Regulation of Hippo pathway by mitogenic growth factors via phosphoinositide 3-kinase and phosphoinositide-dependent kinase-1.
Journal: 2013/April - Proceedings of the National Academy of Sciences of the United States of America
ISSN: 1091-6490
Abstract:
The Hippo signaling pathway inhibits cell growth and regulates organ size through a kinase cascade that leads to the phosphorylation and nuclear exclusion of the growth-promoting transcriptional coactivator Yes-associated protein (YAP)/Yorkie. It mediates contact inhibition of cell growth downstream of cadherin adhesion molecules and other cell surface proteins. Contact inhibition is often antagonized by mitogenic growth factor signaling. We report an important mechanism for this antagonism, inhibition of Hippo pathway signaling by mitogenic growth factors. EGF treatment of immortalized mammary cells triggers the rapid translocation of YAP into the nucleus along with YAP dephosphorylation, both of which depend on Lats, the terminal kinase in the Hippo pathway. A small-molecule inhibitor screen of downstream effector pathways shows that EGF receptor inhibits the Hippo pathway through activation of PI3-kinase (PI3K) and phosphoinositide-dependent kinase (PDK1), but independent of AKT activity. The PI3K-PDK1 pathway also mediates YAP nuclear translocation downstream of lysophosphatidic acid and serum as a result of constitutive oncogenic activation of PI3K. PDK1 associates with the core Hippo pathway-kinase complex through the scaffold protein Salvador. The entire Hippo core complex dissociates in response to EGF signaling in a PI3K-PDK1-dependent manner, leading to inactivation of Lats, dephosphorylation of YAP, and YAP nuclear accumulation and transcriptional activation of its target gene, CTGF. These findings show that an important activity of mitogenic signaling pathways is to inactivate the growth-inhibitory Hippo pathway and provide a mechanism for antagonism between contact inhibition and growth factor action.
Relations:
Content
Citations
(92)
References
(42)
Grants
(35)
Chemicals
(11)
Genes
(1)
Organisms
(1)
Processes
(3)
Anatomy
(1)
Similar articles
Articles by the same authors
Discussion board
Proc Natl Acad Sci U S A 110(7): 2569-2574

Regulation of Hippo pathway by mitogenic growth factors via phosphoinositide 3-kinase and phosphoinositide-dependent kinase-1

EGF Treatment Inhibits Hippo Pathway in Confluent MCF-10A Cells.

MCF-10A is an immortalized human mammary epithelial cell line that is contact-inhibited at high cell density via the Hippo signaling pathway (5, 10, 11). In serum-starved, contact-inhibited MCF-10A cells, the nuclear effector of the Hippo pathway, YAP, is excluded from the nucleus. However, EGF treatment triggered rapid YAP nuclear accumulation in most cells within 30 min (Fig. 1A). This phenomenon was not unique to MCF10A and mammary cells, but was observed for A431 epidermoid and HeLa cervical carcinoma cell lines as well (Fig. S1A).

An external file that holds a picture, illustration, etc.
Object name is pnas.1216462110fig01.jpg

EGF treatment inhibits Hippo signaling pathway in confluent serum-starved MCF-10A cells. (A) EGF treatment for 30 min induces YAP nuclear accumulation in confluent serum-starved MCF-10A cells, observed by confocal immunofluoresence microscopy. Nuclear staining with TOPRO3 is shown below for each panel. (Scale bar: 20 μm.) The percentage of cells with nuclear staining in the control and EGF groups was compared using the two-sided Fisher’s exact test. (B) ChIP assay demonstrates that EGF treatment induces YAP binding to the CTGF promoter region. PCR shows that CTGF promoter fragment is enriched in the cells treated with EGF for 30 min compared with the control treatment. Mouse IgG serves as the antibody control, and β-actin gene serves as the internal control. (C) EGF treatment increases CTGF mRNA expression in confluent serum-starved MCF-10A cells, as measured by RT-PCR. Equal amounts of total RNA were serially diluted as the template for cDNA synthesis and RT-PCR using CTGF primer or GAPDH primer. (D) MCF-10A cells depleted of YAP by siRNA treatment proliferate less than control MCF-10A cells after EGF treatment. Error bar represents mean ± SEM; n = 6. (E) EGF treatment reduces phospho YAP Ser127 in MCF-10A cells, as shown by Western blot analysis, using α-tubulin as an internal loading control. (F) Quantification of Western blot intensity shown in E by ImageJ. Statistical significance was calculated using the Student t test. Error bar represents mean + SEM; n = 3. *P ≤ 0.0005. (G) Lats1 overexpression inhibits EGF-mediated YAP nuclear accumulation in MCF-10A cells. GFP vector or GFP-Lats1 was transiently transfected into MCF-10A cells by electroporation. The percentage of cells with YAP nuclear staining was compared in indicated groups. Statistical significance was calculated using the two-sided Fisher exact test.

To determine whether YAP nuclear translocation is associated with its transcriptional activity, we performed ChIP analysis for promoter-binding activity. YAP is known to associate with the DNA-binding protein TEAD to regulate expression of the connective tissue growth factor (CTGF) gene (12). EGF treatment triggered YAP binding to the CTGF promoter relative to control treatment (Fig. 1B). EGF treatment also increased CTGF mRNA expression compared with control treatment, as determined by RT-PCR (Fig. 1C). To determine whether YAP has a role in EGF-stimulated growth of MCF-10A cells, we knocked down the expression of YAP by siRNA in MCF-10A cells. Three different YAP siRNAs effectively depleted YAP expression for several days compared with control siRNA (Fig. S2A). EGF failed to increase cell numbers in the YAP knockdown group compared with control (Fig. 1D). Taken together, these results show that EGF treatment triggers YAP nuclear accumulation and transcriptional activity, which is critical for EGF-stimulated cell proliferation in MCF-10A cells.

We next examined whether EGF regulates YAP nuclear accumulation through the Hippo pathway. The core Hippo pathway kinase Lats phosphorylates YAP directly at Ser127, which causes YAP cytoplasmic retention (5). EGF treatment reduced overall YAP phosphorylation and YAP phosphorylation at Ser127 in a time-dependent manner in confluent serum-starved MCF-10A cells (Fig. 1 E and F and Fig. S2B), suggesting that EGF treatment regulates YAP through inhibition of Lats activity. To test whether a decrease in Lats activity mediates EGF regulation of YAP localization, we overexpressed Lats in MCF-10A cells. EGF treatment induced YAP nuclear accumulation in 69% of control vector-transfected cells, whereas only 39% of Lats1-transfected cells showed nuclear YAP staining (Fig. 1G). These data indicate that EGF treatment causes a decrease in Lats-mediated phosphorylation of YAP and stimulates YAP nuclear accumulation in a Lats-dependent manner. Given that Lats is a defining core kinase of the Hippo pathway, EGF treatment regulates YAP, at least in part, through inhibition of the Hippo pathway.

EGF Inhibits Hippo Pathway Through PI3K and PDK1, but Independent of AKT Activity.

We used numerous specific pharmacologic inhibitors of its downstream pathways (13) to examine how EGFR affects the Hippo pathway. Confluent, serum-starved MCF-10A cells were pretreated with different inhibitors for 30 min, followed by EGF treatment for another 30 min, and YAP nuclear accumulation was determined by confocal microscopy. Inhibitors of PI3K significantly blocked EGF-mediated YAP nuclear accumulation as effectively as the EGFR inhibitor Iressa (Fig. 2A and Fig. S3B). Inhibitors of the PI3K downstream effector PDK1 were also able to block YAP nuclear accumulation (Fig. 2A and Fig. S3B). Importantly, dose-dependence studies showed that both sets of inhibitors acted at low concentrations expected for specific effects on these enzymes (Fig. S4). In contrast, inhibitors of another major kinase downstream of PI3K, AKT, had no effect on YAP nuclear accumulation (Fig. 2A). None of the other inhibitors, including Src family kinase inhibitor, MEK inhibitors, JAK2 inhibitor, PKC inhibitors, Ras/farnesyltransferase inhibitor, and PKA inhibitors had any effect on EGF-mediated YAP nuclear accumulation (Fig. S3). Moreover, both the PI3K inhibitor and the PDK1 inhibitor blocked the reduction in YAP Ser127 phosphorylation caused by EGF treatment (Fig. 2B). These results demonstrate that EGFR regulates the Hippo pathway through PI3K-PDK1 activity.

An external file that holds a picture, illustration, etc.
Object name is pnas.1216462110fig02.jpg

EGF inhibits the Hippo pathway through the PI3K-PDK1 pathway, independent of AKT activity. (A) Inhibitor screening of the EGF signaling pathway in MCF-10A cells by confocal microscopy of YAP nuclear accumulation. Cells in c–i received a 30-min inhibitor pretreatment followed by a 30-min EGF treatment. a, no treatment; b, EGF treatment alone; c, Wortmannin; d, {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002; e, PDK1 inhibitor II; f, BX795; g, AKT inhibitor V; i, Iressa. (Scale bar: 20 μm.) (B) Pretreatment with PI3K (Wortmannin) or PDK1 inhibitor (PDK1 inhibitor II) blocks the effect of EGF on phospho-YAP Ser127 by Western blot analysis. The bar graph indicates the Western blot band intensity. Statistical significance was calculated using the Student t test. Error bar represents mean ± SEM; n = 4. *P < 0.0001; **P < 0.005.

Because PI3K can be activated by growth factors other than EGF, we tested whether this PI3K-PDK1–mediated regulation of the Hippo pathway is specific to EGFR signaling. Lysophosphatidic acid (LPA) or horse serum, which can activate PI3K signaling independent of EGFR signaling (14), both caused YAP nuclear accumulation within 30 min of treatment of MCF-10A cells (Fig. 3A). Importantly, PI3K and PDK1 inhibitors blocked YAP nuclear accumulation caused by LPA or serum treatment. Pretreatment of the cells by the EGFR inhibitor Iressa had no effect on YAP nuclear accumulation induced by LPA or serum, ruling out an indirect effect via EGFR (Fig. 3A) (15). A different human mammary cell line, MCF-7, harbors a constitutive active E545K mutation in the PI3K gene, PIK3CA (16). In contrast to MCF-10A cells, YAP remained in the nucleus in confluent MCF-7 cells even after serum starvation (Fig. 3B). Importantly, the PI3K or PDK1 inhibitors caused YAP to accumulate in the cytoplasm of MCF-7 cells, demonstrating that constitutive activation of PI3K and PDK1 activity was responsible for YAP nuclear accumulation in confluent, serum-starved MCF-7 cells. Two colon cancer cell lines, HCT-116 and HT29, also carry activating mutations in the PIK3CA gene (17), and both the PI3K and PDK1 inhibitors similarly caused accumulation of YAP in the cytoplasm of both cell lines (Fig. S1B). These data suggest that many, if not all, extracellular stimuli or oncogenic mutations that activate PI3K-PDK1 signaling will inhibit Hippo pathway signaling and trigger YAP nuclear accumulation.

An external file that holds a picture, illustration, etc.
Object name is pnas.1216462110fig03.jpg

PI3K activated by different growth factors or constitutive mutation causes YAP nuclear accumulation. (A) Similar to EGF treatment (Fig. 1), LPA treatment or serum treatment induced YAP nuclear accumulation through PI3K-PDK1 pathway in confluent serum-starved MCF-10A cells, independent of EGFR activity. Cells in c–f received a 30-min inhibitor treatment, followed by a 30-min LPA treatment. a, no treatment; b, LPA treatment only; c, Wortmannin; d, PDK1 inhibitor II; e, BX795; f, Iressa. Cells in h–k received a 30-min inhibitor treatment, followed by a 30-min serum treatment. g, serum treatment only; h, Wortmannin; i, PDK1 inhibitor II; j, BX795; k, Iressa. YAP intracellular localization was determined by confocal microscopy. (Scale bar: 20 μm.) (B) Inhibition of PI3K or PDK1 activity blocks YAP nuclear accumulation caused by PIK3CA constitutive mutation in MCF-7 cells. Serum-starved MCF-7 cells were treated with PI3K inhibitor (Wortmannin) or PDK1 inhibitor (BX795) for 4 h, after which YAP intracellular localization was determined by confocal microscopy. (Scale bar: 20 μm.)

PDK1 Forms a Complex with Hippo Pathway Molecules in Confluent Serum-Starved Cells, and EGF Treatment Dissociates the PDK1-Hippo Complex and the Entire Hippo Pathway-Kinase Complex.

Components of the Hippo kinase cascade, including Mst, Sav1, and Lats, form a complex (2, 1820). These proteins coimmunoprecipitate with endogenous PDK1 in confluent, serum-starved cells (Fig. 4A), demonstrating that PDK1 is in the same complex as Hippo pathway kinases. EGF treatment for 30 min disrupted the PDK1 interaction with these Hippo pathway components, as evidenced by the loss of Lats1, Mst, or Sav1 in the PDK1 immunoprecipitates. Importantly, the PI3K inhibitor and PDK1 inhibitor blocked the effect of EGF on dissociation of PDK1 from the Hippo complex. We also performed a reciprocal coimmunoprecipitation (co-IP) using anti-Lats1 antibody and found that PDK1, as well as Mst and Sav1, coimmunoprecipitated with endogenous Lats1 in serum-starved cells. The interaction of Lats with all of these components was disrupted after 30 min of EGF treatment (Fig. 4B), and pretreatment with the PI3K inhibitor blocked the effect of EGF on complex dissociation. In contrast, the Sav1–Mst interaction assessed by co-IP was not affected by EGF treatment (Fig. S5). These co-IP experiments identify PDK1 as a component of the Hippo pathway complex. They also show that brief EGF treatment causes dissociation of the complex in a way that would be expected to disrupt the kinase cascade owing to the disruption of Lats from Mst and Sav. Finally, these data show that EGF-induced complex dissociation depends on the activities of PI3K and PDK1, suggesting that PDK1 regulates dissociation.

An external file that holds a picture, illustration, etc.
Object name is pnas.1216462110fig04.jpg

PDK1 forms a complex with Hippo pathway components, and EGF treatment dissociates the Hippo-kinase complex. Confluent serum-starved MCF-10A cells were untreated, treated with EGF for 30 min, or treated with EGF after inhibitor pretreatment as indicated. (A) EGF treatment disrupts endogenous PDK1 binding to the Hippo complex proteins (Lats1, Mst, and Sav1) in MCF-10A cells, as determined by co-IP. The precipitated PDK1 immunocomplex and corresponding input samples were subjected to Western blot analysis with the indicated antibody. (B) EGF treatment disrupts endogenous Lats1 binding to PDK1, Mst, and Sav1 in MCF-10A cells, as determined by co-IP. The precipitated Lats1 immunocomplexes and corresponding input samples were subjected to Western blot analysis with the indicated antibody.

To better understand how PDK1 interacts with the Hippo complex, we studied the PDK1–Lats interaction by deletion mutagenesis and co-IP analysis (Fig. S6A). Deletion of the Lats PPxY motif completely disrupted the Lats–PDK1 association in HEK293T cells (Fig. S6B). However, the PPxY motif binds to molecules containing the WW domain, which is absent in PDK1. Thus, Lats may associate with PDK1 indirectly via the WW domain-containing molecule Sav1. Indeed, we found that overexpression of Sav1 increased the binding between PDK1 and Lats by 2.6-fold compared with vector control (Fig. 5A). Sav1 binds to Mst through their SARAH domains (18), and we found that Sav1 increased the PDK1–Mst association in a SARAH domain-dependent manner (Fig. S6C), suggesting that Sav1 mediates the association between PDK1 and Mst.

An external file that holds a picture, illustration, etc.
Object name is pnas.1216462110fig05.jpg

PDK1 interacts with Lats1 and Mst through scaffold protein Sav1. (A) Sav1 expression facilitates Lats1–PDK1 interaction, determined by coexpression of exogenous proteins in HEK293T cells and co-IP. The graph shows quantification of Western blots. Error bar represents mean ± SEM; n = 4. Statistical significance was calculated using the Student t test. (B) siRNA- mediated knocked down of Sav1 used in C. in MCF-10A cells. The bar graph shows Sav1 levels normalized to β-actin (n = 5). Error bar represents mean ± SEM. Statistical significance was calculated using the Student t test. (C) Sav1 mediates PDK1–Hippo complex binding in MCF-10A cells. Lysates from control siRNA or Sav1 siRNA-treated cells was incubated with anti-PDK1 antibody. Co-IP and input samples were subjected to Western blot analysis with indicated antibodies.

To further test the idea that Sav1 is the central molecule that mediates the interaction of PDK1 with other Hippo components, we knocked down Sav1 by siRNA in MCF-10A cells. Although PDK1–Lats1 and PDK1–Mst associations were detected in control siRNA-transfected cells, loss of Sav1 abolished the PDK1–Lats and PDK1–Mst associations (Fig. 5 B and C). Analysis of the interactions of a series of Sav1 deletion mutants with PDK1 in HEK293T cells showed that amino acid residues 145–162 of Sav1 molecule are critical for the PDK1–Sav1 interaction (Fig. S7). Thus, Sav1 is the central molecule that mediates the interaction of PDK1 with the Hippo complex.

PDK1 Pleckstrin Homology Domain Is Important for EGF-Mediated Hippo Inhibition.

PDK1 comprises an N-terminal kinase domain and a C-terminal pleckstrin homology (PH) domain, and is recruited to the plasma membrane on growth factor stimulation through PH domain binding to PtdIns(3,4,5)P3, which leads to PDK1 activation (2123). To determine whether PDK1 PH domain-mediated membrane recruitment and activation is important for the function of PDK1 in EGF regulation of the Hippo pathway, we generated a PDK1 PH domain mutant, PDK1-RRR472/473/474LLL (PDK1-R472/3/4L), which cannot bind PtdIns(3,4,5)P3 and shows impaired kinase activity (21, 23, 24). Expression of the PDK1-R472/3/4L in confluent serum-starved MCF-10A cells reduced YAP nuclear accumulation in response to EGF treatment compared with WT PDK1 (Fig. 6). PDK1-R472/3/4L was expressed at only 10% of the level of WT PDK1 in MCF-10A cells (Fig. S8), consistent with observations in a previous study (24), which explains its only partial effect on EGF-stimulated YAP nuclear accumulation.

An external file that holds a picture, illustration, etc.
Object name is pnas.1216462110fig06.jpg

PDK1 PH domain is important for EGF-mediated Hippo pathway inactivation. (A) Schema showing overall PDK1 structure and the PDK1 PH domain mutant (RRR472/473/474LLL). (B) PDK1 PH domain defective mutant (PDK1-R472/3/4L) inhibits EGF-stimulated YAP nuclear accumulation in MCF-10A cells. GFP control vector, GFP-PDK1-wt, or GFP-PDK1-R472/3/4L was transfected into MCF-10A cells by electroporation. Serum-starved cells were treated with EGF. The statistical significance of differences in YAP nuclear localization was calculated using the two-sided Fisher exact test. (C) PDK1 PH domain-defective mutant (PDK1-R472/3/4L) causes YAP cytoplasmic retention in MCF-7 cells. GFP-PDK1-wt or GFP-PDK1-R472/3/4L was transfected into MCF-7 cells by electroporation. The statistical significance of differences in YAP nuclear localization was calculated using the two-sided Fisher exact test. (D) PDK1 PH domain-defective mutant binds to Sav1 more abundantly than WT PDK1 in HEK293T cells, determined by co-IP analysis of coexpressed exogenous proteins. Statistical significance was calculated using the Student t test. Error bar represents mean ± SEM; n = 4.

We also performed this experiment in MCF-7 cells, and found that PDK1-R472/3/4L significantly decreased YAP nuclear staining compared with WT PDK1 (Fig. 6C). We used co-IP to examine how the PH domain influences the interaction of PDK1 with the Hippo complex. Although PDK1-R472/3/4L was expressed at only 20% of the level of WT PDK1 in HEK293T cells, more Sav1 bound to PDK1-R472/3/4L than to WT PDK1 (Fig. 6D). We obtained similar results for the PDK1-R472/3/4L mutant and Lats1 association (Fig. S8B). These findings suggest that PDK1 affects the Hippo pathway complex as a result of membrane recruitment on EGF treatment.

We propose a model to explain our findings (Fig. 7). In serum-starved confluent cells, PDK1 remains in the cytoplasm and forms a complex with active Hippo pathway kinases that phosphorylate YAP to retain it in the cytoplasm. When growth factors are added, PDK1 is recruited onto plasma membrane, leading to Hippo complex dissociation. Complex dissociation inactivates Lats, leading to YAP dephosphorylation and nuclear accumulation.

An external file that holds a picture, illustration, etc.
Object name is pnas.1216462110fig07.jpg

Model for growth factor regulation of the Hippo pathway. In confluent cells in the absence of growth factors, PDK1 forms a complex with Hippo pathway proteins (Lats, Mst, and Sav1), and the Hippo pathway is active. Mst phosphorylates Mob and Lats, which phosphorylates YAP. YAP is retained in the cytoplasm, and cell growth is arrested. In the presence of growth factors (EGF, LPA, or serum), PDK1 is recruited on to the membrane, and the PDK1-Hippo complex dissociates, preventing regulation of Lats by Mst, which eventually leads to YAP nuclear accumulation and cell proliferation.

Cell Culture and Treatment.

Confluent MCF-10A cells were serum-starved with basal medium supplemented with 1 μg/mL anti–amphiregulin-neutralizing antibody for 24 h. Cells were pretreated with indicated inhibitors for 30 min, and then treated with 20 ng/mL EGF (PeproTech), 25 μM lysophosphatidic acid (LPA; Sigma-Aldrich), or 5% horse serum in basal DMEM/F12 medium (Invitrogen). Other cells were cultured as described previously (10). Antibodies, reagents, plasmids, and transfections are described in detail in SI Materials and Methods.

Immunofluoresence Staining and Confocal Microscopy.

Cells were fixed with 4% paraformaldehyde and then permeabilized with 0.1% Triton X-100. Primary and secondary antibodies were diluted in blocking buffer (PBS with 3% BSA). TOPRO3 was added for nuclear staining. Slides were analyzed with a Nikon Eclipse TE2000 confocal microscope.

Immunoprecipitation and Western Blot Analysis.

MCF-10A cells were lysed with hypotonic buffer [10 mM Hepes (pH 7.4), 1 mM EDTA, 150 mM NaCl] supplemented with protease and phosphatase inhibitors. HEK293T cells were lysed with Nonidet P-40 buffer [150 mM NaCl, 1.0% Nonidet P-40, 50 mM Tris (pH 8.0)] at 24 h after transfection. Immunoprecipitation and Western blot analyses are described in detail in SI Materials and Methods.

ChIP-PCR.

ChIP was performed as described by Braunstein et al. (42) and as detailed in SI Materials and Methods.

Supplementary Material

Supporting Information:
University of Virginia School of Medicine, Charlottesville, VA, 22908
To whom correspondence should be addressed. E-mail: ude.ainigriv@renibmug.
Edited by Joan S. Brugge, Harvard Medical School, Boston, MA, and approved December 28, 2012 (received for review September 21, 2012)

Author contributions: R.F. and B.M.G. designed research; R.F. and N.-G.K. performed research; N.-G.K. contributed new reagents/analytic tools; R.F. and B.M.G. analyzed data; and R.F. and B.M.G. wrote the paper.

Edited by Joan S. Brugge, Harvard Medical School, Boston, MA, and approved December 28, 2012 (received for review September 21, 2012)

Abstract

The Hippo signaling pathway inhibits cell growth and regulates organ size through a kinase cascade that leads to the phosphorylation and nuclear exclusion of the growth-promoting transcriptional coactivator Yes-associated protein (YAP)/Yorkie. It mediates contact inhibition of cell growth downstream of cadherin adhesion molecules and other cell surface proteins. Contact inhibition is often antagonized by mitogenic growth factor signaling. We report an important mechanism for this antagonism, inhibition of Hippo pathway signaling by mitogenic growth factors. EGF treatment of immortalized mammary cells triggers the rapid translocation of YAP into the nucleus along with YAP dephosphorylation, both of which depend on Lats, the terminal kinase in the Hippo pathway. A small-molecule inhibitor screen of downstream effector pathways shows that EGF receptor inhibits the Hippo pathway through activation of PI3-kinase (PI3K) and phosphoinositide-dependent kinase (PDK1), but independent of AKT activity. The PI3K-PDK1 pathway also mediates YAP nuclear translocation downstream of lysophosphatidic acid and serum as a result of constitutive oncogenic activation of PI3K. PDK1 associates with the core Hippo pathway-kinase complex through the scaffold protein Salvador. The entire Hippo core complex dissociates in response to EGF signaling in a PI3K-PDK1–dependent manner, leading to inactivation of Lats, dephosphorylation of YAP, and YAP nuclear accumulation and transcriptional activation of its target gene, CTGF. These findings show that an important activity of mitogenic signaling pathways is to inactivate the growth-inhibitory Hippo pathway and provide a mechanism for antagonism between contact inhibition and growth factor action.

Keywords: MCF-10A, MCF-7, Wortmannin
Abstract

Much is known about the roles of mitogenic growth factors in regulating cell proliferation. More recently, there has been progress in understanding the mechanisms underlying physical constraints on cell growth, such as contact inhibition, mechanotransduction, and the role of cell polarity. In particular, the Hippo signaling pathway is thought to mediate growth inhibition through some of these processes.

The Hippo signaling pathway is an evolutionarily conserved pathway that inhibits cell proliferation, and loss of Hippo signaling leads to organ overgrowth and cancer development (14). The core of the pathway consists of a kinase cascade and associated coactivators and scaffold proteins. The Mst/Hippo kinase in complex with Sav1 phosphorylates and activates the downstream kinase Lats. Lats in turn phosphorylates the growth-promoting transcriptional activator Yes-associated protein (YAP) on Ser127, leading to its cytoplasmic retention (5). When the Hippo pathway is inactive, YAP accumulates in the nucleus and activates the transcription of several growth-promoting genes (5).

Cadherin adhesion proteins have been proposed to mediate contact inhibition of growth by antagonizing growth signaling of receptor tyrosine kinases (RTKs). Cadherins may interact directly with RTKs (6, 7) or via tumor-suppressor proteins merlin/NF2 and NHERF (8). We previously reported that inhibition of EGF receptor (EGFR) phosphorylation by Src family kinases might play a role in cadherin-mediated contact inhibition of growth (9). We recently discovered that cadherin-mediated contact, independent of other cell interactions, inhibits cell growth through activation of the Hippo signaling pathway (10). Given that our earlier work implicated inhibition of RTK signaling activity in the cadherin-mediated contact inhibition of growth, we wondered whether the two growth inhibitory activities are related. In the present study, we found a direct relationship between mitogenic growth factor pathways and the growth-inhibitory Hippo pathway, and identified the general mechanism for this interaction.

Click here to view.

Acknowledgments

We thank Ken Irvine for sharing his related findings in Drosophila with us before publication. This work was supported by National Institutes of Health Grant R01 GM098615 (to B.M.G.).

Acknowledgments

Footnotes

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1216462110/-/DCSupplemental.

Footnotes

References

  • 1. Overholtzer M, et al Transforming properties of YAP, a candidate oncogene on the chromosome 11q22 amplicon. Proc Natl Acad Sci USA. 2006;103(33):12405–12410.[Google Scholar]
  • 2. Pan DThe hippo signaling pathway in development and cancer. Dev Cell. 2010;19(4):491–505.[Google Scholar]
  • 3. Steinhardt AA, et al Expression of Yes-associated protein in common solid tumors. Hum Pathol. 2008;39(11):1582–1589.[Google Scholar]
  • 4. Dong J, et al Elucidation of a universal size-control mechanism in Drosophila and mammals. Cell. 2007;130(6):1120–1133.[Google Scholar]
  • 5. Zhao B, et al Inactivation of YAP oncoprotein by the Hippo pathway is involved in cell contact inhibition and tissue growth control. Genes Dev. 2007;21(21):2747–2761.[Google Scholar]
  • 6. Qian X, Karpova T, Sheppard AM, McNally J, Lowy DRE-cadherin-mediated adhesion inhibits ligand-dependent activation of diverse receptor tyrosine kinases. EMBO J. 2004;23(8):1739–1748.[Google Scholar]
  • 7. Takahashi K, Suzuki KDensity-dependent inhibition of growth involves prevention of EGF receptor activation by E-cadherin-mediated cell-cell adhesion. Exp Cell Res. 1996;226(1):214–222.[PubMed][Google Scholar]
  • 8. Curto M, Cole BK, Lallemand D, Liu CH, McClatchey AIContact-dependent inhibition of EGFR signaling by Nf2/Merlin. J Cell Biol. 2007;177(5):893–903.[Google Scholar]
  • 9. Perrais M, Chen X, Perez-Moreno M, Gumbiner BME-cadherin homophilic ligation inhibits cell growth and epidermal growth factor receptor signaling independently of other cell interactions. Mol Biol Cell. 2007;18(6):2013–2025.[Google Scholar]
  • 10. Kim NG, Koh E, Chen X, Gumbiner BME-cadherin mediates contact inhibition of proliferation through Hippo signaling-pathway components. Proc Natl Acad Sci USA. 2011;108(29):11930–11935.[Google Scholar]
  • 11. Debnath J, Muthuswamy SK, Brugge JSMorphogenesis and oncogenesis of MCF-10A mammary epithelial acini grown in three-dimensional basement membrane cultures. Methods. 2003;30(3):256–268.[PubMed][Google Scholar]
  • 12. Zhao B, et al TEAD mediates YAP-dependent gene induction and growth control. Genes Dev. 2008;22(14):1962–1971.[Google Scholar]
  • 13. Lemmon MA, Schlessinger JCell signaling by receptor tyrosine kinases. Cell. 2010;141(7):1117–1134.[Google Scholar]
  • 14. Mills GB, Moolenaar WHThe emerging role of lysophosphatidic acid in cancer. Nat Rev Cancer. 2003;3(8):582–591.[PubMed][Google Scholar]
  • 15. Daub H, Weiss FU, Wallasch C, Ullrich ARole of transactivation of the EGF receptor in signalling by G-protein-coupled receptors. Nature. 1996;379(6565):557–560.[PubMed][Google Scholar]
  • 16. Stemke-Hale K, et al An integrative genomic and proteomic analysis of PIK3CA, PTEN, and AKT mutations in breast cancer. Cancer Res. 2008;68(15):6084–6091.[Google Scholar]
  • 17. Ikediobi ON, et al Mutation analysis of 24 known cancer genes in the NCI-60 cell line set. Mol Cancer Ther. 2006;5(11):2606–2612.[Google Scholar]
  • 18. Callus BA, Verhagen AM, Vaux DLAssociation of mammalian sterile twenty kinases, Mst1 and Mst2, with hSalvador via C-terminal coiled-coil domains, leads to its stabilization and phosphorylation. FEBS J. 2006;273(18):4264–4276.[PubMed][Google Scholar]
  • 19. Harvey KF, Pfleger CM, Hariharan IKThe Drosophila Mst ortholog, hippo, restricts growth and cell proliferation and promotes apoptosis. Cell. 2003;114(4):457–467.[PubMed][Google Scholar]
  • 20. Tapon N, et al salvador promotes both cell cycle exit and apoptosis in Drosophila and is mutated in human cancer cell lines. Cell. 2002;110(4):467–478.[PubMed][Google Scholar]
  • 21. Anderson KE, Coadwell J, Stephens LR, Hawkins PTTranslocation of PDK-1 to the plasma membrane is important in allowing PDK-1 to activate protein kinase B. Curr Biol. 1998;8(12):684–691.[PubMed][Google Scholar]
  • 22. Filippa N, Sable CL, Hemmings BA, Van Obberghen EEffect of phosphoinositide-dependent kinase 1 on protein kinase B translocation and its subsequent activation. Mol Cell Biol. 2000;20(15):5712–5721.[Google Scholar]
  • 23. Toker A, Newton ACCellular signaling: Pivoting around PDK-1. Cell. 2000;103(2):185–188.[PubMed][Google Scholar]
  • 24. McManus EJ, et al The in vivo role of PtdIns(3,4,5)P3 binding to PDK1 PH domain defined by knockin mutation. EMBO J. 2004;23(10):2071–2082.[Google Scholar]
  • 25. Dupont S, et al Role of YAP/TAZ in mechanotransduction. Nature. 2011;474(7350):179–183.[PubMed][Google Scholar]
  • 26. Zhao B, et al Angiomotin is a novel Hippo pathway component that inhibits YAP oncoprotein. Genes Dev. 2011;25(1):51–63.[Google Scholar]
  • 27. Cho E, et al Delineation of a Fat tumor suppressor pathway. Nat Genet. 2006;38(10):1142–1150.[PubMed][Google Scholar]
  • 28. Robinson BS, Huang J, Hong Y, Moberg KHCrumbs regulates Salvador/Warts/Hippo signaling in Drosophila via the FERM-domain protein Expanded. Curr Biol. 2010;20(7):582–590.[Google Scholar]
  • 29. Grzeschik NA, Parsons LM, Allott ML, Harvey KF, Richardson HELgl, aPKC, and Crumbs regulate the Salvador/Warts/Hippo pathway through two distinct mechanisms. Curr Biol. 2010;20(7):573–581.[PubMed][Google Scholar]
  • 30. Varelas X, et al The Crumbs complex couples cell density sensing to Hippo-dependent control of the TGF-β-SMAD pathway. Dev Cell. 2010;19(6):831–844.[PubMed][Google Scholar]
  • 31. Silvis MR, et al α-catenin is a tumor suppressor that controls cell accumulation by regulating the localization and activity of the transcriptional coactivator Yap1. Sci Signal. 2011;4(174):ra33.[Google Scholar]
  • 32. Schlegelmilch K, et al Yap1 acts downstream of α-catenin to control epidermal proliferation. Cell. 2011;144(5):782–795.[Google Scholar]
  • 33. Straßburger K, Tiebe M, Pinna F, Breuhahn K, Teleman AAInsulin/IGF signaling drives cell proliferation in part via Yorkie/YAP. Dev Biol. 2012;367(2):187–196.[PubMed][Google Scholar]
  • 34. Yu FX, et al Regulation of the Hippo-YAP pathway by G-protein-coupled receptor signaling. Cell. 2012;150(4):780–791.[Google Scholar]
  • 35. Pearce LR, Komander D, Alessi DRThe nuts and bolts of AGC protein kinases. Nat Rev Mol Cell Biol. 2010;11(1):9–22.[PubMed][Google Scholar]
  • 36. Lee JH, et al A crucial role of WW45 in developing epithelial tissues in the mouse. EMBO J. 2008;27(8):1231–1242.[Google Scholar]
  • 37. Zhang J, et al YAP-dependent induction of amphiregulin identifies a non-cell-autonomous component of the Hippo pathway. Nat Cell Biol. 2009;11(12):1444–1450.[Google Scholar]
  • 38. Xin M, et al Regulation of insulin-like growth factor signaling by Yap governs cardiomyocyte proliferation and embryonic heart size. Sci Signal. 2011;4(196):ra70.[Google Scholar]
  • 39. Engelman JA, Luo J, Cantley LCThe evolution of phosphatidylinositol 3-kinases as regulators of growth and metabolism. Nat Rev Genet. 2006;7(8):606–619.[PubMed][Google Scholar]
  • 40. Slamon DJ, et al Human breast cancer: Correlation of relapse and survival with amplification of the HER-2/neu oncogene. Science. 1987;235(4785):177–182.[PubMed][Google Scholar]
  • 41. King CR, Kraus MH, Aaronson SAAmplification of a novel v-erbB-related gene in a human mammary carcinoma. Science. 1985;229(4717):974–976.[PubMed][Google Scholar]
  • 42. Braunstein M, Rose AB, Holmes SG, Allis CD, Broach JRTranscriptional silencing in yeast is associated with reduced nucleosome acetylation. Genes Dev. 1993;7(4):592–604.[PubMed][Google Scholar]
Collaboration tool especially designed for Life Science professionals.Drag-and-drop any entity to your messages.