The G protein Gα_s acts as a tumor suppressor in sonic hedgehog signaling-driven tumorigenesis

ABSTRACT

G protein-coupled receptors (GPCRs) are critical players in tumor growth and progression. The redundant roles of GPCRs in tumor development confound effective treatment; therefore, targeting a single common signaling component downstream of these receptors may be efficacious. GPCRs transmit signals through heterotrimeric G proteins composed of Gα and Gβγ subunits. Hyperactive Gα_s signaling can mediate tumor progression in some tissues; however, recent work in medulloblastoma and basal cell carcinoma revealed that Gα_s can also function as a tumor suppressor in neoplasms derived from ectoderm cells including neural and epidermal stem/progenitor cells. In these stem-cell compartments, signaling through Gα_s suppresses self-renewal by inhibiting the Sonic Hedgehog (SHH) and Hippo pathways. The loss of GNAS, which encodes Gα_s, leads to activation of these pathways, over-proliferation of progenitor cells, and tumor formation. Gα_s activates the cAMP-dependent protein kinase A (PKA) signaling pathway and inhibits activation of SHH effectors Smoothened-Gli. In addition, Gα_s-cAMP-PKA activation negatively regulates the Hippo pathway by blocking the NF2-LATS1/2-Yap signaling. In this review, we will address the novel function of the signaling network regulated by Gα_s in suppression of SHH-driven tumorigenesis and the therapeutic approaches that can be envisioned to harness this pathway to inhibit tumor growth and progression.

KEYWORDS:

• Basal cell carcinoma
• cAMP-dependent PKA
• GPCRs
• G-protein
• Gα_s
• GNAS
• Hedgehog signaling
• Hippo signaling
• medulloblastoma
• Stem cells

Introduction

The G protein coupled receptor (GPCR) signaling pathway plays critical roles in development, normal physiology, and disease. GPCRs transmit extracellular signals through heterotrimeric G proteins, which consist of three main subunits, Gα, Gβ, and Gγ. GDP-bound Gα is associated with Gβγ in an inactive state. Binding of ligands to GPCR causes exchange of GDP for GTP on Gα, leading to its dissociation from a membrane-anchored Gβγ complex. Downstream signaling from both Gα and Gβγ subunits is maintained until the GTPase activity of Gα hydrolyzes bound GTP to GDP, a process accelerated by Regulators of G protein Signaling (RGS) proteins.^1-3 G proteins have been linked to modulation of tumor growth, invasion, and metastasis, making this an important pathway for cancer therapy.^4-9 The Gα protein subunits differ in function and sequence homology. Gα_s activates adenylyl cyclase and increases cytosolic cAMP levels; Gα_i inhibits adenylyl cyclase and decreases intracellular cAMP levels; Gα_q/11 activates phospholipase C; Gα₁₂ and Gα₁₃ regulate RhoA signaling via Rho-GEFs.^1-3 Heterotrimeric G proteins therefore represent a point of signaling convergence from multiple GPCRs, and they exert a pivotal role in mediating the functions of various GPCRs in cell growth and tumorigenesis.

Of these G protein subunits, Gα_s, encoded by GNAS, is one of the most frequently mutated genes in cancer.¹⁰ Many of these mutations in GNAS trigger gain-of-function of GPCR signaling that leads to enhanced intracellular cAMP levels, increased cell growth, and metastasis of human cancers.¹ In contrast, activating mutations in the opposing subunit, Gα_i, decrease cAMP levels and are associated with adrenal cortical cancers and ovarian sex-cord tumors.¹¹ Thus, both elevation and reduction of cAMP levels may be oncogenic; it appears that an imbalance of intracellular cAMP may lead to an oncogenic transformation in a context-specific manner.⁹ Recently, a series of genetic studies pointed to a critical role of GNAS in tumor suppression.^12-14 Genetic loss of a single GNAS allele in neural and skin progenitor cells causes medulloblastoma (MB) and basal cell carcinoma (BCC) respectively with full penetrance.^12,13 In this review, we will discuss the role of Gα_s as a tumor suppressor by exploring underlying mechanisms whereby Gα_s signaling regulates tumorigenesis through cAMP-dependent PKA, Sonic Hedgehog (SHH), and Hippo-LATS signaling pathways. We will further discuss how to target this novel tumor suppressive pathway for cancer treatment.

GNAS is a tumor suppressor gene in medulloblastoma

MBs are the most common malignant brain tumor in children, accounting for approximately 25% of all pediatric brain cancers. At present, molecular events and signaling pathways that drive the initiation and progression of these tumors are not fully understood. Mutations in genes encoding SHH signaling components Patched1, Smoothened (SMO), and Suppressor-of-fused (SUFU) account for approximately half of sporadic human SHH-subgroup MBs,^15,16 leading to hyperactivation of the SHH signaling pathway.

Analysis of two independent cohorts of SHH-associated MB patients in Boston and Heidelberg revealed that low expression of GNAS is correlated with significantly reduced overall survival.¹² Moreover, a recent report indicated that an infant carrying a homozygous nonsense mutation in GNAS developed aggressive MB.¹⁷ These observations suggest that low expression or loss of GNAS specifically defines a subset of aggressive SHH-group MBs.

The loss of a single Gnas gene in neural progenitor cells is sufficient to initiate formation MB-like tumors in animal models.¹² The deletion of Gnas alleles in human glial fibrillary acidic protein (GFAP) promoter-expressing neural stem/progenitor cells, atonal homolog 1 (Atoh1) promoter-expressing cerebellar granular neuron progenitor cells (GNPs), or progenitors that express oligodendrocyte transcription factor 1 gene (Olig1) leads to an expansion of granule neuron progenitors and ultimately to formation of malignant SHH-associated MB in mice.¹² The tumors in these mice developed from anatomically distinct progenitors of the developing hindbrain recapitulating their human counterparts. Thus, Gnas is a critical determinant of progenitor cell competency and proliferation for MB initiation across disparate cells of origin. The identification of Olig1⁺ progenitor cells in the dorsal brainstem as the cellular origin for a subset of an anatomically distinct SHH-associated MB highlights the tumor heterogeneity with regard to cellular origin and anatomical location.

Gα_s suppresses progenitor self-renewal and tumor formation in basal cell carcinoma

SHH signaling activation has been implicated in the etiology of the most common human cancer, basal cell carcinoma.¹⁸ Mutations in the Patched gene, which negatively regulates SHH-SMO signaling have been identified in sporadic BCCs as well as those from patients with the rare genetic syndrome nevoid BCC.¹⁸ When Gnas is knocked out in murine stem cells of the skin under an epidermal stem cell-specific promoter, the promoter that drives Keratin 14 expression, epidermal stem cells undergo uncontrolled proliferation, leading to the tumor lesions that resemble superficial and nodular human basal cell carcinoma.¹³ Conversely, overexpression of Gα_s in these same cells leads to premature differentiation of hair follicle stem cells and basal cells.¹³ Thus, in both neural and skin progenitor populations, Gα_s acts as a brake on excessive self-renewal or proliferation of progenitor cells.

GNAS methylation, which results in a low level of GNAS expression, has also been linked to poor prognosis in neuroblastoma.¹⁹ Neuroblastoma is a neuroendocrine tumor, which arises from the neural crest cell lineage of the sympathetic nervous system. Thus, the tumor-suppressive action of Gα_s is not limited to primordial neural progenitor cells in the cerebellum and hindbrain. Thus, current evidence suggests a broader role for Gα_s in inhibiting multiple cancer types. One potential mechanism for the effect of GNAS loss in neural and epidermal progenitors is alteration in SHH and Hippo signaling pathways.

Gα_s controls tumor formation by activating the PKA-cAMP signaling axis

Gα_s suppresses SHH signal transduction through different cellular mechanisms. In the canonical signaling pathway, Gα_s activation stimulates adenylyl cyclase activity to produce cAMP, which in turn activates the cAMP-dependent PKA. PKA is a major signaling effector of Gα_s downstream of cAMP activation.^20,21 Activation of PKA has been shown to inhibit SHH signaling in a variety of cell types. PKA phosphorylates and inactivates Gli transcription factors, the SHH downstream effectors, and recruits the ubiquitin ligase β-TRCP. β-TRCP ubiquitinates Gli1 and Gli2, leading to their degradation, and enhances Gli3 processing into a Gli3R repressor form, thereby inhibiting SHH signaling.^22-24

The Gα_s-cAMP-PKA signaling axis has an important role in suppression of MB and BCC tumors.^12,13 The loss of Gα_s in the progenitor cells of the cerebellum and hindbrain leads to a decrease in intracellular cAMP levels and a reciprocal increase in SHH downstream target expression, leading to MB formation. Conversely, elevation of Gα_s signaling effectors cAMP by either forskolin (an adenylyl cyclase agonist) or rolipram (a selective inhibitor of phosphodiesterase-4, PDE-4), which blocks cAMP degradation,^25,26 inhibits SHH signaling activation and reduces tumor cell proliferation and tumor size in the Gnas mutation-induced MB model.¹² Similarly, in basal stem cells of the skin, inhibition of PKA increases Gli-mediated transcription in vitro and leads to tumor formation, which phenocopies the tumorigenic phenotype in Gnas-mutant mice.¹³ In addition, activation of cAMP-PKA via forskolin suppresses tumor growth in a K14-Rosa26-SmoM2 model of basal cell carcinoma.²⁷ Thus, there appears to be an inverse correlation between levels of cAMP/PKA activation and SHH signaling induced Gli-transcription-associated tumor growth. Because the loss of Gnas occurs independently of changes in other Hedgehog signaling components,¹² this Gα_s-mediated signaling pathway may not only represent a novel mechanism for regulating the Hedgehog pathway but also underlie the drug resistance in MB treated with SMO antagonists alone.^28,29

In addition to activation of PKA-cAMP intracellular events in murine cerebellar GNPs, Gα_s activity also modulates SHH signaling component trafficking in the primary cilium, a structure believed to be a center for Hedgehog signaling.^30,31 Strikingly, Gα_s protein is highly enriched at the primary cilium of GNPs.¹² Depletion of Gα_s promotes the translocation of Gli2, a SHH downstream effector, onto the tip of primary cilia,¹² which activates the SHH signaling cascade. This is consistent with a role of PKA in restraining Gli2 activation.^32,33 Gα_s can inhibit both ciliary translocation of SMO and Gli2 accumulation at the tip of primary cilia while maintaining the positioning of the SMO inhibitory protein Patched1 at the primary cilium.¹² This effect of Gα_s on hedgehog signaling component trafficking provides an additional level of regulation of SHH signaling. Therefore, dual-mode regulation of both SHH signaling component trafficking at the primary cilia and cAMP-PKA mediated signaling cascade by Gα_s activity reinforces the inhibition of SHH signaling activation and MB tumorigenesis (Fig. 1A).

Gα_s-PKA signaling suppresses Hippo signaling-mediated cell proliferation

In the Hippo pathway, signaling through kinases LATS1/2 and MST1/2 leads to phosphorylation and inactivation of the transcription factors Taz and Yap, the Hippo effectors that promote cell proliferation. Deletion of Gnas leads to an increase in Yap1 expression in BCC ¹³ and MB murine tumor models.¹² Consistently, in other mouse models of SHH-signaling induced MB, Yap1 is upregulated in cerebellar progenitor cells that express neural stem cell markers CD15 and nestin.³⁴

Inhibition of Yap1 expression in keratinocytes in the keratin 14-Cre-Gnas mouse BCC model leads to a profound reduction in colony formation.¹³ This reduction appears to be even stronger than that caused by the loss of Gli1, pointing to a prominent role for the Hippo pathway in driving cell proliferation and self-renewal in tumors derived from epidermal progenitors.¹³ Elevation of cAMP-PKA signaling induced by Gα_s activity leads to LATS1 phosphorylation and activation. LATS1 in turn phosphorylates Yap1 to induce the cytoplasmic retention of Yap1 and thereby keep it in a transcriptionally inactive state. Furthermore, in a human keratinocyte cell line, inhibition of expression of LATS1/2 and NF2 (a co-regulator of LATS1/2) diminishes the cAMP-induced Yap1 phosphorylation,¹³ suggesting that cAMP-dependent PKA can act on NF2/LATS1/2 to activate Hippo-LATS1/2 signaling to suppress Yap1 transcriptional activity (Fig. 1B).

Loss of Gnas activates tumorigenic signaling and unmasks oncogenic activity of heterotrimeric G proteins

The coordinated and balanced activity of heterotrimeric G protein-mediated GPCR signaling regulates SHH and Hippo signaling to ensure proper tissue development and homeostasis by preventing uncontrolled cell growth.^13,14,35 The loss of Gα_s may therefore disrupt the balance between pro-proliferative and pro-differentiation G proteins, leading to excessive signaling through pro-proliferative G proteins.

One of the potential oncogenic heterotrimeric G proteins that may regulate SHH signaling is Gα_i. Gα_i counteracts Gα_s signaling by inhibiting production of intracellular cAMP. The GPCR-like SMO can interact with Gα_i to activate Gli-dependent transcription in NIH 3T3 fibroblasts and in Drosophila.^36-38 Although the existence of SMO-Gα_i coupling has been controversial,^39,40 it might represent a non-canonical branch of the pathway that activates Rac-RhoA-dependent signaling to enhance cell migration and proliferation.⁴¹

Intriguingly, Gα_i2 and Gα_i3 are expressed in the external granular layer of rat cerebella and localized to the primary cilium. The loss of these heterotrimeric G proteins suppresses SHH-induced proliferation of cerebellar GNPs,⁴² suggesting a potential role for Gα_i signaling in MB formation. Recently, a cilia-enriched orphan GPCR Gpr175 (which has also been called Tpra1 or Tpra40) was shown to inhibit cAMP levels and activate SHH signaling through Gα_i.⁴³ These findings are consistent with a model where Gα_ssuppresses, while Gα_i promotes, oncogenic signaling in the primary cilium.

Recent studies indicate that Hippo signaling through Yap is suppressed by Gα_s and is activated by a panoply of other heterotrimeric G proteins including Gα_12/13, Gα_q/11, Gα₁₄, Gα₁₅, and Gα_i.^14,35,44 Of these, the most potent activators of Yap transcription are Gα_12/13 and Gα_q/11. Signaling through the lysophospholipid (LPA) receptor, a GPCR that couples to Gα_12/13, drives serum-induced Yap transcription.¹⁴ In addition, Gα₁₂, Gα₁₁, Gα_q, and Gα_i also regulate the activity of LATS1/2 kinases in Hek293T cells and uveal melanoma cells.^14,35,44 indicating a complex interplay between GPCRs and Hippo signaling. It is worth noting that EDG4, a member of the LPA receptor family, is overexpressed in Wnt and SHH subgroup MBs.⁴⁵ The functions of these heterotrimeric G proteins in MB formation remain to be defined. Nonetheless, the balance between GPCR signaling through adenylyl cyclase activator and suppressor heterotrimeric G proteins at least likely regulates tumorigenic events.

Loss of GNAS in neural and skin progenitors leads to tumor formation with full penetrance, suggesting a role of Gα_s as a potent regulator of cell proliferation in SHH-signaling dependent progenitors originating from the neural tube and surface ectoderm during early lineage progression. In contrast, cancers in which hyperactivation of GNAS is oncogenic, such as in thyroid cancers and pituitary adenomas, arise from terminally differentiated cells derived from the endoderm.¹ This may suggest a potential correlation between cell type or stage in lineage progression and the effect of cAMP on tumorigenesis. Unraveling which cancers will respond positively, as opposed to negatively, to cAMP elevation is critical to the safe clinical application.

Therapeutic targeting of Gα_s-cAMP-PKA signaling suppresses tumor growth

The studies of signaling events following dysregulation of heterotrimeric G proteins identified cAMP as a convergent downstream signaling node, making it an attractive target for tumor suppression. Several Gα_s-coupled GPCRs that inhibit SHH target gene expression have been identified including GPR161 and PAC1.^46,47 These receptors activate PKA resulting in an increase in intracellular cAMP levels. Activation of the ciliary GPR161 elevates cAMP, leading to PKA activation and repression of Gli1/2 transcription.⁴⁶ At present, the role of GPR161 in tumor formation remains undefined. The PAC1 receptor, which binds the PACAP ligand also resulting in increased cAMP levels, has been shown to inhibit SHH signaling and Gli activation by PKA.⁴⁷ Reduced levels of PACAP enhance MB incidence in Patched heterozygous mice,⁴⁸ suggesting that signaling through PAC1 blocks the proliferation of GNPs during cerebellar development and MB formation.

Gα_i-protein coupled receptors have been shown to synergize with Hedgehog signaling. CXCL12 stimulation of CXCR4, a Gαi coupled chemokine receptor, results in a significant reduction of intracellular cAMP levels and enhances the growth of SHH-driven medulloblastoma carrying an activated SmoA1 mutation^49,50 suggesting that CXCR4 activation maximizes proliferation of SHH-driven tumors. Inhibition of CXCR4 signaling via small molecule inhibitors AMD 3100 and AMD 3465 elevates cAMP levels and suppresses the growth of MB xenografts in vivo,⁵⁰ suggesting that dual inhibition of SHH and CXCR4 pathways may be beneficial for treating CXCR4-expressing SHH subtype MBs.

Phosphodiesterases, the enzymes responsible for the degradation of cAMP, have been shown to regulate MB growth.⁵¹ Treatment with rolipram, an inhibitor of PDE4, suppresses SHH signaling and the growth of MB in Gnas-mutant mice without major changes in cerebellar architecture.¹² An unbiased in vivo chemical genetic screen identified that PDE4 inhibitors such as eggmanone exert a potent inhibitory effect on Hedgehog signaling.⁵² PDE4 inhibition decreases the viability of the DAOY cell, an MB cell line.⁵³ In addition, blocking of PDE4D by roflumilast suppresses the growth of MB tumors resistant to the SHH antagonist vismodegib in mice,⁵⁴ whereas overexpression of PDE4A1, an isoform of PDE4, enhances the growth of DAOY cells in a mouse xenograft model.⁵⁵ Collectively, these studies suggest that PDE4, at least the A and D subtypes, represents a potential therapeutic target for SHH-dependent cancers. What is particularly exciting is that a number of PDE4 inhibitors such as rolipram and roflumilast have been used clinically for other indications and are well-tolerated while affording an avenue to tackle SHH antagonist resistance, raising hope in treating an otherwise challenging type of cancer (Fig. 1).

Concluding remarks

GPCR-Gα_s signaling has long been considered an oncogenic pathway in human cancer; however, recent studies defined a novel tumor suppressive action of the Gα_s protein in MB and BCC, suggesting that Gα_s may function as a tumor suppressor in certain contexts. Future studies will determine whether GNAS plays a tumor-suppressive role in other primordial tumors of the developing nervous system such as pineoblastoma, supratentorial primitive neuroectodermal tumor, and neuroblastoma, the solid cancers most commonly observed in childhood. Targeting PDE4 with cAMP-raising agents has been shown to afford additional efficacy when combined with inhibitors of SMO to diminish the growth of MB cells ¹² and to suppress the growth of vismodegib-resistant MB in mice.⁵⁴ This suggests that in combination with existing therapies, cAMP-raising agents might be repurposed to overcome multi-drug resistance in treatment of SHH-associated MBs.^56,57 Given that signaling control mediated via Gα proteins such as Gα_s may be a point of signaling convergence for numerous GPCRs, targeting of Gα_s and downstream pathway components such as cAMP-PKA may circumvent the drug resistance seen with SMO antagonists alone ^28,29,58 and could be beneficial in the treatment of an array SHH-driven tumors including MB, BCC, small cell lung cancer, and pancreatic cancer.^22,29,59,60

Figure 1. Gαs-cAMP-PKA signaling suppresses progenitor proliferation and SHH-driven tumorigenesis. (A) A schematic diagram depicts the role of Gα_s as a molecular switch that controls SHH-Gli and Hippo-Yap signaling activation. Gα_s is highly enriched in the primary cilia of GNPs and blocks SMO-Gli ciliary translocation to block SMO activation. These Gα_s-mediated intracellular cascades inhibit SHH-driven tumorigenic processes. Inactivation of Gα_s activity in cerebellar and epidermal progenitors leads to activation of SHH-Gli and Hippo-Yap signaling and is sufficient to promote progenitor expansion and initiate MB and BCC formation, respectively. (B) GPCR-mediated Gα_s activation, counterbalanced by Gα_iactivity, increases cAMP levels and subsequently activates cAMP-dependent PKA signaling, leading to phosphorylation of Gli and Yap, the effectors of canonical SHH and Hippo signaling, respectively, and inactivation of their transcriptional activity for cell proliferation and tumorigenesis.

Disclosure of potential conflicts of interest

No potential conflicts of interest were disclosed.

Acknowledgments

The authors would like to Drs. Xuelian He and Ed Hurlock for critical comments and Xianyao Zhou for assistance with the manuscript.

Funding

This study was funded in part by grants from the US National Institutes of Health (R01 NS078092 and R01 NS075243) to QRL.