Alpha 1-antichymotrypsin contributes to stem cell characteristics and enhances tumorigenicity of glioblastoma

Abstract

Background: Glioblastomas (GBMs) are the main primary brain tumors in adults with almost 100% recurrence rate. Patients with lateral ventricle proximal GBMs (LV-GBMs) exhibit worse survival compared to distal locations for unknown reasons. One hypothesis is the proximity of these tumors to the cerebrospinal fluid (CSF) and its chemical cues that can regulate cellular phenotype. We therefore investigated the role of CSF on GBM gene expression and the role of a CSF-induced gene, SERPINA3, in GBM malignancy in vitro and in vivo.

Methods: We utilized human CSF and GBM brain tumor-initiating cells (BTICs). We determined the impact of SERPINA3 expression in glioma patients using The Cancer Genome Atlas (TCGA) database. SERPINA3 expression changes were evaluated at mRNA and protein levels. The effects of knockdown (KD) and overexpression (OE) of SERPINA3 on cell migration, viability and cell proliferation were evaluated. Stem cell characteristics on KD cells were evaluated by differentiation and colony formation experiments. Tumor growth was studied by intracranial and flank injections.

Results: GBM-CSF increased BTIC migration accompanied by upregulation of the SERPINA3 gene. In patient samples and TCGA data, we observed SERPINA3 to correlate directly with brain tumor grade and indirectly with GBM patient survival. SERPINA3 KD induced a decrease in cell proliferation, migration, invasion, and stem cell characteristics, while SERPINA3 OE increased cell migration. In vivo, SERPINA3 KD BTICs showed increased survival in a murine model.

Conclusions: SERPINA3 plays a key role in GBM malignancy and its inhibition results in a better outcome using GBM preclinical models.

Keywords: SERPINA3; alpha 1-antichymotrypsin; brain tumor; cerebrospinal fluid; glioblastoma.

Figure 1.

GBM-CSF effects on SERPINA3 expression and migratory capacity in BTICs. (A) GBM-CSF increases the migratory response of 1A and 965 BTICs compared to noncancer CSF. (B) RNA expression microarray showed that SERPINA3 was one of the genes upregulated on BTICs after conditioning of GBM-CSF. (C) Confirmation of higher levels of SERPINA3 mRNA by RT-qPCR following GBM-CSF stimulus in different BTICs. (D) SERPINA3 KD impairs the migration response of 965 and 1A BTICs to CSF stimulation. *P < .05, **P < .01. BTIC, brain tumor-initiating cell; GBM-CSF, glioblastoma-cerebrospinal fluid; KD, knockdown.

Figure 2.

SERPINA3 and ACT expression in cancer samples and relation to patient survival. (A) Higher expression of SERPINA3 in GBM tissues compared to noncancer as measured by RT-qPCR in samples from the Mayo Clinic Biobank. (B, C) Immunoblot quantification of multiple GBM tissues expressing higher levels of ACT than control samples from Mayo Clinic Biobank. (D) TCGA dataset depicting higher levels of SERPINA3 mRNA in GBM samples. (E) Correlation between increased SERPINA3 mRNA expression and glioma grade (TCGA data). (F) Survival analysis of TCGA dataset on glioma patients reveals that patients with high SERPINA3 expression have significantly worse outcome. *P < .05, **P < .01. ACT, α1-antichymotrypsin; GBM, glioblastoma; TCGA, The Cancer Genome Atlas.

Figure 3.

SERPINA3 effect on proliferation and cancer stem cell characteristics. (A) BTICs with SERPINA3 KD exhibit lower sphere formation capacity than controls (1A and 965 cells). (B) S-phase of cell cycle and (C) colony formation capacity are decreased after SERPINA3 KD in BTICs 965 and 1A. (D) The CD133⁺ population of BTICs has increased expression of SERPINA3 mRNA by RT-qPCR (965, 1A, and 612 cells). (E) SERPINA3 KD decreases CD133⁺, CD44, and CD49f expression in BTICs (965 and 1A cells) (F). *P < 0.05, **P < .01. BTIC, brain tumor-initiating cell; KD, knockdown.

Figure 4.

SERPINA3 effects on cellular migration and invasion. (A) SERPINA3 KD decreased migration (965, 1A, and 612 cells) and (B) invasion (965 and 1A) capacity of BTICs. (C) Recombinant ACT (4 µM) increased cellular migration in WT cells (965, 1A, and 612 cells), (D) but does not rescue SERPINA3 KD cells migration capacity (965 and 1A cells). (E, F) SERPINA3 OE increases 965 and 1A BTICs migration and invasion. *P < .05, **P < .01. ACT, α1-antichymotrypsin; BTIC, brain tumor-initiating cell; KD, knockdown; OE, overexpression; WT, wild type.

Figure 5.

Effects of SERPINA3 KD in vivo. (A) Mice injected intracranially with 1A SERPINA3 KD cells survived longer than the control groups. (B) SERPINA3 KD groups showed decreased Ki67 proliferation index and (C) invasive capacity than the controls. (D) In vivo ELDA analysis showing lower tumor formation capacity in SERPINA3 KD BTICs than the controls. (E, F) ELDA in vivo tumors in the SERPINA3 KD group weighed less and had a decreased Ki67 proliferation index than the controls. *P < .05, **P < .01. Scale 2 mm and 200 µm. BTIC, brain tumor-initiating cell; ELDA, extreme limiting dilution assay; KD, knockdown.

Figure 6.

Effect of SERPINA3 expression on ERK and MEK kinases. The active forms of the mitogen activated protein kinases ERK1/2 and MEK1/2 are decreased upon KD of SERPINA3 (965 cells). KD, knockdown.