Characterization of primary cilia in patient-derived glioma stem cells

Primary cilia are highly conserved eukaryotic organelles that serve as a ‘’cellular antenna’’ for signaling functions. Dysfunctions or defects in primary cilia are associated with numerous human diseases. Typically, the mother centriole, transforms into a structure called basal body, which then templates the formation of the primary cilium. Abnormal cilia are implicated in cancer progressions such as in breast, pancreatic, brain, and prostate cancers. Glioblastoma multiforme (GBM) is one of the most frequent lethal primary brain tumors. GBM is characterized by extreme heterogeneity, rapid growth, and efficient invasion of neoplastic cells, called Glioblastoma Stem-like Cells (GSCs). GSCs represent a subpopulation of the cells that are resistant to treatment and are suspected to be driving forces for the disease recurrence. Until now, there is no effective treatment for GBM. The average median survival time of the patients from the initial diagnosis is 12-15 months. GBM cells appear to lose cilia, which can contribute to the malignant phenotype. However, the mechanism of suppressed ciliogenesis is not fully characterized yet. Thus, the aims of this thesis were (i) to understand the possible mechanisms that can suppress the ciliogenesis in GSCs, (ii) to investigate the pathways that could restore the ciliogenesis and, (iii) characterize GSCs after cilium induction. Primary cilium assembly and disassembly are a dynamic process, which is coupled with the cell cycle. At the onset of cilium disassembly, the Centrosomal-P4.1-associated protein (CPAP) provides a scaffold for the cilium-disassembly complex (CDC) proteins, including NDE1, OFD1, NEK2, and CPAP. These proteins are recruited to the ciliary base to ensure timely cilium disassembly and promote cell cycle progression. Hence, using multidisciplinary approaches, the current doctoral thesis investigated primary cilia dynamics in multiple patient-derived GSCs. The experiments revealed that elevated levels and recruitment of CDC components lead to suppressing of the ciliogenesis and an increase in the cell cycle progression. Moreover, depletion of different CDC proteins induced ciliogenesis in GSCs in which PDGFR-α level is elevated. Among the CDC proteins, NEK2 depletion induced the maximum frequencies of ciliation in GSCs. Furthermore, inducible overexpression of the catalytically inactive NEK2 in GSCs was sufficient to induce cilia irreversibly. Importantly, both functional, including transcriptomic analyzes, showed that cilium induction switched GSCs from self-renewal to differentiation state. Taken together, the current work provides evidence for a novel mechanism to induce ciliogenesis in patient-derived GSCs and suggests that the cilium induction can potentially serve as a new strategy to intervene in GSCs proliferation

Rapid and Efficient Invasion Assay of Glioblastoma in Human Brain Organoids

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Recent Zika Virus Isolates Induce Premature Differentiation of Neural Progenitors in Human Brain Organoids

The recent Zika virus (ZIKV) epidemic is associated with microcephaly in newborns. Although the connection between ZIKV and neurodevelopmental defects is widely recognized, the underlying mechanisms are poorly understood. Here we show that two recently isolated strains of ZIKV, an American strain from an infected fetal brain (FB-GWUH-2016) and a closely-related Asian strain (H/PF/2013), productively infect human iPSC-derived brain organoids. Both of these strains readily target to and replicate in proliferating ventricular zone (VZ) apical progenitors. The main phenotypic effect was premature differentiation of neural progenitors associated with centrosome perturbation, even during early stages of infection, leading to progenitor depletion, disruption of the VZ, impaired neurogenesis, and cortical thinning. The infection pattern and cellular outcome differ from those seen with the extensively passaged ZIKV strain MR766. The structural changes we see after infection with these more recently isolated viral strains closely resemble those seen in ZIKV-associated microcephaly.Peer reviewe

Losers of Primary Cilia Gain the Benefit of survival

In this issue, Zhao and colleagues demonstrate that loss of primary cilia in medulloblastoma cells confers resistance to the Smoothened (SMO) inhibitor sonidegib. When treated with sonidegib, medulloblastoma cells lost their cilia and gained resistance. Surprisingly, loss of cilia is associated with recurrent mutations in ciliogenesis genes that are eventually able to drive drug resistance. These findings uncover a previously unknown mechanism of cancer cells in gaining a persister-like state against anticancer agents at the expense of losing primary cilia. (C) 2017 AACR

Cilium induction triggers differentiation of glioma stem cells

Glioblastoma multiforme (GBM) possesses glioma stem cells (GSCs) that promote self-renewal, tumor propagation, and relapse. Understanding the mechanisms of GSCs self-renewal can offer targeted therapeutic interventions. However, insufficient knowledge of GSCs' fundamental biology is a significant bottleneck hindering these efforts. Here, we show that patient-derived GSCs recruit elevated levels of proteins that ensure the temporal cilium disassembly, leading to suppressed ciliogenesis. Depleting the cilia disassembly complex components is sufficient to induce ciliogenesis in a subset of GSCs via relocating platelet-derived growth factor receptor-alpha (PDGFR-a) to a newly induced cilium. Importantly, restoring ciliogenesis enabled GSCs to switch from self-renewal to differentiation. Finally, using an organoid-based glioma invasion assay and brain xenografts in mice, we establish that ciliogenesis-induced differentiation can prevent the infiltration of GSCs into the brain. Our findings illustrate a role for cilium as a molecular switch in determining GSCs' fate and suggest cilium induction as an attractive strategy to intervene in GSCs proliferation

E-cadherin integrates mechanotransduction and EGFR signaling to control junctional tissue polarization and tight junction positioning

Generation of a barrier in multi-layered epithelia like the epidermis requires restricted positioning of functional tight junctions (TJ) to the most suprabasal viable layer. This positioning necessitates tissue-level polarization of junctions and the cytoskeleton through unknown mechanisms. Using quantitative whole-mount imaging, genetic ablation, and traction force microscopy and atomic force microscopy, we find that ubiquitously localized E-cadherin coordinates tissue polarization of tension-bearing adherens junction (AJ) and F-actin organization to allow formation of an apical TJ network only in the uppermost viable layer. Molecularly, E-cadherin localizes and tunes EGFR activity and junctional tension to inhibit premature TJ complex formation in lower layers while promoting increased tension and TJ stability in the granular layer 2. In conclusion, our data identify an E-cadherin-dependent mechanical circuit that integrates adhesion, contractile forces and biochemical signaling to drive the polarized organization of junctional tension necessary to build an in vivo epithelial barrier