Abstract
BackgroundChemoresistance 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.MethodsBioinformatic 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.ResultsZEB1 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.ConclusionOur 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.
Highlights
Chemoresistance is a major obstacle in successfully treating cancers, and the mechanisms responsible for drug resistance are still far from understood
Zinc finger E-box-binding homeobox 1 (ZEB1) transcriptionally regulates Carbonic anhydrase 9 (CA9) expression in tongue cancer cells 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
These results demonstrate that ZEB1 can directly bind to the CA9 promoter to transcriptionally regulate CA9 expression
Summary
Chemoresistance is a major obstacle in successfully treating cancers, and the mechanisms responsible for drug resistance are still far from understood. As one of the standard therapeutic approaches, chemotherapy mostly based on pingyangmycin (PYM) and/or cisplatin (cDDP), plays an important role in tongue cancer treatment and brings many benefits including reducing tumor size, inhibiting distant metastasis and prolonging patient survival [3]. Resistance to anticancer agents is a major obstacle for successful chemotherapy in tongue cancer, as it can result in more aggressive tumor behavior and worse clinical outcome [4,5]. The mechanisms responsible for drug resistance in cancer have been the subject of intense studies for decades, the clinical causes of drug resistance remain poorly understood. There is, an urgent need to clarify these mechanisms and to explore alternative therapeutic strategies to overcome drug resistance in tongue cancer treatment
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