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Low expression of the X-linked ribosomal protein S4 in human serous epithelial ovarian cancer is associated with a poor prognosis
© Tsofack et al.; licensee BioMed Central Ltd. 2013
Received: 6 November 2012
Accepted: 20 June 2013
Published: 22 June 2013
The X-linked ribosomal protein S4 (RPS4X), which is involved in cellular translation and proliferation, has previously been identified as a partner of the overexpressed multifunctional protein YB-1 in several breast cancer cells. Depletion of RPS4X results in consistent resistance to cisplatin in such cell lines.
As platinum-based chemotherapy is a standard first line therapy used to treat patients with ovarian cancer, we evaluated the prognostic value of RPS4X and YB-1 at the protein level in specimen from 192 high-grade serous epithelial ovarian cancer patients.
Immunohistochemistry studies indicated that high expression of RPS4X was associated with a lower risk of death and later disease progression (HR = 0.713, P = 0.001 and HR = 0.761, P = 0.001, respectively) as compared to low expression of RPS4X. In contrast, YB-1 was not significantly associated with either recurrence or survival time in this cohort. Finally, the depletion of RPS4X with different siRNAs in two different ovarian cancer cell lines reduced their proliferative growth rate but more importantly increased their resistance to cisplatin.
Altogether, these results suggest that the levels of RPS4X could be a good indicator for resistance to platinum-based therapy and a prognostic marker for ovarian cancer. Our study also showed that RPS4X is an independent prognostic factor in patients with serous epithelial ovarian cancer.
Epithelial ovarian cancer (EOC) is a disease responsible for more cancer deaths among women in the Western world than all other gynecologic malignancies. Because of its asymptomatic nature, ovarian cancer is characterized at presentation with advanced disease having spread primarily via an intraperitoneal route. An initial surgical approach is essential for proper staging of the disease process and for aggressive cytoreduction, which in turn improves the response to chemotherapy and survival . Chemotherapy has had an increasingly important role in the effective treatment of ovarian cancer. The reference standard for postsurgical ovarian cancer first-line chemotherapy has been the use of a platinum–taxane combination . Although the standard platinum-taxane regimen results in a response rate of 80% in advanced ovarian cancer patients, most of these patients relapse after a median period of 18 months, due to the emergence of tumor resistance to these conventional drugs [3–5]. Thus, there is an immediate need for the identification of pharmacogenomic markers to identify patients unlikely to respond, those that will relapse rapidly, or patients at risk for severe toxicity.
In recent years, several studies have reported the involvement of YB-1 in patient survival and cisplatin resistance in ovarian cancers [6, 7]. The YB-1 protein is a multifunctional protein that affects the transcription, splicing, and translation of specific mRNAs [8–11]. Increased expression of YB-1 is associated with a poor prognosis in ovarian cancer . YB-1 binds preferentially to cisplatin-modified DNA  and interacts with several DNA repair proteins [13, 14]. Although YB-1 affects several biological processes, it is still unknown which ones are important for cisplatin resistance. In a recent study of breast cancer cell lines, we identified the proteins that interact directly to YB-1 and impact on cisplatin response upon depletion . Interestingly, we found that the small ribosomal protein 4X (RPS4X) increases cisplatin resistance upon depletion with specific small interference RNAs. As platinum-based compounds are used in the treatment of ovarian cancers, we sought to correlate the levels of RPS4X in clinical samples with patient survival and disease progression.
In this work, we determined by immunohistochemistry the levels of both RPS4X and YB-1 in ovarian cancer samples from patients who were treated with a platinum-based chemotherapeutic regimen after their surgery. RPS4X not only correlated with stage, but low levels of RPS4X also correlated with poor survival and disease progression. These results indicate that RPS4X could be a predictive and prognostic marker in ovarian cancer.
Ethics approval for specimen collection and the study were obtained by the local institutional ethics board (Comité d’éthique de la recherche du Centre hospitalier de l’Université de Montréal).
Patients and tissue specimens
Description of the high-grade serous ovarian carcinomas (HGSOC) tissue array
N = 192
N = 192
Disease free survival, month
Overall survival, month
Tissue microarray (TMA)
Areas of tumor were selected based on review of a hematoxylin-eosin-stained slide. All samples were fixed with formalin and embedded in paraffin following a standard procedure. Formalin fixed paraffin embedded tumor blocks were then biopsied using a 0.6 mm diameter tissue arrayer and resultant cores were arrayed into a grid in a recipient paraffin block. It has previously been demonstrated using several different antibodies that the quality of the core samples on this TMA was suitable for immunohistochemistry and statistical analyses confirmed that the age of the paraffin blocks was not a confounder in these studies . The tissue array was composed of 260 ovarian cancer samples from patients that never received chemotherapy before their surgery and 11 samples of areas from normal fallopian tubes of cancer patients. After review of the clinical data 68 patients were excluded from the final analysis, as they did not meet the study inclusion criteria. For the RPS4X immunostaining study, two core samples on the TMA were damaged and thus excluded (thus N = 190). For the YB-1 immunostaining study, six core samples were excluded for similar reason (thus N = 186). The completed tissue array was sectioned, stained with hematoxylin-eosin and received another pathology review to confirm tumor content .
The TMA of formalin fixed paraffin embedded tumors was sectioned at 4 μm and slides were stained using the BenchMark XT automated stainer (Ventana Medical System Inc.). The optimal concentration for each primary antibody was determined by serial dilutions. The rabbit polyclonal antibody against human RPS4X (14799-1-AP) was purchased from ProteinTech Group, Inc. (Chicago IL). A polyclonal antibody against the N-terminus portion of YB-1 (ab12148) was purchased from Abcam, Inc. (Cambridge, MA) [19, 20]. The rabbit monoclonal antibody against Ki67 (RM-9106) was purchased from Lab Vision (Fremont, CA). Nuclei were counterstained with hematoxylin. Antigen retrieval was carried out with Cell Conditioning 1 (Ventana Medical System Inc.; #950–124) for 30 min (YB-1 and RPS4X) or 60 min (Ki67). Pre-diluted antibody was automatically dispensed, and the slides were incubated at 37°C for 60 min (YB-1 and RPS4X) or 44 min (Ki67). Reactions were carried out using the UltraView DAB detection kit (Ventana Medical System Inc.; #760–091). Slides were counterstained with hematoxylin (Ventana Medical System Inc.; #760–2021). All sections were scanned with a 20x 0.75NA objective with a resolution of 0.3225 μm. Substitution of the primary antibody with phosphate buffered saline served as a negative control.
Tumor sections were scanned, digitally conserved, and manually visualized. For RPS4X and YB-1, a score was given to each core according to the staining intensity of the cytoplasm in the epithelial cells from 1 (weak) to 5 (strong). For both markers, no cores presented negative staining. For Ki67, cores were scored for the percentage (rounded to the nearest 5%) of total staining. Each array was independently analyzed in a blind study by two independent observers. We use the inter-rating correlation (Cronbach’s Alpha) to evaluate the overall correlation between the observers as described previously . Inter-rating correlation was >75% for all three proteins. The average score from the two independent observers, for each respective core, was used for analysis.
YB-1 and RPS4X knock down
The human OVCAR-3 and SK-OV-3 serous ovarian cancer cell lines were obtained from the American Type Culture Collection (ATCC). The OVCAR-3 cells were maintained in RPMI media supplemented 15% Fetal Bovine Serum (FBS) and 1% Penicillin-Streptomycin (Invitrogen, Carlsbad, CA) at 37°C in atmosphere of 5% CO2. The SK-OV-3 cells were maintained in DMEM supplemented with 10% FBS and 1% Penicillin-Streptomycin. To deplete RPS4X or YB-1 proteins in cells, small interference RNA (siRNA) molecules were transfected with the Lipofectamine 2000 as described by the manufacturer (Invitrogen, Carlsbad, CA). The knock down efficiency was confirmed by western blot analyses with antibodies against YB-1, RPS4X, and β-actin as control. Horseradish peroxidase-conjugated secondary antibodies (anti-rabbit IgG: NAV934V and anti-mouse IgG: NA931V) were purchased from GE Healthcare Limited (Piscataway, NJ). The siRNA sequences against YB-1 are 5′-AAGAAGAAAUAUGAAAUUCCA-3′ for the siRNA-A molecule and 5′-CUGCAAGCACCUGUUAAUAAA-3′ for siRNA-B. The siRNA sequences against RPS4X are 5′-CAGAUCUUUGUACGUAAUUAA-3′ for the siRPS4X-A molecule and 5′-CGGGAGAGAAUUUCCGUCUGA-3′ for siRPS4X-D. A scrambled control siRNA was purchased from Invitrogen (Carlsbad, CA).
To obtain the growth curves of transfected cells, 10,000 OVCAR-3 or 50,000 SK-OV-3 transfected cells were plated in 60 mm dishes and counted with a hemacytometer by the trypan blue exclusion technique every other day. Experiments were performed in triplicate.
All transfected and untransfected cells were lysed in RIPA buffer [50 mM Tris–HCl (pH 7.5), 150 mM NaCl, 1% NP-40, 0.1% SDS, 0.5% sodium deoxycholate] for SDS-PAGE analyses. Proteins from SDS-PAGE were transferred onto Amersham Hybond-P membranes (GE Healthcare Limited, Piscataway, NJ). Membranes were blocked one hour at room temperature in PBS containing 5% milk/0.1% Tween, washed in PBS-Tween (0.1%), and incubated overnight with the primary antibodies in PBS containing 5% milk overnight at 4°C. Blots were washed the next day in PBS-Tween and incubated two hours at room temperature with horseradish peroxidase-conjugated secondary antibody in PBS containing 5% milk. Blots were washed with PBS-Tween and proteins were revealed with chemiluminescence reagents (ECL Plus; GE Healthcare Limited, Piscataway, NJ). Immunoprecipitation of GFP (Green Fluorescent Protein) and GFP-YB-1 constructs were performed as described previously . Protein bands on western blots were quantified using LI-COR Image Studio software 2.0 (LI-COR Biosciences, Lincoln, NE). β-actin was used as a control for protein loading. The background signal for each band was determined using an identical area to the target band covering a region in the same lane where no protein signal was observed. Results were determined by calculating a ratio of target protein signal (minus background) over β-actin signal (minus background).
SK-OV-3 cells were plated on coverslips and transfected the next day with control siRNA or siRNA sequences against RPS4Xm RNA. Three days later cells were fixed in 4% paraformaldehyde for 20 min at room temperature (RT) and permeabilized with 0.15% Triton X-100 at RT for 10 min. After washing with PBS, cells were blocked with 3% BSA at room temperature for 30 min. After blocking, the antibody against RPS4X was diluted in 1% blocking buffer (1:100) and applied to the coverslips for an overnight incubation at 4°C. The next day, coverslips were washed with PBS and incubated with rhodamine-secondary antibody (Santa Cruz) for 1 h30 min in the dark at RT. After washing, coverslips were stained with DAPI 10 min, washed, and mounted on glass slides. Slides were viewed at 60X magnification (1.4NA oil-immersion 60X objective) and zoomed 2X for image acquisition on a Nikon inverted diaphot confocal microscope equipped with Krypton/Argon lasers (488 and 568 nm). Images were captured with a BioRad MRC1024 confocal microscopy system. Finally, images were analyzed (colored and merge) using the Fiji-win32 software.
FACS and FITC-Annexin V analyses
Cells were transfected with either control siRNA or siRNA against RPS4X. After 72 h, cells were fixed in 50% ethanol overnight. Cells were then washed in phosphate-buffered saline (PBS) and incubated for 30 min at 37°C in a buffer containing propidium iodide and RNAses. Cells were then analyzed on a Beckman-Coulter Epics Elite ESP (Cambridge, MA, USA) flow activated cell sorter. Data were analyzed with the MultiCycle software (Phoenix Flow System, San Diego, CA, USA). To estimate apoptosis and/or necrosis, we used the FITC Annexin V apoptosis detection kit I (BD Biosiences, Palo Alto, CA). Transfected cells were treated 48 h with the indicated concentration of cisplatin and then harvested to measure apoptosis/necrosis following the manufacturer’s instructions.
Cisplatin treatment and sulforhodamine B colorimetric assay
Cells were transfected with the indicated siRNAs and allowed to grow for 24 hours. The next day, 10,000 cells were seeded per well on a 96-well plate and incubated at 37°C for 24 hours. Different concentrations (0–40 μM) of cisplatin were added to the cells in triplicate and cells were then allowed to grow for an additional 48 hours. Cells were fixed with tricholoroacetic acid (10% w/v) and stained 30 min with sulforhodamine B as described .
The Spearman correlation (two-tailed) and non-parametric Wilcoxon-Mann–Whitney test were used to estimate the correlation with clinicopathological variables and markers as continuous variables. Survival curves were calculated according to Kaplan-Meier method coupled with a log-rank test for survival analysis. Since survival times were positively skewed, we took the median as the threshold value for each marker (YB-1 and RPS4X). Univariable and multivariable Cox proportional hazard models were used to estimate the hazard ratio for each marker as continuous variables. All statistical analyses were done using Statistical Package for the Social Sciences software version 16.0 (SPSS, Inc.), and statistical significance was set at P < 0.05.
The R software version 2.10.1 (http://www.r-project.org/) was used to estimate the growth rate, the IC50, and the associated standard deviation. Briefly, the growth curves were fitted to a mathematical model of the form y = x0*(1 + r)t, where x0 represents the 50,000 transfected cells plated on day 0, r represents the growth rate, and t represents the time unit (days). The dose response curves were fitted to a standard exponential decay mathematical model of the form y = y0 + A*ekx where y0 represents the minimal normalized intensity, A the intensity at time 0 and k is the decay rate.
RPS4X and YB-1 expression in ovarian cancer samples
Spearman correlation test (two-tailed) for YB-1 and PRS4X expression (intensity) in EOC tissues and clinical data of patients
Ki67 % total
Cox regression analyses representing the statistical association between RPS4X expression and outcome in patients with high-grade serous ovarian cancer patients
To summarize, all our statistical analyses indicate that high expression of RPS4X is associated with less aggressive ovarian tumors, slower disease progression, and with less deaths associated with this disease.
Impact of RPS4X depletion on the growth of two serous epithelial ovarian cancer cell lines
Depletion of RPS4X in OVCAR-3 and SK-OV-3 cells induces cisplatin resistance
We next analyzed the impact of cisplatin on cell death in transfected cells with a FITC-Annexin V assay. OVCAR-3 cells transfected with a control siRNA showed a 14% increase in apoptosis when treated 48 hours with 2 μM cisplatin (Figure 5). There was no significant increase in necrosis. In contrast, RPS4X-depleted OVCAR-3 cells did not exhibit an increase in apoptosis or necrosis after 48 hours of cisplatin treatment. Similarly, SK-OV-3 cells transfected with a control siRNA showed a 30% and 2% increase in apoptosis and necrosis respectively when treated for 48 hours with 15 μM cisplatin (Figure 5). In contrast, RPS4X-depleted SK-OV-3 cells showed only a 7% increase in apoptosis after 48 hours of cisplatin treatment (Figure 5). There was no increase in necrosis. Altogether these results indicate that RPS4X-depleted ovarian cancer cells are resistant to apoptosis induced by cisplatin.
RPS4X interacts with YB-1 in ovarian cancer cells
We previously showed that RPS4X interacts with a tagged YB-1 in a breast cancer cell line . To confirm this interaction in an ovarian cancer cell line, GFP-YB-1 and a control GFP expression vectors were transfected into SK-OV-3 cells. The next day the GFP-YB-1 construct was precipitated with an antibody against the GFP tag and the presence of RPS4X in the immunoprecipitate was detected by immunoblotting (see Additional file 4: Figure S3). Endogenous RPS4X was only found in the GFP-YB-1 immunoprecipitate indicating an interaction between RPS4X and YB-1 in ovarian cancer cells as well.
The expression of YB-1 in ovarian carcinomas has been correlated with a poor prognosis in several studies including one focused on serous ovarian cancer [7, 23]. In contrast, there is one published report indicating no relationship between ovarian cancer patient survival and YB-1 expression . Such contrasting results may be due to the small numbers of ovarian tumor samples, specifically of the serous type (less than 40 samples of both low and high grades), that were used in past studies [7, 23, 24]. Another confounding parameter in the interpretation of the results is the anti-YB-1 antibodies used in the different studies. Antibodies recognizing epitopes on the C-terminus [7, 24] or the N-terminus portion of the YB-1 protein (our study) as well as the immunohistochemistry protocol can impact staining . Finally, as our study focused exclusively on high-grade serous epithelial ovarian cancers, it is possible that within this subset of serous cancer YB-1 has little prognostic value. In contrast, the level of RPS4X may be a better prognostic biomarker than YB-1 in serous epithelial ovarian cancers.
Our recent analyses on YB-1 in breast cancer cell lines resistant to cisplatin have indicated an interaction between RPS4X and YB-1 . As platinum-based regimen is a major treatment for ovarian cancer, we sought to determine whether the expression of RPS4X could have prognostic significance in this cancer type. In this study, we showed by immunohistochemistry that high expression of RPS4X correlated with overall survival and disease free progression. Low expression of RPS4X correlated significantly with tumor stage. These results suggest that RPS4X is a potential prognostic marker for high-grade serous epithelial ovarian cancer at the protein level. To our knowledge, there is no published study on RPS4X levels in ovarian cancers. RPS4X will need to be validated in an independent cohort of patients to confirm its clinical utility. In addition, a more quantitative way of measuring RPS4X expression, as for example real-time quantitative RT-PCR, could be envisioned.
An important aspect of RPS4X protein expression is its association with cisplatin resistance in different cell lines. The SK-OV-3 cell line is more resistant to cisplatin than the OVCAR-3 cell line [26, 27]. Interestingly, the expression of endogenous RPS4X protein is lower in the more cisplatin resistant SK-OV-3 cell line than the OVCAR-3 cell line. In addition, a depletion of RPS4X in both the OVCAR-3 and SK-OV-3 ovarian cancer cell lines induced cisplatin resistance and is consistant with our previous data on RPS4X depleted breast cancer cell lines resistant to cisplatin . Such results suggest that RPS4X would also have predictive values with regards to platinum-based chemotherapy. A major challenge with platinum-based regimen is that ovarian cancers can be either intrinsically resistant to treatments or will become resistant during therapy . As the immunohistochemistry study was performed on serous high-grade ovarian tumors from patients who had not received chemotherapeutic treatment, the patients showing low expression of RPS4X in their tumor tissues at surgery could correlate with an intrinsic resistance to platinum-based drugs. More precisely, cancer cells with low expression of RPS4X present in high-grade tumors that have never been in contact with platinum would correspond to cells exhibiting a pre-existing mechanism for resistance to such a drug. The exact mechanism by which a depletion of RPS4X confers cisplatin resistance is not known. One hypothesis is that depletion of RPS4X could induce a ribosomal stress which in turn leads to a slower growth rate as observed in siRPS4X transfected ovarian cancer cell lines. It has been suggested that a reduced growth rate could constitute a significant event in the survival of cancer cells following a major stress like cisplatin treatment [28, 29]. Finally, differential translation of not only several survival factors in addition to proteins critical in the control of apoptosis during cisplatin response may be affected as well in RPS4X-depleted cells. Large-scale proteomic analyses may help identifying such critical regulators in RPS4X-depleted cisplatin resistant cells. In addition, a thorough analysis of the impact of RPS4X levels on different types of reagents used in chemotherapy is also required.
To conclude, we have established that RPS4X is a new promising prognostic marker for patients with high-grade serous ovarian cancer. More importantly, if RPS4X is shown to be predictive of cisplatin response either alone or in combination with other markers, this could be useful when selecting first line therapies for patients with serous ovarian cancer.
Clinical specimens collection was supported by the Banque de tissus et de données of the Réseau de recherche sur le cancer of the Fonds de recherche du Québec – Santé which is affiliated with the Canadian Tumour Repository Network. This work was supported in part by the Canadian Institutes of Health Research and the Cancer Research Society, Inc. to ML. SPT is a scholar of the Quebec-Clinical Research Organization in Cancer Consortium, financed by the Pfizer- FRSQ Innovation Award.
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