miR-200 expression regulates epithelial-to-mesenchymal transition in bladder cancer cells and reverses resistance to epidermal growth factor receptor therapy

Liana Adam, Meng Zhong, Woonyoung Choi, Wei Qi, Milena Nicoloso, Ameeta Arora, George Calin, Hua Wang, Arlene Siefker-Radtke, David McConkey, Menashe Bar-Eli, Colin Dinney

Research output: Contribution to journalArticle

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Abstract

Purpose: The epithelial-to-mesenchymal transition (EMT) is a cell developmentregulated process in which noncoding RNAs act as crucial modulators. Recent studies have implied that EMT may contribute to resistance to epidermal growth factor receptor (EGFR)-directed therapy. The aims of this study were to determine the potential role of microRNAs (miRNA) in controlling EMT and the role of EMT in inducing the sensitivity of human bladder cancer cells to the inhibitory effects of the anti-EGFRtherapy. Experimental Design: miRNA array screening and real-time reverse transcription-PCR were used to identify and validate the differential expression of miRNAs involved in EMT in nine bladder cancer cell lines. A list of potential miR-200 direct targets was identified through the TargetScan database. The precursor of miR-200b and miR-200c was expressed in UMUC3 and T24 cells using a retrovirus or a lentivirus construct, respectively. Protein expression and signaling pathway modulation, as well as intracellular distribution of EGFRan d ERRFI-1, were validated through Western blot analysis and confocal microscopy, whereas ERRFI-1 direct target of miR-200 members was validated by using the wild-type and mutant 3′-untranslated region/ERRFI-1/luciferse reporters. Results: We identified a tight association between the expression of miRNAs of the miR-200 family, epithelial phenotype, and sensitivity to EGFRinhibitors -induced growth inhibition in bladder carcinoma cell lines. Stable expression of miR-200 in mesenchymal UMUC3 cells increased E-cadherin levels, decreased expression of ZEB1, ZEB2, ERRFI-1, and cell migration, and increased sensitivity to EGFR-blocking agents. The changes in EGFR sensitivity by silencing or forced expression of ERRFI-1 or by miR-200 expression have also been validated in additional cell lines, UMUC5 and T24. Finally, luciferase assays using 3′-untranslated region/ERRFI-1/luciferase and miR-200 cotransfections showed that the direct down-regulation of ERRFI-1 was miR-200-dependent because mutations in the two putative miR-200-binding sites have rescued the inhibitory effect. Conclusions: Members of the miR-200 family appear to control the EMT process and sensitivity to EGFRthe rapy in bladder cancer cells and the expression of miR-200 is sufficient to restore EGFR dependency at least in some of the mesenchymal bladder cancer cells. The targets of miR-200 include ERRFI-1, which is a novel regulator of EGFR-independent growth.

Original languageEnglish
Pages (from-to)5060-5072
Number of pages13
JournalClinical Cancer Research
Volume15
Issue number16
DOIs
Publication statusPublished - Aug 15 2009

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Epithelial-Mesenchymal Transition
Epidermal Growth Factor Receptor
Urinary Bladder Neoplasms
MicroRNAs
3' Untranslated Regions
Luciferases
Cell Line
Therapeutics
Lentivirus
Untranslated RNA
Retroviridae
Cadherins
Growth
Confocal Microscopy
Reverse Transcription
Cell Movement
Urinary Bladder
Research Design
Down-Regulation
Western Blotting

ASJC Scopus subject areas

  • Cancer Research
  • Oncology

Cite this

miR-200 expression regulates epithelial-to-mesenchymal transition in bladder cancer cells and reverses resistance to epidermal growth factor receptor therapy. / Adam, Liana; Zhong, Meng; Choi, Woonyoung; Qi, Wei; Nicoloso, Milena; Arora, Ameeta; Calin, George; Wang, Hua; Siefker-Radtke, Arlene; McConkey, David; Bar-Eli, Menashe; Dinney, Colin.

In: Clinical Cancer Research, Vol. 15, No. 16, 15.08.2009, p. 5060-5072.

Research output: Contribution to journalArticle

Adam, L, Zhong, M, Choi, W, Qi, W, Nicoloso, M, Arora, A, Calin, G, Wang, H, Siefker-Radtke, A, McConkey, D, Bar-Eli, M & Dinney, C 2009, 'miR-200 expression regulates epithelial-to-mesenchymal transition in bladder cancer cells and reverses resistance to epidermal growth factor receptor therapy', Clinical Cancer Research, vol. 15, no. 16, pp. 5060-5072. https://doi.org/10.1158/1078-0432.CCR-08-2245
Adam, Liana ; Zhong, Meng ; Choi, Woonyoung ; Qi, Wei ; Nicoloso, Milena ; Arora, Ameeta ; Calin, George ; Wang, Hua ; Siefker-Radtke, Arlene ; McConkey, David ; Bar-Eli, Menashe ; Dinney, Colin. / miR-200 expression regulates epithelial-to-mesenchymal transition in bladder cancer cells and reverses resistance to epidermal growth factor receptor therapy. In: Clinical Cancer Research. 2009 ; Vol. 15, No. 16. pp. 5060-5072.
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abstract = "Purpose: The epithelial-to-mesenchymal transition (EMT) is a cell developmentregulated process in which noncoding RNAs act as crucial modulators. Recent studies have implied that EMT may contribute to resistance to epidermal growth factor receptor (EGFR)-directed therapy. The aims of this study were to determine the potential role of microRNAs (miRNA) in controlling EMT and the role of EMT in inducing the sensitivity of human bladder cancer cells to the inhibitory effects of the anti-EGFRtherapy. Experimental Design: miRNA array screening and real-time reverse transcription-PCR were used to identify and validate the differential expression of miRNAs involved in EMT in nine bladder cancer cell lines. A list of potential miR-200 direct targets was identified through the TargetScan database. The precursor of miR-200b and miR-200c was expressed in UMUC3 and T24 cells using a retrovirus or a lentivirus construct, respectively. Protein expression and signaling pathway modulation, as well as intracellular distribution of EGFRan d ERRFI-1, were validated through Western blot analysis and confocal microscopy, whereas ERRFI-1 direct target of miR-200 members was validated by using the wild-type and mutant 3′-untranslated region/ERRFI-1/luciferse reporters. Results: We identified a tight association between the expression of miRNAs of the miR-200 family, epithelial phenotype, and sensitivity to EGFRinhibitors -induced growth inhibition in bladder carcinoma cell lines. Stable expression of miR-200 in mesenchymal UMUC3 cells increased E-cadherin levels, decreased expression of ZEB1, ZEB2, ERRFI-1, and cell migration, and increased sensitivity to EGFR-blocking agents. The changes in EGFR sensitivity by silencing or forced expression of ERRFI-1 or by miR-200 expression have also been validated in additional cell lines, UMUC5 and T24. Finally, luciferase assays using 3′-untranslated region/ERRFI-1/luciferase and miR-200 cotransfections showed that the direct down-regulation of ERRFI-1 was miR-200-dependent because mutations in the two putative miR-200-binding sites have rescued the inhibitory effect. Conclusions: Members of the miR-200 family appear to control the EMT process and sensitivity to EGFRthe rapy in bladder cancer cells and the expression of miR-200 is sufficient to restore EGFR dependency at least in some of the mesenchymal bladder cancer cells. The targets of miR-200 include ERRFI-1, which is a novel regulator of EGFR-independent growth.",
author = "Liana Adam and Meng Zhong and Woonyoung Choi and Wei Qi and Milena Nicoloso and Ameeta Arora and George Calin and Hua Wang and Arlene Siefker-Radtke and David McConkey and Menashe Bar-Eli and Colin Dinney",
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T1 - miR-200 expression regulates epithelial-to-mesenchymal transition in bladder cancer cells and reverses resistance to epidermal growth factor receptor therapy

AU - Adam, Liana

AU - Zhong, Meng

AU - Choi, Woonyoung

AU - Qi, Wei

AU - Nicoloso, Milena

AU - Arora, Ameeta

AU - Calin, George

AU - Wang, Hua

AU - Siefker-Radtke, Arlene

AU - McConkey, David

AU - Bar-Eli, Menashe

AU - Dinney, Colin

PY - 2009/8/15

Y1 - 2009/8/15

N2 - Purpose: The epithelial-to-mesenchymal transition (EMT) is a cell developmentregulated process in which noncoding RNAs act as crucial modulators. Recent studies have implied that EMT may contribute to resistance to epidermal growth factor receptor (EGFR)-directed therapy. The aims of this study were to determine the potential role of microRNAs (miRNA) in controlling EMT and the role of EMT in inducing the sensitivity of human bladder cancer cells to the inhibitory effects of the anti-EGFRtherapy. Experimental Design: miRNA array screening and real-time reverse transcription-PCR were used to identify and validate the differential expression of miRNAs involved in EMT in nine bladder cancer cell lines. A list of potential miR-200 direct targets was identified through the TargetScan database. The precursor of miR-200b and miR-200c was expressed in UMUC3 and T24 cells using a retrovirus or a lentivirus construct, respectively. Protein expression and signaling pathway modulation, as well as intracellular distribution of EGFRan d ERRFI-1, were validated through Western blot analysis and confocal microscopy, whereas ERRFI-1 direct target of miR-200 members was validated by using the wild-type and mutant 3′-untranslated region/ERRFI-1/luciferse reporters. Results: We identified a tight association between the expression of miRNAs of the miR-200 family, epithelial phenotype, and sensitivity to EGFRinhibitors -induced growth inhibition in bladder carcinoma cell lines. Stable expression of miR-200 in mesenchymal UMUC3 cells increased E-cadherin levels, decreased expression of ZEB1, ZEB2, ERRFI-1, and cell migration, and increased sensitivity to EGFR-blocking agents. The changes in EGFR sensitivity by silencing or forced expression of ERRFI-1 or by miR-200 expression have also been validated in additional cell lines, UMUC5 and T24. Finally, luciferase assays using 3′-untranslated region/ERRFI-1/luciferase and miR-200 cotransfections showed that the direct down-regulation of ERRFI-1 was miR-200-dependent because mutations in the two putative miR-200-binding sites have rescued the inhibitory effect. Conclusions: Members of the miR-200 family appear to control the EMT process and sensitivity to EGFRthe rapy in bladder cancer cells and the expression of miR-200 is sufficient to restore EGFR dependency at least in some of the mesenchymal bladder cancer cells. The targets of miR-200 include ERRFI-1, which is a novel regulator of EGFR-independent growth.

AB - Purpose: The epithelial-to-mesenchymal transition (EMT) is a cell developmentregulated process in which noncoding RNAs act as crucial modulators. Recent studies have implied that EMT may contribute to resistance to epidermal growth factor receptor (EGFR)-directed therapy. The aims of this study were to determine the potential role of microRNAs (miRNA) in controlling EMT and the role of EMT in inducing the sensitivity of human bladder cancer cells to the inhibitory effects of the anti-EGFRtherapy. Experimental Design: miRNA array screening and real-time reverse transcription-PCR were used to identify and validate the differential expression of miRNAs involved in EMT in nine bladder cancer cell lines. A list of potential miR-200 direct targets was identified through the TargetScan database. The precursor of miR-200b and miR-200c was expressed in UMUC3 and T24 cells using a retrovirus or a lentivirus construct, respectively. Protein expression and signaling pathway modulation, as well as intracellular distribution of EGFRan d ERRFI-1, were validated through Western blot analysis and confocal microscopy, whereas ERRFI-1 direct target of miR-200 members was validated by using the wild-type and mutant 3′-untranslated region/ERRFI-1/luciferse reporters. Results: We identified a tight association between the expression of miRNAs of the miR-200 family, epithelial phenotype, and sensitivity to EGFRinhibitors -induced growth inhibition in bladder carcinoma cell lines. Stable expression of miR-200 in mesenchymal UMUC3 cells increased E-cadherin levels, decreased expression of ZEB1, ZEB2, ERRFI-1, and cell migration, and increased sensitivity to EGFR-blocking agents. The changes in EGFR sensitivity by silencing or forced expression of ERRFI-1 or by miR-200 expression have also been validated in additional cell lines, UMUC5 and T24. Finally, luciferase assays using 3′-untranslated region/ERRFI-1/luciferase and miR-200 cotransfections showed that the direct down-regulation of ERRFI-1 was miR-200-dependent because mutations in the two putative miR-200-binding sites have rescued the inhibitory effect. Conclusions: Members of the miR-200 family appear to control the EMT process and sensitivity to EGFRthe rapy in bladder cancer cells and the expression of miR-200 is sufficient to restore EGFR dependency at least in some of the mesenchymal bladder cancer cells. The targets of miR-200 include ERRFI-1, which is a novel regulator of EGFR-independent growth.

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