Emerging role of the Hippo pathway in autophagy

Autophagy is a dynamic circulatory system that occurs in all eukaryotic cells. Cytoplasmic material is transported to lysosomes for degradation and recovery through autophagy. This provides energy and macromolecular precursors for cell renewal and homeostasis. The Hippo-YAP pathway has significant biological properties in controlling organ size, tissue homeostasis, and regeneration. Recently, the Hippo-YAP axis has been extensively referred to as the pathophysiological processes regulating autophagy. Understanding the cellular and molecular basis of these processes is crucial for identifying disease pathogenesis and novel therapeutic targets. Here we review recent findings from Drosophila models to organisms. We particularly emphasize the regulation between Hippo core components and autophagy, which is involved in normal cellular regulation and the pathogenesis of human diseases, and its application to disease treatment.

The Hippo pathway and autophagy have complex and reciprocal interactions. These bidirectional links coordinate the autophagic flux to the overall microenvironmental signal and regulate homeostasis and tumorigenesis.
• Autophagy is involved in the biological effects of the Hippo pathway and vice versa. Hippo-mediated modalities profoundly influence autophagic flux and are extensively involved in the intracellular quality control, tissue homeostasis, regeneration, development, and differentiation.

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The association between the Hippo pathway and autophagy is relevant to the pathogenesis of a wide range of human diseases, from metabolic and neurodegenerative diseases, cardiovascular diseases to a variety of human solid tumors.

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The emerging link between the Hippo pathway and autophagy means that targeting the Hippo-YAPautophagy axis may provide new insights to prevent or promote autophagy in a variety of contexts, influencing metabolic reprogramming, cellular mechanical signals, mitochondrial quality control, and YAP/TAZ transcriptional activity. Introduction Autophagy (also known as macroautophagy) is an accommodative process that occurs in different forms of cell stresses, including starvation, hypoxia, infection, high reactive oxygen content, and endoplasmic reticulum stress 1 . During this process, cells capture intracellular proteins and organelles, and transport them to lysosomes for degradation and export the products of autophagic degradation from lysosomes to the cytoplasm for recycling 2,3 . The first genetic screening of autophagy was conducted in Ohsumi's laboratory, which analyzed this process in yeast and identified 15 autophagy-related proteins (ATGs) 4 . So far, over 30 ATGs have been identified 5 . The autophagy pathway is frequently divided into various individual stages: initiation, vesicle nucleation, elongation of the autophagy membrane, fusion with lysosomes, and degradation of intravesicular products ( Fig. 1) 6 . Previously, autophagy has been considered a non-selective process. Recent studies have shown that autophagy can selectively eliminate harmful cytosols such as invading pathogens, dysfunctional organelles, and protein aggregates (called selective autophagy, including lipophagy, mitophagy, xenophagy, and aggrephagy), thereby contributing to the protection of cells in various environmental and metabolic stress 3,7 . Autophagy is strongly associated with neurodegeneration, cancer, metabolic diseases, immune and heart diseases, especially the role of autophagy in cancer 7 . Autophagy plays a dual role in cancer. Under tumorigenesis pressure, autophagy can clear oncogenic protein substrates and toxic unfolded proteins, inhibiting tissue damage and genomic instability [8][9][10] . Conversely, after tumor formation, increased autophagic flux often allows tumor cells to survive and grow 9,11 . This makes autophagy an interesting target for pharmacologists and clinicians.

Open questions
The Hippo-Yes-associated protein (YAP) pathway is an evolutionarily conserved pathway that controls organ size and tissue homeostasis 12 . The core kinase cassettes of the mammalian Hippo-YAP pathway consist of the mammalian sterile 20-like protein kinase 1 (STK3/MST2 and STK4/MST1) and an adapter protein, salvador family WW domain-containing protein 1 (SAV1) 13,14 , which Fig. 1 Schematic diagram of the autophagy process in mammalian cells. The mTOR complex 1 (mTORC1) contributes to the initiation of autophagy, integrates upstream signals such as PI3K/Akt pathway, AMPK, P53, and Bcl-2 protein family, which play different regulatory roles in autophagy 135 . The ULK1 complex induces vesicle nucleation and translocates to a characteristic endoplasmic reticulum (ER) structure called omegasome, where it phosphorylates PI3KC3 complex I to produce phosphatidylinositol-3-phosphate (PI3P) in omegasome. Specifically, Beclin1, a Bcl-2-homology (BH)-3 domain-only protein, is phosphorylated by ULK1 and acts as a scaffold for the PI3KC3 complex I, which facilitates localization of autophagy proteins to the phagophore. Atg9 is a transmembrane protein, which participates in the early stage of phagophore formation. PI3P recruits specific autophagy effectors, such as WIPIs (mammalian homolog of yeast Atg18) and zinc finger FYVE-type containing 1 (DFCP1). WIPIs directly binds to ATG16L1 under the regulation of the ubiquitin-like conjugation system to form the ATG12-ATG5-ATG16L1 complex and LC3 (mammalian homolog of yeast Atg8)-phosphatidylethanolamine (PE) binding. Ultimately, the isolation membrane is elongated and closed to form the autophagosome 136 . This binding reaction results in the conversion of LC3-I to LC3-II, a common autophagosome marker. When the autophagosome matures, it sheds the ATG proteins and fuses with the lysosome to produce autophagolysosome. Both the inner membrane of the autophagic vesicle and the luminal contents are degraded by lysosomal hydrolases (cathepsins B, D, and L). The resulting monomer molecules (such as amino acids and lipids) are recycled into the cytoplasm for reuse 137 . The pointed and blunt arrowheads indicate activation and inhibitory interactions, respectively. Ub, ubiquitin. may phosphorylate and activate the large tumor suppressor kinase 1/2 (LATS1/2) 15 . Adapter protein MOB kinase activator 1A (MOB1A) and MOB1B are also involved in the phosphorylation process 16 . YAP and PDZbinding motif (TAZ, also known as WW domaincontaining transcription regulator 1) are the major downstream transcription coactivators of the Hippo pathway. The phosphorylation of YAP/TAZ by the upstream kinase cascades MST1/2-LATS1/2 promotes the interaction of YAP and TAZ with cytoskeletal proteins, retains YAP and TAZ in the cytoplasm, and prevents their importation into the nucleus for transcriptional activation 15,17 . In contrast, when dephosphorylated, YAP can enter the nucleus and bind to the transcription factor TEA domain family member (TEADs) to control the expression of target genes 15,18 . YAP and TAZ rapidly shuttle between the nucleus and the cytoplasm by complex upstream components. LATS1/2mediated phosphorylation limits the rate at which YAP and TAZ are imported into the nucleus. In addition, tethering of YAP and TAZ to the cytoskeletal proteins inhibit them as cellular mechanotransduction receptors 19 . The NDR (nuclear Dbf2-related) protein kinase family, including NDR1/STK38 (Serine/Threonine Kinase 38) and NDR2/STK38L (Serine/Threonine Kinase 38 Like), has identified additional kinases of Hippo signaling, similar to the LATS1/2 status in the Hippo signaling pathway ( Fig. 2A) 20,21 . It is established that the Hippo-YAP pathway is regulated by cell-cell contact, cell polarity, cellular mechanotransduction, and G proteincoupled receptor ligands. However, recent studies have shown that autophagy has a series of crosstalk with the Hippo-YAP pathway. In physiological settings, the two conserved pathways, autophagy and Hippo-YAP signaling are essential in the protection of homeostasis. It has been shown that the deletion of autophagy-related genes interacting with the Hippo kinase cascades is associated with an accrued propensity of laboratory animals to spontaneously develop various disorders (Table 1). In this review, we summarize the regulation of autophagy by the Hippo-YAP pathway and discuss the multidisciplinary function of Hippo-YAP-autophagy in cells and various disorders.

Mechanism of autophagy
Autophagy initiation begins with the activation of the Unc-51-like autophagy activating kinase 1 (ULK1, also known as ATG1) complex, including ULK1, ULK2, RB1 inducible coiled-coil 1 (FIP200), and ATG13. This leads to the recruitment of ATGs to the specific subcellular location called the phagophore assembly site, which activates class III phosphatidylinositol-3-kinase (PI3KC3) complex I, including VPS34, Beclin1 (mammalian homolog of yeast Atg6), p150 (mammalian homolog of yeast VPS15), and Atg14 or ultraviolet radiation resistance-associated gene protein (also known as P63), and nucleation of an annular structure of the isolation membrane, called phagophore 22,23 . The ATG5-ATG12 complex conjugates with ATG16 to expand the autophagosome membrane, causing the phagosome to expand into a sphere. The enzymolysis of Atg4 to LC3 (Atg8 family protein) produces cytoplasmic LC3-I, which conjugates to lipid phosphatidylethanolamine to form LC3-II and then recruits to the autophagosome membrane. Eventually, the autophagosome fuses with the lysosome and the contents are degraded, thereby enabling cellular metabolic pathways and the renewal of specific organelles ( Fig. 1) 1,23-26 . Furthermore, p62 (also known as SQSTM1) is an autophagic modifier of the LC3 family that acts as a bridge between LC3 and ubiquitinated substrates 27,28 . p62-bound polyubiquitinated proteins are integrated into the completed autophagosome and degraded in autolysosomes 29,30 . Whereas increased p62 levels are associated with autophagy inhibition, decreased p62 levels are associated with autophagy activation, revealing that the steady-state levels of this protein could reflect the state of autophagy [29][30][31] . Thus, p62 combined with LC3 completely monitors the autophagic flux 32 .

Crosstalk between the Hippo pathway and autophagy in Drosophila
Hippo signaling is essential for proper growth control in Drosophila and the loss of hippo (MST1/2 in mammals) causes tissue overgrowth 33 . Interestingly, autophagy induction actively suppresses hippo-induced tissue overgrowth. Meanwhile, Atg1 overexpression inhibits Yorkie (YAP in mammals), further suppressing epithelial overgrowth and cell proliferation 34 . Mechanistically, Atg1/ ULK1 phosphorylates Yorkie at two serine residues, S74 and S97, thereby blocking transcriptional activation and inhibition of Yorkie activity. Atg1-mediated phosphorylation is an additional inhibitory input independent of the Hippo-Warts pathway 35 .
Steroid hormones are critical signaling molecules for growth regulation 36 . Warts (LATS1/2 in mammals) regulate steroid hormone production through an autophagydependent pathway (also called lipophagy). Precisely, Warts control the production of Drosophila steroid ecdysone through their effector microRNA bantam, which responds to nutrients, thus mobilizing the transport of the steroid precursor cholesterol 37 . Notably, YAP (mammalian Yki homolog) regulates steroidogenesis in tumor cells 38 , indicating that the regulation of steroidogenesis by the Wts-Yki pathway may be an evolutionarily conserved mechanism.
Trc (NDR1 in mammals) acts as a conserved regulator of autophagy and is required for early autophagosome formation in fly larvae 39 . Kibra, upstream components of the Drosophila Hippo pathway, act as autophagy regulatory factors required for proper autophagy function 40 . Drosophila protocadherin Fat (Ft) is a cell adhesion molecule in the Hippo pathway that regulates growth and planar cell polarity 41 . Ft mutations cause neurodegenerative changes through autophagy defects; autophagosomes accumulate in the Ft mutant photoreceptors, which are filled with partially degraded material and damaged mitochondria 41 . In conclusion, the core components of the Drosophila Hippo pathway are involved in the regulation of autophagy at multiple levels and several crosstalks exist in these two conserved pathways (Fig. 2B).
Regulation of autophagy by the Hippo pathway core kinase cassettes in mammals MST1/2 protein kinases Posttranslational modifications caused by the Hippo pathway kinases have become a powerful means to regulate autophagy in mammals. STK3/MST2 and STK4/MST1 are critical components of the Hippo pathway, which play a pivotal role in organ size control and tumor suppression. Recent studies have shown that STK3/STK4 can also be involved in the regulation of autophagy, which dynamically interacts with the Atg8 family of autophagy proteins in vitro. Specifically, they both phosphorylate LC3 at threonine 50. A Schematics diagram of the Hippo pathway. In mammalian cells, phosphorylation of MST1/2 activates LATS1/2, which then phosphorylates YAP/TAZ at different Ser residues. Notably, STK38 can directly phosphorylate YAP. Phosphorylated YAP/TAZ is inhibited mainly through two mechanisms: (i) cytoplasmic retention through 14-3-3 binding and (ii) proteasome degradation. Inversely, inhibition of Hippo kinase leads to nuclear accumulation of YAP/TAZ, which bind to TEADs and other transcription factors. B Schematic diagram of the crosstalk between the Hippo pathway and autophagy in Drosophila. Typically, autophagy inhibits overgrowth of epithelial tissue. When the Hippo function is reduced, this mechanism is restricted. Atg1 phosphorylates Yorkie in a Hippo-Warts-independent manner, blocks the binding of Scalloped (TEADs in mammals) and decreases the activity of Yorkie. In addition, Warts (LATS1/2 in mammals) regulates autophagy via the EcR and Tor pathways. Trc (NDR1 in mammals) promotes the formation of autophagosome. Furthermore, Kibra (WWC1/2 in mammals) and Ft (FAT1-4 in mammals) are novel autophagyregulated genes that promote ATG8-mediated elongation of the isolated membrane. The Ft mutant increases autophagy flux. The pointed and blunt arrowheads indicate activation and inhibitory interactions, respectively. Abbreviations: EcR, ecdysone receptor; Tor, target of rapamycin kinase; Trc, tricornered. Table 1 The core components of Hippo pathway affect various disorders via autophagy.
Author, year Disorders Experimental models Effector cell Effects Zhang et al. 45 Lin et al. 138 47,48 . The loss of RASSF1A can also lead to the blockage of the autophagic flux 49 . The regulation of autophagy by MST1/2 is involved in several human diseases (see Table 1).

NDR protein kinases
NDR1/2 (STK38/STK38L) is regulated through alterations in the subcellular localization and phosphorylation status, which influence cell cycle, apoptosis, and autophagy in mammalian cells 21 . Furthermore, STK38/STK38L acts as a major stress response and plays an essential role in autophagy. Precisely, STK38 regulates itself and XPO1 (exportin-1) nuclear export by phosphorylating XPO1 on serine 1055, thereby supporting autophagy regulator Beclin1 and Hippo effector YAP shuttle into the cytoplasm 50 . STK38 is also a new binding of Beclin1, which promotes autophagosome formation in mammalian cells. Conversely, STK38-depleted cells reduced PI3KC3 complex I (Beclin1-ATG14-Vps34) and PI3P formation, resulting in reduced autophagosome formation 39 . Moreover, STK38 regulates the chaperone-assisted selective autophagy (CASA), which initiates the CASA complex (including Hsc70, HspB8, synaptopodin-2 (SYNPO2), and the cochaperone BAG3) and mediates the degradation of misfolded, damaged, and aggregation-prone proteins 51 . STK38 further inhibits BAG3-mediated autophagy in a kinase activity-independent manner, which relies on the remodeling of BAG3 chaperone complexes and disrupts the interaction of HspB8 and SYNPO2 52 . The underlying mechanism by which Hippo pathway core kinase cassettes regulate autophagy is shown in Fig. 3.

Crosstalk between transcriptional coactivators YAP/TAZ and autophagy
Autophagy acts as a downstream regulator of YAP/TAZ. Although YAP/TAZ controls autophagic flux by regulating the degradation of autophagosomes, YAP/TAZ is also essential for the maturation of autophagosomes into autolysosomes 53,54 . The use of autophagy inhibitors or endogenous knockdown of autophagy-related genes (e.g., ATG7/ 10 or ATG16L1) can inhibit YAP-mediated cell proliferation. Similarly, double YAP/TAZ knockdown and verteporfin (the inhibitors of YAP/TAZ 55 ) treatment significantly impaired autophagy 53,54 . These demonstrated that the Hippo pathway maintains autophagy. In YAP/TAZ-activated cells, especially the aggressive solid tumor cells, the autophagic flux may be increased, thereby enhancing proliferation, invasion and metastasis of these cells. The underlying mechanism by which YAP/TAZ regulates autophagy is shown in Fig. 4.

Contact inhibition
Contact inhibition is a fundamental characteristic of normal cells. However, the loss of contact inhibition is an important feature of cancer cells 56 . The mechanical signals exerted by the physical state of cells in tissues and contact inhibition have recently been linked to the Hippo-YAP axis 18,57 . The decreased proliferation and cell survival resulting from contact inhibition is partly autophagy-dependent 54 . The Hippo pathway serves as the functionality of YAP and TAZ by regulating their subcellular localization and protein levels 58 . At high cell density, YAP/TAZ is redistributed into the cytoplasm and becomes inactive 18,59 , failing to regulate the expression of myosin II gene. This results in a drastic reduction in the formation of the F-actin stress fibers, ultimately impairing autophagosome formation 54 . Conversely, at low cell density, YAP/TAZ localizes in the nucleus and becomes active, resulting in increased F-actin formation, thus promoting autophagosome formation. In conclusion, YAP/TAZ regulates autophagy. The inhibition of YAP/TAZ function, caused by high cell density, reduces basal autophagy 54 . These signal crosstalks regulate the autophagy-dependent clearance of aggregation-prone proteins, survival under metabolic stress, as well as cell proliferation and differentiation 54,60 . and inhibits Vps34 kinase activity, thereby preventing the formation of autophagosome. RASSF1A promotes the initiation and maturation of autophagy by regulating MST1. In addition, MST1 mediates the interaction between Beclin1 and Bcl-2 thereby inducing apoptosis. B STK38 phosphorylation of XPO1 on S1055 is vital for the nuclear export of crucial intracellular signal sensors such as Beclin1, YAP1, and Centrin1. Cytoplasmic STK38 interacts with Beclin1 and promotes the formation of the Beclin1-ATG14-Vps34 complex, leading to the formation of PI3P. CASA is activated in mechanically stressed cells and tissues under the regulation ofSTK38. STK38 disrupts the interaction of BAG3 with HSPB8 and SYNPO2. Moreover, CASA activation is independent of the STK38 targets BECN1. Fig. 4 Schematic diagram showing the role of YAP and TAZ in autophagy. A When cells are at low density and on a stiff extracellular matrix (ECM), Factin level is elevated leading to activation and nuclear import of YAP/TAZ, and upregulation of YAP/TAZ targets (such as myosin II and Armus). Activation of YAP/TAZ promotes F-actin accumulation. Cell mechanics control autophagic flux by regulating the transcriptional activity of YAP/TAZ. The YAP/TAZ-autophagy axis regulates a series of biological processes, such as proliferation, apoptosis, differentiation and phenotypic plasticity. B Loss of Atg7 or LRPPRC decreases autophagic flux. As an autophagic substrate, YAP cannot be degraded by autophagy, which increases nuclear localization of YAP. Activated YAP triggers accumulation of p27, which in turn leads to cellular polyploidy. lncRNA-ATB influence autophagy by participating in the transcriptional regulation of ATG5. In addition, lncRNA-ATB promotes autophagy by regulating YAP activation. Nogo-B interacts with ATG5 to promote lipophagy leading to LPC-dependent inhibition of YAP phosphorylation and enhances the oncogenic activity of YAP. YAP promotes metastasis via the mitophagy-SERCA-CaMKII pathways and cofilin/F-actin/ lamellipodium axis. YAP binds to JNK in the cytoplasm, inducing JNK phosphorylation and nuclear localization, enhancing Bnip3 transcriptional activity. The Bnip3-induced mitophagy leads to mitochondrial dysfunction and ATP deficiency. Insufficient ATP inactivates SERCA and triggers [Ca2+] i overload; [Ca2+] i which phosphorylates CaMKII and inactivates cofilin, ultimately leading to F-actin degradation and abrogation of lamellipodium-based migration. Cardiolipin (CL) is a phospholipid found in the inner mitochondrial membrane. TAZ is required for catalyzation of CL. When mitochondria are damaged, cardiolipin is externalized and LC3 contains CL-binding sites to initiate mitophagy, thereby maintaining mitochondrial quality control. CaMKII, Ca/calmodulin-dependent protein kinases ΙΙ; CL, cardiolipin; LPA, lysophosphatidic acid; LPC, lysophosphatidylcholine; SERCA, sarco/endoplasmic reticulum Ca2 + -ATPase.

Mechanotransduction
Cells sense microenvironmental factors through a mechanotransduction system. They translate these stimuli into biochemical signals that control cell growth, differentiation, and cancer progression. Notably, YAP/TAZ is the medium for the mechanical cues indicated by the microenvironment 18 . YAP/TAZ is essential in the degradation of autophagosomes in both the steady-state and induced autophagy contexts. Autophagy is a downstream event of the YAP/TAZ mechanotransduction: mechanical signals act as the upstream inputs to control YAP/TAZmediated transcriptional activation of the TBC1D family members (such as Armus). This promotes the fusion of autophagosomal vesicles with lysosomes and regulates autophagy efficiency. Similar to the YAP/TAZ knockdown effect, low mechanical signal input slows down the autophagic flux 53 . Overall, mechanical signals can control autophagic flux through the regulation of YAP/TAZ transcriptional activity.

Cancer stem cell
Autophagy is critical in quality control, remodeling, and metabolic functions of adult and cancer stem cells (CSCs) 61 . Similarly, YAP/TAZ regulates the biological properties of the stem cells during normal organ development and tumorigenesis 62 . The YAP/TAZ-autophagy connection maintains the transformed properties of the tumor cells. It also influences the acquisition of CSC status in benign cells 53 . Increased levels of YAP/TAZ result in the transformation of terminally differentiated epithelial cells (such as primary mammary gland cells) into stem/progenitor cells 63 . These reprogramming events require YAP/TAZ-dependent regulation of autophagy. YAP expression greatly increases autophagosome clearance in induced differentiated cells, whereas inactivated autophagy-related genes impair the YAP-mediated reprogramming steps 53 . These data suggest that YAP/ TAZ requires an effective autophagic flux to maintain CSC-inducing phenotypic plasticity.

Apoptosis
YAP overexpressed in multiple human solid tumors and inhibited apoptosis 64,65 . YAP located in the nucleus interacts with p73 and promotes apoptosis in response to DNA damage, suggesting a dual role of YAP on apoptosis 66 . The role of YAP in inhibiting cell apoptosis is at least partially autophagy-dependent. In ovarian and breast cancer cells, YAP knockdown increased cisplatin-induced apoptosis by decreasing autophagy 67,68 . Besides, YAP maintains mitophagy, the selective degradation of mitochondria by autophagy, which can block the caspase-9 apoptotic pathway, contributing to the gastric cancer cell survival and migration 69 . In tuberous sclerosis complex (TSC) 1-TSC2-deficient cells, the autophagic system impairs the degradation of YAP, leading to YAP accumulation. This subsequently causes abnormal proliferation, inducing apoptosis 70 . The interaction between autophagy and YAP is important in the control and modulation of apoptosis and apoptotic thresholds.

Metastasis
The migration of cancer cells into the circulatory or lymphatic system to form metastases is an extremely complex process in which the Hippo-YAP-autophagy axis is extensively involved [71][72][73] . Recent functional studies suggest that YAP mediates cancer metastasis via the modulation of actin dynamics 74,75 and the control of transcriptional activity 76,77 , and along with the long noncoding RNA (lncRNA)-dependent manner 78,79 . Once the cancer cells spread to the systemic circulation and colonize distant organs, autophagic flux is induced to respond to the stressful microenvironments, including hypoxia, nutritional deficiencies and the extracellular matrix detachment 80,81 . F-actin polymerization drives the cellular membrane extension in lamellipodia, leading to cytoskeletal rearrangement, thus promoting migration 74,82 . YAP deficiency promoted the phosphorylation of JNK (c-Jun N-terminal kinases), which activated Bnip3 transcriptional activity and contributed to the Bnip3-required mitophagy. Higher Bnip3 caused mitochondrial dysfunction and ATP shortage, degraded F-actin via SERCA/[Ca2 +] i/CaMKII/cofilin axis, and attenuated lamellipodiumbased migration 75 . In the triple-negative breast cancer (TNBC) cells, autophagy promoted YAP nuclear localization, promoting TNBC cell migration and invasion 83 .

Hepatocarcinogenesis
YAP and TAZ are widely activated in human malignancies, which plays a vital role in tumorigenesis and the growth of most solid tumors 84 . For example, overexpression of YAP causes prominent hepatomegaly and induces tumor stem cell attributes, and hepatocarcinogenesis 85,86 . Autophagy maintains hepatic organ size and differentiation and, when autophagy is impaired, YAP is a driver of tissue remodeling and tumorigenesis 87 . In vivo, the liver-specific Atg7-deletion (Atg7 knockout (KO)) mice showed an 8.5-fold increase in the relative liver weight compared to the control mice at three months. Dysplastic nodules appeared at 8 months, whereas hepatocellular carcinoma (HCC) developed at 12 months. Meanwhile, the Atg7/YAP double KO mice attenuated hepatomegaly and hepatocarcinogenesis with significantly lower tumor size and number than the Atg7-KO mice 88 .
As expected, knockdown of Atg7 or Atg5 reduced autophagic flux. Interestingly, shAtg7 or shAtg5 induced nuclear translocation of YAP leading to the activation of TEAD4. Furthermore, YAP colocalized with autophagosomes, so that the cytoplasmic degradation of YAP was at least partially autophagy-dependent 88,89 . As YAP is an essential downstream mediator of tissue remodeling, progenitor cell activation, tumorigenesis, and drug resistance in the autophagy-deficient liver, the concomitant loss of YAP attenuates these abnormalities 88,90 . LRPPRC is a mitochondrion-associated protein 91 . The loss of LRPPRC expression promotes hepatocarcinogenesis 92 . Specifically, the deletion of LRPPRC leads to liver-specific YAP nuclear accumulation and induces accumulation of the cyclin-dependent kinase inhibitor p27, which in turn leads to cell polyploidy 92,93 . Concurrently, the deletion of mitochondrion-associated protein LRPPRC reduces p62 protein levels and impairs autophagic maturation. In vitro, LRPPRC knockdown synergistically enhances the diethylamine-induced genomic instability and hepatocarcinogenesis 92 . The lncRNAs are associated with clinicopathological parameters of HCC and can be used as biomarkers for HCC diagnosis 94 . Precisely, lncRNA-ATB activates the YAP-dependent autophagy and increases the expression of ATG5. The high expression of lncRNA-ATB is associated with poor prognosis and pathological characteristics of HCC 95 . Nogo-B, an endoplasmic reticulum residential protein, is highly expressed and promotes tumorigenesis in HCC. Mechanistically, Nogo-B interacts with ATG5 to encourage droplet lipid degradation and induces lipophagy-mediated oxidized lowdensity lipoprotein metabolism and subsequent lysophosphatidic acid-stimulated YAP oncogenic activity 96 .

Mitochondrial quality control
Mitophagy removes damaged mitochondria through autophagy, which is essential for mitochondrial quality control, metabolic homeostasis, and energy supply 97 . Notably, TAZ is required for mitophagy but not autophagosome biogenesis 98 . TAZ is a phospholipid transacylase that catalyzes the remodeling of cardiolipin, a mitochondrial endosomal phospholipid. The redistribution of cardiolipin controls the initiation of mitophagy 98,99 . Mechanistically, TAZ knockdown and inducible TAZ depletion prevent LC3 vesicles from recognizing mitophagosomes, thereby inhibiting mitophagy initiation. This leads to impaired oxidative phosphorylation and oxidative stress. Thus, TAZ is required for the initiation of mitophagy. It is involved in mitochondrial quality control. Mutations of the TAZ gene can cause Barth syndrome 98,100 .

Hippo-YAP-autophagy axis in clinical applications
Autophagy is an attractive therapeutic target in numerous diseases. As autophagy has a wide correlation with normal homeostasis, targeting it is particularly challenging. In contrast, the role of the Hippo pathway in cancer is widely described. It plays a vital role in tissue renewal and repair. Therefore, targeting the Hippo-YAP-autophagy axis might provide several promising targets. The emerging link between the Hippo pathway and autophagy is now largely implicated in pathophysiological processes, such as cancer, metabolic and neurodegenerative diseases, and cardiovascular diseases (Fig. 5) [101][102][103] . Here we introduce some small molecules or drugs that target the Hippo core components autophagy regulatory network (Table 2).

Cancer
Autophagy is involved in several tumor progression stages, including tumorigenesis, progression, and malignant status maintenance 73 . In the early stages of tumorigenesis, autophagy maintains genome stability by removing oncogenic protein substrates, toxic unfolded proteins, and damaged organelles. This prevents chronic tissue damage, cell damage, and inflammation. Moreover, autophagy inhibits the accumulation of carcinogenic p62 protein aggregates, thereby promoting tumor suppression [104][105][106] . At an advanced tumor stage, the autophagic flux increases to cope with various environmental pressures, including hypoxia, nutritional deficiencies, DNA damage, metabolic stress, and chemotherapy. This maintains the survival and growth of tumor cells, and promotes tumor invasion and metastasis 9,73,107 . However, when blocking or promoting autophagy at different stages of tumor development, the biological effects on the tumor cell behavior may vary.
As previously mentioned, YAP acts as an autophagic substrate. The expression of YAP protein and YAP target genes is regulated by the autophagic flux 88,89 . Thus, some small molecules that induce autophagy can reduce the oncogenic activity of YAP/TAZ. Curcumin, a natural polyphenolic compound 108 , induces autophagy in colon cancer cells, further inhibiting cell proliferation and YAP expression 109 . Silibinin, a flavonolignan from the seeds of milk thistle, induced glioblastoma cell apoptosis and autophagy via inhibition of mammalian target of rapamycin and YAP 110 . Shikonin is the main bioactive ingredient extracted from the root of Lithospermum erythrorhizon 111 , which exerts anti-colon cancer effects similar to silibinin and inhibits YAP activity by inducing autophagy 112 .
As of May 2020, a search for "autophagy and cancer" on ClinicalTrials.gov revealed 72 studies focusing on the inhibition and evaluation of autophagy to improve the clinical prognosis for cancer patients. Targeted drugs either as single agents or in combinations can exert antitumor effects by enhancing both apoptotic and toxic autophagic processes 113 . For instance, neratinib (ERBB1/2/4 inhibitor) enhanced [pazopanib (the kinase inhibitor) + entinostat (histone deacetylase inhibitor)] lethality against sarcoma and other tumor cell types in vitro and in vivo. Specifically, the triplet combination increases the phosphorylation of YAP/TAZ and promotes the conversion of LC3 and expression of Beclin1 and ATG13, which together enhance autophagosome formation 114,115 . The mammalian STK 26/ MST4 stimulates ATG4B activity and increases autophagic flux by phosphorylating ATG4B 116 . The MST4-MOB4 complex can disrupt the assembly of the MST1-MOB1 complex by alternative pairing, thereby increasing YAP activity 117 . Neratinib degrades MST4 via autophagy, enhancing LATS1/2 phosphorylation, and is also required for YAP/TAZ inactivation 118 .
Typically, although LATS1 plays a tumor suppressor role in the Hippo pathway, it also exerts a pro-survival function in the HCC cells 119 . Sorafenib (Srf), a multi-kinase inhibitor that promotes autophagy, is the standard treatment for advanced HCC 120,121 . The blockade of LATS1 expression resulted in increased Srf-induced apoptosis and decreased cell viability in vitro, as well as reduced tumor growth in vivo. LATS1 promotes K27-ubiquitination of Beclin1 on lysines K32 and K263, which inhibits autophagy induction and autophagic flux in HCC cells after Srf treatment. In Srfnonrespondent patient, LATS1 expression is significantly increased, suggesting that LATS1 is a clinically relevant biomarker for Srf sensitivity 119 . The revelation of LATS1 functionally independent of the kinase activity in autophagy regulation requires consideration for targeted LATS1 kinases therapy.

Non-cancerous diseases
Autophagy plays a pivotal role in protein quality control, especially in maintaining metabolic homeostasis 122 . Dihydrotanshinone I, a natural monomeric compound isolated from Salvia miltiorrhiza Bunge, can improve liver function and reduce liver fibrosis. The underlying mechanism is associated with the cytoplasmic retention of Fig. 5 The core components of hippo pathway affect various disorders via autophagy. Hippo-YAP axis regulates autophagy and affects the development of disease progression. Inhibition of disease progression (beneficial process) is shown in blue, whereas promotion of disease progression (harmful process) is shown in red. In many diseases, autophagy clears dysfunctional mitochondria and protein aggregates. As two conserved signaling pathways, the Hippo pathway and autophagy intersect in the regulation of cell death and proliferation, tumorigenesis, and survival and growth of tumor cells. ALS, amyotrophic lateral sclerosis; DCM, diabetic cardiomyopathy; HCC, hepatocellular carcinoma; I/R, ischemiareperfusion; NAFLD, non-alcoholic fatty liver disease; PTC, papillary thyroid carcinoma; SCI, spinal cord injury; TSC, tuberous sclerosis complex.
YAP, thereby causing downregulation of fibrogenic gene expression, which stimulates autophagic flux and accelerates the degradation of the liver collagen 123 .
Autophagy offers promising targets for the prevention and treatment of cardiovascular diseases 122 . It has been reported that HMGB1, a chromosomal protein, acts as an autophagy sensor and induces autophagy after prolonged cellular stress 124,125 . The expression of HMGB1 is highly correlated with YAP activity, which is involved in tumorigenesis and acquisition of the tumor stem cell characteristics 126,127 . Adriamycin, an anthracycline chemotherapy drug, can also cause cardiotoxicity 128 . Specifically, adriamycin upregulates HMGB1 expression and induces cardiomyocyte autophagy followed by cardiac damage, whereas YAP reverses adriamycin-induced cardiac damage by downregulating HMGB1 129 . Melatonin regulates autophagy and has both chronobiotic and cytoprotective properties 130 . Melatonin significantly alleviates left ventricle remodeling and cardiac dysfunction in dilated cardiomyopathy by inducing autophagy and alleviating mitochondrial dysfunction, which is partially dependent on MST1/Sirt3 signaling 131,132 .
Autophagy is essential for maintaining proteostasis and a healthy mitochondrial pool, especially in maintaining the homeostasis of non-dividing nerve cells 133 . Melatonin plays a cytoprotective role in a variety of neurodegenerative diseases 130 . In subarachnoid hemorrhage-induced rats, melatonin can regulate the homeostasis between apoptosis and autophagy by inhibiting the ROS-MST1 pathway 134 .

Conclusions and perspectives
The understanding of the regulatory network between the Hippo-YAP pathway and autophagy has gradually been enriched in recent years. In this review, we show that these two highly conserved signaling pathways are widely involved in pathophysiological processes such as apoptosis, cell proliferation, cell differentiation, and metabolism, and can influence the pathogenesis of human diseases. This multidisciplinary view improves our understanding of why these two signaling pathways have been preserved throughout evolution. However, variations in the activity between autophagy and the Hippo-YAP pathway in different tissue types, tumor microenvironments, and disease states are some of the fundamental puzzles yet to be resolved. In addition, the paradoxical effect of autophagy in cancer makes autophagy-targeted therapy in cancer controversial. However, the Hippo pathway dysregulation occurs in a wide range of human cancers. This is essential in the development of novel and more specific drugs. For example, a combination of Hippo pathway-targeted drugs with autophagy inhibitors and inducers may be potential therapies for various human diseases. A better understanding and targeting of the Hippo-YAP-autophagy axis is an auspicious direction.