title: ZEB1 transcriptionally regulated carbonic anhydrase 9 mediates the chemoresistance of tongue cancer via maintaining intracellular pH.
abstract: BACKGROUND
  Chemoresistance is a major obstacle in successfully treating cancers, and the mechanisms responsible for drug resistance are still far from understood. Carbonic anhydrase 9 (CA9) has been shown to be upregulated in the drug-resistant tongue cancer cell line Tca8113/PYM and to be associated with drug resistance. However, the mechanisms regulating CA9 expression and its role in drug resistance remain unclear.
  
  
  METHODS
  Bioinformatic and experimental analysis involving ChIP and luciferase reporter assays were used to validate Zinc finger E-box-binding homeobox 1 (ZEB1) as a transcriptional regulator of CA9. Gene expression and protein levels were evaluated by quantitative RT-PCR and western blotting, respectively. Sensitivity to chemotherapy was examined using the MTS assay and Hoechst staining and analysis caspase-3 activity to evaluate changes in apoptosis. Intracellular pH (pHi) was measured using fluorescent pH-indicator BCECF-AM. Protein expression in patient tissue samples was examined by immunohistochemistry and survival of tongue cancer patients from which these samples were derived was also analyzed.
  
  
  RESULTS
  ZEB1 bound to the promoter of CA9 to positively regulate CA9 expression in tongue cancer cells. Knockdown of CA9 using short interfering RNA (siRNA) abolished the chemoresistance resulting from ZEB1 overexpression in Tca8113 and SCC-25 cells, and CA9 overexpression attenuated chemosensitivity induced by ZEB1 knockdown in Tca8113/PYM cells. CA9 knockdown also prevented maintenance of pHi mediated by overexpression of ZEB1 in Tca8113 and SCC-25 cells following chemotherapy, associated with increased apoptosis and caspase-3 activation. Conversely, ectopic expression of CA9 suppressed decrease in pHi mediated by ZEB1 knockdown in Tca8113/PYM cells following chemotherapy, accompanied by decreased apoptosis and caspase-3 activation. Importantly, a positive correlation was observed between ZEB1 and CA9 protein expression in tongue cancer tissues, and expression of these proteins associated with a poor prognosis for patients.
  
  
  CONCLUSIONS
  Our finding that tumor cells regulate pHi in response to chemotherapy provides new insights into mechanisms of drug resistance during cancer treatment. Identification of the ZEB1-CA9 signaling axis as a biomarker of poor prognosis in tongue cancer will be valuable in future development of therapeutic strategies aimed at improving treatment efficacy, especially in terms of drug resistance associated with this disease.
authors.affiliation: Cancer Hospital and Cancer Research Institute of Guangzhou Medical University, Hengzhigang Road 78#, Guangzhou, 510095, Guangdong, China. zhengguopei@126.com.
authors.forename: Guopei
authors.initials: G
authors.lastname: Zheng
authors.affiliation: Cancer Hospital and Cancer Research Institute of Guangzhou Medical University, Hengzhigang Road 78#, Guangzhou, 510095, Guangdong, China. 834252642@qq.com.
authors.forename: Cong
authors.initials: C
authors.lastname: Peng
authors.affiliation: Cancer Hospital and Cancer Research Institute of Guangzhou Medical University, Hengzhigang Road 78#, Guangzhou, 510095, Guangdong, China. jxt1231994@163.com.
authors.forename: Xiaoting
authors.initials: X
authors.lastname: Jia
authors.affiliation: Cancer Hospital and Cancer Research Institute of Guangzhou Medical University, Hengzhigang Road 78#, Guangzhou, 510095, Guangdong, China. guyx189@163.com.
authors.forename: Yixue
authors.initials: Y
authors.lastname: Gu
authors.affiliation: Cancer Hospital and Cancer Research Institute of Guangzhou Medical University, Hengzhigang Road 78#, Guangzhou, 510095, Guangdong, China. zjiezhang@126.com.
authors.forename: Zhijie
authors.initials: Z
authors.lastname: Zhang
authors.affiliation: Cancer Hospital and Cancer Research Institute of Guangzhou Medical University, Hengzhigang Road 78#, Guangzhou, 510095, Guangdong, China. jxncdyg215@126.com.
authors.forename: Yingen
authors.initials: Y
authors.lastname: Deng
authors.affiliation: Cancer Hospital and Cancer Research Institute of Guangzhou Medical University, Hengzhigang Road 78#, Guangzhou, 510095, Guangdong, China. chareswck@163.com.
authors.forename: Chengkun
authors.initials: C
authors.lastname: Wang
authors.affiliation: Cancer Hospital and Cancer Research Institute of Guangzhou Medical University, Hengzhigang Road 78#, Guangzhou, 510095, Guangdong, China. Fionali0731@126.com.
authors.forename: Nan
authors.initials: N
authors.lastname: Li
authors.affiliation: Cancer Hospital and Cancer Research Institute of Guangzhou Medical University, Hengzhigang Road 78#, Guangzhou, 510095, Guangdong, China. yinjiang20@gmail.com.
authors.forename: Jiang
authors.initials: J
authors.lastname: Yin
authors.affiliation: Cancer Hospital and Cancer Research Institute of Guangzhou Medical University, Hengzhigang Road 78#, Guangzhou, 510095, Guangdong, China. smilenew@gmail.com.
authors.forename: Xiaorong
authors.initials: X
authors.lastname: Liu
authors.affiliation: Cancer Hospital and Cancer Research Institute of Guangzhou Medical University, Hengzhigang Road 78#, Guangzhou, 510095, Guangdong, China. 51315094@qq.com.
authors.forename: Minying
authors.initials: M
authors.lastname: Lu
authors.affiliation: Department of Breast Oncology, Sun Yat-Sen University Cancer Center, State Key Laboratory of Oncology in South China, Collaborative Innovation Center for Cancer Medicine, East Dongfeng Road 651#, Guangzhou, 510060, Guangdong, China. tanghl@sysucc.org.cn.
authors.forename: Hailin
authors.initials: H
authors.lastname: Tang
authors.affiliation: Cancer Hospital and Cancer Research Institute of Guangzhou Medical University, Hengzhigang Road 78#, Guangzhou, 510095, Guangdong, China. hezhimin2005@yahoo.com.
authors.forename: Zhimin
authors.initials: Z
authors.lastname: He
mesh_terms: D000951:Antigens, Neoplasm / Q000235:genetics*; D017209:Apoptosis / Q000235:genetics; D000071231:Carbonic Anhydrase IX; D002256:Carbonic Anhydrases / Q000235:genetics*; D053148:Caspase 3 / Q000235:genetics; D002460:Cell Line; D045744:Cell Line, Tumor; D019008:Drug Resistance, Neoplasm / Q000235:genetics*; D015972:Gene Expression Regulation, Neoplastic / Q000235:genetics; D057809:HEK293 Cells; D018398:Homeodomain Proteins / Q000235:genetics*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D034741:RNA, Small Interfering / Q000235:genetics; D014062:Tongue Neoplasms / Q000235:genetics*; D014157:Transcription Factors / Q000235:genetics*; D014158:Transcription, Genetic / Q000235:genetics*; D000071799:Zinc Finger E-box-Binding Homeobox 1
chemical_list: D000951:Antigens, Neoplasm; D018398:Homeodomain Proteins; D034741:RNA, Small Interfering; D014157:Transcription Factors; C486307:ZEB1 protein, human; D000071799:Zinc Finger E-box-Binding Homeobox 1; D053148:Caspase 3; C089226:CA9 protein, human; D000071231:Carbonic Anhydrase IX; D002256:Carbonic Anhydrases