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Abstract

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References
  8. Supporting Information

Yes-associated protein (YAP), the downstream effecter of the Hippo-signaling pathway as well as cyclic adenosine monophosphate response element-binding protein (CREB), has been linked to hepatocarcinogenesis. However, little is known about whether and how YAP and CREB interact with each other. In this study, we found that YAP-CREB interaction is critical for liver cancer cell survival and maintenance of transformative phenotypes, both in vitro and in vivo. Moreover, both CREB and YAP proteins are highly expressed in a subset of human liver cancer samples and are closely correlated. Mechanistically, CREB promotes YAP transcriptional output through binding to −608/−439, a novel region from the YAP promoter. By contrast, YAP promotes protein stabilization of CREB through interaction with mitogen-activated protein kinase 14 (MAPK14/p38) and beta-transducin repeat containing E3 ubiquitin protein ligase (BTRC). Gain-of-function and loss-of-function studies demonstrated that phosphorylation of CREB by MAPK14/p38 at ser133 ultimately leads to its degradation. Such effects can be enhanced by BTRC through phosphorylation of MAPK14/p38 at Thr180/Tyr182. However, YAP negatively controls phosphorylation of MAPK14/p38 through inhibition of BTRC expression. Conclusion: There is a novel positive autoregulatory feedback loop underlying the interaction between YAP and CREB in liver cancer, suggesting that YAP and CREB form a nexus to integrate the protein kinase A, Hippo/YAP, and MAPK14/p38 pathways in cancer cells and thus may be helpful in the development of effective diagnosis and treatment strategies against liver cancer. (Hepatology 2013;53:1011–1020)

Abbreviations
Abs

antibodies

BTRC

beta-transducin repeat containing E3 ubiquitin protein ligase

cAMP

cyclic adenosine monophosphate

ChIP

chromatin immunoprecipitation

CHX

cycloheximide

Co-IP

coimmunoprecipitation

CRE

cAMP responsive element

CREB

cAMP response element-binding protein

GAPDH

glyceraldehyde 3-phosphate dehydrogenase

HCC

hepatocellular carcinoma

hEGF

human epithelial growth factor

IF

immunofluorescence

IHC

immunohistochemistry

MAPK/p38

mitogen-activated protein kinase 14

MKK

MAPK kinase

mRNA

messenger RNA

MTT

methyl thiazol tetrazolium

nt

nucleotide

PI3K

phosphoinositide 3 kinase

PKA

protein kinase A

pTEN

phosphatase and tensin homolog

qPCR

quantitative polymerase chain reaction

SC

subcutaneously

SD

standard deviation

shRNAs

short hairpin RNAs

TMA

tissue microarray

YAP

Yes-associated protein

Liver cancer is the fifth-most common cancer worldwide and the third-leading cause of cancer death.[1] The treatment options for these hepatic malignancies are extremely limited, mainly because the mechanisms of pathogenesis of these cancers are not completely known. Recently, the dysfunctional Hippo/Yes-associated protein (YAP)-signaling pathway has been linked to hepatocarcinogenesis.[2] Transgenic mice with liver-targeted YAP overexpression demonstrated a dramatic increase in liver size and eventually developed tumors.[3] In addition, clinical studies revealed that YAP was overexpressed in 62% of hepatocellular carcinoma (HCC) patients and was an independent predictor associated with poor disease-free survival and overall survival in HCC.[4] In view of the vital roles that YAP plays in the development of liver cancer, it was extremely important to understand how YAP is up-regulated in tumor.

Numerous studies have shown that cyclic adenosine monophosphate (cAMP) response element-binding protein (CREB) may be involved in liver cancer development. CREB is a ubiquitous transcription factor that activates the transcriptional activity of various promoters through its binding site.[5] The phosphorylation of CREB at ser133 potentiates its transcription activity by recruitment of several transcription coactivators, thereby stimulating expression of target genes that are involved in many cellular activities, from proliferation to apoptosis.[6] It was reported that total and active CREB (p-CREB) significantly increased in HCC, compared to pair-matched normal liver samples.[7] Our previous work also revealed that CREB up-regulates an HCC highly associated long noncoding RNA, HULC expression through interaction with microRNA-372,[8] suggesting the important role of CREB in liver cancer.

In the present study, we highlighted the role of mutual interaction between YAP and CREB in liver tumorigenesis. We found that CREB up-regulated YAP transcription by binding to a novel site in the YAP promoter region. Moreover, we revealed that YAP inhibited the degradation of CREB mediated by mitogen-activated protein kinase 14 (MAPK14/p38) in HCC cells, thus providing a positive feedback loop to promote cellular YAP and CREB output. Our data also showed that the two proteins were closely correlated in tumor samples, suggesting the important role of their feedback loop in liver cancer. Taken together, this work summarizes a novel link between two major oncoproteins and a potential mechanism for liver tumorigenesis.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References
  8. Supporting Information
Cell Culture and Vectors

HepG2, Bel-7402, SMMC-7721, and HEK-293T cells were cultured in Dulbecco's modified Eagle's medium. Cells were treated by H89 (20 uM; Cayman Chemical Company, Ann Arbor, MI), forskolin (50 uM; Cayman), wortmannin (50 uM; Cayman), LY294002 (20 uM; Cell Signaling Technology, Danvers, MA), SB203580 (20 uM; Cell Signaling Technology), SB202190 (5-20 uM; Santa Cruz Biotechnology, Santa Cruz, CA), MG132 (25 uM; Cayman), or human epithelial growth factor (hEGF) (10 ng/mL; Sigma-Aldrich, St Louis, MO) 5-24 hours before harvest. Short hairpin RNAs (shRNAs) were cloned into pLKO.1 lentiviral vectors. Complementary DNA fragments encoding human YAP, CREB, MAPK14/p38, and beta-transducin repeat containing E3 ubiquitin protein ligase (BTRC) were cloned into pGIPZ-based lentiviral vector and pcDNA3.1(+), respectively, and the primers used are listed in Supporting Table 1. shRNA- and protein-expressing plasmids for phosphatase and tensin homolog (pTEN) were gifts from Dr. Xuqian Fang (Shanghai Jiaotong University, Shanghai, China). YAP-Flag and LATS1-Flag expression plasmids were constructed as described previously.[9]

Immunohistochemistry, Immunofluorescence, and Immunoblotting

For immunohistochemistry (IHC), human liver cancer tissue microarray (TMA) slides were purchased from U.S. Biomax (Rockville, MD). Slides were incubated in primary antibodies (Abs) against CREB (#1496; Epitomics, Burlingame, CA) and YAP65 (#2060; Epitomics).

For immunofluorescence (IF), cells were incubated in primary Abs against YAP (#4912; Cell Signaling Technology, or sc-101199; Santa Cruz Biotechnology), CREB (#9197; Cell Signaling Technology), p-p38 (sc-7973; Santa Cruz Biotechnology), p38 (#9218; Cell Signaling Technology, or sc-271120; Santa Cruz Biotechnology), or BTRC (#4394; Cell Signaling Technology).

For immunoblotting, primary Abs used were Flag (#F3165; Sigma-Aldrich, or #2368; Cell Signaling Technology), HA (#3724 or #2367; Cell Signaling Technology), YAP (#2060; Epitomics), ubiquitin (#3933; Cell Signaling Technology), BTRC (#4394; Cell Signaling Technology), CREB (#9197; Cell Signaling Technology), p-CREB (#9198; Cell Signaling Technology), p-38 (#9218; Cell Signaling Technology), p-p38 (#4511; Cell Signaling Technology), MKK3 (#1728; Epitomics), MKK6 (#1640; Epitomics), p-MKK3/6 (#9236; Cell Signaling Technology), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (#5174; Cell Signaling Technology). Relative protein expression levels were quantified by specific protein/GAPDH ratio, which are presented as mean ± standard deviation (SD) from three independent experiments (listed in Supporting Table 2).

Cell Proliferation, Caspase3/7 Activity, Soft-Agar Assays, and Quantitative Reverse-Transcriptase Polymerase Chain Reaction

Cell proliferation was measure by a methyl thiazol tetrazolium (MTT)-based proliferation assay, as described before.[10] Caspase-3/7 activity was determined using the Caspase-Glo 3/7 assay system (Promega, Madison, WI). Anchorage-independent soft-agar growth assay and quantitative reverse-transcriptase polymerase chain reaction (qPCR) was performed as previously described.[10]

Immunoprecipitation

Cell lysates were incubated with indicated Abs and protein A/G beads (Life Technologies Corporation, Carlsbad, CA) overnight. Immunoprecipitates were washed five times and then subjected to immunoblotting analysis.

Luciferase Reporter Analysis

Luciferase reporter constructs containing the YAP promoter region were cloned into pGL3-based vectors, then stably cotransfected with a Renila luciferase expression plasmid into cells. Luciferase activities were analyzed using a dual-luciferase reporter kit (Promega).

Chromatin Immunoprecipitation

Chromatin immunoprecipitation (ChIP) was performed using the ChIP-IT express kit from Active Motif (Carlsbad, CA). Protein-DNA complexes were incubated with 3 μg of anti-CREB Abs (#1496; Epitomics).

Xenograft Mouse Model

HepG2 cells (5× 106) expressing shRNA or protein, as indicated, were subcutaneously (SC) injected into athymic nude mice (Bikai, Shanghai, China). Tumor size was measured every 6 days using a caliper, and tumor volume was calculated as 0.5 × L × W2, with L indicating length and W indicating width. Mice were euthanized at 45 days after injection.

Results

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References
  8. Supporting Information
Both YAP and CREB Are Critical for Cell Survival and Maintenance of Transformative Phenotype

We examined whether YAP and CREB were important for liver cancer cells. YAP- or CREB-specific shRNAs with high knockdown efficiency (Supporting Fig. 1) were used to silence expression in both Bel-7402 and HepG2 cells. We found that inhibition of either YAP or CREB decreased cell proliferation, compared to control, as measured by an MTT-based assay and Ki-67 immunostaining (Fig. 1A and data not shown). Furthermore, we found that both YAP and CREB knockdown impaired the ability of these cells to form colonies in soft agar (Fig. 1B), whereas they markedly increased apoptosis, as shown by increased caspase 3/7 activity and caspase 3 cleavage (Fig. 1C and data not shown).

image

Figure 1. Both YAP and CREB are required for live cancer cell function.

(A and B) Relative cell proliferation and transformation activities measured by MTT (A) and anchorage-independent soft agar colony formation assays (B) in Bel-7402 and HepG2 cells expressing shRNA constructs against either YAP or CREB with or without ectopic expression of CREB or YAP. (C) Knockdown of YAP or CREB induces apoptosis, which can be rescued by overexpression of CREB or YAP, as measured by caspase 3/7 activity. Data are indicated as mean ± SD of three independent experiments. *P < 0.05; **P < 0.01 using the Student t test.

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Furthermore, we found that reduced cell survival and transformative phenotype by shRNA against YAP or CREB could be partially rescued by simultaneous ectopic expression of CREB or YAP (Fig. 1A-C). These data indicate that interaction between YAP and CREB is important for human liver cancer cell growth and survival.

CREB Enhances YAP Transcription

An interesting question arising from our data was how YAP and CREB regulate with each other. We observed that YAP messenger RNA (mRNA) was reduced by the protein kinase A (PKA)/CREB inhibitor, H89 (Fig. 2A), whereas it was induced by the PKA/CREB activator, forskolin (Fig. 2B). YAP protein was also compromised, as measured by immunoblotting analysis (Fig. 2C).

image

Figure 2. CREB activates YAP transcription. (A and B) PKA/CREB induces YAP. YAP mRNA expression tested by qPCR under treatment of either dimethyl sulfoxide (DMSO), H89 (20 uM) (A), or forskolin (50 uM) (B) for 24 hours. (C) Immunoblotting analysis of YAP (S, shorter exposure; L, longer exposure) as well as CREB proteins in HepG2 cells treated by different chemicals, as indicated, for 24 hours. (D) YAP promoter analysis. Luciferase activities were tested for cells stably cotransfected with reporter plasmids containing truncated versions of the YAP promoter region and Renilla reporter. (E) PKA /CREB induces the YAP promoter. Samples were harvested for cells stably transfected with luciferase reporters, as indicated, after chemical incubation for 24 hours. (F) CREB binds to YAP promoter. ChIP from HepG2 cells was performed with control immunoglobulin G IgG or CREB Ab, as indicated. The presence of CREB binding was detected by qPCR using primers either covering the CREB-binding region (R2) or the negative unrelated region (R1). *P < 0.05 versus control, analyzed by the Student t test.

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Then, we investigated the mechanism underlying how YAP is regulated by CREB. A recent study[11] suggests that CREB binds to nucleotide (nt) −232/+115 containing a CRE of YAP promoter in HCC cells. However; our luciferase reporter gene assays revealed that YAP promoter activity was greatly enhanced by nt −608/−439 (Fig. 2D), which is also sensitive to both H89 and forskolin (Fig. 2E), because promoter activities from −608-Luc, but not −439-Luc, could be reduced by H89, whereas it was induced by forskolin (Fig 2E), suggesting the potential role of a novel cAMP responsive element (CRE) at this region. Furthermore, −608-Luc was inhibited when CREB was knocked down, whereas it was activated when CREB was ectopically expressed (Supporting Fig. 2A). Similarly, YAP protein can be up-regulated by CREB (Supporting Fig. 2B). Then, ChIP analysis was performed and it was demonstrated that CREB was able to bind to −608/−439 (R2); however, no enrichment was detected at an unrelated region (R1) (Fig. 2F). Taken together, YAP transcription is controlled by CREB through a novel promoter region.

YAP Is a Pivotal Regulator That Maintains Both CREB Protein Expression and Activity

In both Bel-7402 and SMMC-7721 cells with YAP knocked down, we found that transcription of known CREB target genes, such as Rab25,[12] HULC,[8] as well as the YAP target gene, CTGF,[13] was significantly inhibited (Fig. 3A), suggesting that YAP regulates CREB transcriptional activity. In addition, we observed that CREB correlated with YAP expression in almost all the cell lines detected, with highest YAP and CREB protein expression in HepG2 cells (Fig. 3B).

image

Figure 3. YAP enhances CREB protein independent of transcription. (A) Regulation of CREB or YAP target genes. qPCR analysis was performed for cells expressing shRNA against GFP (control) or YAP. (B) CREB and YAP protein expression patterns were tested by immunoblotting analysis in different cell lines, as indicated. (C) YAP stabilizes CREB. Immunoblotting analysis of CREB and YAP in control and YAP-shRNA-expressing HepG2 and Bel-7402 cells. (D) Immunoblotting analysis of CREB in HepG2 and Bel-7402 cells transiently transfected with control or increased concentration of YAP-Flag expression plasmids. (E) YAP dose not affect CREB transcription. CREB mRNA was tested by qPCR in cells expressing shRNA against GFP or YAP. (F) YAP affects CREB activity. Luciferase activities from HULC core promoter reporter with (WT) or without (Mut) CRE were measured for HepG2 cells expressing shRNA against either GFP or YAP. *P < 0.05 versus control, analyzed using the Student t test.

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Then, we tested whether YAP also regulates CREB protein expression. We found that cells with YAP knocked down had a much lower level of CREB, as compared to control, in both Bel-7402 and HepG2 cells (Fig. 3C). Furthermore, CREB was dose dependently up-regulated by an increasing amount of YAP (Fig. 3D). Surprisingly, YAP knockdown did not significantly affect CREB mRNA levels (Fig. 3E), thus ruling out the possibility that YAP regulates CREB transcription. As previously described, that CREB is critical for HULC promoter activity,[8] we used this luciferase reporter system to confirm our hypothesis that YAP regulates CREB activity. We found that promoter activities from WT (with CRE) was greatly inhibited, whereas no obvious changes were detected from the Mut (without CRE) one in HepG2 cells with YAP knocked down, compared to the control (Fig. 3F). Collectively, these experiments demonstrate that YAP may play an important role in maintaining CREB protein expression and activity.

YAP Interacts With CREB

We found that both YAP and CREB gave strong signals and colocalized in both HCC cells (Fig. 4A) and HCC tissues (Fig. 4D) by IF assays. To confirm the interaction between YAP and CREB, we performed reciprocal coimmunoprecipitation (Co-IP) experiments and found that exogenous CREB-HA could be readily pulled down by YAP-Flag and vice versa (Fig. 4B). Co-IP for endogenous YAP and CREB proteins in Bel-7402 and HepG2 cells also demonstrated that these two proteins readily coimmunoprecipitated (Fig. 4C).

image

Figure 4. Interaction between YAP and CREB. (A) Colocalization of YAP and CREB in HCC cell lines. Cells were harvested for IF analysis by both anti-CREB and anti-YAP Abs. Scale bar, 15 uM. (B) YAP binds to CREB. CREB-HA was cotransfected with YAP-Flag into HEK293T cells, as indicated. YAP and CREB associations were examined by reciprocal Co-IP, as indicated. (C) Endogenous YAP was immunoprecipitated with anti-YAP Abs, and Co-IP of CREB is shown by anti-CREB immunobloting. A control immunoglobulin G (IgG) was used as the negative control for IP. (D) Colocalization of YAP and CREB in HCC tissues. Representative IF images of YAP and CREB staining in two HCC cases. Scale bar, 15 uM. (E and F) CREB correlates with YAP. Representative IHC images of CREB and YAP staining from TMA analysis (E). Statistical analysis of the TMA data is shown in (F).

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To further uncover the relationship between YAP and CREB, we performed IHC using TMA on greater than 400 human liver cancer samples. We found that both CREB and YAP proteins are highly expressed in a subset of human liver cancers and closely correlated with each other (Fig. 4E,F). Taken together, these experiments establish a close relationship between YAP and CREB in liver cancer.

MAPK14/p38 Contributes to CREB Degradation

To reveal how YAP regulates CREB at the protein level, the mechanism underlying CREB degradation needed to be elucidated. We found that CREB protein could be down-regulated by both of the two phosphoinositide 3 kinase (PI3K) inhibitors, LY294002 and wortmannin. Surprisingly, the phosphorylated form of CREB at Ser133 was up-regulated (Fig. 5A). Using PI3K activator hEGF, an opposite expression pattern of CREB and p-CREB was observed (Supporting Fig. 3A). pTEN is known as an endogenous PI3K inhibitor. In HepG2 cells with pTEN overexpressed, we found that, inconsistent with the above-mentioned two chemical inhibitors, p-CREB was up-regulated, whereas total CREB was down-regulated (Supporting Fig. 3B). On the contrary, reduction of p-CREB with induction of total CREB was detected in Bel-7402 cells with pTEN knocked down (Supporting Fig. 3C), suggesting that PI3K inhibits CREB phosphorylation, but protects CREB from degradation. As reported previously, CREB can be phosphorylated at ser133 by p38.[14] By Co-IP assays, we found that p-p38 and CREB interacted with each other in HepG2 cells (Supporting Fig. 4). Such interaction was enhanced by both LY294002 and wortmannin, as detected by IF (Fig. 5B) and Co-IP assays (Fig. 5C), respectively. However, in HepG2 cells under treatment of either LY294002 or wortmannin, p-p38 was slightly up-regulated, whereas total p38 was slightly down-regulated (Fig. 5A). To investigate whether p38 contributes to the phosphorylation and degradation of CREB, two specific p38 inhibitors (SB203580 and SB202190) were used. We found that p-CREB was inhibited, whereas total CREB was induced (Fig. 5D). Consistently, reduced p-CREB with induced total CREB was detected in HepG2 cells with p38 knocked down (Fig. 5E). By contrast, opposite results were observed in HepG2 cells when p38 was overexpressed (Fig. 5F), suggesting that p38 phosphorylates CREB, which, ultimately, leads to CREB degradation.

image

Figure 5. MAPK14/p38 contributes to CREB degradation. (A) PI3K inhibits phosphorylation of CREB. Immunoblotting analysis of p-CREB/CREB and p-p38/p38 in HepG2 cells induced by either dimethyl sulfoxide (DMSO) or two PI3K inhibitors, wortmannin (50 uM) or LY294002 (20 uM), for 24 hours. (B and C) Blocking of PI3K promotes CREB binding to p-p38. HepG2 cells treated by either wortmannin (50 uM) or LY294002 (20 uM) for 24 hours were harvested for IF analysis using both anti-CREB and anti-p-p38 Abs. Scale bar, 15 uM. (B) Immunoprecipitated with anti-p-p38 Abs. Co-IP of CREB is shown by anti-CREB immunobloting (C). (D) Inhibition of p38 reduces p-CREB, but induces CREB. Immunoblotting analysis of p-CREB/CREB in HepG2 cells treated with either DMSO or two p38 inhibitors, SB203580 (20 uM, left panel) or increasing concentration of SB202190 (5-20 uM, right panel), for 24 hours. (E and F) Alteration of p38 regulates CREB. Immunoblotting analysis of p-CREB/CREB in HepG2 cells expressing shRNA against either GFP (control) or p38 (E), or increasing concentration of p38 (F).

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To address the point that phosphorylation of CREB leads to subsequent degradation, Ser133 phosphorylation-deficient mutation (S133A) was introduced. We found that S133A degraded much slower than that of wild type (WT) (Supporting Fig. 5A). Furthermore, S133A was insensitive to p38 overexpression, compared to WT (Fig. S5B), suggesting that ser133 contributes to CREB degradation.

YAP Stabilizes CREB by Inhibiting Phospho-MAPK14/p38

Because YAP regulates CREB independent of transcription (Fig. 3), we assessed whether YAP regulates CREB expression in HCC cells by interaction with p38. We found that when HepG2 cells were treated with the protein synthesis inhibitor, cycloheximide (CHX), the CREB protein was unstable, with a half-life of approximately 2 hours. However, CREB was stabilized when YAP was ectopically expressed (Fig. 6A). In addition, in HepG2 cells with YAP knocked down, we detected a more significant accumulation of ubiquitinated CREB, compared to the nonsilencing control (Fig. 6B, lane 3, compared to lane 1).

image

Figure 6. YAP stabilizes CREB by inhibitory to phospho-MAPK14/p38. (A) YAP protects CREB. HepG2 cells were stably transfected with or without YAP expression plasmid, and protein synthesis was blocked by treatment of CHX (50 ug/mL) for the indicated time. Relative CREB protein levels were quantified by CREB/GAPDH ratio. (B) YAP knockdown induces ubiquitination of CREB. Ubiquitination of CREB in HepG2 cells expressing shRNA against green fluorescent protein (GFP; control) or YAP. Cells were treated with or without MG132 (25 uM) for 5 hours before harvest. Endogenous CREB was immunoprecipitated, and immunoblotting analysis was done using anti-CREB or anti-Ub Ab. (C) YAP affects phosphorylation of p38. Immunoblotting analysis of different proteins, as indicated, in HepG2 cells expressing shRNA against GFP (control) or YAP. (D) LATS induces phosphorylation of p38. Immunoblotting analysis of different proteins, as indicated, in HepG2 cells transiently transfected with increasing concentration of LATS1-Flag expressing plasmids. (E) Colocalization of YAP and p38. Cells were harvested for IF analysis by both anti-CREB and anti-YAP Abs. Scale bar, 15 uM.

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Then, we tested whether YAP regulates p38 phosphorylation. We found that cells with YAP knocked down had a much higher level of phosphorylation of p38 at Thr180/Tyr182 (p-p38), as compared to the control. However, unlike p-p38, total p38 was slightly down-regulated (Fig. 6C). As reported, activation of p-p38 occurs through its upstream kinases, MAPK kinase (MKK)3/6.[15] Therefore, we examined whether YAP regulates p-p38 through interacting with MKK3/6, and found that both p-MKK3/6 and total-MKK3/6 were unaffected by silencing of YAP (Fig. 6C), suggesting that YAP does not regulate the upstream canonical signaling of MAPK14/p38.

Phosphorylation of YAP by the upstream Hippo pathway kinases (such as LATSs) results in its degradation and blockage of activity.[16] We detected that degradation of YAP by overexpression of LATS1 led to up-regulation of p-p38 (Fig. 6D), which suggests the cross-talk between the MAPK14/p38 and Hippo pathways. Next, colocalization of YAP and p38 was detected by IF (Fig. 6E), which supports the conclusion that these two proteins interact with each other. Furthermore, Co-IP experiments also revealed that YAP binds to p38 (Fig. 6F). Taken together, interaction between YAP and p38 may prevent CREB from degradation.

YAP Inhibits BTRC, a Mediator of p38 Activity

As reported, p38-CK2 complex associates with BTRC, an F-box E3 ligase, and leads to the degradation of nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha in fibroblasts.[17] Also, YAP-BTRC interaction was described previously.[18] Therefore, we hypothesized that YAP protects p38-mediated CREB degradation through BTRC. Colocalization of endogenous p38 and BTRC was visualized by an IF assay (Fig. 7A). Interaction of both of the two proteins was also revealed by a Co-IP assay (Fig. 7B), suggesting that BTRC, p38, and YAP may form a complex in liver cancer cells. An intriguing aspect is that silencing of BTRC reduced p-p38 and p-CREB, whereas it induced total p38 and CREB (Fig. 7C), suggesting that BTRC may play differential roles in the phosphorylation and degradation of both p38 and CREB. Consistently, overexpression of BTRC led to up-regulation of p-p38 and p-CREB, but down-regulation of both total p38 and CREB (Fig. 7D). To address how YAP works on BTRC expression, BTRC was examined in HepG2 cells with either YAP knocked down or ectopically expressed. We found that BTRC was up-regulated by knockdown of YAP (Fig. 7E), whereas it was down-regulated by overexpression of YAP (Fig. 7F).

image

Figure 7. YAP inhibits BTRC-induced phosphorylation of MAPK14/p38.

(A) Colocalization of BTRC and p38. Cells were harvested for IF analysis by both anti-BTRC and anti-p38 Abs. Scale bar, 15 uM. (B) p-p38/p38 binds to endogenous BTRC. Endogenous BTRC was immunoprecipitated in HepG2 cells, and Co-IP of p-p38/p38 is shown by immunoblotting. (C and D) BTRC regulates p-p38/p38. Immunoblotting analysis of p-p38/p38 and p-CREB/CREB in HepG2 cells expressing shRNA against either green fluorescent protein (control) or BTRC (sh#1+sh#2) (C), or BTRC-HA (D). (E and F) YAP inhibits BTRC. Immunoblotting analysis of different proteins, as indicated, in HepG2 cells expressing either shRNA against YAP (E) or YAP-FLAG (F).

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The Interaction Between YAP and CREB Promotes HCC Tumor Growth

On the basis of the interaction between YAP and CREB, we investigated the growth of HepG2 clones after injection into athymic mice. Compared to the control, HepG2 cells with either YAP or CREB knocked down effectively prevented tumor growth, but such effects could be rescued by simultaneously overexpressing either CREB or YAP in a nude mouse model (Fig. 8A). Thus, we confirmed such a close relationship in vivo.

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Figure 8. Mutual promotion of YAP and CREB in the xenograft model and the possible mechanism involved. (A) Ectopic expression of CREB or YAP rescues silencing of YAP or CREB in vivo. Tumor volumes were measured for 42 days after SC injection (n = 5 per group). (B) Possible mechanism underlying mutual interaction between YAP and CREB in liver cancer.

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Discussion

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References
  8. Supporting Information

Although much is known about its posttranslational modification, the transcriptional regulation of YAP, as well as the cross-talk between YAP and other pathways, is still poorly understood. In the present study, we show that YAP-CREB interaction is critical for liver cancer cells, both in vitro and in vivo, through a positive autoregulatory feedback loop. We revealed that YAP inhibited the degradation of CREB mediated by BTRC and p38, and the accumulation of CREB, in turn, stimulated YAP transcription. Moreover, both CREB and YAP proteins are highly expressed in a subset of human liver cancer samples and are closely correlated, suggesting an important role of this feedback loop in liver cancers.

Without a DNA-binding domain, YAP has to work through target transcription factors, such as TEAD family proteins Runx2, Smads, and so on.[16] Because of the fact that there were no co-occupancies of YAP and CREB at CRE of YAP, Rab25, and HULC promoters by ChIP assay (data not shown), we believe that YAP proteins do not act as cotranscription factors to CREB, but rather as regulators to CREB activity (Fig. 3). In a recent study, Skouloudaki and Walz[19] reported that YAP recruits tyrosine kinase c-Abl, antagonizes the function of Nedd4.2, an E3 ubiquitin-ligase, and thus protects AMOTL1 from degradation. Similarly, our findings reveal a new role of YAP in protecting another protein CREB from degradation. Phosphorylation on CREB ser133 by MAPK14/p38 kinase primes subsequent CREB degradation (Fig. 5 and Supporting Fig. 5), which can be blocked by YAP (Fig. 6). YAP controls phosphorylation of MAPK14/p38 through BTRC (Fig. 7), an E3 ligase that interacts with and mediates YAP unbiquitination and degradation.[18] Conversely, we first uncovered that this interaction also facilitates BTRC degradation (Fig. 7). Noubissi et al.[20] reported that β-catenin stabilizes BTRC mRNA by enhancing an RNA-binding protein CRD-BP, expression through promoter binding and, ultimately, elevates BTRC protein levels. Also, Imajo et al.[21] demonstrated that YAP suppresses the nuclear translocation of β-catenin by directly binding to it in the cytoplasm, thereby inhibiting β-catenin. YAP keeps the balance of BTRC expression through interaction with β-catenin. On the other hand, alteration of Wnt/β-catenin signaling activities leads to significant activation of MAPK14/p38.[22] Additionally, induction of BTRC expression results in an accelerated degradation of β-catenin.[23] These studies may explain the ability of BTRC in controlling the phosphorylation of MAPK14/p38 (Fig. 7).

In conclusion, we found that YAP and CREB are aberrantly up-regulated in liver tumor samples. Both YAP and CREB are critical for cell survival and maintenance of transformative phenotype. We further found a positive feedback for both YAP and CREB in liver cancers. We showed that CREB loaded onto promoters of YAP to drive transcription. Up-regulation of YAP protects CREB from p38-mediated degradation through inhibition of BTRC. Accumulation of CREB, in turn, promotes the transcription of YAP (Fig. 8B). To our knowledge, our results establish a new signaling mechanism by which the interaction between YAP and CREB promotes HCC tumor growth. The breaking up of this mutual interaction may serve as a crucial target in liver cancer therapy.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References
  8. Supporting Information

The authors thank Tingjun Ye, Xiangfan Liu, Xuqian Fang, Jiafei Lin, and Jiabin Sun of Shanghai Jiaotong University for their technical assistance.

References

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References
  8. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References
  8. Supporting Information

Additional Supporting Information may be found in the online version of this article.

FilenameFormatSizeDescription
hep26420-sup-0001-suppfigs.doc722KSupporting Information Figures.
hep26420-sup-0002-supptab1.doc55KTable. S1. Primers and shRNA sequences
hep26420-sup-0003-supptab2.doc200KSupplementary Table S2 Data from Immunoblotting. Immunoblots of this study have been quantitated, and data are being shown as mean±SD from three independent experiments.

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