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EMX2 Is a Predictive Marker for Adjuvant Chemotherapy in Lung Squamous Cell Carcinomas.

Yue D, Li H, Che J, Zhang Y, Tolani B, Mo M, Zhang H, Zheng Q, Yang Y, Cheng R, Jin JQ, Luh TW, Yang C, Tseng HH, Giroux-Leprieur E, Woodard GA, Hao X, Wang C, Jablons DM, He B - PLoS ONE (2015)

Bottom Line: Current staging methods do not adequately predict outcome for this disease.Positive EMX2 expression was significantly associated with improved overall survival in stage I lung SCC patients, and in stage II/IIIA lung SCC patients receiving adjuvant chemotherapy.EMX2 expression is down-regulated in lung SCC and its down-regulation is associated with chemo-resistance in lung SCC cells, possibly through regulation of Epithelial-to-Mesenchymal Transition (EMT).

View Article: PubMed Central - PubMed

Affiliation: Department of Lung Cancer, Lung Cancer Center, Tianjin Medical University Cancer Institute and Hospital, National Clinical Research Center of Cancer, Key Laboratory of Cancer Prevention and Therapy, Tianjin 300060, China; Thoracic Oncology Program, Department of Surgery, Helen Diller Family Comprehensive Cancer Center, University of California San Francisco, San Francisco, CA 94115, United States of America.

ABSTRACT

Background: Squamous cell carcinomas (SCC) account for approximately 30% of non-small cell lung cancer (NSCLC). Current staging methods do not adequately predict outcome for this disease. EMX2 is a homeo-domain containing transcription factor known to regulate a key developmental pathway. This study assessed the significance of EMX2 as a prognostic and predictive marker for resectable lung SCC.

Methods: Two independent cohorts of patients with lung SCC undergoing surgical resection were studied. EMX2 protein expression was examined by immunohistochemistry, Western blot, or immunofluorescence. EMX2 expression levels in tissue specimens were scored and correlated with patient outcomes. Chemo-sensitivity of lung SCC cell lines stably transfected with EMX2 shRNAs to cisplatin, carboplatin, and docetaxel was examined in vitro.

Results: EMX2 expression was down-regulated in lung SCC tissue samples compared to their matched adjacent normal tissues. Positive EMX2 expression was significantly associated with improved overall survival in stage I lung SCC patients, and in stage II/IIIA lung SCC patients receiving adjuvant chemotherapy. EMX2 expression was also associated with expression of EMT markers in both lung SCC cell lines and tissue samples. Knock-down of EMX2 expression in lung SCC cells promoted chemo-resistance and cell migration.

Conclusions: EMX2 expression is down-regulated in lung SCC and its down-regulation is associated with chemo-resistance in lung SCC cells, possibly through regulation of Epithelial-to-Mesenchymal Transition (EMT). EMX2 may serve as a novel prognostic marker for stage I lung SCC patients and a prediction marker for stage II/IIIA lung SCC patients receiving adjuvant chemotherapy.

No MeSH data available.


Related in: MedlinePlus

EMX2 may regulate EMT in lung SCC cell lines.(a) IF staining of EMX2 (red), E-Cadherin (green) and β-Catenin (green) in H2170 cells stably transfected with control shRNA or EMX2 shRNAs. (b) IF staining of EMX2 (red) and β-Catenin (green) in H1703 cells transiently transfected with EMX2 cDNA. Two representative fields were shown where the transfected cells had dramatic co-up-regulation of EMX2 and membrane β-Catenin, compared to the non-transfected cells in the fields. DAPI (blue) was used to stain nuclei of those cells for both panels (a) and (b). (c) Wound healing assays of lung SCC H2170 cells stably transfected with EMX2 shRNAs. Representative images shown at 0hr and 24hr were taken under a light microscope (100X). (d) Quantification of the wound healing assays. The migration distance of cells stably transfected with control shRNA was set as 100%. A p value of 0.001 or less was indicated as ***. (e) Western blot of EMT markers N-Cadherin and Vimentin and EMT regulators SNAIL and SLUG in lung SCC cell lines H1703 and H2170 stably transfected with different EMX2 shRNA constructs. Actin served as a loading control.
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pone.0132134.g004: EMX2 may regulate EMT in lung SCC cell lines.(a) IF staining of EMX2 (red), E-Cadherin (green) and β-Catenin (green) in H2170 cells stably transfected with control shRNA or EMX2 shRNAs. (b) IF staining of EMX2 (red) and β-Catenin (green) in H1703 cells transiently transfected with EMX2 cDNA. Two representative fields were shown where the transfected cells had dramatic co-up-regulation of EMX2 and membrane β-Catenin, compared to the non-transfected cells in the fields. DAPI (blue) was used to stain nuclei of those cells for both panels (a) and (b). (c) Wound healing assays of lung SCC H2170 cells stably transfected with EMX2 shRNAs. Representative images shown at 0hr and 24hr were taken under a light microscope (100X). (d) Quantification of the wound healing assays. The migration distance of cells stably transfected with control shRNA was set as 100%. A p value of 0.001 or less was indicated as ***. (e) Western blot of EMT markers N-Cadherin and Vimentin and EMT regulators SNAIL and SLUG in lung SCC cell lines H1703 and H2170 stably transfected with different EMX2 shRNA constructs. Actin served as a loading control.

Mentions: To investigate whether EMX2 expression could predict benefit from adjuvant chemotherapy, we analyzed IHC staining of EMX2 in these stage II/IIIA lung SCC specimens. We then established stable lung SCC cell lines after transfection with two different EMX2 shRNAs and subsequent G418 selection, and confirmed down-regulation of EMX2 expression in these stable transfectants (Fig 3a and Fig 4e). Next we treated these stable lines with three chemo drugs that are commonly used for the treatment of lung cancer, cisplatin, carboplatin, and docetaxel, respectively. We found that down-regulation of EMX2 expression by shRNA made H1703 and H2170 cells significantly more resistant to all three chemo treatments with elevated IC50 values compared to the control cells stably transfected with a non-silencing shRNA construct (Fig 3b, S1 Table). These results support our observation that EMX2 is associated with improved survival in lung SCC patients receiving adjuvant chemotherapy (Fig 2).


EMX2 Is a Predictive Marker for Adjuvant Chemotherapy in Lung Squamous Cell Carcinomas.

Yue D, Li H, Che J, Zhang Y, Tolani B, Mo M, Zhang H, Zheng Q, Yang Y, Cheng R, Jin JQ, Luh TW, Yang C, Tseng HH, Giroux-Leprieur E, Woodard GA, Hao X, Wang C, Jablons DM, He B - PLoS ONE (2015)

EMX2 may regulate EMT in lung SCC cell lines.(a) IF staining of EMX2 (red), E-Cadherin (green) and β-Catenin (green) in H2170 cells stably transfected with control shRNA or EMX2 shRNAs. (b) IF staining of EMX2 (red) and β-Catenin (green) in H1703 cells transiently transfected with EMX2 cDNA. Two representative fields were shown where the transfected cells had dramatic co-up-regulation of EMX2 and membrane β-Catenin, compared to the non-transfected cells in the fields. DAPI (blue) was used to stain nuclei of those cells for both panels (a) and (b). (c) Wound healing assays of lung SCC H2170 cells stably transfected with EMX2 shRNAs. Representative images shown at 0hr and 24hr were taken under a light microscope (100X). (d) Quantification of the wound healing assays. The migration distance of cells stably transfected with control shRNA was set as 100%. A p value of 0.001 or less was indicated as ***. (e) Western blot of EMT markers N-Cadherin and Vimentin and EMT regulators SNAIL and SLUG in lung SCC cell lines H1703 and H2170 stably transfected with different EMX2 shRNA constructs. Actin served as a loading control.
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pone.0132134.g004: EMX2 may regulate EMT in lung SCC cell lines.(a) IF staining of EMX2 (red), E-Cadherin (green) and β-Catenin (green) in H2170 cells stably transfected with control shRNA or EMX2 shRNAs. (b) IF staining of EMX2 (red) and β-Catenin (green) in H1703 cells transiently transfected with EMX2 cDNA. Two representative fields were shown where the transfected cells had dramatic co-up-regulation of EMX2 and membrane β-Catenin, compared to the non-transfected cells in the fields. DAPI (blue) was used to stain nuclei of those cells for both panels (a) and (b). (c) Wound healing assays of lung SCC H2170 cells stably transfected with EMX2 shRNAs. Representative images shown at 0hr and 24hr were taken under a light microscope (100X). (d) Quantification of the wound healing assays. The migration distance of cells stably transfected with control shRNA was set as 100%. A p value of 0.001 or less was indicated as ***. (e) Western blot of EMT markers N-Cadherin and Vimentin and EMT regulators SNAIL and SLUG in lung SCC cell lines H1703 and H2170 stably transfected with different EMX2 shRNA constructs. Actin served as a loading control.
Mentions: To investigate whether EMX2 expression could predict benefit from adjuvant chemotherapy, we analyzed IHC staining of EMX2 in these stage II/IIIA lung SCC specimens. We then established stable lung SCC cell lines after transfection with two different EMX2 shRNAs and subsequent G418 selection, and confirmed down-regulation of EMX2 expression in these stable transfectants (Fig 3a and Fig 4e). Next we treated these stable lines with three chemo drugs that are commonly used for the treatment of lung cancer, cisplatin, carboplatin, and docetaxel, respectively. We found that down-regulation of EMX2 expression by shRNA made H1703 and H2170 cells significantly more resistant to all three chemo treatments with elevated IC50 values compared to the control cells stably transfected with a non-silencing shRNA construct (Fig 3b, S1 Table). These results support our observation that EMX2 is associated with improved survival in lung SCC patients receiving adjuvant chemotherapy (Fig 2).

Bottom Line: Current staging methods do not adequately predict outcome for this disease.Positive EMX2 expression was significantly associated with improved overall survival in stage I lung SCC patients, and in stage II/IIIA lung SCC patients receiving adjuvant chemotherapy.EMX2 expression is down-regulated in lung SCC and its down-regulation is associated with chemo-resistance in lung SCC cells, possibly through regulation of Epithelial-to-Mesenchymal Transition (EMT).

View Article: PubMed Central - PubMed

Affiliation: Department of Lung Cancer, Lung Cancer Center, Tianjin Medical University Cancer Institute and Hospital, National Clinical Research Center of Cancer, Key Laboratory of Cancer Prevention and Therapy, Tianjin 300060, China; Thoracic Oncology Program, Department of Surgery, Helen Diller Family Comprehensive Cancer Center, University of California San Francisco, San Francisco, CA 94115, United States of America.

ABSTRACT

Background: Squamous cell carcinomas (SCC) account for approximately 30% of non-small cell lung cancer (NSCLC). Current staging methods do not adequately predict outcome for this disease. EMX2 is a homeo-domain containing transcription factor known to regulate a key developmental pathway. This study assessed the significance of EMX2 as a prognostic and predictive marker for resectable lung SCC.

Methods: Two independent cohorts of patients with lung SCC undergoing surgical resection were studied. EMX2 protein expression was examined by immunohistochemistry, Western blot, or immunofluorescence. EMX2 expression levels in tissue specimens were scored and correlated with patient outcomes. Chemo-sensitivity of lung SCC cell lines stably transfected with EMX2 shRNAs to cisplatin, carboplatin, and docetaxel was examined in vitro.

Results: EMX2 expression was down-regulated in lung SCC tissue samples compared to their matched adjacent normal tissues. Positive EMX2 expression was significantly associated with improved overall survival in stage I lung SCC patients, and in stage II/IIIA lung SCC patients receiving adjuvant chemotherapy. EMX2 expression was also associated with expression of EMT markers in both lung SCC cell lines and tissue samples. Knock-down of EMX2 expression in lung SCC cells promoted chemo-resistance and cell migration.

Conclusions: EMX2 expression is down-regulated in lung SCC and its down-regulation is associated with chemo-resistance in lung SCC cells, possibly through regulation of Epithelial-to-Mesenchymal Transition (EMT). EMX2 may serve as a novel prognostic marker for stage I lung SCC patients and a prediction marker for stage II/IIIA lung SCC patients receiving adjuvant chemotherapy.

No MeSH data available.


Related in: MedlinePlus