# Analysis of the Role of the SRC Tyrosine Kinase and Podoplanin in the Process of Entosis

## TL;DR

This study investigates how the proteins SRC and podoplanin (PDPN) influence entosis, a process where cancer cells engulf each other, which could help in understanding tumor progression and treatment.

## Contribution

The study identifies PDPN as a novel anti-entotic factor and suggests a PDPN-ERM axis as a molecular driver of entosis.

## Key findings

- PDPN-deficient cells are more prone to form entotic structures.
- SRC-deficient cells show suppressed entotic formation linked to increased PDPN expression.
- The PDPN-ERM axis appears to regulate entosis development.

## Abstract

Diagnosis and treatment of cancer constitutes a significant social and clinical problem due to the limited number of effective therapeutic strategies, resulting, among other reasons, from the still poorly understood biology of tumors. It is considered that one of the processes by which cancer cells might survive the host immune response and chemotherapies is their ability to form “entosis” (cell-in-cell structures), the main features of which are the presence of one cell inside another and a crescent-shaped nucleus surrounding the inner cell. Entotic figures are observed in selected cell lines/tissues derived from aggressive tumors, and a high number of entoses are associated with a more advanced tumor stage and worse prognosis. Therefore, our goal was to gain new knowledge concerning entosis by defining new molecular drivers of this process. We believe that, in the end, the identified pro-entotic factors may be clinically beneficial as specific biomarkers indicating the biological status/advancement of the tumor.

Background: Over the last years, the phenomenon of entosis, a form of cell-in-cell structure, has been highlighted in various tumors, including poorly treatable breast or pancreatic cancers. Nevertheless, not only the biological properties, but also the molecular drivers of entosis remain unclear. Here, we evaluated SRC tyrosine kinase, a key proto-oncogene, and podoplanin (PDPN), a membrane glycoprotein, as potential regulators of entotic cell formation. Methods: In the study, two entosis-competent cell lines, BxPC-3 and MFC-7, originating from pancreatic and breast cancers, respectively, were used. SRC or PDPN genes were silenced using dedicated siRNA and the frequency of entotic structure formation was assessed using fluorescent staining and confocal imaging. Results: It was found that BxPC-3 cells deficient in PDPN are more prone to form entotic structures and that over 90% of all entotic figures formed by mixed PDPN+ and PDPN- BxPC-3 cells involved PDPN-silenced cells. The SRC data supports this observation, as the suppressed entotic formation ability presented by SRC-deficient cells was linked with increased expression of PDPN. Even though the observed effects were mainly limited to BxPC-3 cells, as PDPN expression in MCF-7 cells is restricted, overall, the obtained data suggest a strong anti-entotic function of PDPN. Additionally, the performed Western blotting indicated the activation of ezrin-radixin-moesin (ERM) proteins in PDPN-deficient cells. Conclusions: Taken together, these data suggest that the negatively controlled PDPN-ERM axis may act as a molecular factor controlling the development of entotic structures and cells with naturally low PDPN expression may be more liable to form entoses.