EGFR Inhibition in Glioma Cells Modulates Rho Signaling to Inhibit Cell Motility and Invasion and Cooperates with Temozolomide to Reduce Cell Growth

Enforced EGFR activation upon gene amplification and/or mutation is a common hallmark of malignant glioma. Small molecule EGFR tyrosine kinase inhibitors, such as erlotinib (Tarceva), have shown some activity in a subset of glioma patients in recent trials, although the reported data on the cellular basis of glioma cell responsiveness to these compounds have been contradictory. Here we have used a panel of human glioma cell lines, including cells with amplified or mutant EGFR, to further characterize the cellular effects of EGFR inhibition with erlotinib. Dose-response and cellular growth assays indicate that erlotinib reduces cell proliferation in all tested cell lines without inducing cytotoxic effects. Flow cytometric analyses confirm that EGFR inhibition does not induce apoptosis in glioma cells, leading to cell cycle arrest in G1. Interestingly, erlotinib also prevents spontaneous multicellular tumour spheroid growth in U87MG cells and cooperates with sub-optimal doses of temozolomide (TMZ) to reduce multicellular tumour spheroid growth. This cooperation appears to be schedule-dependent, since pre-treatment with erlotinib protects against TMZ-induced cytotoxicity whereas concomitant treatment results in a cooperative effect. Cell cycle arrest in erlotinib-treated cells is associated with an inhibition of ERK and Akt signaling, resulting in cyclin D1 downregulation, an increase in p27kip1 levels and pRB hypophosphorylation. Interestingly, EGFR inhibition also perturbs Rho GTPase signaling and cellular morphology, leading to Rho/ROCK-dependent formation of actin stress fibres and the inhibition of glioma cell motility and invasion

Erlotinib prevents multicellular tumour spheroid formation and induces G₁ arrest in glioma cells.

<p>(A) Glioma cell lines were left untreated (control) or were treated for 24 h with 10 µM erlotinib (erlotinib). Cells were harvested and their DNA content analyzed by flow cytometry as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038770#s2" target="_blank">Materials and Methods</a>. The cell cycle distribution is shown for each experimental condition. (B) The graph summarizes the flow cytometry data obtained in all glioma cell lines, indicating the cell cycle distribution in control and 24 h erlotinib-treated conditions for each cell line.</p

EGFR inhibition reduces glioma cell motility and invasion.

<p>(A) Representative phase-contrast micrographs of U87MG cells left untreated or treated with 10 µM erlotinib as indicated, before (upper panel) and after (lower panel) performing wound healing assays as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038770#s2" target="_blank">Materials and Methods</a>. (B) Representation of the mean ± SD rate of motility, from three independent experiments performed in sextuplicate, expressed as the percentage of U87MG cell motility relative to untreated cells. The differences between control and erlotinib treatment are statistically significant (Student's <i>t</i>-test: *<i>P</i><0.05 and **<i>P</i><0.01, respectively). (C) U87MG cells were seeded onto Matrigel-coated transwells in the absence (−) or presence (+) or 10 µM erlotinib to perform invasion assays as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038770#s2" target="_blank">Materials and Methods</a>. The graph represents the mean ± SD rate of invasion from three independent experiments performed in duplicate, expressed as the percentage of invasion relative to untreated cells. The differences between control and erlotinib treatment are statistically significant (Student's <i>t</i>-test: *<i>P</i><0.05 and ***<i>P</i><0.001, respectively).</p

EGFR inhibition cooperates with temozolomide to inhibit glioma cell growth.

<p>(A) LN229, U251 and HS683 cells were left untreated or were treated for 24 h with erlotinib and subsequently exposed to vehicle or TMZ for 3 h, plated and after 7–10 days the remaining colonies were stained and counted as indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038770#s2" target="_blank">Materials and Methods</a>. The mean ± SD values from three independent experiments, each conducted in duplicate, are shown in the graph, representing the number of clones relative to untreated cells. The differences between combined treatment and either treatment alone are statistically significant (Student's <i>t</i>-test: *<i>P</i><0.05 and **<i>P</i><0.01, respectively). (B) U251 and U87MG cells were plated in 96-well plates, left untreated or treated as indicated for 48 h and cell viability monitored as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038770#s2" target="_blank">Materials and Methods</a>. The mean ± SD values from three independent experiments, each conducted in duplicate, are shown in the graph, representing the percentage of viable cells relative to untreated cells. The differences between combined treatment and either treatment alone are statistically significant (Student's <i>t</i>-test: **<i>P</i><0.01). (C) Representative phase-contrast micrographs of U87MG cells treated as indicated and left for 4–6 days to allow formation of MCTS. The graph indicates the mean ± SD values of MCTS formation from three independent experiments, each conducted in duplicate, expressed as the percentage of MCTS relative to untreated cells. The differences between combined treatment and either treatment alone are statistically significant (Student's <i>t</i>-test: ***<i>P</i><0.001).</p

EGFR inhibition alters the expression levels of key cell cycle regulators.

<p>(A) The indicated human glioma cell lines were harvested and the expression levels of the indicated proteins were analyzed by western blotting with specific antibodies. (B) LN229 and T98G cells were treated with 10 µM erlotinib for the indicated time, harvested and the expression levels of the indicated proteins were analyzed by western blotting with specific antibodies. (C) As in B, but LN229, T98G and U373 cells were treated as indicated.</p

EGFR inhibition leads to actin cytoskeleton reorganization through Rho GTPase modulation.

<p>(A) Representative phase-contrast micrographs of U87MG cells left untreated (control) or treated for 24 h with 10 µM erlotinib (erlotinib). (B) U87MG cells grown on coverslips were left untreated (control) or were treated for 24 h with 10 µM erlotinib (erlotinib), fixed and stained with TRITC-labelled phalloidin. Bar, 5 µm. (C) U87MG cells were treated as indicated, harvested and RhoA and Rac1 activation were analyzed by GST-Rhotekin and GST-PBD pulldown, respectively, followed by western blotting with anti-RhoA and anti-Rac1 antibodies (upper panel). An aliquot of each lysate was also loaded in another gel to analyze total RhoA and total Rac1 levels (bottom panel). The graphs represent the quantified mean ± SD Rho/Rac activation values (Rho-GTP/Total Rho and Rac-GTP/Total Rac), relative to untreated cells, from three independent experiments.</p

Erlotinib-induced effects on cell morphology and motility require Rho/ROCK activity.

<p>(A) Representative phase-contrast micrographs of U87MG cells left untreated (control) or treated for 24 h with 10 µM erlotinib alone or in the presence of 0,5 µg/ml C3 or 0,5 µM H-1152. (B) U87MG cells grown on coverslips were left untreated (control) or were treated for 24 h with 10 µM erlotinib alone or in the presence of 0,5 µg/ml C3 or 0,5 µM H-1152, fixed and stained with TRITC-labelled phalloidin. Bar, 10 µm. (C) Representative phase-contrast micrographs of U87MG cells left untreated or treated as indicated, before (upper panel) and after (lower panel) performing wound healing assays as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038770#s2" target="_blank">Materials and Methods</a>. The graph represents the mean ± SD rate of motility, from three independent experiments performed in sextuplicate, expressed as the percentage of U87MG cell motility relative to untreated cells. The differences in motility between cells treated alone with erlotinib or together with C3 or H-1152 are statistically significant (Student's <i>t</i>-test: *<i>P</i><0.05 and ***<i>P</i><0.001, respectively).</p

EGFR inhibition is effective in glioma cells with amplified or mutant EGFR.

<p>(A) SKMG-3 and U87ΔEGFR cells were treated for 72 h with the indicated concentrations of erlotinib. The mean ± SD values from three independent experiments, each conducted in duplicate, are shown in the graph, representing the percentage of viable cells relative to untreated conditions. The differences between control and erlotinib treatment are statistically significant (Student's <i>t</i>-test: *<i>P</i><0.05, **<i>P</i><0.01 and ***<i>P</i><0.001, respectively). (B) SKMG-3 and U87ΔEGFR cells were left untreated (untreated) or treated with 10 µM erlotinib (erlotinib) and the number of cells counted every 24 h. The mean ± SD values from three independent experiments, each conducted in duplicate, are shown in the graph, representing the fold increase in cell growth in untreated and erlotinib-treated conditions at the indicated time-points. (C) Representative phase-contrast micrographs of U87ΔEGFR cells left for 6 days to allow formation of multicellular tumour spheroids (MCTS), untreated (control) or treated with 10 µM erlotinib (erlotinib). The graph indicates the mean ± SD values of MCTS formation from three independent experiments, each conducted in duplicate, expressed as the percentage of MCTS relative to untreated cells. The differences between control and erlotinib treatment are statistically significant (Student's <i>t</i>-test: ***<i>P</i><0.001). (D) U87ΔEGFR cells were left untreated or treated as indicated and grown for 6 days to allow formation of multicellular tumour spheroids (MCTS). The graph indicates the mean ± SD values of MCTS formation from three independent experiments, each conducted in duplicate, expressed as the percentage of MCTS relative to untreated cells. The differences between combined treatments and either treatment alone are statistically significant (Student's <i>t</i>-test: *<i>P</i><0.05). (E) Representative phase-contrast micrographs of U87ΔEGFR (left panel) and SKMG-3 (right panel) cells left untreated or treated with 10 µM erlotinib as indicated, before (upper panel) and after (lower panel) performing wound healing assays as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038770#s2" target="_blank">Materials and Methods</a>. (F) Representation of the mean ± SD rate of motility, from three independent experiments performed in sextuplicate, expressed as the percentage of cell motility in each of the indicated conditions relative to untreated cells. The differences between control and erlotinib treatment are statistically significant (Student's <i>t</i>-test: *<i>P</i><0.05 and **<i>P</i><0.01, respectively). (G) U87ΔEGFR and SKMG-3 cells were seeded onto Matrigel-coated transwells in the absence or presence of 10 µM erlotinib to perform invasion assays as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038770#s2" target="_blank">Materials and Methods</a>. The graph represents the mean ± SD rate of invasion from three independent experiments performed in duplicate, expressed as the percentage of invasion relative to untreated cells. The differences between control and erlotinib treatment are statistically significant (Student's <i>t</i>-test: ***<i>P</i><0.001).</p