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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 cooperates with temozolomide to inhibit glioma cell growth.(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 Materials and Methods. 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 t-test: *P<0.05 and **P<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 Materials and Methods. 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 t-test: **P<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 t-test: ***P<0.001).
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pone-0038770-g003: EGFR inhibition cooperates with temozolomide to inhibit glioma cell growth.(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 Materials and Methods. 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 t-test: *P<0.05 and **P<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 Materials and Methods. 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 t-test: **P<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 t-test: ***P<0.001).

Mentions: The current therapy for glioma patients involves the use of the alkylating agent temozolomide (TMZ) in combination with radiotherapy. We therefore investigated whether erlotinib could potentiate the antiproliferative effects of TMZ in glioma cells. For this purpose we used different experimental strategies. First, we performed clonogenic assays upon TMZ treatment of control or erlotinib-treated cells to assess if EGFR inhibition could potentiate TMZ-induced genotoxicity. LN229, U251 and HS683 cells pre-treated for 24 h with erlotinib recovered some of their clonogenic ability when re-plated in the absence of erlotinib (Figure 3A). In contrast, a short exposure to TMZ (3 h) dramatically compromised their clonogenic capacity (Figure 3A). Interestingly, erlotinib pre-treatment protected cells from TMZ-induced genotoxicity (Figure 3A). To extend these observations, we next monitored cell proliferation in MTT-based assays. In order to test whether erlotinib could cooperate with TMZ we used sub-optimal doses of both erlotinib (1 µM) and TMZ (25 µM). As expected, neither erlotinib nor TMZ at sub-optimal doses were able to significantly reduce cellular growth (Figure 3B). However, when used in combination, erlotinib was able to cooperate with TMZ to reduce cell proliferation in both U87MG and U251 cells (Figure 3B). In order to validate these results, we performed multicellular tumour spheroid formation assays using U87MG cells. To this end, U87MG cells were treated with sub-optimal doses of erlotinib (1 µM) and TMZ (25 µM), alone or in combination, and the formation of multicellular tumour spheroids was assessed. Similarly to control cells, cells treated with sub-optimal doses of erlotinib or TMZ alone gave rise to a high number of spheroids (Figures 3C). In sharp contrast, the combination of sub-optimal doses of erlotinib and TMZ dramatically reduced spheroid formation, similarly to the standard erlotinib treatment (Figures 3C). These results suggest that the combination of low doses of erlotinib and TMZ can cooperate to reduce glioma cell proliferation.


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 cooperates with temozolomide to inhibit glioma cell growth.(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 Materials and Methods. 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 t-test: *P<0.05 and **P<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 Materials and Methods. 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 t-test: **P<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 t-test: ***P<0.001).
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Related In: Results  -  Collection

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pone-0038770-g003: EGFR inhibition cooperates with temozolomide to inhibit glioma cell growth.(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 Materials and Methods. 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 t-test: *P<0.05 and **P<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 Materials and Methods. 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 t-test: **P<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 t-test: ***P<0.001).
Mentions: The current therapy for glioma patients involves the use of the alkylating agent temozolomide (TMZ) in combination with radiotherapy. We therefore investigated whether erlotinib could potentiate the antiproliferative effects of TMZ in glioma cells. For this purpose we used different experimental strategies. First, we performed clonogenic assays upon TMZ treatment of control or erlotinib-treated cells to assess if EGFR inhibition could potentiate TMZ-induced genotoxicity. LN229, U251 and HS683 cells pre-treated for 24 h with erlotinib recovered some of their clonogenic ability when re-plated in the absence of erlotinib (Figure 3A). In contrast, a short exposure to TMZ (3 h) dramatically compromised their clonogenic capacity (Figure 3A). Interestingly, erlotinib pre-treatment protected cells from TMZ-induced genotoxicity (Figure 3A). To extend these observations, we next monitored cell proliferation in MTT-based assays. In order to test whether erlotinib could cooperate with TMZ we used sub-optimal doses of both erlotinib (1 µM) and TMZ (25 µM). As expected, neither erlotinib nor TMZ at sub-optimal doses were able to significantly reduce cellular growth (Figure 3B). However, when used in combination, erlotinib was able to cooperate with TMZ to reduce cell proliferation in both U87MG and U251 cells (Figure 3B). In order to validate these results, we performed multicellular tumour spheroid formation assays using U87MG cells. To this end, U87MG cells were treated with sub-optimal doses of erlotinib (1 µM) and TMZ (25 µM), alone or in combination, and the formation of multicellular tumour spheroids was assessed. Similarly to control cells, cells treated with sub-optimal doses of erlotinib or TMZ alone gave rise to a high number of spheroids (Figures 3C). In sharp contrast, the combination of sub-optimal doses of erlotinib and TMZ dramatically reduced spheroid formation, similarly to the standard erlotinib treatment (Figures 3C). These results suggest that the combination of low doses of erlotinib and TMZ can cooperate to reduce glioma cell proliferation.

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