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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 reduces glioma cell motility and invasion.(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 Materials and Methods. (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 t-test: *P<0.05 and **P<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 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<0.05 and ***P<0.001, respectively).
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pone-0038770-g006: EGFR inhibition reduces glioma cell motility and invasion.(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 Materials and Methods. (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 t-test: *P<0.05 and **P<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 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<0.05 and ***P<0.001, respectively).

Mentions: Actin cytoskeleton reorganization in response to Rho GTPase signaling plays a crucial role in the regulation of cell motility [16]. We therefore investigated whether EGFR inhibition could also modulate glioma cell motility. For this purpose, we performed wound-healing assays in untreated and erlotinib-treated U87MG cells. Control U87MG cells were highly motile and migrated very efficiently in wound-healing assays (Figure 6A). Interestingly, erlotinib clearly inhibited cell motility in these assays (Figure 6A and 6B). Cell motility inhibition in response to erlotinib was also clearly observed in both T98G and LN229 cells (Figure 6A and 6B). Since increased motility and invasiveness are hallmarks of malignant glioma cells, we also tested whether EGFR inhibition could reduce glioma cell invasion in a 3D context. To this end, we monitored cell invasion using matrigel-coated transwells. In agreement with our previous data, whilst untreated glioma cells were highly invasive, cell invasion was strongly inhibited in erlotinib-treated cells (Figure 6C).


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 reduces glioma cell motility and invasion.(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 Materials and Methods. (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 t-test: *P<0.05 and **P<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 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<0.05 and ***P<0.001, respectively).
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pone-0038770-g006: EGFR inhibition reduces glioma cell motility and invasion.(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 Materials and Methods. (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 t-test: *P<0.05 and **P<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 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<0.05 and ***P<0.001, respectively).
Mentions: Actin cytoskeleton reorganization in response to Rho GTPase signaling plays a crucial role in the regulation of cell motility [16]. We therefore investigated whether EGFR inhibition could also modulate glioma cell motility. For this purpose, we performed wound-healing assays in untreated and erlotinib-treated U87MG cells. Control U87MG cells were highly motile and migrated very efficiently in wound-healing assays (Figure 6A). Interestingly, erlotinib clearly inhibited cell motility in these assays (Figure 6A and 6B). Cell motility inhibition in response to erlotinib was also clearly observed in both T98G and LN229 cells (Figure 6A and 6B). Since increased motility and invasiveness are hallmarks of malignant glioma cells, we also tested whether EGFR inhibition could reduce glioma cell invasion in a 3D context. To this end, we monitored cell invasion using matrigel-coated transwells. In agreement with our previous data, whilst untreated glioma cells were highly invasive, cell invasion was strongly inhibited in erlotinib-treated cells (Figure 6C).

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