Abstract
Background/Aim: Endothelin-1 (ET-1) is overexpressed in many types of cancer, inhibiting the release of the microRNA 15a (miR-15a) and inducing the production of Mxi-2. Our aim was to identify a molecular complex regulating p53 activity in prostate cancer (PCa). Materials and Methods: DU145 cells were treated with ET-1, MAPK p38 inhibitor, Endothelin A receptor inhibitor (ETAR inhibitor) and Endothelin B receptor inhibitor (ETBR inhibitor). Extracts were analysed using Western Blot, immunoprecipitation and qRT–PCR. Furthermore, prostate cancer patient samples were analysed using qRT–PCR and ELISA. Results: The hypothesised molecular complex was identified, with miR-15a, microRNA 1285 (miR-1285) and Mxi-2 levels up-regulated in patients in relation to increasing aggressiveness of PCa. Conclusion: A complex composed of Argonaut 2 (Ago2)/Mxi-2/miR-1285 is involved in PCa. The expression of Mxi-2 correlates with increasing PCa aggressiveness and might be used as a non-invasive marker for the diagnosis and progression of PCa.
- Prostate cancer
- Endothelin-1
- Mxi-2
- Ago2
- p53
Endothelin-1 (ET-1) is a potent vasoconstrictor and multifunctional protein in cancer. It is one of the most studied proteins in the family of multifunctional proteins (1). ET-1 is able to bind to two different G-protein coupled receptors, called Endothelin A and B receptor (ETAR and ETBR). The binding to the ETAR and its subsequent activation results in cell proliferation, hault of apoptosis, angiogenesis, invasion, and metastatic spread (2) and activates a survival pathway, whereas binding to the ETBR causes clearance of ET-1 and apoptosis. In prostate cancer, ET-1 is produced in prostate epithelial cells and is associated with progression of prostate cancer (2).
ET-1 is a transcriptional target gene of the tumor suppressor protein p53 (3), which plays a major role in tumor development (4). In general, p53 can respond to many stress signals, especially to DNA damage, and plays a major role in the regulation of the cell cycle (5). In addition, p53 is needed for a proper stress response to protect the cell from malignant transformations (6). In prostate cancer, a mutation in p53 is associated with tumor development and progression (7, 8). Mutant p53 functions as an oncogene, which allows tumour growth and survival advantages (9).
The transcription factor activity of p53 can be activated by Protein kinase C (PKC) α (10), a member of the protein kinase family (11). PKCα is able to migrate into the nucleus via the transcription complex, composed of MAPK p38α and NFĸB p65, where it binds to the pri-miR 15a, inhibiting its maturation. This migration process is regulated by the presence of ET-1 (11). As recently described, miR-15a binds to the intron region of MAPK p38α introducing a stop codon and resulting in a C-terminal truncated protein, called Mxi-2 (12).
In this article, we show that ET-1 induces a signalling pathway that influences the presence of p53. This pathway involves Mxi-2, Ago2 and miR-1285. In the literature it is known that miR-1285 binds directly to the 3’ UTR of the mRNA of p53 (13), which results in the down-regulation of p53. ET-1 inhibits the migration of PKCα into the nucleus, which leads to the maturation of miR-15a. Subsequently, the mature miR-15a migrates back into the nucleus, inducing the production of Mxi-2 (Figure 1) (12).
Materials and Methods
Cell culture and treatments. DU145 cells (ATCC, Rockville, MD, USA) were cultured in Roswell Park Memorial Institute Medium (RPMI) (Life Technology GmbH, Dramstadt, Germany) supplemented with 10% FCS (PAN-Biotech GmbH, Aidenbach, Germany) and 1% Penicillin/Streptomycin (Invitrogen Cooperation, Karlsruhe, Germany) at 37°C and 5% CO2. For the experiments, cells were incubated for 24 h with serum free medium. Cells were treated with Endothelin-1 (50 nM, Sigma-Aldrich, Deisenhofen, Germany) BQ123 (ETAR antagonist, 100 nM, Sigma-Aldrich), BQ788 (ETBR antagonist, 100 μM, Sigma-Aldrich) and SB203580 (MAPK p38 inhibitor, 100 μM, Merck, Darmstadt, Germany).
Total protein extraction and nuclear/cytoplasmic isolation. Total protein extracts were isolated from the treated cells and controls according to the manufacturer's protocol (RIPA lysis kit, Santa Cruz, Heidelberg, Germany). Nuclear and cytoplasmic extracts were isolated according to the manufacturer's protocol (Nuclear extraction kit, Active motif, Rixwnsart, Germany). Protein was quantified via the Bradford assay (AppliChem GmbH, Darmstadt, Germany) with bovine serum albumin as standard.
Western blot. The protein concentration was determined as described previously. Proteins (25 μg) were separated by 10% SDS-Page and blotted on a Nitrocellulose membrane (Whatman GmbH, Dassel, Germany). Membranes were blocked in 3% TBST-Milk and for Mxi-2 in blotting buffer (NanoTools Antibodies, Tenigen, Germany). Membranes were incubated with specific antibodies against MAPK p38 α, β (Cayman Chemical, Ann Arbour, MI, USA), Mxi-2 (NanoTools Antibodies), Ago2 (Santa Cruz), PKCα (Santa Cruz) and p53 (Santa Cruz). All specific primary antibodies were diluted 1:500 and incubated for 1h at room temperature or over – night at 4°C. For visualisation an antibody conjugated with HRP was used in an enhanced chemiluminescence assay according to the manufacturer's protocol. The Chemostar (Intas Science Imaging, Göttingen, Germany) was used for analysis.
Immunoprecipitation. For immunoprecipitation, an Mxi-2, Ago2 or a miR-1285-FITC agarose-conjugated antibody (all from Santa Cruz) was used. The assay was performed according to the Santa Cruz protocol with 500 μg of total cellular protein. The final washing step was done 3 times using 1x PBS (PAN-Biotech GmbH) and the samples were diluted in 20 μl of electrophoresis buffer. After a boiling step of 3 min, the samples were used for Western Blot analysis.
ELISA. The ELISA was performed in non–coated 96 well plates (Brand GmbH & Co KG, Wertheim, Germany) and the 50 μl of patients' serum were incubated for 1 h at room temperature. The washing step was done 3 times using 1 x PBS followed by an incubation step with a Mxi-2 specific antibody (1:1,000) (NanoTools, Antibodies) for 1 h at room temperature or overnight at 4°C. After washing twice, the secondary antibody (1:5,000) (Biomol GmbH, Hamburg, Germany) was incubated for 1h at room temperature or overnight at 4°C. After washing twice again, step 50 μl substrate solution (Invitrogen Corporation) was added and the reaction was stopped according to the manufacturer's protocol (Bethyl Laboratories. Inc, Mongomery, TX, USA). The ELISA read at 450 nm by the FluroStar Omega (BGM Lebtech, Ortenberg, Germany).
Total RNA isolation. RNA was isolated using the RNeasy kit from Qiagen GmbH, Hilden, Germany according to the manufacturer's protocol. Quantification of the isolated RNA was done via NanoDrop technology (Thermo Fisher Scientific Inc., Rockford, IL, USA) (14).
Reverse transcriptase PCR. The cDNA was obtained from 250 ng RNA using random primers and SuperScript III reverse transcriptase according to the manufactory's protocol (Invitrogen Cooperation).
qRT – PCR. For real-time PCR analysis of the expression of miR-1285 (Forward 5’-TAGCAGCACATAATGGTTTGTG-3’) (Invitrogen Cooperation) and miR-1285 (Forward 5’-GAUCUCACUUUGUUGCCCAGG-3’) (Invitrogen Cooperation) we used 1 μl of the converted cDNA (transcribed from 250 ng RNA). For quantitative analysis, beta–actin or miRNA 5s (Forward 5’-GGCCAUACCACCCUGAACGC-3’) (Invitrogen Cooperation) were measured. The procedure has been previously described von Brandenstein et al. (11, 14).
Patients and statistics. All patient tissue and serum samples were collected at the University Hospital in Cologne in the corresponding biobanks. The patient sampling complied with the Declaration of Helsinki and a local ethics committee approval was obtained (BioMASOTA, University Hospital of Cologne, file reference: 12-163). The analyzed patient material (n=26 patient tissues and n=68 patient serum samples) were divided into 6 different groups: i) the control groups were men without any cancer history (n=5 tissue and n=10 serum samples), ii) the low risk patients had a Gleason score of 6 (n=5 tissue and n=6 serum samples), iii) intermediate risk were patients with a Gleason 7 (n=5 tissue and n=20 serum samples) and iv) high risk patients had Gleason 8-10 (n=4 tissue and n=14 serum samples). In addition, patients with all kinds of metastasis (n=4 tissue and n=8 serum samples) and all types of recurrence (n=3 tissue and n=10 serum samples) were analyzed. The patients with metastases and recurrence were under hormone therapy. Overview of the patients' groups can be seen in Table I.
All experiments were performed in triplicates. The statistical analysis was done using the GraphPrism 5 (Graphpad software, La Jolla, CA, USA). A paired t-test with one-way ANOVA was performed. The alpha–value was 0.05 and the results are visualized as boxplots (***p<0.0001, **p<0.001, *p<0.01).
Results
Concentrations of proteins of interest were determined in DU145 cells before and after stimulation. Cells were stimulated with i) ET-1, ii) SB203580 (MAPK 38 antagonist), iii) BQ123 (ETAR antagonist), iv) BQ788 (ETBR antagonist) and v) in combinations. Unstimulated DU145 cells were used as controls. Depending on the stimulation, either cytoplasmic or nuclear proteins were isolated (Figures 2 and 3). In the cytoplasmic fraction (Figure 2) concentrations of the proteins of interest changed upon stimulation of the cells. Concentration of Mxi-2 and MAPK p38 concentration was significantly decreased after SB203580 stimulation and in combination with ET-1. Mxi-2 levels were more affected compared to the MAPK p38. Even the presence of SB203580 in DU145 cells resulted in a decrease of Mxi-2. The concentration of Mxi-2 was significantly decreased after blocking ETAR or ETBR in combination with ET-1. The p53 concentration was also affected by stimulation, and was significantly increased after blocking the ET receptors and the MAPK p38 in combination with ET-1.
Since nuclear PKCα plays an important protein regarding the production of mature miRNA 15a (11), its concentration was determined via western blot (Figure 3), showing a significant decrease by the blockage of the ETAR in combination with ET-1.
A complex formation consisting of Mxi-2, Ago2 and miR-1285 was identified using immunoprecipitation (Figure 4).
To investigate the clinical significance of the regulation mechanism, the expression of the miR-15a and 1285 via qRT-PCR were determined in patients' biopsies (Figures 5A and B). In Figure 5 it can be seen that both miRNAs increased significantly by raising the aggressiveness of the prostate cancer as well as the Mxi-2 protein level.
Discussion
The signalling pathway composed of Mxi-2/Ago2 and miR-1285 influences the presence of p53 by binding to the 3’UTR of its mRNA. The same regulatory complex has also been found in breast cancer, where it also regulates the processing of p53 mRNA (13).
As mentioned, Mxi-2 is a truncated variant of MAPK p38 (12) and already known to be affected by SB203580 (15). Mxi-2 has a unique C-terminal ending with the first 280 amino acids identical to MAPK p38α (12). Stimulation with SB203580 has a bigger impact on Mxi-2 than on MAPK p38, due to a higher down-regulation of Mxi-2, compared to MAPK p38.
Blocking Mxi-2 has a positive effect on p53 concentration in the DU145 cells. This phenomenon can be explained by the fact that the identified regulatory mechanism cannot form and, therefore, no down-regulation of p53 occurs. The same positive effect can also be seen after blocking one of the two ET receptors, since the regulatory mechanism is again blocked and PKCα migrates into the nucleus, were it binds to the pri-miR-15a resulting in the down-regulation of mature miR-15a (11, 16). Similar results were obtained in the present study, where we showed that by blocking one of the two ET receptors PKCα concentrations is significantly increased.
For determining the biological significance of the regulatory mechanism in prostate cancer, the expression of the miR-15a and 1285 were determined in patient material. The expression of both miRNAs was significantly raised with increasing aggressiveness of the prostate cancer as defined by an increasing Gleason score. The significant increase in the miR-15a indicates prostate tumour growth (17). The overexpression of miR-15a correlates with increased levels of Mxi-2, which is a necessary complex partner of the described regulation complex. Overexpression of miR-1285 is also essential for complex formation, since this miRNA binds to the 3’ untranslated region (UTR) of the corresponding mRNA. The increased production of this regulatory mechanism results in an increased down-regulation of p53 in aggressive prostate cancers in agreement with the literature (7, 8).
Mxi-2 ELISA results from patient's sera show a significant increase relative to the prostate cancer aggressiveness. Mxi-2 might be a secretory protein found in DU145 cells, as many other secretory proteins (18). Since the prediction of prostate cancer can be difficult, Mxi-2 could may be used as biomarker; however, the number of serum samples needs to be increased. In addition, the role of Mxi-2 should be further investigated and compared to other potential biomarkers (19).
Here, we present a new regulatory mechanism, which plays a role in the down-regulation of p53 in prostate cancer. This down-regulation of p53 in aggressive prostate cancer correlates with findings in the literature (7, 8).
The newly identified complex seems to be of importance in prostate cancer with increasing aggressivness. The Mxi-2 concentration in patient serum might be used as non-invasive biomarker for prostate cancer.
Footnotes
* These Authors contributed equally to this study.
Authors' Contributions
MVB and AH designed the study and directed the project. BK carried out the experiments. BK wrote the manuscript with the support of AH, PP, KR, MVB. HG, JF, AH and MVB supervised the findings of this work. All Authors performed the analysis, designed the figures and discussed the results.
Conflicts of Interest
The Authors declare no conflicts of interest.
- Received July 2, 2020.
- Revision received July 31, 2020.
- Accepted August 4, 2020.
- Copyright© 2020, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved