GPCR-Hippo Signaling in Cancer

The Hippo signaling pathway is involved in tissue size regulation and tumorigenesis. Genetic deletion or aberrant expression of some Hippo pathway genes lead to enhanced cell proliferation, tumorigenesis, and cancer metastasis. Recently, multiple studies have identified a wide range of upstream regulators of the Hippo pathway, including mechanical cues and ligands of G protein-coupled receptors (GPCRs). Through the activation related G proteins and possibly rearrangements of actin cytoskeleton, GPCR signaling can potently modulate the phosphorylation states and activity of YAP and TAZ, two homologous oncogenic transcriptional co-activators, and major effectors of the Hippo pathway. Herein, we summarize the network, regulation, and functions of GPCR-Hippo signaling, and we will also discuss potential anti-cancer therapies targeting GPCR-YAP signaling.

β1and β2-adrenergic receptors Catecholamines (e.g., dobutamine) Gαs ↓ Breast cancer [106] GPCR ligands regulate the Hippo pathway differentially ( Figure 1). It has been established that the effect of GPCR ligands on YAP/TAZ activity is dependent on the type of downstream G proteins activated [32]. GPCRs coupled with Gα 12/13 , Gα q/11 , or Gα i/o , such as LPA and thrombin receptors, will activate YAP/TAZ; on the contrary, GPCRs coupled with Gα s , such as epinephrine and glucagon receptors, will inhibit YAP/TAZ [32]. The function of GPCRs and G proteins on the Hippo signaling is most likely depended on protein kinases (such as PKA and PKC), Rho GTPases, and remodeling of the actin cytoskeleton [4]. PKA has been proposed to mediate upstream signals by repressing actin fiber formation or phosphorylating LATS1/2 directly [107][108][109]. The effect of PKC appears to be isoform-specific, for instance canonical PKC isoforms induce YAP/TAZ activity, whereas novel PKC isoforms repress YAP/TAZ activity [110]. The isoform-specific effect of PKC towards YAP/TAZ may explain cell type-dependent response to PKC activation, as the expression of PKC isoforms vary across different cell types. It seems like MST1/2 is not a direct target of GPCR signaling but the phosphorylation level of LATS1/2 is sensitive to different GPCR ligands [32]. In the absence of MST1/2, MAP4Ks may be responsible for LATS1/2 phosphorylation, as the deletion of MST1/2 and MAP4Ks together abolished the regulation of LATS1/2 phosphorylation by GPCR signaling [5]. Collectively, G proteins and related kinases relay the GPCR signaling to regulate dynamics of the actin cytoskeleton, which, in turn, can be sensed by the Hippo pathway. How different states of actin cytoskeleton sensed by Hippo pathway components remains unclear.
Some seven-(pass)-transmembrane domain receptors, such as Frizzled and smoothened (SMO), are not considered as typical GPCRs. Recent evidence suggests that these atypical GPCRs also regulate the Hippo pathway in a G protein-dependent manner. For instance, Wnt ligands and their receptors (Frizzle proteins) can repress LATS1/2 activity and lead to enhanced YAP/TAZ activity [111]. In addition, Hedgehog (Hh) ligands, via SMO -Gα s -cAMP-PKA signaling axis, lead to repression of YAP/TAZ [112]. Thus, atypical GPCRs can regulate the Hippo pathway and also contribute to the crosstalk between Hippo and other important pathways (such as Wnt and Hedgehog) in development and cancer.
The regulation of Hippo pathway by GPCR signaling can also be fine-tuned by additional signals. For instance, it has been shown that the effect of GPCR on YAP/TAZ activity is enhanced when insulin is present, and PI3K and PKD downstream of insulin receptor are involved in this regulation [113]. Moreover, MAPK signaling has also been shown to modulate the Hippo pathway [114]. Hence, the crosstalk between the GPCR-Hippo signaling axis with other pathways should be explored in the future.
Polypeptides and secreting proteins, such as thrombin, glucagon, Angiotensin II, and Endothelin, also modulate YAP/TAZ activity via GPCR signaling [32, [84][85][86][87]. These signals, either locally or from a long range, represent major constitutes of cell niche or microenvironment, suggesting that the Hippo pathway is regulated collectively by signals surrounding a given cell. Adhesion GPCRs link cells to their neighbors and probably cell matrix [88], these receptors may also link physical signals to the Hippo pathway.  GPCRs modulate Hippo-YAP signaling via GPCRs-G-protein-cytoskeleton axis. The molecular scheme of GPCR-Hippo signaling is shown, including activating and inhibitory regulation of YAP/TAZ. GPCRs and G proteins activating YAP/TAZ are marked with red and those inhibiting YAP/TAZ are in blue.

Widespread Alternations of GPCR-YAP Signaling Axis in Cancer
Aberrant GPCR signaling is an important mechanism in cancer development [115]. Different effectors, such as MAPK signaling, mediate the aberrant GPCR signaling to promote tumorigenesis [116]. As a new downstream branch of GPCR signaling, the Hippo tumor suppressor pathway may also play a role in this process. Moreover, aberrant GPCR signaling represents a potential mechanism responsible for prevalent YAP/TAZ activation in human cancers (Table 1).
Cancer genome sequencing analyses revealed that mutations in GPCRs and G proteins are widespread and frequent in multiple tumor types [117]. It has been reported that GNAQ and GNA11 encoding the alpha subunits of Gα q and Gα 11 , respectively, are frequently mutated at Arg183 or Gly209 in uveal melanoma and blue nevi [118,119], the mutations at Arg183 and Gly209 result in constitutive activation of Gα q/11 , and several downstream effectors including YAP/TAZ are activated, leading to tumorigenesis [120,121]. Mutations in GNAS has been discovered in human medulloblastoma [122] and it has been demonstrated in mice that GNAS loss leads to YAP activation and tumorigenesis [109,123]. Thus, YAP activation might be a common mechanism underlying cancer associated mutations on G proteins. However, mutations at Gly227 on GNAS contribute to the development of hormone-secreting pituitary tumors and thyroid adenomas [124,125], in principle this mutation will inactivate YAP/TAZ [126].
Mutations in genes encoding GPCRs are observed in approximately 20% of cancers, including mutations in TSHR in thyroid cancer, luteinizing hormone receptor (LHCGR), and follicle stimulating hormone receptor (FSHR) in breast, lung, and colon cancers [127]. Smoothened (SMO) is also frequently mutated in cancers arising at the colon, central nervous system, and many other cancers types [128]. Mutations observed in the family of GPCR adhesion receptors, the majority of which are still orphan, resulted in constitutive activation of the receptors, leading to pathological conditions [129]. Moreover, mutated glutamate receptors, such as GRM8, GRM1, and GRM3, have been implicated in squamous non-small cell lung cancer (NSCLC) and melanomas [126]. Currently, the effect of these mutations on the Hippo pathway has not been systematically examined.
Aberrant expression of GPCRs and their ligands may also contribute to tumorigenesis. Elevated PAR1 (a thrombin receptor) expression is associated with poor differentiation and metastasis of breast cancer [130]. Increased expression of G protein-coupled estrogen receptors (GPER) and TAZ activation are detected at the early stage of breast tumor development [89]. Additionally, aberrant expression of LPA receptors may elicit cancer initiation and progression in breast cancer and ovarian cancer via activation of the Rho-dependent transduction pathway [131]. Abnormal G-protein coupled hormone receptor also involved in several adrenal diseases, including tumor and hyperplasia [132]. Kaposi's sarcoma (KS) is caused by infection of human herpesvirus 8 (HHV-8, also known as KSHV) and a viral GPCR (vGPCR) encoded in HHV-8 genome can activate TAZ, which is essential for the development of KS [133]. Recently, an orphan G protein-coupled receptor GPRC5A has been identified as a hypoxia-induced protein, which protects hypoxic tumor cells from apoptosis via the HIF-GPRC5A-RhoA-YAP axis [134]. For GPCR ligands, the levels of LPA has been considered as a marker for ovarian cancer, and high circulating angiotensin II is associated with carcinogenesis, prognosis, and drug resistance in several malignancies, such as colorectal cancer, hepatocellular carcinoma, melanoma, ovarian cancer, and breast cancer [135][136][137][138][139][140][141]. Together, abnormal expression of GPCRs and GPCR ligands are associated with the development of different cancers and YAP/TAZ activation may, at least in part, participate in the tumorigenic process.

Potential Cancer Therapies Targeting GPCR-Hippo Signaling Axis
Given the frequent dysregulation of GPCR signaling and Hippo pathway in cancer, the GPCR-Hippo signaling axis may serve as a therapeutic target for cancer treatment. As the Hippo pathway is a downstream branch of the GPCR signaling, cancers initiated by aberrant GPCR signaling might be treated by modulating the Hippo pathway, especially YAP/TAZ activity. Meanwhile, for cancers with a dependency on high YAP/TAZ activity, drugs targeting GPCRs and G proteins may reduce YAP/TAZ activation and slowdown cancer progression.
The Hippo pathway can be modulated at multiple levels [69]. As the function of Hippo pathway is mediated mainly by YAP/TAZ and associated transcription factors TEAD1-4, different approaches have been developed to disrupt YAP/TAZ-TEAD interaction. Liu and colleagues discovered the porphyrin family compounds, such as verteporfin (VP), can effectively block YAP-TEAD interaction [142]. A VGLL4-mimicking peptide has also been developed to displace YAP from TEAD [61]. Recently, it has been reported that the pocket in TEAD critical for YAP/TAZ binding is palmitoylated and targeting TEAD palmitoylation is a new approach to repress YAP/TAZ activity [143][144][145][146]. Both VP and VGLL4-mimicking peptides have been used in vitro and in vivo to repress YAP/TAZ activity, tissue growth, and tumorigenesis, whereas further improvement of these drugs might be required for clinical use.
GPCRs represent a major target of currently available drugs, and some GPCR-based drugs could be repositioned to block YAP/TAZ activity. For instance, Gα s -targeted molecules may repress YAP/TAZ activity in a way that is similar to epinephrine, dobutamine, and glucagon [106,147]. In contrast, antagonizing or depleting Gα 12/13 -, Gα q/11 -, or Gα i/o -mediated signals, such as using phosphatase-resistant LPA analogues and monoclonal antibodies specific for LPA or S1P [117,148], may also limit YAP/TAZ activity. It is noteworthy that some GPCR-based drugs, like β-blockers and dopamine, will significantly affect heart and psychiatric functions, thus, side effects must be considered before using these drugs in cancer therapies [149,150].
Proteins transmitting GPCR signaling can also be targeted to manipulate the Hippo pathway. Recently, cyclic depsipeptide FR900359 has been shown to target mutant Gα q/11 and repress downstream effectors MAPK and YAP [151,152]. The activity of PKA is associated with cellular cyclic-AMP (cAMP) levels, Forskolin or phosphodiesterase inhibitors, such as Rolipram, have been shown to induce PKA activity and repress YAP/TAZ [112,148]. PKC inhibitors can also repress YAP/TAZ activity in a cell type-dependent manner [121]. Rho GTPases are central for the regulation of Hippo pathway by GPCR signaling, it has been shown that statins, inhibitors of HMG-CoA reductase (HMGCR), can indirectly inactivate Rho GTPases and reduce YAP/TAZ nuclear localization [153,154]. As GPCR-Hippo signaling is a complex signaling network, drugs targeting GPCRs, G proteins, or downstream signaling nodes may affect effectors other than YAP/TAZ, hence, the specificity towards the Hippo pathway will be compromised when using these drugs.

Conclusions
In the last few years, Hippo pathway has been under the spotlight due to its function in organ size control and tumorigenesis. GPCRs, the largest cell membrane receptor family, regulate an array of downstream signal pathways, including Hippo pathway effectors YAP/TAZ. Aberrant GPCR and YAP/TAZ activation have been observed in pathogenesis of several types of cancer. Although further in-depth studies are still required to address issues relevant to side effects and pharmacodynamics, it is clear that GPCR-Hippo signaling axis will be a promising target for anticancer therapy.