Leukemia-Associated Rho Guanine Nucleotide Exchange Factor and Ras Homolog Family Member C Play a Role in Glioblastoma Cell Invasion and Resistance

Abstract

Glioblastoma (GBM) is the most common primary malignant brain cancer in adults. A hallmark of GBM is aggressive invasion of tumor cells into the surrounding normal brain. Both the current standard of care and targeted therapies have largely failed to specifically address this issue. Therefore, identifying key regulators of GBM cell migration and invasion is important. The leukemia-associated Rho guanine nucleotide exchange factor (LARG) has previously been implicated in cell invasion in other tumor types; however, its role in GBM pathobiology remains undefined. Herein, we report that the expression levels of LARG and ras homolog family members C (RhoC), and A (RhoA) increase with glial tumor grade and are highest in GBM. LARG and RhoC protein expression is more prominent in invading cells, whereas RhoA expression is largely restricted to cells in the tumor core. Knockdown of LARG by siRNA inhibits GBM cell migration in vitro and invasion ex vivo in organotypic brain slices. Moreover, siRNA-mediated silencing of RhoC suppresses GBM cell migration in vitro and invasion ex vivo, whereas depletion of RhoA enhances GBM cell migration and invasion, supporting a role for LARG and RhoC in GBM cell migration and invasion. Depletion of LARG increases the sensitivity of GBM cells to temozolomide treatment. Collectively, these results suggest that LARG and RhoC may represent unappreciated targets to inhibit glioma invasion.

Figure 1

The expression of leukemia-associated Rho guanine nucleotide exchange factor (LARG), ras homolog family member A (RhoA), and ras homolog family member C (RhoC) increases with glial tumor grades. A: Immunohistochemistry (IHC) analysis of LARG, RhoC, and RhoA expression in low-grade astrocytoma (LGA), anaplastic astrocytoma (AA), and glioblastoma (GBM). B: Dot plot shows the distribution of total IHC scores for LARG, RhoC, and RhoA in all patient specimens. n = 10 LGA and AA (A); n = 30 GBM (A). ∗P < 0.05, ∗∗P < 0.01. Scale bars: 100 μm (A, top and middle rows); 500 μm (A, bottom row).

Figure 2

Immunohistochemistry analysis of the expression of leukemia-associated rho guanine nucleotide exchange factor (LARG), ras homolog family member C (RhoC), and ras homolog family member A (RhoA) in the tumor core and invading edge in glioblastoma (GBM). LARG, RhoC, and RhoA are expressed in the tumor core. LARG and RhoC are positively stained in invading cells, but not RhoA. Arrows indicate invasive GBM cells at the tumor edge. Scale bars = 100 μm.

Figure 3

Depletion of leukemia-associated rho guanine nucleotide exchange factor (LARG, also known as ARHGEF12) inhibits glioblastoma cell migration in vitro and invasion ex vivo. A–C: Top panels: U87 (A), glioblastoma (GBM) 10 (B), and GBM43 (C) cells were transfected with the indicated siRNAs and then assayed for migration through 8-μm pore size membranes. Migrated cells were visualized by DAPI staining 24 hours after plating and quantified. Bottom panels: Lysates of cells transfected with indicated siRNAs were blotted with the indicated antibodies. D: U87 cells stably expressing green fluorescent protein were transfected with the indicated siRNAs for 24 hours. Cells were subsequently implanted into the bilateral putamen on rat organotypic brain slices and observed after 48 hours. Depth of invasion was quantified by confocal microscopy. Data represent the depth of invasion. Immunoblot (IB) analysis confirms the efficiency of knockdown of LARG using two independent siRNAs. Data are expressed as means ± SD (A–C, top panels, and D). n = 4 to 6 independent experiments (D). ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001 versus siCTRL; ^†P < 0.05, ^††P < 0.01 versus luc. a.u., arbitrary unit; luc, luciferase; PDZ-RhoGEF, rho guanine nucleotide exchange factor 11; siCTRL, control siRNA; siLARG, small interfering RNA targeting LARG.

Figure 4

Leukemia-associated rho guanine nucleotide exchange factor (LARG) can promote guanine nucleotide exchange on ras homolog family members A (RhoA) and C (RhoC). HEK293 cells (A) and U87 cells (B) were transfected with human influenza hemagglutinin (HA)-epitope–tagged LARG or empty pcDNA expression vector (pcDNA) for 24 hours and then serum starved overnight. Rho activities in cell lysates were measured using glutathione S-transferase (GST)-Rhotekin–rho binding domain (RBD) pull-down assay. pcDNA, empty pcDNA expression vector.

Figure 5

Ras homolog family members A (RhoA, also known as RHOA) and C (RhoC, also known as RHOC) have differing roles in cell migration in vitro and cell invasion ex vivo. A–C: Cell migration assay for U87 cells (A), glioblastoma (GBM) 10 cells (B), and GBM43 cells (C) transfected with indicated siRNAs. Twenty-four hours after transfection with siRNAs, cells were serum starved overnight, seeded onto the upper chamber of 8-μm pore size transwell inserts, and allowed to migrate for 24 hours. Migrated cells were visualized by DAPI staining and quantified. Immunoblotting confirms the knockdown of RhoA and RhoC. D: U87 cells stably expressing green fluorescent protein were transfected with the indicated siRNAs. At 24 hours after transfection, cells were seeded onto the bilateral putamen of rat brain slices. Depth of invasion was quantified 48 hours after seeding by confocal microscopy. Data represent the depth of invasion. Immunoblot (IB) analysis confirms the efficiency of knockdown of RhoA and RhoC using two independent siRNAs. Data are expressed as means ± SD (D). n = 4 to 6 independent experiments (D). ∗∗P < 0.01, ∗∗∗P < 0.001 versus siCTRL; ^††P < 0.01, ^†††P < 0.001 versus luc. a.u., arbitrary unit; Luc, luciferase; siCTRL, control siRNA; siRho, small interfering RNA targeting rho.

Figure 6

Depletion of ras homolog family member A (RhoA, also known as RHOA) results in the decreased phosphorylation of myosin II. U87 cells (A), glioblastoma (GBM) 10 cells (C), and GBM43 cells (E) were transfected with either nonsilencing siRNA or siRNAs targeting leukemia-associated rho guanine nucleotide exchange factor (LARG, also known as ARHGEF12). Forty-eight hours after transfection, cells were serum starved overnight and directly lysed in sample buffer. The lysates were immunoblotted with the indicated antibodies. U87 cells (B), GBM10 cells (D), and GBM43 cells (F) were transfected with either nonsilencing siRNA or siRNAs targeting RhoA or ras homolog family member C (RhoC, also known as RHOC). Forty-eight hours after transfection, cells were serum starved overnight and directly lysed in sample buffer. The lysates were immunoblotted with the indicated antibodies. IB, immunoblot; MLC, myosin light chain; pMLC, phosphorylation of MLC; siCTRL, control siRNA; siLARG, small interfering RNA targeting LARG.

Figure 7

Depletion of leukemia-associated rho guanine nucleotide exchange factor (LARG, also known as ARHGEF12) sensitizes glioblastoma (GBM) 10 and GBM43 cells to temozolomide (TMZ) treatment. GBM10 cells (A) and GBM43 cells (B) were transfected with either nonsilencing siRNA or siRNAs targeting LARG. Forty-eight hours after transfection, cells were treated with vehicle or 500 μmol/L TMZ. After 24 hours, cells were harvested, 1500 cells were replated in triplicate, and the surviving fraction was assessed by colony formation 10 days later. ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001 siCTRL, control siRNA.

Figure 8

Leukemia-associated rho guanine nucleotide exchange factor (LARG) regulates ras homolog family member C (RhoC) to enhance glioblastoma cell migration and invasion. RhoA, ras homolog family member A.