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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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The ZEB1–CA9 axis regulates chemosensitivity in tongue cancer cells. (A and B) MTS cell proliferation assays showed that ZEB1 overexpression promoted resistance in Tca8113 and SSC-25 cells in response to PYM and cDDP, and that knockdown of CA9 abolished this effect. (C) Knockdown of ZEB1 enhanced the sensitivity of Tca8113/PYM cells to PYM and cDDP, while overexpression of CA9 attenuated this effect. * p < 0.01.
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Fig2: The ZEB1–CA9 axis regulates chemosensitivity in tongue cancer cells. (A and B) MTS cell proliferation assays showed that ZEB1 overexpression promoted resistance in Tca8113 and SSC-25 cells in response to PYM and cDDP, and that knockdown of CA9 abolished this effect. (C) Knockdown of ZEB1 enhanced the sensitivity of Tca8113/PYM cells to PYM and cDDP, while overexpression of CA9 attenuated this effect. * p < 0.01.

Mentions: We have previously shown that CA9 is involved in drug resistance in tongue cancer cells, and have shown here that ZEB1 positively regulates CA9 expression. Next, we wished to determine whether ZEB1 modulates chemosensitivity in tongue cancer and whether it exerts its effect via regulating CA9 expression. We found that ectopic ZEB1 expression enhanced the resistance of Tca8113 and SCC-25 cells to PYM or cDDP, with the marked increase of IC50 values (Figure 2A and B). Conversely, ZEB1 knockdown increased the sensitivity of Tca8113/PYM cells to PYM and cDDP, with the significant decrease of IC50 values (Figure 2C). Moreover, CA9 knockdown markedly impaired ZEB1-mediated drug resistance to PYM or cDDP in Tca8113 and SCC-25 cells (Figure 2A and B), while overexpression of CA9 in Tca8113/PYM cells impaired the effect of ZEB1 knockdown in response to chemotherapy (Figure 2C), suggesting CA9 is responsible for ZEB1-mediated drug resistance in tongue cancer cells.Figure 2


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)

The ZEB1–CA9 axis regulates chemosensitivity in tongue cancer cells. (A and B) MTS cell proliferation assays showed that ZEB1 overexpression promoted resistance in Tca8113 and SSC-25 cells in response to PYM and cDDP, and that knockdown of CA9 abolished this effect. (C) Knockdown of ZEB1 enhanced the sensitivity of Tca8113/PYM cells to PYM and cDDP, while overexpression of CA9 attenuated this effect. * p < 0.01.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4404088&req=5

Fig2: The ZEB1–CA9 axis regulates chemosensitivity in tongue cancer cells. (A and B) MTS cell proliferation assays showed that ZEB1 overexpression promoted resistance in Tca8113 and SSC-25 cells in response to PYM and cDDP, and that knockdown of CA9 abolished this effect. (C) Knockdown of ZEB1 enhanced the sensitivity of Tca8113/PYM cells to PYM and cDDP, while overexpression of CA9 attenuated this effect. * p < 0.01.
Mentions: We have previously shown that CA9 is involved in drug resistance in tongue cancer cells, and have shown here that ZEB1 positively regulates CA9 expression. Next, we wished to determine whether ZEB1 modulates chemosensitivity in tongue cancer and whether it exerts its effect via regulating CA9 expression. We found that ectopic ZEB1 expression enhanced the resistance of Tca8113 and SCC-25 cells to PYM or cDDP, with the marked increase of IC50 values (Figure 2A and B). Conversely, ZEB1 knockdown increased the sensitivity of Tca8113/PYM cells to PYM and cDDP, with the significant decrease of IC50 values (Figure 2C). Moreover, CA9 knockdown markedly impaired ZEB1-mediated drug resistance to PYM or cDDP in Tca8113 and SCC-25 cells (Figure 2A and B), while overexpression of CA9 in Tca8113/PYM cells impaired the effect of ZEB1 knockdown in response to chemotherapy (Figure 2C), suggesting CA9 is responsible for ZEB1-mediated drug resistance in tongue cancer cells.Figure 2

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