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Abstract
Neuroblastoma, the most common extra-cranial solid tumour in children, accounts for 15% of paediatric cancer
deaths. Survival for patients with distant metastases carries a very poor prognosis. This highlights the need to
address metastatic neuroblastoma, the mechanisms of which are poorly understood. Metastasis, the movement
of disease from one site to another, involves cell cytoskeletal remodelling. The overexpression of the
microtubule destabilising protein, stathmin, is an important contributor of metastasis in neuroblastoma. There is
limited knowledge of stathmin’s role in neuroblastoma metastasis. This study evaluated stathmin’s contribution
in specific steps of metastasis, how stathmin exerts these effects and whether stathmin can be therapeutically
targeted in metastatic neuroblastoma.
Stathmin suppression reduced cell migration and invasion, and 3D tumour spheroid invasion into 3D
extracellular matrices, without influencing anoikis. Moreover, stathmin suppression reduced transendothelial
migration (TEM). Additionally, to examine TEM in vivo, stathmin suppression reduced whole body, lung,
kidney and liver metastasis. Treatment of neuroblastoma cells with ROCK inhibitors returned TEM to control
levels highlighting that stathmin regulates TEM through ROCK. Stathmin suppression increased the activation
of upstream RhoA. Examination of the microtubule-bound GEF-H1 was found not to be influenced by stathmin
levels, highlighting that stathmin influences RhoA activation via another means. Interestingly, restoration of
stathmin levels with either wild-type stathmin or 4E:mutant (defective tubulin-binding ability) stathmin returned
cell migration back to controls indicating that stathmin influences migration in neuroblastoma cells
independently of tubulin binding.
To target stathmin therapeutically in metastatic neuroblastoma, stathmin siRNA was delivered, using
InvivofectamineTM, to suppress stathmin in vivo. Stathmin siRNA was delivered and partially reduced (~30%)
stathmin expression in tumour cells. This level of stathmin suppression did not influence metastasis.
In conclusion, this study demonstrates that stathmin regulates neuroblastoma cell migration and invasion, and
TEM via RhoA activation. Furthermore, stathmin’s influence on cell migration is independent of tubulin
binding. Further investigation is required to determine how stathmin is regulating these phenotypes in a
microtubule independent manner and how to enhance stathmin therapeutic targeting through exploitation of key
metastasis-related signalling pathways.