P-049 Verteporfin suppresses cell proliferation, survival and migration of TCam-2 Human Seminoma Cells via inhibits the YAP-TEAD complex
Authors: Önel T , Yıldırım E , Yaba A 
Published: June 2023

Abstract

Study question
Does verteporfin administration effect on proliferation, survival and migration of TCam-2 human seminoma cells?

Summary answer
Verteporfin may be a promising anticancer agent for TCam-2 human seminoma cells via inhibition of cell viability, proliferation, migration and stimulation of apoptosis.

What is known already
Seminoma is germ cell tumor of the testis and TCam-2 cells is the first seminoma-derived cell line that retains many of the characteristic traits of seminoma. The Hippo signalling is a evolutionarily highly conserved pathway that controls organ size, tissue homeostasis, and cancer development. In mammals, the Hippo pathway consists of the serine/threonine kinases Mammalian Sterile 20-protein Kinase 1 and 2 (MST1/2), Large Tumor Suppressor Homologues 1 and 2 (LATS1/2), Yes-Associated Protein (YAP), and its transcriptional cofactor TAZ. Verteporfin inhibits YAP–TEAD complex, blocking cell proliferation and survival.

Study design, size, duration
TCam-2 cells were treated with different concentrations of verteporfin (1, 5, 10, 20 and 40 µM) for 24, 48 and 72 hours (h).

Participants/materials, setting, methods
TCam-2 cells were treated with verteporfin at different doses and incubation times. Afterwards, protein expression of Hippo pathway proteins was determined by western blot, mRNA levels were analyzed by qRT-PCR and protein localizations were evaluated by immunoflourescence staining. Cell migration was determinated with wound-healing scratch assay; cell viability, early-late apoptosis and necrosis rates were evaluated by flow cytometry.

Main results and the role of chance
We detected that 40 μM verteporfin decreased LATS1/2 and p-LATS1/2 protein expression after 48h and 72h treatment (p &lt; 0.05, **p&lt;0.01). MST1/2 and p-MST1/2 was significantly decreased in groups treated with 20 and 40 µM verteporfin after 48 and 72 h of treatment (p &lt; 0.05, ****p&lt;0.0001). YAP expression decreased at 5, 20 and 40 μM verteporfin treatment after 48h and all verteporfin concentrations after 72h (**p&lt;0.01, ***p&lt;0.001, ****p&lt;0.0001). p-YAP expression significantly decreased at all verteporfin concentrations after 48h and only at 40 μM concentrations after 72h (**p&lt;0.01). TEAD4 expression decreased at all verteporfin concentrations after 48 and 72h (****p&lt;0.0001). After 48 and 72h of verteporfin treatment, no significant difference was observed in the expression of the MST1, MST2, LATS1, LATS2 and YAP1 mRNA levels in TCam-2 cells, while TEAD4 mRNA expression level was significantly decreased at 20 and 40 µM compared to control (**p&lt;0.01, ***p&lt;0.001). We also showed that Hippo signaling pathway proteins localized in the cell nucleus especially YAP1 and TEAD4, were translocated to cytoplasm depending on the increasing dose of verteporfin in TCam-2 cells. Verteporfin decreased cell proliferation (****p&lt;0.0001), migration and cell viability (****p&lt;0.0001), as well as increased the early-late apoptosis (**p&lt;0.01, ***p&lt;0.001, ****p&lt;0.0001) in TCam-2 human seminoma cells dose-dependent manner.

Limitations, reasons for caution
The TCam-2 cell line was gifted by Prof. Dr. Hubert Schorle (Department of Developmental Pathology and Institute of Molecular Diagnostic Pathology, Faculty of Medicine, Germany, Bonn) to our laboratory. There is no limitation in the scope of the study.

Wider implications of the findings
We determined for the first time the effect of verteporfin treatment on the protein expression and mRNA levels of Hippo signaling pathway in TCam-2 human seminoma cells. We suggested that Hippo pathway and verteporfin relationship may be a therapeutic target for seminoma by affecting cell proliferation, survival, migration and apoptosis.

Trial registration number
not applicable