O-GlcNAcylation: An Emerging Protein Modification Regulating the Hippo Pathway

Simple Summary The contact point between the Hippo pathway, which serves as a central hub for various external environments, and O-GlcNAcylation, which is a non-canonical glycosylation process acting as a dynamic regulator in various signal transduction pathways, has recently been identified. This review aims to summarize the function of O-GlcNAcylation as an intrinsic and extrinsic regulator of the Hippo pathway. Abstract The balance between cellular proliferation and apoptosis and the regulation of cell differentiation must be established to maintain tissue homeostasis. These cellular responses involve the kinase cascade-mediated Hippo pathway as a crucial regulator. Hence, Hippo pathway dysregulation is implicated in diverse diseases, including cancer. O-GlcNAcylation is a non-canonical glycosylation that affects multiple signaling pathways through its interplay with phosphorylation in the nucleus and cytoplasm. An abnormal increase in the O-GlcNAcylation levels in various cancer cells is a potent factor in Hippo pathway dysregulation. Intriguingly, Hippo pathway dysregulation also disrupts O-GlcNAc homeostasis, leading to a persistent elevation of O-GlcNAcylation levels, which is potentially pathogenic in several diseases. Therefore, O-GlcNAcylation is gaining attention as a protein modification that regulates the Hippo pathway. This review presents a framework on how O-GlcNAcylation regulates the Hippo pathway and forms a self-perpetuating cycle with it. The pathological significance of this self-perpetuating cycle and clinical strategies for targeting O-GlcNAcylation that causes Hippo pathway dysregulation are also discussed.


Hippo Pathway as an Essential Cellular Hub
The Hippo pathway is controlled by diverse mechanical or chemical upstream inputs rather than exclusive molecules ( Figure 1). Cell adhesion enhances Hippo signaling by attracting MST1/2 and LATS1/2 closer to each other or sequestering YAP/TAZ at cell junctions [52]. Mechanical cues regulate the Hippo pathway by actin remodeling, and extracellular signaling molecules such as hormones, growth factors, and lysophosphatidic acid (LPA) modulate the Hippo pathway via their receptors or G protein-coupled receptors (GPCRs) [52,53]. The Hippo pathway is also controlled by stresses such as glucose deprivation [54][55][56], hypoxia [57], endoplasmic reticulum stress [55], heat shock [58][59][60], osmotic stress [61], and oxidative stress [62,63]. Recently, the striatin-interacting phosphatase and kinase (STRIPAK) complex was reported as an upstream regulator of the Hippo pathway. It dephosphorylates and suppresses MST1 in a manner dependent on Ras homolog family member A (RhoA) [64]. The STRIPAK mechanism can explain how upstream signals such as LPA and serum connect to one another to regulate the Hippo signaling pathway, but their correlation with other stimuli needs to be further investigated. Schematic representation of the mammalian Hippo pathway. The Hippo kinase cascade, composed of Ser/Thr kinases MST1/2 and LATS1/2, adaptor proteins SAV1 and MOB1, and effectors YAP/TAZ, is regulated by various stimuli, including cell-cell junctions, cellular stresses, mechanical cues, and multiple extracellular signaling molecules. A "+" indicates a stimulus that increases the activity of the Hippo pathway, and a "−" indicates a stimulus that decreases activity. The Hippo pathway phosphorylates YAP/TAZ, leading to their cytoplasmic sequestration and proteasomal degradation. Dephosphorylated YAP/TAZ translocate into the nucleus and act as transcriptional cofactors, thereby controlling cellular responses such as proliferation, survival, and metastasis and affecting stemness, regeneration, organ size, and tissue homeostasis.

Hippo Pathway as an Essential Cellular Hub
The Hippo pathway is controlled by diverse mechanical or chemical upstream inputs rather than exclusive molecules ( Figure 1). Cell adhesion enhances Hippo signaling by attracting MST1/2 and LATS1/2 closer to each other or sequestering YAP/TAZ at cell junctions [52]. Mechanical cues regulate the Hippo pathway by actin remodeling, and extracellular signaling molecules such as hormones, growth factors, and lysophosphatidic acid (LPA) modulate the Hippo pathway via their receptors or G protein-coupled receptors (GPCRs) [52,53]. The Hippo pathway is also controlled by stresses such as glucose deprivation [54][55][56], hypoxia [57], endoplasmic reticulum stress [55], heat shock [58][59][60], osmotic stress [61], and oxidative stress [62,63]. Recently, the striatin-interacting phosphatase and kinase (STRIPAK) complex was reported as an upstream regulator of the Hippo pathway. It dephosphorylates and suppresses MST1 in a manner dependent on Ras homolog family member A (RhoA) [64]. The STRIPAK mechanism can explain how upstream signals such as LPA and serum connect to one another to regulate the Hippo signaling pathway, but their correlation with other stimuli needs to be further investigated.

O-GlcNAcylation
YAP/TAZ hyperactivation is a common feature of cancer cells, but the genetic mutations of the core components in the Hippo pathway are rarely found in patients with cancer [4,16,[82][83][84]. This observation begs the question of what causes Hippo pathway dysregulation in cancer cells? The abnormal elevation of intracellular O-GlcNAcylation in cancer cells is one possible answer.

OGT and OGA: The Sole Enzymes Responsible for the Intracellular O-GlcNAcylation Cycle
O-GlcNAc modifications of thousands of intracellular proteins are reversibly and dynamically regulated by two enzymes, namely OGT and OGA [85]. OGT is an exclusive enzyme involved in O-GlcNAcylation [90,93]. Human OGT (hOGT) contains 2.5-13.5 tetratricopeptide repeats (TPRs), a linker domain, and C-terminal catalytic domains [94,95]. Three hOGT variants are derived by alternative splicing and multiple transcription start sites ( Figure 3A). Among them, nucleocytoplasmic OGT (ncOGT) is the longest (with 13.5 TPR repeats) and most abundant OGT variant. The shortest OGT (sOGT) possesses 2.5 TPR repeats. ncOGT and sOGT are both found in the nucleus and cytoplasm. Mitochondrial OGT (mOGT) contains nine TPR repeats, and its location is due to a mitochondrial targeting sequence (MTS) in the N-terminal region. The substrate recognition of OGT relies on TPR repeats [96]. These conserved tandem repeats of 34 amino acids form a superhelix structure, and the asparagine ladder in the superhelix mediates the recognition between OGT and its diverse substrates [97]. Moreover, diverse adapter proteins that recruit OGT to specific substrates depending on the cellular conditions confer OGT substrate selectivity and substrate diversity [98].

OGT and OGA: The Sole Enzymes Responsible for the Intracellular O-GlcNAcylation Cycle
O-GlcNAc modifications of thousands of intracellular proteins are reversibly and dynamically regulated by two enzymes, namely OGT and OGA [85]. OGT is an exclusive enzyme involved in O-GlcNAcylation [90,93]. Human OGT (hOGT) contains 2.5-13.5 tetratricopeptide repeats (TPRs), a linker domain, and C-terminal catalytic domains [94,95]. Three hOGT variants are derived by alternative splicing and multiple transcription start sites ( Figure 3A). Among them, nucleocytoplasmic OGT (ncOGT) is the longest (with 13.5 TPR repeats) and most abundant OGT variant. The shortest OGT (sOGT) possesses 2.5 TPR repeats. ncOGT and sOGT are both found in the nucleus and cytoplasm. Mitochondrial OGT (mOGT) contains nine TPR repeats, and its location is due to a mitochondrial targeting sequence (MTS) in the N-terminal region. The substrate recognition of OGT relies on TPR repeats [96]. These conserved tandem repeats of 34 amino acids form a superhelix structure, and the asparagine ladder in the superhelix mediates the recognition between OGT and its diverse substrates [97]. Moreover, diverse adapter proteins that recruit OGT to specific substrates depending on the cellular conditions confer OGT substrate selectivity and substrate diversity [98].
Similar to OGT, OGA recognizes diverse substrates. Human OGA has two distinct splice variants ( Figure 3B): nucleocytoplasmic OGA (ncOGA), which is located primarily in the cytoplasm, and short OGA (sOGA), which is located primarily in the nucleus and lipid droplets [99,100]. Both OGA variants possess an N-terminal hydrolase catalytic domain that hydrolyzes O-GlcNAc modifications. However, sOGA does not have the C-terminal histone acetyltransferase (HAT)-like domain and part of the stalk domain. The stalk domain participates in forming an OGA homodimer in which a potential substrate- Similar to OGT, OGA recognizes diverse substrates. Human OGA has two distinct splice variants ( Figure 3B): nucleocytoplasmic OGA (ncOGA), which is located primarily in the cytoplasm, and short OGA (sOGA), which is located primarily in the nucleus and lipid droplets [99,100]. Both OGA variants possess an N-terminal hydrolase catalytic domain that hydrolyzes O-GlcNAc modifications. However, sOGA does not have the C-terminal histone acetyltransferase (HAT)-like domain and part of the stalk domain. The stalk domain participates in forming an OGA homodimer in which a potential substrate-binding cleft is created by covering the catalytic domain of the sister monomer OGA [101][102][103]. Through this substrate-binding cleft, binding to a GlcNAc moiety and sequence-independent peptide backbone interactions with the substrate are possible [101][102][103]. In addition, sequencedependent side chain interactions can occur within the substrate-binding cleft [104]. Hence, interactions within the OGA substrate-binding cleft likely allow OGA to differentially regulate the O-GlcNAcylation turnover rate for various substrates [104]. Due to the lack of these interactions in sOGA, the hydrolase activity of sOGA is much weaker than that of ncOGA. Human OGA was expected to possess histone acetyltransferase activity due to the similarity of its HAT-like domain to GCN5-related N-acetyltransferase (GNAT) [105]. However, the P-loop motif that supports acetyl-CoA binding is absent from the HAT-like domain of hOGA [106,107]. Thus, the HAT-like domain of OGA is a pseudo-HAT, but its function remains unclear. Although these structural studies have provided insights into the interactions of OGA with various substrates, further studies are needed to explain clearly how OGA is regulated to recognize substrates and investigate the functional roles of the HAT-like domain in substrate recognition by OGA.
Cancers 2022, 14, 3013 6 of 17 nuclear import of OGT by facilitating its interaction with importin α5 [110]. OGT O-Glc-NAcylation at Ser3 and Ser4 also decreases OGT activity by competing with GSK3β-mediated phosphorylation, which enhances the OGT activity [111]. However, OGA O-Glc-NAcylation at Ser405 reduces its stability and enzymatic activity [112,113] (Figure 3D). Hence, additional research is needed to support the hypothesis that the O-GlcNAcylation of OGA is involved in maintaining O-GlcNAc homeostasis.  O-GlcNAcylation affects the cellular processes involved in gene expression and signal transduction by regulating chromatin remodeling and protein stability, activity, localization, and protein-protein interactions [98,108,109]. In particular, it plays a role in many cellular signaling pathways through its reciprocal effects with phosphorylation [14,15]. Thus, O-GlcNAc homeostasis must be maintained within cells to sustain normal cellular functions, and it is accomplished by OGT and OGA [98]. OGT and OGA are mutually regulated in terms of the gene transcription levels, protein activity, and protein stability [98]. The O-GlcNAcylation of OGT and OGA is also thought to play a role in maintaining O-GlcNAc homeostasis [98]. OGT O-GlcNAcylation decreases the overall level of intracytoplasmic O-GlcNAcylation ( Figure 3C). OGT O-GlcNAcylation at Ser389 promotes the nuclear import of OGT by facilitating its interaction with importin α5 [110]. OGT O-GlcNAcylation at Ser3 and Ser4 also decreases OGT activity by competing with GSK3β-mediated phosphorylation, which enhances the OGT activity [111]. However, OGA O-GlcNAcylation at Ser405 reduces its stability and enzymatic activity [112,113] (Figure 3D). Hence, additional research is needed to support the hypothesis that the O-GlcNAcylation of OGA is involved in maintaining O-GlcNAc homeostasis.

Effect of O-GlcNAcylation on the Hippo Pathway
Glucose is a major factor regulating the Hippo pathway. As a representative energy sensor, AMP-activated protein kinase (AMPK) activated in response to glucose deprivation induces YAP phosphorylation in a LATS-dependent and LATS-independent manner; consequently, it interferes with the binding of YAP to TEAD [54,55]. Glucose metabolism enhances YAP/TAZ transcriptional activity. Phosphofructokinase 1 (PFK1), the key enzyme in the first step of glycolysis, binds to TEAD and functionally cooperates with YAP/TAZ [114]. Glucose metabolism and the Hippo pathway are also connected by O-GlcNAcylation synthesized by the HBP, which branches from glycolysis. YAP activity is enhanced by an increase in cellular O-GlcNAcylation levels via OGT overexpression or treatment with PUGNAc, an OGA inhibitor [9,10]; conversely, such activity is attenuated by a decrease in O-GlcNAcylation via OGT knockdown or treatment with OSMI, an OGT inhibitor [9,12].
Cancer cells enhance the glucose uptake to meet the increased energy and metabolism demands for cell growth and proliferation [115,116]. In cancer cells, excessive glucose uptake and increased GFAT, the rate-limiting enzyme in the HBP, cause an increase in UDP-GlcNAc from the HBP flux [117,118]. Together with an increase in UDP-GlcNAc synthesis, OGT overexpression in cancer cells causes aberrant hyper-O-GlcNAcylation [21][22][23]. O-GlcNAcylation enhances YAP/TAZ activity and YAP/TAZ induce an increase in the cellular O-GlcNAcylation levels [9,10,12,79,80]. This mutual relationship drives a selfperpetuating cycle that sustains aberrant hyper-O-GlcNAcylation and Hippo pathway dysregulation [12]. Hence, O-GlcNAcylation should be studied as a factor regulating the Hippo pathway. In this section, we describe O-GlcNAcylation associated with Hippo pathway dysregulation and suggest potential mechanisms through which O-GlcNAcylation affects the Hippo pathway by integrating the results of studies on O-GlcNAcylation in intracellular signaling pathways that crosstalk with the Hippo pathway.

Mechanism by Which O-GlcNAcylation Induces Hippo Pathway Dysregulation
Since YAP O-GlcNAcylation was reported in 2017, several studies have been conducted on the O-GlcNAcylation of core components in the Hippo pathway kinase cascade [9][10][11][12]. Subsequently, the O-GlcNAcylation of MST1 and LATS2 has been confirmed [12]. However, the O-GlcNAcylation of MST2 and LATS1, whose sequences are similar to those of MST1 and LATS2, has not been detected [9,12]. Furthermore, the O-GlcNAcylation of TAZ, SAV, and MOB has not been observed [9,11] Although the effect of MST O-GlcNAcylation on the Hippo pathway is unclear, the O-GlcNAcylation of YAP and LATS2 is closely associated with Hippo pathway dysregulation ( Figure 4). The O-GlcNAcylation of LATS2 at Thr436 interferes with the interaction between LATS2 and MOB1, decreasing the LATS2 activity by inhibiting MST-mediated phosphorylation [12]. Thus, the O-GlcNAcylation of LATS2 increases the activity of YAP/TAZ [12]. The O-GlcNAcylation of YAP at Ser109 or Thr241 also enhances the activity of YAP by inhibiting its interaction with LATS1 [9,10].
In addition to the O-GlcNAcylation of the core components in the Hippo kinase cascade, the O-GlcNAcylation of angiomotin (AMOT) and LDL receptor-related protein 6 (LRP6) is possibly implicated in Hippo pathway dysregulation [11,13] (Figure 4). AMOT affects cancer growth and invasion via several signaling pathways: mTOR, MAPK, Wnt signaling, and the Hippo pathway [119]. However, studies have yet to verify whether AMOT acts as an oncoprotein or a tumor suppressor, because its effect on tumor growth differs depending on the cancer cell type [119]. For example, the effect of AMOT on the Hippo pathway varies depending on the cell type. AMOT acts as an oncoprotein by increasing the activity of YAP in hepatic carcinoma, but it acts as a tumor suppressor by repressing the activation of YAP target genes in ovarian cancer [119]. AMOT has two isoforms, namely AMOT-p130 and AMOT-p80, due to alternative splicing. AMOT-p130, which can interact with YAP via PPxY motifs in its N-terminal region, undergoes O-GlcNAcylation [11,120]. In liver cancer cells, the effect of AMOT on the Hippo pathway depends on the concentration of glucose, a major source of UDP-GlcNAc. AMOT functions as a YAP suppressor under normal glucose conditions, but under high glucose conditions, AMOT induces the nuclear accumulation of YAP, thereby enhancing the pro-tumorigenic function of YAP [11]. LRP6, a co-receptor of canonical Wnt signaling, also affects the Hippo pathway by binding to Merlin [121,122]. This interaction suppresses the Hippo pathway by reducing the interaction between Merlin and LATS1/2 [13,121]. Under nutrient starvation conditions, such as serum-or glucose-free culture, LRP6 O-GlcNAcylation decreases, and the endocytosis-mediated lysosomal degradation of LRP6 increases. As a result, more Merlin becomes available to interact with LATS1/2, and the YAP activity decreases [13]. These results further support that O-GlcNAcylation indirectly attenuates the Hippo pathway. Further research that identifies O-GlcNAcylation sites in AMOT and LRP6 is needed to elucidate the function of AMOT and LRP6 O-GlcNAcylation.

O-GlcNAcylation in Cellular Signaling Pathways That Crosstalk with the Hippo Pathway
The Hippo pathway crosstalks with multiple cellular signaling pathways, such as Wnt, TGFβ, GPCR, and Notch [65]. Because some of these signaling pathways are regulated by O-GlcNAcylation, the Hippo pathway is expected to be indirectly affected by O- Excessive YAP/TAZ activation can be prevented, because YAP triggers the transcription of Merlin and LATS2, which are negative regulators of YAP and TAZ [79,80]. However, this negative feedback loop can be blocked by LATS2 O-GlcNAcylation and, even if more LATS2 is recruited to the MST-MOB1 complex by an increase in Merlin and LATS2 transcription, LATS2 O-GlcNAcylation inhibits the interaction between the MST-MOB1 complex and LATS2 [12]. Hence, abnormally increased O-GlcNAcylation can disrupt Hippo pathway homeostasis, leading to persistent YAP and TAZ hyperactivation. Interestingly, activated YAP also promotes glucose uptake by enhancing the GLUT3 gene expression and increases HBP-stimulating intracellular metabolites, such as glutamine, acetyl-CoA, and Fru-6-P [10,54]. In addition, YAP enhances OGT transcription, which increases the overall intracellular O-GlcNAcylation levels [9,10]. In summary, aberrantly increased O-GlcNAcylation induces a positive feedback loop that sustains a hyper-O-GlcNAcylation state via Hippo pathway dysregulation. These findings imply that increased O-GlcNAcylation triggers Hippo pathway dysregulation in cancer cells and maintains a hyper-O-GlcNAcylation state, leading to tumor growth and metastasis. In xenograft mouse experiments that observe the effects of YAP and LATS2 O-GlcNAcylation on tumor growth, tumors from grafts expressing an O-GlcNAcylation-deficient YAP (S109A or T241A) or LATS2 (T436A) mutant are significantly smaller than those from grafts expressing wild-type YAP or LATS2 [9,10,12].

O-GlcNAcylation in Cellular Signaling Pathways That Crosstalk with the Hippo Pathway
The Hippo pathway crosstalks with multiple cellular signaling pathways, such as Wnt, TGFβ, GPCR, and Notch [65]. Because some of these signaling pathways are regulated by O-GlcNAcylation, the Hippo pathway is expected to be indirectly affected by O-GlcNAcylation ( Figure 5). For example, β-catenin, an essential mediator of the canonical Wnt signaling pathway, is O-GlcNAcylated at Thr41 in its N-terminus [123]. This O-GlcNAcylation increases β-catenin stability by competing with ubiquitination-inducing phosphorylation that occurs in the absence of a Wnt activity [123]. Activated β-catenin induces the transcriptional upregulation of YAP by forming a β-catenin/TCF4 complex that binds to a DNA enhancer element within YAP in colorectal cancer cells [124]. Phosphorylated β-catenin also induces the proteasomal degradation of TAZ by bridging TAZ to β-TrCP, a ubiquitin ligase [66]. Hence, O-GlcNAcylation may indirectly enhance YAP/TAZ activity by controlling Wnt signaling. Likewise, the O-GlcNAcylation of Smad4, an important regulator of the TGFβ signaling pathway, at Thr63 prevents the GSK3β-mediated proteasomal degradation of Smad4, inducing the TGFβ signaling pathway [125]. SnoN, a target gene of TGFβ signaling, stabilizes TAZ by preventing phosphorylation by LATS [68]. Thus, Smad4 O-GlcNAcylation likely promotes YAP/TAZ activity by inducing TGFβ signaling. However, the O-GlcNAcylation of PKC decreases the TGFβRII expression by diminishing PKC activity; as a result, TGFβ signaling is reduced [126,127]. Therefore, the effect of O-GlcNAcylation on the Hippo pathway via TGFβ signaling may vary depending on OGT target proteins. PKA, a protein kinase that bridges the Hippo pathway and GPCR-Gαs signaling by enhancing LATS1/2 activity through the direct phosphorylation of LATS1/2 or the suppression of actin fiber formation, is also O-GlcNAcylated [128][129][130]; consequently, PKA kinase activities are enhanced [131]. Therefore, the O-GlcNAcylation of PKA may enhance the Hippo pathway through the GPCR signaling pathway. NOTCH1 O-GlcNAcylation induces the release of the Notch intracellular domain (NICD) by enhancing DLL1-NOTCH and DLL4-NOTCH1 interaction [132]. NICD promotes YAP/TAZ stability, thereby enhancing YAP/TAZ activity [71][72][73]. Hence, O-GlcNAcylation may improve YAP/TAZ stability by regulating NOTCH signaling. Collectively, these studies imply that O-GlcNAcylation indirectly affects the Hippo pathway by regulating its associated pathways. However, these conclusions are derived by integrating individual findings from multiple studies; thus, confirmatory studies are needed. Table 1

Conclusions
Since the discovery of the Hippo pathway in the early 21st century, its phosphorylation-mediated signaling has been elucidated. Although phosphorylation is the primary mechanism of Hippo pathway regulation, it is affected by several PTMs, such as ubiquitination, acetylation, methylation, sumoylation, and O-GlcNAcylation [9][10][11][12]133]. Particularly, O-GlcNAcylation, which can crosstalk with phosphorylation, causes Hippo pathway dysregulation, leading to continuous YAP/TAZ hyperactivation. In addition, hyperactivated YAP increases intracellular glucose and HBP-stimulated metabolite concentrations and promotes OGT gene expression, which abnormally increases intracellular O-GlcNAcylation. This mutual relationship between O-GlcNAcylation and the Hippo pathway causes a self-perpetuating cycle that disrupts intracellular O-GlcNAc homeostasis, thereby sustaining aberrant hyper-O-GlcNAcylation and Hippo pathway dysregulation. The O-GlcNAcylation of β-catenin, a mediator of the canonical Wnt signaling pathway, competes with ubiquitinylation-inducing β-catenin phosphorylation, which may stabilize β-catenin and TAZ and promote β-catenin/TCF4 complex-mediated YAP expression. Smad4 O-GlcNAcylation enhances the TGF-β/SMAD signaling pathway, which can upregulate SnoN gene expression, thereby inactivating LATS1/2 by stabilizing Smad4. PKC O-GlcNAcylation is possibly related to TGF-β signaling in a way that TGFβRII expression is decreased by reducing PKC activities. In Gαs-coupled GPCR signaling, PKA O-GlcNAcylation may enhance LATS1/2 activity by increasing PKA-mediated LATS1/2 phosphorylation or inhibiting actin fiber formation. NOTCH1 O-GlcNAcylation elicits the release of NICD, which can stabilize YAP/TAZ, by enhancing the interaction between NOTCH1 and DLL1 or DLL4.

Conclusions
Since the discovery of the Hippo pathway in the early 21st century, its phosphorylationmediated signaling has been elucidated. Although phosphorylation is the primary mechanism of Hippo pathway regulation, it is affected by several PTMs, such as ubiquitination, acetylation, methylation, sumoylation, and O-GlcNAcylation [9][10][11][12]133]. Particularly, O-GlcNAcylation, which can crosstalk with phosphorylation, causes Hippo pathway dysregulation, leading to continuous YAP/TAZ hyperactivation. In addition, hyperactivated YAP increases intracellular glucose and HBP-stimulated metabolite concentrations and promotes OGT gene expression, which abnormally increases intracellular O-GlcNAcylation. This mutual relationship between O-GlcNAcylation and the Hippo pathway causes a selfperpetuating cycle that disrupts intracellular O-GlcNAc homeostasis, thereby sustaining aberrant hyper-O-GlcNAcylation and Hippo pathway dysregulation. Hippo pathway dysregulation and aberrant increases in intracellular O-GlcNAcylation have been observed in cancer cells derived from various tissues, and they contribute to carcinogenesis and cancer progression. Thus, Hippo pathway components and O-GlcNAcylation regulatory enzymes (OGT and OGA) are potential targets for cancer diagnosis and treatment. Currently, Hippo pathway-targeting compounds, such as Verteporfin, and various OGT-and OGA-targeting molecular probes, such as OSMI-1 and Thiamet-G, are being developed. However, the systemic application of these compounds causes severe side effects because OGT and OGA exclusively control the O-GlcNAcylation of numerous vital intracellular proteins and the Hippo pathway is also involved in tissue and organ growth, development, regeneration, repair, and immune modulation. With the development of compounds targeting specific O-GlcNAcylation that induces Hippo pathway dysregulation, new cancer treatment approaches can be established. Such compounds can be developed into a wide range of medical applications due to the diversity of diseases associated with O-GlcNAc homeostasis disruption and Hippo pathway dysregulation, including inappropriate immune responses, excessive fibrosis, and metabolic disorders. YAP O-GlcNAcylation disturbs YAP-LATS1 interactions; LATS2 O-GlcNAcylation enhances YAP/TAZ activity and stability and blocks the negative feedback loop of the Hippo pathway, resulting in persistent YAP/TAZ hyperactivation. Hence, YAP O-GlcNAcylation and LATS2 O-GlcNAcylation are excellent target candidates.
Our understanding of how OGT and OGA select target proteins differently depending on the cellular environment is insufficient, and the technology that targets only the O-GlcNAcylation of specific target proteins is not secure. Moreover, the fundamental biology underlying the interactions between O-GlcNAcylation and the Hippo pathway needs additional research. With additional knowledge about the mutual relationship between O-GlcNAcylation and the Hippo pathway and the development of techniques for detecting and regulating the O-GlcNAcylation of specific target proteins, more therapeutics and regenerative medicine products can be discovered to cure human diseases.

Data Availability Statement:
The data used to support the findings of this study are available from the corresponding author upon reasonable request.

Conflicts of Interest:
The authors declare no conflict of interest.