br SOX OCT and KLF have been

SOX2, OCT4, and KLF4 have been shown to cooperate in promoting self-renewal and maintaining the pluripotent state of embryonic stem Lactacystin (ESCs).48 This tightly-controlled gene
circuitry regulates developmental processes through spatial and temporal activation or repression of various downstream targets including transcription factors.49,50 However, in cancer cells this circuitry may be dysregulated resulting in aberrant transcrip-tional products and associated pathologic signal transduction effects.51 While downregulation of KLF4 may occur in tandem with SOX2, the reverse has been shown with SOX2 and OCT4, the result of a negative-feedback loop.52,53 Although, upregu-lation of OCT4 in the CL-siSOX2 group may prove problematic to the long-term suppression of the pluripotent state of any residual tumor, it is refreshing to observe that CL-siSOX2 + cisplatin completely abolished OCT4; thereby, sup-porting the rationale for the use of CL-siSOX2 in adjunctive therapy instead of as a stand-alone treatment.
Upregulation of SOX2 is associated with activation of mediators of tumor resistance.54–56 We observed that SOX2 overexpression promoted activation of Wnt/Catenin oncogenic signaling. However, CL-siSOX2 abrogated Wnt/Catenin-mediated resistance by down-regulation of Wnt3a, Wnt5a/b, phosphorylated β-catenin, and downstream targets, ABCG2 and Dvl2. We observed enhanced down-regulation of ABCG2 in the CL-siSOX2 + cisplatin group compared to individual treat-ments, which further supports a mutually-exclusive mechanism for the delayed progression of cisplatin-induced loss of body weight. Activation of β-catenin by SOX2 is through inhibition of its interaction with E-cadherin whose expression is mutually inhibitory to N-cadherin. SOX2 binds to the promoter region of Slug with resultant loss of E-cadherin function and enhancement of epithelial-to-mesenchymal transition (EMT).45,57 CL-siSOX2 facilitated reversal of EMT by upregulating E-cadherin, while downregulating Slug and N-cadherin. Toll-like receptors (TLR) activate pro-inflammatory NF-κB signaling resulting in en-hanced tumor proliferation. Inhibition of canonical IKK/NF-κB activation has been shown to sensitize cancer cells to therapy.58 SOX2 down-regulation was associated with decreased expres-sion of TLR1, TLR9 and IKKγ. Additionally, CL-siSOX2 disrupted the TGFβ/SMAD/SOX2 regulatory network by diminishing their expression together with Bcl-2 and Survivin.59
In conclusion, this study demonstrates the successful engineering of an efficient siRNA-loaded cationic lipoplex, for the therapeutic interference of SOX2 oncogenic activity in a mouse xenograft model of H1650 CSC/SP lung tumor. The study also defines the molecular events involved in attenuating tumor growth though SOX2 interference, resulting in the inhibition of markers of tumor growth, metastasis, invasion and chemoresistance. Consistent with other studies using specific siRNA targeting alone or in combination with other therapies such as radiation, chemotherapy, and/or targeted therapy,44,60 our results demonstrate the effectiveness of SOX2 siRNA in shrinking tumor volume in mice, either alone or in combination with cisplatin.
Appendix A. Supplementary data
2. Bora-Singhal N, Perumal D, Nguyen J, Chellappan S. Gli1-Mediated Regulation of Sox2 Facilitates Self-Renewal of Stem-Like Cells and Confers Resistance to EGFR Inhibitors in Non-Small Cell Lung Cancer. Neoplasia 2015;17(7):538-51.
3. Goldberg SB, Oxnard GR, Digumarthy S, Muzikansky A, Jackman DM, Lennes IT, et al. Chemotherapy With Erlotinib or Chemotherapy Alone in Advanced Non-Small Cell Lung Cancer With Acquired Resistance to EGFR Tyrosine Kinase Inhibitors. Oncologist 2013.