ENDOCRINE SOCIETY
OXFORD
YAP1 Is a Prognostic Marker and Its Inhibition Reduces Tumor Progression in Adrenocortical Tumors
Candy C. B. More,1 Ana Carolina Bueno,1 Cesar A. O. Rojas,2 Mônica F. Stecchini,1 Fernando S. Ramalho,3 Silvia R. Brandalise,4 Izilda A. Cardinalli,4 José Andres Yunes, 4 Thais Junqueira,4 Carlos A. Scrideli,5 Margaret Castro,6 and Sonir R. R. Antonini10D
1Department of Pediatrics, Ribeirao Preto Medical School, University of Sao Paulo, Ribeirao Preto, SP 14049-900, Brazil
2Hematology Division, LIM31, Medical School, University of Sao Paulo, Ribeirao Preto, SP 01246-903, Brazil
3Department of Pathology, Ribeirao Preto Medical School, University of Sao Paulo, Ribeirao Preto, SP 14049-900, Brazil
4Boldrini Children’s Center, State University of Campinas, Campinas, SP 13083-210, Brazil
5 Department of Pediatrics - Pediatric Oncology Division, Ribeirao Preto Medical School, University of Sao Paulo, Ribeirao Preto, SP 14049-900, Brazil 6Department of Internal Medicine, Ribeirao Preto Medical School, University of Sao Paulo, Ribeirao Preto, SP 14049-900, Brazil
Correspondence: Sonir R. Antonini, MD, PHD, Departamento de Pediatria/FMRP-USP, University of Sao Paulo, Avenida Bandeirantes, 3900 - Monte Alegre, CEP14049-900, Ribeirao Preto, SP, Brazil. Email: antonini@fmrp.usp.br.
Abstract
Context: Adrenocortical cancer (ACC) is rare and aggressive, with YAP1 overexpression associated with poor outcomes in pediatric patients. Objective: In this study, we investigated the mechanisms by which YAP1 drives ACC progression and explored it as a potential target therapy. Methods: YAP1 expression and methylation in ACC were analyzed from pediatric and adult cohorts. The role of YAP1 on ACC progression was examined in vitro using an adrenocortical cell line. Also, was evaluated the YAP1’s influence on ß-catenin. The effect of YAP1 pharmacological inhibition was assessed on tumor growth in a murine xenograft model of ACC.
Results: High YAP1 expression was associated with lower survival in all cohorts. The YAP1 methylation signature was associated with patients’ prognosis. Inhibition of YAP1 reduced ACC cell viability through cell cycle arrest in the GO/G1 phase, and inhibited the epithelial-mesenchymal transition and cell invasion. YAP1 modulated ß-catenin protein levels and transcription activity, whereas ß-catenin partially mediated the effect of YAP1 on adrenocortical tumorigenesis. In vivo, verteporfin impaired tumor growth and Ki67 immunoreactivity in xenografts.
Conclusion: YAP1 is a potential novel prognostic marker in patients with ACC. Its deregulation contributes to adrenocortical tumorigenesis partially through crosstalk between Hippo/YAP1 and Wnt/B-catenin pathways. YAP1 inhibition is a new antitumor target.
Key Words: adrenocortical tumor, Hippo pathway, YAP1, B-catenin, epithelial-mesenchymal transition
Abbreviations: ACC, adrenocortical cancer; COG, Children’s Oncology Group; GSEA, gene set enrichment analysis; PBS, phosphate-buffered saline; VP, verteporfin.
Adrenocortical cancer (ACC) is rare and usually occurs in children under 5 years and in adults between 40 and 60 years of age (1, 2). Currently, tumor surgical removal remains the only curative treatment for these patients, being effective only in the early disease stages. Thus, advanced ACC is con- sidered a cancer with a poor prognosis. Different risk factors have been identified for ACC (3, 4), but the mechanisms in- volved in ACC pathogenesis are not fully understood. Therefore, it is necessary to identify new therapeutic targets for patients with advanced or relapsing disease.
Several genetic alterations have been found in ACC, espe- cially in TP53 and CTNNB1 (the ß-catenin encoding gene). Activating mutations in CTNNB1 exon 3 have been found in 15% to 36% and 6% of adult and pediatric ACCs, respect- ively (5, 6). These mutations induce loss of phosphorylation in a specific serine/threonine-rich domain, which activates ß-catenin, leading to its nuclear accumulation. ß-Catenin acti- vates the nuclear TCF/LEF transcription factors and induces
target gene expression such as CCND1, MYC, and AXIN2. ß-Catenin activation is associated with poor prognosis in adult and pediatric patients with ACC (5, 6). Besides, other al- terations in the Wnt/pathway have been reported in ACC, such as homozygous deletions in ZNRF3 and mutations in APC and ZNRF3 genes, which are also associated with B-catenin nuclear accumulation (3, 4, 7). However, in several ACCs without alterations in the Wnt pathway, activation of B-catenin was observed, suggesting that alterations in other pathways would induce this activation.
The Hippo pathway is conserved in mammals and regulates organ size and cell differentiation (8). Its deregulation has been associated with the development of different types of cancer and patients’ unfavorable prognosis (8-10). The main components of the Hippo pathway are the core kinases MST1/2 and LATS1/2 and their downstream effectors YAP1/TAZ. This pathway can be regulated by cell inter- action, mechanical stress, and other mechanisms. When the
Hippo pathway is activated, it induces a phosphorylation cas- cade of the core kinases, which phosphorylate YAP1 at the p.Ser127 residue, leading to its cytoplasmic retention and pro- teasomal degradation. In contrast, when the Hippo pathway is inactivated, YAP1 is translocated to the cell nucleus, where it interacts with the transcription factor TEAD, inducing the expression of target genes involved in cell proliferation and tumorigenesis, like CTGF and CYR61 (11).
YAP1 upregulation and nuclear overexpression were shown in different types of cancers (12, 13). In pediatric ACC, we demonstrated that YAP1 may be found in the cell nucleus, and its overexpression is associated with worse pa- tient survival (14).
In vitro assays highlighted the importance of YAP1 in the tumorigenesis process of several types of cancers (15-17). It is known that YAP1 regulates Wnt/B-catenin signaling by the action of Dishevelled (DVL) proteins (18). When DVLs are phosphorylated, they serve as crucial negative regu- lators of the GSK3b-destruction complex, resulting in the sta- bilization of ß-catenin (19). In the Hippo pathway, the YAP1- TEAD interaction is thought to be the most direct drug target to suppress YAP1-induced tumor growth, although other mechanisms of inhibition have been described. Verteporfin (VP) is an FDA-approved drug for macular degeneration treatment that was identified as an inhibitor of YAP1- TEAD interaction (20). VP has been indicated as a promising drug in various cancers (21). Whether and how YAP1 is in- volved in adrenocortical tumorigenesis remains poorly under- stood. However, it has been shown that YAP1 has a crucial role in adrenal development in male mice (22).
In this study, in order to evaluate the role of YAP1 and the effect of its inhibition on adrenocortical tumorigenesis, we as- sessed different cohorts of patients with ACC and used H295R ACC cells to identify some of the mechanisms that could be involved.
Materials and Methods
Patients and Samples
Primary pediatric ACC samples were obtained from patients followed in 2 university-based reference centers in Southeast Brazil: Ribeirao Preto Medical School-University of Sao Paulo (FMRP-USP) and Boldrini Children’s Cancer Center- State University of Campinas (BCC-SUC). Patient diagnostic evaluation and follow-up were performed as previously reported (23). Clinical and molecular characteristics of the patients were retrieved from our database and are available in supplemental data (Table S1 (24).) We excluded patients with synchronous bi- lateral tumors. A total of 50 samples were subjected to transcrip- tome and 57 samples to methylome evaluation.
The samples were obtained in accordance with the Declaration of Helsinki, and the study was approved by the Ethical Committee from FMRP-USP (#5856/2013) and BCC-SUC (#1.7-050809). The patients were included after written informed consent from their parents or legal guardians.
Transcriptome Analysis
Brazilian cohort (FMRP-USP/BCC-SUC cohort)
YAP1 expression levels were obtained from the transcriptome data from 50 pediatric adrenocortical tumors (ACTs) from the FMRP-USP/BCC-SUC cohort. Briefly, the tumors’ RNA was extracted using the QIAmp RNeasy Mini kit (Qiagen, RE,
Germany), following the protocol indicated by the manufac- turer. RNA samples were quantified in a Qubit fluorometer (Thermo Fisher Scientific, MA, USA); RNA quality was eval- uated by electrophoresis using the TapeStation 4200 (Agilent Technologies, CA, USA), and RNA integrity (RNA integrity number) values ≥7 were considered adequate.
Sequencing and data preanalysis were performed at the University of Southern California Keck Genomics Platform (USC-KCP, Los Angeles, CA, USA), and the detailed protocol is shown in supplemental material (24).
Public available data
We evaluated tumor gene expression data obtained from 2 other pediatric cohorts (both microarray data), as well as from a cohort of adult patients (RNA-seq data), whose data is publicly available, as follows:
1. Expression data collected by the International Pediatric Adrenocortical Tumor Registry (IPACTR) that in- cluded normal adrenals (n = 7), adrenocortical adenomas (n =5), and ACC (n=18) (GEO: GSE75415) (25), were used to compare the expression levels between pediatric normal adrenals, adrenocortical adenomas and ACC. Expression data are presented as fluorescence intensity.
2. Expression and event-free survival data from pediatric patients treated under the Children’s Oncology Group (COG) protocol, including ACC (n=34) (GEO: GSE76019) (26). Expression data are presented as fluor- escence intensity .
3. RNA-seq and clinical data from 79 ACC from adult pa- tients obtained from the TCGA project were retrieved from the Broad Institute Fire-Browse (http://firebrowse. org/) portal and the cBioPortal for Cancer Genomics Portal (https://www.cbioportal.org).
The illuminahiseq_rnaseqv2-RSEM_genes_normalized (MD5) data were log2-transformed.
Survival receiver operating characteristic curves and the Youden index were used to calculate optimal cutoffs to dichot- omize patients in “high” and “low” YAP1 expression groups for all cohorts. Additionally, quartile categorization was per- formed for the 3 cohorts (Figs. S1A, S1B, and S11C (24)).
Mutation and multivariate analysis
Mutation analysis was performed in the TCGA data from adult ACC using the cBioPortal platform (https://www. cbioportal.org/). Univariate and multivariate survival ana- lyses were carried out using the Cox regression proportional hazard using the Survival analysis R package.
Gene set enrichment analysis
Gene set enrichment analysis (GSEA) (RRID:SCR_003199) was performed using the data from TCGA, FMRP-USP/ BCC-SUC, and GSE76019 cohorts (26) using the GSEA 3.0 software (http:/software.broadinstitute.org/gsea/index.jsp) (27). Gene sets with a false discovery ratio <0.01 were consid- ered enriched. The enrichment was performed using Pearson correlation of YAP1 expression.
DNA methylation profiling
The YAP1 methylation profiling enrolled the samples (n = 57) from the FMRP-USP/BCC-SUC pediatric ACC DNA
methylation profiling study, which fully describes the DNA methylation evaluation (23). The methylation values from YAP1-associated probes (n = 17) were retrieved and subjected to unsupervised hierarchical clustering analysis considering Euclidean distance and the Ward method in R. We also eval- uated YAP1 methylation profile of pediatric samples from IPACTR (GSE131350) (28) and adult samples from TCGA.
In vitro Analysis
Cell lines
Human ACC cell line H295R (RRID:CVCL_0458) and mouse ACC cell line Y1 (RRID:CVCL_0585) were kindly provided by Professor Claudimara Lotfi (USP). HeLa human endocervical adenocarcinoma cells (RRID:CVCL_0030) were kindly pro- vided by Dr. Paulo Peitl Jr, PhD, and Dr. Beatriz Paixao, PhD (DNAapta). The H295R cell line was cultured in RPMI 1640 medium (GIBCO, Life Technologies, California, USA) supplemented with 2% fetal bovine serum (Sigma Aldrich, St. Louis, MO, USA), 1% penicillin-streptomycin (GIBCO Life Technologies), and 1% insulin-transferrin-selenium (BD Biosciences, New Jersey, USA). HeLa and Y1 cell lines were cul- tured in Dulbecco’s modified Eagle’s medium (Sigma Aldrich) supplemented with 10% fetal bovine serum and 1% penicillin- streptomycin. All cell lines were cultured in monolayer and main- tained under standard conditions at 37 ℃ with 5% CO2. The cell lines had been recently authenticated by short tandem repeat (STR) profiling and tested for mycoplasma contamination as previously described (29).
siRNA, pharmacological reagent, gene expression assays, and antibodies
siYAP1 (M-012200-00-0005), siCTNNB1 (L-003482-00-0005 5), or silencer negative control (siNonTargeting; D-001810- 10-20) (all ON-TARGETplus siRNA, Dharmacon GE) were used to transfect H295R cells using DharmaFECT 1 transfection reagent (T-2001-03, Dharmacon GE). VP was purchased from Sigma Aldrich (VP; SML0534) and was used always in darkness.
The following monoclonal antibodies were used: anti- goat antirabbit IgG-HRP (RRID:AB_631746; Santa Cruz Biotechnology), anti-m-IgGk BP-HRP (RRID:AB_2687626; Santa Cruz Biotechnology), anti-GAPDH (RRID:AB_ 627678; Santa Cruz Biotechnology), anti-Dvl3 (RRID: AB_627434; Santa Cruz Biotechnology), anti-twist (RRID: AB_1130910; Santa Cruz Biotechnology), antivimentin (RRID:AB_10917747; Santa Cruz Biotechnology), antisnail (RRID:AB_10709902; Santa Cruz Biotechnology), anti-ß-catenin (RRID:AB_397555; BD Biosciences), anti-E-cadherin (RRID: AB_397580; BD Biosciences), anti-N-cadherin (RRID:AB_ 10696943; abcam), anti-YAP1-phospho S127 (RRID: AB_1524578; Abcam), anti-YAP1 (RRID:AB_2219140; Abcam), anticyclin D1 (RRID:AB_2750906; Abcam), anti- fibrilarin (RRID:AB_2278087; Cell Signaling Technology), and anti-Ki67 (RRID:AB_2620142, Cell Signaling Technology). The TaqMan gene expression assays and antibodies used in this study are listed in Tables S2 and S3 (24), respectively.
Cell viability assay
To evaluate cell viability, the cells were plated at an initial dens- ity of 3 × 104 (H295R), 2x 104 (Y1), or 1 x 104 (HeLa) cells per well in 96-well plates and incubated for 48 hours. The cell cul- tures were treated with VP at final concentrations of 0.5, 1, 2.5,
5, 10, and 20 µM and further incubated for another 48 hours. MTS-based assay CellTiter 96 AQueous One-Solution- Reagent (Promega, Wisconsin, USA) was used according to the manufacturer’s instructions. At least 3 independent experi- ments were carried out in triplicate for each cell type.
Colony formation assay
H295R cells were transiently transfected for 24 hours or treated with VP for 48 hours and then resuspended and seeded for anchorage-dependent growth. To this, a cell culture me- dium containing 0.7% agarose (18300012, Gibco) was added into 6-well plates at 1.5 mL/well in the bottom layer. After the bottom layer solidification, another 1.5 mL of culture medium containing 0.4% agarose and 2 × 104 treated cells were added to the top. Then, 1 mL of culture medium was added at the top. The plates were kept in the cell culture incubator for 2 to 3 weeks, and the medium was changed every 4 days. The colonies were visualized, and pictures were taken under a Zeiss microscope. Three independent experiments were car- ried out in triplicate.
Invasion assay
Cell invasion was performed using BioCoat Matrigel Invasion chambers (354480, Corning, NY, USA) according to the man- ufacturer’s protocol. H295R cells were seeded at 3 x 105 cells/ insert. First, the cells were transfected for 24 hours or treated with VP (10 uM) for 48 hours. Then, the cells were suspended in 500 µL serum-free RPMI and placed into the upper com- partment of each chamber (8 um pores). The well was filled with 700 µL RPMI supplemented with 5% SR3 (S2640, Sigma Aldrich) as a chemo-attractant. The plates were then in- cubated at 37 °℃ for 72 hours. Inserts were then fixed and stained with violet crystal. Images were taken under a light microscope (Zeiss), and the cells in 5 different fields of each well were averaged at 100x. Three independent experiments were carried out in triplicate.
Cell cycle assay
H295R cells were harvested and transiently transfected for 72 hours. Then, the cells were suspended, and 2 x 105 cells/ treatment were washed twice in phosphate-buffered saline (PBS), fixed in ice-cold absolute ethanol, and kept at -20 ℃. The cells were treated with 200 uL of staining solution (propi- dium iodide, 3.4 mM Tris-Cl [pH 7.4], 50 µg/mL RNase A, and 0.2% IGEPAL buffer). Cell populations in different cycle phases were measured with the BD FACSCanto flow cytome- try system (BD Biosciences). Three independent experiments were carried out in triplicate.
Immunofluorescence
H295R cells (1x105) were seeded on coverslips in 24-well plates and treated with VP (10 µM) for 48 hours. After treat- ment, the cells were washed twice with PBS 1X, fixed with 4% paraformaldehyde, and blocked with bovine serum albumin (BSA) 2%. The cells were then incubated with the primary antibodies overnight in a wet camera at 4 ℃, followed by sec- ondary antibodies for 2 hours in a wet camera at room tem- perature in darkness. The cells’ nuclei were counterstained with 4’,6-diamidino-2-phenylindole (DAPi; #4083, Cell Signaling Technology). Immunofluorescence was visualized with a confocal microscope Zeiss Observer.z1 and evaluated
using the Icy software (GLPv3). Three independent experi- ments were carried out in triplicate.
RNA extraction and quantitative real-time PCR
Total RNA from H295R cells (1 x 105) transfected or treated with VP (10 uM) was isolated using the TRIzol Reagent (Invitrogen) according to the manufacturer’s protocol. RNA was quantified by spectrometry, measuring the absorbance at 260 nm (NanoDrop, ThermoScientific). The reverse tran- scription reaction was performed using the High-capacity cDNA Reverse Transcription Kit (AppliedBiosystems) and the MultiScribe enzyme following the manufacturer’s protocol.
Quantitative real-time PCR was performed on an ABI7500 Sequence Detection System (Applied Biosystems, CA, USA) using specific TaqMan gene expression assays. The ß-glucuronidase gene (GUSB) was used as an internal control gene, as previously described (14). Relative mRNA expression levels were deter- mined using the 2-44Ct (Ct: cycle threshold) method.
Western blotting
H295R cells (1 x 106 cells) treated were washed in PBS, lysed with CelLytic M Reagent (C2978, Sigma Aldrich) with prote- ase and phosphatase inhibitor cocktails (P8340 and P5726, Sigma Aldrich), following the manufacturer’s protocol, to ob- tain total protein. Nuclear and cytoplasmic protein fractions were extracted using the CelLytic NuCLEAR Extraction Kit (NXTRACT, Sigma Aldrich) with the aforementioned protease and phosphatase inhibitor cocktails, following the manufacturer’s protocol. Equal amounts (20 µg) of protein per treatment were separated by sodium dodecyl sulfate- polyacrylamide gel electrophoresis and transferred to nitrocel- lulose membranes. Immunodetection was conducted by incu- bating the membranes with primary antibodies overnight at 4 ℃, followed by HRP-conjugated secondary antibodies at room temperature for 1 hour, and developing the protein complexes with enhanced chemiluminescence (Immun-Star WesternC Chemiluminescence Kit) on a ChemiDoc XRS + System (Bio-Rad, Hercules, CA, USA). Acquired bands were analyzed using the Image Lab software (Bio-Rad) (RRID: SCR_014210).
Tumor xenograft and pharmacological treatment
The mouse experiments were performed according to pro- tocols approved by the FMRP-USP Ethics Committee on Animal Experimentation (#196/2017) and by the National Biosafety Technical Committee (CTNBio, #297/2017.016.01) following the ARRIVE (RRID:SCR_018719) guidelines. Mouse transplantation and xenograft development were performed according to the protocol previously described by our group (30). Briefly, a total of 2.5 x 106 H295R cells were suspended in 100 µL DPBS: Matrigel (1:1, 354262BD, Biosciences) and injected subcutaneously into both flanks of the 8- to 10-week-old female athymic NOD/SCID- gamma (NSG) mice (IMSR Cat# JAX_005557, RRID: IMSR_JAX:005557). The treatment began once the xeno- grafts were established (V = 100 mm3). Thus, the female mice were randomly assigned to VP (n = 6) or vehicle (DMSO, n = 6) groups. VP was given at 100 mg/kg per mouse weight every 2 days by intraperitoneal injections (100 µL) for 4 weeks. Mice were weighed weekly. The tumor volume (V) was deter- mined using the formula: V (mm3) = (D x d2)/2, where D is the largest and d is the smallest perpendicular diameters (mm),
measured with a caliper (Starrett). At the end of the treat- ment, the mice were anesthetized and sacrificed. The xenografts were excised, weighted, and formalin-fixed for immunohisto- chemical analysis.
Histological and immunostaining analysis
The excised xenografts were embedded in paraffin, transverse- ly sectioned into 5-um-thick sections and mounted on glass slides. For the histological analysis, the sections were stained with hematoxylin-eosin.
Immunohistochemical analysis was performed using a monoclonal antibody against human Ki67 (1:250, RRID: AB_2620142, Cell Signaling Technology). An experienced pathologist analyzed the histology and immunostaining of the tissue sections.
Statistical analysis
Continuous or discrete variables were represented as mean and/or median, and range or as a percentage, as described in fig- ures and table legends. Mann-Whitney, Kruskal-Wallis, Student’s t-test, ANOVA, Fisher’s exact test, chi-squared test, or Pearson’s correlation test were used as appropriate. Survival curves were analyzed according to the Kaplan-Meier method and were compared by means of the log-rank test. Univariate and multivariate analyses were performed using Cox proportional-hazards regression analysis. P-values were considered statistically significant at *P <. 05, ** P <. 01, and *** P <. 001. GraphPad Prism software (v.9.0.0, RRID:SCR_ 002798) or R 4.1.1 (The CRAN project, www.r-project.org) software were used for statistical analysis.
Results
YAP1 Expression Is a Marker of Unfavorable Prognosis for Pediatric and Adult Patients With ACC
In order to investigate if YAP1 is associated with ACC progression, we evaluated its gene expression from data in pediatric and adult patient cohorts. The IPACTR/GSE75415 pediatric cohort data analyses demonstrate similar YAP1 ex- pression in normal adrenals and ACC samples (Fig. 1A). The COG pediatric cohort data analyses demonstrated lower event-free survival in pediatric patients with high YAP1 ex- pression (Fig. 1B). In the FMRP-USP/BCC-SUC cohort, no as- sociation between clinical and prognostic characteristics of pediatric patients and YAP1 mRNA expression was observed (Table S4 (24)). However, we only observed deaths in the group of patients with high YAP1 expression (n = 5, 23%), and they were followed up for a shorter period (81 vs 39.6 months, P = . 004). Likewise, we observed that overall survival and progression-free survival were lower in patients with high YAP1 expression (Fig. 1C and 1D).
The analysis of TCGA data demonstrated that high YAP1 expression was also associated with poor prognosis in adult patients (Fig. 1E and 1F, respectively). The multivariate Cox regression analysis showed that YAP1 mRNA expression is an independent factor related to overall survival, where high YAP1 expression remained with a hazard ratio of 4 (P =. 002) (Fig. 1G). Once more, in the group of patients with high YAP1 expression were observed a greater number of deaths and a follow-up for a shorter period (Table S5 (24)). These results suggest high YAP1 expression as a marker of unfavorable prognosis in patients with ACC.
A
B
C
COG
500-
FMRP/USP-Boldrini
n.s.
YAP1 Signal Intensity
100%
100%
400-
n.s.
Event-free survival (%)
75%
Overall survival (%)
75%
300-
50%
50%
200-
100-
25%
YAP1 expression
25%
YAP1 expression
Low (n=19)
Log-rank
Low (n=28)
Log-rank
0%
High (n=15)
p = 0.0018
0
0%
High (n=22)
p = 0.0021
Normal (n=7)
ACA (n=5)
ACC (n=18)
0
20
40
60
80
0
50
100
150
200
Months
Months
D
E
F
FMRP/USP-Boldrini
TCGA
TCGA
100%
100%
YAP1 expression
100%
YAP1 expression
Disease-free survival (%)
75%
Overall survival (%)
Low (n=60)
Progression-free survival (%)
75%
High (n=19)
Low (n=60)
75%
High (n=18)
50%
50%
50%
25%
YAP1 expression
25%
25%
Low (n=28)
Log-rank
Log-rank
Log-rank
0%
High (n=22)
p = 0.0092
0%
p < 0.0001
0%
p = 0.0041
0
50
100
150
200
Months
0
50
100
150
0
50
100
150
Months
Months
G
| Variable | Univariate analysis | Multivariate analysis | ||||
|---|---|---|---|---|---|---|
| HR | CI 95% | p-value | HR | CI 95% | p-value | |
| YAP1 expression (High) | 4.22 | 1.87 - 9.52 | 0.0005 | 3.94 | 1.61 - 9.64 | 0.003 |
| CTNNB1 mutations (gain of function) | 3.07 | 1.28 -7.34 | 0.0117 | 1.41 | 0.4 - 4.9 | 0.598 |
| TP53 mutations (loss of function) | 4.71 | 2.04 - 10.9 | 0.0003 | 2.76 | 0.78 - 9.85 | 0.117 |
| Age (>50 years) | 1.97 | 0.908 - 4.27 | 0.086 | 2.61 | 1.013 - 6.70 | 0.047 |
| Sex (female) | 0.944 | 0.438 - 2.04 | 0.884 | 0.48 | 0.17 - 1.36 | 0.167 |
| Tumor stage | ||||||
| II | 2.38 | 0.29 - 19.4 | 0.418 | 2.36 | 0.26 - 21.21 | 0.443 |
| III | 8.7 | 1.05 - 72.3 | 0.045 | 6.56 | 0.67 - 64.44 | 0.107 |
| IV | 20.2 | 2.47 - 165 | 0.005 | 10.03 | 0.96 - 104.23 | 0.054 |
| Autonomous cortisol secretion | 2.1 | 0.96 - 4.58 | 0.062 | 2.70 | 0.90 - 8.12 | 0.076 |
Figure 1. Survival outcome associated with YAP1 expression in pediatric and adult patients with adrenocortical tumors (ACTs). (A) YAP1 gene expression in normal adrenals (n = 7), adrenocortical adenomas (AAC, n = 5) and adrenocortical carcinomas (CACs, n = 18) of pediatric patients from the International Pediatric Adrenocortical Tumors Registry (IPACTR), obtained from public microarray data available in the Gene Expression Omnibus database (GEO, accession number GSE75415). (B) Kaplan-Meier survival curve considering YAP1 expression and event-free survival in pediatric patients with adrenocortical tumors followed by the Children’s Oncology Group (COG). Public microarray data is available in the GEO database (Accession number GSE76019). (C, D) Kaplan-Meier survival curves considering YAP1 expression and overall survival and progression-free survival in pediatric patients with adrenocortical tumors from the FMRP-USP/BCC (n = 50 and n = 40, respectively. (E, F) Kaplan-Meier survival curves considering YAP1 expression and total survival and disease-free survival in adult patients with carcinoma adrenocortical from the TCGA database. (G) Univariate and multivariate analysis considering clinical data and YAP1 expression of adult patients with ACC. Multivariate analysis was performed using Cox regression to identify predictive prognostic factors for overall survival in adult patients with adrenocortical carcinoma (ACC) based on the data available from TCGA. HR, hazard ratio. YAP1 oncogene expression (“High expression” vs “Low expression”), age in years (greater than 50 years), tumor staging (ENSAT), sex (women), and ACT autonomous cortisol secretion.
Next, we evaluated which pathways were enriched in the ACC samples from these cohorts. GSEA demonstrated that high YAP1 expression positively enriched of several pathways involved in the progression of tumorigenesis, such as path- ways related to cell proliferation, cell cycle progression, tumor invasion, and epithelial-mesenchymal transition (EMT) (Fig. 2A; Table S6 (24)). Additionally, the high YAP1 expres- sion was positively correlated with Wnt/ß-catenin signaling, both in pediatric and adult samples (Fig. 2A), suggesting an as- sociation between Wnt/B-catenin activation and tumorigenic processes with high YAP1 expression in ACC.
Genetic Variations in Hippo and Wnt/B-Catenin Pathways Associated With High YAP1 Expression in ACC
To identify which variations could be associated with the YAP1 expression in ACTs, we compared the TCGA variation data from adult ACC samples in the high vs the low expression groups. At least 1 mutation was found in 10 patients from the high and 17 from the low groups (Fig. S2A (24)). We found a tendency toward a higher frequency of CTNNB1 mutations in the high YAP1 expression group (Fig. 2B), and a significant Spearman correlation was found between the expression of YAP1 and CTNNB1 in this dataset (Fig. S2B (24)).
Since methylation regulates gene expression in several tu- mors, we evaluated if a specific methylation profile could regu- late YAP1 expression in patients with ACC. Unsupervised hierarchical clustering analysis of YAP1-annotated methyla- tion values from the FMRP-USP/BCC-SUC, TCGA, and IPACTR cohort revealed 2 groups of tumors, namely YAP1-1 and YAP1-2 (Table S7 and Figs. S3A, S4A, and S54A (24)). No differences were shown in median methylation values, and the 2 groups of methylation signatures displayed similar YAP1 mRNA levels in the 3 cohorts (Figs. S3B-C, S4B, and S5B-C (24)).
In Vitro Study
YAP1 inhibition reduces ACC cell viability through Hippo pathway activation and cell cycle arrest in G0/G1 phase
To investigate whether YAP1 activity impacts ACC prolifer- ation, we evaluated the viability of H295R cells after treat- ment. We found that VP reduced H295R cell viability in a time- and concentration-dependent manner (Fig. 3A). Since VP’s IC50 after 48 hours of treatment was 9.7 µM in dark- ness, we fixed the 10 uM concentration in all upcoming ex- periments. We next evaluated whether VP action on cell viability was specific to adrenal cells with ß-catenin activation (H295 cells) and compared VP’s effect in H295R with those observed in HeLa (cervix tumor) and Y1 cells (mouse ACC, without ß-catenin activation) (Fig. 3B), and, differently from H295 cells, we found no effect in HeLa or Y1 cells’ viability. These results suggest YAP1’s influence in tumor proliferation occurs in ACC cells with Wnt/B-catenin activation.
Due to the reduction in H295R cell viability after YAP1 inhibition by VP and YAP1 knockdown by siRNA, as pre- viously demonstrated by our group (14), we investigated the effects of YAP1 inhibition in the cells’ cycle progres- sion. Knocking down YAP1 induced cell cycle arrest at the G0/G1 phase (Fig. 3C) through inhibiting cyclin D1 (Fig. 3D and 3E). In line with this effect, VP treatment re- duced cyclin D1 and CDK2 gene expression in H295R cells
(Fig. 3F and 3G). These data indicate that YAP1 inhibition blocks the transition between G0/G1 and S/G2 phases by regulating cyclin D1.
Additionally, YAP1 inhibition by VP impacted the Hippo pathway’s signaling in H295R cells, resulting in upregulation of its core kinases LATS1, STK3 (encoding MST2), and STK4 (encoding MST1) (Fig. 3H), and in downregulation of TEAD4 and YAP1’s target gene CCN2 (also known as CTGF) (Fig. 3H). Interestingly, YAP1 gene expression was not altered, but YAP1 and pYAP1 (Ser127) protein abundan- cies were reduced (Fig. 3J). In addition, YAP1 was preferably located in the cytoplasm after VP treatment (Fig. 3I). These findings indicate that, upon VP’s influence, the activation of the Hippo pathway in ACC cells results in YAP1 translocation to the cytoplasm.
Crosstalk between YAP and Wnt/B-catenin pathway in ACC cells
Interaction between the Wnt/B-catenin pathway and high YAP1 levels in ACC was demonstrated by our GSEA data (Fig. 2A). In this context, we evaluated the impact of YAP1 in- hibition on Wnt/B-catenin signaling. We found considerably lower ß-catenin levels after VP treatment, mainly at the nu- clear level (Fig. 4A-4D). Meanwhile, YAP1 knockdown in- creased CTNNB1 expression but did not alter ß-catenin abundance (Fig. 4E and 4F). Considering some bona fide B-catenin target genes, YAP1 knockdown impaired CCND1 expression (Fig. 4E) and abundance-previously showed in Fig. 3E-but not MYC mRNA expression. Additionally, YAP1 inhibition reduced the abundance of DVL3, but no ef- fects were found on its gene expression (Fig. 4G and 4H).
Furthermore, once we evaluated the effects of CTNNB1 knockdown in H295R cells, we observed no differences in the expression of Hippo pathway core kinases nor YAP1 ex- pression and abundance (Fig. 5I and 5J). In contrast, CCN2/ CTGF expression was upregulated (Fig. 5A). These results support the interaction between YAP1 and ß-catenin and show a regulatory role of YAP1 on ß-catenin transcription ac- tivity in ACC.
YAP1 promotes EMT in ACC cells
To gain insight into the processes involved in the poor progno- sis of patients with ACC allocated in the high YAP1 expression group and its enrichment on the tumorigenesis hallmark EMT, as we showed by GSEA (Fig. 2A), we evaluate whether YAP1 inhibition affects EMT. Inhibition of YAP1 in H295R cells showed reduced mesenchymal markers N-cadherin and vimen- tin and their transcription factors SNAIL and TWIST (Fig. 5A and 5B), indicating the involvement of YAP1 in the EMT pro- cess and the potential effect of VP treatment on EMT reversal in ACC cells.
Inhibition of YAP reduces invasiveness and transformation in ACC cells.
Considering that the inhibition of YAP1 is involved in the rever- sal of EMT, we evaluated its effect on ACC cell invasion. VP treatment resulted in a decreased percentage of cells with inva- sive capacity (100 ±28.3 vs 14.34±6.8; P <. 05; Fig. 5C), and the YAP1 knockdown showed a tendency to the same effect (103.1 ±28.53 vs 40±7; P =. 09; Fig. 5D). Additionally, we evaluated the cell transformation by the ability of cells to grow independently of anchorage. YAP1 knockdown and VP
A
Brazil
COG
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GOMF: Single stranded dna helicase activity
REAC TOME: Activation of the pre replicative complex
REAC TOME: Cell extracellular matrix interactions
HALLMARK: E2F targets
HALLMARK: G2M checkpoint
FDR q-value
HALLMARK: Mitotic spindle
REACTOME: Integrin cell surface interactions
0.03
REAC TOME: Formation of the beta catenin TCF transactivating complex
0.02
GOBP: Negative regulation of cell substrate adhesion
REACTOME: Degradation of the extracellular matrix
0.01
GOBP: DNA dependent DNA replication
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GOBP: Regulation of transcription by RNA polymerase I
REACTOME: ECM proteoglycans
Count
HALLMARK: Epithelial mesenchymal transition
40
REAC TOME: Collagen degradation
80
REAC TOME: Cell junction organization
120
HALLMARK: Hypoxia
KEGG: ECM receptor interaction
REAC TOME: Extracellular matrix organization
GOBP: Branching morphogenesis of an epithelial tube
0.4
0.6
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Gene ratio
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Mutation frequency (%)
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(B) Low
15
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CTNNB1
FAT1
FAT3
FAT4
TAOK1
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PAK3
TP53
| Genes | High expression (n=19) | Low expression (n=60) | p- Total (n=79) valu e | ||||
|---|---|---|---|---|---|---|---|
| n | % | n | % | n | % | ||
| CTNNB1 | 6 | 31.58 | 7 | 11.67 | 13 | 16.46 | 0.071 |
| FAT1 | 1 | 5.26 | 2 | 3.33 | 3 | 3.79 | 0.567 |
| FAT3 | 2 | 10.53 | 0 | 0 | 2 | 2.53 | 0.056 |
| FAT4 | 3 | 15.79 | 3 | 5.0 | 6 | 7.59 | 0.147 |
| TAOK1 | 1 | 5.26 | 0 | 0 | 1 | 1.27 | 0.241 |
| MAPK1 | 1 | 5.26 | 0 | 0 | 1 | 1.27 | 0.241 |
| PAK3 | 3 | 15.79 | 0 | 0 | 3 | 3.79 | 0.017 |
| TP53 | 5 | 26.32 | 11 | 18.33 | 16 | 20.25 | 0.516 |
Figure 2. GSEA analysis of the 3 ACT cohorts according to YAP1 expression. (A) Representative pathway enrichment analysis results in the 3 cohorts evaluated. The dot plots were built considering the pathways with the highest gene ratios according to the order in the gene ratio. Count represents the number of genes in the list of the same pathway. In the legend, the darkest red is related to the lowest q value of false discovery ratio. (B) Number of samples (%) with mutations in genes related to the Hippo pathway and in the CTNNB1 and TP53 genes in the YAP1 “high expression” and “low expression” groups using adult patient data from the TCGA-ACC. The image was obtained using the cBioPortal for Cancer Genomics portal.
A
B
140-
120-
24h
140
Cell viability (%)
100-
48h
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H295
72h
Cell viability (%)
T
100-
T
T
HeLa
80-
80-
*
Y1
60-
60-
40-
40-
20-
IC50 - 48h = 9.721 µM
20-
0
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0
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48h
72h
20
Verteporfin (LM)
Hours after verteporfin treatment (10 μ.Μ)
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E
Relative mRNA expression (2-4ACt)
2.5-
*
DMSO
YAP1
YAP1 + DAPI
2.0-
VP 10 UM
1.5-
*
*
n.s
T
DMSO
1.0-
I
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0.0
YAP1
LATS2
STK4
STK3
TEAD4
CTGF
5
Relative quantification (GAPDH)
1.5-
D
DMSO
VP
DMSO
75 kDa
pYAP
1.0
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T
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VP 10UM
49 kDa
YAP
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0.5
37 kDa
GAPDH
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T
0.0
YAP1
pYAP1
F
G
H
100
W
GO-G1
1.5-
Cell population (%)
80
**
S
CCND1 expression
G2-M
60
4Act)
1.0-
**
SiNT siYAP1
40
~
0.5-
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34 kDa
20
GAPDH
37 kDa
0
SİNT
SİYAP1
0.0
SINT SİYAP1
I
J
mRNA Gene expression (2-4ACt)
1.5-
DMSO
VP 10 µM
n.s.
1.0-
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DMSO VP
T
Cyclin D1
34 kDa
0.5-
GAPDH
37 kDa
0.0
CCND1
CCNE1
CDK2
A
Total Beta-catenin
D
B-Catenin + DAPI
Fluorescence intensity (a.u)
15000-
CTNNB1 Gene expression
1.5-
*
10000
DMSO
(2-44Ct)
1.0
5000
0.5-
0.0
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DMSO
DMSO
VP
VP 10UM
B
1.5
VP 10uM
Beta-catenin Relative quantification (Beta-catenin/GAPDH)
DMSO
VP
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90 kDa
B-catenin
37 kDa
GAPDH
0.5-
0.0
DMSO
VP
Beta-catenin Relative quantification
1.5-
C
Nucleus
Cytoplasm
Fold Intesity
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*
DMSO
VP
DMSO
VP
Beta-catenin
Beta-catenin
0.5
Fibrillarin
GAPDH
0.0
DMSO
VP
DMSO
VP
Nucleus
Cytoplasm
E
F
Gene mRNA expression (2-44Ct)
1.5-
SİNT
Beta-catenin Relative quantification (Beta-catenin/GAPDH)
**
SİYAP1
T
SiNT
1.5
siYAP1
YAP1
49 kDa
1.0-
T
T
I
T
1.0-
n.s.
**
T
pYAP1
75 kDa
0.5
Beta-catenin
90 kDa
0.5-
0.0
YAP1
CTNNB1
CCND1
MYC
DVL3
GAPDH
37 kDa
0.0
SINT
SİYAP1
G
H
3-
SINT
siYAP1
DMSO
VP
Gene mRNA expression
T
SİNT
90 kDa
DVL3
90 kDa
DVL3
siCTNNB1
2
(2-4ACt)
37 kDa
GAPDH
37 kDa
GAPDH
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I
=
T
I
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DVL3 Relative quantification (DVL3/GAPDH)
1.5
DVL3 Relative quantification (DVL3/GAPDH)
1.5
1.0
1.0
0
YAP1
CTNNB1
CTGF
CCND1
LATS2
STK3
STK4
TEAD4
MYC
CYR61
0.5
0.5
J
*
SINT
SiCTNNB1
75 kDa
pYAP1
0.0
SiNT
SİYAP1
0.0
DMSO
VP
49 kDa
YAP1
90 kDa
Beta-catenin
37 kDa
GAPDH
A
B
C
SİNT
SİYAP1
DMSO
VP
140-
N-cadherin
Invasive cells (%)
120
N-cadherin
100
Snail
80
Vimentin
DMSO
100X
60
Vimentin
40
Twist
20
Twist
0
DMSO
VP
GAPDH
GAPDH
VP 10uM
100X
D
E
Relative number of colony (%)
120-
100-
80-
140-
60-
Invasive cells (%)
120-
40-
100
20-
SINT
100x
80-
GO
DMSO
100X
VP 10UM
100X
0
DMSO
VP
40
Relative number of colony (%)
20
100-
0
SINT
siYAP1
80-
60-
SİYAP1
100X
40-
20-
SINT
100X
Sİ YAP1
100X
F
0
H
SINT
SIYAP1
1000-
400
Tumor volume (mm3)
G
DMSO
VP
800-
Tumor volume (mm3)
300-
Control
600
200
VP
400-
100
2
3
2
13
200
r=0.9146
p < 0.0001
0
0-
0
N
4
7
9
11
14
16
19
21
23
25
28
Days after treatment
0.0
0.2
0.4
0.6
0.8
1.0
Tumor weight (g)
J
DMSO
VP
110-
Overall survival (%)
100
20-
90-
Control
Positive cell Ki67 (%)
80-
VP
15
*
70-
Logrank p=0.0302
10
HR (Control) = 9.18
60-
n (Control) = 10
n (VP) = 10
5
50
0
5
10
15
20
25
30
Days after starting treatment
0
Control
VP
400X
400X
treatment inhibited the transformation capacity of H295R ACC (Fig. 5E), highlighting the relevant role of YAP1 in the cell transformation process and that ACC cell invasiveness po- tential may be through regulation of EMT process.
In vivo Study
VP reduces tumor proliferation in a murine ACC xenograft model
To evaluate whether VP treatment could reduce tumor growth in vivo, we use a xenograft model using the H295R ACC cell line. The tumors reached a volume of 100 mm3 after 7 weeks of the transplant, and then the treatment began. We observed that VP restricted tumor growth from the third week of treat- ment, and at the end of 4 weeks of treatment, the VP group showed a reduction of approximately 50% in the tumors’ volume (control: 356 ±69 vs VP: 184±59 mm3; P <. 001) (Fig. 5F and 5G). Notably, there was a positive correlation be- tween tumor weight and volume (Fig. 5H), without systemic effects when comparing VP-treated and untreated animals (Fig. S6A (24)). Besides, VP-treated animals had increased overall survival (Fig. 5I).
Histological analysis showed similar tumor characteristics in both groups, including highly proliferative areas, as demon- strated by frequent atypical mitoses and few apoptotic bodies (Fig. S6B (24)). The Wieneke index adapted was performed, where only 4 items were analyzed: mitosis rate, presence of atypical mitoses, and presence of necrosis and sinusoidal inva- sion. In most cases, the index was a maximum of 4 (60% in control and 33.3% in VP-treated tumors). The higher index was observed because the tumors generally exhibit anaplasia, with high rates of mitosis, atypical mitoses, and, consequent- ly, necrosis. However, in the case of tumors treated with VP, the frequency of the index between 0 and 3 was higher than the control group (33.3% vs 10%, respectively. Additionally, the proliferation marker Ki67 was significantly reduced on VP-treated tumors compared with the controls (Fig. 5J). Together, these results indicate that VP treatment can reduce ACC tumor growth.
Discussion
The tumorigenesis process in ACC still needs to be understood, particularly concerning ß-catenin activation. Recently, the oncogene YAP1 has been identified as a critical factor in tumorigenesis in several types of cancer, with high expression and nuclear localization associated with unfavorable prognosis (12, 13). In this study, the high expression of YAP1 was associ- ated with lower survival in pediatric and adult cohorts of ACC (14). Also, we showed that YAP1 may serve as an independent marker of poor prognosis in adult patients. While pediatric and adult ACCs have some distinct characteristics (31, 32), our data suggest similarities in YAP1 expression and its ability to classify tumors with unfavorable prognosis.
Inhibition of YAP1 activity has been shown to decrease cell growth in some types of cancers (20, 21). YAP1 inhibition by YAP1 knockdown (14) or VP treatment, as shown in this study, reduced the viability of ACC cells with active ß-catenin, marking the first report of VP-mediated growth inhibition in ACC cells. However, this effect was not observed in the Y1 adrenal tumor or HeLa cell lines, likely due to the absence of the p.S45P mutation in the CTNNB1 gene, which leads to constitutive acti- vation of ß-catenin in H295R cells (33). Supporting this (34),
demonstrated that cells with an active Wnt/B-catenin pathway require a YAP1-TBX5 complex for transformation and survival, underscoring the role of Hippo/YAP and Wnt/ß-catenin path- way interactions in tumorigenesis. In our study, VP-induced cell cycle arrest at the GO/G1 phase was accompanied by reduced cyclin D1 expression, a direct target of ß-catenin. Additionally, VP treatment increased the expression of key Hippo pathway kinases, suggesting Hippo pathway activation and subsequent YAP1 degradation. Similar findings were reported by (35) in endometrial cancer cells, where VP increased NF2 and LATS1 expression. These kinases (MST1-MST1 and LATS1-LATS2) phosphorylate YAP1, inducing cytoplasmic retention and deg- radation, thus blocking its transcriptional activity (36). Besides, it is known that these kinases are involved in the control of the cell cycle. In a study using the NIH3T3 cells, ectopic LATS2 suppressed the tumor’s development in mice by inhib- ition of the G1-S transition (36). Likewise, the simultaneous de- letion of MST1/2 in the liver results in increased polyploidy, elevated p53 levels and liver enlargement (37). So, these results showed the potential effects on upstream Hippo mediators. Further studies are needed to confirm these findings.
We observed that YAP1 knockdown did not affect B-catenin abundance in ACC cells, likely due to a CTNNB1 mutation that causes an amino acid substitution at Ser45, al- lowing ß-catenin to escape cytoplasmic degradation requiring YAP1 (38). Consistent with our findings, other studies re- ported that YAP1 knockdown or knockout does not affect B-catenin stability or levels (18, 34). However, YAP1 silencing reduced ß-catenin transcriptional activity. In colon cancer cells, a nuclear YAP/B-catenin/TCF4 complex induces the transcription of target genes like CCND1 and LGR5 (39). In contrast, YAP1 inhibition in basal breast cancer cells de- creases ß-catenin target gene expression and shows the inter- action between YAP/TEAD and ß-catenin enhancers or promoters (40). Interestingly, reducing YAP1 activity with VP led to ß-catenin transcriptional inactivation and degrad- ation, possibly via ubiquitination at an alternative phosphor- ylation site.
Interestingly, CTNNB1 knockdown increased transcrip- tional activation of YAP1 but not YAP1 abundance in ACC cells. In hepatoblastoma cell lines harboring deletions of the exon 3 of CTNNB1, YAP1 knockdown only downregu- lated some of ß-catenin’s target genes, such as CCND1 and MYC, but not AXIN2 or DKK1 (41). On the other hand, CTNNB1 knockdown downregulated the expression of a YAP1 target, Cyr61, but did not affect CTGF/CCN2 expres- sion (41). It has been speculated that YAP1 and ß-catenin bind to activate the transcription of shared genes in the nucleus but are negatively associated in the cytoplasm (38-40, 42). Thus, regulation of ß-catenin target gene expression by YAP1 is context- and tissue specific. Hence, our results indicate cross- talk between YAP1 and the Wnt/B-catenin pathway in ACC cells with active ß-catenin. However, further studies are needed to reveal the mechanisms by which the direct inter- action between YAP1 and ß-catenin regulates their activity.
DVL acts as a central scaffold protein that transduces sig- nals from Wnt receptors to downstream effectors, including B-catenin. Upon activation of the Wnt pathway, DVL inhibits GSK3ß-mediated phosphorylation of ß-catenin, preventing its ubiquitination and subsequent degradation via the prote- asome. This process leads to the stabilization and nuclear translocation of ß-catenin (18, 43). Our findings show that both YAP1 knockdown and VP treatment led to a significant
decrease in DVL3 protein levels, suggesting that DVL3 expres- sion is tightly regulated by YAP1-mediated ß-catenin transcrip- tional activation. These results underscore the importance of the YAP1-B-catenin axis in controlling Wnt pathway compo- nents like DVL3 and highlight a potential mechanism through which VP could modulate ß-catenin signaling independently of direct YAP1 transcriptional inhibition.
Our study demonstrated that increased expression of YAP1 is associated with worse prognosis, metastasis/recurrence, and deaths in pediatric and adult patients with ACC. In these tu- mors, higher YAP1 expression was significantly associated with critical oncogenic processes, such as collagen degrad- ation, EMT, downregulation of cell adhesion, and cell inva- sion. These processes are critical for cancer progression and metastatic colonization. Thus, our data suggest that YAP1 ex- pression can be a molecular prognostic marker in adult ACC and in pACT. However, markers of prognostic significance should ideally be, in addition to molecular data, also assessed using immunohistochemistry, as it has been the gold standard in clinical practice. The present study did not assess YAP1 tis- sue expression by immunohistochemistry. Thus, future work in ours and additional cohorts will be important to confirm this data. However, using immunohistochemistry, we previ- ously showed consistent YAP1 cytoplasmic and nuclear stain- ing in YAP1 positive pACT (14). In addition, it will be interesting to compare YAP1 expression and outcome associ- ation between low- and high-grade ACCs in future work ac- cording to the updated WHO classification.
In vitro, we also found that inhibiting YAP1 activity de- creased cell transformation in ACC cells, consistent with find- ings in other cancer types (34). Specifically, in ACC, little is known about the process of metastasis and EMT. A previous study showed that mesenchymal markers and their transcrip- tional factors are expressed in both normal adrenal and ACC but were highly expressed in ACCs (44). Our study found in- creased vimentin abundance in ACC cells, which was reduced by YAP1 knockdown and VP treatment. Additionally, we ob- served a reduction of N-cadherin, TWIST, and SNAIL levels after YAP1 inhibition. Thus, our findings suggest that YAP1 is involved in acquiring mesenchymal characteristics in ACC cells, making it a potential therapeutic target for more aggres- sive ACC with metastatic features.
Currently, there are no approved targeted therapies for pa- tients with ACC. Therefore, identifying new targets and therap- ies for patients with advanced or recurrent disease is essential (45). Our murine xenograft model of ACC showed that treat- ment with VP significantly reduced tumor growth by impairing cell proliferation, which has been observed in other types of tumors (20, 21, 46-48), supporting YAP1 as a potential thera- peutic target for patients with ACC. Additionally, further stud- ies will be necessary to evaluate the combination of inhibitors to YAP1 and ß-catenin since there seems to be a cooperation be- tween these 2 proteins in ACC. A study in hepatoblastoma showed that combined treatment using VP with PNU-74654 (an inhibitor of ß-catenin/TCF) led to a substantial decrease in proliferation and increased apoptosis when compared with each treatment alone (41).
In summary, our study highlights the role of YAP1 as a new potential molecular prognostic marker for patients with ACC. We also shed light on how YAP1 activation contributes to adrenocortical tumorigenesis and suggest that YAP1 is a po- tential therapeutic target for developing new compounds to treat patients with ACC.
Acknowledgments
We thank Cleide Silva, PhD, for her technical support to our xenograft animals in the bacterium. Also, we thank Juliana Vargas, PhD, and Rui Milton, PhD, for their support with the immunofluorescence assay and confocal microscope.
Funding
This work was funded by the Sao Paulo Research Foundation (FAPESP) grants 14/03989-6 (M.C. and S.R.A.), 15/19663-5 (S.R.A.), 17/17737-7 (C.C.B.M.), and 19/00860-6 (A.C.B.), and by CAPES, CNPq, and FAEPA-HC-FMRP-USP.
Author Contributions
Study concept and design: C.B.M., A.C.B., and S.R.R.A .; data acquisition and analysis: C.B.M., C.O.R., M.F.S., F.S.R., and A.C.B .; bioinformatics analysis: C.B.M. and C.O.R .; resour- ces: M.F.S., F.S.R., S.R.B., I.A.C., J.A.Y., T.J., and M.C .; writ- ing of manuscript: C.B.M., A.C.B., C.O.R., and S.R.R.A .; critical review of manuscript: all authors; supervision: A.C.B. and S.R.R.A.
Disclosures
The authors declare no conflicts of interest.
Data Availability
The complete methylation data are publicly available from the NCBI’s GEO under the accession number GSE179175.
Statement of Significance
YAP1 is a novel prognostic marker in patients with ACC. Its deregulation contributes to adrenocortical tumorigenesis par- tially through crosstalk between Hippo/YAP1 and Wnt/ B-catenin pathways. YAP1 inhibition is a new antitumor target.
References
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