Suppression of microRNA-9 by mutant EGFR signaling upregulates FOXP1 to enhance glioblastoma tumorigenicity

Abstract

The EGF receptor (EGFR) is amplified and mutated in glioblastoma, in which its common mutation (ΔEGFR, also called EGFRvIII) has a variety of activities that promote growth and inhibit death, thereby conferring a strong tumor-enhancing effect. This range of activities suggested to us that ΔEGFR might exert its influence through pleiotropic effectors, and we hypothesized that microRNAs might serve such a function. Here, we report that ΔEGFR specifically suppresses one such microRNA, namely miR-9, through the Ras/PI3K/AKT axis that it is known to activate. Correspondingly, expression of miR-9 antagonizes the tumor growth advantage conferred by ΔEGFR. Silencing of FOXP1, a miR-9 target, inhibits ΔEGFR-dependent tumor growth and, conversely, de-repression of FOXP1, as a consequence of miR-9 inhibition, increases tumorigenicity. FOXP1 was sufficient to increase tumor growth in the absence of oncogenic ΔEGFR signaling. The significance of these findings is underscored by our finding that high FOXP1 expression predicts poor survival in a cohort of 131 patients with glioblastoma. Collectively, these data suggest a novel regulatory mechanism by which ΔEGFR suppression of miR-9 upregulates FOXP1 to increase tumorigenicity.

Figure 1

Validation of miR-9 repression by ΔEGFR. (A) and (B) Northern blotting for miR-9 in triplicate glioma cell extracts validates the downregulation of miR-9 in U87 and U373 ΔEGFR cells (p-value ≤ 0.001). (C) Northern blotting and qRT-PCR show miR-9 downregulation in mouse ΔEGFR expressing astrocytes (p-value ≤ 0.002). (D) Inhibition of ΔEGFR kinase activity in U373Δ cells (left panel, 1µM gefitinib) and AstrocytesΔ (right panel, 0.5µM and 1µM treated) induces miR-9 expression. (E) Prolonged stimulation of U87wtEGFR cells with EGF does not suppress miR-9 expression.

Figure 2

ΔEGFR downregulates pri-miR-9-2. (A) ΔEGFR negatively regulates expression of the mouse and human primary transcript, pri-miR-9-2, encoding for miR-9 (p value ≤ 0.001). (B) The rate of pri-miR-9-2 degradation is not accelerated by ΔEGFR. MiR-9 expression was analyzed in U373 cells and mouse astrocytes treated for 6 hrs with Actinomycin C to block transcription. (C) ΔEGFR downregulates miR-9* expression. Northern blotting revealed the suppression of miR-9*, generated from pri-miR-9-2, in ΔEGFR cells.

Figure 3

The Ras/PI3K/AKT axis is required for miR-9 repression. (A) MiR-9 expression was determined in mutant ΔEGFR alleles bearing tyrosine to phenylalanine substitutions. The U87DY5 mutant with impaired binding to Ras shows upregulation of miR-9 (p-value ≤ 0.005). (B) SOS1 silencing upregulates miR-9 in U87ΔEGFR cells (p-value ≤ 0.004). (C) H-Ras G12V suppresses miR-9 (p-value ≤ 0.006). (D, left panels) Treatment of U87ΔEGFR cells with a Raf inhibitor does not modulate miR-9. (D, middle and right panels) Introduction of wild type PTEN (p-value ≤ 0.004) or dead kinase AKT (DKAKT, p-value ≤ 0.04), relieved the suppression of miR-9 in U87ΔEGFR cells.

Figure 4

MiR-9 targets FOXP1. (A) FOXP1 expression inversely correlates with miR-9 expression. Parental and DK cells with higher miR-9 expression exhibit decreased FOXP1 protein levels relative to ΔEGFR cells showing low miR-9 expression. (B) Overexpression of miR-9 in U87Δ and AstrocytesΔ downregulate FOXP1 expression. (C) Inhibition of miR-9 activity using the miRZipmiR-9 vector upregulates FOXP1. (D) U87ΔEGFR cells were co-transfected with control or miR-9 mimics and FOXP1 3’ UTR luciferase reporter, miR-9 and mutant miR-9 reporters. MiR-9 mimics repressed the FOXP1 and miR-9 reporters (p ≤ 0.02) but not the mutant miR-9 reporters. (E) U373 cells were co-transfected with anti-miR-9 oligonucleotides and the FOXP1 3’ UTR luciferase reporter, miR-9 and mutant miR-9 reporters. Inhibition of miR-9 activity relieved the repression of FOXP1 and miR-9 reporters (p ≤ 0.03).

Figure 5

MiR-9 and FOXP1 regulate tumorigenicity. (A) U87ΔEGFR cells were infected with pSuper vector (pS) and pSmiR-9-1 vector to upregulate miR-9 (top panel). Mice were subcutaneously implanted with U87DK, U87ΔpS and U87ΔmiR-9 cells. The growth of U87DK and U87ΔmiR-9 tumors was significantly slower compared to U87ΔpS tumors (p-value ≤ 0.01). (B) Mice implanted with U87miRZip control cells developed significantly smaller tumors relative to mice implanted with U87miRZip miR-9 cells showing impaired miR-9 activity (p-value ≤ 0.03). (C) Knock-down of FOXP1 dramatically impaired the growth of U87ΔEGFR tumors (p-value ≤ 0.02). (D) Overexpression of FOXP1 increases the tumorigenic capacity of U373 and U251 cells (p-value ≤ 0.02). (E) Kaplan-Meir survival curve analysis reveals that high FOXP1 predicts poor survival in a cohort of 131 GBM patients.