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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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Erlotinib-induced effects on cell morphology and motility require Rho/ROCK activity.(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 Materials and Methods. 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 t-test: *P<0.05 and ***P<0.001, respectively).
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pone-0038770-g007: Erlotinib-induced effects on cell morphology and motility require Rho/ROCK activity.(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 Materials and Methods. 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 t-test: *P<0.05 and ***P<0.001, respectively).

Mentions: Our previous observations suggest that the increase in Rho activity and actomyosin contractility are responsible for the reduction in cell motility observed in response to EGFR inhibition. In order to confirm this hypothesis, we treated cells with erlotinib and tested whether Rho/ROCK inhibition could restore cell morphology and motility to similar conditions as those observed in untreated cells. As opposed to control cells, erlotinib-treated cells showed their distinctive morphology, characterized by the presence of larger, more homogeneous and firmly attached cells (Figure 7A). Interestingly, treatment of U87MG cells with either a Rho inhibitor (the C3 exoenzyme) or a small-molecule ROCK inhibitor (H-1152) together with erlotinib resulted in a very similar cellular morphology to that of control cells (Figure 7A). Accordingly, whereas erlotinib induced the formation of thick and robust stress fibres, co-treatment with either C3 or H-1152 completely prevented the formation of actin stress fibres (Figure 7B). We finally investigated whether Rho/ROCK inhibition could restore cell motility in erlotinib-treated glioma cells. To this end, we performed wound healing assays using U87MG cells in the presence of erlotinib alone or together with C3 or H-1152. In agreement with our previous data, EGFR inhibition led to a marked decrease in the rate of cell motility (Figure 7C). In sharp contrast, inhibition of either Rho or ROCK prevented erlotinib-induced reduction in cell motility, restoring the motility rate to that of untreated cells (Figure 7C). Rho/ROCK inhibitors also restored cell motility in erlotinib-treated T98G and LN229 cells (data not shown). Taken together, these results confirm that EGFR inhibitors alter glioma cell morphology and motility through the activation of the Rho/ROCK signaling pathway.


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)

Erlotinib-induced effects on cell morphology and motility require Rho/ROCK activity.(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 Materials and Methods. 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 t-test: *P<0.05 and ***P<0.001, respectively).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3368887&req=5

pone-0038770-g007: Erlotinib-induced effects on cell morphology and motility require Rho/ROCK activity.(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 Materials and Methods. 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 t-test: *P<0.05 and ***P<0.001, respectively).
Mentions: Our previous observations suggest that the increase in Rho activity and actomyosin contractility are responsible for the reduction in cell motility observed in response to EGFR inhibition. In order to confirm this hypothesis, we treated cells with erlotinib and tested whether Rho/ROCK inhibition could restore cell morphology and motility to similar conditions as those observed in untreated cells. As opposed to control cells, erlotinib-treated cells showed their distinctive morphology, characterized by the presence of larger, more homogeneous and firmly attached cells (Figure 7A). Interestingly, treatment of U87MG cells with either a Rho inhibitor (the C3 exoenzyme) or a small-molecule ROCK inhibitor (H-1152) together with erlotinib resulted in a very similar cellular morphology to that of control cells (Figure 7A). Accordingly, whereas erlotinib induced the formation of thick and robust stress fibres, co-treatment with either C3 or H-1152 completely prevented the formation of actin stress fibres (Figure 7B). We finally investigated whether Rho/ROCK inhibition could restore cell motility in erlotinib-treated glioma cells. To this end, we performed wound healing assays using U87MG cells in the presence of erlotinib alone or together with C3 or H-1152. In agreement with our previous data, EGFR inhibition led to a marked decrease in the rate of cell motility (Figure 7C). In sharp contrast, inhibition of either Rho or ROCK prevented erlotinib-induced reduction in cell motility, restoring the motility rate to that of untreated cells (Figure 7C). Rho/ROCK inhibitors also restored cell motility in erlotinib-treated T98G and LN229 cells (data not shown). Taken together, these results confirm that EGFR inhibitors alter glioma cell morphology and motility through the activation of the Rho/ROCK signaling pathway.

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