Research ArticleExperimental Studies

SOX18 Expression Predicts Response to Platinum-based Chemotherapy in Ovarian Cancer

BARTOSZ PULA, CHRISTOPHER KOBIERZYCKI, DANIEL SOLINSKI, MATUESZ OLBROMSKI, EWA NOWAK-MARKWITZ, MAREK SPACZYNSKI, WITOLD KEDZIA, MACIEJ ZABEL and PIOTR DZIEGIEL
Anticancer Research August 2014, 34 (8) 4029-4037;

Abstract

Background: SOX18 is a transcription factor known to be involved in blood and lymphatic vessel, hair follicle development, and wound healing processes. In addition, it has been reported that SOX18 may influence cancer growth. The role of SOX18 expression in ovarian cancer (OC) has not been determined. Materials and Methods: SOX18 expression was assessed in 85 OC cases using immunohistochemical methods and in ovarian cancer cell lines on the mRNA and protein level. Results: SOX18 was expressed in cancer cell nuclei as well as the cytoplasm. Higher nuclear SOX18 expression was associated with presence of residual disease following surgical treatment (p=0.0158) and advanced disease stage (p=0.0056). Univariate survival analysis revealed that high SOX18 (p=0.0125) expression, presence of residual disease (p<0.0001) and advanced disease stage (p<0.0324) predicted poor patient outcome. Conclusion: SOX18 may be a new predictive marker for OC.

Ovarian cancer (OC) is the leading cause of death of all the gynecological malignancies, as most of women are diagnosed at advanced clinical stages due to unspecific or subtle symptoms of this malignancy (1). Ovarian tumors are also classified in five major subtypes designed as follows: serous, mucinous, endometrioid, clear cell and transitional cell (Brenner type). Tumors in each type can be further sub-divided into benign, intermediate (borderline) and malignant (2). So far the clinicopathological factors as clinical advancement stage, histological type and residual disease after surgery are the most important prognostic factors (1, 3).

The SOX (Sex-Determining Region on the Y chromosome-related High Mobility Group Box) genes are a well-conserved family of genes encoding various transcription factors. The high-mobility group (HMG) domain, which is present in all the family members, specifically binds to the 5’-A/TA/TCAAA/TG-3’ DNA sequence motif (4, 5). It was shown that SOX proteins may interact directly with DNA through binding to its minor groove or indirectly through co-activators resulting in formation of various complexes (6). The SOX family is divided into ten groups (A-J) based on their amino acid homology (4, 7-9). SOX7, SOX17 and SOX18 are the members of group F and were shown to participate in cardiovascular system development in different animal species (4, 10). Interestingly, SOX7 and SOX17 may overtake the functions of SOX18 during these processes (11, 12). Loss of SOX18 function has been associated with defective blood and lymphatic vessel formation, resulting in the hypotrichosis-lymphedema-tel-angiectasia syndrome (13-15).

In addition to its role in the vascular development, it was shown that SOX18 may influence tumor growth (16-18). Recently, we noted that SOX18 was expressed in cancer cells of invasive ductal breast cancer (IDC) paraffin sections, as well as in established cancer cell lines. Moreover, its expression correlated positively with the malignancy grade of the tumors and human endothelial growth factor receptor-2 (HER2) expression (18). SOX18 expression was also found in some of the normal tissues of the gastrointestinal tract and in cancer-derived cell lines from breast, gastric, pancreatic cancers, and melanoma (17, 19). In gastric cancer tissues SOX18 expression was specifically found in gastric tumor stroma and was correlated with poor patient outcome (20). SOX18 was also shown to affect proliferation of human breast cancer MCF-7 cells and vascular smooth muscle cells (VASC) of human coronary atherosclerotic lesions. Inhibition of SOX18 expression resulted in a decreased proliferation rate of these cells (17, 21). Moreover, in VASC SOX18 expression co-localized with the proliferating cell nuclear antigen protein (PCNA) (21).

Until now no studies determining the role SOX18 expression in OC have been performed. Therefore, based on the impact of SOX18 on the progression of gastric cancer and IDC, utilizing immunohistochemical (IHC) methods we aimed to evaluate its expression in a subset of OC and correlated its expression with patients' clinicopathological data. Due to the observed involvement of SOX18 in cellular proliferation, we additionally related its expression to the immunoreactivity of the Ki-67 antigen.

Materials and Methods

Patients. The study was conducted on 85 archival paraffin embedded OC samples collected during radical ovariectomy in the Department of Gynecologic Oncology at the University of Medical Sciences in Poznan. All patients received surgical and chemical treatment according to running protocols. Debulking surgery led to an optimal cytoreduction of the tumor masses in 22 out of 85 (25.9%) cases, however, in 63 (74.1%) radical removal of the tumor was not possible and presence of a residual disease was confirmed during histopathological examination. All of the patients received chemotherapy based on platinum compounds (cisplatin or carboplatin) in combinations with doxorubicin, paclitaxel or cyclophosphamide. In serous OC optimal cytoreduction could be performed in 14 (21.2%) patients. Mean patients' age at the time of the diagnosis was 50.9±9.14 (median: 50, range 31-79). Histological types and malignancy grade (G) of the tumors were determined according to the World Health Organization (WHO) criteria in the routinely 6-μm-thick hematoxylin and eosin (H&E) stained sections (22). The serous OC was the most frequently diagnosed type (66/85 cases, 77.6%). The remaining 19 cases (22.4%) were diagnosed as following types: endometrioid (14/85 cases (16.4%), mucinous (3/85 cases, 3.6%) and clear cell (2/85 cases, 2.4%) cancers. The patients' clinicopathological data are summarized in Table I. During this time out of the 85 observed patients 66 (77.6%) patients died of the disease, whereas 70 (82.4%) suffered recurrence or did not respond to the treatment. In the serous OC group 56 (84.8%) died and 57 (86.4%) suffered from recurrence or therapy failure.

IHC. IHC reactions were performed as previously described on 4-μm-thick tumor paraffin sections using Dako Autostainer Link 48 (Dako, Glostrup, Denmark), murine monoclonal antibodies directed against SOX18 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and Ki-67 antigen (clone MIB-1, ready-to-use, Dako) to ensure repeatable reaction conditions (18). For both studied antigens, deparaffinization and antigen retrieval were performed using Antigen Retrieval Solution (pH 9.0 for SOX18 or pH 6.0 for Ki-67; 97°C, 20 min; Dako) in PT Link Rinse Station (Dako). The sections were then washed in tris buffered saline (TBS)/0.05% Tween buffer and endogenous peroxidase was blocked using EnVision FLEX Peroxidase-Blocking Reagent (5 min at room temperature; RT, Dako) followed by a washing step with TBS/0.05% Tween. Primary antibodies were applied for 20 min at RT and then washed in TBS/0.05% Tween. Following this EnVision FLEX/HRP, secondary antibodies were applied (20 min at RT; Dako). Diaminobenzidine (DAB, Dako) was utilized as the peroxidase substrate and the sections were incubated for 10 min at RT. Finally, the sections were counterstained with Mayer's haematoxylin, dehydrated in alcohols (70%, 96%, 99.8%) and xylene and mounted in the SUB-X Mounting Medium (Dako). Negative controls were designed by omitting the incubation with primary antibody, whereas positive controls for SOX18 and Ki-67 were previously positively stained IDC sections (18). Moreover, SOX18 expression in vessels served as internal staining control.

View this table:
Table I.

Clinicopathological characteristics of the whole study cohort (85 cases), as well as serous type of ovarian cancer (66 cases).

Evaluation of IHC reactions. The IHC sections were evaluated using a BX-41 light microscope (Olympus, Tokyo, Japan) by two pathologists who were blinded to the patients' clinical data. Nuclear SOX18 and Ki-67 expression in OC cancer cells was assessed as described earlier (23). In each whole tissue section three fields with potentially the highest number of tumor cells yielding a positive nuclear reaction were selected (hot spots). The percentage of positive cells in each hot-spot was evaluated under ×400 magnification and the brown-labeled cell nuclei of cancer cells were counted. The general result for every sample was an average of three hot-spot percentages.

Cytoplasmic SOX18 expression was evaluated using a 12-point semi-quantitative IRS (Immunoreactive Score) scale according to Remmele and Stegner, which takes into account the percentage of cells with a positive reaction (0: absence of cells with positive reaction, 1: 1-10% of cells, 2: 11-50%, 3: 51-80%, 4: over 80% of cells) and its intensity (0: no reaction, 1: low intensity of the reaction, 2: moderate intensity of the reaction, 3: intense reaction). The final score is the product of the two assessed parameters (24).

Cell lines. Human OC cell lines SK-OV-3 (Sigma, St. Louis, MI, USA) and OVCAR3 (ATCC, Washington, CO, USA) were used in the study. SK-OV-3 were cultured in McCoy's 5A Medium (Sigma) supplemented with 15% fetal calf serum (FCS; Lonza, Basel, Switzerland). OVCAR3 were cultured in Dulbecco's modified Eagle's medium (Lonza) supplemented with 10% FCS and 0.01 mg/ml insulin (Sigma). Human immortalized ovarian surface epithelial cells (IOSE 364; kind gift from prof. Nelly Auersperg from the Canadian Ovarian Tissue Bank) were cultured in MCDB 105 and 199 media (both Sigma) at a 1:1 ratio supplemented with 5% FCS. In all the studied cell lines, 1mM L-glutamine and antibiotics were also added to the media.

RNA isolation and real-time polymerase chain reaction (PCR). Total RNA from the cultured cell lines was isolated using the RNeasy Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. The protocol included on-column DNase digestion to eliminate the genomic DNA. The quality of the RNA samples was evaluated with the use of agarose gels and staining with ethidium bromide. 18S and 28S bands were visualized under UV light. Concentration and quality of the isolated RNA was measured in the NanoDrop1000 (NanoDrop Technologies, Wilmington, DE, USA). First-strand cDNA was synthesized using the QuantiTect Reverse Transcription Kit (Qiagen).

The relative SOX18 mRNA expression levels were determined by quantitative real-time PCR using the 7900HT Fast Real-Time PCR System and a TaqMan Gene Expression Master Mix (Applied Biosystems, Foster City, CA, USA), according to the manufacturer's protocol. The primers used were SOX18 Hs00746079_s1 and ACTB Hs99999903_m1 for β-actin (Applied Biosystems). The latter served as a reference for determining SOX18 expression in the analyzed cancer cell lines. The reactions were carried out in triplicate using the previously established conditions: initial denaturation at 94°C for 120 sec, followed by 40 cycles of denaturation at 94°C for 15 sec, and annealing and elongation at 60°C for 60 sec (18). The relative mRNA expression levels of the SOX F genes were calculated by the ΔΔCt method (25).

SDS-PAGE and western-blot. Whole-protein lysates were obtained using a CellLytic Buffer with the addition of protease inhibitors (both from Sigma, Munich, Germany), quantified using the BCA protein assay (Pierce, Rockford, IL, USA) and resolved on 10% SDS-PAGE gels according to Laemmli (26). Following electrophoresis, the samples were transferred to polyvinylidene fluoride (PVDF) membranes (Immobilon; Millipore, Bedford, MA, USA) and incubated with the anti-SOX18 antibody (Santa Cruz Biotechnology) overnight at 4°C. Next, the membranes were incubated with a secondary anti-mouse antibody conjugated with horseradish peroxidase (Jacksons Immunoresearch, Mill Valley, CA, USA) for 1 h at RT, rinsed, and treated with the Immun-Star-HRP Chemiluminescent Kit (Biorad, Hercules, CA, USA). β-actin, detected with anti-human β-actin antibody (Abcam, Cambridge, UK), was used as an internal control to normalize the amounts of SOX18 on the same membrane.

Statistical analysis. Prism 5.0 (GraphPad, La Jolla, CA, USA) was used to analyze the data. The Mann-Whitney and Kruskal-Wallis with post hoc Dunn's multiple comparison tests were was used to compare the groups of data that did not meet the assumptions of the parametric test. Correlations between patients' age and expression of the studied markers was analyzed by the Pearson's correlation test. The Spearman correlation test was used to assess the relationship between IHC parameters. The Kaplan-Meier method and the log-rank test were used to determine the significance of patients' disease specific survival (DSS) and disease free survival (DFS). For each variable, the hazard ratio and 95% confidence interval (95% CI) were estimated. In all the analyses, results were considered statistically significant when p<0.05.

Results

SOX18 expression in OC tissues. Differentiated SOX18 expression was observed in OC cancer cell nuclei (mean 66.92±19.67, median 72.08) as well as cytoplasm (mean IRS 1.48±1.78, median IRS 1) (Figure 1A-D). Although, in all the analyzed cases a nuclear expression of SOX18 was observed, its cytoplasmic expression was noted only in 44 out of 85 analyzed cases (51.78%). Nuclear SOX18 expression was also noted in vessels surrounding the nests of OC cancer cells (Figure 1E). Nuclear Ki-67 antigen expression was observed in all of analyzed cases (mean= 53.83±21.83, median=55.2) (Figure 1F).

Significant positive correlation of SOX18 nuclear and cytoplasmic expression was noted in the whole patient cohort (r=0.27, p=0.012), however, in the serous OC type was close to significance (r=0.21, p=0.085; both by the Spearman correlation test). No correlations were noted between nuclear and cytoplasmic SOX18 expression and the labeling index of the Ki-67 antigen-positive OC cells.

SOX18 expression in OC tissues in regard to patients' clinicopathological data. Statistical analysis revealed that significantly higher nuclear, but not cytoplasmic, SOX18 expression was observed in cases with presence of residual disease following surgical treatment compared to patients with optimal cytoreduction (p=0.0158, Mann-Whitney test). Similarly, cases in advanced disease stage (Fédération Internationale de Gynécologie et d'Obstétrique – FIGO III-IV) had significantly higher SOX18 expression in comparison to cases diagnosed in less advanced FIGO I-II stages (p=0.0056, Mann-Whitney test) (Figure 2). No significant differences were noted in cytoplasmic and nuclear SOX18 expression with regard to malignancy grade of the tumors and histological types (Table II). No significant SOX18 expression was also noted in regard to presence of residual disease after surgery, stage and malignancy grade in serous OC patients (data not shown). No correlation between nuclear, as well as cytoplasmic SOX18 expression with patients' age at diagnosis was noted (data not shown, Pearson's correlation test). Significant differences were observed in Ki-67 antigen expression depending on malignancy grade in all analyzed OC cases (p=0.0225) and serous OC type (p=0.0276; both Kruskal-Wallis test). Post hoc analysis revealed that Ki-67 antigen expression was significantly higher in the G2 and G3 compared to G1 (p<0.05 in both analyses, Dunn's multiple comparison test) cases in the whole study cohort. In the serous OC type, only the difference between G1 and G3 cases was found to be significant (p<0.05, Dunn's multiple comparison test) (Figure 3).

SOX18 expression in analyzed cell lines. Real-time PCR analysis revealed that the lowest mean SOX18 mRNA levels were noted in IOSE 364 (RQ 1.12±0.14), whereas in SK-OV-3 (RQ 15.6±1.55) and OVCAR3 (RQ 2.65±0.64) OC cell lines its mRNA expression was higher (Figure 4A). SOX18 expression was noted in the studied cell lines also on the protein level. The highest SOX18 protein levels were observed in IOSE 364 cells. Lower SOX18 expression was noted in the two studied OC cell lines SK-OV-3 and OVCAR3 (94% and 67% of its expression in IOSE 364 cells, respectively) (Figure 4B, C).

Figure 1.
Figure 1.

Low (A, B) and high (C, D) nuclear (A, C) and cytoplasmic (B, D) SOX18 expression in ovarian cancer cells. Nuclear SOX18 expression in endothelium of two blood vessels surrounded by nests of ovarian cancer cells (E). Nuclear Ki-67 antigen expression in ovarian cancer cells (F). Magnification ×200.

Figure 2.
Figure 2.

SOX18 expression in regard to presence of residual disease following surgical treatment (A) and disease stage (B). *p<0.05, **p<0.01, Mann-Whitney test.

Survival analysis. Median expression of the analyzed IHC antigens (cytoplasmic and nuclear SOX18 and Ki-67 antigen) were used to dichotomize the patients for the purpose of survival analysis. Using the Mantel-Cox test, high nuclear SOX18 (>72.08% of positive cells; p=0.0125), advanced clinical stage (III-IV; p=0.0342) and residual disease (p<0.0001) were found to be associated with the patients' shorter DSS in the whole study group (Figure 5) (Table III). Moreover, in the same patients, residual disease (p=0.0002) and advanced clinical stage (p=0.0426) were also found to correlate with patients' shorter DFS (Table III). In the 66 serous OC patients low Ki-67 antigen (≤55.20% of positive cells; p=0.0071) and presence of residual disease (p=0.0020) were associated with patients' shorter DSS. Similarly, these factors were also found to be correlated with patients' shorter DFS (p=0.0041 and p=0.0465, respectively; Table III).

Discussion

To date prognostic significance of SOX18 expression has been assessed only in two studies. In the study of Eom et al. SOX18 expression in the gastric tumor stroma, but not in cancer cells, correlated with patients' poor survival (20). Recently, we have shown in a group of 122 IDC patients that SOX18 expression in cancer cells was associated with higher malignancy grade and HER2 positivity (18). The two above mentioned clinicopathological traits were shown to be associated with the patients' poor prognosis. Although, SOX18 expression in IDC cancer cells did not predict patients' poor outcome, the high vascular density of SOX18-positive peritumoral vessels was associated with patient' poor prognosis (18).

Figure 3.
Figure 3.

Ki-67 antigen expression in regard to malignancy grade of the tumors in the whole patient cohort (A) and serous ovarian carcinomas (B). *p<0.05, Dunn's multiple comparison test.

In the present study we found that SOX18 was expressed in the nuclei and cytoplasm of ovarian cancer cells. Furthermore, SOX18 expression was also noted on mRNA and protein level in immortalized ovarian surface epithelial cells (IOSE 364) and two OC cell lines SK-OV-3 and OVCAR3 indirectly confirming the results obtained from SOX18 immunostained sections. Interestingly, we have also noted its particularly high protein expression in IOSE 364 cells, although in our previous studies in the immortalized normal breast cells (HME1-hTERT) we have noted virtually no SOX18 expression (18). SOX18 protein expression in IOSE 364 cells was comparable to that noted in OVCAR3 cells and higher to the one observed in SK-OV-3 cells. Nevertheless, SOX18 mRNA levels were higher in OC cells in relation to its levels in the benign ovarian surface epithelial cells. Currently, the observed differentiated SOX18 expression on mRNA and protein in the above mentioned cells requires further research to elucidate the regulatory mechanism of its expression.

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Table II.

The relationship between SOX18 expression in cancer cells and selected clinicopathological parameters in 85 ovarian cancer patients. The data are presented as mean±standard deviation (median).

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Table III.

Univariate Cox proportional hazards analysis in 85 ovarian cancers and 66 ovarian serous type cancers. Significant p-values are given in bold print.

Statistical analysis revealed that high nuclear, but not cytoplasmic, SOX18 expression was associated with presence of residual disease, advanced clinical stage and patients' shorter DSS and DFS, confirming the importance of nuclear localization of SOX18 in OC cancer cells. Moreover, the results obtained in this study are in accordance with our previous observations as SOX18 expression was found mostly in nuclei of IDC cancer cells in paraffin sections (18). To note, in both studies the same antibody was used for performing IHC reactions. Previous reports indicated that SOX18 may affect proliferation of normal as well as tumor cells, however, in the present study we did not find any correlations of its expression with that of the Ki-67 antigen (17, 21). Increasing Ki-67 antigen expression was observed with increasing malignancy grade of the tumors in the whole study cohort, as well as in serous OC patients only, pointing to a reliable selection towards the aim of our study. A similar relationship has been noted in the literature. This tendency is observed not only in ovarian cancer but also in other tumors (23, 27-30). Similarly to other reports, we noted an association between high Ki-67 antigen expression in serous OC and patients' good prognosis (31, 32).

Figure 4.
Figure 4.

Real-time polymerase chain reaction analysis of SOX18 mRNA expression in human immortalized ovarian surface epithelial cells (IOSE 364) and ovarian cancer cell lines OVCAR3 and SK-OV-3 using the ΔΔCt method. SOX18 levels were normalized against 18s RNA and the IOSE 364 cell line was assigned as a calibrator sample (A). SOX18 and β-actin expression in above mentioned cell lines (B). Densitometric measurement of the bands of nuclear fractions of the analyzed cell lines. The results were normalized to β-actin and IOSE 364 cells were used as calibrator sample. The results as percentage of mean optical density and standard deviation (SD) (C).

Figure 5.
Figure 5.

Kaplan-Meier survival curves of disease specific survival in regard to nuclear SOX18 expression (A), disease stage (B) and residual disease (C). *p<0.05, ****p<0.0001, Mantel-Cox test.

Based on our study, it seems that SOX18 may be a potential target in ovarian cancer therapy. Interestingly, as all of the patients received platinum-based chemotherapy (cisplatin or carboplatin) it may be speculated that SOX18 mediates resistance to these therapeutic agents. To note, in this study SOX18 expression was found in SK-OV-3 and OVCAR3 cell lines, which are regarded as platinum resistant (33). The determination of SOX18 as new predictive marker of response to platinum agents however requires further research.

Although, as in our previous study, we have noted SOX18 immunoexpression in the nuclei of intra-tumoral vessels endothelial cells, we did not investigated the relationship of SOX18-positive vessel density as this will be addressed in our next study (18). Based on the previously obtained results, it seems that assessment of SOX18-postive vessel density may be of great importance as peri-tumoral SOX18-positive, but not CD31-, Lyve-1- and podoplanin (D2-40)-positive vessel density, was found to correlate with patients' poor outcome (18). Furthermore, Duong et al. showed that SOX18 expression increases in lymphatic endothelial cells during melanoma growth (34). SOX18 may be a promising marker for tumor neolymphangiogenesis activity, but more detailed studies are required to clarify this hypothesis.

In summary, we showed for the first time that the SOX18 protein is expressed in ovarian cancer cells (paraffin tumor sections as well cell lines). We found that increased SOX18 expression in these cells correlated with patients' poor outcome, as determined by the univariate survival analysis. As our study was performed on a limited subset of OC patients, further studies are required to fully-determine the prognostic and predictive significance of SOX18 expression in OC.

Acknowledgements

This study was supported by a grant of the National Science Centre decision no. DEC-2012/05/N/NZ4/02517. The Authors thank Mrs. Aleksandra Piotrowska and Ms Agata Zemla from the Department of Histology and Embryology, Wroclaw Medical University for their technical assistance.

  • Received April 28, 2014.
  • Revision received June 11, 2014.
  • Accepted June 12, 2014.

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SOX18 Expression Predicts Response to Platinum-based Chemotherapy in Ovarian Cancer
BARTOSZ PULA, CHRISTOPHER KOBIERZYCKI, DANIEL SOLINSKI, MATUESZ OLBROMSKI, EWA NOWAK-MARKWITZ, MAREK SPACZYNSKI, WITOLD KEDZIA, MACIEJ ZABEL, PIOTR DZIEGIEL
Anticancer Research Aug 2014, 34 (8) 4029-4037;
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