The protein kinase LKB1 promotes self-renewal and blocks invasiveness in glioblastoma.

The role of liver kinase B1 (LKB1) in glioblastoma (GBM) development remains poorly understood. LKB1 may regulate GBM cell metabolism and has been suggested to promote glioma invasiveness. After analyzing LKB1 expression in GBM patient mRNA databases and in tumor tissue via multiparametric immunohistochemistry, we observed that LKB1 was localized and enriched in GBM tumor cells that co-expressed SOX2 and NESTIN stemness markers. Thus, LKB1-specific immunohistochemistry can potentially reveal subpopulations of stem-like cells, advancing GBM patient molecular pathology. We further analyzed the functions of LKB1 in patient-derived GBM cultures under defined serum-free conditions. Silencing of endogenous LKB1 impaired 3D-gliomasphere frequency and promoted GBM cell invasion in vitro and in the zebrafish collagenous tail after extravasation of circulating GBM cells. Moreover, loss of LKB1 function revealed mitochondrial dysfunction resulting in decreased ATP levels. Treatment with the clinically used drug metformin impaired 3D-gliomasphere formation and enhanced cytotoxicity induced by temozolomide, the primary chemotherapeutic drug against GBM. The IC50 of temozolomide in the GBM cultures was significantly decreased in the presence of metformin. This combinatorial effect was further enhanced after LKB1 silencing, which at least partially, was due to increased apoptosis. The expression of genes involved in the maintenance of tumor stemness, such as growth factors and their receptors, including members of the platelet-derived growth factor (PDGF) family, was suppressed after LKB1 silencing. The defect in gliomasphere growth caused by LKB1 silencing was bypassed after supplementing the cells with exogenous PFDGF-BB. Our data support the parallel roles of LKB1 in maintaining mitochondrial homeostasis, 3D-gliomasphere survival, and hindering migration in GBM. Thus, the natural loss of, or pharmacological interference with LKB1 function, may be associated with benefits in patient survival but could result in tumor spread.

The polarity protein Par3 coordinates positively self-renewal and negatively invasiveness in glioblastoma.

Glioblastoma (GBM) is a brain malignancy characterized by invasiveness to the surrounding brain tissue and by stem-like cells, which propagate the tumor and may also regulate invasiveness. During brain development, polarity proteins, such as Par3, regulate asymmetric cell division of neuro-glial progenitors and neurite motility. We, therefore, studied the role of the Par3 protein (encoded by PARD3) in GBM. GBM patient transcriptomic data and patient-derived culture analysis indicated diverse levels of expression of PARD3 across and independent from subtypes. Multiplex immunolocalization in GBM tumors identified Par3 protein enrichment in SOX2-, CD133-, and NESTIN-positive (stem-like) cells. Analysis of GBM cultures of the three subtypes (proneural, classical, mesenchymal), revealed decreased gliomasphere forming capacity and enhanced invasiveness upon silencing Par3. GBM cultures with suppressed Par3 showed low expression of stemness (SOX2 and NESTIN) but higher expression of differentiation (GFAP) genes. Moreover, Par3 silencing reduced the expression of a set of genes encoding mitochondrial enzymes that generate ATP. Accordingly, silencing Par3 reduced ATP production and concomitantly increased reactive oxygen species. The latter was required for the enhanced migration observed upon silencing of Par3 as anti-oxidants blocked the enhanced migration. These findings support the notion that Par3 exerts homeostatic redox control, which could limit the tumor cell-derived pool of oxygen radicals, and thereby the tumorigenicity of GBM.

The protein kinase SIK downregulates the polarity protein Par3.

The multifunctional cytokine transforming growth factor ß (TGFß) controls homeostasis and disease during embryonic and adult life. TGFß alters epithelial cell differentiation by inducing epithelial-mesenchymal transition (EMT), which involves downregulation of several cell-cell junctional constituents. Little is understood about the mechanism of tight junction disassembly by TGFß. We found that one of the newly identified gene targets of TGFß, encoding the serine/threonine kinase salt-inducible kinase 1 (SIK), controls tight junction dynamics. We provide bioinformatic and biochemical evidence that SIK can potentially phosphorylate the polarity complex protein Par3, an established regulator of tight junction assembly. SIK associates with Par3, and induces degradation of Par3 that can be prevented by proteasomal and lysosomal inhibition or by mutation of Ser885, a putative phosphorylation site on Par3. Functionally, this mechanism impacts on tight junction downregulation. Furthermore, SIK contributes to the loss of epithelial polarity and examination of advanced and invasive human cancers of diverse origin displayed high levels of SIK expression and a corresponding low expression of Par3 protein. High SIK mRNA expression also correlates with lower chance for survival in various carcinomas. In specific human breast cancer samples, aneuploidy of tumor cells best correlated with cytoplasmic SIK distribution, and SIK expression correlated with TGFß/Smad signaling activity and low or undetectable expression of Par3. Our model suggests that SIK can act directly on the polarity protein Par3 to regulate tight junction assembly.

TGF-ß Family Signaling in Epithelial Differentiation and Epithelial-Mesenchymal Transition.

Epithelia exist in the animal body since the onset of embryonic development; they generate tissue barriers and specify organs and glands. Through epithelial-mesenchymal transitions (EMTs), epithelia generate mesenchymal cells that form new tissues and promote healing or disease manifestation when epithelial homeostasis is challenged physiologically or pathologically. Transforming growth factor-ßs (TGF-ßs), activins, bone morphogenetic proteins (BMPs), and growth and differentiation factors (GDFs) have been implicated in the regulation of epithelial differentiation. These TGF-ß family ligands are expressed and secreted at sites where the epithelium interacts with the mesenchyme and provide paracrine queues from the mesenchyme to the neighboring epithelium, helping the specification of differentiated epithelial cell types within an organ. TGF-ß ligands signal via Smads and cooperating kinase pathways and control the expression or activities of key transcription factors that promote either epithelial differentiation or mesenchymal transitions. In this review, we discuss evidence that illustrates how TGF-ß family ligands contribute to epithelial differentiation and induce mesenchymal transitions, by focusing on the embryonic ectoderm and tissues that form the external mammalian body lining.