A Novel Role of BIRC3 in Stemness Reprogramming of Glioblastoma

Abstract

Stemness reprogramming remains a largely unaddressed principal cause of lethality in glioblastoma (GBM). It is therefore of utmost importance to identify and target mechanisms that are essential for GBM stemness and self-renewal. Previously, we implicated BIRC3 as an essential mediator of therapeutic resistance and survival adaptation in GBM. In this study, we present novel evidence that BIRC3 has an essential noncanonical role in GBM self-renewal and stemness reprogramming. We demonstrate that BIRC3 drives stemness reprogramming of human GBM cell lines, mouse GBM cell lines and patient-derived GBM stem cells (GSCs) through regulation of BMP4 signaling axis. Specifically, BIRC3 induces stemness reprogramming in GBM through downstream inactivation of BMP4 signaling. RNA-Seq interrogation of the stemness reprogramming hypoxic (pseudopalisading necrosis and perinecrosis) niche in GBM patient tissues further validated the high BIRC3/low BMP4 expression correlation. BIRC3 knockout upregulated BMP4 expression and prevented stemness reprogramming of GBM models. Furthermore, siRNA silencing of BMP4 restored stemness reprogramming of BIRC3 knockout in GBM models. In vivo silencing of BIRC3 suppressed tumor initiation and progression in GBM orthotopic intracranial xenografts. The stemness reprograming of both GSCs and non-GSCs populations highlights the impact of BIRC3 on intra-tumoral cellular heterogeneity GBM. Our study has identified a novel function of BIRC3 that can be targeted to reverse stemness programming of GBM.

Keywords: BIRC3; BMP4; GBM; brain tumor; cancer stem cell; stemness.

Figure 1

Expression of BIRC3 correlates with stem cell markers expression and self-renewal in both human and mouse GBM cells (A). Protein expression of BIRC3 in U251 and U87 GBM cells. Each cell line includes control, BIRC3 overexpression (BIRC3-OE) and BIRC3 knockout (BIRC3-KO) groups. Specific antibodies as indicated. β-actin acts as internal control. (B,C). Control, BIRC3-OE or BIRC3-KO of U251 and U87 cells were seeded in 6 well plates and cultured in neurosphere formation medium. The number of neurospheres were observed and calculated under microscope. (B): Representative images are under 4× magnification (top raw) and 20× magnification (bottom raw). (C): Representative images are under 4× magnification (top raw) and 10× magnification (bottom raw). n = 5, * p < 0.05. (D). CD133 and ABCG2 mRNA expression analyzed by real-time PCR in U251/U87 control, BIRC3-OE and BIRC3-KO cells. n = 3, * p < 0.05. (E). Immunofluorescence staining of Nestin in U251/U87 BIRC3-OE and BIRC3-KO cells. Blue: DAPI; Green: Nestin. (F). Protein expression of mBIRC3 in CT-2A mouse GBM cells including control, BIRC3-OE and BIRC3-KO groups. Specific antibodies as indicated. β-actin acts as internal control. (G). Control, BIRC3-OE or BIRC3-KO of CT-2A cells were seeded in 6 well plates and cultured in neurosphere formation medium. The number of neurospheres were observed and calculated under microscope. Representative images are under 10× magnification. n = 5, * p < 0.05. (H). Mouse CD133 and ABCG2 mRNA expression were analyzed by real-time PCR in CT-2A cells. n = 3, * p < 0.05.

Figure 2

Human GBM stem cell self-renewal is regulated by BIRC3 expression. (A). Protein expression of BIRC3 in three different GSCs. Each GSC includes control, BIRC3-OE and BIRC3-KO groups. Specific antibodies as indicated. β-actin acts as internal control. (B). CD133 and ABCG2 mRNA expression analyzed by real time PCR in control, BIRC3-OE and BIRC3-KO GSCs. n = 3, * p < 0.05. (C). Control, BIRC3-OE or BIRC3-KO of differentiated GSCs were seeded in 6-well plate and cultured in neurosphere formation medium. The number of neurospheres were observed and calculated by microscope. Representative images are under 4× magnification. n = 5, * p < 0.05. (D). CD133 and ABCG2 mRNA expression analyzed by real time PCR in control, BIRC3-OE and BIRC3-KO of differentiated GSCs. n = 3, * p < 0.05. (E). Immunofluorescence staining of Nestin in differentiated BIRC3-OE and BIRC3-KO GSCs.

Figure 3

BIRC3 directs BMP4 signaling inhibition in GBM. (A). Analysis of correlation between BIRC3 and BMP4 expression in different regions of GBM using IVY dataset. Analyzed regions include hyperplastic blood vessels, microvascular proliferation region, perinecrotic zone and pseudopalisading cells region. (B–D). Human BMP4 mRNA expression analyzed by real time PCR in control, BIRC3-OE and BIRC3-KO cells including U251/U87 GBM cell lines, GSCs and differentiated GSCs. n = 3, * p < 0.05. (E). Mouse BMP4 mRNA expression analyzed by real time PCR in control, BIRC3-OE and BIRC3-CT-2A cells. n = 3, * p < 0.05. (F). Protein expression of SMAD1, SMAD5 and phosphorylated SMAD1/5 in U251/U87 control, BIRC3-OE and BIRC3-KO cells. Specific antibodies as indicated. β-actin acts as internal control.

Figure 4

BIRC3 impacts GBM cell self-renewal and is dependent on BMP4 suppression. (A). Human BMP4 mRNA expression analyzed by real time PCR in control, BIRC3-KO, and BMP4-siRNA silenced BIRC3-KO U251/U87 GBM cell lines. n = 3, * p < 0.05. (B). Human CD133 mRNA expression analyzed by real time PCR in control, BIRC3-KO, and BMP4-siRNA silenced BIRC3-KO U251/U87 GBM cell lines. n = 3, * p < 0.05. (C). Human ABCG2 mRNA expression analyzed by real time PCR in control, BIRC3-KO, and BMP4-siRNA silenced BIRC3-KO U251/U87 GBM cell lines. n = 3, * p < 0.05. (D). Protein expression of SMAD1, SMAD5 and phosphorylated SMAD1/5 in control, BIRC3-KO, and BMP4-siRNA silenced BIRC3-KO U251/U87 GBM cell lines. Specific antibodies as indicated. β-actin acts as internal control. (E). Control or BIRC3-KO of U251 cells were seeded in 6 well plates and cultured in neurosphere formation medium. The BIRC3-KO cells had been treated with control siRNA and BMP4 siRNA separately 1 day before seeding. The number of neurospheres were observed and calculated by microscope. Representative images are under 4× magnification (top raw) and 10× magnification (bottom raw). n = 5, * p < 0.05.

Figure 5

BIRC3 influences tumor initiation and progression in GBM orthotopic xenograft model. GBM Intracranial models with control, BIRC3-OE and BIRC3-KO U251 cells. (A). Horizontal axial MRI scan of mouse brain tumors 4 weeks after implantation. Two of BIRC3-OE mice were already dead at 4 weeks. (B). Tumor size calculation from MRI scan. n = 5. (C). Kaplan-Meier survival curve of U251 control BIRC3-OE and BIRC3-KO intracranial injection mice. n = 5 mice/group. (D). Mice were sacrificed at different timepoints and brain tissues of U251 control and BIRC3-OE groups were fixed in 10% neutral formalin. H&E staining and BIRC3 immunohistochemistry was performed as described in the Material and Methods Section 4. Five mice were included in this histological study and similar results were observed in each animal. (E). When mice were sacrificed, part of tumor tissues were isolated. mRNA from tumor tissues were extracted. BMP4, CD133 and ABCG2 mRNA expression analyzed by real-time PCR in extracted tumor tissues. n = 3, * p < 0.05.