J Neurooncol (2014) 118:201–203 DOI 10.1007/s11060-014-1406-5
LETTER TO THE EDITOR
Activation of ErbB2- ErbB3 signaling pathway supports potential therapeutic activity of ErbB inhibitors in AT/RT Simone Treiger Sredni • Kashyap Patel • Felipe D’Almeida Costa • Maria de Fa´tima Bonaldo Tadanori Tomita
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Received: 16 September 2013 / Accepted: 17 February 2014 / Published online: 27 February 2014 Ó Springer Science+Business Media New York 2014
To the Editor, With interest, we read Fouladi and colleagues’ results from a molecular biology and phase II trial of lapatinib in children with refractory CNS malignancies [1]. We share the authors’ interest in finding new therapeutic options for pediatric CNS tumors that are refractory to treatment. Our focus of interest is atypical teratoid rhabdoid tumors (AT/ RTs), which are associated with significantly worse overall survival when compared to other pediatric CNS tumors. Here, we would like to share our results obtained through high throughput gene expression (GE) profiling in a series of frozen tumor samples that underwent surgical excision at our institution. Our results suggest that ErbB inhibitors, including lapatinid, could be beneficial to treat AT/RT patients. Because only one AT/RT patient was enrolled in the phase I trial [2] of the above mentioned study, and no AT/RT patient was included in the phase II trial [1], we put together our experience with other studies that found favorable activity of lapatinib in AT/RT [3] to advocate for the inclusion of AT/RT patients in future trials. Six AT/RT and six medulloblastoma (MB) frozen tissue samples were included in our analysis. Informed consents were obtained and the study was approved by our S. T. Sredni (&) K. Patel T. Tomita Division of Pediatric Neurosurgery, Ann and Robert H. Lurie Children’s Hospital of Chicago, 225 E. Chicago Avenue, Box # 28, Chicago, IL 60611, USA e-mail:
[email protected];
[email protected] S. T. Sredni M. de Fa´tima Bonaldo T. Tomita Feinberg School of Medicine, Northwestern University, Chicago, IL, USA F. D’AlmeidaCosta AC Camargo Cancer Center, Sa˜o Paulo, Brazil
Institutional Review Board (IRB#2009-13778). GE profiling was performed using Illumina HT-12 BeadChip arrays (Illumina, Inc., CA, USA), fold change (FC) was determined by calculating the ratio of the average expression between groups, and a t test was applied to access the significance of differentially expressed genes between groups. Ingenuity Pathway Analysis (www.ingenuity.com) was used to identify enriched biological functions and activated pathways. The expression of selected genes was validated by real-time PCR and protein expression was investigated by immunohistochemistry in an independent set of tumors. ELISA assay was performed in two rhabdoid tumor cell lines (G401 and MON). Unsupervised hierarchical clustering showed clear distinction between AT/RT and MB samples (Fig. 1a). Among the 1,002 significantly differentially expressed transcripts (-2.0 B FC C 2.0 and p value B 0.005) the most up-regulated genes in AT/RT, included ErbB2 (FC = 5.15, p value \ 0.0001), ErbB3 (FC = 3.61, p value = 0.03), TYK2 (FC = 2.25, p value = 0.0009), SHC (FC = 11.82, p value \ 0.0001), RRas (FC = 3.20, p value = 0.0001), MAP2K2/ MEK2 (FC = 2.20, p value = 0.0004), STAT3 (FC = 2.28, p value = 0.001), c-Myc (FC = 4.94, p value = 0.018) and CyclinD1 (FC = 11.88, p value = 0.00018). These results point towards consistent downstream activation of genes from the ErbB2-ErbB3 signaling pathway in AT/RTs (Fig. 1b, c). Immunohistochemical expression of ErbB3, MAP2K2/MEK, STAT3, Ras, c-Myc, phospho-STAT3, phospho-EGFR and phospho-AKT was detected in an independent set of rhabdoid tumors. In the same set of tumors, ErbB2 was detected only in the cytoplasm of tumor cells. (Fig. 1d). The phosphorylated forms of AKT and ERK1/2 were detected by ELISA in both cell lines. In addition both cell lines were treated with increasing concentrations and increasing time of exposure to lapatinib.
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Fig. 1 a Heatmap and dendogram of the unsupervised hierarchical clustering of 6 AT/RT and 6 MB for the 2,000 probes with higher coefficient of variability (standard deviation/average expression). b Graphic representation of ErbB2-ErbB3 signaling activation in AT/RT when compared to MB. Molecules highlighted in red are significantly overexpressed in AT/RT. Image generated by Ingenuity Pathway Analysis System (IPA). c Immunohistochemical detection of
ErbB2 (clone CB11), ErbB3 (clone rb-9211), MAP2K2 (clone NBP21767), RAS (clone E201), c-Myc (clone 9E10.3), phospho-STAT3 (clone Ps727), phospho-EGFR (clone Y38) and phospho-AKT (clone 587F11)—magnification 940. d Graph representing average of cell death among replicates when 150,000 G401/MON cells were treated with lapatinib at the dilutions of 1, 10 and 50 lM for 8, 12 and 24 h
Progressive cell death was observed as determined morphologically by cell rounding, shrinkage and detachment from the bottom of the plate (Fig. 1e). It is well recognized that the ErbB family of transmembrane RTKs plays an important role in the pathogenesis of many cancers. In our study, we observed overexpression of ErbB2 and ErbB3 in AT/RTs, accompanied by ErbB2-ErbB3 downstream activation of the Ras/Raf/MEK/ ERK pathway as reflected by activation of the oncogene Ras, the protein kinase MAP2K2 and the transcription factor c-Myc. The JAK/STAT pathway also indicates activation, which is shown by the overexpression of the signal transducer and transcription activator STAT3. High expression of CyclinD1 that is also downstream of ErbB2-
ErbB3 and is well recognized to be overexpressed in AT/ RTs, further reinforces the biological significance of our findings. Recently, Singh and collaborators demonstrated that lapatinib inhibited cell migration, initiated apoptosis in vitro, and showed effective antitumor activity in xenografts [3]. In Fouladi’s phase II study [1] the authors performed a detailed study and demonstrated that although lapatinib presented little activity in the cohort studied, the drug has the ability to cross the brain-blood barrier and is well tolerated by children. Altogether, it has been proposed by both our group and by Singh et al. [3] that AT/RT in vitro and in vivo responds to lapatinib, suggesting that AT/RT, a highly lethal and untreatable tumor, presents the
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potential to respond to this drug. Therefore, we would like to advocate for the inclusion of patients with AT/RT in upcoming trials using ErbB inhibitors. Acknowledgments We would like to thank the Rally Foundation for Childhood Cancer Research in memory of Hailey Trainer and Dr. Ralph & Marian C. Falk Medical Research Trust for funding; Dr. Fernando A. Soares for scientific support, Dr. Olivier Delattre for providing the MON cells, Chiang-Ching Huang for statistical support and Abby L. Halpern for technical and editorial assistance. Conflicts of interest
None
References
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1. Fouladi M, Stewart CF, Blaney SM, Onar-Thomas A, Schaiquevich P, Packer RJ, Goldman S, Geyer JR, Gajjar A, Kun LE,
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