RACO‐1 modulates Hippo signalling in oesophageal squamous cell carcinoma

Abstract Oesophageal cancer is one of the most lethal malignancies worldwide, whereas the 5‐year survival is less than 20%. Although the detailed carcinogenic mechanisms are not totally clear, recent genomic sequencing data showed dysregulation of Hippo signalling could be a critical factor for oesophageal squamous cell carcinoma (ESCC) progression. Therefore, understanding of the molecular mechanisms that control Hippo signalling activity is of great importance to improve ESCC diagnostics and therapeutics. Our current study revealed RACO‐1 as an inhibitory protein for YAP/TEAD axis. Depletion of RACO‐1 increases the protein level of YAP and expression of YAP/TEAD target gene. Besides, RACO‐1 silencing could promote ESCC cell invasion and migration, which effect could be rescued by YAP depletion in ESCC cells. Immunoprecipitation showed that RACO‐1 associated with YAP and promote ubiquitination and degradation of YAP at k48 poly‐ubiquitination site. Our research discovered a new regulator of Hippo signalling via modulating YAP stability. RACO‐1 could be a promising factor, which serves cancer diagnostics and therapeutics in ESCC patients.


| INTRODUC TI ON
Oesophageal cancer ranks NO. 8 in cancer incidence and cancer-related death in the world. 1 Approximately 60% of newly diagnosed oesophageal cancer cases are found in China. 2 However, oesophageal squamous cell carcinoma (ESCC) is the major subtype of oesophageal cancer in China, whereas adenocarcinoma is the dominant type in Western countries. Although there are more than 300 000 new diagnosed cases in China, the rate of oesophageal cancer varies widely in China mainland. For example, there is significantly higher oesophageal cancer incidence in northern part of Henan province, compared with neighbourhood area. 3 Besides, the known risk factors such as tobacco, alcohol and lack of nutrition recently progress of genomic-based sequencing showed that the genetic alternations also play critical roles in the oncogenic process. The genomic sequencing and profiling data showed that the deficit of inhibitory control in Hippo signalling is familiar in ESCC patients. 4 The cancer biology studies illustrated that silencing or inhibiting YAP/TEAD axis dramatically inhibited ESCC cell growth and invasion. 5,6 However, the detail of the mechanism that controls Hippo signalling activity, especially YAP/TEAD trans-activation turnover, is still not totally clear. As the particular role of Hippo signalling in ESCC carcinogenesis, it is important to reveal the adjustment mechanism of YAP/TEAD in ESCC.
Hippo signalling was first detected in Drosophila through genetic screening. 7 More investigation showed that Hippo signalling functions to the tissue haemostasis and organ size control, which depended on a fine balance between cell proliferation and apoptosis. 8 Kinase cascade is the key to Hippo signalling. MST1/2 (Mammalian sterile 20-like kinase) and SAV1 functions as a complex to phosphorylate LATS1/2 (large tumour suppressor kinase), which subsequently phosphorylates YAP/TAZ and promotes YAP/TAZ cytosol retention and proteolysis. 9 In addition, when the Hippo signal is turned off, the unphosphorylated YAP/TAZ shifts to the nucleus, activating some transcription factors such as TEADs. 10 The over-activation of YAP/ TEAD axis was observed in several human cancers. 11 The molecular biology studies showed that YAP/TEAD activation contributed to several cancer biological behaviours, such as tumour growth, cancer invasion, and 'stemness' maintenance. 12 In ESCC studies, Hippo signalling abnormalities, such as YAP gene amplification and FAT mutations were observed in 40% of oesophageal tumour cases. 4 YAP level is increased in oesophageal cancer samples and correlates with cancer metastasis and advanced cancer stage. 13 As the critical role of Hippo signalling in oesophageal cancer, further understanding of the control of Hippo signalling and YAP turnover are of great importance to improve ESCC cancer diagnosis and therapy. Here, we identified a novel inhibitor RACO-1 for YAP/TEAD axis of Hippo signalling in ESCC. RACO-1 (RING domain AP-1 co-activator-1, RING finger 187) is a RING finger protein, which functions as an E3 ubiquitin ligase in several cellular processes. 14 Our current study identifies RACO-1 as an endogenous inhibitor for YAP/TEAD axis of Hippo signalling in ESCC. RACO-1 promotes YAP poly-ubiquitination and degradation, which subsequently leads to transcriptional suppression of YAP/TEAD target genes in ESCC.

| Plasmids and siRNA
The Myc-RACO-1 plasmid was acquired from Origene. The HA-K48 and HA-K63 Ubi plasmids were acquired from Henan Collaborative Innovation Center of Molecular Diagnosis and Laboratory Medicine. 15

| RNA extraction and qPCR analysis
RNeasy Plus Mini Kits were used to extract total RNA (Qiagen). Realtime PCR was performed as previously described. 16 36B4 was used for internal control. 17 The primer sequences were as follows: RACO-

| Quantification of cell viability
EC9706 cells were transfected with siRACO-1 or siControl in 12well plates. After transfection for 24 hours, cells were collected and counted. Five thousand cells per well were placed into 96-well plates. Cell proliferation was measured using the WST-1 as described previously. 18 Absorbance at 450 nm (OD450) was measured at the same time-point.

| Transwell assay
Cell migration test was performed by Transwell chamber as before.
EC9706 cells and NEC cells were both transfected with siRACO-1 or siControl, and were used in the migration experiment. Stimulating cell migration through starvation, medium containing 10% foetal bovine serum was added to the bottom of the chamber and serum-free medium was added to the chamber. Twelve hours later, migrating cells were immobilized, stained with 1% crystal violet and counted in three random fields with an optical microscope. and Anti-Actin (GB12001, Servicebio). Membranes were washed and then incubated in secondary antibodies peroxidase-conjugated AffiniPure Goat Anti-Mouse IgG or Goat Anti-Rabbit IgG.

| Western blotting
Enhanced chemiluminescence system was used to observe the cell membrane.

| Poly-ubiquitination detection assay
To directly detect the enriched K48-ubiquitinated and K63-

| Immunofluorescence assay
EC9706 cells were fixed with 4% paraformaldehyde for 10 minutes, 0.5% Triton perforated for 5 minutes, after that rinsed with PBS for three times and then sealed with 5% BSA at room temperature for 1.5 hours. Rabbit anti-RACO-1 polyclonal antibody (HAP030098, Sigma) and mouse anti-YAP monoclonal antibodies (SC-101199, Santa Cruz) were used to incubated and then combined with fluorescence secondary antibody (Invitrogen). As negative controls, the samples were incubated with the secondary antibodies without primary antibodies. After stained with DAPI, images were observed under laser scanning confocal microscopy (Nikon C2+/si+ Japan).

| Statistics
Student's t test and Pearson's correlation coefficient were used for comparisons. A P-value of < .05 was considered to be significant.

| RACO-1 depletion promoted invasion and migration in ESCC cells
The NEC and EC9706 cells were used to carry out most of the experiments to illustrate the role of RACO-1 in ESCC cells.

RACO-1 was depleted in NEC cells via two independent siRNAs
to avoid off-target effect. The knocking-down efficiency was shown in Figure 1A and B. In order to measure cancer cell migration capacity, transwell assay was carried out in EC9706 and NEC cells. The transwell assay showed that RACO-1 depletion significantly increased migration capacity in EC9706 and NEC cells ( Figure 1C-F). The wound-healing assay illustrated that RACO-1 depletion accelerated the wound-healing speed in both EC9706 and NEC cells ( Figure 1G-J). However, RACO-1 depletion inhibited ESCC cell proliferation, which is consistent with previous studies. 14

| RACO-1 inhibited YAP/TEAD axis of Hippo signalling in ESCC cells
Then, we analysed the role of RACO-1in Hippo signalling activity in ESCC cells. RACO-1 depletion could increase the protein level of YAP in EC9706 and NEC cells (Figure 2A,B). While transient overexpression ofRACO-1 reduced the protein level of YAP in EC9706 cells ( Figure 2C).
We further checked the classic target gene of YAP in ESCC cells. Our qPCR results indicated that RACO-1 depletion could enhance YAP target gene expression (CTGF and CYR61) in both EC9706 and NEC cells ( Figure 2D and E). Accordantly, RACO-1 overexpression decreased the expression of YAP target genes in EC9706 cells ( Figure 2F).

| RACO-1 inhibited ESCC cell migration and invasion through Hippo/YAP signalling
Previous data showed that RACO-1 inhibited YAP level and its target gene expression together with Hippo signalling-related phenotype.

| RACO-1 inhibited YAP stability in ESCC cells
The

| RACO-1 interacts with YAP and promoted YAP poly-ubiquitination
We performed more experiments to uncover the underlying mechanism between YAP and RACO-1.  Figure 5B). In order to detect whether YAP is degraded inside the nucleus. We used leptomycin B (LMB), a specific inhibitor of nuclear export, treated cells for 6 hours on the basis of the CHX assay, YAP was stabilized in the nucleus exposed to LMB, and the result showed that LMB treatment significantly decreased YAP half-life ( Figure S2A,B), and the ubiquitinated YAP was also improved when the transfected cells were treated with LMB ( Figure S2C). Thus, we further extracted the cytosol and nuclear for the ubiquitin-based immunoprecipitation assay, the experiment showed that RACO-1 ubiquitinated YAP in the nuclear ( Figure S1C). YAP-5SA is a mutation of the five phosphorylation sites of YAP plasmid. We tested YAP ubiquitination level in YAP-5SA condition, excluding the phosphorylation effect, and the result showed that RACO-1 inducing YAP ubiquitination is Hippo-independent ( Figure S2D). There are some studies illustrated that K48-linked ubiquitination promoted the degradation of YAP, whereas K63-linked ubiquitination enhanced YAP nuclear localization and trans-activation function. 19 We further analysed

| D ISCUSS I ON
Our study showed that RACO1 as an ubiquitin ligase promotes YAP K48-linked ubiquitination and proteasome-dependent degradation in ESCC cells ( Figure 6). Besides, RACO-1 inhibits ESCC cell migration and invasion via the suppression of Hippo/YAP signalling. Our study provides a novel mechanism between RACO-1 and Hippo signalling, which may be a hopeful marker for cancer diagnostics and therapeutics.

F I G U R E 3 RACO-1 inhibits cell migration and invasion through Hippo/YAP signalling in ESCC cells. A, The increased YAP protein level
by RACO-1 knocking down could be rescued by YAP depletion. EC9706 cells were transfected with siControl or siRACO-1. After 24 h, cells were transfected with siYAP or siControl. After 48 h, cells were harvested for Western blot analysis. RACO-1 and YAP levels were determined by Western blot. Actin was used as internal control. B, RACO-1 depletion increased Hippo target gene expression, which effect could be reversed by YAP knocking down. EC9706 cells were transfected with siControl or siRACO-1. After 24 h, cells were transfected with siYAP or siControl. After 48 h, total RNA was extracted for gene expression analysis. Each group was done in triplicates. *P < .05, **P < .01, ***P < .001 for target gene expression comparison. C and D, RACO-1 depletion increased ESCC cell migration capacity, which effect could be reversed by YAP knocking down. EC9706 cells were transfected with siControl or siRACO-1. After 24 h, cells were transfected with siYAP or siControl. After another 24 h, cancer cells were seeded into the chamber for transwell assay. The cell number was counted and Data are presented as ±SD. **P < .01, ***P < .001 (Student's t test). E and F, Wound-healing assay indicated that RACO-1 depletion increased ESCC cell migration capacity, which effect could be reversed by YAP knocking down. EC9706 cells were transfected with siControl or siRACO-1. After 24 h, cells were transfected with siYAP or siControl. Quantification of wound closure at the indicated time-points. Data are presented as ±SD. **P < .01, ***P < .001 (Student's t test) Previous studies identified that YAP/TEAD axis of Hippo signalling contributed to carcinogenesis of server human cancers. 11 For example, YAP gene amplification was found in liver cancer, triple-negative breast cancer and ESCC. 13,20 Besides, YAP/TAZ could trans-activate many transcriptional factors, including RUNX, TEAD and AP1 family members. 10 can promote the phosphorylation of YAP at multiple sites (S127, S381), thus promoting the binding of YAP to 14-3-3 proteins. 24 This action causes YAP to remain in the cytoplasm and leads to protein degradation.
In contrast, recent studies have shown that ubiquitin modification of YAP also plays an important role in Hippo signal regulation.
For example, the SCF β-TRCP complex can interact with the YAP to facilitate its proteasome-mediated degradation. 25 In addition, FBW7, as a RING E3 ubiquitin ligase, can also cause ubiquitination and degradation of YAP at the k48 site. 26

ACK N OWLED G EM ENTS
We thank all the members of Laboratory of Molecular Oncology in Xinxiang University for sharing valuable material and research support.

CO N FLI C T O F I NTE R E S T
The authors have no conflict of interest.

E TH I C A L A PPROVA L
No ethic issues were involved in the study.

DATA AVA I L A B I L I T Y S TAT E M E N T
Additional data and materials may be requested from the corresponding author on reasonable request.