Background
Prostate cancer (PCa) is one of the most common causes of cancer death in the world with the highest incidence rate and the second highest mortality rate among the male population in the United States in 2018 [
1].
The Hippo signalling pathway plays an important role in the development of prostate cancer [
2‐
5]. The Hippo signalling pathway contains 13 core proteins including MST1 / 2, SAV1, LATS1 / 2, MOB1A, MOB1B, YAP1, TAZ and TEAD1–4 [
6]. YAP1 acts as a downstream transcriptional coactivator of the Hippo pathway. Its abnormal expression causes malignant proliferation and metastasis, induces epithelial-mesenchymal transition, and produces possible cancer drug resistance [
7‐
9]. In addition, since YAP1 is active in cancer cells, it can regulate a variety of cancer genes or form complexes with them and then jointly regulate the downstream target genes.
The impact of the tumour microenvironment (TME) on cancer has recently drawn much attention [
10,
11]. As one of the key components of the tumour microenvironment, cancer-associated fibroblasts (CAFs) play a significant role on tumour progression and metastasis [
12,
13]. The activation of normal fibroblasts into CAFs can result in the secretion of abundant tumour-promoting factors and facilitate the malignant behaviour of tumour cells through a complicated paracrine signal network. For example, CAFs rely on the activation of HIF-1 to secrete carbonic anhydrase (CAIX), which raises the acidity of extracellular matrix; CAFs secrete MMP-2/9 [
14], therefore inducing the epithelial-mesenchymal transition (EMT) in tumour cells and enhancing the migration ability of tumour cells [
15]. We aimed to discover the regulatory function of YAP1 in PCa related fibroblasts cells as well as the association between YAP1 and the conversion from NF to CAF in PCa.
This study found that in prostate cancer stromal cells, YAP1, FAP and α-SMA expression levels were significantly elevated compared to those of normal cells. We further conclude that due to the high expression of YAP1, normal fibroblasts are activated into CAFs. During this process, SRC is regulated by the YAP1/TEAD1 complex, which leads to the activation of downstream actins and cytoskeletal proteins. After conversion, CAFs significantly enhance the proliferation and invasion of tumour epithelial cells. We believe that, as one of the indicators of the biological behaviour of malignant tumours, YAP1 is increased in the stroma of the PCa and there could be a certain reference value for the diagnosis of cancer. This may indicate that the disease progresses to an advanced stage or even metastasizes. As a result, detecting the expression level of YAP1 in prostate cancer stromal cells may be an early indicator for the disease prognosis. YAP1 may be used as a potential target for new targeted cancer therapy.
Materials and methods
Ethical approval for the study protocol
This study was approved by the Ethics Committee of the Second Hospital of Tianjin Medical University, Tianjin, China. (No. KY2019K077 and No. YN2019Y70). Written informed consent was obtained from all patients, and the study was conducted in accordance with the Declaration of Helsinki.
Human samples
The prostate tissue specimens that were used in this study were surgical specimens from PCa patients with complete clinicopathological data. Benign prostatic hyperplasia tissues were acquired by transurethral resections of the prostate (n = 12), and prostate cancer tissue specimens (n = 25) were acquired by radical prostatectomy. These samples were paraffin-embedded and subjected to IHC and IF assays.
Antibody
The following antibodies were used in this study for western blot, immunohistochemistry, immunofluorescence staining and immunoprecipitation: YAP1 (Santa Cruz Biotechnology, sc-376,830, 1:100 dilution for western blot; 1:50 dilution for immunohistochemistry and 1:50 dilution for Immunofluorescence staining; Abcam, ab52771, 1:20 dilution for IP), p-YAP1 (Abcam, ab76252, 1:10000 dilution for western blot), α-SMA (Abcam, ab5694, 1:200 dilution for western blot; 1:100 dilution for immunohistochemistry and 1:100 dilution for immunofluorescence staining), FAP (Abcam, ab53066, 1:1000 dilution for western blot and 1:100 dilution for immunofluorescence staining), SRC (Signalway Antibody, #40790, 1:1000 dilution for western blot, 1:100 dilution for immunohistochemistry and 1:100 dilution for immunofluorescence staining), p-SRC (Abcam, ab4816, 1:1000 dilution for western blot), TEAD1 (Abcam, ab133533, 1:20 dilution for IP and 1:500 dilution for western blot), GAPDH (Sungene Biotech, KM9002, 1:5000 dilution for western blot).
Cell culture and cell lines
The prostate and prostate cancer cell lines that were used in this experiment, including TrampC1, RM1, CAF and NF, were all derived from Dr. Chang, George Whipple Lab for Cancer Research, and these four types of cells are of mouse origin [
16,
17]. TrampC1 and RM1 were cultured in RPMI-1640 medium (Gibco, Waltham, MA USA) containing 10% foetal bovine serum (Gibco, Waltham, MA USA) and culture conditions of 37 °C with 5% CO
2. CAF and NF were cultured in DMEM (Gibco, Waltham, MA USA) containing 10% foetal bovine serum (Gibco, Waltham, MA USA) and incubated at 37 °C with 5% CO
2.
In this experiment, human prostate cancer hCAF and human prostate hNF were taken from the primary culture of urological surgical specimens from the Second Hospital of Tianjin Medical University. The hCAF and hNF samples were cultured in DMEM (Gibco, Waltham, MA USA) containing 10% foetal bovine serum (Gibco, Waltham, MA USA) and incubated at 37 °C with 5% CO2.
MTT assay
A 5 g/mL concentration of MTT solution was prepared and stored at 4 °C in the dark. The cells to be tested were seeded in a 96-well plate (cell number 2 × 103 cells/well, medium 100 μL/well). The samples were incubated for 3–6 days at 37 °C with 5% CO2. Then, 50 μL of MTT solution were added to each well and incubated at 37 °C for 4 h. The supernatant was aspirated, and 150 μL of DMSO were added to each well and shaken on a plate shaker. The microplate reader measures the optical density OD value of each well at a wavelength of 570 nm. The value-added active fold lines were drawn using GraphPad Prism 5 software (GraphPad Software, La Jolla, CA, USA).
Western blot
Total cellular proteins were extracted using RIPA (Thermo Scientific, 89,901). The prepared protein sample was added to the gel lane of the separation gel, and electrophoresis was performed using a constant voltage. After the end of the electrophoresis, the protein was transferred using a PVDF membrane. After the completion of the electroporation, the PVDF membrane was sealed with skim milk for 60 min. After TBST was washed, the primary antibody was incubated overnight at 4 °C. The primary antibody was washed away by TBST, and the secondary antibody solution corresponding to the primary antibody was added and incubated at room temperature for 1 h. The secondary antibody was washed with TBST and prepared for exposure. The prepared developing solutions A and B are mixed in proportion (Immobilon Western, Chemiluminescent HRP Substrate, Millipore Corporation, Billerica, MA, USA), and the mixed liquid is dropped on the corresponding molecular weight strip of the PVDF film and placed in an exposure machine for exposure.
Immunohistochemistry
After the specimen was fixed with a formalin solution, wax block preparation was performed and anti-separation sections prepared. Dewaxing was performed for water and antigen retrieval by conventional methods. Then, 3% H2O2 was added to the specimen to remove the endogenous peroxidase in the specimen. After washing with PBS, the primary antibody was added dropwise and incubated at 4 °C for 12–18 h. After rewarming, the secondary antibody was added to the specimen and incubated at 37 °C for 30 min. After the PBS was washed again, the pre-configured DAB solution was added dropwise, and after the staining was completed, the sections were rinsed in PBS buffer in time. After washing with tap water, the nucleus was counterstained by adding haematoxylin working solution. The results were observed under a microscope, the positive rate was counted, and the results were analysed.
Transwell invasion assay
Matrigel was pre-treated to 4 °C until liquified. Matrigel was diluted with pre-cooled serum-free 1640 medium (the dilution ratio was 1:3), and 60 μL of the diluted gel were added to a 24-well plate (Corning Costar, 3524) in a Transwell chamber (BD FALCON, 353097) for 6 h. The cells were suspended in serum-free medium. A total of 105 cells were added to each Transwell upper chamber, and 500 μL of foetal bovine serum-containing medium or conditioned medium were placed in the Transwell lower chamber. The cell culture was performed as described above. After 24 h, the cells on the upper surface of the Transwell membrane were removed with a cotton swab, and the cells on the lower surface of the Transwell membrane were fixed and stained. The cells on each Transwell membrane were photographed and counted. The statistical results were drawn.
In vitro transfection
The following shRNA plasmids were used in this study for in vitro transfection: YAP1 Mouse shRNA Plasmid, CAT#: TG502437, Origene; TEAD1 Mouse shRNA Plasmid, CAT#: TL513813, Origene; shRNA vector, CAT#: TR30007, Origene; YAP1 Mouse Tagged ORF Clone, CAT#: MR226049, TrueORF®; and TEAD1 Mouse Tagged ORF Clone, CAT#: MR206462, TrueORF®. The siRNA sequences (Supplementary Table
1) were synthesized by RiboBio (Guangzhou, China), and a scrambled siRNA (RiboBio) was used as the negative control. The cells were transfected with liposomes. The plasmid was mixed with Transfection Reagent 1:1–1:4 and added to opti-MEM for 30 min. The above mixture was added to the medium of the cells. The new medium was replaced after 24–48 h. The transfected cells were screened using G418. A stable transfected cell line was finally obtained.
Immunofluorescence staining
The cells were seeded in glass slides and treated with paraformaldehyde and Triton, and the primary antibody was incubated overnight at 4 °C. After the secondary antibody was added dropwise, it was incubated at room temperature for 1 h, and after washing with PBS, the nuclei were stained with DAPI. The photograph was taken under an Olympus FV1000D confocal microscope [
18].
RNA isolation and quantitative RT-PCR analysis
The total RNA was extracted using Trizol reagent according to the manufacturer’s protocol. The RNA was reverse transcribed using a reverse transcription kit (RevertAid First Strand cDNA Synthesis Kit, Thermo Scientific, Waltham, MA USA) to obtain cDNA. The mRNA reverse transcription-PCR (RT-PCR) primers for YAP1, α-SMA, FAP, SRC, MYL9, F-actin and paxillin were purchased from Applied Biosystems. The primer sequences are shown in Supplementary Table
2. The expression of the mRNAs in the quantitative RT-PCR analysis was determined by an Applied Biosystems 7900 Real Time PCR System (Thermo Scientific, Waltham, MA USA). Small nucleolar RNA U6 was used as an internal reference for normalization.
Co-immunoprecipitation (co-IP)
A small amount of cell lysate was used as the input. The remaining lysate was added to the YAP1 or TEAD1 antibody and placed on a shaker at 4 °C overnight. The pretreated protein A agarose magnetic beads were added to the lysate to fully couple the YAP1 or TEAD1 antibody to the protein A agarose magnetic beads. After the immunoprecipitation reaction, the agarose beads were collected. The agarose beads were washed 3 times with the lysis buffer. The SDS loading buffer was added to the liquid and heated at 95 °C for 5 min. Analysis was performed using western blot.
Chromatin immunoprecipitation (CHIP)
The prepared cells were subjected to the YAP1 or TEAD1 CHIP assay using the EpiQuik Chromatin Immunoprecipitation Kit (Epigentek, Farmingdale, NY, USA) according to the protocol [
18]. PCR was performed using primers specific for the YAP1 or TEAD1 binding regions in the SRC promoter. The primer sequences of the promoter region are shown in Supplementary Table
2.
Luciferase reporter assays
Luciferase reporter constructs (MCS-firefly_Luciferase and TK promoter-Renilla_Luciferase) were processed by Genechem (Shanghai Co., Ltd.). The 293 T cells were transfected with the SRC luciferase reporter constructs (MCS-firefly_Luciferase) with or without YAP1 and TEAD1 overexpressing plasmid. Luciferase activity was normalized to Renilla luciferase activity. A Dual-Luciferase Reporter Assay System (Promega) was applied to measure the luciferase value according to the manufacturer’s instructions [
18]. Three independent assays were performed in triplicate.
In vivo experiments
The animal studies were approved by the Second Hospital of Tianjin Medical University, Tianjin, China. Male nude mice (6 weeks old, n = 12) were purchased from Beijing HFK Bioscience Co. Ltd. (Beijing, China). The animal studies were approved by Tianjin Institute of Urology, Tianjin, China. Male nude mice (6 weeks old, n = 20) were purchased from Beijing HFK Bioscience Co. Ltd. (Beijing, China). Subcutaneous tumour growth assays were performed with CA, CAFshYAP1, NF and NFoverexpressYAP1 stable cell lines. We mixed each of the four stable cell lines previously described in the paper with the epithelial cell TrampC1 at a ratio of 1:1 (1 × 106 stromal cells mixed 1 × 106 epithelial cells) to obtain 4 different experimental groups: CAF TrampC1, CAFshYAP1 TrampC1, NF TrampC1, and NFoverexpressYAP1 TrampC1. Five BALB/c nude mice are in each group. After 2 weeks, 20 of the injected mice developed tumours. The tumours were harvested under standard institutionally approved processes. The tumour samples were paraffin fixed and processed for IHC analysis.
Statistical analysis
SPSS 22 statistical software (SPSS, IBM Corporation, Armonk, NY, USA) was used for the statistical analysis. A one-way analysis of variance was used for multiple comparisons. A paired t-test was used for comparison between different treatment groups and control groups. GraphPad Prism 5 software was used to draw the graphics. P < 0.05 indicates a statistically significant difference in results. P < 0.05 was marked as *, P < 0.01 was marked as **, P < 0.001 was marked as ***, and no significant difference was expressed as n.s.
Discussion
The TME plays an important role in the occurrence and development of solid tumours. An increasing number of studies have begun to focus on the TME. Cell types in the TME include neuroendocrine cells, fat cells, endothelial cells, mesenchymal cells, immune inflammatory cells, and fibroblasts [
37]. The normal fibroblasts are usually quiescent. When their intrinsic signalling pathway is abnormal, they are induced to convert into cancer-associated fibroblasts (CAFs). CAFs are characterized by increased expression of markers such as α-SMA, fibroblast activation protein (FAP), fibroblast-specific protein 1 (FSP1 or S100A4), vimentin and platelet-derived growth factor receptor (PDGFR)-α and β [
38,
39]. CAFs can be derived from different cell types, such as NFs, epithelial cells after the EMT, endothelial cells through the endothelial-mesenchymal transition (EndMT), bone marrow-derived cells (BMDCs), adipocytes and stellate cells [
40]. The interaction between CAFs and tumour epithelial cells has led to the failure of tumour treatment.
Yes-associated protein was discovered by Sudol et al. in 1994 as a 65 kda protein containing the WW domain [
41]. Among many pathogenic proteins, the YAP1 protein functions as a transcriptional coactivator and a connexin [
42]. YAP1 is capable of cell proliferation, induction of the epithelial-mesenchymal transition (EMT), enhancement of cell migration/invasion, and inhibition of apoptosis. When YAP acts as a transcriptional coactivator, it needs to interact with the transcription factor TEAD to function. The TEAD protein alone does not induce gene expression and requires additional coactivators to achieve its transcriptional potential [
36]. TEAD1 is involved in the regulation of prostate epithelial cell differentiation and epithelial morphogenesis. TEAD1 expression levels are higher in PC3 cells and tissue specimens, which is associated with poor prognosis in prostate cancer patients [
43]. SRC is a non-receptor tyrosine protein kinase. Studies have shown that Src protein overexpression and sustained activation are found in solid tumours such as breast cancer [
44], colon cancer [
45] and pancreatic cancer [
46]. In addition, the high activation of SRC is also present in prostate cancer tissues [
47,
48]. SRC regulates the expression levels of actin and cytoskeletal proteins in cells [
28]. Actin and cytoskeletal proteins are extremely active during the conversion of NFs to CAFs. The link between YAP1 and SRC in prostate cancer specifically indicates that the prostate cancer stroma has not been explicitly implicated. This study demonstrates the role of YAP1, TEAD1 and SRC in the conversion of NFs to CAFs in prostate cancer.
We first found that the expression level of YAP1 in the prostate cancer stroma was significantly higher than that in BPH, and the amount of CAF in the prostate cancer stroma increased with the increase in the YAP1 expression level (Fig.
1). Through the detection of the CAF-specific protein markers SMA and FAP, we found that when the expression of YAP1 in NF was increased, NFs were converted to CAFs. Its function may be similar to that of CAF (i.e., promote the proliferation and invasion of epithelial cells). In contrast, when the level of YAP1 expression in CAFs is lowered, it reverses the above conversion and attenuates the function of promoting proliferation and invasion (Fig.
2). We further confirmed that YAP1 forms a protein complex with TEAD1, which together regulate the transcription of SRC in fibroblasts. SRC regulates the downstream actin and cytoskeletal proteins (such as MYL9, F-actin and paxillin), which ultimately leads to the conversion of NFs to CAFs (Fig.
3). We have demonstrated that fibroblasts with high expression of YAP1 can promote tumour proliferation in vivo (Fig.
4). Clinical features have shown that cancer metastasis occurs almost exclusively in prostate cancer patients who have a high expression of YAP1 in stromal cells (Table
1). Therefore, the high expression of YAP1 may indicate a poor prognosis.
At present, the diagnosis and treatment of prostate cancer is a recognized problem in the world. Abnormal expression of proteins such as PSA and AR has been used as a biomarker for the diagnosis or treatment of prostate cancer. However, some malignant prostate cancers, such as metastatic prostate cancer, do not have an appropriate biomarker as a diagnostic basis. Through our research, we believe that YAP1 in stromal cells has the potential to serve as a diagnostic marker or therapeutic target for prostate cancer. Since we demonstrated that the upregulation of YAP1 in stromal cells leads to the proliferation and metastasis of prostate cancer cells, we draw the following two conclusions: 1. by detecting the expression of YAP1 in the tumour stroma of patients with prostate cancer, we can predict the trend by which prostate cancer develops, and 2. the clinical application of verteporfin (VP) can prevent the proliferation or metastasis of prostatic tumours. In this regard, the research group will continue to conduct in-depth research and strive to use YAP1 as a therapeutic target for prostate cancer. In the follow-up study, the research team continued to explore the effect of tumour epithelial cells on mesenchymal cells. A very real possibility is that the exosomes that are released by epithelial cells are absorbed by mesenchymal cells and that both NFs and CAFs can absorb the exosomes. YAP1 expression levels increase when NFs are affected by exosomal-derived nucleic acids, and then the NFs are converted into CAFs. CAFs also maintain high expression levels of YAP1 due to the influence of related factors from the exosomes, thereby maintaining their own characteristics and functions. In this manner, CAFs will continue to affect epithelial cells, forming a positive feedback loop from the epithelial cells to mesenchymal cells and then back to epithelial cells. This positive feedback loop allows tumours to proliferate. In this feedback loop, YAP1 plays as a decisive factor and is necessary for CAFs to promote tumour proliferation and invasion. We will continue to study this positive feedback loop and determine the mechanism of YAP1 in the tumour microenvironment.
In summary, we confirmed a new mechanism of YAP1 in the prostate cancer stroma. The function of YAP1 was critical for the formation of CAF and maintenance of its own characteristics. Of particular note is that YAP1 is a key factor in converting NFs into CAFs. The YAP1 protein in the tumour stroma can be used as a potential target for tumour diagnosis and treatment. The expression of YAP1 in the tumour stroma may indicate the trend and prognosis of the disease, which provides new ideas and directions for cancer treatment in the future.
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