Sujuan Guo

Connexin 43 Inhibition Sensitizes Chemoresistant Glioblastoma Cells to Temozolomide.

Resistance of glioblastoma (GBM) to the front-line chemotherapeutic agent temozolomide (TMZ) continues to challenge GBM treatment efforts. The repair of TMZ-induced DNA damage by O-6-methylguanine-DNA methyltransferase (MGMT) confers one mechanism of TMZ resistance. Paradoxically, MGMT-deficient GBM patients survive longer despite still developing resistance to TMZ. Recent studies indicate that the gap junction protein connexin 43 (Cx43) renders GBM cells resistant to TMZ through its carboxyl terminus (CT). In this study, we report insights into how Cx43 promotes TMZ resistance. Cx43 levels were inversely correlated with TMZ sensitivity of GBM cells, including GBM stem cells. Moreover, Cx43 levels inversely correlated with patient survival, including as observed in MGMT-deficient GBM patients. Addition of the C-terminal peptide mimetic αCT1, a selective inhibitor of Cx43 channels, sensitized human MGMT-deficient and TMZ-resistant GBM cells to TMZ treatment. Moreover, combining αCT1 with TMZ-blocked AKT/mTOR signaling, induced autophagy and apoptosis in TMZ-resistant GBM cells. Our findings suggest that Cx43 may offer a biomarker to predict the survival of patients with MGMT-independent TMZ resistance and that combining a Cx43 inhibitor with TMZ could enhance therapeutic responses in GBM, and perhaps other TMZ-resistant cancers.

Real-Time Visualization of Nanoparticles Interacting with Glioblastoma Stem Cells

Nanoparticle-based therapy represents a novel and promising approach to treat glioblastoma, the most common and lethal malignant brain cancer. Although similar therapies have achieved significant cytotoxicity in cultured glioblastoma or glioblastoma stem cells (GSCs), the lack of an appropriate approach to monitor interactions between cells and nanoparticle-based therapies impedes their further clinical application in human patients. To address this critical issue, we first obtained NOTCH1 positive GSCs from patient-derived primary cultures. We then developed a new imaging approach to directly observe the dynamic nature of nanoparticles at the molecular level using in situ transmission electron microscopy (TEM). Utilizing these tools we were able to visualize real-time movements of nanoparticles interacting with GSCs for the first time. Overall, we show strong proof-of-concept results that real-time visualization of nanoparticles in single cells can be achieved at the nanoscale using TEM, thereby providing a powerful platform for the development of nanotherapeutics.

Patient-derived glioblastoma stem cells respond differentially to targeted therapies

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.

PIK3CB/p110β is a selective survival factor for glioblastoma

Background Glioblastoma (GBM) is difficult to treat. Phosphoinositide 3-kinase (PI3K) is an attractive therapeutic target for GBM; however, targeting this pathway to effectively treat GBM is not successful because the roles of PI3K isoforms remain to be defined. The aim of this study is to determine whether PIK3CB/p110β, but not other PI3K isoforms, is a biomarker for GBM recurrence and important for cell survival. Methods Gene expression and clinical relevance of PI3K genes in GBM patients were analyzed using online databases. Expression/activity of PI3K isoforms was determined using immunoblotting. PI3K genes were inhibited using short hairpin RNAs or isoform-selective inhibitors. Cell viability/growth was assessed by the MTS assay and trypan blue exclusion assay. Apoptosis was monitored using the caspase activity assay. Mouse GBM xenograft models were used to gauge drug efficacy. Results PIK3CB/p110β was the only PI3K catalytic isoform that significantly correlated with high incidence rate, risk, and poor survival of recurrent GBM. PIK3CA/p110α, PIK3CB/p110β, and PIK3CD/p110δ were differentially expressed in GBM cell lines and primary tumor cells derived from patient specimens, whereas PIK3CG/p110γ was barely detected. PIK3CB/p110β protein levels presented a stronger association with the activities of PI3K signaling than other PI3K isoforms. Blocking p110β deactivated PI3K signaling, whereas inhibition of other PI3K isoforms had no effect. Specific inhibitors of PIK3CB/p110β, but not other PI3K isoforms, remarkably suppressed viability and growth of GBM cells and xenograft tumors in mice, with minimal cytotoxic effects on astrocytes. Conclusions PIK3CB/p110β is a biomarker for GBM recurrence and selectively important for GBM cell survival.

Detecting Autophagy and Autophagy Flux in Chronic Myeloid Leukemia Cells Using a Cyto-ID Fluorescence Spectrophotometric Assay

Autophagy is a catabolic process whereby cellular components are degraded to fuel cells for longer survival during stress. Hence, autophagy plays a vital role in determining cell fate and is central for homeostasis and pathogenesis of many human diseases including chronic myeloid leukemia (CML). It has been well established that autophagy is important for the leukemogenesis as well as drug resistance in CML. Thus, autophagy is an intriguing therapeutic target. However, current approaches that detect autophagy lack reliability and often fail to provide quantitative measurements. To overcome this hurdle and facilitate the development of autophagy-related therapies, we have recently developed an autophagy assay termed as the Cyto-ID fluorescence spectrophotometric assay. This method uses a cationic fluorescence dye, Cyto-ID, which specifically labels autophagic compartments and is detected by a spectrophotometer to permit a large-scale and quantitative analysis. As such, it allows rapid, reliable, and quantitative detection of autophagy and estimation of autophagy flux. In this chapter, we further provide technical details of this method and step-by-step protocols for measuring autophagy or autophagy flux in CML cell lines as well as primary hematopoietic cells.

A large-scale RNA interference screen identifies genes that regulate autophagy at different stages

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 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.

A Diphtheria Toxin Negative Selection in RNA Interference Screening

RNA interference (RNAi) screening is a powerful technique for understanding the molecular biology of cancer and searching drug targets. Genes and their upstream activators that are essential for the survival of cancer cells often dictate cancer formation/progression. Hence, they are preferable therapeutic targets. Identifying these genes using RNAi is, however, problematic because knocking them down leads to cell death. Here we describe a diphtheria toxin (DT) negative selection method to circumvent the problem of cell death in RNAi screening. DT fails to kill mouse cells due to the lack of functional DT receptor (DTR). Thus, we first prepare a construct encoding a human functional DTR driven by the promoter of mouse Atf5, a gene essential for the survival of malignant glioma. Then a DT-sensitive mouse malignant glioma cell line is established by over-expressing this DTR. Finally, an RNAi screen is performed in this cell line and genes that activate Atf5 expression are identified. The negative selection approach described here allows RNAi screening to be used for identifying genes controlling cell survival in cancers or perhaps other human diseases with potential in therapeutic intervention.

Casein Kinase 1 Epsilon Regulates Glioblastoma Cell Survival

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.

Abstract 336: PIK3CB/p110B is a survival factor in glioblastoma

Glioblastoma multiforme (GBM) is lethal even after surgical removal of the tumor, radiation, and chemotherapy. Residual tumor cells form an intractable tumor in nearly all patients within two years. Recurrent GBM is incurable due to resistance to current therapies. Inhibitors of PI3K (phosphatidylinositol-4,5-biphosphate 3-kinase)-a signaling pathway that causally contributes to tumor formation/recurrence-have been used to treat recurrent GBMs and achieved modest clinical effect. This is perhaps attributed to non-selective inhibition of PI3K isoforms, which yields intolerable toxicity. Class IA PI3K isoforms include three catalytic subunits (PIK3CA, B, or D that encodes p110α, β, or δ) and three regulatory subunits (PIK3R1-3 that encodes p85 isoforms). Our recent work indicates that PIK3CB levels positively correlate with the chances/risk of GBM recurrence, while being inversely associated with patient prognosis. This suggests that PIK3CB/p110β is important for GBM cell survival. To test this hypothesis, we first measured the expression of PI3K isoforms in a panel of 9 GBM cell lines. We found that U87MG, SF295, and U251 expressed much higher levels of p110β, coinciding with the levels of phosphorylated AKT. We then knocked down PIK3CA, B, and D in human U87MG cells and found that only depletion of PIK3CB/p110β resulted in an inactivation of downstream AKT. Moreover, knockdown of PIK3CB/p110β, but not other isoforms, induced substantial growth inhibition in U87MG, SF295 and U251 cells. This is congruent with the result that inhibition of PIK3CB/p110β activated apoptosis in U87MG cells. We also report that treatment of p110βhigh GBM cells with isoform specific inhibitors selectively represses the viability of these cells. Finally, we report that ectopic expression of p110β, but not p110α or δ, partially rescued U87MG cells from growth inhibition induced by TGX-221, a p110β-selective inhibitor. Collectively, our results demonstrate that PIK3CB/p110β is an important selective survival factor in GBM, underscoring the divergent roles of PI3K isoforms in GBM disease progression/recurrence and future therapeutic intervention. Citation Format: Kevin J. Pridham, Sujuan Guo, Zhi Sheng. PIK3CB/p110B is a survival factor in glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 336. doi:10.1158/1538-7445.AM2017-336

Abstract 4765: Targeting glioblastoma cancer stem cells with a novel Connexin43 mimetic peptide

Glioblastoma (GBM) is a highly malignant and lethal cancer of the central nervous system. Despite intensive research efforts, there has been limited progress in improving patient outcome. The current therapy for GBM patients includes surgical resection, radiotherapy and cytotoxic chemotherapy with temozolomide (TMZ), conferring a median survival time of only 14.6 months. Failure to generate more effective treatment strategies is due in part to the cellular heterogeneity within GBM tumors, which comprise a sub-population of GBM cancer stem cells (GSCs) characterized by self-renewal characteristics and resistance to TMZ. Recent work, including our own, has demonstrated that levels of the gap junction protein Connexin43 (Cx43) correlate with GBM TMZ resistance. Increased Cx43 levels occur in GSCs compared to parental GBM cells, and patients with high levels of Cx43 mRNA and low levels of MGMT, an enzyme that repairs TMZ-induced DNA lesions, have a significantly shorter life span. In contrast, Cx43 has also been associated with anti-proliferative effects in glioma and reduced levels of Cx43 protein are reported in high-grade gliomas. In addition to forming gap junctions, Cx43 can regulate cell proliferation, migration, and apoptosis, through distinct channel-independent mechanisms. Therefore, altering the localization and/or activity of Cx43 rather than Cx43 expression alone may represent a more targeted strategy in GBM treatment. Using super-resolution microscopy, we find intracellular Cx43 decorating microtubules in GSCs demonstrating for the first time such clustering in situ. Regulation of Cx43 function is primarily associated with multiple sites for post-translational modification and protein-protein interaction within the Cx43 carboxy-terminus (CT) which includes a tubulin binding domain. We have developed a peptide named JM2 (juxtamembrane 2) composed of the Cx43 CT amino acids encompassing the microtubule-binding sequence fused to an antennapedia cell penetration domain for cellular uptake. Super-resolution microscopy and biochemical analysis confirm JM2 specific interaction with microtubules concomitantly with a loss of Cx43 interaction with microtubules in GSCs derived from patient tumors. In addition, we observe that JM2 decreases Cx43 gap junction plaque formation and cell-cell communication in GSCs and limits microtubule dynamics. Importantly, JM2 decreases cell survival in TMZ-resistant GSCs and GSC neurosphere formation in vitro, and GSC-derived tumor growth in vivo. Our current research includes the development of JM2-loaded biodegradable nanoparticles for sustained JM2 delivery in preparation for future clinical trials. In conclusion, we have developed a Cx43 mimetic peptide that significantly decreases the tumorigenic potential of GSCs and represents a novel and potent therapeutic opportunity to alter Cx43 activity in targeting chemoresistant GSCs for GBM treatment. Citation Format: Samy Lamouille, James W. Smyth, Laurie O9Rourke, Pratik Kanabur, Sujuan Guo, Jane Jourdan, Zhi Sheng, Robert G. Gourdie. Targeting glioblastoma cancer stem cells with a novel Connexin43 mimetic peptide [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4765. doi:10.1158/1538-7445.AM2017-4765