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Investigation of the LKB1–TSSK1B axis and GPR54 signaling in the Hippo-YAP pathway-mediated tumorigenesis

  • 김초롱 (김초롱(Cho-Long Kim), 아주대학교 일반대학원)

초록/요약

The Hippo signaling pathway is a signaling cascade that plays a crucial role in regulating cell growth, proliferation, and organ size in mammals, including humans. It was initially discovered in Drosophila melanogaster (fruit flies) and later found to have similar functions in mammals. The Hippo pathway comprises several key components, including mammalian STE20-like kinase 1/2 (MST1/2), the protein Salvador homolog 1 (SAV1), MOB kinase activator 1A/B (MOB1A/B), large tumor suppressor kinase 1/2 (LATS1/2), Yes-associated protein 1 (YAP), Transcriptional coactivator with PDZ-binding motif (TAZ), and the transcriptional enhanced associated domain (TEAD) family. At its core, YAP and TAZ serve as canonical transcriptional coactivators and act as effectors within the Hippo pathway. When the Hippo pathway is inactivated, YAP and TAZ are not phosphorylated and translocate into the nucleus where they interact with transcriptional factors such as the TEAD family to regulate the expression of target genes involved in cell proliferation, survival, metastasis, and differentiation. Conversely, when the Hippo pathway is activated, MST1/2 kinases are activated and phosphorylated. The phosphorylated MST1/2 kinases then activate LATS1/2 kinases, enhancing their kinase activity. Subsequently, the activated LATS1/2 kinases phosphorylate YAP and TAZ, causing them to remain in the cytoplasm. Eventually, the phosphorylated YAP and TAZ bind to 14-3-3 proteins, which leads to their degradation. Moreover, dysregulation of the Hippo pathway has been implicated in various aspects of cancer development and progression, including tumor growth, metastasis, and resistance to therapy. Aberrant activation of YAP and TAZ can promote uncontrolled cell proliferation, inhibit apoptosis, and enhance tumor invasion and metastasis. Consequently, targeting components of the Hippo pathway is considered a potential strategy for therapeutic intervention. Therefore, it is considered that potential therapeutic strategies and further research are needed to regulate the excessively activated YAP and TAZ. In part I of this study, testis-specific serine/threonine kinase 1B (TSSK1B), a member of the calcium/calmodulin-dependent protein kinase (CAMK) superfamily, is identified as a negative regulator of YAP, suppressing cellular proliferation and oncogenic transformation. Two pathways were identified that inhibit YAP activity through phosphorylation. First, the liver kinase B1 (LKB1), a serine/threonine kinase, directly activates the TSSK1B kinase, leading to the phosphorylation of YAP at Ser94. Second, activated TSSK1B phosphorylates LATS1/2 kinases, indirectly phosphorylating YAP at Ser127. Additionally, xenograft experiments showed that TSSK1B-mediated phosphorylation resulted in suppressed tumor growth. These studies reveal that the LKB1–TSSK1B axis directly phosphorylates YAP at Ser94 and impedes tumor growth through anti-cancer effects. Part II of this study investigated the role of GPR54, a G protein-coupled receptor known to be overexpressed in triple-negative breast cancer (TNBC), to identify potential therapeutic targets for this highly aggressive and treatment refractory form of breast cancer. GPR54 activates Gαq/11 proteins, which in turn activate RhoA and inhibits the LATS1/2 kinases, leading to the activation of YAP. Moreover, this activation of YAP not only upregulates YAP target genes but also increases mRNA levels of GPR54 and KISS1 in MDA-MB-231 cells. Furthermore, GPR54 signaling triggers YAP/TAZ-dependent tumorigenic potentials, such as invasion, migration, proliferation, and anchorage-independent growth. These findings suggest that activation of YAP via GPR54 signaling establishes a positive feedback loop in TNBC, which promotes tumorigenic potential. Consequently, GPR54 signaling contributes to the growth and development of TNBC. Thus, targeting GPR54 to inhibit YAP activity could represent a promising therapeutic strategy for treating TNBC. Keywords: Hippo-YAP pathway, YAP/TAZ, phosphorylation, TSSK1B, LKB1, LATS1/2, GPR54, Kisspeptin, GPCR, TNBC, RhoA, actin polymerization

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목차

Introduction 1
PART I. The LKB1-TSSK1B axis inhibits tumor growth through YAP phosphorylation 5
I. Introduction 6
II. Aims of study 9
III. Materials and methods 12
A. Cell culture and transfection 12
B. Virus production and infection 12
C. Western blotting and immuoprecipitation (IP) 12
D. Subcellular fractionation 13
E. Luciferase assay 13
F. Recombinant protein purification 13
G. In vitro kinase assay 14
H. Immunofluorescence staining 14
I. Mutagenesis 15
J. Generation of knockout cell lines 15
K. TA cloning 16
L. shRNA transduction 16
M. Sulforhodamine B (SRB) assy 16
N. Soft agar assay 17
O. Xenograft 17
P. Antibodies and Reagents 17
Q. RNA extraction and quantitative real-time polymerase chain reactions (qRT-PCR) 18
R. Statistical analysis 19
IV. Results 20
1. TSSK1B triggers phosphorylation of YAP, leading to the suppression of its transcriptional activity 20
2. Endogenous TSSK1B modulates YAP phosphorylation and target genes activation 23
3. Endogenous TSSK1B modulates YAP phosphorylation through the Hippo pathway stimulation 26
4. TSSK1B induces YAP phosphorylation through upregulation of LATS1/2 activity via MAP4K4/6/7- and MST1/2-dependent mechanism 29
5. LKB1, the upstream kinase of TSSK1B, induces TSSK1B phosphorylation and mediate subcellular localization 32
6. LKB1-mediated activation of TSSK1B induces phosphorylation of YAP and modifies its subcellular localization 36
7. TSSK1B phosphorylates YAP independent of AMPKα1/2 39
8. TSSK1B directly phosphorylates YAP at Ser94 to regulate its activity 42
9. TSSK1B suppresses tumorigenic potential of YAP without LATS1/2 dependency 45
V. Discussion 50

PART II. GPR54 signaling induces YAP activation through a positive feedback loop in breast cancer 53
I. Introduction 54
II. Aims of study 57
III. Materials and methods 61
A. Cell culture and transfection 61
B. Antibodies and Reagents 61
C. Virus production and transduction 62
D. Western blot and immuoprecipitation (IP) 62
E. In vitro kinase assay 62
F. Luciferase assay 63
G. RNA isolation and quantitative real-time polymerase chain reactions (qRT-PCR) 63
H. Immunofluorescence staining 64
I. Site-directed mutagenesis 64
J. Generation of knockout cell lines using CRISPR/Cas9 system 65
K. TA cloning 65
L. siRNA transfection 66
M. shRNA transduction 66
N. Sulforhodamine B (SRB) assay 66
O. Soft agar assay 67
P. Clonogenic assay 67
Q. Chromatin immunoprecipitation (ChIP-IP) 67
R. Invasion assay 68
S. Migration assay 69
T. Statistical analysis 69
IV. Results 70
1. GPR54 signaling enhances YAP-TEAD transcriptional activity 70
2. Wild-type of GPR54 signaling facilitating YAP activation 73
3. GPR54 signaling enhances YAP activity through Gαq/11 and RhoA activation 76
4. GPR54 signaling inhibits LATS1/2 kinase activity 81
5. GPR54 signaling has a positive feedback loop with the Hippo-YAP pathway 84
6. GPR54 signaling facilitates cancer progression through the Hippo-YAP pathway 88
7. Verteporfin and p234 antagonist regulate YAP activity via GPR54 signaling 91
8. Deletion of YAP attenuates oncogenic potentials by GPR54 signaling 94
V. Discussion 97
Conclusion 100
Reference 102
국문요약 111

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