REST-dependent downregulation of von Hippel-Lindau tumor suppressor promotes autophagy in SHH-medulloblastoma

Abstract

The RE1 silencing transcription factor (REST) is a driver of sonic hedgehog (SHH) medulloblastoma genesis. Our previous studies showed that REST enhances cell proliferation, metastasis and vascular growth and blocks neuronal differentiation to drive progression of SHH medulloblastoma tumors. Here, we demonstrate that REST promotes autophagy, a pathway that is found to be significantly enriched in human medulloblastoma tumors relative to normal cerebella. In SHH medulloblastoma tumor xenografts, REST elevation is strongly correlated with increased expression of the hypoxia-inducible factor 1-alpha (HIF1α)-a positive regulator of autophagy, and with reduced expression of the von Hippel-Lindau (VHL) tumor suppressor protein - a component of an E3 ligase complex that ubiquitinates HIF1α. Human SHH-medulloblastoma tumors with higher REST expression exhibit nuclear localization of HIF1α, in contrast to its cytoplasmic localization in low-REST tumors. In vitro, REST knockdown promotes an increase in VHL levels and a decrease in cytoplasmic HIF1α protein levels, and autophagy flux. In contrast, REST elevation causes a decline in VHL levels, as well as its interaction with HIF1α, resulting in a reduction in HIF1α ubiquitination and an increase in autophagy flux. These data suggest that REST elevation promotes autophagy in SHH medulloblastoma cells by modulating HIF1α ubiquitination and stability in a VHL-dependent manner. Thus, our study is one of the first to connect VHL to REST-dependent control of autophagy in a subset of medulloblastomas.

Keywords: Autophagy; Hypoxia-inducible factor 1-alpha (HIF1α); Medulloblastoma; REST; Ubiquitination; Von Hippel-Lindau (VHL).

Figure 1

Autophagy is an enriched pathway in MB. (a) Pathway enrichment analysis shows enriched pathways in MB subgroups compared to normal samples. Analysis was performed with the GSE124814 dataset. (b) Clustering analysis was done using the R2 genome analysis and visualization platform to examine the expression patterns of autophagy-related genes across four distinct subgroups of MBs (GSE85217). (c) Box plot showing mRNA expression of REST in SHH-MB patients (GSE85217) categorized as high REST (HR) and low REST (LR) samples. (d) Heatmap shows the clustering of HR and LR SHH-MB samples based on the expression of autophagy-related genes in GSE85217.

Figure 2

REST regulates autophagy in SHH MB. (a) Immunohistochemical staining of patient-derived orthotopic xenografts (PDOX) with high and low REST expression for autophagy markers, LC3B and p62. Scale bar: [20 µm for H&E and 10 µm for immunohistochemistry]. (b) Western blot analysis of autophagy markers in PDOX samples, RCMB-018 and RCMB-056. The densitometry ratios of LC3B-II/LC3B-I are shown below the Western blot images. Western blot of SHH-MB cell lines, Daoy and UW228 to show (c) basal levels of REST, LC3B-I, LC3B-II and p62 and changes in their levels following (d) REST knockdown and (e) overexpression. The densitometry ratio of LC3B-II/LC3B-I (top row) and p62/Actin (bottom row) is shown below the Western blot image. (f) Immunofluorescence images of parental and REST-overexpressing isogenic cells showing LC3B staining. Zoomed-in images highlight punctate staining of LC3B in REST-overexpressing cells. Scale bar: [25 µm] (g) Transmission Electron Microscopy (TEM) images displaying immunogold labeling of LC3B in parental and REST-overexpressing UW228 cells. The zoomed-in images show the localization of LC3B in an autophagosome (arrow), (scale bar = 200 nm (50000X)).

Figure 3

HIF1α is required for REST-dependent autophagy in SHH MBs. (a) Volcano plot showing the expression of positive regulators of autophagy including HIF1α in HR compared to LR SHH-MB samples (GSE85217). (b) Box plot showing mRNA expression of HIF1α in HR and LR SHH-MB samples (GSE85217). (c) Scatter plot of correlation of REST mRNA expression and HIF1α mRNA expression (GSE85217; n = 223). (d) Immunohistochemical staining of HIF1α in SHH-MB PDOX with high and low expression of REST. Scale bar: [20 µm]. Western blot of SHH-MB cell lines, Daoy and UW228 to show (e) the basal levels of REST and HIF1α, and changes in their levels following (f) REST knockdown or (g) overexpression. Western blot to show the change in HIF1α, LC3B-I, LC3B-II and p62 level after siRNA (h) or pharmacological (Chrysin) ablation of HIF1α in parental and (i) REST-overexpressing Daoy and UW228 cells. Densitometry ratios of LC3B-II/LC3B-I (top row) and p62/Actin (bottom row) are shown below the Western blot images.

Figure 4

REST elevation causes a decline in VHL expression. (a) Scatter plot of correlation of REST mRNA expression and VHL mRNA expression (GSE85217; n = 223). (b) Box plot to show significant decrease (p = 0.0006) of VHL expression in HR samples compared to LR SHH-MBs (GSE85217). (c) Kaplan–Meier plot to demonstrate significant differences in overall survival of SHH-MB patients based on the relative expression of REST and VHL in the tumors (GSE85217). (d) Immunohistochemical staining of VHL in SHH-MB PDOX with high and low expression of REST. Scale bar: [20 µm]. (e) Western blot showing expression of VHL in Daoy and UW228 cell lines. Changes in VHL levels were studied by Western blotting after siRNA-mediated REST silencing (f) and REST overexpression (g) in Daoy and UW228 cells. (h) Co-immunoprecipitation of HIF1α and VHL in isogenic parental and REST overexpressing Daoy and UW228 cells. Cell lysates were subjected to immunoprecipitation with anti-HIF1α antibody followed by Western blot analysis with anti-ubiquitin (Ub) and anti-VHL antibodies.

Figure 5

Overview of REST-mediated control of autophagy in SHH-MBs. Model figure shows REST promotes HIF1α stabilization by preventing its VHL-mediated ubiquitination and proteasomal degradation (Created with BioRender.com).