Doublecortin reduces glioma tumor progression via blocking mitosis by mitotic spindle catastrophe and inhibition of glioma cell invasion by depolymerization of actin.

A93 We recently demonstrated that doublcortin (DCX) suppresses glioma tumor growth in nude rats by blocking G2-M phase of cell cycle progression in glioma U87 cell. Interestingly, the microtubule (MT) associated proteins including DCX that inhibit tumors, are either absent or mutated in many tumors including glioma. The MTs play a critical role during mitosis. We therefore sought to examine the effect of DCX on the dynamic instability of MTs in mitotic spindle checkpoint of U87 cells. Immunostaining data revealed that the mitotic spindles of DCX transfected U87 cells underwent catastrophe. In contrast, mitotic spindle formation was normal in control U87 (DCX negative) and neurabin II null HEK293T cells transfected with or without DCX. Sequential immuno precipitation (IP) and Western blot analysis of total cell lysates showed that interaction between kinesin-13 (a major MT depolymerizer) and PP1 (a key inactivator of MT depolymerizer) was abolished in DCX synthesizing U87 cells, but re-established in the cells by knocking down either DCX or neurabin II with their siRNA or even by dephosphorylating DCX with Jun N-terminal kinase (JNK) inhibitor or alkaline phosphatase treatment. In contrast, Phophorylated DCX (P-DCX) is associated with neurabin II and PP1 in DCX synthesizing U87 cells. These data demonstrated that P-DCX acts as a competitive inhibitor of PP1 for association between Kinesin-13 and PP1. To complete mitosis, activation (phosphorylation) of kinesin is required to be balanced by its inactivation (dephosphorylation). Immunostaining data also confirmed that PP1 was associated with neurabin II and DCX, and colocalized in the cytosol of DCX expressing U87 cells, whereas, kinesin-13 localized in the nucleus, remained active and induced catastrophe in mitotic spindles that leads to growth arrest in G2-M phase of cell cycle progression of DCX synthesizing U87 cells in a novel neurabin II dependent pathway.
 >DCX targets PP1 that plays a major role in actin polymerization. Immunostaining data revealed that DCX transfection induced depolymerization of actin filaments in U87 cells. In contrast, DCX transfection had no effect on actin filaments in neurabin II null HEK293T cells. Dynamic alterations in the organization of the actin cytoskeleton play a critical role in tumor-associated progression such as invasion and metastasis. Migration assay showed that DCX transfection significantly inhibited glioma U87 cell invasion along with downregulation of matrix metalloproteinase-2 (MMP2) and MMP-9, markers for glioma cell invasion. This effect of DCX on reduction of glioma invasion was reversed by either DCXsiRNA or neurabin II siRNA transfection or JNK inhibitors or alkaline phosphatase treatment. These data demonstrated that the expression of both neurabin II and P-DCX were required to inhibit glioma invasion. P-DCX interacts with neurabin II and PP1 in the cytosol. Localization of DCX-NrbII-PP1 complex in the cytosol causes inhibition of phosphatase activities of PP1 that are involved in two mechanistic links of reduction of glioma tumor-associated progressions- 1) catastrophe in mitotic spindle checkpoint that leads to block mitosis and 2) depolymerization of actin that leads to inhibition of glioma cell invasion.