EGFR inhibition in glioma cells modulates Rho signaling to inhibit cell motility and invasion and cooperates with temozolomide to reduce cell growth
Authors: Elena Thomàs-Moyà
Silvia Fernández de Mattos
José Rodríguez
Guillem Ramis
Priam Villalonga
Journal: PloS one (2012)
Discipline: Multidisciplinary
Keywords: EGFR activation; glioma; erlotinib; cell proliferation; apoptosis
Affiliations: 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. 

Retracted: Yes

PubPeer comments:
 Figure 4B. https: imgur.com Uw7ZwBn , 
 Figures 4B and C. https: imgur.com Z9E9Zhr , 
 2019 retraction. https: journals.plos.org plosone article?id=10.1371 journal.pone.0222210 After publication of this article [1], the following concerns were raised: In Fig 4B, pRB panel (LN229) lanes 4,5 appear similar. The 0h and 4h ERK data for LN229 cells in Fig 4B appear similar to the 0h and 24h ERK data for LN229 cells in Fig 4C, when aspect ratio is adjusted. The 0h and 24h ERK data for T98G cells appear similar in Fig 4B and 4C, although the corresponding data for p-ERK and other proteins differ. The western blot experiments shown in Fig 4 did not include general loading controls, and the authors did not report total Rb controls for the phospho-Rb (pRb) experiments. The authors provided pRB and ERK p-ERK data from replication experiments and the available primary images underlying the western blots in Fig 4B, C, but the original data are no longer available for the pRB (LN229) and cyclin D1 (LN229, T98G) blots in Fig 4B; or for the cyclin D1 (U373) and ERK (LN229, T98G, U373) blots in Fig 4C. A member of our Editorial Board evaluated the data provided by authors and noted that they support the conclusions. However, owing to the lack of original primary data supporting the panels in question we have been unable to resolve the concerns raised around the pRb data in Fig 4B or the ERK data reported in Fig 4C. Furthermore, the lack of protein loading controls for the experiments in Fig 4 calls into question the validity of the results. In light of these concerns, the PLOS ONE Editors retract this article. In our assessment of this case, it also came to light that two cell lines used in the study (U87MG, U373) may be misidentified (2u20134). SFdM and PV did not agree with the retraction. GR, ETM, and JR did not respond. References 1. Ramis G, Thomu00e0s-Moyu00e0 E, Fernu00e1ndez de Mattos S, Rodru00edguez J, Villalonga P (2012) EGFR Inhibition in Glioma Cells Modulates Rho Signaling to Inhibit Cell Motility and Invasion and Cooperates with Temozolomide to Reduce Cell Growth. PLoS ONE 7(6): e38770. pmid:22701710 2. https: web.expasy.org cellosaurus CVCL_2219 (accessed 8 22 2019) 3. https: web.expasy.org cellosaurus CVCL_0022 (accessed 8 22 2019) 4. Allen M, Bjerke M, Edlund H, Nelander S, Westermark B. (2016) Origin of the U87MG glioma cell line: Good news and bad news. Sci Transl Med. 8(354):354re3. pmid:27582061 , 
 #3 The retraction has been corrected in: https: journals.plos.org plosone article?id=10.1371 journal.pone.0230016 The retraction notice [1] for this article [2] included the following statements: u2018The western blot experiments shown in Fig 4 did not include general loading controls, and the authors did not report total Rb controls for the phospho-Rb (pRb) experimentsu2019, u2018Furthermore, the lack of protein loading controls for the experiments in Figure 4 call into question the validity of the results.u2019 After publication of the notice, it was clarified that in Figure 4 of [2] u201cpRbu201d referred to total retinoblastoma protein rather than a phosphorylated form of this protein. As such, this point in the retraction notice is incorrect: u201cthe authors did not report total Rb controls for the phospho-Rb (pRb) experiments.u201d In addition, the authors noted that the total ERK and total Akt panels in Figure 4 provided information about protein loading across lanes. Additional loading controls were not requested during the re-evaluation of the article, the authors noted that such controls could in principle have been provided had they been requested. ,