(P 322) A NEW PLAYER IN SOFT TISSUE SARCOMA PROGRESSION: WNT5A IS A PROGNOSIS BIOMARKER AND A POTENTIAL ACTIONABLE TARGET

Objective: Soft tissue sarcomas (STS) are a heterogeneous group of mesenchymal tumors with more than 70 histological subtypes, accounting for around 1% of all adult malignancies. It is estimated that up to 50% of patients with localized STS develop tumor relapse after surgery, with increased risk of metastasis and poor outcomes. Therapeutic options for STS patients have not evolved much over time, being surgery the mainstay treatment, accompanied by radiotherapy or/and chemotherapy in both neoadjuvant and adjuvant settings. Targeted therapies for STS are very limited and only available for some STS subtypes. Hence, it is crucial to identify reliable biomarkers for early diagnosis, risk stratification, prognosis prediction and with therapeutic potential to improve STS patient outcomes. The goal of our study was to evaluate the potential value of WNT5A, one of the WNT family secreted ligands with controversial roles among different malignancies, as a new STS biomarker and therapeutic target.

Methods: Publicly accessible data from The Cancer Genome Atlas was extracted to assess the potential significance of WNT5A in STS (265 STS samples from Firehose Legacy dataset). We established WNT5A high and WNT5A low groups based on the obtained mRNA cut-off values for overall survival (OS) and disease-free survival (DFS) through Youden index analysis. Survival rates were evaluated by Kaplan-Meier curves. Then, in vitro WNT5A silencing experiments were performed using four different STS cell lines by reverse transfection. Human WNT5A siRNA and Control siRNA-A used were purchased from Santa Cruz Biotechnology. STS cells were also treated with 20 µM of the WNT5A antagonist, Box5 (Sigma-Aldrich). CellTiter 96® AQueous One Solution (Promega), Wound Healing assay and clonogenic assay were used to explore the effects of WNT5A downregulation and Box5 inhibitor on STS cell proliferation, migration and colony formation. The expression of epithelial to mesenchymal transition markers was analyzed by RT-qPCR. Finally, exosomes were isolated from pre- and post-surgery liquid biopsy samples of four STS patients using ExoGAG commercial kit. Exosomic WNT5A levels were quantified by enzyme-linked immunosorbent assay (ELISA) kit.

Results: OS and DFS of WNT5A high STS patients were enormously shorter than that of WNT5A low STS patients when we correlated WNT5A mRNA expression with patient outcomes of a Sarcoma TCGA dataset. To study the potential of WNT5A as a therapeutic target, we performed WNT5A gene silencing or inhibited WNT5A signaling by using Box5 in four WNT5A expressing STS cell lines. Results showed a significant decrease in STS cell migration and colony formation capacity when we impaired WNT5A expression or signaling with the specific inhibitor. In all the cases, antitumoral effects were more pronounced in WNT5A silencing experiments than in Box5 treatment experiments. STS cell viability was not altered due to WNT5A expression or signaling inhibition. To shed light on the molecular mechanisms of the obtained results, the expression of the EMT markers vimentin (VIM) and E-cadherin (CDH1) was analyzed. STS cell lines suffered a slight decrease in vimentin expression levels. At the same time, two experienced a significant increase in CDH1 expression levels, supporting the hypothesis that STS cell lines become more phenotypically epithelial-like during WNT5A gene silencing. Finally, exosomes were isolated from liquid biopsies of four STS patients before surgery (baseline) and at three months follow-up. Baseline samples consisted of three primary tumors and one recurrence, while after three months, three patients were disease-free and one developed a recurrence. Exosome-derived WNT5A levels were quantified in pre- and post-surgery samples. In the three cases of disease-free STS patients at follow-up, WNT5A levels decreased compared to baseline samples. Interestingly, the patient having a relapse at three months presented a more than double increase in WNT5A levels compared to the ones found in the matched primary tumor. Samples corresponding to STS recurrences expressed higher WNT5A levels than STS primary tumors, being these levels also higher than that detected in disease-free samples.

Conclusion: Elevated WNT5A expression level in STS patients is indicative of poor prognosis. WNT5A gene silencing and WNT5A targeting with the specific antagonist Box5 impair STS cell migration and colony formation in vitro. Moreover, WNT5A can be detected in liquid biopsies-derived exosomes of STS patients and an increase in its levels during the follow-up could serve as a prognostic biomarker of recurrence. All in all, WNT5A emerges as a promising therapeutic strategy to mitigate STS tumors invasion and dissemination and as a new prognostic and predictive biomarker of recurrence in STS.