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ZEB1 transcriptionally regulated carbonic anhydrase 9 mediates the chemoresistance of tongue cancer via maintaining intracellular pH.

Zheng G, Peng C, Jia X, Gu Y, Zhang Z, Deng Y, Wang C, Li N, Yin J, Liu X, Lu M, Tang H, He Z - Mol. Cancer (2015)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Cancer Hospital and Cancer Research Institute of Guangzhou Medical University, Hengzhigang Road 78#, Guangzhou, 510095, Guangdong, China. zhengguopei@126.com.

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.

Conclusion: 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.

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CA9 is upregulated by ZEB1 in tongue cancer cells. (A) A schematic representation of ZEB1 binding sites with the E-box sequence (CACCTG) in the 3kb putative CA9 promoter. The first base of the 3kb strand is defined as ‘1’. (B) Chromatin immunoprecipitation assays identified ZEB1 binding sites within the putative CA9 promoter. Primers specific for sites B, C and E yielded PCR reaction products from ZEB1–DNA immunoprecipitates. The input represents DNA directly after lysis. The PCR reaction product for immunoprecipitates obtained using the RNA Polymerase antibody represents the positive control. (C and D) Changes in CA9 mRNA and protein expression following the inhibition of ZEB1 in tongue cancer cell lines were evaluated by qRT-PCR and western blotting, respectively. (E) Luciferase activity driven by the putative CA9 promoter was higher in Tca8113/PYM cells (which exhibit endogenous ZEB1 overexpression) than in Tca8113 and SCC-25 cells. (F) Reporter assays revealed changes in luciferase activity after inhibition of ZEB1 expression in tongue cancer cells. (G) ZEB1 promoted luciferase activity driven by the putative CA9 promoter in HEK283T cells. * p < 0.01.
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Fig1: CA9 is upregulated by ZEB1 in tongue cancer cells. (A) A schematic representation of ZEB1 binding sites with the E-box sequence (CACCTG) in the 3kb putative CA9 promoter. The first base of the 3kb strand is defined as ‘1’. (B) Chromatin immunoprecipitation assays identified ZEB1 binding sites within the putative CA9 promoter. Primers specific for sites B, C and E yielded PCR reaction products from ZEB1–DNA immunoprecipitates. The input represents DNA directly after lysis. The PCR reaction product for immunoprecipitates obtained using the RNA Polymerase antibody represents the positive control. (C and D) Changes in CA9 mRNA and protein expression following the inhibition of ZEB1 in tongue cancer cell lines were evaluated by qRT-PCR and western blotting, respectively. (E) Luciferase activity driven by the putative CA9 promoter was higher in Tca8113/PYM cells (which exhibit endogenous ZEB1 overexpression) than in Tca8113 and SCC-25 cells. (F) Reporter assays revealed changes in luciferase activity after inhibition of ZEB1 expression in tongue cancer cells. (G) ZEB1 promoted luciferase activity driven by the putative CA9 promoter in HEK283T cells. * p < 0.01.

Mentions: We have previously shown that both CA9 mRNA and protein expression is upregulated in the PYM-induced multidrug-resistant tongue cancer cell line Tca8113/PYM. To determine whether transcriptional regulation contributes to CA9 upregulation in Tca8113/PYM cells, we analyzed the response elements of a cohort of transcription factors located within a three kilobase region upstream of the first exon of the CA9 gene. Using the JASPAR database (http://jaspar.binf.ku.dk) we identified five putative ZEB1 binding sites within this region, conforming to the optimal recognition sequence of ZEB1 (CACCTG) (Figure 1A). To confirm the direct association of ZEB1 with the CA9 promoter, we performed a ChIP assay in Tca8113/PYM cells for all putative ZEB1 binding sites within the three kilobase region. ChIP results revealed that ZEB1 bound most significantly to sites B, C and E within the potential CA9 promoter (Figure 1B). As expected, ectopic expression of ZEB1 using the pLEX-ZEB1 construct enhanced both CA9 mRNA and protein expression in Tca8113 and SCC-25 cell lines (Figure 1C and D). Conversely, CA9 mRNA and protein expression decreased following knockdown of ZEB1 in Tca8113/PYM cells using ZEB1-specific siRNAs (Figure 1C and D). To investigate further the effects of ZEB1 on CA9 expression, the putative three kilobase CA9 promoter was cloned into a luciferase reporter vector and expression assays subsequently performed. As expected, CA9 promoter-driven luciferase activity was much higher in Tca8113/PYM cells than in Tca8113 and SCC-25 cells (Figure 1E). In addition, ZEB1 was found to significantly enhanced luciferase activity driven by the CA9 promoter in HEK293T cells (Figure 1G). These results demonstrate that ZEB1 can directly bind to the CA9 promoter to transcriptionally regulate CA9 expression.Figure 1


ZEB1 transcriptionally regulated carbonic anhydrase 9 mediates the chemoresistance of tongue cancer via maintaining intracellular pH.

Zheng G, Peng C, Jia X, Gu Y, Zhang Z, Deng Y, Wang C, Li N, Yin J, Liu X, Lu M, Tang H, He Z - Mol. Cancer (2015)

CA9 is upregulated by ZEB1 in tongue cancer cells. (A) A schematic representation of ZEB1 binding sites with the E-box sequence (CACCTG) in the 3kb putative CA9 promoter. The first base of the 3kb strand is defined as ‘1’. (B) Chromatin immunoprecipitation assays identified ZEB1 binding sites within the putative CA9 promoter. Primers specific for sites B, C and E yielded PCR reaction products from ZEB1–DNA immunoprecipitates. The input represents DNA directly after lysis. The PCR reaction product for immunoprecipitates obtained using the RNA Polymerase antibody represents the positive control. (C and D) Changes in CA9 mRNA and protein expression following the inhibition of ZEB1 in tongue cancer cell lines were evaluated by qRT-PCR and western blotting, respectively. (E) Luciferase activity driven by the putative CA9 promoter was higher in Tca8113/PYM cells (which exhibit endogenous ZEB1 overexpression) than in Tca8113 and SCC-25 cells. (F) Reporter assays revealed changes in luciferase activity after inhibition of ZEB1 expression in tongue cancer cells. (G) ZEB1 promoted luciferase activity driven by the putative CA9 promoter in HEK283T cells. * p < 0.01.
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Fig1: CA9 is upregulated by ZEB1 in tongue cancer cells. (A) A schematic representation of ZEB1 binding sites with the E-box sequence (CACCTG) in the 3kb putative CA9 promoter. The first base of the 3kb strand is defined as ‘1’. (B) Chromatin immunoprecipitation assays identified ZEB1 binding sites within the putative CA9 promoter. Primers specific for sites B, C and E yielded PCR reaction products from ZEB1–DNA immunoprecipitates. The input represents DNA directly after lysis. The PCR reaction product for immunoprecipitates obtained using the RNA Polymerase antibody represents the positive control. (C and D) Changes in CA9 mRNA and protein expression following the inhibition of ZEB1 in tongue cancer cell lines were evaluated by qRT-PCR and western blotting, respectively. (E) Luciferase activity driven by the putative CA9 promoter was higher in Tca8113/PYM cells (which exhibit endogenous ZEB1 overexpression) than in Tca8113 and SCC-25 cells. (F) Reporter assays revealed changes in luciferase activity after inhibition of ZEB1 expression in tongue cancer cells. (G) ZEB1 promoted luciferase activity driven by the putative CA9 promoter in HEK283T cells. * p < 0.01.
Mentions: We have previously shown that both CA9 mRNA and protein expression is upregulated in the PYM-induced multidrug-resistant tongue cancer cell line Tca8113/PYM. To determine whether transcriptional regulation contributes to CA9 upregulation in Tca8113/PYM cells, we analyzed the response elements of a cohort of transcription factors located within a three kilobase region upstream of the first exon of the CA9 gene. Using the JASPAR database (http://jaspar.binf.ku.dk) we identified five putative ZEB1 binding sites within this region, conforming to the optimal recognition sequence of ZEB1 (CACCTG) (Figure 1A). To confirm the direct association of ZEB1 with the CA9 promoter, we performed a ChIP assay in Tca8113/PYM cells for all putative ZEB1 binding sites within the three kilobase region. ChIP results revealed that ZEB1 bound most significantly to sites B, C and E within the potential CA9 promoter (Figure 1B). As expected, ectopic expression of ZEB1 using the pLEX-ZEB1 construct enhanced both CA9 mRNA and protein expression in Tca8113 and SCC-25 cell lines (Figure 1C and D). Conversely, CA9 mRNA and protein expression decreased following knockdown of ZEB1 in Tca8113/PYM cells using ZEB1-specific siRNAs (Figure 1C and D). To investigate further the effects of ZEB1 on CA9 expression, the putative three kilobase CA9 promoter was cloned into a luciferase reporter vector and expression assays subsequently performed. As expected, CA9 promoter-driven luciferase activity was much higher in Tca8113/PYM cells than in Tca8113 and SCC-25 cells (Figure 1E). In addition, ZEB1 was found to significantly enhanced luciferase activity driven by the CA9 promoter in HEK293T cells (Figure 1G). These results demonstrate that ZEB1 can directly bind to the CA9 promoter to transcriptionally regulate CA9 expression.Figure 1

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Cancer Hospital and Cancer Research Institute of Guangzhou Medical University, Hengzhigang Road 78#, Guangzhou, 510095, Guangdong, China. zhengguopei@126.com.

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.

Conclusion: 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.

Show MeSH
Related in: MedlinePlus