To aid development of immunotherapy for poor outcome Group A ependymoma (EPN) in children we sought to identify the molecular pathway underlying the adverse pro-inflammatory phenotype associated with this molecular subgroup. We have previously shown the importance of immunological factors in EPN outcome and that an anti-tumor immunophenotype was critical in survival whereas a pro-inflammatory phenotype was associated with a poor outcome. Moreover we showed a strong correlation between these immunophenotypes and the newly recognized molecular subgroups of posterior fossa EPN. We showed that Group A EPN have an adverse pro-inflammatory immunophenotype at presentation which adversely affects outcome even at relapse.
We contrasted the gene expression profiles of patient tumor samples from Group A EPN to the more clinically favorable Group B EPN, as well as a broader panel of pediatric and adult brain tumors and normal brain, to identify potential molecular pathways that might be associated with the Group A immunophenotype. The results of these studies were validated using functional assays in short-term cultures of patient tumor samples and novel EPN cell lines.
Strikingly we found a robust expression signature implicating the interleukin-6/signal transduced and activator of transcription 3 (IL6/STAT3) pathway in the Group A tumors. Comparison of transcriptomic profiles of EPN Group A (n=17) and Group B (n=20) demonstrated significant overexpression both IL6 (19.7-fold) and STAT3 (1.6 fold) in Group A. High overexpression of STAT3 driven genes IL8 (30-fold), SOCS3 (14-fold) and BIRC3 (8.5-fold) provided additional proof of IL6/STAT3 pathway activation in Group A. Furthermore, IL6 and IL8 gene expression was shown to be significantly higher (FDR<0.05) than any other type of pediatric or adult brain tumor or normal brain, suggesting that this phenotype is unique to Group A EPN. Western blot analysis of whole tumor samples from Groups A and B showed a significantly elevated ratio of pSTAT3 to total STAT3 in the Group A tumors compared to the Group B, denoting activation of STAT3. Measurement of IL6 secretion from primary ex-vivo tumor cells, isolated by flow sorting of disaggregated surgical tumor samples, showed a significantly higher secretion by tumors cells from Group A patients compared to Group B. To further investigate the role of the IL6/STAT3 pathway, we utilized a novel EPN cell line (811) established from a multiple recurrent EPN, with the transcriptome characteristics of a Group A EPN. This cell line secretes large amounts of IL6, considerably more than glioblastoma (GBM) cell line U87 in which the IL6/STAT3 pathway has been extensively studied. This cell line induced a contact-independent polarization of myeloid cells to a pro-inflammatory phenotype. HLA-matched donor CD14+ that we treated with 811 conditioned media showed a 9.2-fold increase in IL8 secretion (p=0.027). Demonstrating the mitogenic role of STAT3 activation in EPN, we showed 811 is more sensitive to STAT3 inhibition by S31-201 (IC50 = 81uM) than breast cancer and GBM cell lines. PhosphoSTAT3 and the downstream targets of the pathway, SOCS3 and Mcl-1 are significantly reduced in a dose dependent manner in response to S31-201. S3I-201 treatment of 811 and an EPN short-term cell line were shown to cause a significant dose-dependent increase in Caspase-3/7 cleavage demonstrating apoptosis as a result of STAT3 inhibition.
To conclude: The IL6/STAT3 pathway is active in Group A EPN and is important in maintaining tumor growth in a pro-inflammatory microenvironment. Effective design of Group A-targeted immunotherapy for children with EPN may require alleviation of this potentially immunosuppressive pathway.
Citation Format: Andrea Griesinger, Andrew M. Donson, Diane Birks, Vladimir Amani, Rebecca Josephson, Kathleen Dorris, Philip Reigan, Michael Handler, Nicholas Foreman. Activation of the IL6/STAT3 pathway in childhood ependymoma is associated with a pro-inflammatory tumor microenvironment and a poor prognosis. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr B69.
- ©2015 American Association for Cancer Research.