The Hippo signaling pathway is a signaling pathway that has been found to play an important role in the regulation of multicellular biological organ size in recent years. Patient sample analysis and mouse model studies indicate that dysregulation of the Hippo signaling pathway plays a key role in the different stages of cancer development. The Hippo signaling pathway senses a variety of external stimuli such as the mechanical environment, G-protein coupled receptor signaling, and cellular energy levels, and is activated by protein kinase chains. then, the transcriptional coactivator YAP (Yes-associated protein) and its homologous protein TAZ (transcriptional coactivator with PDZ-binding motif) are directly phosphorylated, resulting in their cytoplasmic retention and degradation.
In general, this signaling pathway has three interrelated components: upstream regulatory signaling, the hippo core kinase cascade complex, and the downstream nuclear transcriptional regulatory complex. Among them, hippo upstream regulatory signals or abnormal activity of the core kinase cascade complex can lead to excessive organ growth and multiple tumors.
Figure 1 Regulation and functions of the Hippo pathway
Upstream signaling of Hippo
Activation of MST1/2
MST1/2 is a pro-apoptotic kinase that is activated by caspase-specific cysteine proteolytic enzymes under apoptotic signals. SAV1 directly binds to the SARAH domain of the MST1/2 protein via the SARAH domain, thereby activating MST1/2. In mammalian cells, MST1/2 can also be activated by binding to the RASSF (Ras-association domain family) protein, possibly due to altered MST1/2 subcellular localization.
In the protein kinase chain of the Hippo signaling pathway, MST1/2 activates LATS1/2 through a variety of mechanisms. Activation of LATS1/2 requires phosphorylation of a conserved serine or threonine residue within its activation loop and its C-terminal hydrophobic motif. MST1/2 phosphorylates the C-terminal hydrophobic motif of LATS1/2, which promotes autoactivation of the activation loop of LATS1/2. MST1/2 phosphorylates MOB1, which promotes the binding of MOB1 to the inhibitory region of LATS1/2, and activates the inhibition of LATS1/2 activity. In addition, crystal structure analysis indicated that MOB1 was phosphorylated by MST1/2 to form MST1/2-MOB1 complex, which was then combined with LATS1/2 to form MST1/2-MOB1-LATS1/2 ternary complex. When MST1/2 phosphorylates LATS1/2 and MOB1, phosphorylated MOB1 drives the ternary complex to dissociate. The activated LATS1/2 phosphorylation transcripts a specific amino acid residue of the auxiliary activator YAP/TAZ, resulting in its activity being inhibited. In addition to MST1/2, MAP4Ks (mitogen-activated protein kinase kinase kinase kinase) can also phosphorylate and activate LATS1/2.
MAP4Ks, like MST1/2, are also STE20 family protein kinases. MAP4K1/2/3/5 (a homologous protein of Happyhour in Drosophila) and MAP4K4/6/7 (a homologous protein of Misshapen in Drosophila) directly phosphorylate the hydrophobic motif of LATS1/2, activating LATS1/2. In HEK293 cells, increased levels of YAP/TAZ phosphorylation by serum starvation were significantly affected by MAP4K4/6/7 knockouts than by MST1/2 knockouts. It can be seen that MAP4Ks play a more important role in the regulation of Hippo signaling pathway than MST1/2 in some cases. However, to completely suppress the response of Hippo signaling pathway to cell contact inhibition, energy stress, serum starvation, etc., it is necessary to knock out MST1/2 and MAP4Ks simultaneously. It can be seen that the regulation of LATS1/2 by MST1/2 and MAP4Ks is functionally redundant and complementary.
LPA (lysophosphatidic acid) and S1P (sphingosine 1-phosphophate) are key factors regulating YAP/TAZ activity, and LPA and S1P through their corresponding G-protein coupled receptor GPCRs and its downstream Rho GTPase activates YAP/TAZ. In ovarian cancer cells, LPA does not affect the kinase activity of the upstream kinases MST1/2 and LATS1/2 of the Hippo signal pathway, but rather the protein phosphorylase PP1A downstream of Rho A mediates YAP dephosphorylation. GPCR also mediates the regulation of YAP/TAZ activity by the ligand thrombin of PARs (protease activated receptors).
Rho GTPase-mediated GPCR regulates YAP, and GPCR signals coupled with Gα12/13, Gαq/11, and Gαi/o to activate Rho GTPase, which activates YAP activities such as LPA, S1P, thrombin, and estrogen. Conversely, GPCR signals coupled to Gαs to inhibit RhoGTPase and YAP activity, such as epinephrine and glucagon. It can be seen that YAP/TAZ is regulated by numerous secretion signals and related GPCRs, thereby linking the Hippo signaling pathway with more extracellular signals and exerting more physiological functions.
The function of the Hippo signaling pathway in organ size regulation is highly conserved in mammals and has been validated in a variety of mouse models. Specific knockout of Yap in the intestine has no obvious defects in its structure and size. Loss of Yap function has no effect on mammary gland development during adolescence, but it severely impedes mammary gland growth during pregnancy. Knockout of Yap in the liver of mice, although leading to bile duct defects, is not affected by liver size, which may be due to the activation of Taz by Yap deletion.
Core kinase cascade complex
The core members of most Hippo signaling pathways are found in Drosophila through genetic screening of tumor suppressor genes. The Hippo core kinase cascade is a series of serine/threonine phosphokinases. It inhibits cell proliferation and promotes apoptosis by phosphorylation of the transcriptional cofactor Yki or Yki, a homolog of YAP and TAZ in mammals. In Drosophila, this kinase complex is composed of two kinases, the serine/threonine Ste20-like kinase Hippo and the Nulear Dbf-2-related family kinase Wts. In addition, the scaffold protein Sav and the tumor suppressor Mats (Mob as tumor suppressor) form a complex with Hippo and Wts, respectively, which play a role in inhibiting cell growth.
Downstream transcriptional regulator
The output of the biological signal of the Hippo signaling pathway is dependent on the transcriptional regulation of the cell, which inhibits cell growth, promotes apoptosis, and determines cell fate. In Drosophila, the transcriptional cofactor Yki, when not inhibited by Hippo signaling, enters the nucleus and binds to transcription factors, promotes cell proliferation, and inhibits apoptosis. The transcription factor that was first reported to bind to Yki is the transcription factor Scalloped of the TEA domain transcription factor (TEAD)/TEF (PAR bZIP transcription factor) family. The N-terminus contains a DNA-binding domain, and the C-terminus binds to Yki, thereby promoting the expression of apoptosis-inhibiting factor-1.
Similar to the downstream target genes of Drosophila, anti-apoptotic and proliferative target genes are also present at the downstream of the mammalian Hippo signaling pathway, such as connective tissue growth factor, cysteine-rich angiogenesis-inducing factor 61, ankyrin repeat domain gene 1, amphiregulin, insect virus XIAP repeat-containing protein 5, fibroblast growth factor and GLI family zinc finger protein 2 and so on.
Hippo signaling pathway and tumor
Abnormalities in the growth regulation mechanism of tumor cells themselves have been recognized as an important feature of tumorigenesis. At present, mouse models and human tumor tissues or cell lines have shown that Hippo signaling pathway plays an important role in cancer development. There is increasing evidence that the Hippo signaling pathway is involved in the development of a variety of different tumors. AP can promote the proliferation and migration of prostate epithelial cells and promote the malignant transformation of epithelial cells.
Mutations or expression levels of the YAP gene are up-regulated in renal and bladder cancer cells, and high expression of YAP promotes the growth and migration of these tumor cells. During pancreatic carcinogenesis, YAP mediates epithelial-mesenchymal transition and colonization of cancer cells. The absence of Sav1 or MST1 /2 will cause the liver to become larger and form a tumor. Other studies have found that the liver of YAP transgenic mice proliferates excessively and eventually leads to the formation of liver cancer. The Hippo signaling pathway is seen as a new pathway that is potentially involved in tumor pathogenesis and has become a new hotspot for targeted therapy.
Studies found that the Hippo signaling pathway is also involved in the regulation of tumor immunity. It was found that LATS1/2, which has been recognized as a tumor suppressor gene in the Hippo signaling pathway, strongly induces an anti-tumor immune response when knocked out, and induces immune rejection of tumor cells and inhibits tumor growth in an isogenic mouse transplantation model. Mechanism analysis revealed knockdown of LATS1/2 in tumor cells, stimulating secretion of nucleic acid-enriched exosomes. The toll-like receptor MYD88/TRIF-mediated type I interferon response is then triggered, stimulating multiple components in the host immune response, and ultimately activating T cells. Activated T cells assist tumor-specific cytotoxic T cell responses and B cell antibody production, killing tumor cells and inhibiting tumor formation. This suggests that inhibition of Hippo signaling pathway activity in tumor cells may be an effective method to induce anti-tumor immunity. The latest research found that long-chain non-coding RNA (lncRNA) can also promote bone turnover in breast cancer cells by affecting the activity of the Hippo signaling pathway.
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