Céline Saraiva Gonçalves - ICVS, University of Minho
Title: WNT6 regulation in glioblastoma: mechanistic, functional and clinical implications
Abstract:
Background: Glioblastoma (GBM) is the most common and most malignant type of glioma, a heterogeneous group of primary brain tumors. While the clinical outcome of GBM patients is unpredictable, patients are equally treated with a standardized approach. Thus, the identification of new biomarkers is crucial. HOXA9 overexpression in GBM is associated with poor prognosis and a more aggressive tumor phenotype. We recently found that HOXA9 transcriptionally activates the WNT pathway; here, we explore how WNT6, a WNT ligand/activator, may contribute to the malignant behavior of GBM.
Material and Methods: Gene set enrichment analysis (GSEA) was used to query the HOXA9 transcriptome. Quantitative PCR, Western blot, chromatin immunoprecipitation (ChIP), methylation-specific PCR (MSP), and immunohistochemistry were performed in GBM cell lines, in vivo xenografts, or in patient samples to study WNT6 at various molecular levels. The functional effects of WNT6 in cell viability (MTT/Trypan blue), proliferation (BrdU), invasion (transwell matrigel), migration (ibidi inserts), angiogenesis (Chick Chorioallantoic Membrane), cell death after treatment with temozolomide (TMZ; Annexin/PI staining) and stemness capacity (limiting dilution assay) were assessed after silencing WNT6 with shRNA. U373+/- WNT6 cells were intra-cranially implanted in NSG mice to evaluate implications in survival. TCGA dataset was assessed for WNT6 status and clinicopathological correlations.
Results: We found that the Wnt pathway is over-activated in HOXA9-positive GBM cells. Specifically, WNT6 is a direct transcriptional target of HOXA9 and is overexpressed in a subset of GBM patients. Additionally, we observed that WNT6 expression correlates with higher glioma grades and with the GBM proneural subtype, whose patients do not benefit from more intensive therapies. Interestingly, we demonstrated that WNT6 expression is also regulated by DNA methylation in GBM patients. In vitro, WNT6-positive cells showed increased viability, migration, invasion and resistance to TMZ, and decreased cell death, when comparing to their negative counterparts. When cultured in stem-cell conditions, WNT6-positive cells show increased viability and capacity to form neurospheres than WNT6-negative cells. In addition, mice bearing WNT6-positive tumors presented faster glioma-related symptomatology and a significantly shorter overall survival (p=0.0042). Importantly, we provide the first evidence of the clinical prognostic value of WNT6 in GBM patients from TCGA and at the protein level in a cohort of Brazilians patients, implicating high levels of WNT6 as a novel independent negative prognostic marker.
Title: WNT6 regulation in glioblastoma: mechanistic, functional and clinical implications
Abstract:
Background: Glioblastoma (GBM) is the most common and most malignant type of glioma, a heterogeneous group of primary brain tumors. While the clinical outcome of GBM patients is unpredictable, patients are equally treated with a standardized approach. Thus, the identification of new biomarkers is crucial. HOXA9 overexpression in GBM is associated with poor prognosis and a more aggressive tumor phenotype. We recently found that HOXA9 transcriptionally activates the WNT pathway; here, we explore how WNT6, a WNT ligand/activator, may contribute to the malignant behavior of GBM.
Material and Methods: Gene set enrichment analysis (GSEA) was used to query the HOXA9 transcriptome. Quantitative PCR, Western blot, chromatin immunoprecipitation (ChIP), methylation-specific PCR (MSP), and immunohistochemistry were performed in GBM cell lines, in vivo xenografts, or in patient samples to study WNT6 at various molecular levels. The functional effects of WNT6 in cell viability (MTT/Trypan blue), proliferation (BrdU), invasion (transwell matrigel), migration (ibidi inserts), angiogenesis (Chick Chorioallantoic Membrane), cell death after treatment with temozolomide (TMZ; Annexin/PI staining) and stemness capacity (limiting dilution assay) were assessed after silencing WNT6 with shRNA. U373+/- WNT6 cells were intra-cranially implanted in NSG mice to evaluate implications in survival. TCGA dataset was assessed for WNT6 status and clinicopathological correlations.
Results: We found that the Wnt pathway is over-activated in HOXA9-positive GBM cells. Specifically, WNT6 is a direct transcriptional target of HOXA9 and is overexpressed in a subset of GBM patients. Additionally, we observed that WNT6 expression correlates with higher glioma grades and with the GBM proneural subtype, whose patients do not benefit from more intensive therapies. Interestingly, we demonstrated that WNT6 expression is also regulated by DNA methylation in GBM patients. In vitro, WNT6-positive cells showed increased viability, migration, invasion and resistance to TMZ, and decreased cell death, when comparing to their negative counterparts. When cultured in stem-cell conditions, WNT6-positive cells show increased viability and capacity to form neurospheres than WNT6-negative cells. In addition, mice bearing WNT6-positive tumors presented faster glioma-related symptomatology and a significantly shorter overall survival (p=0.0042). Importantly, we provide the first evidence of the clinical prognostic value of WNT6 in GBM patients from TCGA and at the protein level in a cohort of Brazilians patients, implicating high levels of WNT6 as a novel independent negative prognostic marker.