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Crosstalk Between SOX2 and TGF-Β Signaling Regulates EGFR-TKI Tolerance and Lung Cancer Dissemination

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Crosstalk Between SOX2 and TGF-Β Signaling Regulates EGFR-TKI Tolerance and Lung Cancer Dissemination Author Manuscript Published OnlineFirst on August 19, 2020; DOI: 10.1158/0008-5472.CAN-19-3228 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Crosstalk between SOX2 and TGF-β signaling regulates EGFR-TKI tolerance and lung cancer dissemination Ming-Han Kuo1,10, An-Chun Lee1,10, Shih-Hsin Hsiao2,10, Sey-En Lin3,4, Yu-Fan Chiu1, Li-Hao Yang1, Chia-Cherng Yu5, Shih-Hwa Chiou6,7, Hsien-Neng Huang8, Jen-Chung Ko9*, Yu-Ting Chou1* 1Institute of Biotechnology, National Tsing Hua University, Hsinchu, Taiwan; 2Division of Pulmonary Medicine, Department of Internal Medicine, Taipei Medical University Hospital, Taipei, Taiwan; 3Department of Pathology, Taipei Medical University Hospital, Taipei, Taiwan; 4Department of Pathology, Taipei Municipal Wan Fang Hospital, Taipei, Taiwan; 5Department of Medical Research, National Taiwan University Hospital, Taipei, Taiwan; 6Institute of Pharmacology, National Yang-Ming University, Taipei, Taiwan; 7Department of Medical Research, Taipei Veterans General Hospital, Taipei, Taiwan; 8Department of pathology, National Taiwan University Hospital, Hsin-Chu Branch, Hsinchu, Taiwan; 9Department of Internal Medicine, National Taiwan University Hospital, Hsin-Chu Branch, Hsinchu, Taiwan;10Co-first authors *Corresponding authors Jen-Chung Ko, National Taiwan University Hospital, Hsin-Chu Branch, No. 25, Lane 442, Sec. 1, Jingguo Rd., Hsinchu 30013, Taiwan. E-mail: [email protected] Yu-Ting Chou, National Tsing Hua University, No. 101, Sec. 2, Kuang-Fu Rd., Hsinchu 30013, Taiwan. E-mail: [email protected] Running title: Interplay of SOX2 and TGF- on EGFR–TKI tolerance Conflicts of interest The authors disclose no potential conflicts of interest. This work was supported by National Tsing Hua University and Ministry of Science and Technology, Executive Yuan, Taiwan. 1 Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 2020 American Association for Cancer Research. Author Manuscript Published OnlineFirst on August 19, 2020; DOI: 10.1158/0008-5472.CAN-19-3228 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Abstract Regulation of the stemness factor SOX2 by cytokine stimuli controls self-renewal and differentiation in cells. Activating mutations in epidermal growth factor receptor (EGFR) are proven therapeutic targets for tyrosine kinase inhibitors (TKI) in lung adenocarcinoma, but acquired resistance to TKI inevitably occurs. The mechanism by which stemness and differentiation signaling emerge in lung cancers to affect TKI tolerance and lung cancer dissemination has yet to be elucidated. Here we report that crosstalk between SOX2 and TGF-β signaling affects lung cancer cell plasticity and TKI tolerance. TKI treatment favored selection of lung cancer cells displaying mesenchymal morphology with deficient SOX2 expression, whereas SOX2 expression promoted TKI sensitivity and inhibited the mesenchymal phenotype. Preselection of EGFR-mutant lung cancer cells with the mesenchymal phenotype diminished SOX2 expression and TKI sensitivity, whereas SOX2 silencing induced vimentin but suppressed BCL2L11 expression and promoted TKI tolerance. TGF-β stimulation downregulated SOX2 and induced epithelial-to-mesenchymal transdifferentiation accompanied by increased TKI tolerance, which can interfere with ectopic SOX2 expression. SOX2-positive lung cancer cells exhibited a lower dissemination capacity than their SOX2-negative counterparts. Tumors expressing low SOX2 and high vimentin 2 Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 2020 American Association for Cancer Research. Author Manuscript Published OnlineFirst on August 19, 2020; DOI: 10.1158/0008-5472.CAN-19-3228 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. signature were associated with worse survival outcomes in patients with EGFR mutations. These findings provide insights into how cancer cell plasticity regulated by SOX2 and TGF-β signaling affects EGFR-TKI tolerance and lung cancer dissemination. Significance Findings suggest the potential of SOX2 as a prognostic marker in EGFR-mutant lung cancer, as SOX2-mediated cell plasticity regulated by TGF-β stimulation and epigenetic control affects EGFR-TKI tolerance and cancer dissemination. 3 Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 2020 American Association for Cancer Research. Author Manuscript Published OnlineFirst on August 19, 2020; DOI: 10.1158/0008-5472.CAN-19-3228 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Introduction SOX2 belongs to the SOX (Sry-related HMG Box) family of proteins and is an important transcription factor that regulates self-renewal in embryonic stem cells (ESCs). Its downregulation in response to cytokine stimulation determines the timing and degree of differentiation (1). Responding to respiratory tract injuries, SOX2 signaling initiates the proliferation and differentiation of lung progenitor cells to maintain tissue homeostasis (2,3). SOX2, in conjunction with OCT4, KLF4, and MYC, can reverse the mesenchymal morphology of fibroblasts and reprogram them into induced pluripotent stem cells (iPSCs) (4,5). Moreover, inhibition of TGF- signaling facilitates the SOX2-mediated reprogramming process of fibroblasts, whereas activation of TGF- blocks the reprogramming process (6,7). Cancer cell plasticity is characterized as a phenotypic switch between the mesenchymal and epithelial states. Said switch is accompanied by signaling pathway alterations and has been proposed to both generate heterogeneity and mediates tumor progression (8,9). SOX2 serves as a nodal epigenetic regulator in determining the mesenchymal-to-epithelial transdifferentiation (MET) of lung cancer cells (9). Additionally, SOX2 promotes epidermal growth factor receptor (EGFR) expression via a positive feedback 4 Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 2020 American Association for Cancer Research. Author Manuscript Published OnlineFirst on August 19, 2020; DOI: 10.1158/0008-5472.CAN-19-3228 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. manner in lung cancer (10). Activating mutations (exon 19 deletions and L858R mutation) in EGFR are predictive markers and therapeutic targets of TKI in the treatment of patients with lung cancer bearing mutations in EGFR (11-16). Despite initial positive responses to treatment, patients eventually develop resistance to EGFR–TKI. The mutations EGFR–T790M and EGFR–C797S confer acquired resistance to gefitinib and osimertinib, respectively (17-21). However, the mutation EGFR– T790M correlates negatively with distant metastasis and confers better patient survival (22-25), suggesting that an EGFR-mutation-independent TKI-resistance mechanism promotes a worse survival outcome. EMT, a reverse process of MET and pre-invasive status in cancer plasticity, has been linked to EGFR–TKI resistance (26-29). Neuroendocrine transformation of adenocarcinoma has also been detected in lung tumors after EGFR–TKI treatment (19). A drug-tolerant state is essential for the development of acquired resistance to EGFR–TKI (30), and deficient expression of BCL2L11 has been linked to EGFR–TKI tolerance in lung cancer (31). Since SOX2 signaling is highly expressed in both stem cells and lung cancer cells, this study aims to test whether the mechanism behind SOX2-regulated stem cell differentiation and reprogramming is shared by lung 5 Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 2020 American Association for Cancer Research. Author Manuscript Published OnlineFirst on August 19, 2020; DOI: 10.1158/0008-5472.CAN-19-3228 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. cancer cells to affect TKI tolerance and cancer dissemination. We further characterized how crosstalk between SOX2 stemness and TGF- cytokine signaling generates lung cancer cell plasticity with distinct TKI tolerance and dissemination patterns. Materials and Methods Cell culture HCC827 and H1975 cells were obtained from Dr. Jeff Wang (Development Center for Biotechnology, Taiwan) and Dr. Wayne Chang (National Health Research Institutes, Taiwan), respectively (9,10). Human fibroblasts and fibroblast-derived iPSCs were described previously (32). HCC827GR and H1975AZDR cells were established in our laboratory by exposure of HCC827 and H1975 to stepwise increased concentrations of gefitinib and osimertinib, respectively (29). All lung cancer cells were tested positive for human origin carrying specific EGFR mutations by EGFR sequencing analysis (Supplementary Fig. S1A-S1C). All lung cancer cells were cultured in RPMI-1640 medium containing L-glutamine (4 mM), sodium pyruvate (1 mM), HEPES (10 mM), and FBS (10%). 6 Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 2020 American Association for Cancer Research. Author Manuscript Published OnlineFirst on August 19, 2020; DOI: 10.1158/0008-5472.CAN-19-3228 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Quantitative real-time PCR (qPCR) and Chromatin immunoprecipitation-qPCR (ChIP-qPCR) The qPCR and ChIP-qPCR assays were performed as described previously (9). Primer sequences and probes used in qPCR are listed in Supplementary Table S1. Detailed materials for the ChIP-qPCR are described in Supplementary Table S2
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