Megalencephalic leukoencephalopathy with subcortical cysts 1 (MLC1) promotes glioblastoma cell invasion in the brain microenvironment

Abstract

Glioblastoma (GBM), or grade IV astrocytoma, is a malignant brain cancer that contains subpopulations of proliferative and invasive cells that coordinately drive primary tumor growth, progression, and recurrence after therapy. Here, we have analyzed functions for megalencephalic leukoencephalopathy with subcortical cysts 1 (Mlc1), an eight-transmembrane protein normally expressed in perivascular brain astrocyte end feet that is essential for neurovascular development and physiology, in the pathogenesis of GBM. We show that Mlc1 is expressed in human stem-like GBM cells (GSCs) and is linked to the development of primary and recurrent GBM. Genetically inhibiting MLC1 in GSCs using RNAi-mediated gene silencing results in diminished growth and invasion in vitro as well as impaired tumor initiation and progression in vivo. Biochemical assays identify the receptor tyrosine kinase Axl and its intracellular signaling effectors as important for MLC1 control of GSC invasive growth. Collectively, these data reveal key functions for MLC1 in promoting GSC growth and invasion, and suggest that targeting the Mlc1 protein or its associated signaling effectors may be a useful therapy for blocking tumor progression in patients with primary or recurrent GBM.

Figure 1.. MLC1 is expressed in primary human GSCs.

(A); Detergent-soluble lysates from six different primary human GSC spheroid cultures were immunoblotted with anti-Mlc1 antibodies. Note the robust expression of Mlc1 protein in four of the GSC samples. (B); Immunoblotting detergent-soluble lysates from six different human GBM cell lines reveals lack of detectable Mlc1 protein expression. As positive controls (+ controls) for these immunoblots, lysates from transfected HEK-293T cells expressing exogenous Mlc1 protein were used. (C); Quantitation of MLC1 mRNA levels reveals enrichment in human GSCs versus several established human GBM cell lines. These data were mined from the cBioPortal genomic database. The z-score for MLC1 mRNA levels was 1.2 standard deviations greater in the classical GBM subtype versus non-cancerous brain tissue samples, ***p<0.001. (D); MLC1 expression is enriched in the classical GBM sub-type, as determined by analysis of mRNA levels in the TCGA database, *p<0.05. Differences between groups were analyzed using two-way analysis of variance (ANOVA) with Bonferroni post-hoc test. (E); Analysis of the cBioPortal genomic database reveals that expression of MLC1 mRNA is most correlative with HEPACAM/GLIALCAM expression in human tumor specimens. Several other genes also show coincident expression, including ITGB8 which like MLC1 is a molecular marker for the classical GBM sub-type.

Figure 2.. MLC1 promotes GSC spheroid formation in vitro.

(A); pGIPZ lentivirus-expressed shRNAs were tested for MLC1 (n=3) gene silencing in infected GSC6–27 cells. All three MLC1 shRNAs silenced gene expression by >90% as determined by anti-Mlc1 immunoblotting. As negative controls for these experiments we used GSC6–27 cells infected with pGIPZ lentivirus expressing non-targeting (NT) shRNAs. (B); GSC6–27 cells infected with control pGIPZ lentivirus formed neurosphere-like spheroids that were significantly larger than spheroids formed from cells expressing MLC1 shRNAs. (C); At 72 hours post-lentivirus infection, GSC6–27 cells expressing MLC1 shRNAs showed a significant reduction in total cell number in comparison to GSC6–27 cells expressing control shRNAs, ***p<0.001. (D); Single cell suspensions were allowed to form spheroids over 7 days. The cross-sectional area of newly formed spheroids was recorded each day, revealing MLC1-dependent defects in spheroid sizes, *p<0.05 and ***p<0.001. Differences between groups were analyzed using two-way ANOVA with Bonferroni post-hoc test. (E); The cross-sectional areas of spheroids were recorded daily for 8 consecutive days. GSC6–27 cells expressing MLC1 shRNAs formed spheroids that were significantly smaller in comparison to GSC6–27 cells expressing control non-targeting (NT) shRNAs. The percentages of total spheroids with measured cross-sectional areas (grouped as <10 μm², 10–20 μm², or >20 μm²). are shown on the y-axes. The days (1–8) on which the sphere cross-sectional areas were measured are indicated on the x-axis.

Figure 3.. MLC1 promotes GSC polarity and invasion in vitro.

(A); GSC6–27 cells expressing MLC1 shRNAs showed diminished invasion through the Matrigel-coated transwells as compared to control pGIPZ-infected cells, *p<0.05. (B); MLC1 regulates the polarity and mesenchymal-like features of cells that have invaded through the Matrigel-coated transwells as determined by analyzing cell shapes, ***p<0.001. Differences between groups (in A, B) were analyzed using two-way ANOVA with Bonferroni post-hoc test. (C); The outlines of hematoxylin-stained GSC6–27 cells expressing non-targeting shRNAs or MLC1 shRNAs that invaded through the Matrigel-coated transwell were traced. Note that GSC6–27 cells expressing MLC1 shRNAs were more epithelial-like, whereas GSC6–27 cells expressing control shRNAs were more elongated and displayed mesenchymal-like shapes. Representative GSC6–27 cell tracings are show in the images on the right. (D); Expression of various proteins with links to cell polarity and the epithelial to mesenchymal transition were analyzed in GSC6–27 cells expressing NT shRNAs or MLC1 shRNAs by immunoblotting detergent-soluble lysates.

Figure 4.. RPPA analysis of MLC1-regulated signaling pathways in GSCs.

(A); Strategy to identify MLC1-dependent changes in protein expression or phosphorylation in cancer cells using RPPA, a high-throughput antibody platform. (B); Bar graph summarizing select proteins that show statistically significant differences in expression and/or phosphorylation in GBM cells expressing MLC1 shRNAs versus control cells. Shown are the top 15 proteins displaying reduced (green) or elevated (red) expression and/or phosphorylation in MLC1 shRNA cells versus NT shRNA cells. (C); Immunoblots of detergent-soluble lysates from GSC6–27 cells expressing MLC1 shRNAs versus control NT shRNAs reveals differential expression and/or phosphorylation of select proteins identified by RPPA.

Figure 5.. MLC1 promotes GSC invasive growth in the brain microenvironment

(A-D); Coronal sections through the striatum of mice harboring tumors formed from GSC6–27 cells expressing control non-targeting (NT) shRNAs (A, B) or MLC1 shRNAs (C, D) were immunofluorescently labeled with anti-GFP antibodies to identify GBM cells. Shown are images of the injected left hemisphere (A, C) and the non-injected right hemisphere (B, D). Note that tumors derived from GSC6–27 cells expressing non-targeting shRNAs showed cell invasion from the injection site (A) into the opposite hemisphere (B). In contrast, GSC6–27 cells expressing MLC1 shRNAs formed tumors (C) but were defective in invasion to the opposing hemisphere (D). (E); Quantitative results showing that MLC1 expression is required for the ability of GSC6–27 cells to invade from the injected striatum (L) to the opposite brain hemisphere (R), *p<0.05. (F, G); Invasive GBM cells (green) traversed across myelin basic protein (MBP)-positive white matter tracts (red) that comprise the corpus callosum. Note that GSC6–27 cells expressing control shRNAs (F) showed robust invasion in the corpus callosum, whereas GBM cells expressing MLC1 shRNAs (G) showed diminished invasion through the corpus callosum. (H); Quantitation of GSC6–27 cell invasion in the corpus callosum, revealing MLC1-dependent defects in white matter invasion in comparison to GSC6–27 cells expressing control shRNAs, ***p<0.001. Differences between NT shRNA and MLC1 shRNA groups were analyzed using two-way ANOVA with Bonferroni post-hoc test.

Figure 6.. Analysis of Mlc1 protein expression in human GBM tissue samples.

(A-D); Analysis of four different fixed human GBM tissue sections by anti-Mlc1 immunohistochemistry showed that Mlc1 protein was expressed in GBM cells throughout the tumor. Note that there is some enriched expression of Mlc1 protein mainly at contact points with cerebral blood vessels. Scale bars, 30 μm. (E); A panel of detergent-soluble lysates from human GBM tissues (n=11) were analyzed by anti-Mlc1 immunoblotting. Note that many GBM samples express Mlc1 protein. The control lane contains a sample of non-cancerous human brain tissue lysate. (F); Quantitation of Mlc1 protein expression in GBM tissue lysates based on densitometry analysis of the immunoblot data in (E), normalized to the α-actinin loading control.

Figure 7.. Analysis of Mlc1 protein expression in primary and recurrent human GBM samples.

(A-J); Anti-Mlc1 immunohistochemical staining of matched primary (A, C, E, G, I) and recurrent (B, D, F, H, J) GBM tissue samples resected from five different patients. Primary tumors were resected prior to therapy, whereas tumor that recurred were resected after standard chemotherapy and radiation. Note that Mlc1 protein is expressed in cancer cells in all 5 primary GBM samples and expression levels are maintained or elevated in matched recurrent GBM samples. These results support important roles for Mlc1 in invasive GSCs, which escape surgical resection and generate recurrent lesions after chemoradiation. Scale bars, 50 μm.

Figure 8.. A model for Mlc1 control of GBM cell polarity and invasive growth in the brain microenvironment.

Mlc1 protein is expressed in GBM cells, where it regulates contact and communication with stromal components in the brain microenvironment to activate pro-invasive signaling pathways. Mlc1-dependent regulation of Axl at the cell surface and its various intracellular signaling proteins drive invasion. Inhibiting Mlc1 functions in GBM cells leads to impaired cell polarity and diminished invasive growth. We propose that Mlc1 interacts with other cell surface proteins including GlialCAM, Aqp4, Trp4, and components of the DGC to modulate the microenvironment and activate signaling pathways that promote GSC invasion.