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

MARK4 (MAP/Microtubule Affinity-Regulating Kinase 4) belongs to a family of serine-threonine 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 adult sub-ventricular zone (SVZ), where stem cells reside, suggest that some GBM of our panel originated from the SVZ. In contrast, the concomitant expression of both MARK4 isoforms in the main substrates of glioma transformation, mature glial cells and progenitors, could explain the origin of the Oligodendrogliomas, Astrocytomas and of some GBM here studied. Furthermore, both MARK4 isoforms are expressed in neurons, extending published data of MARK4S as a neuron-specific marker in mouse CNS. By Immunofluorescence we found that the two MARK4 isoforms localize at centrosomes and midbody of normal and glioma cell lines, showing that MARK4 association with these cell compartments is neither isoform- nor tumor-specific. It has been reported that both normal and cancer stem cells can display centrosome amplification, genetic instability and loss of asymmetry with switch to a prevalent symmetric division, which may converge in malignant transformation and unrestrained growth of stem cells. Interestingly, we previously found MARK4 protein in association with glioma aberrant centrosomes, a hint for a possible role of the kinase in the abnormal mitotic processes of human glioma. Symmetric division, besides promoting the expansion of stem cell numbers, may also be permissive for secondary events leading to aneuploidies, since the machinery that controls asymmetric division also regulates the orientation of mitotic spindles and of centrosomes. Finally, an intriguing finding, delineating MARK4L as a tumor marker through its nucleolar association, comes from the evidence that L isoform, in contrast to MARK4S, is detectable in nucleoli and exclusively in cancer cells. Furthermore, the differential expression of MARK4 isoforms in undifferentiated and glial malignant cells expands the concept of MARK4 splice variants dysregulation in mediating tumor initiation and progression.