Browsing by Author "Guo, Sujuan"

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    Casein Kinase 1 Epsilon Regulates Glioblastoma Cell Survival
    Varghese, Robin T.; Young, Sarah; Pham, Lily; Liang, Yanping; Pridham, Kevin J.; Guo, Sujuan; Murphy, Susan F.; Kelly, Deborah F.; Sheng, Zhi (Nature, 2018-09-11)
    Glioblastoma is the most common malignant brain cancer with a dismal prognosis. The difficulty in treating glioblastoma is largely attributed to the lack of effective therapeutic targets. In our previous work, we identified casein kinase 1 ε (CK1ε, also known as CSNK1E) as a potential survival factor in glioblastoma. However, how CK1ε controls cell survival remains elusive and whether targeting CK1ε is a possible treatment for glioblastoma requires further investigation. Here we report that CK1ε was expressed at the highest level among six CK1 isoforms in glioblastoma and enriched in high-grade glioma, but not glia cells. Depletion of CK1ε remarkably inhibited the growth of glioblastoma cells and suppressed self-renewal of glioblastoma stem cells, while having limited effect on astrocytes. CK1ε deprivation activated β-catenin and induced apoptosis, which was further counteracted by knockdown of β-catenin. The CK1ε inhibitor IC261, but not PF-4800567, activated β-catenin and blocked the growth of glioblastoma cells and glioblastoma stem cells. Congruently, IC261 elicited a robust growth inhibition of human glioblastoma xenografts in mice. Together, our results demonstrate that CK1ε regulates the survival of glioblastoma cells and glioblastoma stem cells through β-catenin signaling, underscoring the importance of targeting CK1ε as an effective treatment for glioblastoma.
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    Connexin 43 confers chemoresistance through activating PI3K
    Pridham, Kevin J.; Shah, Farah; Hutchings, Kasen R.; Sheng, Kevin L.; Guo, Sujuan; Liu, Min; Kanabur, Pratik; Lamouille, Samy Y.; Lewis, Gabrielle; Morales, Marc; Jourdan, L. Jane; Grek, Christina L.; Ghatnekar, Gautam S.; Varghese, Robin T.; Kelly, Deborah F.; Gourdie, Robert G.; Sheng, Zhi (Springer Nature, 2022-01-12)
    Circumventing chemoresistance is crucial for effectively treating cancer including glioblastoma, a lethal brain cancer. The gap junction protein connexin 43 (Cx43) renders glioblastoma resistant to chemotherapy; however, targeting Cx43 is difficult because mechanisms underlying Cx43-mediated chemoresistance remain elusive. Here we report that Cx43, but not other connexins, is highly expressed in a subpopulation of glioblastoma and Cx43 mRNA levels strongly correlate with poor prognosis and chemoresistance in this population, making Cx43 the prime therapeutic target among all connexins. Depleting Cx43 or treating cells with αCT1–a Cx43 peptide inhibitor that sensitizes glioblastoma to the chemotherapy temozolomide–inactivates phosphatidylinositol-3 kinase (PI3K), whereas overexpression of Cx43 activates this signaling. Moreover, αCT1-induced chemo-sensitization is counteracted by a PI3K active mutant. Further research reveals that αCT1 inactivates PI3K without blocking the release of PI3K-activating molecules from membrane channels and that Cx43 selectively binds to the PI3K catalytic subunit β (PIK3CB, also called PI3Kβ or p110β), suggesting that Cx43 activates PIK3CB/p110β independent of its channel functions. To explore the therapeutic potential of simultaneously targeting Cx43 and PIK3CB/p110β, αCT1 is combined with TGX-221 or GSK2636771, two PIK3CB/p110β-selective inhibitors. These two different treatments synergistically inactivate PI3K and sensitize glioblastoma cells to temozolomide in vitro and in vivo. Our study has revealed novel mechanistic insights into Cx43/PI3K-mediated temozolomide resistance in glioblastoma and demonstrated that targeting Cx43 and PIK3CB/p110β together is an effective therapeutic approach for overcoming chemoresistance.
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    A large-scale RNA interference screen identifies genes that regulate autophagy at different stages
    Guo, Sujuan; Pridham, Kevin J.; Virbasius, Ching-Man; He, Bin; Zhang, Liqing; Varmark, Hanne; Green, Michael R.; Sheng, Zhi (Nature Publishing Group, 2018-02-12)
    Dysregulated autophagy is central to the pathogenesis and therapeutic development of cancer. However, how autophagy is regulated in cancer is not well understood and genes that modulate cancer autophagy are not fully defined. To gain more insights into autophagy regulation in cancer, we performed a large-scale RNA interference screen in K562 human chronic myeloid leukemia cells using monodansylcadaverine staining, an autophagy-detecting approach equivalent to immunoblotting of the autophagy marker LC3B or fluorescence microscopy of GFP-LC3B. By coupling monodansylcadaverine staining with fluorescence-activated cell sorting, we successfully isolated autophagic K562 cells where we identified 336 short hairpin RNAs. After candidate validation using Cyto-ID fluorescence spectrophotometry, LC3B immunoblotting, and quantitative RT-PCR, 82 genes were identified as autophagy-regulating genes. 20 genes have been reported previously and the remaining 62 candidates are novel autophagy mediators. Bioinformatic analyses revealed that most candidate genes were involved in molecular pathways regulating autophagy, rather than directly participating in the autophagy process. Further autophagy flux assays revealed that 57 autophagy-regulating genes suppressed autophagy initiation, whereas 21 candidates promoted autophagy maturation. Our RNA interference screen identifies identified genes that regulate autophagy at different stages, which helps decode autophagy regulation in cancer and offers novel avenues to develop autophagy-related therapies for cancer.
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    Patient-derived glioblastoma stem cells respond differentially to targeted therapies
    Kanabur, Pratik; Guo, Sujuan; Simonds, Gary S.; Kelly, Deborah F.; Gourdie, Robert G.; Verbridge, Scott S.; Sheng, Zhi (Impact Journals, 2016-12-27)
    The dismal prognosis of glioblastoma is, at least in part, attributable to the difficulty in eradicating glioblastoma stem cells (GSCs). However, whether this difficulty is caused by the differential responses of GSCs to drugs remains to be determined. To address this, we isolated and characterized ten GSC lines from established cell lines, xenografts, or patient specimens. Six lines formed spheres in a regular culture condition, whereas the remaining four lines grew as monolayer. These adherent lines formed spheres only in plates coated with poly-2-hydroxyethyl methacrylate. The self-renewal capabilities of GSCs varied, with the cell density needed for sphere formation ranging from 4 to 23.8 cells/well. Moreover, a single non-adherent GSC either remained quiescent or divided into two cells in four-seven days. The stem cell identity of GSCs was further verified by the expression of nestin or glial fibrillary acidic protein. Of the two GSC lines that were injected in immunodeficient mice, only one line formed a tumor in two months. The protein levels of NOTCH1 and platelet derived growth factor receptor alpha positively correlated with the responsiveness of GSCs to γ-secretase inhibitor IX or imatinib, two compounds that inhibit these two proteins, respectively. Furthermore, a combination of temozolomide and a connexin 43 inhibitor robustly inhibited the growth of GSCs. Collectively, our results demonstrate that patient-derived GSCs exhibit different growth rates in culture, possess differential capabilities to form a tumor, and have varied responses to targeted therapies. Our findings underscore the importance of patient-derived GSCs in glioblastoma research and therapeutic development.
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    A rapid and high content assay that measures cyto-ID-stained autophagic compartments and estimates autophagy flux with potential clinical applications
    Guo, Sujuan; Liang, Yanping; Murphy, Susan F.; Huang, Angela; Shen, Haihong; Kelly, Deborah F.; Sobrado, Pablo; Sheng, Zhi (Taylor & Francis, 2015-03-01)
    The lack of a rapid and quantitative autophagy assay has substantially hindered the development and implementation of autophagy-targeting therapies for a variety of human diseases. To address this critical issue, we developed a novel autophagy assay using the newly developed Cyto-ID fluorescence dye. We first verified that the Cyto-ID dye specifically labels autophagic compartments with minimal staining of lysosomes and endosomes. We then developed a new Cyto-ID fluorescence spectrophotometric assay that makes it possible to estimate autophagy flux based on measurements of the Cyto-ID-stained autophagic compartments. By comparing to traditional autophagy approaches, we found that this assay yielded a more sensitive, yet less variable, quantification of the stained autophagic compartments and the estimate of autophagy flux. Furthermore, we tested the potential application of this autophagy assay in high throughput research by integrating it into an RNA interference (RNAi) screen and a small molecule screen. The RNAi screen revealed WNK2 and MAP3K6 as autophagy-modulating genes, both of which inhibited the MTOR pathway. Similarly, the small molecule screen identified sanguinarine and actinomycin D as potent autophagy inducers in leukemic cells. Moreover, we successfully detected autophagy responses to kinase inhibitors and chloroquine in normal or leukemic mice using this assay. Collectively, this new Cyto-ID fluorescence spectrophotometric assay provides a rapid, reliable quantification of autophagic compartments and estimation of autophagy flux with potential applications in developing autophagy-related therapies and as a test to monitor autophagy responses in patients being treated with autophagy-modulating drugs.