the molecular basis for de novo versus acquired resistance through BDNF in our system. One interesting result regarding the reduction in Bim expression mediated by BDNF is that Bim has been reported to regulate anoikis, apoptosis induced by detachment, in various cancers [22,23]. Another confounding issue is distinguishing the anti-apoptotic roles of BDNF in anoikis resistance, as opposed to its import in chemotherapy resistance [24,25]. Resistance to many common anticancer drugs, particularly platinum-based agents, often occurs due to enhanced expression of MDR1 [26], which encodes a P-glycoprotein, calcium-dependent efflux pump. [27,28,29]. MDR1 expression has been reported in various types of chemoresistant tumor phenotypes including neuroblastoma, hepatoblastoma, ovarian cancer, and prostate cancer [30]. Epigenetic changes in methylation of its promoter region has been suggested as a mechanism to modulate MDR1 expression [31]. Although change in mRNA stability can contribute to MDR1 expression [32] epigenetic methylation of promoter for MDR1 gene has also been suggested to be a critical mechanism in MDR1 expression at the transcription level and to be associated with poor prognosis in various cancers [33,34,35,36]. In line with our studies, BDNF was shown to induce MDR1 expression in TrkB-overexpressing neuroblastoma cells, at the transcriptional level [37]. These results suggest a possible mechanism underlying up-regulation of MDR1 by TrkB/BDNF through change in both transcription rate and mRNA stability in CDDP-resistant HNSCC. It is well known that BDNF utilizes PI-3K/Akt, MAPK, and Src signaling pathways to induce various physiological functions including anti-apoptosis and cell proliferation, angiogenesis, anoikis and metastasis [9,13,14,38]. The results of current study also revealed that activation of TrkB by BDNF was enhanced in the CR cell lines, and led to robust induction of the Akt, MAPK, and Src signaling pathways that exceeded the effects in the parental lines. Because Akt is a central node in BDNF signaling and its downstream effects [21,39,40,41], we focused on the contribution of Akt in the development of CDDP resistance. Although we cannot entirely exclude the relevance of MAPK and Src here, our results do not support major roles for these pathways in acquired chemore- sistance. Modulation of XiAP and MDR1 expression by BDNF was blocked by both pharmacological and genetic modulation of Akt, suggesting that Akt is responsible for BDNF-induced apoptotic resistance in HNSCC models. In distinction to this, Akt inhibition failed to modulate the BDNF-regulation of Bim expression, perhaps indicated that a bypass mechanism exists for Bim regulation through TrkB. Previously, BDNF-induced BIM down-regulation was found not to be an Akt-dependent effect, but rather a MAPK- dependent pathway [18,21]. However, the Akt pathway was reported to regulate Bim expression negatively by modulating Forkhead transcription factor in nerve growth factor depravation References 1. Jemal A, Siegel R, Xu J, Ward E (2010) Cancer statistics, 2010. CA Cancer J Clin 60: 277–300. 2. Arriagada R, Bergman B, Dunant A, Le Chevalier T, Pignon JP, et al. (2004) Cisplatin-based adjuvant chemotherapy in patients with completely resected non-small-cell lung cancer. N Engl J Med 350: 351–360. 3. Cooper JS, Pajak TF, Forastiere AA, Jacobs J, Campbell BH, et al. (2004) Postoperative concurrent radiotherapy and chemotherapy for high-risk squa- mous-cell carcinoma of the head and neck. N Engl J Med 350: 1937–1944. 4. Coppin CM, Gospodarowicz MK, James K, Tannock IF, Zee B, et al. 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In conclusion, our findings reveal novel regulatory mechanisms for chemotherapy resistance directed in part by BDNF-TrkB axis in a model of therapy resistant HNSCC. In the current study, we found: (1) both TrkB and BDNF were up-regulated in CDDP resistant HNSCC cell lines (2) a molecular phenotype consistent with apoptotic resistance was associated with acquired CDDP resistance expression (3) exogenous BDNF can induce a CDDP- resistance in CDDP-sensitive cells (4) modulation of TrkB expression resulted in XiAP, Bim, and MDR1 dysregulation, which contributed to CDDP resistance (5) a phenotype that was driven in part by dysfunction Akt signaling mediates CDDP resistance in HNSCCs. Taken together, we have demonstrated a BDNF-TrkB mediated mechanism that underlies CDDP resistance development in HNSCC. These findings may provide insights into potential molecular-based approaches to re-introduce chemotherapy sensi- tivity in tumors that have developed acquired resistance in HNSCC. Supporting Information Table S1 STR profile analysis of parental and chemo- resistant cells derived from OSC-19 and HN-5 HNSCCs. (DOC) Figure S1 A, CDDP-resistant HNSCC cells showed up- regulation of Bcl-XL and survivin expression but failed to show modulation of Bcl-2 and PUMA expression. B, Small molecule inhibitor of TrkB, AZ64, attenuated Bcl-XL expression in CDDP- resistant HNSCC cell lines. C, Small molecule inhibitor of TrkB, AZ64, sensitized OSC-19CR to cisplatin. (TIF) Acknowledgemnts The authors wish to thank Ms. Terri Astin for administrative assistance. STR DNA fingerprinting was done by the Cancer Center Support grant funded Characterized Cell Line core, NCI # CA16672. Author Contributions Conceived and designed the experiments: JL TJ MEK. Performed the experiments: JL TJ MEK. Analyzed the data: JL TJ MEK. Contributed reagents/materials/analysis tools: JL TJ MEK. Wrote the paper: JL TJ MEK. 7. Ho R, Eggert A, Hishiki T, Minturn JE, Ikegaki N, et al. 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(2010) Theraputic targeting of Trk supresses tumor proliferation and enhances cisplatin activity in HNSCC. Cancer Biol Ther 10. PLoS ONE | www.plosone.org 10 January 2012 | Volume 7 | Issue 1 | e30246