Role of SOX9 protein in ovarian carcinoma: A molecular insight

Methods

A cross-sectional study was conducted by the department of Biochemistry, All India Institute of Medical Sciences Bathinda, Punjab, India on patients diagnosed with ovarian carcinoma by histopathological evidence during the study period (October 2022 to June 2024), after ethical clearance from the Institutional Ethics Committee.

Estimation of SOX 9 protein by ELISA

SOX9 protein in tissue extract obtained from study samples and the cell lysates from SK OV3 cells was determined by ELISA method using human transcription factor SOX-9 ELISA Kit (BioTechno Labs). A standard curve of SOX9 was plotted as shown in Figure 1.

Close

Fig. 1.

Standard curve of ELISA of SOX 9 standards of different concentrations. X–axis shows concentration of standard and y–axis shows absorbance. The equation and R² value displayed on the graph. Using the equation, concentration of SOX 9 protein was estimated in all the samples including the tissue lysates and cell lysate.

Export to PPT

Results

We have enrolled 60 cases with ovarian carcinoma and 60 controls based on the eligibility criteria. Of 60 cases with ovarian carcinoma, majority (n=44,73%) had serous carcinoma. Mucinous carcinoma germ cell tumour and others were diagnosed in 5, 6 and 5 patients, respectively. Of 60 patients 37 (61.7%) were post menopausal. Menopausal status was significantly associated with risk of malignancy (OR 2.41; 95% CI: 1.15-5.02; P=0.018).

Tissue protein levels in study group vs. control group are presented in Table I.

Median (IQR) of CA125 levels in study group was 116.5 (54.9-319.25 U/mL). The mean level of CA125 levels in tissues of high-grade serous carcinoma was 613.9±138.7 U/mL compared to 36.0±19.1 U/mL in low grade serous carcinoma (P<0.001). Table II compares various biomarker levels between high-grade and low-grade serous ovarian carcinoma.

Correlation of SOX 9 protein levels with CA 125

CA125 and SOX 9 levels in the study group were found to have positive correlation coefficient of 0.37 and P value of 0.004 as in Figure 2. CA 125 was significantly associated with SOX 9 levels. There is a significant positive expression of SOX 9 protein in the ovarian cancer cell line, acting as supportive evidence for the findings of increased SOX 9 protein in ovarian cancer tissues.

Close

Fig. 2.

Showing the scatter plot of CA125 and SOX 9 among the patients of ovarian cancer, showing a positive correlation between them.

Export to PPT

Discussion

SOX9 is a nuclear protein which is regulated by the gene Sox 9 that controls cell differentiation and pluripotency and regulates tissue homeostasis in adults.⁶ The present study concluded that SOX 9 protein is significantly overexpressed in ovarian carcinoma tissues when compared to the non-malignant controls, with markedly higher levels in high-grade tumours. SOX9 expression also showed a positive correlation with CA 125 levels and was consistently expressed in SK-OV3 ovarian carcinoma cells. Collectively, these findings highlight the biological relevance of SOX9 in ovarian tumourigenesis and its potential as a diagnostic and prognostic biomarker.

Several studies support our findings that SOX9 is upregulated in malignant ovarian tissues. Jo A et al⁷ and Sherman-Samis et al⁸ have reported higher expression of SOX 9 levels in high grade ovarian carcinoma, where it was associated with poor clinical outcome, also suggested the use of SOX 9 as a prognostic tool and perhaps a novel therapeutic target for further exploration. Similarly, Malki et al⁹ demonstrated SOX9 induction in ovarian cancer cell lines and confirmed its presence in human tumours.⁹ Siu et al¹⁰ showed that signalling cascades involving Erk1/2 and Nanog can drive SOX9 overexpression, contributing to invasion and metastasis. Recent evidence indicates that SOX9 is involved in chemoresistance through miR-34c/SOX9 regulatory axis, further indicating its clinical relevance.^11,12

SOX9 is known to influence multiple oncogenic pathways. Accumulating evidence shows their involvement in tumour initiation and progression. Onder et al⁶ evaluated immunohistochemistry markers such as FOXL2, SOX9 and β-catenin status in differentiating sex cord tumour with annular tubules (SCTAT) from other sex-cord stromal tumour and concluded that diffuse and strong expression of SOX 9 was a distinguishing feature.⁶ This strongly implicates the diagnostic utility of SOX9 in these tumours.¹² Raspaglio et al¹³ observed that higher levels of SOX9 are seen in biopsies of human Sertoli tumours, where its co-expression with Ki-67 and BCL-2 suggested a link with proliferative and anti-apoptotic signalling. Substantially, they reported that overall survival is significantly affected by SOX9 expression.¹³

Mechanistically, SOX9 modulates PGD2 linked pathways, represses Wnt/β-catenin signalling and cooperates with Snail/Slug to induce EMT (epithelial mesenchymal transition), thereby enhancing invasiveness.^14-16 Our findings of elevated SOX9 in high grade tumours align with these mechanisms, suggesting that its overexpression may accelerate the tumour progression and resistance to apoptosis. Aldaz et al¹⁷ further implied that the oncogenicity of SOX9 is through the upregulation of BMI 1 expression which represses the tumour suppressors like p21CIP and p16INK4A. SOX 9 induces tumour progression by promoting cell proliferation and evading senescence and apoptosis. Silencing of SOX9 showed reduced cell viability and cell proliferation and induced senescence, abrogating proliferation of ovarian cancer cells.¹⁸

It is also important to acknowledge that SOX9 exhibits context-dependent roles, functioning as an oncogene in ovarian and breast cancer, but reported as a tumour suppressor in cervical cancer.^19,20 This duality underscores the complexity of SOX9 biology and the need for tissue-specific investigations. In ovarian carcinoma however, the preponderance of evidence, including our results, support its oncogenic activity. SOX 9 has the potential to be the novel marker, but the translation into a diagnostic or prognostic tool would further require validation, including studies on serum or minimally invasive tissue specimen, larger sample size and longitudinal study designs.

The strengths of our study include the use of both clinical tissue samples and SK-OV3 cell lines, ensuring translational relevance and reproducibility of SOX9 expression. The present study also integrated correlation with CA 125, a standard biomarker which strengthens the findings. Limitations include relatively small sample size, the cross-sectional design, and the restriction to tissue-based measurements, which precludes direct application as a non-invasive diagnostic tool. Further studies should evaluate SOX9 in serum, ascitic fluid or circulating tumour cells to validate its clinical utility in early detection and monitoring.

In summary, our findings reinforce SOX9 as an important player in the molecular tumourigenesis of ovarian cancer, particularly in high grade tumours, where its expression correlates with disease severity. The high levels of SOX9 in early stages can prove its diagnostic ability with a sensitivity of 81%. By linking SOX9 with established oncogenic pathways such as Wnt/β-catenin inhibition, EMT promotion and BMI1 mediated repression of tumour suppressors, our study provides a mechanistic context to its overexpression in ovarian carcinoma. SOX 9 is yet to be explored as a therapeutic target, meriting its further investigation in larger, multicentric cohort studies.