DIFFERENTIAL INVOLVEMENT OF S AND L ISOFORMS OF THE CENTROSOMAL MARK4 KINASE IN GLIOMA INITIATION AND PROGRESSION

Abstract

2 MARK4 (MAP/Microtubule Affinity-Regulating Kinase 4) belongs to a family of serinethreonine kinases that are able to phosphorylate the Microtubule Associated Proteins, causing their detachment from the microtubules and thus increasing microtubule dynamics. MARK4 gene is ubiquitously expressed and encodes at least two alternatively spliced isoforms, L and S, which differ in the C-terminal region and are differentially regulated in human tissues, especially in the Central Nervous System (CNS). MARK4S predominance in normal brain has been related to a putative role in neuronal differentiation; MARK4L has been found up-regulated in hepatocarcinoma cells and highly expressed in gliomas, suggesting an involvement of the kinase in cycling cells. Gliomas are the most common tumors of the CNS and are characterized by elevated cellular heterogeneity, which has been imputed to the presence of different cells (mature cells / progenitors / stem cells) from which each glioma originates. To further investigate MARK4 up-regulation in glial tumors, we analyzed a panel of 35 glioma tissue samples and 21 glioma cell lines (both low and high malignancy grade) and 6 glioblastoma-derived cancer stem cell populations (GBM CSC; glioblastoma is a IV grade glioma). Human neural progenitors, total human normal brain and mouse neural stem cells (NSC, isolated from the sub-ventricular zone) were also included in the study. A quantitative approach (Real-time PCR; q-PCR) was applied for mRNA analyses whereas MARK4 protein levels and localization were tested by Immunoblotting (IB), Immunohistochemistry (IHC) and Immunofluorescence (IF). We first carried out mutational analysis of the main MARK4 domains, but it didn’t reveal any genomic alteration, as did not the previously performed array-CGH analysis. Integrated approaches of q-PCR, IB and IHC studies show that, although MARK4S and L have a heterogeneous expression within and across different glioma subtypes (consistent with the intrinsic cell heterogeneity of these brain tumors), MARK4L is the prevalent isoform in near all the glioma samples. Conversely, MARK4S mRNA levels display a significant decrease inversely correlating with malignancy grade and are also hardly detectable in both neural and GBM-derived cancer stem cell populations. Therefore, a higher MARK4L prevalence in parallel to low levels of MARK4S characterizes highly undifferentiated cells, such as NSC, and highly malignant cells, such as GBM CSC and glioblastomas, favouring the hypothesis that the ratio between the two MARK4 isoforms is strictly regulated along neural differentiation and may be subverted in gliomagenesis. These findings, together with the observation that in normal brain only the L isoform localizes in the embryonic ventricular zone (VZ) and