YAP promotes the proliferation of neuroblastoma cells through decreasing the nuclear location of p27Kip1 mediated by Akt

Abstract Objective We aimed to investigate the roles and underlying mechanisms of YAP in the proliferation of neuroblastoma cells. Methods The expression level of YAP was evaluated by Western blotting and immunocytochemistry. Cell viability, cell proliferation and growth were detected by CCK‐8, PH3 and Ki67 immunostaining, and the real‐time cell analyser system. The nuclear and cytoplasmic proteins of p27Kip1 were dissociated by the nuclear‐cytosol extraction kit and were detected by Western blotting and immunocytochemistry. mRNA levels of Akt, CDK5 and CRM1 were determined by qRT‐PCR. Results YAP was enriched in SH‐SY5Y cells (a human neuroblastoma cell line). Knock‐down of YAP in SH‐SY5Y cells or SK‐N‐SH cell line (another human neuroblastoma cell line) significantly decreased cell viability, inhibited cell proliferation and growth. Mechanistically, knock‐down of YAP increased the nuclear location of p27Kip1, whereas serum‐induced YAP activation decreased the nuclear location of p27Kip1 and was required for cell proliferation. Meanwhile, overexpression of YAP in these serum‐starved SH‐SY5Y cells decreased the nuclear location of p27Kip1, promoted cell proliferation and overexpression of p27Kip1 in YAP‐activated cells inhibited cell proliferation. Furthermore, knock‐down of YAP reduced Akt mRNA and protein levels. Overexpression of Akt in YAP‐downregulated cells decreased the nuclear location of p27Kip1 and accelerated the proliferation of SH‐SY5Y cells. Conclusions Our studies suggest that YAP promotes the proliferation of neuroblastoma cells through negatively controlling the nuclear location of p27Kip1 mediated by Akt.


| INTRODUC TI ON
The Hippo signalling pathway is a critical regulator of stem cell self-renewal, tissue regeneration and organ size. [1][2][3][4][5] Dysfunction of this classical pathway will lead to several diseases such as tumours including lung cancer, liver cancer, breast cancer, colorectal cancer and gliomas. 6,7 As the principle effector of the Hippo pathway and a key transcriptional co-factor, YAP plays important roles in organ size control through regulating cell differentiation and proliferation. [8][9][10] Uncontrolled cell proliferation caused by excessive YAP activation will give rise to brain tumours that are usually fatal, such as neuroblastoma and medulloblastoma. [11][12][13] Neuroblastoma (NB) is one of the most common extracranial solid tumours in children. 14 So far, the role of YAP in neuroblastoma has rarely been studied. 13 In recent decades, YAP is found to participate in the proliferation of neuroblastoma. Some studies have indirectly suggested that YAP and TAZ are activated in neuroblastoma and may be closely related to neuroblastoma invasion and metastasis. 15,16 For example, in recurrent neuroblastoma, a mutation in PTPN14 as a negative regulator of YAP is detected, thus, YAP activity may be increased in recurrent neuroblastoma. 15 TAZ can promote the transformation of epithelial cells into mesenchymal cells and promote neuroblastoma invasion and metastasis. 16 As a co-transcription factor of TAZ, YAP may also have the similar effects on the invasion and metastasis of neuroblastoma. Moreover, YAP is highly expressed in neuroblastoma and the expression level is correlated with advanced tumour staging.
Downregulation of YAP significantly impairs neuroblastoma proliferation, tumorigenesis and invasion in vitro. 13 In addition, injection of the YAP inhibitor, peptide 17, dramatically prevents the subcutaneous tumour growth of neuroblastoma. 13 Although these evidences indicate that YAP promotes the tumorigenesis of neuroblastoma, the detailed mechanism about how YAP modulates the proliferation of neuroblastoma cells remains unclear.
Cell proliferation is closely related to the cell cycle. Cyclindependent kinases inhibitors (CDKIs), such as p27 Kip1 and p21 Cip1 , by binding to CDK, inhibit the kinase activity of most CDKs, thereby inhibit cell proliferation and exert anti-tumour activity. 17 There are evidences showing that p27 kip1 is related to neuroblastoma as well as p16, p21 and p53. [18][19][20][21] Decreased transcript levels of p27 Kip1 increases human susceptibility to neuroblastoma, 22 positive expression of p27 Kip1 increases survival in patients with neuroblastoma 18 and accumulation of p27 Kip1 inhibits the growth of human neuroblastoma cells. 23 Several studies have shown that p27 Kip1 can be an important regulatory target of YAP, affecting cell proliferation; however, the conclusions seem to be contradictory to each other. It is reported that exogenous YAP could induce cell proliferation, enhance cyclin D1 expression and reduce p27 kip1 /p21 cip1 levels in contact-inhibited HCEC monolayers and post-confluent B4G12 cells. 24 This means that low p27 kip1 level is conducive to cell proliferation, which is the widely accepted view. [25][26][27] However, there is also evidence showing that low p27 kip1 level can cause defect in cell cycle progression, and the expression of p27 kip1 can also be regulated by YAP. 28 The nuclear and cytoplasmic location of p27 kip1 might be the critical reason that affects cell proliferation, rather than the total p27 kip1 expression. [29][30][31] As an inhibitor of DNA duplication and cell division, p27 Kip1 protein locates in the cytoplasm as well as in the nucleus and exerts its anti-proliferative action inside the nucleus.
Accumulation of nuclear p27 Kip1 prevents cell proliferation. 29,32 However, it remains unclear whether YAP regulates the proliferation of neuroblastoma through controlling the nuclear distribution of p27 Kip1 .
In this study, we found YAP was enriched in SH-SY5Y cells, a cell line of neuroblastoma. Knock-down of YAP in SH-SY5Y cells slowed down cell proliferation, reduced Akt mRNA and protein levels and increased the nuclear location of p27 Kip1 . Overexpression of Akt in YAP-inactivated cells decreased the nuclear location of p27 Kip1 and increased cell proliferation. Our results suggest that YAP regulates the proliferation of neuroblastoma cells through decreasing the nuclear distribution of p27 Kip1 via Akt. Therefore, our findings suggest that nuclear p27 Kip1 entrapment by targeting YAP-Akt signalling may be a potential therapeutic strategy for neuroblastoma. serum-starved SH-SY5Y cells decreased the nuclear location of p27 Kip1 , promoted cell proliferation and overexpression of p27 Kip1 in YAP-activated cells inhibited cell proliferation. Furthermore, knock-down of YAP reduced Akt mRNA and protein levels.

| cDNA constructs
Overexpression of Akt in YAP-downregulated cells decreased the nuclear location of p27 Kip1 and accelerated the proliferation of SH-SY5Y cells.

Conclusions:
Our studies suggest that YAP promotes the proliferation of neuroblastoma cells through negatively controlling the nuclear location of p27 Kip1 mediated by Akt.

| Cell culture and transfection
Primary astrocyte cultures were prepared from the cerebral cortex of P1-P3 mice as described previously. 9 In brief, cerebral neocortex was dissected, chopped, and then incubated with 0.125% trypsin

| Western blotting
Western blotting was carried out as described previously. 9 Briefly, cultured cells were lysed by ice-cold RIPA Buffer (P0013B; Beyotime) and incubated at 4°C for 30 minutes. Following centrifugation at 12 000 × g for 10 minutes, proteins were extracted with 5× loading buffer and boiled at 100°C for 8-10 minutes. The protein samples then were separated using 10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and were transferred onto nitrocellulose membranes (Life Sciences). After blocking in TBST containing 5% skim milk for 1 hour, the immunoblots were incubated with different primary antibodies as shown in above tables at 4°C overnight. Subsequently, the membranes were washed three times in TBST, and incubated with the horseradish peroxidase (HRP)conjugated secondary antibodies for 1 hour. After washing in TBST for another three times, the protein signals were detected using the ECL detection kit (Bio-Rad). Blots were analysed using Quantity One software (Bio-Rad).

| Immunocytochemistry
The protocols used for immunofluorescence staining and quantitative analysis were described previously. 9 Briefly, cultured cells were rinsed once with PBS, fixed in 4% paraformaldehyde for 20 minutes.
Then, they were blocked and permeabilized with 0.1% Triton X-100 in PBS containing 5% bovine serum albumin (BSA) at room temperature for 1 hour. Subsequently, cells were incubated with primary antibodies as shown above tables at 4°C overnight, washed three times in PBS and then with secondary antibodies at room temperature for 1 hour. After washing in PBS for another three times, cells were mounted. Images were acquired by using a fluorescence microscopy (NIKON). The density of fluorescence was measured by Image J software.

| Cell counting Kit-8 (CCK-8) assay
Cell viability was measured by using CCK-8 cell counting kit (A311-01/02; Vazyme Biotech). In brief, the transfected SH-SY5Y cells were seeded into 96-well plates at a density of 2000 cells/well and cultured for 24-48 hours. Subsequently, 10 μL CCK-8 solution was added to each well and incubated at 37°C for 2 hours. The optical density at 450 nm, which was indicative of a positive correlation with cell viability, was measured using a microplate reader (Varioskan Flash; Thermo Scientific).

| Growth curve
The growth curves for SH-SY5Y cells transfected with control-shRNA or YAP-shRNA were generated by using the real-time cell analyser system (IncuCyte S3). The atmosphere was maintained at 37°C, 95% O 2 and 5% CO 2 during recordings. Briefly, about 2-4 × 10 5 viable cells were seeded per well of a six-well plate and recorded for 48 hours. Data were reported as confluence and were defined as the percentage of the cell density at different time points over the cell density at 48 hours, which was auto-calculated by the offline software of IncuCyte S3.

| RNA extraction and quantitative real-time PCR (qRT-PCR)
To determine the mRNA expression levels of genes, total RNA was extracted from cells using TRIzol™ reagent (15596026; Ambion) according to the protocol provided by the manufacturer. A total of

| Cytosol-nuclei fractionation
We used the nuclear-cytosol extraction kit to dissociate the cytoplasmic and nuclear proteins (#P1200; Applygen) according to the manufacturer's instructions. Fractions were analysed by SDS-PAGE and Western blot with specific antibodies.

| Statistical analysis
All data values were presented as mean ± SEM derived from at least three independent experiments. GraphPad Prism software was used for statistical analysis. For comparison between two groups, we used unpaired t test; for comparison between three groups, we applied one-way ANOVA with the Bonferroni post hoc multiple comparison test; for the analysis of the growth curve, two-way ANOVA was utilized. A P value of <.05 was considered to be statistically significant.

| YAP was enriched in the neuroblastoma cell line
To examine the roles of YAP in neuroblastoma cells, we firstly detected the expression level of YAP proteins in SH-SY5Y cells and control cells, such as astrocytes and three human glioma cell lines, A172 cells, U87 cells and DBTRG cells. Our previous studies have shown that YAP was highly expressed in cultured astrocytes. 8 As shown in Figure 1A,B, Western blot results showed that YAP was highly expressed in SH-SY5Y cells and was significantly higher than the primary cultured astrocytes and other glioma cell lines. Further immunostaining results showed that YAP was mainly expressed in the nucleus of SH-SY5Y cells, astrocytes and other glioma cell lines ( Figure 1C,D). These results suggest that YAP is enriched in the neuroblastoma cell line, which may be involved in the proliferation of neuroblastoma cells.

| Knock-down of YAP reduced the proliferation of SH-SY5Y cells
To examine whether YAP was required for the proliferation of SH-SY5Y cells, SH-SY5Y cells were transfected with YAP-shRNA constructs to downregulate YAP expression. As shown in Figure 2A,B and Figure S1, the protein level of YAP was decreased significantly by YAP-shRNA constructs, compared with control-shRNA constructs. Interestingly

| Knock-down of YAP increased the nuclear location of p27 Kip1 in SH-SY5Y cells
Since the subcellular localization of p27 Kip1 can affect cell proliferation, 29,30 we next examined whether YAP enhanced cell proliferation by controlling the nuclear/cytoplasmic location of p27 Kip1 . As shown in Figure 3A,B, knock-down of YAP in SH-SY5Y cells significantly in- at Ser10 facilitates its binding to the carrier protein for nuclear export and subsequent transport from the nucleus to the cytoplasm, 32 we next tested the level of p-p27 kip1 -Ser10 in YAP-downregulated SH-SY5Y cells. As shown in Figure S3A,B, YAP knock-down significantly decreased the level of p-p27 kip1 -Ser10, indicating more p27 kip1 molecules will accumulate in the nucleus. These results suggest that knock-down of YAP increases the nuclear location of p27 Kip1 , which may inhibit the proliferation of SH-SY5Y cells.

| Activation of YAP promoted serum-induced cell proliferation via decreasing the nuclear location of p27 Kip1
Since several previous studies have shown that serum treatment can increase YAP expression, promote the nuclear translocation of YAP and activate YAP, 9,36 thus we next tested whether activation of YAP by serum can promote cell proliferation via decreasing the nuclear location of p27 Kip1 . As shown in Figure 4A Figure 4H).
Similarly, we further quantified the nuclear and cytoplasmic location of p27 Kip1 by immunostaining and nuclear-cytosol fractionation. As expected, immunostaining showed that the nucleus/ cytoplasm ratio of YAP was significantly increased, whereas the nucleus/cytoplasm ratio of p27 Kip1 was significantly decreased by serum treatment ( Figure 4I). Again, the nuclear-cytosol dissociation experiment showed increased nuclear location and nucleus/ cytoplasm ratio of YAP and reduced nuclear location and nucleus/ cytoplasm ratio of p27 Kip1 by serum treatment, compared to cells cultured in serum-free media ( Figure 4J,K). In addition, we tested the level of p-p27 kip1 -Ser10 in YAP-overexpressed SH-SY5Y cells.
As shown in Figure S3C,D, YAP overexpression significantly increased the level of p-p27 kip1 -Ser10, indicating that less p27 kip1 molecules will stay in the nucleus. Taken together, these results strongly suggest that activation of YAP decreases the nuclear location of p27 Kip1 in SH-SY5Y cells, which may promote cell proliferation.
We next examined whether the decrease of nuclear location of p27 Kip1 by YAP activation was required for serum-induced cell proliferation. As shown in Figure 5A Figure 5E,F, Figure S4). Moreover, as we see, overexpressed p27 Kip1 was mostly expressed in the nucleus ( Figure S4), which reminded us that nuclear location of p27 Kip1 may be the real reason that inhibits cell proliferation. Taken together, these results suggest that YAP may promote cell proliferation via decreasing the nuclear location of p27 Kip1 .

| Overexpression of YAP restored the nuclear location of p27 Kip1 in serum-starved SH-SY5Y cells
To examine whether YAP activation is sufficient to restore the nu-

| YAP negatively regulated the nuclear location of p27 Kip1 in SH-SY5Y cells through Akt
How does YAP regulate the subcellular location of p27 Kip1 in SH-

SY5Y cells? Previous studies have shown that Akt can promote
the nuclear export of p27 Kip1 and hinder nuclear import of p27 Kip1 through phosphorylation 37 ; thus, we next examined whether YAP

| D ISCUSS I ON
Here, we present evidence for YAP function in neuroblastoma proliferation and propose a working model, as depicted in Figure 8. In YAP is highly expressed in human glioma specimens, compared to non-tumour human brain tissues. 38 Immunohistochemical will be performed to test these possibilities in future. In our study, surprisingly, we found that the total level of p27 kip1 was decreased when YAP was knock-down ( Figure S8). Although decreased level of total p27 kip1 would promote the proliferation of SH-SY5Y cells according to traditional concepts, the effects of increased nuclear accumulation of p27 kip1 might be more than the effects of decreased level of total p27 kip1 , which may be the main reason that caused reduced proliferation of SH-SY5Y cells by YAP knock-down. This view is supported by the publications that showing nuclear p27 kip1 inhibits cell proliferation, and cytoplasmic p27 kip1 promotes cell proliferation. [29][30][31]37 To know why knock-down of YAP led to a decrease in total expression level of p27 kip1 , further studies are needed.
We also noticed that serum treatment activated YAP and increased p27 kip1 level in SH-SY5Y cells ( Figure 4); however, when YAP was overexpressed, the p27 kip1 level in both nucleus and cytoplasm seemed significantly reduced ( Figure 6). This may be the reason that Moreover, as we know, YAP is a transcriptional co-activator, it has no DNA-binding domain and cannot bind to DNA directly. 52 Therefore, the transcriptional expression of target genes regulated by YAP requires DNA-binding transcription factors. The transcription factor of TEAD family member is the major binding molecule of YAP. 5,33,[53][54][55] The hydrogen bond formed by YAP S94 and TEAD1 Y406 was critical for YAP/TEAD-mediated tissue development and homeostasis. 56 Actually, the functions of YAP are dependent on TEAD at most conditions. There are several reports showing that YAP promotes cell proliferation through TEAD. 5,57-60 Therefore, there is great possibility that the regulation of Akt by YAP is dependent on TEAD family members.
Nevertheless, to know whether the regulation of Akt by YAP is dependent on TEAD family members exactly, further experiments involving YAP-TEAD inhibitors are needed.
The nuclear p27 Kip1 can be regulated by nuclear export, [29][30][31][32]61 nuclear import 62,63 and nuclear degradation. 37,64,65 Currently, the nuclear export of p27 Kip1 is mainly determined by phosphorylation of p27 Kip1 on serine. 10 Phosphorylation of p27 Kip1 on serine 10 by Akt, CDK5 and KIS (Kinase Interacting Stathmin) in the early G1 phase is necessary for its binding to a carrier protein CRM1 for nuclear export, which promotes cell proliferation subsequently. [29][30][31][32] However, in our study, we found that the mRNA level of CDK5 has no significant change, and CRM1 exhibited increased mRNA level in YAP knock-down cells, indicating that they are not direct target genes of YAP, and accumulation of p27 Kip1 in the nucleus of YAP knock-down cells may not be mediated by CDK5 or CRM1.
In the early G1 phase, Thr157 and Thr198 of p27 Kip1 are phosphorylated by Akt, p90RSK1 (p90 ribosomal protein S6 kinases), SGK (serum and glucocorticoid-inducible kinase), AMPK and PIM (although this phosphorylation is relatively rare), which will prevent the nuclear transfer of p27 Kip1 . 62 Moreover, Akt induces phosphorylation of Thr157 and Thr198 to form a recognition motif for 14-3-3 protein to prevent nuclear translocation of p27 Kip1 . 63 Thus, Akt might also implicate in the nuclear and cytoplasmic distribution of p27 Kip1 . Interestingly, in our study, we found that the mRNA and protein level of Akt were both decreased by YAP Mitogenic factors such as serum promote the nuclear translocation of YAP, which initiates the transcription of target genes such as Akt. Furthermore, upregulation of Akt expression promotes the nuclear export of p27 Kip1 and decreases the nuclear location of p27 Kip1 , which enhances proliferation So far, no evidence has shown that YAP-Akt-p27 kip1 signalling is involved in the proliferation of neuroblastoma in vivo, as well as YAP-Akt, Akt-p27 kip1 and YAP-p27 kip1 signalling. However, there are in vivo results suggesting that YAP, Akt and p27 kip1 are related to neuroblastoma. 13,22,66 YAP is significantly higher in the malignant neuroblastoma tissues of human, compared to the peritumoral tissues. 13 Inhibition of the Akt signalling pathway shows therapeutic efficacy in neuroblastoma xenografts in vivo. 66 Decrease of transcript levels of p27 Kip1 increases human susceptibility to neuroblastoma. 22 In summary, our results indicate that YAP promotes proliferation of neuroblastoma cells through decreasing the nuclear location of p27 Kip1 mediated by Akt. Disruption of the balance of p27 Kip1 distribution between nuclear and cytoplasmic distribution by YAP activation will influence cell cycle and cell proliferation.
Our study provides a new mechanism for the proliferation of neuroblastoma cells, and suggest that nuclear p27 Kip1 entrapment may be a potential therapeutic strategy for anti-proliferation in neural tumour cells.

ACK N OWLED G EM ENTS
We thank Prof. Chuanshu Huang (New York University School of Medicine) for the pEGFP-p27 Kip1 plasmid.

CO N FLI C T S O F I NTE R E S T
The authors declare no potential conflicts of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.