CIRCULATING TUMOR SIGNALING MOLECULES WNT - Β-CATENIN AND WISP1, IL-8 AS PROGNOSTIC BIOMARKERS IN OVARIAN CANCER

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Introduction. Epithelial ovarian cancer (OC) is the most lethal gynecological malignancy, with a 5-year survival rate of 20-30% [1], with virtually no specific symptoms in the early stages. Multiple signaling pathways, including complex Wnt signaling, are involved in the progression of OC. To date, Wnt activity has been shown to correlate with the degree of malignancy, chemoresistance, epithelial-mesenchymal transition, and poor prognosis in patients with OC [2]. β-catenin (CTNNB1) plays a dual role in the cell. It is a key effector of the Wnt/β-catenin signaling pathway, being a transcriptional coactivator of TCF/LEF target genes, and acts as a key component of cadherin-based cell adhesion. Literature data confirm the existence of membrane, cytoplasmic, and nuclear pools of CTNNB1 [3]. Dysregulation of Wnt/β-catenin signaling can be caused by mutations in various components of the pathway. β-catenin mutations are considered to be the most common alterations in cancer [4]. Wnt/β-catenin signaling in tumors has been shown to be associated with the ability to support cell proliferation and preserve stemness. A role for Wnt/β-catenin signaling has also been shown in angiogenesis, chemotherapy resistance, tumor metastasis and metabolism, and in the regulation of the tumor microenvironment [5]. Altered serum β-catenin levels have been shown in colorectal cancer [6], osteonecrosis [7], and liver diseases [8]. WISP1 (CCN4), a secreted protein of the CCN family, is a downstream target gene of Wnt/β-catenin signaling. WISP1 is expressed in the heart, placenta, kidneys, spleen, lungs, ovaries, brain and as a matricellular protein is able to modulate mitosis, cell growth arrest and migration, apoptosis and adhesion. It has been shown that aberrant expression of WISP is associated with various pathologies, including cancer. Moreover, WISP proteins can exhibit both pro- and anti-oncogenic properties [9]. Increased expression of WISP was detected in ovarian cancer tissues and cells and correlated with a poor prognosis [10]. IL-8 is a chemotactic protein secreted by several cell types, including neoplastic cells [11]. It has been shown that its pathological overexpression correlates with an unfavorable prognosis in cancer patients, sensitivity to chemotherapy and persists throughout the disease. Moreover, the expression of IL-8 receptors in neutrophils, macrophages and endothelial cells shows that autocrine and paracrine activity of IL-8 stimulates signaling pathways, including Wnt/β-catenin signaling, promoting proliferation, angiogenesis and migration of endothelial cells. It has been established that the molecular mechanisms of IL-8 action contribute to carcinogenesis, tumor development and metastasis [11].

The aim of the study was to assess the prognostic significance of circulating Wnt signaling molecules - β-catenin, WISP1 and IL-8 in advanced OC.

Material and methods. The study included 30 patients (median age 60 years) treated at the Ulyanovsk Regional Clinical Oncology Dispensary in 2020-2023. The inclusion criteria were newly diagnosed ovarian cancer at stage III-IV according to FIGO (ascites form), cytologically verified serous high-grade adenocarcinoma, general condition according to the ECOG (Eastern Cooperative Oncology Group) scale of 0±2 points, signed informed consent and life expectancy of more than 2 months. At the first stage of treatment, patients received neoadjuvant chemotherapy (CT) according to the TP (paclitaxel + cisplatin) regimen from 2 to 4 courses with an interval of 3 weeks. Subsequently, cytoreductive surgery and adjuvant chemotherapy were performed. Blood was collected before the start of specific antitumor treatment. The comparison group was the plasma of somatically healthy women comparable in age to the experimental group (median - 56 years). The study was approved by the local ethics committee of the Institute of Medicine, Ecology and Physical Culture of Ulyanovsk State University (protocol No. 6 dated June 15, 2020). The ELISA method was used to assess the level of IL-8 (Kit A-8762, Interleukin-8-ELISA-BEST, Vector-Best JSC, Russia), beta-catenin (β-catenin) (Kit SEB021Hu Cloud-Clone Corp., PRC) and WISP1 (Kit SEG895Hu Cloud-Clone Corp., PRC) in plasma. During dynamic observation during and after completion of chemotherapy according to the TR regimen, patients were divided into 2 groups: I - progression against the background of CT (platinum-resistant group) and early relapse; II – relapse after 6 or more months after the end of chemotherapy (platinum-sensitive group). The sets of quantitative parameters, the distribution of which differed from the normal one, were described using the median (Me) values ​​and the lower and upper quartiles (Q0.25-Q0.75). The study was performed prospectively, and the progression-free time (PFT) was estimated to assess the risks of early relapse in common forms of OC. The analysis of patients' PFT was performed using the Cox and Kaplan-Meier regression methods. To identify categories based on the values ​​used in the forecasting, a cut-point was determined. The construction of a prognostic model of the risk of malignant neoplasm outcome was performed using the binary logistic regression method. The statistical significance of the resulting model was determined using the χ2 criterion. The quality of the prognostic model obtained using ROC analysis was assessed based on the values ​​of the area under the ROC curve with the standard error and 95% confidence interval (CI) and the level of statistical significance (Jamovi 2.4.14).

Results and discussion. As a result of the conducted studies, significant changes in the level of β-catenin, WISP1 and IL-8 in the blood plasma of patients with advanced ovarian cancer were found compared to the control (Table 1).

Table 1 The level of β-catenin, WISP1 and IL-8 in the plasma of patients with advanced ovarian cancer (Me (Q1-Q3))

groups		Parameters
		β-catenin, pg/ml	WISP1, pg/ml	IL-8, pg/ml
control, n=12		12,0 (7,85-13,8)	342,0 (302,0-378,0)	42,6 (35,5-45,5)
ovarian cancer	III stage, n=14	17,3 (15,9-44,9) p1=0,003	358,0 (320,0-445,0) p1=0,283	26,7 (25,0-28,1) p1=0,059
	IV stage, n=16	19,9 (17,8-23,1) p1=0,001 p2=0,032	440,0 (434,0-522,0) p1=0,001 p2=0,007	115,0 (109,0-131,0) p1=0,001 p2=0,001
	platinum sensitive group, n=13	17,9 (16,9-20,4) p1=0,042	440,0 (348,0-483,0) p1=0,021	55,9 (27,0-108,0) p1=0,033
	platinum-resistant group, n=17	17,7 (15,6-19,2) p1=0,045	435,0 (377,0-494,0) p1=0,013	109,0 (26,5-12,5) p1=0,029

 

Note: p₁ – level of statistical significance of differences compared with similar parameters in healthy donors, p₂ – the level of statistical significance of differences compared to the indicators at the previous stage of the disease.

At the same time, no significant differences were found between the studied parameters in the blood plasma of patients with refractory and sensitive tumors: β-catenin level (p=0.740); WISP1 level (p=0.695) and IL-8 level (p=0.449). Analysis of the obtained data allowed us to establish a statistically significant increase in the blood plasma of patients with stage IV of the disease compared to patients with stage III OC: β-catenin level (p=0.032), WISP1 (p=0.007) and IL-8 (p=0.001). Discussion. A significant amount of data confirms the importance of Wnt/β-catenin signaling in the stemness of OC, progression to malignant tumor and resistance to chemotherapy. CTNNB1 (catenin-β1), on the one hand, is the main effector operating at lower levels of Wnt signaling. On the other hand, it is a key component of cadherin-based adhesive compounds. The activation of β-catenin, both at the gene and protein levels in neoplastic processes, is evidenced by the results of a number of in vitro studies in cervical cancer, colon cancer, ovarian cancer, B-cell lymphoma, and lung cancer. The level of β-catenin in blood plasma was assessed in osteonecrosis. In this case, an increased level of the biomarker correlated with the stages of acute necrosis of the femoral head [7]. When determining β-catenin in liver pathology, a correlation was shown between its level and the parameters of the necroinflammatory process, with the phase of viral hepatitis B disease, which allows predicting the development of hepatocellular carcinoma. Serum overexpression of β-catenin is, according to a number of authors, a potential marker for detection in patients with colorectal cancer (CRC). β-catenin levels were elevated in CRC but did not correlate with disease stage [6].

We have established an elevated level of β-catenin in the blood serum of patients with widespread ovarian cancer. Moreover, the indicators at clinical stage IV of the disease were significantly (p=0.032) higher than the indicators at stage III. The multivariate Cox model shows the possibility of assessing the risk of developing stage IV of the disease based on the level of β-catenin in blood plasma (HR 0.90 (0.81-0.99, p=0.033). Thus, the level of β-catenin in blood plasma can be considered as a prognostic factor in assessing the progression of OC. Cytosolic β-catenin is a key mediator between the activation of Wnt signaling and increased expression of WISP1, a secreted protein of the CCN family. WISP1 is expressed in many organs, including the ovaries, and is involved in the occurrence and development of malignant neoplasms of various localizations. Increased expression of WISP1 in OC tumor tissue has been established, which correlated with a poor prognosis [12]. An increased level of WISP1 in the blood serum was found in prostate cancer [13]. We have found that the level of WISP in the blood serum of patients at clinical stage III of OC reliably does not differ from that in the control (p=0.283). However, with progression in patients at stage IV OC, the serum WISP level increases sharply and significantly and exceeds both the control (p=0.001) and the level at stage III of the disease (p=0.007). In the univariate logistic regression analysis for differential diagnosis between stage III and IV OC, statistical significance was jointly demonstrated by β-catenin (OR 0.718 95% CI 0.503-1.024, p=0.047) and plasma WISP1 (OR 0.980 95% CI 0.968-0.993, p=0.002). The area under the curve (AUC) of this model was 0.883, and stage IV ovarian cancer can be diagnosed with 91.3% probability (Spec.=1.00, Sens.=0.80) (Fig. 1). The risk of developing stage IV ovarian cancer increases with simultaneously elevated levels of β-catenin (more than 28.1 pg/ml) and WISP1 (more than 445 pg/ml) in blood plasma (R2=0.451, χ²=28.4, p=0.001).

Figure 1. ROC curve for the regression model of differential diagnosis of stage III and IV ovarian cancer taking into account β-catenin and WISP1 in blood plasma

It has been previously shown that IL-8, as an inflammatory chemokine, promotes oncogenesis in malignant neoplasms of a number of localizations. In laboratory conditions, IL-8 promoted the migration of cancer cells, triggered the epithelial-mesenchymal transition program, and activated the Wnt/β-catenin signaling pathway. It has also been established that in the tissues of progressive serous OC, the level of IL-8 expression is associated with the stage of OC, its malignancy, and metastases in the lymph nodes [14]. When we assessed the chemokine IL-8 levels in the blood serum, we found a significant decrease in its level compared to the control in patients with stage III OC (p=0.059) and a sharp increase at stage IV OC both compared to the control (p=0.001) and compared to the IL-8 level in patients at stage III of the disease (p=0.001). We found that with an increase in the serum IL-8 level above 87.3 pg/ml, the median PFT is 9.8 months [95% CI 8.1-14]; if the serum level is below 87.3 pg/ml, the median PFT is 13.2 months [95% CI 11.8-55] (Fig. 2).

Figure 2. Progression-free time curve of patients with ovarian cancer depending on the level of IL-8 (cut-point = 87.3 pg/ml) in blood plasma (Long-rank = 0.001)

Moreover, with an increase in the level of circulating IL-8 by 1 pg/ml, the odds ratio (OR) increases by 1.02 times (1.0-1.04, p<0.001). Conclusions. The established cutoff point for the IL-8 level for patients with a median PFS of 9.8 months and 13.2 months was 87.3 pg/ml IL-8. The level of β-catenin in blood plasma can be considered as a prognostic factor in assessing the risk of metastasis. At the same time, for the differential diagnosis of stages III and IV of OC, β-catenin and WISP-1 in blood plasma demonstrated statistical significance together.

 

About the authors

Tatyana Vladimirovna Abakumova

Ulyanovsk State University

Email: taty-abakumova@yandex.ru
ORCID iD: 0000-0001-7559-5246
SPIN-code: 8564-4253
Scopus Author ID: 37103623900

PhD, MD (Biology), Professor of Department of Physiology and Pathophysiology

 

Russian Federation, Ulyanovsk, 42, Leo Tolstoy Str.

Dinara R. Dolgova

Ulyanovsk State University

Email: dolgova.dinara@yandex.ru
ORCID iD: 0000-0001-5475-7031
SPIN-code: 7093-3564
Scopus Author ID: 55378365200

Candidate of Biological Sciences, Associate Professor, Associate Professor of the Department of Physiology and Pathophysiology

Russian Federation, Ulyanovsk< 42, Leo Tolstoy Str.

Inna Ivanovna Antoneeva

Ulyanovsk State University;
Regional Clinical Oncology Center of Ulyanovsk

Email: aii72@mail.ru
ORCID iD: 0000-0002-1525-2070
SPIN-code: 5305-5108
Scopus Author ID: 56529650400

PhD, MD (Medical), Professor, Professor of Department of Physiology and Pathophysiology; Head, Department of Oncogynecology

 

 

Russian Federation, Ulyanovsk, 42, Leo Tolstoy Str.; Ulyanovsk, 90, 12th September Str.

Ilseya R. Myagdieva

Ulyanovsk State University

Email: Ilseya2015@yandex.ru
ORCID iD: 0000-0002-3908-0840
SPIN-code: 1240-5547
Scopus Author ID: 58249336400

Senior Lecturer of Department of Physiology and Pathophysiology

Russian Federation, Ulyanovsk, 42, Leo Tolstoy Str.

Snezhanna O. Gening

LLC BestDoctor, Moscow

Email: sgening@bk.ru
ORCID iD: 0000-0001-6970-6659
SPIN-code: 2496-2467
Scopus Author ID: 55151451500

candidate of medical sciences, PhD

Russian Federation, Moscow, st. Vyatskaya, 27 building 15

Tatyana Petrovna Gening

Ulyanovsk State University

Email: Naum-53@yandex.ru
ORCID iD: 0000-0002-5117-1382
SPIN-code: 7285-8939
Scopus Author ID: 6507217338

PhD, MD (Biology), Professor, Head of Department of Physiology and Pathophysiology

 

Russian Federation, Ulyanovsk, 42, Leo Tolstoy Str.

Elizaveta Viktorovna Burkhanova

Ulyanovsk State University

Author for correspondence.
Email: burkhanova_e@inbox.ru
ORCID iD: 0009-0008-6955-9204
SPIN-code: 4883-1274

student of the Faculty of Medicine, "General Medicine"

Russian Federation, Ulyanovsk, 42, Leo Tolstoy Str.

References

Supplementary files