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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 alters the expression levels of key cell cycle regulators.(A) The indicated human glioma cell lines were harvested and the expression levels of the indicated proteins were analyzed by western blotting with specific antibodies. (B) LN229 and T98G cells were treated with 10 µM erlotinib for the indicated time, harvested and the expression levels of the indicated proteins were analyzed by western blotting with specific antibodies. (C) As in B, but LN229, T98G and U373 cells were treated as indicated.
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pone-0038770-g004: EGFR inhibition alters the expression levels of key cell cycle regulators.(A) The indicated human glioma cell lines were harvested and the expression levels of the indicated proteins were analyzed by western blotting with specific antibodies. (B) LN229 and T98G cells were treated with 10 µM erlotinib for the indicated time, harvested and the expression levels of the indicated proteins were analyzed by western blotting with specific antibodies. (C) As in B, but LN229, T98G and U373 cells were treated as indicated.

Mentions: We next investigated the molecular mechanisms responsible for the cell cycle arrest induced by EGFR inhibition, monitoring alterations on relevant signaling intermediates and cell cycle regulatory proteins in glioma cells (Figure 4A). For this purpose we performed a time-course experiment upon erlotinib treatment in LN229 and T98G cells. Erlotinib induced a rapid inhibition of ERK phosphorylation and an inhibition of Akt phosphorylation that was apparent upon longer term treatment (Figure 4B). Erlotinib also induced a significant downregulation of cyclin D1 and similarly reduced the levels of p21cip1 (Figure 4B). In contrast, Erlotinib increased the levels of the cyclin-dependent kinase inhibitor p27kip1 (Figure 4B). In agreement with these observations, erlotinib inhibited pRb phosphorylation (Figure 4B). We next investigated whether the observed molecular events correlated with the sensitivity to erlotinib in different glioma cell lines. To this end, we compared the aforementioned molecular alterations in three representative glioma cell lines (LN229, T98G and U373) with high, medium and low sensitivity to erlotinib. Erlotinib clearly inhibited both ERK and Akt phosphorylation in LN229 cells, which also showed a marked downregulation of cyclin D1 and a strong increase in p27kip1 levels (Figure 4C). However, p27kip1 levels did not increase in T98G cells, which show slightly lower sensitivity to erlotinib (Figure 4C). The behaviour of U373 cells, the least sensitive cell line, was different to that of LN229 and T98G cells. In U373 cells, erlotinib did not alter cyclin D1 levels nor Akt or ERK phosphorylation, although it induced p27kip1 upregulation (Figure 4C). Interestingly, the sensitivity to erlotinib could not be correlated with the expression levels of EGFR (Figure 4A). Taken together, our results suggest that the inhibition of signaling pathways converging on cell cycle regulatory elements mediate the antiproliferative effects of erlotinib in glioma cells.


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 alters the expression levels of key cell cycle regulators.(A) The indicated human glioma cell lines were harvested and the expression levels of the indicated proteins were analyzed by western blotting with specific antibodies. (B) LN229 and T98G cells were treated with 10 µM erlotinib for the indicated time, harvested and the expression levels of the indicated proteins were analyzed by western blotting with specific antibodies. (C) As in B, but LN229, T98G and U373 cells were treated as indicated.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3368887&req=5

pone-0038770-g004: EGFR inhibition alters the expression levels of key cell cycle regulators.(A) The indicated human glioma cell lines were harvested and the expression levels of the indicated proteins were analyzed by western blotting with specific antibodies. (B) LN229 and T98G cells were treated with 10 µM erlotinib for the indicated time, harvested and the expression levels of the indicated proteins were analyzed by western blotting with specific antibodies. (C) As in B, but LN229, T98G and U373 cells were treated as indicated.
Mentions: We next investigated the molecular mechanisms responsible for the cell cycle arrest induced by EGFR inhibition, monitoring alterations on relevant signaling intermediates and cell cycle regulatory proteins in glioma cells (Figure 4A). For this purpose we performed a time-course experiment upon erlotinib treatment in LN229 and T98G cells. Erlotinib induced a rapid inhibition of ERK phosphorylation and an inhibition of Akt phosphorylation that was apparent upon longer term treatment (Figure 4B). Erlotinib also induced a significant downregulation of cyclin D1 and similarly reduced the levels of p21cip1 (Figure 4B). In contrast, Erlotinib increased the levels of the cyclin-dependent kinase inhibitor p27kip1 (Figure 4B). In agreement with these observations, erlotinib inhibited pRb phosphorylation (Figure 4B). We next investigated whether the observed molecular events correlated with the sensitivity to erlotinib in different glioma cell lines. To this end, we compared the aforementioned molecular alterations in three representative glioma cell lines (LN229, T98G and U373) with high, medium and low sensitivity to erlotinib. Erlotinib clearly inhibited both ERK and Akt phosphorylation in LN229 cells, which also showed a marked downregulation of cyclin D1 and a strong increase in p27kip1 levels (Figure 4C). However, p27kip1 levels did not increase in T98G cells, which show slightly lower sensitivity to erlotinib (Figure 4C). The behaviour of U373 cells, the least sensitive cell line, was different to that of LN229 and T98G cells. In U373 cells, erlotinib did not alter cyclin D1 levels nor Akt or ERK phosphorylation, although it induced p27kip1 upregulation (Figure 4C). Interestingly, the sensitivity to erlotinib could not be correlated with the expression levels of EGFR (Figure 4A). Taken together, our results suggest that the inhibition of signaling pathways converging on cell cycle regulatory elements mediate the antiproliferative effects of erlotinib in glioma cells.

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