Parry, Phillip V.; Engh, Johnathan A.
Gliomas are the most common primary brain tumor in adults and are categorized into 1 of 4 WHO grades based on histopathological characteristics.1 The aggressive, infiltrative nature of high-grade gliomas portends a grave prognosis despite aggressive chemotherapy, radiation, and surgical resection. Current efforts for developing novel and effective treatment options for gliomas have focused on molecular targets that interrupt tumor proliferation. However, these techniques require a thorough understanding of the cell signaling mechanisms of gliomas. Previous research has delineated a number of proteins critical for normal growth and development that includes regulation of ligand-mediated signaling, cell adhesion, and formation of the extracellular matrix for cell migration.2,3 The leucine-rich repeat containing G protein-coupled receptor 5 (LGR5), has been recently shown to play a key functional role in normal cellular development.4 When abnormal expression of LGR5 is present, tumorigenesis has been shown to occur through activation of the Wnt pathway. However, the exact mechanisms of LGR5 activity in glioma cells have not been elucidated.
Recent research published in the October edition of Oncology Reports from Zhang et al in China have confirmed the expression of LGR5 in human gliomas and its correlation with pathological grade and proliferation.5 Additionally, they demonstrate compelling evidence of tumor growth inhibition using RNA interference against LGR5 in both in vitro and in vivo models.
The investigators collected 54 gliomas of varying grade from patients who presented for initial surgical resection and had no prior treatments with radiation or chemotherapy. All of the specimens were subjected to immunohistochemical assays for LGR5. LGR5 immunoreactivity scores (IRS), LGR5 proliferative indices (PI), and the percentage of Ki-67 positive cells were quantified. Both the LGR5 IRS and PI correlated with increasing WHO grade. The investigators then studied the LGR5 expression levels in 3 glioma cell lines (U118, U87, and U251) in vitro and compared them to LGR5 levels expressed in normal human astrocytes. The results demonstrated higher LGR5 expression in the glioma cell lines compared to the normal astrocytes.
The team then investigated the role of LGR5 in the malignant transformation of gliomas. The U87 cell lines were transfected with RNAi against LGR5, which resulted in a significant and specific decrease in the endogenous expression of LGR5. In order to evaluate the effect of LGR5 on the growth of U87 cells, the researchers conducted viability assays using U87, U87-negative control (NC), and U87-knockdown (KD) cells. Their data showed a marked growth inhibition of U87-KD cells compared to the other groups. Additionally, there were no significant differences in cell growth between the U87 and U87-NC cells.
Knockdown of LGR5 also decreased the number and size of colony formation in U87-KD cells compared to U87 and U87-NC, indicating that LGR5 expression is necessary for anchorage-dependent growth in glioma. Furthermore, U87-KD cells also had impaired capacity for self-renewal as indicated by a reduced capacity to generate tumorsphere formation compared to control cell lines.
Finally, the researchers investigated the tumor-forming capacity of U87-KD and control cell lines in an in vivo murine model. Live imaging using fluorescence was used to detect tumor growth (Figure) from mice that had intracranial orthotopic xenografts of tumor cells. The signal emitted from U87-KD cells was significantly less compared to control models.
Figure. LGR5 depleti...Image Tools
Zhang et al provide invaluable new information about the molecular characteristics of gliomas using both in vitro and in vivo models, in which knockdown LGR5 resulted in suppression of glioma formation. These results help clarify the molecular signaling pathways required for glial tumorigenesis and demonstrate that LGR5 may be a novel target for glioma treatment. Hopefully, the preclinical and clinical trials needed to establish the feasibility of this approach in humans will translate into a more effective treatment strategy against these tenacious tumors.
1. Louis DN, Ohgaki H, Wiestler OD, Cavenee WK. WHO Classification of Tumours of the Central Nervous System. 4th ed. Lyon, France: IARC Press; 2007.
2. Barker N, van Ed JH, Kuipers J, Kujala P, et al.. Identification of stem cells in small intestine and colon by marker gene LGR5. Nature. 2007;449(7165):1003–1007.
3. Barker N, Clevers H. Leucine-rich repeat containing G-protein-coupled receptors as markers of adult stem cells. Gastroenterology. 2010;138(5):1681–1696.
4. Barker N, Rookmaaker MD, Kujala P, Ng A, et al.. LGR5+ve
stem/progenitor cells contribute to nephron formation during kidney development. Cell Rep. 2010;2(3):540–552.
5. Wang D, Zhou J, Cungang F, Jiao F, et al.. Knockdown of LGR5 suppresses the proliferation of glioma cells in vitro and in vivo. Oncol Rep. 2013;31(1):41–49.