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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 leads to actin cytoskeleton reorganization through Rho GTPase modulation.(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.
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pone-0038770-g005: EGFR inhibition leads to actin cytoskeleton reorganization through Rho GTPase modulation.(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.

Mentions: Our initial observations suggested that EGFR inhibition induced morphological changes in glioma cells (Figure 1A). We used U87MG cells to further investigate this alteration, since in these cells erlotinib exerted a robust effect on cell morphology. Untreated U87MG cells were relatively small, morphologically heterogeneous and loosely attached, with many cells eventually rounding up (Figure 5A). In contrast, erlotinib-treated U87MG cells were significantly larger, more homogeneous, firmly attached and well spread (Figure 5A). These results suggested that EGFR inhibition induced a profound cytoskeletal rearrangement. We thus evaluated the organization of the actin cytoskeleton in control and erlotinib-treated U87MG cells. F-actin staining indicated that polymerized actin was mostly located within the cell periphery in untreated U87MG cells, which were largely devoid of actin stress fibres (Figure 5B). Interestingly, erlotinib increased actomyosin contractility, as indicated by the assembly of actin stress fibres (Figure 5B). Since the dynamic regulation of the actin cytoskeleton is controlled by Rho GTPases [15], we monitored the activity of both Rho and Rac upon EGFR inhibition. Basal Rho activity is low in U87MG cells, but erlotinib induced a rapid and sustained activation of Rho, in agreement with the observed increase in actin stress fibres (Figure 5C). In sharp contrast, erlotinib dramatically reduced Rac activity in U87MG cells (Figure 5C). These results indicate that EGFR inhibition promotes the reorganization of the actin cytoskeleton through the modulation of Rho GTPase signaling.


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 leads to actin cytoskeleton reorganization through Rho GTPase modulation.(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.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3368887&req=5

pone-0038770-g005: EGFR inhibition leads to actin cytoskeleton reorganization through Rho GTPase modulation.(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.
Mentions: Our initial observations suggested that EGFR inhibition induced morphological changes in glioma cells (Figure 1A). We used U87MG cells to further investigate this alteration, since in these cells erlotinib exerted a robust effect on cell morphology. Untreated U87MG cells were relatively small, morphologically heterogeneous and loosely attached, with many cells eventually rounding up (Figure 5A). In contrast, erlotinib-treated U87MG cells were significantly larger, more homogeneous, firmly attached and well spread (Figure 5A). These results suggested that EGFR inhibition induced a profound cytoskeletal rearrangement. We thus evaluated the organization of the actin cytoskeleton in control and erlotinib-treated U87MG cells. F-actin staining indicated that polymerized actin was mostly located within the cell periphery in untreated U87MG cells, which were largely devoid of actin stress fibres (Figure 5B). Interestingly, erlotinib increased actomyosin contractility, as indicated by the assembly of actin stress fibres (Figure 5B). Since the dynamic regulation of the actin cytoskeleton is controlled by Rho GTPases [15], we monitored the activity of both Rho and Rac upon EGFR inhibition. Basal Rho activity is low in U87MG cells, but erlotinib induced a rapid and sustained activation of Rho, in agreement with the observed increase in actin stress fibres (Figure 5C). In sharp contrast, erlotinib dramatically reduced Rac activity in U87MG cells (Figure 5C). These results indicate that EGFR inhibition promotes the reorganization of the actin cytoskeleton through the modulation of Rho GTPase signaling.

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