Suppression of dual-specificity phosphatase–2 by hypoxia increases chemoresistance and malignancy in human cancer cells
Shih-Chieh Lin, Chun-Wei Chien, Jenq-Chang Lee, Yi-Chun Yeh, Keng-Fu Hsu, Yen-Yu Lai, Shao-Chieh Lin, Shaw-Jenq Tsai
Shih-Chieh Lin, Chun-Wei Chien, Jenq-Chang Lee, Yi-Chun Yeh, Keng-Fu Hsu, Yen-Yu Lai, Shao-Chieh Lin, Shaw-Jenq Tsai
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Research Article Oncology

Suppression of dual-specificity phosphatase–2 by hypoxia increases chemoresistance and malignancy in human cancer cells

Abstract

Hypoxia inducible factor–1 (HIF-1) is the master transcriptional regulator of the cellular response to altered oxygen levels. HIF-1α protein is elevated in most solid tumors and contributes to poor disease outcome by promoting tumor progression, metastasis, and resistance to chemotherapy. To date, the relationship between HIF-1 and these processes, particularly chemoresistance, has remained largely unexplored. Here, we show that expression of the MAPK-specific phosphatase dual-specificity phosphatase–2 (DUSP2) is markedly reduced or completely absent in many human cancers and that its level of expression inversely correlates with that of HIF-1α and with cancer malignancy. Analysis of human cancer cell lines indicated that HIF-1α inhibited DUSP2 transcription, which resulted in prolonged phosphorylation of ERK and, hence, increased chemoresistance. Knockdown of DUSP2 increased drug resistance under normoxia, while forced expression of DUSP2 abolished hypoxia-induced chemoresistance. Further, reexpression of DUSP2 during cancer progression caused tumor regression and markedly increased drug sensitivity in mice xenografted with human tumor cell lines. Furthermore, a variety of genes involved in drug response, angiogenesis, cell survival, and apoptosis were found to be downregulated by DUSP2. Our results demonstrate that DUSP2 is a key downstream regulator of HIF-1–mediated tumor progression and chemoresistance. DUSP2 therefore may represent a novel drug target of particular relevance in tumors resistant to conventional chemotherapy.

Authors

Shih-Chieh Lin, Chun-Wei Chien, Jenq-Chang Lee, Yi-Chun Yeh, Keng-Fu Hsu, Yen-Yu Lai, Shao-Chieh Lin, Shaw-Jenq Tsai

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Figure 2

DUSP2 is inhibited by HIF-1α.

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DUSP2 is inhibited by HIF-1α. (A) Cancer cell lines were exposed to norm...
(A) Cancer cell lines were exposed to normoxia and hypoxia for 24 hours, and levels of DUSP2 mRNA were quantified by RT-qPCR. (B) Representative Western blots of cells cultured under normoxia or hypoxia (H) for 24 hours showing levels of DUSP2, HIF-1α, and HIF-1β. (C) Levels of DUSP2 mRNA in cancer cells treated with DFO (10 mM), DMOG (1 mM), or vehicle (Control) for 24 hours. (D) Representative Western blots of HeLa cells with HIF-1α or HIF-2α knockdown showing levels of HIF proteins (indicated by arrows). (E and F) Levels of DUSP2 mRNA and protein in HIF-1α or HIF-2α knockdown cells are shown. (G) Representative Western blots of HeLa cells transiently transfected with wild-type HIF-1α, P402A/P564A double-mutated HIF-1α (Dm), or empty vector (pcDNA6.0) under normoxia showing the levels of DUSP2, HIF-1α, and HIF-1β. (H) Schematic drawing of human DUSP2 promoter (upper panel). Core HRE is indicated in bold. Mutated bases are shown above. Promoter activity of HeLa cells transiently transfected with reporter constructs as indicated and cultured under normoxia, normoxia treated with DFO, or hypoxia for 24 hours (lower panel). Mu-core, core HRE mutated construct; Mu-flank, sequence flanking core HRE mutated construct. (I) Promoter activity of HeLa and Hep3B cells transiently transfected with reporter constructs as indicated and cultured under normoxia for 24 hours with or without overexpression of HIF-1α–Dm. (J) ChIP-PCR results of HeLa cells exposed to normoxia or hypoxia for 4 hours. HRE, primers amplified flanking HRE region; Distal, primers amplified a fragment of 2,000 bp upstream of HRE. *P < 0.05.