 1) degradation in an autophagy-dependent manner [44,45]. SQSTM1-mediated NFE2L2 activation further promotes MDM2 (MDM2 proto-oncogene) expression, which leads to cell proliferation by stimulating NOTCH (notch receptor) and suppressing TP53 signaling in vitro and in vivo [43]. In addition to NFE2L2, it remains unknown whether other types of SQSTM1-mediated protein degradation contribute to this KRAS-driven PDAC.

Although TP53 mutations accelerate KRAS-mediated pancreatic tumorigenesis, the function of TP53 in autophagy depends on the tumor stage [46]. Genetic ablation of pancreatic autophagy leads to increased tumor cell initiation, whereas inhibition of autophagy by CQ or RNAi limits the pancreatic tumor growth in a TP53-independent manner [47]. The conditional knockout of Atg7 in mice accelerates the formation of KRAS-driven PanINs in the early stages of progression but limits the development of PDAC by inducing apoptosis in the later stages [48], indicating a different role for autophagy in tumor formation and development. In addition, combined knockout of Tp53 (we refer to both the human TP53 and mouse Trp53 gene as TP53 for simplicity) and the inhibition of autophagy (by knockout of Atg7 or Atg5, or using CQ) lead to the rapid death of mice with KRAS mutations [48]. The onset of tumors in these mice accelerate, and the evolving tumors are metabolically rewired [48]. When autophagy is insufficient, PDAC genetically engineered mouse models with homozygous Tp53 deletion or loss of heterozygosity/LOH may exhibit a tumor-promoting phenotype [49]. Similar to TP53, PTEN (phosphatase and tensin homolog) also functions as a tumor suppressor, and its loss has been observed in PDAC. The conditional knockout of Atg7 with Pten hemizygosity in the pancreas promotes tumor formation and earlier death in mice [50]. Collectively, these findings indicate a complex relationship between KRAS, TP53, PTEN, autophagy, and apoptosis, which affects tumor development [48,50] Figure 2.

Figure 2. Dual role of autophagy regulators in KRAS-driven pancreatic tumorigenesis in mice. Abbreviations: ATG, autophagy related; AGER, advanced glycosylation end-product specific receptor; BNIP3L/NIX, BCL2 interacting protein 3 like; HMGB1, high mobility group box 1; PINK, PTEN induced kinase 1; PRKN, parkin RBR E3 ubiquitin protein ligase; PTEN, phosphatase and tensin homolog; TP53, tumor protein p53

Mitophagy is the selective degradation of mitochondria by autophagy, and this process is regulated by different mitochondrial or cytosolic proteins, such as PINK1 (PTEN induced kinase 1), PRKN/PARK2 (parkin RBR E3 ubiquitin protein ligase), and BNIP3L/NIX (BCL2 interacting protein 3 like) [51]. Interestingly, the knockout of Pink1 or Prkn accelerates, and the depletion of Bnip3l prevents, KRAS-driven PDAC in mice by regulating iron or redox homeostasis, respectively [52,53]. These proteins also participate in mitochondrial quality control in a mitophagy-independent manner. The differences in the phenotypes of PINK1, PRKN, and BNIP3L in KRAS-driven PDAC animal models indicate that their regulation of pancreatic tumorigenesis may depend on their non-mitophagic function.

Cancer stem cells (CSC) are a small part of cells that have the ability to self-renew and differentiate in tumors. KRAS^G12D ablation-resistant pancreatic cancer cells exhibit CSC-like phenotype by increasing mitochondrial function, autophagy, and lysosome activity [54]. Mitophagy appears to affect CSC function in the PDAC tumor microenvironment. This process is partially mediated by ISG15 (ISG15 ubiquitin like modifier), a regulator for mitochondrial dynamics and metabolism in CSCs [55]. The loss of ISG15 leads to impaired mitophagy, resulting in mitochondrial dysfunction and reduced energy production, which ultimately reduces the stemness and metabolic plasticity of CSCs in vitro and xenograft models [55]. However, whether ISG15 plays a role in the regulation of mitophagy in non-CSCs is unclear.

Autophagy in pancreatic cancer progression

Autophagy also plays dual roles in the progression of PDAC by modulating proliferation, invasion and metastasis, metabolism, cell death, or immunity. Established pancreatic cancer has a high level of autophagy in the basal state in pancreatic cancer cell lines and patient tumor tissues [56]. Early studies show that the genetic (e.g., atg7^−/- or hmgb1^−/-) and pharmacological inhibition (e.g., using CQ) of autophagy lead to the generation of reactive oxygen species (ROS), increased DNA damage and metabolic dysfunction, which ultimately inhibits the development of pancreatic cancer in vitro and in vivo, indicating that increased autophagy contributes to the PDAC progression [39,56]. Recent studies suggest that the enhancement of autophagy may also inhibit the progress of PDAC. Below, we discuss the complex autophagy signal network that regulates the features associated with PDAC progression.

Pancreatic stroma

The progression of pancreatic cancer is accompanied by a fibrotic stromal (desmoplastic) response, and pancreatic stellate cells (PSCs) are the main source of collagen in pancreatic stroma. In addition to acinar cells, an increase in autophagy is related to the function of PSCs for PDAC progression in preclinical models or clinical samples [57]. The autophagy level of PSCs in tumor tissue is higher than that of pancreatitis, which may lead to higher expression of extracellular matrix and pro-inflammatory IL6 (interleukin 6) in PSCs [58] Figure 3. Autophagy inhibitors put PSCs in a quiescent state and reduce the expression of extracellular matrix and IL6, thereby inhibiting inflammation and pancreatic tumor growth in cell culture and mice [58]. IL6 is elevated in the serum of patients with pancreatic cancer and is an important proinflammatory and growth signal that activates the STAT3 (signal transducer and activator of transcription 3) pathway during the initiation and development of pancreatic tumors [59,60]. In addition, autophagy-mediated secretion of nutrients from PSCs promotes the metabolism of PDAC in xenograft models [61], highlighting that autophagy mediates metabolic crosstalk between cancer and non-cancer cells in the pancreatic tumor microenvironment.

Figure 3. Role of autophagy in the pancreatic cancer microenvironment. The microenvironment of pancreatic cancer has unique characteristics and increased desmoplastic reactions, and is infiltrated by various immune or neural cells. Communication between cells in the microenvironment of pancreatic tumors depends on increased autophagy-dependent release or degradation of mediators (e.g., IL6, ECM, alanine, MHC-I, and exosomes), which ultimately promote cancer cell proliferation, invasion, or immune escape. Abbreviations: ARG1, arginase 1; ECM, extracellular matrix; IL6, interleukin 6; IL10, interleukin 10; KRAS, KRAS proto-oncogene, GTPase; MHC-I, major histocompatibility complex-I; NBR1, NBR1 autophagy cargo receptor; PDAC, pancreatic ductal adenocarcinoma; PDGF, platelet derived growth factor; PSC, pancreatic stellate cell; RAB27A, RAB27A, member RAS oncogene family; STAT3, signal transducer and activator of transcription 3; T cell, T lymphocyte cell; TCA cycle, tricarboxylic acid cycle; TGFB1, transforming growth factor beta 1

Cell proliferation

Two hallmarks of PDAC are that its proliferation signals are maintained and the growth of cancer cells is promoted. MicroRNAs (MIRs), such as MIR506 and MIR372, inhibit pancreatic cancer growth through targeting BECN1 or ULK1 expression in vitro, respectively [62,63], indicating the importance of MIRs in the modulation of autophagy-related PDAC. This process is further regulated by long noncoding RNAs (lncRNAs), a type of RNA that exceeds 200 nucleotides in length. For example, lncRNA PVT1 competitively replaces the MIR20A-5p sponge, thereby upregulating ULK1, and subsequently induces proliferation and development of PDAC by stimulating autophagy in cell culture and xenograft animal models [64]. MDH1 (malate dehydrogenase 1), an enzyme that catalyzes the oxidation of malate to oxaloacetate in many metabolic pathways, promotes the proliferation of PDAC cells by maintaining ULK1 expression and subsequent autophagy in a hypoxic environment [65]. Unlike MDH1, MMP7 (matrix metallopeptidase 7), a member of a family of zinc-containing enzymes capable of degrading various components of the extracellular matrix, increases autophagic flux and cell proliferation in hypoxic PDAC cells by activating YAP1 (Yes-associated protein 1) [65], indicating that the Hippo signaling pathway may sustain proliferation signals through autophagy under hypoxic conditions.

The regulation of ULK1 protein stability by the UPS affects proliferation signals during PDAC development. The NEDD4L (NEDD4 like E3 ubiquitin protein ligase), an E3 ubiquitin protein ligase, is responsible for ULK1 degradation in PDAC cells [66]. In contrast, the knockout of NEDD4L maintains the level of ULK1, leading to autophagy activation, followed by enhanced mitochondrial respiratory and PDAC cell proliferation [66]. These findings further indicate that ULK1 is an important regulator of autophagy-related cell proliferation in PDAC cells. Collectively, the expression of ULK1 is regulated not only by gene transcription but also by protein degradation pathways, thereby affecting the formation of autophagy-inducing signals.

Immune receptors are also involved in the regulation of autophagy-related proliferation. AGER/RAGE (advanced glycosylation end-product specific receptor) is a member of the immunoglobulin superfamily of cell surface molecules [67]. AGER promotes autophagy by activating the class III PtdIns3K complex, thereby promoting the survival and anti-apoptotic ability of PDAC cells [68]. Moreover, AGER expression promotes ATG5-dependent autophagy, leading to proinflammatory IL6 production and subsequent activation of mitochondrial STAT3 and the production of ATP in vitro [69]. Consequently, the absence of AGER restricts autophagy and STAT3 signaling, thereby inhibiting the development of Kras^G12D/+-driven PDAC in mice [69] Figure 2, indicating that AGER-dependent autophagy promotes PDAC growth by maintaining energy production. AGER mediates the MAPK (mitogen-activated protein kinase) cascade, including MAP2K/MEK (mitogen-activated protein kinase kinase) and MAPK1/ERK1 kinase (mitogen-activated protein kinase 1), which may directly sustain KRAS-mediated proliferative capacity in PDAC cells and mouse models by activating autophagy [70,71]. Additionally, AGER-dependent autophagy increases the formation of neutrophil extracellular traps in animal models of PDAC and clinical patients, which may sustain a hypoxic and inflammatory tumor microenvironment [72]. These findings support a pathological role of AGER in PDAC by regulating autophagy, metabolism, and inflammation.

SHOC2 (SHOC2 leucine rich repeat scaffold protein) is a positive regulator of RAS-MAPK signaling. SHOC2 inhibits MTOR1 activation through RPTOR/raptor (regulatory associated protein of MTOR complex 1), which ultimately causes autophagy and subsequent proliferation of PDAC cells. Excessive RPTOR that competitively binds SHOC2 also blocks MAPK activation, thereby limiting tumor growth [73], indicating that the balance between SHOC2 and RPTOR affects RAS signaling and autophagy during the proliferation of PDAC cells. The tumor suppressor EI24 (EI24 autophagy associated transmembrane protein) is a component of the autophagy pathway that promotes proliferation of PDAC cells in vitro and in vivo [74]. These findings indicate that both oncogenes and tumor suppressors can regulate the autophagy-related proliferation process.

MALAT1 (metastasis associated lung adenocarcinoma transcript 1) is an lncRNA that promotes the proliferation and metastasis of PDAC cells by regulating TIA1 (TIA1 cytotoxic granule associated RNA binding protein) or ELAVL1/HuR (ELAV like RNA binding protein 1) function to induce autophagic flux [75]. Unlike AGER- or MALAT1-mediated autophagy activation, TP53BP2/ASPP2 (tumor protein p53 binding protein 2)-mediated autophagy inhibition limits cell proliferation in various PDAC cell lines by modulating the AMPK-MTOR pathway [76], although the role of TP53BP2 in autophagy in vivo remains unclear. Autophagy inhibition mediated by MAGEA3 (MAGE family member A3; a tumor-specific antigen extensively expressed in solid and hematological malignancies) may promote PDAC development through an unknown mechanism in vitro and in vivo [77]. These findings further support a dual role of autophagy in tumor biology.

Other cells in the tumor microenvironment, such as CSCs, also promote tumor proliferation and development by activating autophagy [58,78]. A high level of autophagy seems to be widely associated with the presence of a stem cell-like phenotype in PDAC, namely stemness [79]. The exact mechanism by which autophagy regulates the stemness, such as through the presence of specific degradation substrates, requires further study.

Cell invasion and metastasis

Cancer cells have a strong ability to invade and metastasize, which is not only an indispensable biological process for tumor progression, but also a major cause of cancer-related death in patients. A large amount of evidence indicates that abnormal autophagy is related to the invasion and metastasis of PDAC in various models. For example, the protein level of ATG5 is positively related to the invasiveness of human pancreatic cancer, whereas the deletion of Atg5 inhibits tumor proliferation and metastasis in PDAC animal models [80]. Unlike the core autophagy component ATG5, TGM2/TG2 (transglutaminase 2; a calcium-dependent enzyme of the transglutaminase family) is a negative regulator of autophagy involved in promoting metastasis in PDAC cells [81]. The increase of TGM2 expression in PDAC cells is mediated by the activation of PRKCD/PKCδ (protein kinase C delta), and the inhibition of TGM2 by PRKCD inhibitors leads to the excessive activation of BECN1-dependent autophagy, thus causing cell death and metastasis inhibition in mice [81,82].

TGFB (transforming growth factor beta) plays an important role in advanced tumors, including promoting tumor metastasis and drug resistance. The autophagy induction effects of TGFB1 (transforming growth factor beta 1) are mediated by TFEB (transcription factor EB), which promotes the migration and metastasis in SMAD4-positive PDAC cells, but not SMAD4-negative cells [83]. The role of TGFB1 in autophagy also depends on the SMAD4 expression level [84]. TGFB1-induced autophagy promotes the proliferation of SMAD4-positive PDAC cells and inhibits their migration [84]. In contrast, TGFB1-induced autophagy inhibits the proliferation of SMAD4-negative PDAC cells and promotes their migration by activating MAPK1 [84]. These findings suggest that SMAD4 may be a checkpoint that distinguishes the different roles of autophagy in TGF family-induced proliferation and migration.

RALGAP (Ral GTPase activating protein) and UBL4A (ubiquitin like 4A) also regulate autophagy-related invasion and metastasis in PDAC through different mechanisms [85,86]. RALGAP is a negative regulator of autophagy by activating MTOR, leading to a decrease in the ability of PDAC cells to migrate and invade [85]. UBL4A inhibits the proliferation and metastasis of PDAC cells by binding to LAMP1 (lysosomal associated membrane protein 1), resulting in lysosomal dysfunction and autophagy inhibition [86]. In addition, QKI (QKI, KH domain containing RNA binding) and MAPK1 play a cell-dependent role in the activation of autophagy in fibroblasts and PSCs, respectively, mediating the mutual interference between stromal cells and cancer cells, which ultimately leads to proliferation, metastasis, and invasion of PDAC cells in vitro, organoid cultures, and xenograft mouse models [87,88]. These findings further suggest that increased autophagy in cells of the tumor microenvironment regulates PDAC invasion.

The epithelial-mesenchymal transition (EMT) is a process in which epithelial cells acquire interstitial characteristics that participate in promoting metastatic events. It is worth noting that, in some cases, the inhibition of autophagy may specifically activate the EMT program in KRAS-mutated PDAC cells [89]. Mechanistically, autophagy inhibition promotes the upregulation of SQSTM1-mediated NFKB/NF-κB (nuclear factor kappa B) activation, thereby increasing the expression of EMT promoters, such as SNAI2 (snail family transcriptional repressor 2) and ZEB1 (zinc finger E-box binding homeobox 1) [89]. These studies also indicate a dual role of autophagy in invasion and metastasis of PDAC.

Cell metabolism

PDAC cells are exposed to an environment with insufficient blood vessels. Therefore, when oxygen and nutrients are exhausted, it is critical for cells to obtain sufficient energy through certain metabolic pathways. In fact, PDAC relies on autophagy to provide enough bioenergy intermediates to maintain metabolism and energy homeostasis through fine-tuned mechanisms. On the one hand, MTORC1 promotes biosynthetic processes and inhibits catabolic pathways, such as autophagy, which is considered a key process for cells to adapt to an environment [90]. TFEB promotes the recruitment of MTORC1 to lysosomes both in vitro and in vivo through regulating the expression of RRAGD (Ras related GTP binding D) [91]. TFEB also promotes gene expression to maintain the metabolic function of lysosomes, which is critical for metabolic adaptation to the availability of nutrients and to support the metabolic requirements during PDAC proliferation [26,91]. On the other hand, excessive autophagy may trigger stress-mediated cell death by activating the NUPR1 (nuclear protein 1, transcriptional regulator)-AURKA (aurora kinase A) pathway during hypoxia and glucose starvation in MiaPACA2, PANC1, and BxPC3 cells [92]. These studies describe the complex relationship between autophagy and PDAC metabolism, and highlight new areas where drug combinations targeting metabolism and autophagy may yield greater benefits. Below, we focus on how autophagy regulates oxidative stress, glucose metabolism, and amino acid metabolism in PDAC cells Figure 4.

Figure 4. Autophagy maintains the metabolism and function of PDAC cells. The autophagy pathway is modulated by different metabolic conditions (e.g., oxidative stress, low glucose, high glucose, and low amino acids) in which cellular components are degraded. During this process, bioenergy intermediates are reused and oxidants are elimated, thereby promoting cell survival. Abbreviations: AGER, advanced glycosylation end-product specific receptor; AMPK, AMP-activated protein kinase; BECN1, beclin 1; GPX1, glutathione peroxidase 1; HMGB1, high mobility group box 1; MTOR, mechanistic target of rapamycin kinase; NFE2L2, nuclear factor, erythroid 2 like 2; NFKB, nuclear factor kappa B; PKM2, M2 splice isoform of PKM (pyruvate kinase M1/2); PINK, PTEN induced kinase 1; PRKN, parkin RBR E3 ubiquitin protein ligase; ROS, reactive oxygen species; SREBF1, sterol regulatory element binding transcription factor 1; STAT3, signal transducer and activator of transcription 3; ULK1, unc-51 like autophagy activating kinase 1

Oxidative stress

Oxidative stress refers to increased levels of ROS in cells, resulting in damage to lipids, proteins, and DNA. It has been associated with various pathologies, including PDAC progression. NFE2L2 is the main antioxidant transcription factor of cells in response to oxidative stress. Because ROS is the initiation signal of autophagy by suppressing MTORC1 activation [93], NFE2L2 plays a role in the modulation of autophagic flux in PDAC cells [94]. Blocking autophagy leads to increased stability of NFE2L2, whereas silencing NFE2L2 enhances the autophagic flux by promoting the formation of autolysosomes under conditions of ROS stimulation [94]. As a danger signal, the release of HMGB1 during oxidative stress can trigger autophagy by binding to AGER in PDAC cells, which contributes to energy metabolism, inflammation, and tumor growth [38,95].

The mucin family consists of glycoproteins that form a physical barrier and protects cancer cells from various types of damage. The hypoxia-mediated abnormal expression of MUC1 (mucin 1, cell surface associated) and MUC4 (mucin 4, cell surface associated) is implicated in PDAC cells growth in vitro [96]. However, ROS-induced autophagy mediates MUC4 degradation and may provide metabolites for tumor cell growth [97]. Although the metal ion-induced Fenton response may increase ROS production, preventing copper absorption also promotes mitochondrial ROS production, which may activate autophagy to protect PDAC cells from death [98]. The PINK1-PRKN pathway protects against mitochondrial oxidative injury via the autophagic degradation of mitochondrial iron importers, such as SLC25A37/mitoferrin-1 (solute carrier family 25 member 37) and SLC25A28/mitoferrin-2 (solute carrier family 25 member 28), thereby inhibiting the occurrence of pancreatic tumors [52]. In addition, the depletion of MYOF (myoferlin; a ferlin family member protein overexpressed in many types of cancer) causes mitochondrial fission, which reduces energy production and triggers autophagy in PDAC cells [99]. Overall, there is an integrated network between mitochondria, ROS, and autophagy in PDAC.

Glucose metabolism

The pancreatic cancer microenvironment has limited nutrients because of its dense stroma tissues and poor blood vessel formation. This characteristic of the microenvironment makes glucose an essential nutrient for PDAC under hypoxic conditions because it can be used as a basic fuel for anaerobic metabolism. Therefore, PDAC cells use autophagy-mediated glycolysis to resist death caused by glucose deprivation [100]. For example, MIR7 may decrease autophagy-derived pools of glucose in PDAC cells by modulation of the activation of the STK11/LKB1 (serine/threonine kinase 11)-AMPK-MTOR pathway [101]. Glucose deprivation also induces autophagy in PDAC cells through ROS, thereby maintaining cancer cell survival [102]. In contrast, GPX1 (glutathione peroxidase 1) inhibits glucose deprivation-induced autophagy by limiting ROS production [102]. Although these findings indicate that GPX1 acts as an anti-autophagic protein in PDAC, the role of other members of the GPX family in PDAC remains unknown.

Several glycolytic genes play a role in the regulation of metabolism-related autophagy. PKM2 (an M2 splice isoform of PKM [pyruvate kinase M1/2]) catalyzes the last step of glycolysis under aerobic conditions, and its expression is downregulated by low glucose in PDAC cells or the subvascular area of PDAC tissues [103]. As a survival mechanism, reduced PKM2 may promote metabolic flux by increasing AMPK expression, thereby promoting low glucose-induced autophagy in PDAC cells [103]. Autophagy positively regulates the expression of mitochondrial GFM1 (G elongation factor mitochondrial 1), providing essential nutrients for cells lacking glycolysis [104,105]. In addition to hypoglycemia, hyperglycemia also triggers autophagy for PDAC tumor growth by upregulating the expression of SREBF1/SREBP1 (sterol regulatory element binding transcription factor 1; an intracellular cholesterol sensor) in vitro and in vivo [106]. These findings indicate that glucose or lipid abnormalities are important signals for autophagy induction.

In addition, KRAS-mediated expression of the lysosomal protein PLAC8 (placenta associated 8) is involved in the formation of autolysosomes, which may maintain the metabolic requirement of PDAC cells during tumor progression [107]. Of note, the acute inhibition of KRAS or its downstream effector MAPK1 also increases the autophagy flux in KRAS-mutant human pancreatic cancer lines and Kras^G12D-driven mouse models [108], indicating that any changes in KRAS levels may lead to autophagy in PDAC. Therefore, even under low-glucose or hypoxic conditions, the combination of inhibiting KRAS signaling (e.g., using MAPK1 inhibitors) and autophagy signaling (e.g., using CQ) may become a powerful anticancer strategy for PDAC [71,108]. The functional role of HIF1A (hypoxia inducible factor 1 subunit alpha; the main regulator of hypoxia) is also expected to regulate autophagy by modulating KRAS and glucose signaling as a feedback mechanism.

Amino acid metabolism

During the development of PDAC, certain amino acids (e.g., branched-chain amino acids and glutamine) undergo significant changes, providing nutrition for cancer cell proliferation and invasion. Compared with the pharmacological inhibition of MTORC1, amino acid starvation is a powerful condition to induce autophagy in PDAC cells by activating ULK1 [28]. The increase in autophagy plays a protective role in preserving the energy deficiency caused by amino acid starvation.

Glutamine is an important source of nitrogen and carbon in cancer cells and produces energy through mitochondria [109,110]. Glutamine is used to produce the tricarboxylic acid cycle intermediate alpha-ketoglutarate (αKG) through a series of mitochondrial enzymes (e.g., GLUD [glutamate dehydrogenase], GOT1 [glutamic-oxaloacetic transaminase 1], and GOT2 [glutamic-oxaloacetic transaminase 2]) to maintain the mitochondrial carbon pool. This biochemical process is called glutaminolysis and is one of the anti-aging strategies of metabolic stress [111]. Human PDAC cells rely on GOT1-mediated glutaminolysis to obtain glutamine to promote tumor growth and development [112]. Reprogramming of glutamine metabolism in PDAC is further regulated by oncogenic KRAS-mediated transcriptional regulation [112]. Increased glutaminolysis-mediated αKG production leads to MTORC1 lysosomal translocation and activation by stimulating GTP loading of RRAGB (Ras related GTP binding B) and lysosomal translocation, thereby inhibiting autophagy [113]. As a defense mechanism, the autophagy-mediated degradation of cellular materials provides amino acids (including glutamine) for reuse in PDAC cells during glutamine deprivation [114]. Under the latter conditions, autophagy activation is further regulated by TFEB-induced macropinocytosis (a type of endocytosis involving a nonspecific uptake of extracellular materials, such as nutrients) [114,115]. Abnormal glutamine enhanced by glutaminolysis can also lead to proliferation inhibition and apoptosis of PDAC cell lines by activating MTORC1 and consequently inhibiting autophagy [116]. These findings provide a framework for explaining the dynamic balance between glutamine metabolism, autophagy, and apoptosis in PDAC cells.

Branched-chain amino acids contain three essential amino acids (leucine, isoleucine, and valine), which cannot be synthesized in cells, so they must be obtained from food. Leucine is a branched-chain amino acid and is described as an MTOR agonist that promotes PDAC growth. Leucine reuse depends on the cooperation between autophagy and lysosomal proteolysis in human PDAC cell lines and murine pancreatic tumor cells isolated from Kras^G12D/+;tp53^−/− mice [117]. SLC38A9 (solute carrier family 38 member 9) is not only a lysosomal arginine sensor, but also a high-affinity transporter for lysosomal proteolysis-produced leucine to exit lysosomes and activate MTORC1 in PDAC cells [117]. Because SLC38A9 is a part of the lysosomal amino acid-sensing machinery, it is unclear whether other components play a similar role in the regulation of PDAC leucine metabolism.

PSCs in the tumor microenvironment also participate in the interaction between autophagy and amino acid metabolism to promote the growth of PDAC cells [61]. Autophagy-mediated alanine release in PSCs can be used by PDAC cells, which promotes the tricarboxylic acid cycle or the biosynthesis of nonessential amino acids and lipids, possibly through the transamination of alanine into pyruvate Figure 3 [61]. This autophagy-mediated feedback mechanism between PSCs and cancer cells supports the nutritional requirements of pancreatic cancer and promotes tumor growth [61]. Autophagy can also mediate protein release in a nonclassical manner under certain circumstances. Whether autophagy releases endogenous proteins to mediate crosstalk between cells within the tumor microenvironment remains to be understood.

Cell death

Historically, autophagy has been defined as a type of cell death based on morphological classification [118]. Currently, autophagy plays a dual role in cell death and survival, depending on whether the degradation substrate is an anti-injury or pro-injury protein [119]. Induced autophagy is common and is triggered by various cell death stimuli, such as metabolic stress, oxidative damage, and anticancer drugs. It is worth noting that the term “autophagic cell death” is used to describe the phenomenon of increased autophagy accompanied by cell death, and the term “autophagy-dependent cell death” is a type of cell death that depends on the autophagy machinery [120]. The ability to resist cell death is a hallmark of cancer and can be regulated by autophagy that depends on stimulation [10].

Apoptosis

Apoptosis represents the main type of regulated cell death, including death due to the classical caspase family-mediated cell death receptor or mitochondrial pathway. Although targeted apoptosis (e.g., using gemcitabine) is the most commonly used nonsurgical treatment for PDAC, the PDAC cells can develop different mechanisms, including autophagy, to resist apoptosis [68]. For example, 3-methyladenine (3 MA), an inhibitor of both the class I phosphoinositide 3-kinase and the class III PtdIns3K, inhibits autophagy and induces apoptosis following H₂O₂ exposure in PDAC cells [121], indicating an anti-apoptosis role of autophagy. As mentioned above, AGER is an autophagy promoter in PDAC cells. NFKB pathway-mediated AGER upregulation increases autophagy flux and limits apoptosis in PDAC cells in response to oxidative stress or chemotherapy (e.g., gemcitabine and oxaliplatin) [68]. In contrast, the knockdown of AGER inhibits autophagy in PDAC cells by activating MTORC1 or inhibiting BECN1, while increasing mitochondrial apoptosis by inducing TP53 phosphorylation [68]. AGER also binds to mutant KRAS to maintain HIF1A activation in PDAC cells under hypoxic conditions [122]. The inhibition of AGER expression by the compound scalarin inhibits autophagy and limits pancreatic tumor growth in PANC1 and MiaPACA2 cell lines [123]. Although these studies indicate that AGER is a potential molecular switch between autophagy and apoptosis in a hypoxic pancreatic tumor microenvironment, it is unclear whether AGER-dependent autophagy inhibits apoptosis by directly degrading caspase effectors.

UCPs (uncoupling proteins) are a superfamily of mitochondrial carrier proteins anchored on the inner mitochondrial membrane. These proteins are highly expressed in various tumors (including PDAC) and mediate tumorigenesis and development [124]. UCP2 promotes autophagy by inhibiting the AKT-MTOR pathway and activating BECN1, thereby inhibiting apoptosis or other types of cell death caused by ROS accumulation [125]. The proteasome inhibitor bortezomib used clinically shows anticancer activity by inducing ROS-dependent apoptosis [126]. Similar to blocking NFE2L2, autophagy inhibitors also enhance bortezomib-induced apoptosis in PDAC cells [127], indicating that autophagy plays a pro-survival role in the regulation of bortezomib activity.

Epigenetic regulators, such as USP22 (ubiquitin specific peptidase 22) and PTBP3 (polypyrimidine tract binding protein 3), are implicated in autophagy-mediated gemcitabine resistance [128,129]. USP22 is a component of Spt-Ada-Gcn5-acetyltransferase/SAGA, a transcription regulation histone acetylation complex that regulates gene transcription at the epigenetic level, thereby promoting cell survival and apoptosis resistance [130]. In PDAC patients, USP22 expression in tumors is associated with increased MAP1LC3 expression and poor prognosis. In PDAC cell lines, USP22 contributes to cell proliferation and gemcitabine resistance by activating MAPK1-mediated autophagy [128]. Similarly, the RNA-splicing protein PTBP3 is also upregulated in tumors of PDAC patients as well as in animal models [129]. PTBP3-mediated autophagy under hypoxic conditions leads to subsequent gemcitabine resistance of PDAC cells [129]. However, the direct target genes of these epigenetic regulators mediate autophagy, and subsequent resistance to chemotherapy remains unknown.

Compared with cancer cells, pancreatic CSCs are more resistant to gemcitabine-induced apoptosis [131]. This process may be mediated by increased autophagic flux under hypoxic conditions [132]. In contrast, the administration of autophagy inhibitors may trigger apoptosis in hypoxic CSCs, and ultimately suppresses the development of PDAC in mouse models [132]. In addition to CSCs, blocking autophagy also enhances apoptosis in PSCs during chemotherapy in vitro and in vivo [133]. Therefore, targeting autophagy is an attractive strategy to inhibit self-renewed CSCs and activated fibroblasts in the pancreatic tumor microenvironment.

Necroptosis

Necroptosis is an alternative type of regulated cell death, mimicking the morphological characteristics of apoptosis and necrosis [134]. During classic necroptosis, activated RIPK1 (receptor interacting serine/threonine kinase 1) and RIPK3 (receptor interacting serine/threonine kinase 3) interact with other proteins to produce the necrosome, which then induces MLKL (mixed lineage kinase domain like pseudokinase) to form large octamers. The oligomeric MLKL translocates to the plasma membrane and forms pores that damage the membrane, and then releases intracellular contents (namely DAMPs), affecting the surrounding cells and environment [135]. These necroptosis regulators are not only highly expressed in human and murine PDAC, but can also be induced by the chemotherapeutic agent gemcitabine.

Dysfunctional necroptosis plays a dual role in PDAC, which may be affected by the status of autophagy [136]. On the one hand, RIPK1-RIPK3-driven necroptosis induces adaptive immunosuppression by the release of DAMPs (e.g., CXCL1 [C-X-C motif chemokine ligand 1], CXCL5 [C-X-C motif chemokine ligand 5], and SAP130 [Sin3A associated protein 130]) that mediate pancreatic tumorigenesis [137]. In addition, the depletion of RIPK3 results in mice with conditional deletion of the essential autophagy gene Atg7 being more sensitive to experimental pancreatitis [35]. Although pancreatitis is a recognized risk factor for pancreatic cancer, it is unclear whether knocking out RIPK3 or other necroptotic regulators will affect pancreatic ATG7 depletion-mediated tumorigenesis. On the other hand, necroptosis-based cancer treatment has been proposed as a strategy to eliminate PDAC when caspase is inhibited or defective. For example, shikonin (a naphthoquinone extracted from Lithospermum) is an inhibitor of PKM2 and can trigger different types of cell death, including RIPK3-dependent necroptosis in PDAC cells [138]. In the absence of glucose, decreased PKM2 promotes survival by inducing autophagy in PDAC [103]. These findings support the notion that autophagy defects can enhance necroptosis, and this process is regulated by PKM2-mediated glycolysis.

In addition to regulating mitotic spindle formation and chromosome segregation, AURKA is also a positive regulator of autophagy in several diseases, including cancer. Pharmacological inhibition of AURKA is one of the strategies for treating tumors and shows promising clinical efficacy. CCT137690 is an AURKA inhibitor and exhibits strong anticancer activity in various PDAC tumor models by inducing necroptosis, but not other types of cell death [139]. AURKA acts as a negative regulator of necroptosis in PDAC cells through the phosphorylation of GSK3B (glycogen synthase kinase 3 beta), thereby inhibiting the phosphorylation of RIPK3 and MLKL [139]. The release of DAMPs (e.g., HMGB1 and ATP) by CCT137690 also improves the antitumor T-cell immunity in the established KRAS-driven PDAC [139]. Thus, DAMPs may play a dual role in the microenvironment of pancreatic tumors, not only inhibiting but also enhancing antitumor immunity.

Ferroptosis

Activated autophagy generally promotes survival in response to stimulation of apoptosis or necrosis. However, in cancer and noncancer cells under the treatment of ferroptosis inducers, the enhancement of autophagy may switch from a pro-survival function into a pro-death function [140–142]. Ferroptosis was initially identified as a mutant RAS-dependent cancer cell death [143], relying on iron accumulation and lipid peroxidation [144]. Although the exact effector of ferroptosis is unknown, it can be specifically induced by small-molecule compounds (e.g., erastin or RSL3) targeting antioxidant SLC7A11 (solute carrier family 7 member 11) or GPX4 (glutathione peroxidase 4), respectively [145]. Consequently, increased ROS production triggers oxidative damage, including lipid peroxidation, which finally causes membrane damage and the release of DAMPs [146]. The interplay between autophagy and lipid metabolism is complex [147]. Several types of selective autophagy (e.g., ferritinophagy [148], lipophagy [149], and clockophagy [150,151]) and chaperone-mediated autophagy [152] contribute to ferroptosis by the degradation of antiferroptosis regulators (e.g., ferritin, lipid droplets, ARNTL/BMAL1 [aryl hydrocarbon receptor nuclear translocator like], and GPX4), thereby promoting oxidative damage Figure 5.

Figure 5. The mechanism of autophagy-dependent ferroptosis in PDAC. Several types of selective autophagy (e.g., ferritinophagy or lipophagy) contribute to ferroptosis in PDAC cells by mediating iron accumulation or lipid peroxidation. Autophagy-dependent ferroptosis plays a dual role in PDAC. On the one hand, small molecule compounds (e.g., erastin or RSL3) or drugs (e.g., rapamycin or zalcitabine) can induce autophagy-dependent ferroptosis for tumor therapy. On the other hand, oxidative stress induces autophagy-dependent ferroptosis in PDAC cells, which leads to the release of mutated KRAS and subsequent macrophage polarization, thereby promoting tumor growth. Abbreviations: AA, arachidonic acid; ACSL4, acyl-CoA synthetase long chain family member 4; ALOX5, arachidonate 5-lipoxygenase; ALOXs, ALOX: arachidonate lipoxygenases; ATF4, activating transcription factor 4; cGAMP, cyclic GMP-AMP; CGAS, cyclic GMP-AMP synthase; CoA, coenzyme A; CTSB, cathepsin B; DUSP1, dual specificity phosphatase 1; FA, fatty acid; Fe²⁺, ferrous iron; Fe³⁺, ferric iron; GPX4, glutathione peroxidase 4; GSH, glutathione; HSPA5, heat shock protein family A (hsp70) member 5; KRAS, KRAS proto-oncogene, GTPase; LPCAT3, lysophosphatidylcholine acyltransferase 3; mtDNA, mitochondrial DNA; MTOR, mechanistic target of rapamycin kinase; NCOA4, nuclear receptor coactivator 4; PE, phosphatidylethanolamine; POLG, DNA polymerase gamma, catalytic subunit; RAB7A, RAB7A, member RAS oncogene family; RAB27A, RAB27A, member RAS oncogene family; STING1/TMEM173, stimulator of interferon response cGAMP interactor 1; system xc⁻, the cystine/glutamate antiporter; TF, transferrin; TFAM, transcription factor A, mitochondrial; TFRC, transferrin receptor

As with necroptosis, ferroptosis seems to play a complex role in the modulation of pancreatic tumor formation and therapy [153–160]. SLC7A11 is a component of the cysteine-glutamic acid transporter, which plays a vital role in the uptake of cysteine and subsequent production of the antioxidant glutathione (GSH). The conditional knockout of Slc7a11 in pancreas, or treatment with cysteine-depletion drugs, inhibits mutant Kras- and Tp53-driven PDAC in mice [156]. However, oxidative stress-related ferroptotic PDAC cells can release mutant KRAS into the extracellular space through autophagy-dependent exosome biogenesis and then be taken up by AGER expressed in macrophages [153]. This ferroptotic cancer cell death-induced cell-to-cell communication eventually leads to macrophage polarization into a pro-tumor type [153]. Therefore, ferroptosis not only inhibits the occurrence of pancreatic tumors by the consumption of cysteine in cancer cells, but also promotes the growth of tumors by DAMP-mediated macrophage polarization in the tumor microenvironment. Given that high iron is a risk factor for many tumors, it is unclear whether chronic iron accumulation-mediated oxidative damage plays a role similar to cysteine starvation in regulating pancreatic tumorigenesis [161].

For established PDAC, the induction of ferroptosis by compounds or drugs significantly inhibits tumor growth. For example, zalcitabine is used to treat human immunodeficiency virus infection. It can also cause autophagy-dependent ferroptosis in PDAC cells by activating mitochondrial DNA stress-mediated STING1/STNG/TMEM173 (stimulator of interferon response cGAMP interactor 1) activation [162]. Moreover, the translocation of lysosomal cysteine protease CTSB (cathepsin B) into the nucleus triggers DNA damage-related STING1 activation and subsequent autophagy-dependent ferroptosis in human PDAC cells [163]. HSPA5/BIP (heat shock protein family A [hsp70] member 5)-mediated GPX4 stability is related to gemcitabine resistance in PDAC cells. Thus, inducing ferroptosis by targeting HSPA5 or GPX4 function may overcome gemcitabine resistance [164]. The activation of DUSP1 (dual specificity phosphatase 1), a phosphatase that plays a role in inhibiting RAS signaling, limits autophagy-dependent ferroptosis in human PDAC cells [160]. In addition, the interaction between MTOR and GPX4 signaling regulates autophagy-dependent ferroptosis in PDAC cells [165]. High-dose rapamycin suppresses the growth of PDAC in vitro or in xenograft models by activating autophagy-dependent ferroptosis [165]. Given that autophagy is highly upregulated in PDAC, pharmacological induction of autophagy-dependent ferroptosis may be a safe target therapy for PDAC.

Immune escape

The host’s immune system can recognize tumor antigens by presenting small antigen peptides to the receptors of cytotoxic T lymphocytes (CTLs) and reject new tumors [166]. However, cancer cells may develop different systems (e.g., decreased antigenicity and impaired immunogenicity) to avoid destruction from the immune system [167]. In addition to the DAMP release discussed above that is regulated by the interaction between autophagy and cell death, dysfunctional autophagy also participates in the formation of tumor immune escape through the degradation of immunomodulators, such as major histocompatibility complex class I (MHC-I), in PDAC Figure 3 [168]. MHC-I on the surface of cancer cells plays a key role in antigen presentation, and immune checkpoints expressed in cancer cells and immune cells may limit CTL. Elevated autophagic degradation of MHC-I through the specific autophagy receptor NBR1 significantly reduces antigenicity [168]. In contrast, pharmacological or genetic inhibition of autophagy can restore the expression of MHC-I in PDAC, thereby increasing antigenicity and subsequent immunogenicity during combined immune checkpoint blockade therapy in vitro and in orthotopic models [168]. These findings suggest that inhibiting selective autophagy-mediated degradation of MHC-I may enhance the anticancer activity of immune checkpoint inhibitors in PDAC by inducing CTL. It is unclear whether other autophagic degradation substrates, especially expressed in antigen-presenting cells (e.g., dendritic cells [DCs] and macrophages) or effector cells (e.g., T and NK cells), are involved in tumor immune regulation. In addition, tumor nerve infiltration is associated with increased autophagy in PDAC patients [169]. An interesting possibility is that this crosstalk between autophagy and the nervous system may allow amplification of immune escape in the microenvironment of PDAC.

Autophagy in pancreatic cancer therapy

Preclinical studies

Chemotherapy

As discussed above, the increase in autophagy in PDAC cells has led to apoptosis resistance to some first-line treatment drugs for PDAC patients, especially gemcitabine and oxaliplatin. The inhibition of autolysosome formation by CQ (a lysosomotropic agent) [56], bafilomycin A₁ (a V-ATPase inhibitor) [56], UPSM (ultra pH-sensitive micelles) [170], or withaferin-A (a SNARE inhibitor) [171] suppress PDAC growth alone or together with other drugs. Natural products (e.g., ancistrolikokine E₃) [172], heavy metal chelators (e.g., N,N,N’,N’-tetrakis[2-pyridylmethyl]-ethylenediamine/TPEN) [173], or organic compounds (e.g., EAD1) [174] also show anticancer activity in PDAC cells by inhibiting autophagy. Together, these findings support the conclusion that the inhibition of autophagy is beneficial for PDAC treatment.

In contrast, the pharmacological induction of autophagy-dependent ferroptosis by targeting SLC7A11 (e.g., erastin and sorafenib), GPX4 (e.g., RSL3), or STING1 (e.g., zalcitabine) pathways also exhibits anticancer effects on PDAC cells in vitro or in xenograft models [155,162,175]. In addition to ferroptosis, the induction of other types of autophagy-dependent cell death in PDAC cells or PSCs by drugs (e.g., tocotrienol, oleandrin, ascorbate, triptolide, and rottlerin) also suppress PDAC growth in vitro or in vivo [176–181] Table 1. The mechanism and effect of these drugs are related to the activation of AMPK, the inhibition of MTOR or the induction of BECN1 expression.

Table 1. Monotherapies that affect autophagy in PDAC

Agent	Model	Autophagy flux	Mechanism	Effect	Ref.
11 k	PANC1 cells; xenografts in mice	↓	N/A	Inhibit autophagy and tumor growth	[182]
Ancistrolikokine E3	PANC1 cells	↓	Inhibit MTOR pathway	Inhibit autophagy and tumor growth	[172]
Aqueous extract of clove	AsPC1 cells	↑	Activate AMPK and ULK1 pathway	Induce autophagy-dependent cell death	[183]
Ascorbate	MiaPACA2, AsPC1, and BxPC3 cells; xenografts in mice	↑	Increase ROS production	Induce autophagy-dependent cell death	[179,184]
BA145	PANC1 cells	↑	Inhibit MTOR pathway	Induce G2/M arrest and cell death	[185]
Cannabinoids	PANC1 cells	N/A	Activate AMPK pathway and inhibit PKM2 function	Inhibit glycolysis and induce autophagy	[186]
CQ	8988 T, 8902, and PANC1 cells; xenografts in mice	↓	Increase ROS production and inhibit autolysosome formation	Inhibit autophagy and tumor growth	[56]
Cucurbitacin I	PANC1 cells	↑	Induce ER stress	Induce autophagy-dependent cell death	[187]
Dandelion root extract	PANC1 and BxPC3 cells	N/A	N/A	Induce autophagy-dependent cell death	[188]
Disulfiram	PANC1 and PDAC6 cells	↑	Activate ERN1-XBP1s pathway	Induce autophagy-mediated apoptosis	[189]
Dp44mT	PANC1, AsPC1, and MiaPACA2 cells	N/A	Decrease HMGCR expression	Induce lysosomal dysfunction	[190]
EAD1	BxPC3 cells	↓	Inhibit autolysosome formation	Inhibit autophagy and tumor growth	[174]
Erastin	MiaPACA2 and CAPAN2 cells	↑	Inhibit SLC7A11 function	Induce autophagy-dependent ferroptotic cell death	[155]
Fisetin	PANC1 cells; xenografts in mice	↓	Increase NUPR1 expression and ER stress	Inhibit autophagy and induce apoptosis	[191]
Gemcitabine	PANC1 and MiaPACA2 cells	↑	Increase VMP1 function	Induce autophagy-dependent apoptosis	[192]
Ginger extract	PANC1, AsPC1, BxPC3, CAPAN2, CFPAC1, Mia PACA2, SW1990, and Panc02 cells; xenografts in mice	↑	Inhibit MTOR and activate MAPK pathway	Induce autophagy-dependent cell death	[193]
IMB-6 G	MiaPACA2 and HUPT3 cells	↑	Increase lysosomal membrane permeabilization	Induce autophagy-mediated apoptosis	[194]
Leflunomide	Patient-derived pancreatic cancer cells; genetically engineered mice	N/A	Induce mitochondrial fusion and mitophagy	Inhibit ATP production and tumor growth	[195]
LZ1	PANC1, BxPC3, AsPC1, CFPAC1, and SW1990 cells; orthotopic mice	↑	Induce NCL (nucleolin) degradation	Induce autophagy-dependent cell death	[196]
Marigold supercritical extract	PANC1 and MiaPACA2 cells	↑	Induce AMPK activation	Induce autophagy-dependent cell death	[197]
Matrine	MiaPACA2 and 8988 T cells; xenografts in mice	↓	Inhibit lysosomal protease function and STAT3 expression	Inhibit autophagy-mediated energy metabolism and tumor growth	[198]
N-(4-(3-aminophenyl) thiazol-2-yl) acetamide 6b	BxPC3 and MiaPACA2 cells	↑	N/A	Induce autophagy-dependent cell death	[199]
Oleandrin	PANC1 cells	↑	Inhibit MTOR and activate MAPK pathway	Induce autophagy-dependent cell death	[178]
Parthenolide	PANC1 and BxPC3 cells	↑	Increase BECN1 and SQSTM1 expression	Induce autophagy-mediated apoptosis	[200]
Penfluridol	PANC1, BxPC3, and AsPC1 cells; xenografts in mice	↑	N/A	Induce autophagy-mediated apoptosis	[201]
Phycocyanin	Capan1, PANC1, and BxPC3 cells; xenografts in mice	↑	Inhibit MTOR pathway and increase BECN1 expression	Induce autophagy-dependent cell death	[202]
Polyoxomolybdates	AsPC1 cells; xenograft in mice	↑	N/A	Induce autophagy-dependent cell death	[203]
Pyrrolidine 20	MiaPACA2 and BxPC3 cells	↑	N/A	Induce autophagy-mediated apoptosis	[204]
Rapamycin	PC2 cells	↑	Increase BECN1 expression	Induce autophagy-dependent cell death	[205]
Rottlerin	Human pancreatic CSCs	↑	Inhibit MTOR pathway and increase BECN1 expression	Induce autophagy-dependent apoptosis	[181]
RSL3	MiaPACA2 cells	↑	Inhibit GPX4 function	Induce autophagy-dependent ferroptotic cell death	[175]
Scalarin	PANC1 and MiaPACA2 cells	N/A	Inhibit AGER expression	Inhibit autophagy and tumor growth	[123]
Sorafenib	MiaPACA2 and CAPAN2 cells	↑	Inhibit SLC7A11 function	Induce autophagy-dependent ferroptotic cell death	[155]
Tocotrienol-rich fraction	PSCs (rat)	↑	Activate BECN1 pathway	Induce mitochondrial dysfunction and cell death	[176,177]
TPEN	PANC1, 8988 T, and BxPC3 cells	↓	Increase ROS production and inhibit lysosomal function	Inhibit autophagy and induce cell death	[173]
Triptolide	S2013 and S2VP10 cells	↑	Inhibit MTOR and activate MAPK pathway	Induce autophagy-dependent cell death	[180]
Ultra pH-sensitive micelles (UPSM)	PANC1 and MiaPACA2 cells; subcutaneous and orthotopic mice	N/A	Inhibit lysosomal catabolism and reduce the generation of amino acids	Inhibit lysosomal catabolism and induce apoptosis	[170]
Zalcitabine	PANC1 and Capan2 cells; xenografts in mice	↑	Activate STING1-mediated DNA-sensing pathway	Induce autophagy-dependent ferroptotic cell death	[162]
Zn-CuO NPs	AsPC1 and MiaPACA2 cells; xenografts in mice	↑	Activate AMPK and increase the level of BECN1 and p-ULK1	Induce autophagy and suppress tumor growth	[206]

In most cases (especially as adjuvant or neoadjuvant therapy), combination therapy is more effective than monotherapy for PDAC treatment. Combination therapy inhibits PDAC by decreasing autophagy activation or increasing autophagy activation Table 2. In addition to agents that target lysosomes (e.g., CQ or bafilomycin A₁), the pharmacological blocking of autophagy by inhibiting AMPK (using lumican) [207] or increasing the accumulation of lysosomal-associated palmitoylated proteins (using DQ661) [208] enhance the anticancer activity of gemcitabine or other chemotherapy drugs. Alternatively, excessive autophagy activation may lead to tumor suppression through autophagy-dependent metabolic stress or injury. For example, combined treatment with adrenergic A and phenethyl isothiocyanate activates autophagy-dependent ferroptosis in human PDAC cells [209]. Combined treatment with troglitazone and IFNB/interferon-β induces autophagy-dependent tumor suppression in PDAC cells by inhibiting the phosphorylation of EIF4E (eukaryotic translation initiation factor 4E) [210]. The use of CP-31,398-RITA, gossypol-BRD4770 or dugrin-doxorubicin combination therapy also inhibits PDAC growth through activating autophagy-dependent cell death [211–213]. These findings suggest the complexity of applying autophagy-related drugs in pancreatic tumor chemotherapy.

Table 2. Combination therapies that affect autophagy in PDAC

Agent	Model	Autophagy flux	Mechanism	Effect	Ref.
Alantolactone and oxaliplatin	PANC1 and MiaPACA2 cells; xenografts in mice	↓	Inhibit lysosomal function	Inhibit autophagy and increase chemosensitivity	[214]
Arsenic trioxide (ATO) and JQ1	PANC1, AsPC1, BxPC3, MiaPACA2, and CFPAC1 cells; xenografts in mice	↓	Inhibit NFE2L2 expression and lysosomal function	Inhibit autophagy and induce apoptosis	[215]
Cannabinoid and gemcitabine	PANC1, PaCa44, PaCa3, CFPAC1, T3M4, and MiaPACA2 cells; xenografts in mice	N/A	Increase ROS production	Induce autophagy-dependent cell death	[216]
Chloroquine-modified HES	AsPC1 and MiaPACA2 cells	↓	Inhibit MAPK pathway	Inhibit autophagy and tumor growth	[217]
Cotylenin A and phenethyl isothiocyanate	MiaPACA2, PANC1, and gemcitabine-resistant PANC1 cells	↑	Increase ROS production	Induce autophagy-dependent ferroptotic cell death	[209]
CP-31,398 and RITA and CQ	PaCa3 and PANC1 cells	↓	Activate AMPK pathway and inhibit autolysosome formation	Induce autophagy-dependent cell death	[212]
Deguelin and doxorubicin	PANC1and MiaPACA2 cells	↓	N/A	Inhibit autophagy and increase chemosensitivity	[211]
DQ661 and gemcitabine	KRPC and PDA4662 cells; xenografts in mice	↓	Inhibit lysosomal function	Inhibit autophagy and tumor growth	[208]
Gossypol and BRD4770	PANC1 cells	↑	Increase BNIP3 expression	Induce autophagy-dependent cell death	[213]
Lumican and gemcitabine	PANC1 and MDAPATC53 cells (human); xenografts in mice	↓	Inhibit AMPK function	Inhibit autophagy and increase chemotherapy sensitization	[207]
LW6 and gemcitabine	6606PDA and MiaPACA2 cells; orthotopic mice	↓	N/A	Inhibit autophagy and induce cell death	[218]
Macrolides and epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI)	PANC1 and BxPC3 cells	↓	N/A	Inhibit autophagy and induce cell death	[219]
MPT0L145 and gemcitabine	PANC1 cells	N/A	Inhibit PIK3C3 function	Inhibit autophagy and increase chemosensitivity	[220]
Plectin/MIR9 and doxorubicin	PANC1, CFPAC1, and CAPAN1 cells; xenografts in mice	↓	Inhibit EIF5A2 expression	Inhibit autophagy and increase chemosensitivity	[221]
Plectin/MIR212 nanoparticles and doxorubicin	HPNE, CAPAN1, PANC1, and CFPAC1 cells; xenografts in mice	↑	Inhibit USP9X expression	Induce autophagy-dependent cell death and increase chemosensitivity	[222]
Quercetin and gemcitabine	MiaPACA2, BxPC3, AsPC1, and PANC1cells	↑	Inhibit AGER expression	Induce autophagy-mediated apoptosis	[223]
Rocaglamide A and CQ or bafilomycin A1	MiaPACA2 and PANC1 cells; xenografts in mice	↓	Activate PINK1-PRKN pathway and inhibit autolysosome formation	Inhibit autophagy and induce apoptosis	[224]
Sunitinib and CQ	Panc0203, PANC327, MiaPACA2, Panc02, and KPCP1 cells; orthotopic mice	N/A	Inhibit autolysosome formation and lysosome function	Inhibit autophagy and induce apoptosis	[225]
TPCS2a and CQ	PANC327 cells	↓	Increase ROS production and inhibit autolysosome formation	Inhibit autophagy and increase chemotherapy sensitization	[226]
Troglitazone and IFNB (interferon beta)	BxPC3 cells	↑	Inhibit MTOR pathway	Induce autophagy-dependent cell death	[210]
TR-PTX and HCQ-Lip	BxPC3 and NIH3T3 (rat) cells; xenografts in mice and orthotopic mice	↓	N/A	Inhibit autophagy and crosstalk between tumor cells and fibroblasts	[227]
Ursolic acid and gemcitabine	MiaPACA2 and gemcitabine-resistant MiaPACA2 cells	N/A	Inhibit AGER expression	Induce autophagy-mediated apoptosis and increase chemosensitivity	[228]
VE-822 and CQ	MiaPACA2, PANC327, CFPAC1, and DANG cells; 3D organotypic model; xenografts in mice and orthotopic mice	↓	Inhibit mitochondrial and lysosomal function	Inhibit autophagy and tumor growth	[229]
Verteporfin and gemcitabine	BxPC3, CAPAN2, PANC1, HS766T, and MiaPACA2 cells; xenografts in mice	↓	Inhibit autophagosome formation	Inhibit autophagy and increase chemotherapy sensitization	[230]
Withaferin-A and ER stress aggravators	AsPC1, BxPC3, PANC1, SW1990, and MiaPACA2 cells; xenografts in mice	↓	Inhibit SNARE function	Inhibit autophagy and induce apoptosis	[171]
WP1130 and gemcitabine	PANC1, BxPC3, and MiaPACA2 cells; PDAC xenografts in mice	↓	Inhibit USP9X function	Inhibit autophagy and increase chemosensitivity	[231]
XL765 and CQ	AsPC1, BxPC3, HPAFII, and MiaPACA2 cells; xenografts in mice	↓	Inhibit MTOR pathway and autolysosome formation	Inhibit autophagy and induce apoptosis	[232]
ZST93 and gemcitabine	PANC1 and MiaPACA2 cells; xenografts in mice	↑	N/A	Induce autophagy-dependent cell death and increase chemosensitivity	[233]
Chemical and radiation
Gemcitabine and ionizing radiation	PANC1, AsPC1, and MiaPACA2 cells; xenografts in mice	↑	Increase MAP1LC3 and BECN1 expression	Induce autophagy and suppress tumor growth	[238]
Gemcitabine and noninvasive radiofrequency	PANC1, AsPC1, and MiaPACA2 cells; xenografts in mice and orthotopic mice	↑	N/A	Induce autophagy and increase chemosensitivity	[237]
MG132 and ionizing radiation	MiaPACA2, BxPC3, and PANC1 cells; orthotopic mice	↑	Inhibit TRAF6 expression	Induce autophagy-dependent cell death	[239]
Rapamycin and ionizing radiation	PANC1 cells	↑	Inhibit MTOR pathway	Induce autophagy and increase radiosensitivity	[234]
Seaweed polyphenols and fractionated irradiation	PANC327 and MiaPACA2 cells; xenografts in mice	N/A	Inhibit ATG5, ATG12, and MAP1LC3 expression	Inhibit autophagy and increase radiosensitivity	[240]

Radiotherapy

Radiotherapy uses radiation to destroy cancer cells, and this process is regulated by SMAD4-related autophagy in PDAC cells. In particular, the depletion of SMAD4 increases autophagy, which helps PDAC cells to resist ionizing radiation in vitro or in vivo [235]. The downregulation of MIR23B also contributes to radiotherapy resistance in PDAC cells by activating ATG12-dependent autophagy [236]. In contrast, the overexpression of MIR23B suppresses the expression of ATG12, thus restoring ionizing radiation sensitivity by inhibiting autophagy [236]. These findings support a pro-survival role of autophagy during radiotherapy.

However, the combination of radiotherapy and chemotherapy (e.g., using gemcitabine or rapamycin) can suppress PDAC by activating excessive autophagy-dependent cell death in xenografts and orthotopic tumor models [237,238]. Similarly, the combined use of MG132 (a proteasome inhibitor) and radiation also triggers autophagy-dependent cell death in PDAC cells, and this process is mediated by the inhibition of pro-survival TRAF6 (TNF receptor associated factor 6; an adaptor protein that mediates a wide array of protein-protein interactions) [239]. These findings indicate that feedback between protein degradation and interaction pathways modulate the sensitivity of radiotherapy in PDAC. Interestingly, the combination of seaweed polyphenols and radiation fractionation may inhibit autophagy through unknown molecular mechanisms to protect radiation-resistant PDAC cells [240]. All these studies provide a theoretical basis for targeting autophagy to enhance the response to radiotherapy.

Immunotherapy

As mentioned above, the direct evidence of autophagy-modulated immunotherapy is that autophagy mediates the degradation of MHC-I in PDAC cells [168]. Inhibiting this type of selective autophagy ultimately enhances the anticancer activity of immune checkpoint inhibitors by activating classic CD8⁺ T-cell-mediated antitumor immunity [168]. It is still necessary to know how the different autophagy levels of immune cells (e.g., NK, B, and T cells as well as DCs and macrophages) in the tumor microenvironment regulate antitumor immunity under different conditions. How autophagy regulates immunometabolism, neuroimmunity, and microbial homeostasis in the tumor microenvironment remains an open question. In addition, autophagy-mediated protein release may play a role in mediating the communication between immune and cancer cells.

Clinical studies

Hydroxychloroquine (HCQ) is a derivative of CQ and is used to treat patients with malaria or autoimmune diseases. Both HCQ and CQ enhance the efficacy of many anticancer regimens by disrupting lysosomal function and impairing autophagy in preclinical animal models [241]. Although they are all used as potential autophagy inhibitors, HCQ is only half as toxic as CQ in humans. Thus, in the past few years, clinical research has greatly expanded into cancer-related autophagy using HCQ combined with other chemotherapy for PDAC Table 3. Two randomized trials showed that, compared with nab-paclitaxel and gemcitabine treatment group, additional HCQ combination therapy can improve the response rate [242–244]. Although survival rates are not affected, these studies show that this autophagy inhibition approach can hold promise [242–244]. Some case reports also show that the combined use of HCQ and other drugs (e.g., the MAP2K/MEK-MAPK/ERK inhibitor) has potential benefits for some PDAC patients [71,108,245], but it is still uncertain whether these combination therapies are useful for most patients.

Table 3. Published clinical studies on using HCQ to inhibit autophagy in PDAC

Study’s HCQ dose	Phase	Participants	Result	Ref.
400 or 600 mg twice/day	II	20	Effect of autophagy inhibition with HCQ is inconsistent and has negligible therapeutic efficacy.	[246]
200–1200 mg/day	I/II	35	Pre-operative autophagy inhibition with HCQ is safe. Outcomes are encouraging.	[242]
600 mg twice/day	II	112	Additional HCQ treatment could not improve the primary endpoint of overall survival at 12 months.	[244]
600 or 1200 mg twice/day	II	64	Additional HCQ treatment produces greater pathological tumor response and improves the serum biomarker response and immune activity.	[243]

Conclusion and perspectives

In the past decade, we have made significant progress in our understanding of autophagy as a fundamental cellular homeostasis program, not only in comprehending its regulation networks and signaling pathways, but also in the wide range of pathophysiological effects of autophagy in health and disease. Similarly, our understanding of autophagy in PDAC is increasing dramatically. Impaired autophagy involves various biological processes involved in the occurrence and development of pancreatic cancer, which play complex and important roles not only in cancer cells but also in noncancer cells within the tumor microenvironment. The complex functions of autophagy in PDAC are further regulated by oncogenes (e.g., KRAS) and tumor suppressors (e.g., TP53 and PTEN).

While preclinical studies have achieved promising results in targeting autophagy to treat PDAC, clinical research involving this strategy is uncertain and even frustrating. Although the exact cause of this difference in preclinical and clinical studies is not clear, at least the following four aspects could explain it: First, animals and humans have different genetic backgrounds, which indicate that the model of mouse PDAC may not be able to mimic the pathological process of human PDAC. Second, most of the experimental mice are fed in a sterile environment, which may result in a significant difference in the intestinal microbial composition and immune response between mice and humans. This difference not only affects the activation of the autophagy pathway, but also leads to different microenvironments in pancreatic tumors. Third, the inhibition of autophagy is not the only effect of HCQ. Its potential off-target and even toxic side effects (e.g., causing liver and heart problems) may affect the interpretation of clinical trial results. Fourth, autophagy is also beneficial to normal tissue function in terms of overall maintenance of cellular homeostasis; the inhibition of nonselective autophagy by HCQ may cause normal tissue dysfunction or damage.

Autophagy plays a dual role in the regulation of pancreatic tumorigenesis and treatment, depending on the type of autophagy, the stage of cancer, and the activation by drugs [10]. The duality of autophagy is ultimately reflected in its pro-survival and pro-death functions, which indicates that both autophagy inducers and autophagy inhibitors can be used to treat cancer, including PDAC. Therefore, the development of drugs targeting signaling factors or modulators of selective autophagy, such as specific autophagy receptors or cargos, may be a reasonable and safe clinical strategy to inhibit tumor growth. The accomplishment of this arduous goal requires multidisciplinary cooperation as well as the development of reliable and convenient autophagy detection methods in patients.

Acknowledgments

We thank Dave Primm (Department of Surgery, University of Texas Southwestern Medical Center) for his critical reading of the manuscript. This work was supported by grants from the US National Institutes of Health (R01CA160417 [D.T.], R01CA211070 [R.K.] and GM131919 [D.J.K.]).

Disclosure statement

No potential conflict of interest was reported by the authors.