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Stanniocalcin-1 protein expression profile and mechanisms in proliferation and cell death pathways in prostate cancer
Journal Pre-proof Stanniocalcin-1 protein expression profile and mechanisms in proliferation and cell death pathways in prostate cancer Bruna Pasqualotto Costa, Vanessa Schein, Zhao Rafael, Andressa Schneiders Santos, Lucia Maria Kliemann, Fernanda Bordignon Nunes, João Carlos dos Reis Cardoso, Rute Castelo Félix, Adelino Vicente Mendonça Canário, Ilma Simoni Brum, Gisele Branchini PII:
S0303-7207(19)30361-2
DOI:
https://doi.org/10.1016/j.mce.2019.110659
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MCE 110659
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Molecular and Cellular Endocrinology
Received Date: 19 September 2019 Revised Date:
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Accepted Date: 18 November 2019
Please cite this article as: Costa, B.P., Schein, V., Rafael, Z., Santos, A.S., Kliemann, L.M., Nunes, F.B., Carlos dos Reis Cardoso, Joã., Félix, R.C., Mendonça Canário, A.V., Brum, I.S., Branchini, G., Stanniocalcin-1 protein expression profile and mechanisms in proliferation and cell death pathways in prostate cancer, Molecular and Cellular Endocrinology (2020), doi: https://doi.org/10.1016/ j.mce.2019.110659. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier B.V.
Stanniocalcin-1 protein expression profile and mechanisms in proliferation and cell death pathways in prostate cancer Bruna Pasqualotto Costa1, Vanessa Schein2, Zhao Rafael3, Andressa Schneiders Santos3, Lucia Maria Kliemann4, Fernanda Bordignon Nunes1, João Carlos dos Reis Cardoso5, Rute Castelo Félix5, Adelino Vicente Mendonça Canário5, Ilma Simoni Brum2, Gisele Branchini1. 1
Programa de Pós-Graduação em Patologia, Universidade Federal de Ciências da Saúde de Porto Alegre, Brazil. 2
Departmento de Fisiologia, Instituto de Ciências Básicas da Saúde, Universidade Federal de Ciências da Saúde de Porto Alegre, Porto Alegre, Brazil.
3 4
Universidade Federal de Ciências da Saúde de Porto Alegre, Porto Alegre, Brazil.
Departmento de Patologia, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil. 5
Centre of Marine Sciences (CCMAR), University of Algarve, Campus de Gambelas, Faro, Portugal.
*Corresponding author: Dra. Gisele Branchini Department of Basic Health Sciences, Universidade de Ciências da Saúde de Porto Alegre (UFCSPA), 245 Sarmento Leite, Rio Grande do Sul, Porto Alegre, Brazil. Email address:
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ABSTRACT
Prostate cancer (PCa) is one of the most prevalent male tumours. Stanniocalcin-1 (STC1) is a glycoprotein and, although the role of STC1 in human cancer is poorly understood, it is suggested to be involved in the development and progression of different neoplasms. This study investigated the protein expression profile of STC1 in PCa and benign prostatic hyperplasia (BPH) samples and STC1 signalling during cell proliferation and cell death in vitro using cell lines. We found higher levels of STC1 in PCa when compared to BPH tissue and that STC1 inhibited forskolin stimulation of cAMP in PC-3 cells. A monoclonal antibody against STC1 was effective in reducing cell proliferation, in promoting cell cycle arrest, and in increasing apoptosis in the same cells. Since STC1 acts as a regulator of prostatic tissue signalling, we suggest that this protein is a novel candidate biomarker for prostrate tumour clinical progression and a potential therapeutic target. Key words: prostatic neoplasm; prostate cancer; Stanniocalcin-1; tumour development; Gleason score.
1. INTRODUCTION Prostate cancer (PCa) is one of the most prevalent tumours in males (Attard et al., 2016) and is responsible for significant morbidity and mortality (Chatterjee, 2003). The aggressiveness of PCa is determined by the Gleason score, a grading system based on histopathological analysis of a tissue biopsy used to evaluate the prognosis of PCa according to the degree of damage of the glandular tissue morphology (Gleason and Mellinger, 1974). Elevated Gleason scores are positively correlated with aggressive and invasive tumours (D’Amico et al., 1998). The origin of this neoplasia is controversial and is suggested to develop from both luminal and basal epithelial cells (Goldstein et al., 2010; Wang et al., 2013). Changes or loss of communication between epithelial cells and stroma by aberrant expression of surface receptor signalling molecules can stimulate the malignant transformation of prostatic cells (Liu et al., 2011). Studies evaluating cell patterns and their communication have shown that stromal cells help to direct differentiation and development of epithelial cells and secrete a number of growth factors that can positively or negatively influence prostate growth and differentiation (Schalken and van Leenders, 2003). However, the mechanisms involved in the PCa development, progression and metastasis are still poorly understood, as well as the factors in the tumour microenvironment that may contribute to pathology. Stanniocalcin (STC) is a secreted glycoprotein, that was initially discovered in the Corpuscles of Stannius (CS) of teleost fish (Lu et al., 1994; Wagner et al., 1998). It has been established that most vertebrates possess two STC orthologue genes (STC1 and STC2) with the exception of teleosts which have paralogues of STC1 and STC2 genes (Chang et al., 2003, 1995; Schein et al., 2012; Varghese et al., 1998; Yoshiko et al., 1999; Zhang et al., 1998). Studies in humans have indicated that increased expression of STC1 and STC2 is associated with the development and/or progression of different types of malignant tumours, an observation that led to suggested that they could be used as a tumour markers (Chang et al., 2015; Dai et al., 2016; Hayase et al., 2015; He et al., 2011; Klopfleisch and Gruber, 2009; Koide and Sasaki, 2006; Liu et al., 2010; Pena et al., 2013). Higher levels of STC1 expression have already been correlated with poor cancer prognosis (Chang et al., 2015; Dai et al., 2016; Hayase et al., 2015; He et al., 2011; Klopfleisch and Gruber, 2009; Koide and Sasaki, 2006; Liu et al., 2010; Pena et al.,
2013; Tohmiya et al., 2004). Furthermore, previous research has found increased in STC1 gene and protein expression in prostate carcinoma, suggesting that STC1 may play a significant role in this type of cancer (Bai et al., 2017; Orr et al., 2012). In the present study, we explored the hypothesis that STC1 is associated with PCa development. Firstly, tissue expression pattern of STC1 in patients with PCa was compared to a non-neoplastic tissue with benign prostatic hyperplasia (BPH) and secondly, STC1 signalling was analysed to elucidate the role of this protein in the PCa. We found that STC1 expression is correlated to progression of malignancy and suggest that this protein could be used as a potential therapeutic target for PCa.
2. MATERIALS AND METHODS 2.1. Tissue analysis 2.1.1. Study population Male patients were recruited from the Urology Service of the Hospital de Clínicas de Porto Alegre (HCPA) (Porto Alegre, Brazil) which authorized the use of tissue samples for this study by signing an Informed Consent Form. The PCa tissues were collected from patients submitted to radical prostatectomy, and the BPH tissues collected from patients submitted to open prostatectomy. Patients between 45 and 90 years old of age with a diagnosis of BPH or PCa were the targets of this study. Patients who received hormone therapy or chemotherapy and/or were diagnosed with another concomitant neoplasia were excluded. Immediately after surgical removal, the collected tissue fragments were fixed in 10% formalin for protein expression analysis and for anatomopathological confirmation. A total of thirty-three samples were analysed for protein expression by immunohistochemistry (IHC): 11 of BPH, 11 of PCa Gleason 6 (3+3) and 11 of PCa Gleason 8 (4+4).
2.1.2. Tissue sample processing and immunohistochemistry (IHC) Samples fixed in 10% formalin were processed and included in paraffin blocks. Sections (4 µm) were heated in a water bath in citrate buffer (pH 6.0) for 1 hour at 95 °C for antigen retrieval. Endogenous peroxidase was blocked using 3% H2O2 solution in
methanol for 30 minutes. Protein blocking to avoid non-specific binding was performed with powdered skimmed milk diluted 5% in PBS for 20 min. IHC was performed on tissue histological sections using a polyclonal rabbit antiSTC1 human primary antibody (FL-247: sc-30183, Santa Cruz Biotechnology, Inc.) diluted 1:100 (2 µg/mL) incubated overnight; and a goat anti-rabbit secondary antibody (IgG (H+L) HRP conjugated cat No. AP307P, Chemicon International) diluted 1:200. The reaction was visualized using DAB chromogen (Liquid Dab, K3468, Dako), according to the manufacturer's recommendations, followed by counterstaining with Harris hematoxylin. A blinded evaluation of each histological slide was made by a pathologist to delimit the neoplasia and hyperplasia regions. Three images of each slide were captured using an Olympus BX51 microscope with the QCapture (v. 2.0.11, Teledyne Qimaging, Canada), and analysed using the ImageJ (ImageJ v1.43j; National Institute of Health, Bethesda, MD, http://rsbweb.nih.gov/ij/). The captured images were submitted to the deconvolution analytical procedure (Ruifrok and Johnston, 2001). The final intensity of the DAB was calculated according to the formula: f = 255 - i, where f = final intensity of DAB and i = mean intensity of DAB obtained by the software. The intensity of DAB ranged from 0 (white, no expression) to 255 (dark brown, high expression).
2.2. Cell lines analysis 2.2.1. Cell lines and culture maintenance The prostate cancer cell lines PC-3 (ATCC® CRL-1435™) and LNCaP (ATCC® CRL-1740™) and the normal human prostate cell line PNT1A (ECACC 9501e 2614) (#Hs 505.T, ATCC® CRL-7306TM) were maintained in RPMI medium (Gibco, BRL) supplemented with 10% fetal bovine serum (FBS, Gibco, BRL) and 0.5 mg/mL kanamycin (Invitrogen) at 37 °C in a humidified atmosphere 5 % CO2 incubator.
2.2.2. Immunocytochemistry (ICC) Detection of STC1 protein expression on human cell lines PC-3, LNCaP and PNT1A was done by immunofluorescence assay. Cells were fixed with 4% paraformaldehyde for 20 min, permeabilized with 0.5% Triton X-100, and subsequently
washed with phosphate buffer saline (PBS). The cells were first incubated with the antiSTC1 primary antibody overnight (diluted 1:100, 2 µg/mL) and subsequently incubated with the goat anti-rabbit IgG (H + L) secondary antibody conjugated with the green fluorophore Alexa Fluor 488 (Invitrogen, A-11034) (1:400). Cells were washed with PBS and the nuclei was stained with DAPI (4,6-diamidino-2-phenylindole; Invitrogen). Fluorescence was acquired using a confocal microscope (FV 1000 Spectral Confocal Microscope, Olympus America) at 488 nm and emission of the captured fluorescence at 520 nm.
2.2.3. RNA extraction, cDNA synthesis and quantitative expression Total RNA was extracted from cells using the Trizol reagent (Invitrogen, USA) following the manufacturer’s protocol. Total RNA was quantified using the NanoDrop 1000 Spectrophotometer (Thermo Fisher Scientific, DE) and 1µg of total RNA was reverse-transcribed into complementary DNA (cDNA) using the GoScript™ Reverse Transcription System kit (Promega, USA). Semi-quantitative real-time PCR was performed with the GoTaq® qPCR Master Mix kit (Promega, USA) using the Applied Biosystems™ 7500 Real-Time PCR System (Applied Biosystems, USA). The mean Ct values from triplicate measurements were used to calculate the expression of STC1 normalized to an internal control glyceraldehyde-3-phosphate dehydrogenase (GAPDH) using the 2-∆∆Ct method (Schmittgen and Livak, 2008).
2.2.4. cAMP assay The effect of the recombinant human STC1 (rhSTC1, Biovendor, Czech Republic) on the inhibition of intracellular production of cAMP in PC-3 cells was analysed using the cAMP Dynamic 2 kit (Cisbio, France) following the manufacturer’s protocol. PC-3 cells were seeded at 37 ºC in 384 well plates (1.5x104 cells/well) and incubated with 5 µM forskolin for 30 min (to achieve maximum stimulation of cAMP) (Sigma-Aldrich, USA) or with 5 µM forskolin for 30 min followed by 1 µM rhSTC1 for 30 min (Terra et al., 2015). The reaction was stopped following the manufacturer’s instructions and plates were read using a Biotek Synergy 4 plate reader (Biotek, USA). The results were standardized according to the manufacturer's recommendations for data analysis.
2.2.5. Cell proliferation assay To evaluate the effect of STC1 on cell proliferation, a concentration-response curve was performed using a monoclonal antibody against STC1 (Mab Anti-STC1; human anti-STC1, C-terminal, cat no 57831, ABCAM, USA). PC-3 cells were seeded in 24-well plates (1 x 103 cells/well) and divided into 4 experimental groups: control (no antibody) and different Mab Anti-STC1 concentrations 0.5 µg/ml, 1.0 µg/ml and 5.0 µg/ml. The choice of concentrations was based on studies that used monoclonal antibodies in anti-cancer therapies for human patients (Hendrikx et al., 2017; Weiner, 2015). Evaluation of cell proliferation was performed after 96 h of treatment using the SR-B Assay (Skehan et al., 1990). To obtain the cell growth ratio of each experimental group, final absorption values were adjusted relative to the initial number of clustered cells (Vichai and Kirtikara, 2006). Proliferation in the control group was considered to be 100%. Optical density (OD) was measured at 510 nm using an Anthos Zenyth 200 rt plate spectrophotometer.
2.2.6. Cell cycle and cell death analysis Human PC-3 cells were seeded in 6 well plates (1x104 cells/well) and treated with 1.0 µg/ml Mab Anti-STC1 for 48h. We have used a shorter time of incubation than in the cell proliferation assay, just long enough to trigger the initiation of cell cycle changes and cell death process by Mab treatment instead of the outcome. For cell cycle analysis cells were fixed and stained 48 hours after treatment with propidium iodide (PI 16 µg/ml) (Sigma-Aldrich, USA). For cell death analysis, the cells were stained 48 hours after treatment with Annexin V-PE (PE Annexin V Apoptosis Detection Kit I BD Pharmingen ™, BD Biosciences, USA). Doxorubicin (4 µM) was used as positive control of cell death induction. A total of 10,000 events were collected for each analysis using a FACS Calibur (BD Biosciences, USA) and the data collected was analysed with BD CellQuest software version 5.1.
2.3. Statistical analysis Quantitative data is presented as the mean ± standard deviation (SD). For the cell culture studies, statistical comparisons were made using the generalized estimating equation (GEE) (Twisk, 2004) followed by Bonferroni post-hoc test. Other data was analysed using one-way analysis of variance (ANOVA) followed by the Bonferroni test. All analyses were performed using SPSS 19.0 (IBM SPPS, Armonk, NY, USA) Differences were considered significant when p < 0.05.
3. RESULTS 3.1. Tissue protein expression of STC1 STC1 protein expression was analysed by IHC in PCa and BPH samples. A significantly stronger signal was detected in the PCa tissue Gleason 8 (129.7±12.50, p <0.01, n=11) when compared to PCa tissue Gleason 6 (112.5±12.27 p <0.01, n=11 and BPH (97.80 ±13.38 p <0.01, n=11) (Figure 1).
Figure 1. IHC detection of STC1 in benign prostatic hyperplasia (BPH) and prostate cancer (PCa) tissues. (A) Graphical representation of the intensity of DAB staining in the different tissues. Each histological slide was analysed using the ImageJ software. Intensity of DAB stain (brown areas) was generated by image deconvolution. Data is represented in arbitrary units and the bars represent mean ± standard deviation. “*”p <0.05; “**”p <0.01 (BPH: n = 11; PCa Gleason 6: n = 11; PCa Gleason 8: n = 11). Tissue sections of representative areas of the histological immunolocalization of STC1 in (B) BPH, (D) PCa Gleason 6 (3+3), and (F) PCa Gleason 8 (4+4). Negative control samples (only incubated with goat anti-rabbit secondary antibody) of each tissue were shown in (C), (E), and (G).
3.2. STC1 gene and protein expression in prostate cell lines The STC1 gene was highly expressed in the PC-3 and LNCaP cell lines compared to the normal PNT1A prostate cell line, in which the transcript level was low or undetectable (p <0.01; Figure 2A). This was further confirmed by the detection of STC1 protein in PC-3 and LNCaP when compared to the control cells (Figure 2B). Because of the high levels of STC1 expression the PC-3 cell line was chosen for the subsequent experiments.
Figure 2. STC1 gene and protein expression in PC-3, LNCaP and PNT1A cell lines. (A) Gene expression of STC1 in human prostate cell lines. STC1 was not detectable in PNT1A. The bars represent the mean of 3 independent experiments performed in triplicate ± standard deviation of the mean, "**" p < 0.01. Immunocytochemistry of STC1 protein in prostatic cells (B) PC-3, (D) LNCaP and (F) non-neoplastic prostate cells PNT1A. STC1 is stained in green and the nuclei are stained in blue. Negative control samples (only incubated with goat anti-rabbit secondary antibody) of each tissue were shown in (C), (E), and (G).
3.3. Effect of rhSTC1 on the inhibition of cAMP-production into PC-3 cells The inhibition of forskolin-stimulated cAMP production by 1 µM rhSTC1in PC3 cells was 70.8% (p <0.01, n = 4) (Figure 3) indicating STC1 regulation cAMP PC-3 signalling via the inhibitory G-protein (Gi).
Figure 3. Inhibition of forskolin-stimulated cAMP production by rhSTC1 in PC-3 cells. The bars represent the mean of 4 independent experiments analysed in triplicate ± standard deviation of the mean.”**”p <0.01.
3.4. Effect of Mab Anti-STC1 on PC-3 cell proliferation A significant decrease in cell proliferation rate (at least 50%) was observed at all concentrations of Mab Anti-STC1 tested (0.5 µg/mL, 1.0 µg/mL and 5.0 µg/mL) compared to the control group (p <0.05, n=3). The concentration of 1.0 µg/mL Mab Anti-STC1 was selected to analyse its effects on the cell cycle.
Figure 4. Effect of Mab Anti-STC1 on PC-3 cell proliferation. The bars represent the mean of 3 independent experiments analysed in quadruplicate ± standard deviation of the mean after 96 hours treatment, "*" p <0.05.
3.5. Effect of Mab Anti-STC1 on the cell cycle and cell death of human PC-3 cells PC-3 cells treated for 48 h with 1.0 µg/mL Mab Anti-STC1 showed an increase in the number of cells in G1-phase (a difference of 13.12%) when compared to the control (p <0.01, n=3) (Figure 5A and B). No statistically significant differences were detected in the S and G2-phases (p >0.05, n=3) between the control and the Mab AntiSTC1 treated group (Figure 5B).
Figure 5. Effect of Mab Anti-STC1 (1.0 µg/mL) on the cell cycle of prostate cancer cell line PC-3. (A) Cell cycle distribution: the continuous line represents the control group and the dotted line the treated cells. (B) Distribution of cells (%) between G1, S and G2 phases in the treated and control groups. The bars represent the mean of 3 independent experiments ± standard deviation of the mean, "**"p <0.01.
The percentage of viable cells in the Mab Anti-STC1 group decreased significantly (15.38%) when compared to the control group (p <0.05, n=3) (Figure 6). In the presence of Mab Anti-STC1 the number of cells that underwent apoptosis increased significantly (18.43%) and this corresponds to a 4-fold increase (p <0.01, n=3) in relation to the control group (Figure 6). There were no significant differences in the proportion of cells in necrosis and in late apoptosis between groups (p >0.05, n=3).
Figure 6. Cell death profile of PC-3 prostate cancer cells treated with Mab AntiSTC1 (1.0 µg/ml). (A) Percentage of cells in each quadrant. The bars represent the mean of 3 independent experiments ± standard deviation of the mean, "*" p <0.05; "**" p <0.01. Representative raw scatter plots of the cells in the flow cytometry quadrants of the control (B) and treated (C) groups.
4. DISCUSSION In the present study we have shown that STC1 expression is more abundant in PCa tissue in direct proportion to the severity of malignancy. We have also shown that STC1 inhibits cell cAMP production and that a specific anti-STC1 antibody reduces cell proliferation and increases cell death in prostate cancer cell lines. The expression levels of STC1 in PCa were significantly higher in more aggressive tumours (Gleason ≥ 8) in relation to less aggressive tumours (Gleason score = 6) and BPH tissue. STC1 gene expression was also found to be highly expressed in the prostate cancer lines PC-3 and LNCaP when compared to normal prostate cell line PNT1A, consistent with histological protein expression and previous studies with different classes of human tumors such as colorectal, ovarian, breast, hepatocellular, and thyroid cancer (Chang et al., 2015; Dai et al., 2016; Hayase et al., 2015; He et al., 2011;
Klopfleisch and Gruber, 2009; Koide and Sasaki, 2006; Liu et al., 2010; Pena et al., 2013; Tohmiya et al., 2004). According Orr et al. (2012), the STC1 was localized in areas of tumour stroma and can act as a potential Epithelial-Mesenchymal Transition (EMT) marker. This may indicate that these stromal cell subsets express important regulators of epithelia (Orr et al., 2012). The most accepted theory for the PCa development is from epithelial cells; however, the STC1 signalling could be important for the PCa progression at the both compartments, and the increased staining at more aggressive cancers could be related to the tissue architecture loss through the progression of the disease. Based on our findings, we suggest that STC1 may be a candidate biomarker for the prognosis of prostate cancer in addition or in alternative to the classical tissue morphological methods and the STC1 signalling could be a potential target for future PCa therapies. The rhSTC1 strongly inhibited, by at least 50%, forskolin-stimulated cAMP production in PC-3 cells and this is in agreement with our previous studies on the effect of STC1 on cAMP production using mammalian in HEK293 cells (Terra et al., 2015). Furthermore, once STC1 signalling was blocked by Mab Anti-STC1 in PC-3 prostate cancer cells, cell proliferation was also reduced up to 70%, supporting the hypothesis that this protein is involved in cell growth signalling. This provides further support to an association of STC1 to cell proliferation, as demonstrated by overexpression of STC1 in cells and by gene knock out (Bai et al., 2017; Ma et al., 2015). STC1 is considered a pro-survival factor in differentiated cells, besides being involved in epithelial and mesenchymal signalling and development (Joensuu et al., 2008). PC-3 prostate cancer cells when treated with Mab Anti-STC1 showed an arrest in cell cycle in the G0/G1 phase. This pattern corroborates with studies that evaluated the effect of STC1 silencing in different tumour cells through small interfering RNA (siRNA), where the absence of STC1 led to an accumulation of G0/G1 cells and a reduction in the proliferation of carcinoma cells (Ma et al., 2015), prostate cancer cells (Bai et al., 2017) and ovarian cancer (Liu et al., 2010). In addition, Bai et al. (2017) also demonstrated that, by knocking out STC1 gene in LNCaP and DU145 cells, the reduced cell proliferation was achieved due to changes in the levels of cyclin E1 and Cdk2 proteins (Bai et al., 2017). It is widely known that defects in the apoptotic pathway contribute to tumorigenesis (Wong, 2011) and that this may exert a positive influence on the anti-
apoptotic pathway (Kim et al., 2013), and possibly it is one of the target mechanisms regulated by STC1. Previous studies have shown that STC1 can act as an inhibitor of cellular apoptosis and that when STC1 levels are attenuated due to the blockade of the C-terminal region of STC1, there was an approximately 4-fold increase in cell death via apoptosis (Law et al., 2008; Li et al., 2008). Since in PC-3 (metastatic prostate cancer in bone) cells elevated cAMP levels are positively correlated with the inhibition of cell growth and apoptosis (Bang et al., 1992; Carraway and Mitra, 1998), the decrease in cAMP caused by the presence of rhSTC1 supports this hypothesis that STC1 could be involved in cell proliferation and PCa tumour growth. Thus, the high concentrations of STC1 detected in PCa cells, and possibly the reduced levels of intracellular cAMP evoked by the presence of STC1 may stimulate cell growth and tumour progression. Whilst no specific receptor for STC1 has been identified, it is possible that STC1 intracellular signalling may recruit known cell proliferative pathways such as mitogenactivated protein (MAP) kinase cascade (Schmitt and Stork, 2001). It is still unclear whether STC1 is a cause of tumorigenesis or if it acts as a coreceptor promoting and facilitating the underlying mechanisms involved in establishing signalling and the subsequent progression of neoplastic cell multiplication. As such, one of the limitations of this study was not being able to determine if the STC1 detected by IHC in PCa and BPH tissues was produced locally or in another tissue. If STC1 exerts its effects in prostate cells being produced in other organ, it is possible that after signalling the protein could be internalized and thus detected during staining. Additionally, an aspect to be analysed in future work is the measurement of STC1 concentrations in patient’s blood and urine to further support the possible use of STC1 as a PCa progression biomarker. Nonetheless, this study provides evidence for the use of STC1 staining in tissue samples as a prognostic factor for tumour aggressiveness.
5. CONCLUSION Uncontrolled growth of prostatic cells is influenced by multiple factors including the increased synthesis and secretion of STC1 which induces cell proliferation and reduces apoptosis. Considering that STC1 can act as a regulator of stromal action and paracrine signalling in the prostatic tissue, this protein could be considered as a novel biomarker for tumour progression and clinical follow-up as well as the target for future
therapeutics. Our results also suggests possible ways for therapeutic intervention, such as the use of monoclonal antibodies or gene silencing approaches to neutralize STC1 expression, in order to restore normal intracellular cAMP levels, to inhibit of cell growth and proliferation, and to induce cells to programmed death via apoptosis. However, the main physiological and pathological roles of STC1 have yet to be established. Knowledge of the mechanisms by which STC1 signals and establishes a cellular response is fundamental to understand its association with pathophysiological disorders.
ACKNOWLEDGMENTS We thank Elvis Branchini for his image edition assistance. This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior – Brasil (CAPES) – Finance Code 001 –and Fundo de Incentivo à Pesquisa do Hospital de Clínicas de Porto Alegre (FIPE-HCPA) (Grant number 15-0382) and by the Portuguese Foundation
for
Science
and
Technology
(FCT)
through
project
CCMAR/Multi/04326/2013.
CONFLICT OF INTEREST STATEMENT The authors declare that there are no conflicts of interest.
Ethical approval: All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.
Informed consent: Informed consent was obtained from all individual participants included in the study.
REFERENCES Attard, G., Parker, C., Eeles, R.A., Schröder, F., Tomlins, S.A., Tannock, I., Drake, C.G., de Bono, J.S., 2016. Prostate cancer. Lancet 387, 70–82. https://doi.org/10.1016/S0140-6736(14)61947-4 Bai, Y., Xiao, Y., Dai, Y., Chen, X., Li, D., Tan, X., Zhang, X., 2017. Stanniocalcin 1 promotes cell proliferation via cyclin E1/cyclin‑dependent kinase 2 in human prostate carcinoma. Oncol. Rep. 37, 2465–2471. https://doi.org/10.3892/or.2017.5501 Bang, Y.J., Kim, S.J., Danielpour, D., O’Reilly, M. a, Kim, K.Y., Myers, C.E., Trepel, J.B., 1992. Cyclic AMP induces transforming growth factor beta 2 gene expression and growth arrest in the human androgen-independent prostate carcinoma cell line PC-3. Proc. Natl. Acad. Sci. U. S. A. 89, 3556–60. Carraway, R.E., Mitra, S.P., 1998. Neurotensin enhances agonist-induced cAMP accumulation in PC3 cells via Ca2+-dependent adenylyl cyclase(s). Mol. Cell. Endocrinol. 144, 47–57. https://doi.org/10.1016/S0303-7207(98)00154-3 Chang, A.C.-M., Doherty, J., Huschtscha, L.I., Redvers, R., Restall, C., Reddel, R.R., Anderson, R.L., 2015. STC1 expression is associated with tumor growth and metastasis in breast cancer. Clin. Exp. Metastasis 32, 15–27. https://doi.org/10.1007/s10585-014-9687-9 Chang, A.C.M., Janosi, J., Hulsbeek, M., de Jong, D., Jeffrey, K.J., Noble, J.R., Reddel, R.R., 1995. A novel human cDNA highly homologous to the fish hormone stanniocalcin. Mol. Cell. Endocrinol. 112, 241–247. https://doi.org/10.1016/03037207(95)03601-3 Chang, A.C.M., Jellinek, D.A., Reddel, R.R., 2003. Mammalian stanniocalcins and cancer. Endocr. Relat. Cancer. https://doi.org/10.1677/erc.0.0100359 Chatterjee, B., 2003. The role of the androgen receptor in the development of prostatic hyperplasia and prostate cancer. Mol. Cell. Biochem. 253, 89–101. https://doi.org/10.1023/A:1026057402945 D’Amico, A. V, Whittington, R., Malkowicz, S.B., Schultz, D., Blank, K., Broderick, G.A., Tomaszewski, J.E., Renshaw, A.A., Kaplan, I., Beard, C.J., Wein, A., 1998. Biochemical outcome after radical prostatectomy, external beam radiation therapy, or interstitial radiation therapy for clinically localized prostate cancer. JAMA 280, 969–974. https://doi.org/joc80111 [pii] Dai, D., Wang, Q., Li, X., Liu, J., Ma, X., Xu, W., 2016. Klotho inhibits human follicular thyroid cancer cell growth and promotes apoptosis through regulation of the expression of stanniocalcin-1. Oncol. Rep. 35, 552–558. https://doi.org/10.3892/or.2015.4358 Gleason, D.F., Mellinger, G.T., 1974. Prediction of Prognosis for Prostatic Adenocarcinoma by Combined Histological Grading and Clinical Staging. J. Urol. 111, 58–64. https://doi.org/10.1016/j.juro.2016.10.099 Goldstein, A.S., Huang, J., Guo, C., Garraway, I.P., Witte, O.N., 2010. Identification of a cell of origin for human prostate cancer. Science 329, 568–71. https://doi.org/10.1126/science.1189992 Hayase, S., Sasaki, Y., Matsubara, T., Seo, D., Miyakoshi, M., Murata, T., Ozaki, T.,
Kakudo, K., Kumamoto, K., Ylaya, K., Cheng, S., Thorgeirsson, S.S., Hewitt, S.M., Ward, J.M., Kimura, S., 2015. Expression of stanniocalcin 1 in thyroid side population cells and thyroid cancer cells. Thyroid 25, 425–36. https://doi.org/10.1089/thy.2014.0464 He, L., Wang, T., Gao, Q., Zhao, G., Huang, Y., Yu, L., Hou, Y., 2011. Stanniocalcin-1 promotes tumor angiogenesis through up-regulation of VEGF in gastric cancer cells. J. Biomed. Sci. 18, 39. https://doi.org/10.1186/1423-0127-18-39 Hendrikx, J.J.M.A., Haanen, J.B.A.. G., Voest, E.E., Schellens, J.H.M., Huitema, A.D.R., Beijnen, J.H., 2017. Fixed Dosing of Monoclonal Antibodies in Oncology. Oncologist theoncologist.2017-0167. https://doi.org/10.1634/theoncologist.20170167 Joensuu, K., Heikkilä, P., Andersson, L.C., 2008. Tumor dormancy: Elevated expression of stanniocalcins in late relapsing breast cancer. Cancer Lett. 265, 76– 83. https://doi.org/10.1016/j.canlet.2008.02.022 Kim, S.J., Ko, J.H., Yun, J.H., Kim, J.A., Kim, T.E., Lee, H.J., Kim, S.H., Park, K.H., Oh, J.Y., 2013. Stanniocalcin-1 Protects Retinal Ganglion Cells by Inhibiting Apoptosis and Oxidative Damage. PLoS One 8. https://doi.org/10.1371/journal.pone.0063749 Klopfleisch, R., Gruber, A.D., 2009. Derlin-1 and Stanniocalcin-1 are Differentially Regulated in Metastasizing Canine Mammary Adenocarcinomas. J. Comp. Pathol. 141, 113–120. https://doi.org/10.1016/j.jcpa.2008.09.010 Koide, Y., Sasaki, T., 2006. [Stanniocalcin-1 (STC-1) as a molecular marker for human cancer]. Rinsho Byori 54, 213–220. Law, A.Y.S., Lai, K.P., Lui, W.C., Wan, H.T., Wong, C.K.C., 2008. Histone deacetylase inhibitor-induced cellular apoptosis involves stanniocalcin-1 activation. Exp. Cell Res. 314, 2975–2984. https://doi.org/10.1016/j.yexcr.2008.07.002 Li, K., Dong, D., Yao, L., Dai, D., Gu, X., Guo, L., 2008. Identification of STC1 as an beta-amyloid activated gene in human brain microvascular endothelial cells using cDNA microarray. Biochem Biophys Res Commun 376, 399–403. https://doi.org/S0006-291X(08)01733-6 [pii]\r10.1016/j.bbrc.2008.08.158 Liu, A.Y., Pascal, L.E., Vêncio, R.Z., Vêncio, E.F., 2011. Stromal-epithelial interactions in early neoplasia. Cancer Biomarkers 9, 141–155. https://doi.org/10.3233/CBM-2011-0174 Liu, G., Yang, G., Chang, B., Mercado-Uribe, I., Huang, M., Zheng, J., Bast, R.C., Lin, S.H., Liu, J., 2010. Stanniocalcin 1 and ovarian tumorigenesis. J. Natl. Cancer Inst. 102, 812–827. https://doi.org/10.1093/jnci/djq127 Lu, M., Wagner, G.F., Renfro, J.L., 1994. Stanniocalcin stimulates phosphate reabsorption by flounder renal proximal tubule in primary culture. Am. J. Physiol. 267, R1356–R1362. Ma, X., Gu, L., Li, H., Gao, Y., Li, X., Shen, D., Gong, H., Li, S., Niu, S., Zhang, Y., Fan, Y., Huang, Q., Lyu, X., Zhang, X., 2015. Hypoxia-induced overexpression of stanniocalcin-1 is associated with the metastasis of early stage clear cell renal cell carcinoma. J. Transl. Med. 13, 56. https://doi.org/10.1186/s12967-015-0421-4 Orr, B., Riddick, A.C.P., Stewart, G.D., Anderson, R.A., Franco, O.E., Hayward, S.W.,
Thomson, A.A., 2012. Identification of stromally expressed molecules in the prostate by tag-profiling of cancer-associated fibroblasts, normal fibroblasts and fetal prostate. Oncogene 31, 1130–42. https://doi.org/10.1038/onc.2011.312 Pena, C., Cespedes, M.V., Lindh, M.B., Kiflemariam, S., Mezheyeuski, A., Edqvist, P.H., Hagglof, C., Birgisson, H., Bojmar, L., Jirstrom, K., Sandstrom, P., Olsson, E., Veerla, S., Gallardo, A., Sjoblom, T., Chang, A.C.M., Reddel, R.R., Mangues, R., Augsten, M., Ostman, A., 2013. STC1 expression by cancer-associated fibroblasts drives metastasis of colorectal cancer. Cancer Res. 73, 1287–1297. https://doi.org/10.1158/0008-5472.CAN-12-1875 Ruifrok, A.C., Johnston, D.A., 2001. Quantification of histochemical staining by color deconvolution. Anal. Quant. Cytol. Histol. 23, 291–299. https://doi.org/10.1097/00129039-200303000-00014 Schalken, J.A., van Leenders, G., 2003. Cellular and molecular biology of the prostate: stem cell biology. Urology 62, 11–20. Schein, V., Cardoso, J.C.R., Pinto, P.I.S., Anjos, L., Silva, N., Power, D.M., Canário, A.V.M., 2012. Four stanniocalcin genes in teleost fish: Structure, phylogenetic analysis, tissue distribution and expression during hypercalcemic challenge. Gen. Comp. Endocrinol. 175, 344–356. https://doi.org/10.1016/j.ygcen.2011.11.033 Schmitt, J.M., Stork, P.J.S., 2001. Cyclic AMP-Mediated Inhibition of Cell Growth Requires the Small G Protein Rap1. Mol. Cell. Biol. 21, 3671–3683. https://doi.org/10.1128/mcb.21.11.3671-3683.2001 Schmittgen, T.D., Livak, K.J., 2008. Analyzing real-time PCR data by the comparative CT method. Nat. Protoc. 3, 1101–1108. https://doi.org/10.1038/nprot.2008.73 Skehan, P., Storeng, R., Scudiero, D., Monks, a, McMahon, J., Vistica, D., Warren, J.T., Bokesch, H., Kenney, S., Boyd, M.R., 1990. New colorimetric cytotoxicity assay for anticancer-drug screening. J. Natl. Cancer Inst. 82, 1107–1112. https://doi.org/10.1093/jnci/82.13.1107 Terra, S.R., Cardoso, J.C.R., Félix, R.C., Martins, L.A.M., Souza, D.O.G., Guma, F.C.R., Canário, A.V.M., Schein, V., 2015. STC1 interference on calcitonin family of receptors signaling during osteoblastogenesis via adenylate cyclase inhibition. Mol. Cell. Endocrinol. 403, 78–87. https://doi.org/10.1016/j.mce.2015.01.010 Tohmiya, Y., Koide, Y., Fujimaki, S., Harigae, H., Funato, T., Kaku, M., Ishii, T., Munakata, Y., Kameoka, J., Sasaki, T., 2004. Stanniocalcin-1 as a Novel Marker to Detect Minimal Residual Disease of Human Leukemia. Tohoku J. Exp. Med. 204, 125–133. https://doi.org/10.1620/tjem.204.125 Twisk, J.W.R., 2004. Longitudinal data analysis. A comparison between generalized estimating equations and random coefficient analysis. Eur. J. Epidemiol. 19, 769– 776. https://doi.org/10.1023/B:EJEP.0000036572.00663.f2 Varghese, R., Wong, C.K.C., Deol, H., Wagner, G.F., Dimattia, G.E., 1998. Comparative analysis of mammalian stanniocalcin genes. Endocrinology 139, 4714–4725. https://doi.org/10.1210/en.139.11.4714 Vichai, V., Kirtikara, K., 2006. Sulforhodamine B colorimetric assay for cytotoxicity screening. Nat. Protoc. 1, 1112–1116. https://doi.org/10.1038/nprot.2006.179 Wagner, G.F., Jaworski, E.M., Haddad, M., 1998. Stanniocalcin in the seawater salmon: structure, function, and regulation. Am J Physiol Regul Integr Comp Physiol 274,
R1177-1185. Wang, Z.A., Mitrofanova, A., Bergren, S.K., Abate-Shen, C., Cardiff, R.D., Califano, A., Shen, M.M., 2013. Lineage analysis of basal epithelial cells reveals their unexpected plasticity and supports a cell-of-origin model for prostate cancer heterogeneity. Nat. Cell Biol. 15, 274–283. https://doi.org/10.1038/ncb2697 Weiner, G.J., 2015. Building better monoclonal antibody-based therapeutics. Nat. Rev. Cancer. https://doi.org/10.1038/nrc3930 Wong, R.S.Y., 2011. Apoptosis in cancer: from pathogenesis to treatment. J. Exp. Clin. Cancer Res. 30, 87. https://doi.org/10.1186/1756-9966-30-87 Yoshiko, Y., Son, A., Maeda, S., Igarashi, A., Takano, S., Hu, J., Maeda, N., 1999. Evidence for stanniocalcin gene expression in mammalian bone. Endocrinology 140, 1869–1874. https://doi.org/10.1210/en.140.4.1869 Zhang, K.Z., Westberg, J.A., Paetau, A., Von Boguslawsky, K., Lindsberg, P., Erlander, M., Guo, H., Su, J., Olsen, H.S., Andersson, L.C., 1998. High expression of stanniocalcin in differentiated brain neurons. Am. J. Pathol. 153, 439–445. https://doi.org/10.1016/S0002-9440(10)65587-3
Highlights •
Expression levels of STC1 in PCa were significantly higher in more aggressive tumors.
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STC1 inhibits cAMP production in PCa cells in vitro.
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Specific anti-STC1 antibody treatment reduces cell proliferation in PCa cell line
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Specific anti-STC1 antibody treatment increases apoptosis in PCa cell line
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STC1 signalling can be a potential target for PCa therapies.
S0303-7207(19)30361-2
DOI:
https://doi.org/10.1016/j.mce.2019.110659
Reference:
MCE 110659
To appear in:
Molecular and Cellular Endocrinology
Received Date: 19 September 2019 Revised Date:
18 November 2019
Accepted Date: 18 November 2019
Please cite this article as: Costa, B.P., Schein, V., Rafael, Z., Santos, A.S., Kliemann, L.M., Nunes, F.B., Carlos dos Reis Cardoso, Joã., Félix, R.C., Mendonça Canário, A.V., Brum, I.S., Branchini, G., Stanniocalcin-1 protein expression profile and mechanisms in proliferation and cell death pathways in prostate cancer, Molecular and Cellular Endocrinology (2020), doi: https://doi.org/10.1016/ j.mce.2019.110659. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier B.V.
Stanniocalcin-1 protein expression profile and mechanisms in proliferation and cell death pathways in prostate cancer Bruna Pasqualotto Costa1, Vanessa Schein2, Zhao Rafael3, Andressa Schneiders Santos3, Lucia Maria Kliemann4, Fernanda Bordignon Nunes1, João Carlos dos Reis Cardoso5, Rute Castelo Félix5, Adelino Vicente Mendonça Canário5, Ilma Simoni Brum2, Gisele Branchini1. 1
Programa de Pós-Graduação em Patologia, Universidade Federal de Ciências da Saúde de Porto Alegre, Brazil. 2
Departmento de Fisiologia, Instituto de Ciências Básicas da Saúde, Universidade Federal de Ciências da Saúde de Porto Alegre, Porto Alegre, Brazil.
3 4
Universidade Federal de Ciências da Saúde de Porto Alegre, Porto Alegre, Brazil.
Departmento de Patologia, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil. 5
Centre of Marine Sciences (CCMAR), University of Algarve, Campus de Gambelas, Faro, Portugal.
*Corresponding author: Dra. Gisele Branchini Department of Basic Health Sciences, Universidade de Ciências da Saúde de Porto Alegre (UFCSPA), 245 Sarmento Leite, Rio Grande do Sul, Porto Alegre, Brazil. Email address:
[email protected]
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Phone
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33038753
ABSTRACT
Prostate cancer (PCa) is one of the most prevalent male tumours. Stanniocalcin-1 (STC1) is a glycoprotein and, although the role of STC1 in human cancer is poorly understood, it is suggested to be involved in the development and progression of different neoplasms. This study investigated the protein expression profile of STC1 in PCa and benign prostatic hyperplasia (BPH) samples and STC1 signalling during cell proliferation and cell death in vitro using cell lines. We found higher levels of STC1 in PCa when compared to BPH tissue and that STC1 inhibited forskolin stimulation of cAMP in PC-3 cells. A monoclonal antibody against STC1 was effective in reducing cell proliferation, in promoting cell cycle arrest, and in increasing apoptosis in the same cells. Since STC1 acts as a regulator of prostatic tissue signalling, we suggest that this protein is a novel candidate biomarker for prostrate tumour clinical progression and a potential therapeutic target. Key words: prostatic neoplasm; prostate cancer; Stanniocalcin-1; tumour development; Gleason score.
1. INTRODUCTION Prostate cancer (PCa) is one of the most prevalent tumours in males (Attard et al., 2016) and is responsible for significant morbidity and mortality (Chatterjee, 2003). The aggressiveness of PCa is determined by the Gleason score, a grading system based on histopathological analysis of a tissue biopsy used to evaluate the prognosis of PCa according to the degree of damage of the glandular tissue morphology (Gleason and Mellinger, 1974). Elevated Gleason scores are positively correlated with aggressive and invasive tumours (D’Amico et al., 1998). The origin of this neoplasia is controversial and is suggested to develop from both luminal and basal epithelial cells (Goldstein et al., 2010; Wang et al., 2013). Changes or loss of communication between epithelial cells and stroma by aberrant expression of surface receptor signalling molecules can stimulate the malignant transformation of prostatic cells (Liu et al., 2011). Studies evaluating cell patterns and their communication have shown that stromal cells help to direct differentiation and development of epithelial cells and secrete a number of growth factors that can positively or negatively influence prostate growth and differentiation (Schalken and van Leenders, 2003). However, the mechanisms involved in the PCa development, progression and metastasis are still poorly understood, as well as the factors in the tumour microenvironment that may contribute to pathology. Stanniocalcin (STC) is a secreted glycoprotein, that was initially discovered in the Corpuscles of Stannius (CS) of teleost fish (Lu et al., 1994; Wagner et al., 1998). It has been established that most vertebrates possess two STC orthologue genes (STC1 and STC2) with the exception of teleosts which have paralogues of STC1 and STC2 genes (Chang et al., 2003, 1995; Schein et al., 2012; Varghese et al., 1998; Yoshiko et al., 1999; Zhang et al., 1998). Studies in humans have indicated that increased expression of STC1 and STC2 is associated with the development and/or progression of different types of malignant tumours, an observation that led to suggested that they could be used as a tumour markers (Chang et al., 2015; Dai et al., 2016; Hayase et al., 2015; He et al., 2011; Klopfleisch and Gruber, 2009; Koide and Sasaki, 2006; Liu et al., 2010; Pena et al., 2013). Higher levels of STC1 expression have already been correlated with poor cancer prognosis (Chang et al., 2015; Dai et al., 2016; Hayase et al., 2015; He et al., 2011; Klopfleisch and Gruber, 2009; Koide and Sasaki, 2006; Liu et al., 2010; Pena et al.,
2013; Tohmiya et al., 2004). Furthermore, previous research has found increased in STC1 gene and protein expression in prostate carcinoma, suggesting that STC1 may play a significant role in this type of cancer (Bai et al., 2017; Orr et al., 2012). In the present study, we explored the hypothesis that STC1 is associated with PCa development. Firstly, tissue expression pattern of STC1 in patients with PCa was compared to a non-neoplastic tissue with benign prostatic hyperplasia (BPH) and secondly, STC1 signalling was analysed to elucidate the role of this protein in the PCa. We found that STC1 expression is correlated to progression of malignancy and suggest that this protein could be used as a potential therapeutic target for PCa.
2. MATERIALS AND METHODS 2.1. Tissue analysis 2.1.1. Study population Male patients were recruited from the Urology Service of the Hospital de Clínicas de Porto Alegre (HCPA) (Porto Alegre, Brazil) which authorized the use of tissue samples for this study by signing an Informed Consent Form. The PCa tissues were collected from patients submitted to radical prostatectomy, and the BPH tissues collected from patients submitted to open prostatectomy. Patients between 45 and 90 years old of age with a diagnosis of BPH or PCa were the targets of this study. Patients who received hormone therapy or chemotherapy and/or were diagnosed with another concomitant neoplasia were excluded. Immediately after surgical removal, the collected tissue fragments were fixed in 10% formalin for protein expression analysis and for anatomopathological confirmation. A total of thirty-three samples were analysed for protein expression by immunohistochemistry (IHC): 11 of BPH, 11 of PCa Gleason 6 (3+3) and 11 of PCa Gleason 8 (4+4).
2.1.2. Tissue sample processing and immunohistochemistry (IHC) Samples fixed in 10% formalin were processed and included in paraffin blocks. Sections (4 µm) were heated in a water bath in citrate buffer (pH 6.0) for 1 hour at 95 °C for antigen retrieval. Endogenous peroxidase was blocked using 3% H2O2 solution in
methanol for 30 minutes. Protein blocking to avoid non-specific binding was performed with powdered skimmed milk diluted 5% in PBS for 20 min. IHC was performed on tissue histological sections using a polyclonal rabbit antiSTC1 human primary antibody (FL-247: sc-30183, Santa Cruz Biotechnology, Inc.) diluted 1:100 (2 µg/mL) incubated overnight; and a goat anti-rabbit secondary antibody (IgG (H+L) HRP conjugated cat No. AP307P, Chemicon International) diluted 1:200. The reaction was visualized using DAB chromogen (Liquid Dab, K3468, Dako), according to the manufacturer's recommendations, followed by counterstaining with Harris hematoxylin. A blinded evaluation of each histological slide was made by a pathologist to delimit the neoplasia and hyperplasia regions. Three images of each slide were captured using an Olympus BX51 microscope with the QCapture (v. 2.0.11, Teledyne Qimaging, Canada), and analysed using the ImageJ (ImageJ v1.43j; National Institute of Health, Bethesda, MD, http://rsbweb.nih.gov/ij/). The captured images were submitted to the deconvolution analytical procedure (Ruifrok and Johnston, 2001). The final intensity of the DAB was calculated according to the formula: f = 255 - i, where f = final intensity of DAB and i = mean intensity of DAB obtained by the software. The intensity of DAB ranged from 0 (white, no expression) to 255 (dark brown, high expression).
2.2. Cell lines analysis 2.2.1. Cell lines and culture maintenance The prostate cancer cell lines PC-3 (ATCC® CRL-1435™) and LNCaP (ATCC® CRL-1740™) and the normal human prostate cell line PNT1A (ECACC 9501e 2614) (#Hs 505.T, ATCC® CRL-7306TM) were maintained in RPMI medium (Gibco, BRL) supplemented with 10% fetal bovine serum (FBS, Gibco, BRL) and 0.5 mg/mL kanamycin (Invitrogen) at 37 °C in a humidified atmosphere 5 % CO2 incubator.
2.2.2. Immunocytochemistry (ICC) Detection of STC1 protein expression on human cell lines PC-3, LNCaP and PNT1A was done by immunofluorescence assay. Cells were fixed with 4% paraformaldehyde for 20 min, permeabilized with 0.5% Triton X-100, and subsequently
washed with phosphate buffer saline (PBS). The cells were first incubated with the antiSTC1 primary antibody overnight (diluted 1:100, 2 µg/mL) and subsequently incubated with the goat anti-rabbit IgG (H + L) secondary antibody conjugated with the green fluorophore Alexa Fluor 488 (Invitrogen, A-11034) (1:400). Cells were washed with PBS and the nuclei was stained with DAPI (4,6-diamidino-2-phenylindole; Invitrogen). Fluorescence was acquired using a confocal microscope (FV 1000 Spectral Confocal Microscope, Olympus America) at 488 nm and emission of the captured fluorescence at 520 nm.
2.2.3. RNA extraction, cDNA synthesis and quantitative expression Total RNA was extracted from cells using the Trizol reagent (Invitrogen, USA) following the manufacturer’s protocol. Total RNA was quantified using the NanoDrop 1000 Spectrophotometer (Thermo Fisher Scientific, DE) and 1µg of total RNA was reverse-transcribed into complementary DNA (cDNA) using the GoScript™ Reverse Transcription System kit (Promega, USA). Semi-quantitative real-time PCR was performed with the GoTaq® qPCR Master Mix kit (Promega, USA) using the Applied Biosystems™ 7500 Real-Time PCR System (Applied Biosystems, USA). The mean Ct values from triplicate measurements were used to calculate the expression of STC1 normalized to an internal control glyceraldehyde-3-phosphate dehydrogenase (GAPDH) using the 2-∆∆Ct method (Schmittgen and Livak, 2008).
2.2.4. cAMP assay The effect of the recombinant human STC1 (rhSTC1, Biovendor, Czech Republic) on the inhibition of intracellular production of cAMP in PC-3 cells was analysed using the cAMP Dynamic 2 kit (Cisbio, France) following the manufacturer’s protocol. PC-3 cells were seeded at 37 ºC in 384 well plates (1.5x104 cells/well) and incubated with 5 µM forskolin for 30 min (to achieve maximum stimulation of cAMP) (Sigma-Aldrich, USA) or with 5 µM forskolin for 30 min followed by 1 µM rhSTC1 for 30 min (Terra et al., 2015). The reaction was stopped following the manufacturer’s instructions and plates were read using a Biotek Synergy 4 plate reader (Biotek, USA). The results were standardized according to the manufacturer's recommendations for data analysis.
2.2.5. Cell proliferation assay To evaluate the effect of STC1 on cell proliferation, a concentration-response curve was performed using a monoclonal antibody against STC1 (Mab Anti-STC1; human anti-STC1, C-terminal, cat no 57831, ABCAM, USA). PC-3 cells were seeded in 24-well plates (1 x 103 cells/well) and divided into 4 experimental groups: control (no antibody) and different Mab Anti-STC1 concentrations 0.5 µg/ml, 1.0 µg/ml and 5.0 µg/ml. The choice of concentrations was based on studies that used monoclonal antibodies in anti-cancer therapies for human patients (Hendrikx et al., 2017; Weiner, 2015). Evaluation of cell proliferation was performed after 96 h of treatment using the SR-B Assay (Skehan et al., 1990). To obtain the cell growth ratio of each experimental group, final absorption values were adjusted relative to the initial number of clustered cells (Vichai and Kirtikara, 2006). Proliferation in the control group was considered to be 100%. Optical density (OD) was measured at 510 nm using an Anthos Zenyth 200 rt plate spectrophotometer.
2.2.6. Cell cycle and cell death analysis Human PC-3 cells were seeded in 6 well plates (1x104 cells/well) and treated with 1.0 µg/ml Mab Anti-STC1 for 48h. We have used a shorter time of incubation than in the cell proliferation assay, just long enough to trigger the initiation of cell cycle changes and cell death process by Mab treatment instead of the outcome. For cell cycle analysis cells were fixed and stained 48 hours after treatment with propidium iodide (PI 16 µg/ml) (Sigma-Aldrich, USA). For cell death analysis, the cells were stained 48 hours after treatment with Annexin V-PE (PE Annexin V Apoptosis Detection Kit I BD Pharmingen ™, BD Biosciences, USA). Doxorubicin (4 µM) was used as positive control of cell death induction. A total of 10,000 events were collected for each analysis using a FACS Calibur (BD Biosciences, USA) and the data collected was analysed with BD CellQuest software version 5.1.
2.3. Statistical analysis Quantitative data is presented as the mean ± standard deviation (SD). For the cell culture studies, statistical comparisons were made using the generalized estimating equation (GEE) (Twisk, 2004) followed by Bonferroni post-hoc test. Other data was analysed using one-way analysis of variance (ANOVA) followed by the Bonferroni test. All analyses were performed using SPSS 19.0 (IBM SPPS, Armonk, NY, USA) Differences were considered significant when p < 0.05.
3. RESULTS 3.1. Tissue protein expression of STC1 STC1 protein expression was analysed by IHC in PCa and BPH samples. A significantly stronger signal was detected in the PCa tissue Gleason 8 (129.7±12.50, p <0.01, n=11) when compared to PCa tissue Gleason 6 (112.5±12.27 p <0.01, n=11 and BPH (97.80 ±13.38 p <0.01, n=11) (Figure 1).
Figure 1. IHC detection of STC1 in benign prostatic hyperplasia (BPH) and prostate cancer (PCa) tissues. (A) Graphical representation of the intensity of DAB staining in the different tissues. Each histological slide was analysed using the ImageJ software. Intensity of DAB stain (brown areas) was generated by image deconvolution. Data is represented in arbitrary units and the bars represent mean ± standard deviation. “*”p <0.05; “**”p <0.01 (BPH: n = 11; PCa Gleason 6: n = 11; PCa Gleason 8: n = 11). Tissue sections of representative areas of the histological immunolocalization of STC1 in (B) BPH, (D) PCa Gleason 6 (3+3), and (F) PCa Gleason 8 (4+4). Negative control samples (only incubated with goat anti-rabbit secondary antibody) of each tissue were shown in (C), (E), and (G).
3.2. STC1 gene and protein expression in prostate cell lines The STC1 gene was highly expressed in the PC-3 and LNCaP cell lines compared to the normal PNT1A prostate cell line, in which the transcript level was low or undetectable (p <0.01; Figure 2A). This was further confirmed by the detection of STC1 protein in PC-3 and LNCaP when compared to the control cells (Figure 2B). Because of the high levels of STC1 expression the PC-3 cell line was chosen for the subsequent experiments.
Figure 2. STC1 gene and protein expression in PC-3, LNCaP and PNT1A cell lines. (A) Gene expression of STC1 in human prostate cell lines. STC1 was not detectable in PNT1A. The bars represent the mean of 3 independent experiments performed in triplicate ± standard deviation of the mean, "**" p < 0.01. Immunocytochemistry of STC1 protein in prostatic cells (B) PC-3, (D) LNCaP and (F) non-neoplastic prostate cells PNT1A. STC1 is stained in green and the nuclei are stained in blue. Negative control samples (only incubated with goat anti-rabbit secondary antibody) of each tissue were shown in (C), (E), and (G).
3.3. Effect of rhSTC1 on the inhibition of cAMP-production into PC-3 cells The inhibition of forskolin-stimulated cAMP production by 1 µM rhSTC1in PC3 cells was 70.8% (p <0.01, n = 4) (Figure 3) indicating STC1 regulation cAMP PC-3 signalling via the inhibitory G-protein (Gi).
Figure 3. Inhibition of forskolin-stimulated cAMP production by rhSTC1 in PC-3 cells. The bars represent the mean of 4 independent experiments analysed in triplicate ± standard deviation of the mean.”**”p <0.01.
3.4. Effect of Mab Anti-STC1 on PC-3 cell proliferation A significant decrease in cell proliferation rate (at least 50%) was observed at all concentrations of Mab Anti-STC1 tested (0.5 µg/mL, 1.0 µg/mL and 5.0 µg/mL) compared to the control group (p <0.05, n=3). The concentration of 1.0 µg/mL Mab Anti-STC1 was selected to analyse its effects on the cell cycle.
Figure 4. Effect of Mab Anti-STC1 on PC-3 cell proliferation. The bars represent the mean of 3 independent experiments analysed in quadruplicate ± standard deviation of the mean after 96 hours treatment, "*" p <0.05.
3.5. Effect of Mab Anti-STC1 on the cell cycle and cell death of human PC-3 cells PC-3 cells treated for 48 h with 1.0 µg/mL Mab Anti-STC1 showed an increase in the number of cells in G1-phase (a difference of 13.12%) when compared to the control (p <0.01, n=3) (Figure 5A and B). No statistically significant differences were detected in the S and G2-phases (p >0.05, n=3) between the control and the Mab AntiSTC1 treated group (Figure 5B).
Figure 5. Effect of Mab Anti-STC1 (1.0 µg/mL) on the cell cycle of prostate cancer cell line PC-3. (A) Cell cycle distribution: the continuous line represents the control group and the dotted line the treated cells. (B) Distribution of cells (%) between G1, S and G2 phases in the treated and control groups. The bars represent the mean of 3 independent experiments ± standard deviation of the mean, "**"p <0.01.
The percentage of viable cells in the Mab Anti-STC1 group decreased significantly (15.38%) when compared to the control group (p <0.05, n=3) (Figure 6). In the presence of Mab Anti-STC1 the number of cells that underwent apoptosis increased significantly (18.43%) and this corresponds to a 4-fold increase (p <0.01, n=3) in relation to the control group (Figure 6). There were no significant differences in the proportion of cells in necrosis and in late apoptosis between groups (p >0.05, n=3).
Figure 6. Cell death profile of PC-3 prostate cancer cells treated with Mab AntiSTC1 (1.0 µg/ml). (A) Percentage of cells in each quadrant. The bars represent the mean of 3 independent experiments ± standard deviation of the mean, "*" p <0.05; "**" p <0.01. Representative raw scatter plots of the cells in the flow cytometry quadrants of the control (B) and treated (C) groups.
4. DISCUSSION In the present study we have shown that STC1 expression is more abundant in PCa tissue in direct proportion to the severity of malignancy. We have also shown that STC1 inhibits cell cAMP production and that a specific anti-STC1 antibody reduces cell proliferation and increases cell death in prostate cancer cell lines. The expression levels of STC1 in PCa were significantly higher in more aggressive tumours (Gleason ≥ 8) in relation to less aggressive tumours (Gleason score = 6) and BPH tissue. STC1 gene expression was also found to be highly expressed in the prostate cancer lines PC-3 and LNCaP when compared to normal prostate cell line PNT1A, consistent with histological protein expression and previous studies with different classes of human tumors such as colorectal, ovarian, breast, hepatocellular, and thyroid cancer (Chang et al., 2015; Dai et al., 2016; Hayase et al., 2015; He et al., 2011;
Klopfleisch and Gruber, 2009; Koide and Sasaki, 2006; Liu et al., 2010; Pena et al., 2013; Tohmiya et al., 2004). According Orr et al. (2012), the STC1 was localized in areas of tumour stroma and can act as a potential Epithelial-Mesenchymal Transition (EMT) marker. This may indicate that these stromal cell subsets express important regulators of epithelia (Orr et al., 2012). The most accepted theory for the PCa development is from epithelial cells; however, the STC1 signalling could be important for the PCa progression at the both compartments, and the increased staining at more aggressive cancers could be related to the tissue architecture loss through the progression of the disease. Based on our findings, we suggest that STC1 may be a candidate biomarker for the prognosis of prostate cancer in addition or in alternative to the classical tissue morphological methods and the STC1 signalling could be a potential target for future PCa therapies. The rhSTC1 strongly inhibited, by at least 50%, forskolin-stimulated cAMP production in PC-3 cells and this is in agreement with our previous studies on the effect of STC1 on cAMP production using mammalian in HEK293 cells (Terra et al., 2015). Furthermore, once STC1 signalling was blocked by Mab Anti-STC1 in PC-3 prostate cancer cells, cell proliferation was also reduced up to 70%, supporting the hypothesis that this protein is involved in cell growth signalling. This provides further support to an association of STC1 to cell proliferation, as demonstrated by overexpression of STC1 in cells and by gene knock out (Bai et al., 2017; Ma et al., 2015). STC1 is considered a pro-survival factor in differentiated cells, besides being involved in epithelial and mesenchymal signalling and development (Joensuu et al., 2008). PC-3 prostate cancer cells when treated with Mab Anti-STC1 showed an arrest in cell cycle in the G0/G1 phase. This pattern corroborates with studies that evaluated the effect of STC1 silencing in different tumour cells through small interfering RNA (siRNA), where the absence of STC1 led to an accumulation of G0/G1 cells and a reduction in the proliferation of carcinoma cells (Ma et al., 2015), prostate cancer cells (Bai et al., 2017) and ovarian cancer (Liu et al., 2010). In addition, Bai et al. (2017) also demonstrated that, by knocking out STC1 gene in LNCaP and DU145 cells, the reduced cell proliferation was achieved due to changes in the levels of cyclin E1 and Cdk2 proteins (Bai et al., 2017). It is widely known that defects in the apoptotic pathway contribute to tumorigenesis (Wong, 2011) and that this may exert a positive influence on the anti-
apoptotic pathway (Kim et al., 2013), and possibly it is one of the target mechanisms regulated by STC1. Previous studies have shown that STC1 can act as an inhibitor of cellular apoptosis and that when STC1 levels are attenuated due to the blockade of the C-terminal region of STC1, there was an approximately 4-fold increase in cell death via apoptosis (Law et al., 2008; Li et al., 2008). Since in PC-3 (metastatic prostate cancer in bone) cells elevated cAMP levels are positively correlated with the inhibition of cell growth and apoptosis (Bang et al., 1992; Carraway and Mitra, 1998), the decrease in cAMP caused by the presence of rhSTC1 supports this hypothesis that STC1 could be involved in cell proliferation and PCa tumour growth. Thus, the high concentrations of STC1 detected in PCa cells, and possibly the reduced levels of intracellular cAMP evoked by the presence of STC1 may stimulate cell growth and tumour progression. Whilst no specific receptor for STC1 has been identified, it is possible that STC1 intracellular signalling may recruit known cell proliferative pathways such as mitogenactivated protein (MAP) kinase cascade (Schmitt and Stork, 2001). It is still unclear whether STC1 is a cause of tumorigenesis or if it acts as a coreceptor promoting and facilitating the underlying mechanisms involved in establishing signalling and the subsequent progression of neoplastic cell multiplication. As such, one of the limitations of this study was not being able to determine if the STC1 detected by IHC in PCa and BPH tissues was produced locally or in another tissue. If STC1 exerts its effects in prostate cells being produced in other organ, it is possible that after signalling the protein could be internalized and thus detected during staining. Additionally, an aspect to be analysed in future work is the measurement of STC1 concentrations in patient’s blood and urine to further support the possible use of STC1 as a PCa progression biomarker. Nonetheless, this study provides evidence for the use of STC1 staining in tissue samples as a prognostic factor for tumour aggressiveness.
5. CONCLUSION Uncontrolled growth of prostatic cells is influenced by multiple factors including the increased synthesis and secretion of STC1 which induces cell proliferation and reduces apoptosis. Considering that STC1 can act as a regulator of stromal action and paracrine signalling in the prostatic tissue, this protein could be considered as a novel biomarker for tumour progression and clinical follow-up as well as the target for future
therapeutics. Our results also suggests possible ways for therapeutic intervention, such as the use of monoclonal antibodies or gene silencing approaches to neutralize STC1 expression, in order to restore normal intracellular cAMP levels, to inhibit of cell growth and proliferation, and to induce cells to programmed death via apoptosis. However, the main physiological and pathological roles of STC1 have yet to be established. Knowledge of the mechanisms by which STC1 signals and establishes a cellular response is fundamental to understand its association with pathophysiological disorders.
ACKNOWLEDGMENTS We thank Elvis Branchini for his image edition assistance. This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior – Brasil (CAPES) – Finance Code 001 –and Fundo de Incentivo à Pesquisa do Hospital de Clínicas de Porto Alegre (FIPE-HCPA) (Grant number 15-0382) and by the Portuguese Foundation
for
Science
and
Technology
(FCT)
through
project
CCMAR/Multi/04326/2013.
CONFLICT OF INTEREST STATEMENT The authors declare that there are no conflicts of interest.
Ethical approval: All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.
Informed consent: Informed consent was obtained from all individual participants included in the study.
REFERENCES Attard, G., Parker, C., Eeles, R.A., Schröder, F., Tomlins, S.A., Tannock, I., Drake, C.G., de Bono, J.S., 2016. Prostate cancer. Lancet 387, 70–82. https://doi.org/10.1016/S0140-6736(14)61947-4 Bai, Y., Xiao, Y., Dai, Y., Chen, X., Li, D., Tan, X., Zhang, X., 2017. Stanniocalcin 1 promotes cell proliferation via cyclin E1/cyclin‑dependent kinase 2 in human prostate carcinoma. Oncol. Rep. 37, 2465–2471. https://doi.org/10.3892/or.2017.5501 Bang, Y.J., Kim, S.J., Danielpour, D., O’Reilly, M. a, Kim, K.Y., Myers, C.E., Trepel, J.B., 1992. Cyclic AMP induces transforming growth factor beta 2 gene expression and growth arrest in the human androgen-independent prostate carcinoma cell line PC-3. Proc. Natl. Acad. Sci. U. S. A. 89, 3556–60. Carraway, R.E., Mitra, S.P., 1998. Neurotensin enhances agonist-induced cAMP accumulation in PC3 cells via Ca2+-dependent adenylyl cyclase(s). Mol. Cell. Endocrinol. 144, 47–57. https://doi.org/10.1016/S0303-7207(98)00154-3 Chang, A.C.-M., Doherty, J., Huschtscha, L.I., Redvers, R., Restall, C., Reddel, R.R., Anderson, R.L., 2015. STC1 expression is associated with tumor growth and metastasis in breast cancer. Clin. Exp. Metastasis 32, 15–27. https://doi.org/10.1007/s10585-014-9687-9 Chang, A.C.M., Janosi, J., Hulsbeek, M., de Jong, D., Jeffrey, K.J., Noble, J.R., Reddel, R.R., 1995. A novel human cDNA highly homologous to the fish hormone stanniocalcin. Mol. Cell. Endocrinol. 112, 241–247. https://doi.org/10.1016/03037207(95)03601-3 Chang, A.C.M., Jellinek, D.A., Reddel, R.R., 2003. Mammalian stanniocalcins and cancer. Endocr. Relat. Cancer. https://doi.org/10.1677/erc.0.0100359 Chatterjee, B., 2003. The role of the androgen receptor in the development of prostatic hyperplasia and prostate cancer. Mol. Cell. Biochem. 253, 89–101. https://doi.org/10.1023/A:1026057402945 D’Amico, A. V, Whittington, R., Malkowicz, S.B., Schultz, D., Blank, K., Broderick, G.A., Tomaszewski, J.E., Renshaw, A.A., Kaplan, I., Beard, C.J., Wein, A., 1998. Biochemical outcome after radical prostatectomy, external beam radiation therapy, or interstitial radiation therapy for clinically localized prostate cancer. JAMA 280, 969–974. https://doi.org/joc80111 [pii] Dai, D., Wang, Q., Li, X., Liu, J., Ma, X., Xu, W., 2016. Klotho inhibits human follicular thyroid cancer cell growth and promotes apoptosis through regulation of the expression of stanniocalcin-1. Oncol. Rep. 35, 552–558. https://doi.org/10.3892/or.2015.4358 Gleason, D.F., Mellinger, G.T., 1974. Prediction of Prognosis for Prostatic Adenocarcinoma by Combined Histological Grading and Clinical Staging. J. Urol. 111, 58–64. https://doi.org/10.1016/j.juro.2016.10.099 Goldstein, A.S., Huang, J., Guo, C., Garraway, I.P., Witte, O.N., 2010. Identification of a cell of origin for human prostate cancer. Science 329, 568–71. https://doi.org/10.1126/science.1189992 Hayase, S., Sasaki, Y., Matsubara, T., Seo, D., Miyakoshi, M., Murata, T., Ozaki, T.,
Kakudo, K., Kumamoto, K., Ylaya, K., Cheng, S., Thorgeirsson, S.S., Hewitt, S.M., Ward, J.M., Kimura, S., 2015. Expression of stanniocalcin 1 in thyroid side population cells and thyroid cancer cells. Thyroid 25, 425–36. https://doi.org/10.1089/thy.2014.0464 He, L., Wang, T., Gao, Q., Zhao, G., Huang, Y., Yu, L., Hou, Y., 2011. Stanniocalcin-1 promotes tumor angiogenesis through up-regulation of VEGF in gastric cancer cells. J. Biomed. Sci. 18, 39. https://doi.org/10.1186/1423-0127-18-39 Hendrikx, J.J.M.A., Haanen, J.B.A.. G., Voest, E.E., Schellens, J.H.M., Huitema, A.D.R., Beijnen, J.H., 2017. Fixed Dosing of Monoclonal Antibodies in Oncology. Oncologist theoncologist.2017-0167. https://doi.org/10.1634/theoncologist.20170167 Joensuu, K., Heikkilä, P., Andersson, L.C., 2008. Tumor dormancy: Elevated expression of stanniocalcins in late relapsing breast cancer. Cancer Lett. 265, 76– 83. https://doi.org/10.1016/j.canlet.2008.02.022 Kim, S.J., Ko, J.H., Yun, J.H., Kim, J.A., Kim, T.E., Lee, H.J., Kim, S.H., Park, K.H., Oh, J.Y., 2013. Stanniocalcin-1 Protects Retinal Ganglion Cells by Inhibiting Apoptosis and Oxidative Damage. PLoS One 8. https://doi.org/10.1371/journal.pone.0063749 Klopfleisch, R., Gruber, A.D., 2009. Derlin-1 and Stanniocalcin-1 are Differentially Regulated in Metastasizing Canine Mammary Adenocarcinomas. J. Comp. Pathol. 141, 113–120. https://doi.org/10.1016/j.jcpa.2008.09.010 Koide, Y., Sasaki, T., 2006. [Stanniocalcin-1 (STC-1) as a molecular marker for human cancer]. Rinsho Byori 54, 213–220. Law, A.Y.S., Lai, K.P., Lui, W.C., Wan, H.T., Wong, C.K.C., 2008. Histone deacetylase inhibitor-induced cellular apoptosis involves stanniocalcin-1 activation. Exp. Cell Res. 314, 2975–2984. https://doi.org/10.1016/j.yexcr.2008.07.002 Li, K., Dong, D., Yao, L., Dai, D., Gu, X., Guo, L., 2008. Identification of STC1 as an beta-amyloid activated gene in human brain microvascular endothelial cells using cDNA microarray. Biochem Biophys Res Commun 376, 399–403. https://doi.org/S0006-291X(08)01733-6 [pii]\r10.1016/j.bbrc.2008.08.158 Liu, A.Y., Pascal, L.E., Vêncio, R.Z., Vêncio, E.F., 2011. Stromal-epithelial interactions in early neoplasia. Cancer Biomarkers 9, 141–155. https://doi.org/10.3233/CBM-2011-0174 Liu, G., Yang, G., Chang, B., Mercado-Uribe, I., Huang, M., Zheng, J., Bast, R.C., Lin, S.H., Liu, J., 2010. Stanniocalcin 1 and ovarian tumorigenesis. J. Natl. Cancer Inst. 102, 812–827. https://doi.org/10.1093/jnci/djq127 Lu, M., Wagner, G.F., Renfro, J.L., 1994. Stanniocalcin stimulates phosphate reabsorption by flounder renal proximal tubule in primary culture. Am. J. Physiol. 267, R1356–R1362. Ma, X., Gu, L., Li, H., Gao, Y., Li, X., Shen, D., Gong, H., Li, S., Niu, S., Zhang, Y., Fan, Y., Huang, Q., Lyu, X., Zhang, X., 2015. Hypoxia-induced overexpression of stanniocalcin-1 is associated with the metastasis of early stage clear cell renal cell carcinoma. J. Transl. Med. 13, 56. https://doi.org/10.1186/s12967-015-0421-4 Orr, B., Riddick, A.C.P., Stewart, G.D., Anderson, R.A., Franco, O.E., Hayward, S.W.,
Thomson, A.A., 2012. Identification of stromally expressed molecules in the prostate by tag-profiling of cancer-associated fibroblasts, normal fibroblasts and fetal prostate. Oncogene 31, 1130–42. https://doi.org/10.1038/onc.2011.312 Pena, C., Cespedes, M.V., Lindh, M.B., Kiflemariam, S., Mezheyeuski, A., Edqvist, P.H., Hagglof, C., Birgisson, H., Bojmar, L., Jirstrom, K., Sandstrom, P., Olsson, E., Veerla, S., Gallardo, A., Sjoblom, T., Chang, A.C.M., Reddel, R.R., Mangues, R., Augsten, M., Ostman, A., 2013. STC1 expression by cancer-associated fibroblasts drives metastasis of colorectal cancer. Cancer Res. 73, 1287–1297. https://doi.org/10.1158/0008-5472.CAN-12-1875 Ruifrok, A.C., Johnston, D.A., 2001. Quantification of histochemical staining by color deconvolution. Anal. Quant. Cytol. Histol. 23, 291–299. https://doi.org/10.1097/00129039-200303000-00014 Schalken, J.A., van Leenders, G., 2003. Cellular and molecular biology of the prostate: stem cell biology. Urology 62, 11–20. Schein, V., Cardoso, J.C.R., Pinto, P.I.S., Anjos, L., Silva, N., Power, D.M., Canário, A.V.M., 2012. Four stanniocalcin genes in teleost fish: Structure, phylogenetic analysis, tissue distribution and expression during hypercalcemic challenge. Gen. Comp. Endocrinol. 175, 344–356. https://doi.org/10.1016/j.ygcen.2011.11.033 Schmitt, J.M., Stork, P.J.S., 2001. Cyclic AMP-Mediated Inhibition of Cell Growth Requires the Small G Protein Rap1. Mol. Cell. Biol. 21, 3671–3683. https://doi.org/10.1128/mcb.21.11.3671-3683.2001 Schmittgen, T.D., Livak, K.J., 2008. Analyzing real-time PCR data by the comparative CT method. Nat. Protoc. 3, 1101–1108. https://doi.org/10.1038/nprot.2008.73 Skehan, P., Storeng, R., Scudiero, D., Monks, a, McMahon, J., Vistica, D., Warren, J.T., Bokesch, H., Kenney, S., Boyd, M.R., 1990. New colorimetric cytotoxicity assay for anticancer-drug screening. J. Natl. Cancer Inst. 82, 1107–1112. https://doi.org/10.1093/jnci/82.13.1107 Terra, S.R., Cardoso, J.C.R., Félix, R.C., Martins, L.A.M., Souza, D.O.G., Guma, F.C.R., Canário, A.V.M., Schein, V., 2015. STC1 interference on calcitonin family of receptors signaling during osteoblastogenesis via adenylate cyclase inhibition. Mol. Cell. Endocrinol. 403, 78–87. https://doi.org/10.1016/j.mce.2015.01.010 Tohmiya, Y., Koide, Y., Fujimaki, S., Harigae, H., Funato, T., Kaku, M., Ishii, T., Munakata, Y., Kameoka, J., Sasaki, T., 2004. Stanniocalcin-1 as a Novel Marker to Detect Minimal Residual Disease of Human Leukemia. Tohoku J. Exp. Med. 204, 125–133. https://doi.org/10.1620/tjem.204.125 Twisk, J.W.R., 2004. Longitudinal data analysis. A comparison between generalized estimating equations and random coefficient analysis. Eur. J. Epidemiol. 19, 769– 776. https://doi.org/10.1023/B:EJEP.0000036572.00663.f2 Varghese, R., Wong, C.K.C., Deol, H., Wagner, G.F., Dimattia, G.E., 1998. Comparative analysis of mammalian stanniocalcin genes. Endocrinology 139, 4714–4725. https://doi.org/10.1210/en.139.11.4714 Vichai, V., Kirtikara, K., 2006. Sulforhodamine B colorimetric assay for cytotoxicity screening. Nat. Protoc. 1, 1112–1116. https://doi.org/10.1038/nprot.2006.179 Wagner, G.F., Jaworski, E.M., Haddad, M., 1998. Stanniocalcin in the seawater salmon: structure, function, and regulation. Am J Physiol Regul Integr Comp Physiol 274,
R1177-1185. Wang, Z.A., Mitrofanova, A., Bergren, S.K., Abate-Shen, C., Cardiff, R.D., Califano, A., Shen, M.M., 2013. Lineage analysis of basal epithelial cells reveals their unexpected plasticity and supports a cell-of-origin model for prostate cancer heterogeneity. Nat. Cell Biol. 15, 274–283. https://doi.org/10.1038/ncb2697 Weiner, G.J., 2015. Building better monoclonal antibody-based therapeutics. Nat. Rev. Cancer. https://doi.org/10.1038/nrc3930 Wong, R.S.Y., 2011. Apoptosis in cancer: from pathogenesis to treatment. J. Exp. Clin. Cancer Res. 30, 87. https://doi.org/10.1186/1756-9966-30-87 Yoshiko, Y., Son, A., Maeda, S., Igarashi, A., Takano, S., Hu, J., Maeda, N., 1999. Evidence for stanniocalcin gene expression in mammalian bone. Endocrinology 140, 1869–1874. https://doi.org/10.1210/en.140.4.1869 Zhang, K.Z., Westberg, J.A., Paetau, A., Von Boguslawsky, K., Lindsberg, P., Erlander, M., Guo, H., Su, J., Olsen, H.S., Andersson, L.C., 1998. High expression of stanniocalcin in differentiated brain neurons. Am. J. Pathol. 153, 439–445. https://doi.org/10.1016/S0002-9440(10)65587-3
Highlights •
Expression levels of STC1 in PCa were significantly higher in more aggressive tumors.
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STC1 inhibits cAMP production in PCa cells in vitro.
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Specific anti-STC1 antibody treatment reduces cell proliferation in PCa cell line
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Specific anti-STC1 antibody treatment increases apoptosis in PCa cell line
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STC1 signalling can be a potential target for PCa therapies.