1. Potential involvement of UBE2N, PSMB6 and PPP1CB in prostate cancer
Ivana Maleva Kostovska1, Selim Komina2, Gordana Petrusevska2, Momir Polenakovic1 and Katarina Davalieva1*
1Research Centre for Genetic Engineering and Biotechnology “Georgi D Efremov”, Macedonian Academy of Sciences and Arts, Skopje, Republic of Macedonia
2Institute of Pathology, Medical Faculty, University “St. Cyril and Methodius”, Skopje, Republic of Macedonia *
INTRODUCTION
The comparative proteomics analysis of prostate cancer (PCa) tissues could elucidate the proteins and regulatory pathways involved in this disease. In previous study, we characterized the proteins with differential abundances between tissues from patients with clinically and histologicaly confirmed PCa and benign prostate hyperplasia (BPH) using DIGE for protein separation and MALDI-TOF MS for protein identification. In this study, we have evaluated by Western blot analysis the abundance levels of UBE2N, PSMB6 and PPP1CB in larger and independent cohort of PCa and BPH tissues.
MATERIALS AND METHODS
The samples used in this study were fresh surgical tissues from 28 BPH and 14 PCa patients. Manual micro dissection method was employed to obtain tumor samples with more than 70% of tumor cells. Visualization of immunoreactive bands was detected by using Pierce ECL Western Blotting Substrate while densitometric comparisons were made with ImageJ software. The measured band intensities of tested proteins were normalized to β-actin and expressed as relative intensities.
Table 1. Functional characterization and association with prostate and other types of malignancy of UBE2N, PSMB6 and PPP1CB found with differential abundance between PCa and BPH tissues.
Swis Prot name (Symbol)
Protein Name
Molecular/biological function(s)
Association with PCa (proteomics studies)
Associations with cancer other than PCa (proteomics studies)
Association with cancer (genomics and functional studies)
PSB6_HUMAN (PSMB6)
proteasome (prosome, macropain) subunit, beta type, 6
PSMB6 is part of 20S catalytic core of the proteasome. Involved in an ATP/ubiquitin-dependent non-lysosomal proteolytic pathway for protein degradation. The proteasome controls the cell cycle and other processes through the degradation of critical regulatory components and transcription factors
PSMB6 was found up-regulated in the serum of Gleason score 7 patients vs. GS 5 using 2D DIGE (Byrne JC et al., Journal of proteome research 2009;8: ). PSMB-6 was not differentially expressed between tumor or benign prostate epithelium (Oʼhurley G et al., Appl Immunohistochem Mol Morphol. 2010)
Up-regulated in lung cancer (Lu Z et al., Cell Physiol Biochem. 2014); Up regulated in hypoxia and its overexpression is associated with cell prolifferation (Wang J et al., PLoS One. 2013)
Up-regulated in anaplastic thyroid cancers (Onda M et la., Endocr Relat Cancer. 2004)
UBE2N_HUMAN (UBE2N)
ubiquitin-conjugating enzyme E2N
UBE2N, also known as Ubc13, is a member of the E2 ubiquitin-conjugating enzyme family that mediates the synthesis of Lysine 63-linked polyubiquitination chains. UBE2N is part of ubiquitin proteasome pathway for protein degradation where transfers activated ubiquitin to the target protein. UBE2N has also been suggested to have a critical role in activation of mitogen-activated protein kinases (MAPKs) signaling. Recent studies have revealed the potential role of UBE2N as a novel regulator of p53 by reducing its transcriptional activity.
Up-regulated in triple-negative breast cancer (Muñiz Lino MA et al., J Proteomics. 2014); Up-regulated in neuroblastoma (Cheng J et al., Cell Death Dis. 2014); Up-regulated in diffuse large B-cell lymphoma (Pulvino M et al., Blood. 2012); Differentially expressed in colon cancer cell HCT-15 (Zhao J et al., Protein Pept Lett. 2012)
Associated with lung cancer by SNP's analysis (Kazma R et al., Carcinogenesis. 2012)
PP1B_HUMAN (PPP1CB)
protein phosphatase 1, catalytic subunit, beta isozyme
PP1 associates with over 200 regulatory proteins to form highly specific holoenzymes which dephosphorylate hundreds of biological targets. PP1 is essential for cell division, it participates in the regulation of glycogen metabolism, muscle contractility and protein synthesis. Involved in regulation of ionic conductances and long-term synaptic plasticity. Component of the PTW/PP1 phosphatase complex, which plays a role in the control of chromatin structure and cell cycle progression during the transition from mitosis into interphase.
Expression of PP1CB and CSNK1A1 was significantly greater in human melanoma specimens than normal melanocytes (Sun D et al., Melanoma Res. 2011);
Catalytic subunit genes (PPP1CA, PPP1CB and PPP1CC) have the essential role for the growth of cancer cells such as lung, colorectal, gastric and ovarian (Takakura S et al., Int J Oncol. 2001); Down-regulated in lung squamous cell carcinoma (Shen C et al., Lung Cancer. 2002); PPP1CB silencing in leukemia cells resulted in increased proliferation (Thirunavukkarasu V et al., PNAS 2013)
RESULTS AND DISCUSSION
The Western blot results confirmed the abundance levels of UBE2N, PSMB6 and PPP1CB, obtained by the DIGE experiment (Figure 1). The Mann–Whitney U test showed that the levels of UBE2N and PSMB6 in PCa were significantly higher than in BPH (p < ) while the levels of PPP1CB were significantly lower in PCa (p=0.019) (Figure 2). There are previous observations that UBE2N, PSMB6 and PPP1CB have a role in the molecular mechanisms of some cancers (Table 1). However, up till now, no association or contradictory association of these proteins with PCa has been reported. Our study demonstrated the differential protein abundances of these proteins at tissue level between PCa and BPH, suggesting that they might be related to prostate tumorogenesis. Therefore, this study represents a starting point for further elucidation of their function in PCa initiation and progression.
Figure 2. Protein abundances, expressed as relative band intensities normalized to β-actin were used to visualize the levels of UBE2N, PSMB6 and PPP1CB in PCa and BPH groups. UBE2N and PSMB6 levels in PCa were significantly higher than in BPH while PPP1CB levels in PCa were significantly lower than in BPH (Mann–Whitney U-test, P<0.05). In the box plot analysis, median, 25th and 75th percentiles, standard deviation (+) and outliers (♦) are shown. Box plots were constructed based on a series of 42 tissue samples.
Innovative aspects
Figure 1. The levels of UBE2N, PSMB6 and PPP1CB in PCa and BPH tissues. (A) 2DE profiles obtained by 2D DIGE. Each group was represented by five biological replicates. The red and blue bars indicate PCa and BPH groups respectively. (B) The representative images of Western blots. UBE2N and PSMB6 showed higher abundance while PPP1CB had lower abundance in PCa samples compared to BPH.
Our study confirmed the previous observation of differential protein abundances of UBE2N, PSMB6 and PPP1CB at tissue level between PCa and BPH, suggesting that they might be related to prostate tumorogenesis
ACKNOWLEDGMENTS
This work was supported by the funds for Science of the Macedonian Academy of Sciences and Arts (grant no /1, Biomarker detection in prostate cancer with the use of 2D DIGE/MALDI MS technology)