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EGFR inhibition in glioma cells modulates Rho signaling to inhibit cell motility and invasion and cooperates with temozolomide to reduce cell growth.

Ramis G, Thomàs-Moyà E, Fernández de Mattos S, Rodríguez J, Villalonga P - PLoS ONE (2012)

Bottom Line: 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 p27(kip1) levels and pRB hypophosphorylation.

View Article: PubMed Central - PubMed

Affiliation: Cancer Cell Biology Group, Institut Universitari d'Investigació en Ciències de la Salut (IUNICS), Universitat de les Illes Balears, Illes Balears, Spain.

ABSTRACT
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 G(1). 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 p27(kip1) 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.

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EGFR inhibition is effective in glioma cells with amplified or mutant EGFR.(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 t-test: *P<0.05, **P<0.01 and ***P<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 t-test: ***P<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 t-test: *P<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 Materials and Methods. (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 t-test: *P<0.05 and **P<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 Materials and Methods. 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 t-test: ***P<0.001).
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pone-0038770-g008: EGFR inhibition is effective in glioma cells with amplified or mutant EGFR.(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 t-test: *P<0.05, **P<0.01 and ***P<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 t-test: ***P<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 t-test: *P<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 Materials and Methods. (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 t-test: *P<0.05 and **P<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 Materials and Methods. 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 t-test: ***P<0.001).

Mentions: EGFR is frequently activated through mutation or amplification in malignant gliomas, although commonly-used glioma cell lines lack amplified or mutant EGFR. We therefore investigated whether EGFR inhibition was equally effective in the context of amplified or mutant EGFR. For this purpose we took advantage of the U87MG derivative cell line U87ΔEGFR, stably expressing the truncated and constitutively active EGFR mutant EGFRvIII [6], [10], and the SKMG-3 cell line that maintains endogenous EGFR amplification and expresses high levels of wild-type EGFR [17]. Dose-response and growth curve experiments showed that both cell lines were sensitive to EGFR inhibition, similarly to the previously-characterized standard glioma cell lines (Figure 8A and 8B). In agreement with our previous data, EGFR inhibition also prevented multicellular tumour spheroid formation in U87ΔEGFR cells (Figure 8C). We next used multicellular tumour formation assays to investigate cooperation with TMZ. To this end, U87ΔEGFR cells were treated with sub-optimal doses of erlotinib (1–5 µM) and TMZ (25–50 µM), alone or in combination, and the formation of multicellular tumour spheroids was assessed. Similarly to untreated cells, cells treated with sub-optimal doses of erlotinib or TMZ alone gave rise to a high number of spheroids (Figure 8D). In contrast, the diverse combinations of sub-optimal doses of erlotinib and TMZ clearly reduced spheroid formation, similar to the standard erlotinib treatment (Figure 8D). We also investigated whether EGFR inhibition could reduce cell motility and invasion in the context of activated EGFR using wound-healing and transwell invasion assays, respectively. EGFR inhibition strongly reduced cell motility in both U87ΔEGFR and SKMG-3 cells (Figures 8E and 8F) and this inhibitory effect on cell motility was significantly reverted by Rho/ROCK inhibitors C3 and H-1152 (data not shown). Cell invasion within a 3D matrix was also strongly inhibited in the presence of erlotinib in both cell lines (Figure 8G). Taken together, these results confirm that EGFR inhibition can effectively reduce glioma cell proliferation, motility and invasion in cells with enforced EGFR activation.


EGFR inhibition in glioma cells modulates Rho signaling to inhibit cell motility and invasion and cooperates with temozolomide to reduce cell growth.

Ramis G, Thomàs-Moyà E, Fernández de Mattos S, Rodríguez J, Villalonga P - PLoS ONE (2012)

EGFR inhibition is effective in glioma cells with amplified or mutant EGFR.(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 t-test: *P<0.05, **P<0.01 and ***P<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 t-test: ***P<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 t-test: *P<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 Materials and Methods. (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 t-test: *P<0.05 and **P<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 Materials and Methods. 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 t-test: ***P<0.001).
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getmorefigures.php?uid=PMC3368887&req=5

pone-0038770-g008: EGFR inhibition is effective in glioma cells with amplified or mutant EGFR.(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 t-test: *P<0.05, **P<0.01 and ***P<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 t-test: ***P<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 t-test: *P<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 Materials and Methods. (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 t-test: *P<0.05 and **P<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 Materials and Methods. 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 t-test: ***P<0.001).
Mentions: EGFR is frequently activated through mutation or amplification in malignant gliomas, although commonly-used glioma cell lines lack amplified or mutant EGFR. We therefore investigated whether EGFR inhibition was equally effective in the context of amplified or mutant EGFR. For this purpose we took advantage of the U87MG derivative cell line U87ΔEGFR, stably expressing the truncated and constitutively active EGFR mutant EGFRvIII [6], [10], and the SKMG-3 cell line that maintains endogenous EGFR amplification and expresses high levels of wild-type EGFR [17]. Dose-response and growth curve experiments showed that both cell lines were sensitive to EGFR inhibition, similarly to the previously-characterized standard glioma cell lines (Figure 8A and 8B). In agreement with our previous data, EGFR inhibition also prevented multicellular tumour spheroid formation in U87ΔEGFR cells (Figure 8C). We next used multicellular tumour formation assays to investigate cooperation with TMZ. To this end, U87ΔEGFR cells were treated with sub-optimal doses of erlotinib (1–5 µM) and TMZ (25–50 µM), alone or in combination, and the formation of multicellular tumour spheroids was assessed. Similarly to untreated cells, cells treated with sub-optimal doses of erlotinib or TMZ alone gave rise to a high number of spheroids (Figure 8D). In contrast, the diverse combinations of sub-optimal doses of erlotinib and TMZ clearly reduced spheroid formation, similar to the standard erlotinib treatment (Figure 8D). We also investigated whether EGFR inhibition could reduce cell motility and invasion in the context of activated EGFR using wound-healing and transwell invasion assays, respectively. EGFR inhibition strongly reduced cell motility in both U87ΔEGFR and SKMG-3 cells (Figures 8E and 8F) and this inhibitory effect on cell motility was significantly reverted by Rho/ROCK inhibitors C3 and H-1152 (data not shown). Cell invasion within a 3D matrix was also strongly inhibited in the presence of erlotinib in both cell lines (Figure 8G). Taken together, these results confirm that EGFR inhibition can effectively reduce glioma cell proliferation, motility and invasion in cells with enforced EGFR activation.

Bottom Line: 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 p27(kip1) levels and pRB hypophosphorylation.

View Article: PubMed Central - PubMed

Affiliation: Cancer Cell Biology Group, Institut Universitari d'Investigació en Ciències de la Salut (IUNICS), Universitat de les Illes Balears, Illes Balears, Spain.

ABSTRACT
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 G(1). 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 p27(kip1) 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.

Show MeSH
Related in: MedlinePlus