Elucidation Of The Role Of Plexin-A2 In Tumor Progression

Introduction: The plexin family of receptors includes 9 members divided into 4 subfamilies. They are single pass transmembrane receptors characterized by an intracellular GTPase activating (GAP) domain. Plexins can function as both ligand-binding receptors and as signaling receptors for all semaphorins, which are a family of signaling molecules that were initially identified as axon guidance cues but today they are known also as important regulators of angiogenesis and tumor progression. It was recently discovered that plexin-A2 protects cancer cells from death after human papillomavirus infection.

Materials and Methods: U87MG cells were infected with specific shRNA viral vectors directed for plexin-A2. Additionally, plexin-A2 knock-out U87MG cells were created by using the CRISPR/Cas9 methodology. Plexin-A2 was re-expressed in plexin-A2 knock-out U87MG cells by infection with lentiviral vector encoding full length plexin-A2. U87MG cells were also infected with a truncated plexin-A2 (A2ExTm) containing the extracellular and trans-membrane domain. Proliferation assays were performed by seeding U87MG cells in complete growth medium (10% FCS). The number of adherent cells after a week was determined by using a coulter-counter or by using the WST-1 assay according to vendor instructions. Clonal colony formation assay was done using complete growth medium. After 10 days colonies were fixed in 4% PFA and stained with crystal violet. Tumour formation assay was performed by subcutaneously injecting U87MG control cells or cells that were silenced for plexin A2 expression into Athimic/Nude mice. The development of tumors was measured twice a week. At the end of the experiment the tumors were excised and weighed.

Results and discussion: Inhibition of Plexin-A2 expression strongly inhibits U87MG cell proliferation in vitro as well as tumor formation from such cells in-vivo. Additionally, the proliferation of plexin-A2 knock-out U87MG cells is also strongly inhibited in vitro and is rescued following the expression of the cDNA encoding the full length plexin-A2 in the knock-out cells. The loss and rescue of plexin-A2 expression are accompanied by profound changes in the shape of the cells. U87MG cell proliferation in vitro is also inhibited when the cells are induced to express a truncated plexin-A2 (A2ExTm) containing the extracellular and trans-membrane domain which apparently functions as a dominant-negative inhibitor.

Conclusions: Plexin-A2 expression in U87MG cells enables cell proliferation and tumor progression. Plexin-A2 may represent a novel target for the development of novel anti- tumorigenic drugs and we are currently trying to elucidate the mechanism by which it affects tumor progression.