Homeobox B8 Targets Sterile Alpha Motif Domain-Containing Protein 9 and Drives Glioma Progression

Abstract

Gliomas are the most commonly occurring tumors of the central nervous system. Glioblastoma multiforme (GBM) is the most malignant and aggressive brain cancer in adults. Further understanding of the mechanisms underlying the aggressive nature of GBM is urgently needed. Here we identified homeobox B8 (HOXB8), a member of the homeobox family, as a crucial contributor to the aggressiveness of GBM. Data mining of publicly accessible RNA sequence datasets and our patient cohorts confirmed a higher expression of HOXB8 in the tumor tissue of GBM patients, and a strong positive correlation between the expression level and pathological grading of tumors and a negative correlation between the expression level and the overall survival rate. We next showed that HOXB8 promotes the proliferation and migration of glioblastoma cells and is crucial for the activation of the PI3K/AKT pathway and expression of epithelial-mesenchymal transition-related genes, possibly through direct binding to the promoter of SAMD9 (Sterile Alpha Motif Domain-Containing Protein 9) and activating its transcription. Collectively, we identified HOXB8 as a critical contributor to the aggressiveness of GBM, which provides insights into a potential therapeutic target for GBM and opens new avenues for improving its treatment outcome.

Keywords: Aggressiveness; Glioma; HOXB8; SAMD9; Treatment.

Fig. 1

HOXB8 expression in gliomas in relation to their grades. A HOXB8 expression in gliomas and non-tumor brain tissue from EBI ArrayExpress data (17 non-tumor brain tissues and 75 gliomas). ***P < 0.001. B HOXB8 expression in low-grade and high-grade gliomas from CGGA data (122 low-grade and 179 high-grade). ***P < 0.001. C Overall survival of glioma patients with high and low expression of HOXB8. Data from OncoLnc (http://www.oncolnc.org/). *P < 0.05. D Overall survival of glioma patients with high and low expression of HOXB8. Data from our cohort of patients. *P < 0.05. E Expression of HOX family members including HOXB8 in gliomas from the GSEA database (17 non-tumor controls and 75 gliomas). F Immunohistochemical analysis of HOXB8 expression in gliomas of different pathological grades. Data are presented as the mean ± SD. Scale bars, 50 μm.

Fig. 2

HOXB8 promotes proliferation of glioma cells and affects activation of the AKT/PI3K pathway. A Expression of HOXB8 assessed by western blot in glioma samples. Upper panel, blots of U251, U87, C6, A172, GOS-3 cells; lower panel, statistical analysis of grayscale values. B Proliferation assays in U251 cells (upper) and U87 (lower) with stable expression of shHOXB8. C Proliferation assays in U251 (upper) and U87 (lower) cells with overexpression of HOXB8. D Colony formation assays in U251 cells (left) with stable expression of shHOXB8 and statistical results (right; 3 independent experiments). E Colony formation assays in U87 cells (left) with stable expression of shHOXB8, and statistical results (right: 3 independent experiments). F, G Western blots of PI3K and AKT, and phosphorylation of these proteins in U251 and U87 cells with stable expression of shHOXB8 (F) and overexpression of HOXB8 (G). Data are shown as the mean ± SD and were pooled from 3 independent experiments. *P < 0.05, **P < 0.01.

Fig. 3

HOXB8 promotes migration of glioma cells and upregulates EMT-related genes. A, B Migration (A) and invasion (B) assays in U251 (upper) and U87 (lower) cells with stable expression of shHOXB8. C, D Migration (C) and invasion (D) assays in U251 (upper) and U87 (lower) cells with overexpressed HOXB8. E Western blots of EMT-related proteins (E-cadherin, N-cadherin, vimentin, MMP2, MMP3, MMP9, Slug, and Snail) in U251 and U87 cells with stable expression of shHOXB8. F Western blots of EMT-related proteins E-cadherin, N-cadherin and vimentin in U251 cells with HOXB8 overexpression. Data are shown as the mean ± SD and were pooled from three independent experiments. Scale bars, 100 μm. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 4

SAMD9 is one of the mediators of HOXB8 in glioma cells. A Volcano plot depicting a log transformation plot of the fold-change of different genes by whole genome RNA sequencing in U251 cells transfected with control siRNA and HOXB8 siRNA. B Top differentially-expressed genes in RNA-seq results. C Top differentially-expressed genes in RNA sequencing confirmed in U251 cells by qPCR. *P < 0.05, **P < 0.01. DDe novo motifs recovered by the MD module from the HOXB8 binding sites with a RefSeq gene within 2 kb. E EMSA of biotinylated SMAD9 promoter probes in nuclear extracts from siNC-treated and HOXB8 siRNA-treated U251 cells. Biotinylated probes were detected with HRP-streptavidin. “ck” and “cold” indicate competitor and unlabeled probe negative controls, respectively. Data are shown as the mean ± SD and were pooled from three independent experiments.

Fig. 5

SAMD9 expression in glioma cells and its correlation with HOXB8. A SAMD9 expression in gliomas from GEO data (23 non-tumor brain tissue samples and 157 gliomas). ***P < 0.001. B SAMD9 expression levels in higher and lower grade gliomas from CGGA data (122 low-grade and 179 high-grade). ***P < 0.001. C Overall survival of glioma patients with high and low expression of SAMD9. Data from OncoLnc (http://www.oncolnc.org/). ***P < 0.001. D One normal brain sample and 9 gliomas stained with hematoxylin and eosin (H&E) and for HOXB8, SAMD9, and p-AKT. The arrows show the positive protein expression. E Correlation analysis of HOXB8 and SAMD9 immunohistochemical scores. X-axis, HOXB8 staining score; y-axis, SAMD9 staining score; correlation coefficient r = 0.96; ***P < 0.001. F Correlation analysis of HOXB8 and p-AKT immunohistochemical scores. X-axis, HOXB8 staining score; y-axis, p-AKT staining score; correlation coefficient r = 0.91; ***P < 0.001. G Correlation analysis of SAMD9 and p-AKT immunohistochemical scores. X-axis, SAMD9 staining score; y-axis, p-AKT staining score; correlation coefficient r = 0.96; ***P < 0.001. Data are shown as the mean ± SD. Scale bars, 50 μm.

Fig. 6

SAMD9 mediates the proliferation, migration, and invasion of glioma cells and affects the activation of the AKT/PI3K pathway and expression of EMT-related genes. A, B Transient silencing of SAMD9 in U251 cells (A) and U87 cells (B) decreased the rate of proliferation. C Western blots of phosphorylation of PI3K and AKT in U251 and U87 cells with transiently silenced SAMD9. D Transient silencing of HOXB8 in U251 (upper) and U87 (lower) cells decreased the rate of migration. E Transient silencing of SAMD9 in U251 (upper) and U87 (lower) cells decreased the rate of invasion. F Western blots of EMT-related proteins (E-cadherin, N-cadherin, vimentin, MMP2, MMP3, MMP9, Slug, and Snail) in U251 and U87 cells with transiently silenced SAMD9. Data are shown as the mean ± SD and were pooled from three independent experiments. Scale bars, 100 μm. *P < 0.05, **P < 0.01.

Fig. 7

Working model. Schematic showing the speculation that HOXB8/SAMD9 drives the proliferation, migration, and invasion of glioma cells and involves the PI3K/AKT signaling pathway.