UNIVERSIDADE ESTADUAL PAULISTA “JÚLIO DE MESQUITA FILHO” CAMPOS DE BOTUCATU INSTITUTO DE BIOCIÊNCIAS Sulfiredoxina: um potencial alvo terapêutico para o câncer de próstata avançado Caroline Nascimento Barquilha Orientador: Sérgio Luis Felisbino Trabalho de Conclusão de Curso apresentado como requisito para obtenção do grau de Bacharel em Ciências Biológicas no Instituto de Biociências da Universidade Estadual Paulista “Júlio de Mesquita Filho” – Campus de Botucatu. Botucatu - SP 2017 O presente Trabalho de Conclusão de Curso segue o modelo de Artigo Científico, o qual obedece às normas para submissão de trabalhos da seguinte revista de divulgação da área: Experimental Cell Research Sulfiredoxin: a potential therapeutic target for advanced prostate cancer Caroline Nascimento Barquilha¹, Flávio de Oliveira Lima2, Flávia Karina Delella¹, Sérgio Luis Felisbino¹ 1Department of Morphology, Institute of Biosciences, Sao Paulo State University, Botucatu, SP, Brazil 2Department of Pathology, School of Medicine of Botucatu, Sao Paulo State University, Botucatu, SP, Brazil Corresponding author: Sérgio L Felisbino Sao Paulo State University (UNESP), Institute of Biosciences. Rua Professor Doutor Antonio Celso Wagner Zanin, 18618689 - Botucatu, São Paulo - Brazil. E-mail address: felisbin@ibb.unesp.br ABSTRACT Despite the effectiveness of surgical and anti-androgenic therapies against organ- confined and metastatic prostate cancer (PCa), respectively, new therapeutic approaches are still needed for treatment of advanced metastatic PCa that occasionally becomes resistant to androgen deprivation, chemo and radiation therapies, which are responsible for all deaths related to this disease. Oxidative stress unbalance is involved with cancer development, progression and metastasis, including PCa. Sulfiredoxin, an antioxidant enzyme, has been shown to have elevated expression in the tumorigenic process of different organs. Here, we analyzed the expression of sulfiredoxin in PCa, investigating its potential as a new therapeutic target for PCa. Sulfiredoxin expression was investigated by immunohistochemistry in PCa from transgenic mice conditional knockout for Pten and human prostate cancer and showed an increased expression in undifferentiated adenocarcinoma and high grade Gleason tumors. In the human data base TCGA, 5% of patients with PCa have higher expression of sulfiredoxin and also lower disease free survival rates (P<0,000122). Quantitative PCR showed that tumor cell lines, PC-3 and LNCaP, express higher levels of sulfiredoxin than prostate normal cell line RWPE-1, which are in agreement with results from metadata analysis in the GEO profile from others studies. Our results demonstrate that sulfiredoxin play a role in some subset of PCa, especially in the advanced stages of the tumor. Considering the personalized medicine, our results suggest that inhibition of this enzyme can be an effective strategy of adjuvant treatment to castration-resistant CaP patients with sulfiredoxin upregulation. Keys words: Sulfiredoxin; cancer; prostate; oxidative stress, mouse knockout, PC3 INTRODUCTION Circulating androgens are essential for the normal development of the prostate as well as in the initial development of prostate cancer (PCa) (Hsing, Reichardt, & Stanczyk, 2002). One strategy to treat and regress this tumor involves the removal of the testicular androgens by surgical or chemical castration (Huggins & Hodges, 1972). Despite the advance of anti-hormonal therapies against PCa, new therapeutic approaches have been investigated for treatment of advanced stage tumors that are resistant to androgen deprivation. In this context, oxidative stress has been pointed out as one of the greatest influence associated with age over the prostatic carcinogenic risks (DeWeese, Hruszkewycz, & Marnett, 2001; Minelli, Bellezza, Conte, & Culig, 2009). It is also involved with cancer progression and metastasis, considering that tumor cells are more dependent of antioxidant mechanisms (Gorrini, Harris, & Mak, 2013; Huang, Feng, Oldham, Keating, & Plunkett, 2000). Previous studies have been shown that sulfiredoxin, an antioxidant enzyme, has an important role in oxidative stress balance (Mishra, Jiang, Wu, Chawsheen, & Wei, 2015). Specifically, sulfiredoxin acts in the peroxiredoxin (I-IV) reactivation (Jonsson & Lowther, 2007) which are a group of peroxidases enzymes responsible by reactive oxygen species (ROS) reduction, like hydrogen peroxide and organic peroxides (Cox, Winterbourn, & Hampton, 2009). By reducing the hyperoxided peroxiredoxins, sulfiredoxin prevents them from being degraded (Woo et al., 2005). Besides that, sulfiredoxin can participate of the deglutathionylation of different substrates (Findlay, Tapiero, & Townsend, 2005). Although the role of sulfiredoxin in the tumorgenesis of different organs, such as lung (H. Kim et al., 2016; Wei et al., 2011), colon (Wei et al., 2013), skin (Wu et al., 2014), breast (Hartikainen et al., 2012), is well established, its participation in PCa progression and metastasis is not well described, so we decided to analyze the expression of sulfiredoxin in PCa samples and cell lines, investigating its potential as new therapeutic target for PCa. MATERIALS AND METHODS Transgenic mouse prostates and human prostate tumors tissue microarray Parafin samples of prostatic tumors from genetic engineered mouse model PB- Cre/PtenloxP/loxP, which presents deletion of both alleles of the gene Pten exclusively in the prostatic epithelium (conditional knockout), were obtained from CRUK Cambridge Institute, on behalf David Neal’s Uro-Oncology Group. Human tissue microarray (TMA) was constructed using the prostate of 125 patients who underwent radical prostatectomy between 1980 and 2000, being 115 samples of CaP, confined organ, and 10 samples of adjacent non-neoplasic tissue. One tissue cores of 1 mm diameter were used for each sample. The TMA was donated and analysed by a consultant Pathologist Flávio de Oliveira Lima (FMB- UNESP- Botucatu). Immunohistochemistry Histological sections of normal and tumor prostate samples from mouse and human TMA were used. After antigenic recuperation with sodium citrate buffer (10mM, pH 6.0) using a PASCAL pan, the sections were treated with a solution containing 3% hydrogen peroxide in methanol for 15 minutes. In the sequence, the materials were blocked with 3% powdered milk in PBS for 1 hour at room temperature and incubated overnight at 4oC with primary antibody against sulfiredoxin (Biorbyt, 1:100). After, sections were incubated with a secondary peroxidase-conjugated antibody (Santa Cruz Biotechnology, 1:200), which was detected using diaminobenzidine (Sigma, USA) as chromogen. Slides were counterstained with Harris's hematoxylin. Negative control was performed by excluding the primary antibody incubation step. Cell Lines and Culture Conditions PC-3, LNCaP and RWPE-1 cells were obtained from American Type Cell Culture (Manassas,Virginia, USA). PC-3 and LNCaP cells line were cultivated using the RPMI 1640 medium (GIBCO/Invitrogen™) supplemented with 10% fetal bovine serum, 50μg/ml penicilin, 50μg/ml streptomycin and 0,5μg/ml amphotericin B. RWPE-1 cells were maintained in Keratinocyte Serum Free Medium (Gibco/InvitrogenTM) supplemented with 0.05 mg/ml bovine pituitary extract, 5 ng/ml recombinant human epidermal growth factor (EGF) and 1% antibiotic/antimycotic solution. The medium was changed twice per week. Cells were grown at 37 °C and 5% CO2. For passaging, cells were detached with 0.05% Trypsin (Gibco/Invitrogen™) for 3 min at 37°C, resuspended in growth medium and re- seeded. RNA extraction and RT-qPCR Total RNA from cells was extracted using the Allprep DNA/RNA/Protein extraction kit (QIAGEN, Crawley, UK) according to the manufacturer’s instructions. RNA quantification was determined by Nanovue Spectrophotometer (GE, EUA). cDNA was synthesized using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA). RT-qPCR reactions were performed using the AB7900 Real Time PCR system (Applied Biosystems). Relative gene expression was calculated according to the 2-∆∆CT method; ACTB was used as housekeeping gene. Details of primers used are given in the table 1. Data base Meta-analysis We searched for sulfiredoxin pattern of expression in available data bases from previous published studies. We used GEO (Gene Expression Ominibus) to generate GEO profiles for sulfiredoxin (SRXN1) gene expression at different Gleason grade and Gleason Score of prostate tumors and for different normal and tumoral epithelial prostate cell lines. We also investigated the Genomic and gene expression alterations for SRXN1 gene in the TCGA (The Cancer Genome Atlas, cBioPortal to correlate these alterations with clinical data from patients Statistical analysis One-way analysis of variance (ANOVA) and Tukey–Kramer multicomparison post hoc test was performed to analyze whether differences existed between groups (p<0.05). The statistical tests were performed using Instat (version 3.0; Graph-Pad, Inc., San Diego, CA, USA). RESULTS Sulfiredoxin expression is increased in advanced prostate cancer from mice and human The immunohistochemistry analysis showed a higher immunostaining of sulfiredoxin in the advanced prostate cancer stages from mice, when compared to a normal tissue (figure 1). By tissue micro away analysis of prostate samples from humans, it was observed an increased expression of this enzyme in the undifferentiated adenocarcinomas (figure 2), representing the patients with high Gleason Score prostate cancer. Data already published and available (database GEO - Gene Expression Ominibus) about human gene expression PCa also demonstrated an increased expression of sulfiredoxin in patients with advanced tumor (Gleason Score 8 and 9) in relation to control (figure 3). Patients with sulfiredoxin upregulation have lower disease free time survival rates A human database (TCGA - The Cancer Genome Atlas, cBioPortal) showed that, considering 332 patients with available information, 5% of them presented changes in the SRXN1 gene expression, mainly the upregulation (figure 4). Those patients with alteration in SRXN1 had a lower survival rate with a high statistical significance (figure 5). Prostate cancer cell lines express higher levels of SRXN1 than normal cell line Quantitative PCR (Figure 6) showed that prostatic tumor cell lines, PC-3 and LNCaP, express higher levels of SRXN1 than prostate normal cell line RWPE-1. Among the prostatic tumor cell lines, LNCaP (androgen sensitive) has an increased SRXN1 expression compared to PC-3 (androgen non-sensitive). The analysis of GEO profiles from others studies also showed higher expression of SRXN1 in the LNCaP and PC3 cells than normal cells (figure 7). DISCUSSION SRXN1 expression is modulated by different environmental and intrinsic factors. Oxidative stress is one of the mains triggers to activation of sulfiredoxin signalizing pathways (Mishra, Jiang, Wu, Chawsheen, & Wei, 2015). Singh et al., 2009 observed an induction of sulfiredoxin in lung cells by cigarette smoke exposition. Soriano et al., 2008 described an influence in its modulation related to a dietary. Liver cells treated with D3T also had an increased expression of this enzyme (Kwak et al., 2003). In the presence of reactive oxygen species (ROS), it has been already described that nuclear factor erythroid 2-related factor (Nrf-2) promotes SRXN1 expression, as well activator protein-1 (AP-1) and c-Jun (Soriano et al., 2009; Soriano et al., 2008). Many studies have demonstrated the protective role of sulfiredoxin against oxidative injury. Li, Yu, Wu, Zou, & Zhao, 2013 induced oxidative stress by oxygen peroxide (H2O2) treatment in PC12 cells and observed a protective action by this enzyme. Sulfiredoxin also prevents astrocytes from apoptosis after exposition to H2O2 (Zhou, Yu, Wu, Chen, & Zhao, 2015). Tumor cells have been already described as more vulnerable than normal cells to damages caused by ROS and more dependents of antioxidant mechanisms (Gorrini et al., 2013; Huang et al., 2000), which explains the high levels of sulfiredoxin in LNCaP and PC-3 tumor cells observed in our study. The increased expression of sulfiredoxin antioxidant enzyme has been observed in several types of tumor. Wei et al., 2011 showed a more intense staining for sulfiredoxin in lung tumor samples by immuhistochemistry. Wei et al., 2013 demonstrated a higher expression of this enzyme in human colon carcinoma, showing that knockout animals to sulfiredoxin were resistant to the carcinogenic induction model used from them. It was also described a high expression of sulfiredoxin in human skin squamous cell carcinoma (Wu et al., 2014). These studies reinforce the cytoprotective role of sulfiredoxin against the cell death induced by oxidative stress in tumor cells, but not in adjacent normal tissues. It has been demonstrated that SRXN1 is essential for cancer cell proliferation (Lei, Townsend, & Tew, 2008) and its depletion decreases cell viability (Zhou et al., 2015), suppresses cell migration and inhibits tumor growth (Wei, Jiang, Matthews, & Colburn, 2008; Wei et al., 2011). Gene polymorphism to sulfiredoxin also has effect in the breast cancer development and in the patient survival (Hartikainen et al., 2012). Sulfiredoxin expression was associated with the poor survival of patients with pancreatic adenocarcinoma (Soini et al., 2014). Considering the pathogenic role of sulfiredoxin in human cancer, it has been already pointed as a new potential therapeutic target for this disease. In human skin malignant tumors, Wei et al., 2008 showed that sulfiredoxin inhibition by AP-1 pathway may be a novel strategy to skin cancer prevention and treatment, considering that its knockdown was critical for tumor transformation. H. Kim et al., 2016 and J. Kim et al., 2016 demonstrated that sulfiredoxin inhibition by synthetic inhibitors (J14 and K27) promotes selectively death of A549 pulmonary tumor cells. 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Representative image of immnohistochemistry to Sulfiredoxin in control and tumor tissue micro away from human prostate cancer. Arrows indicate positive stained cells. Figura 3. Expression levels of SRXN1 gene in normal and tumor prostate samples (Gleason score 8 and 9) from patients of the GEO profiles human database (Satake H, 2010). Figure 4. Analysis of main genomic alterations to SRXN1 gene in patients with PCa cataloged by TCGA human database (Cerami et al., 2012; Gao et al., 2013). Figure 5. Kaplan-Meler curve displaying disease free of prostate cancer from patients with or without sulfiredoxin alteration, cataloged by TCGA human database (Cerami et al., 2012; Gao et al., 2013). Figure 6. Graph of RT-qPCR to SRXN1 mRNA levels from RWPE-1, LNCaP and PC-3 cell lines. Figure 7. SRXN1 mRNA levels of different prostate cell lines obtained from patients of GEO profiles human database (Zhao et al., 2005).