CD99 regulation of cancer cell dynamics and tumour formation


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Adam Odell1,Aarren Mannion2,Alison Taylor3,Pam Jones3,Graham Cook3
1York St. John University,2Karolinska Institute,3University of Leeds

Abstract

Background

Secondary breast cancer carriers the greatest risk to patient life and therapeutic targeting of metastasis remains a prominent novel anti-cancer strategy. During metastasis, tumour cells cross endothelial cell barriers via mechanisms akin to extravasating leukocytes, utilising numerous cell surface molecules in the process. CD99 is one such molecule capable of modifying leukocyte transendothelial migration (TEM). However, CD99 is also expressed by tumour cells, suggesting a potential role for this molecule in tumour progression and cancer TEM.

Method

We used RNA interference to deplete CD99 protein from human breast cancer (MDA-MB-231) and primary endothelial (HUVECs) cells. Tumour-endothelial cell interactions and cytoskeletal dynamics were analysed in vitro, using fluorescence microscopy, live cell imaging and impedance-based analyses. CDC42 activity was determined using pull down assays with PAK1 coated beads and immunoblotting. Angiogenesis assays were performed in vitro using co-cultures models. For tumour progression studies, we used a mouse tail vein injection xenograft model with luciferase-expressing MDA-MB-231 cells, complemented by expression analysis of publicly available breast cancer databases.

Results

Breast cancer cell lines expressed moderate to high levels of CD99 protein and tumour cell CD99 was required for cancer cell adhesion to the endothelium. However, the presence of tumour cell CD99 impeded the subsequent TEM of breast cancer cells. Depletion of tumour cell CD99 was associated with cytoskeletal remodelling and increased activity of the Rho family GTPase, CDC42. For endothelial cells, loss of CD99 also increased activity of CDC42, while enhancing endothelial barrier function and reducing tumour cell TEM. Importantly, normal CD99 expression inhibited metastatic breast cancer progression to the lungs in vivo. Furthermore, in patients, we found reduced CD99 expression was associated with metastasis.

Conclusion

Cell surface CD99 negatively regulates the activity of the Rho family GTPase CDC42 in both breast cancer and endothelial cells. For tumour cells, loss of CD99 favours CDC42 activity, TEM and tumour progression. In endothelial cells, CD99 loss strengthened endothelial junctions while reducing tumour cell adhesion and TEM. As a key signal transduction hub, CDC42 activity influences numerous hallmarks of cancer. The functional link between CD99 and CDC42 implicates CD99 in regulating these diverse pathways through alterations in cytoskeletal remodelling.