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DEAD-box protein p68 is regulated by β-catenin/transcription factor 4 to maintain a positive feedback loop in control of breast cancer progression.

Guturi KK, Sarkar M, Bhowmik A, Das N, Ghosh MK - Breast Cancer Res. (2014)

Bottom Line: Protein and mRNA expressions were determined by immunoblotting and quantitative RT-PCR respectively.Promoter activity of p68 was checked using luciferase assay.Furthermore, we have also established a positive feedback regulation for the expression of TCF4 by p68.

View Article: PubMed Central - PubMed

Affiliation: Signal Transduction in Cancer and Stem Cells Laboratory, Cancer Biology and Inflammatory Disorder Division, Council of Scientific and Industrial Research-Indian Institute of Chemical Biology (CSIR-IICB), 4 Raja S C Mullick Road, Jadavpur, Kolkata, 700032, India. kirankumarndkm@gmail.com.

ABSTRACT

Introduction: Nuclear accumulation of β-catenin is important for cancer development and it is found to overlap with p68 (DDX5) immunoreactivity in most breast cancers, as indicated by both clinical investigations and studies in cell lines. In this study, we aim to investigate the regulation of p68 gene expression through β-catenin/transcription factor 4 (TCF4) signaling in breast cancer.

Methods: Formalin-fixed paraffin-embedded sections derived from normal human breast and breast cancer samples were used for immunohistochemical analysis. Protein and mRNA expressions were determined by immunoblotting and quantitative RT-PCR respectively. Promoter activity of p68 was checked using luciferase assay. Occupancy of several factors on the p68 promoter was evaluated using chromatin immunoprecipitation. Finally, a syngeneic mouse model of breast cancer was used to assess physiological significance.

Results: We demonstrated that β-catenin can directly induce transcription of p68 promoter or indirectly through regulation of c-Myc in both human and mouse breast cancer cells. Moreover, by chromatin immunoprecipitation assay, we have found that both β-catenin and TCF4 occupy the endogenous p68 promoter, which is further enhanced by Wnt signaling. Furthermore, we have also established a positive feedback regulation for the expression of TCF4 by p68. To the best of our knowledge, this is the first report on β-catenin/TCF4-mediated p68 gene regulation, which plays an important role in epithelial to mesenchymal transition, as shown in vitro in breast cancer cell lines and in vivo in an animal breast tumour model.

Conclusions: Our findings indicate that Wnt/β-catenin signaling plays an important role in breast cancer progression through p68 upregulation.

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Related in: MedlinePlus

p68 gene expression is controlled by both β-catenin and c-Myc. (a) HEK293T cells were transfected with small interfering RNAs (siRNAs) targeting either β-catenin or c-Myc. Scrambled siRNA-transfected cells were kept as control. After 24 h, cells were transfected with wnt3a and kept for another 24 h. Whole cell lysates (WCLs) were prepared and proteins were analysed by immunoblotting (IB) as shown in the figure. (b, c) β-catenin was knocked down in presence or absence of c-Myc overexpression by ectopic expression of myc-tagged c-Myc in H1299 cells. WCLs and total RNAs of 36 h post-transfected cells were analysed for p68 expression by IB and qRT-PCR. (d, e) 4T1 cells were transfected with siRNA targeting transcription factor 4 (TCF4), c-Myc, p68 or scrambled siRNA independently, and expression of respective proteins and mRNAs were analysed by IB and qRT-PCR after 36 h of transfection.
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Fig3: p68 gene expression is controlled by both β-catenin and c-Myc. (a) HEK293T cells were transfected with small interfering RNAs (siRNAs) targeting either β-catenin or c-Myc. Scrambled siRNA-transfected cells were kept as control. After 24 h, cells were transfected with wnt3a and kept for another 24 h. Whole cell lysates (WCLs) were prepared and proteins were analysed by immunoblotting (IB) as shown in the figure. (b, c) β-catenin was knocked down in presence or absence of c-Myc overexpression by ectopic expression of myc-tagged c-Myc in H1299 cells. WCLs and total RNAs of 36 h post-transfected cells were analysed for p68 expression by IB and qRT-PCR. (d, e) 4T1 cells were transfected with siRNA targeting transcription factor 4 (TCF4), c-Myc, p68 or scrambled siRNA independently, and expression of respective proteins and mRNAs were analysed by IB and qRT-PCR after 36 h of transfection.

Mentions: To decipher the significance of c-Myc in the Wnt signaling-mediated tumourigenesis and its involvement in p68 gene expression, if any, we have knocked down either β-catenin or c-Myc in HEK293T cells followed by transiently overexpressing Wnt3a. We have observed that the resultant knockdown of either β-catenin or c-Myc reduces the Wnt3a-dependent increase in the expression of p68 as well as Wnt/β-catenin target c-Myc (Figure 3a). We have also overexpressed either c-Myc or β-catenin in HEK293T cells. We observed increased expression of p68 without additional signaling by wnt3a overexpression (Figure S5 in Additional file 6). We have further confirmed this regulation by knocking down β-catenin in c-Myc-overexpressed H1299 cells and analysed by IB and qRT-PCR (Figures 3b and c). Consistently, upon knockdown of either TCF4, c-Myc or p68 in mouse 4T1 cells using respective siRNAs, we have found a substantial decrease in the expression levels of p68 (Figures 3d and e). Surprisingly, here we have also observed an unexpected result in p68 knockdown cells where TCF4 was abruptly diminished indicating a relation between them. All these results suggest that both β-catenin/TCF4 and c-Myc are important factors in regulating p68 gene expression.Figure 3


DEAD-box protein p68 is regulated by β-catenin/transcription factor 4 to maintain a positive feedback loop in control of breast cancer progression.

Guturi KK, Sarkar M, Bhowmik A, Das N, Ghosh MK - Breast Cancer Res. (2014)

p68 gene expression is controlled by both β-catenin and c-Myc. (a) HEK293T cells were transfected with small interfering RNAs (siRNAs) targeting either β-catenin or c-Myc. Scrambled siRNA-transfected cells were kept as control. After 24 h, cells were transfected with wnt3a and kept for another 24 h. Whole cell lysates (WCLs) were prepared and proteins were analysed by immunoblotting (IB) as shown in the figure. (b, c) β-catenin was knocked down in presence or absence of c-Myc overexpression by ectopic expression of myc-tagged c-Myc in H1299 cells. WCLs and total RNAs of 36 h post-transfected cells were analysed for p68 expression by IB and qRT-PCR. (d, e) 4T1 cells were transfected with siRNA targeting transcription factor 4 (TCF4), c-Myc, p68 or scrambled siRNA independently, and expression of respective proteins and mRNAs were analysed by IB and qRT-PCR after 36 h of transfection.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4308923&req=5

Fig3: p68 gene expression is controlled by both β-catenin and c-Myc. (a) HEK293T cells were transfected with small interfering RNAs (siRNAs) targeting either β-catenin or c-Myc. Scrambled siRNA-transfected cells were kept as control. After 24 h, cells were transfected with wnt3a and kept for another 24 h. Whole cell lysates (WCLs) were prepared and proteins were analysed by immunoblotting (IB) as shown in the figure. (b, c) β-catenin was knocked down in presence or absence of c-Myc overexpression by ectopic expression of myc-tagged c-Myc in H1299 cells. WCLs and total RNAs of 36 h post-transfected cells were analysed for p68 expression by IB and qRT-PCR. (d, e) 4T1 cells were transfected with siRNA targeting transcription factor 4 (TCF4), c-Myc, p68 or scrambled siRNA independently, and expression of respective proteins and mRNAs were analysed by IB and qRT-PCR after 36 h of transfection.
Mentions: To decipher the significance of c-Myc in the Wnt signaling-mediated tumourigenesis and its involvement in p68 gene expression, if any, we have knocked down either β-catenin or c-Myc in HEK293T cells followed by transiently overexpressing Wnt3a. We have observed that the resultant knockdown of either β-catenin or c-Myc reduces the Wnt3a-dependent increase in the expression of p68 as well as Wnt/β-catenin target c-Myc (Figure 3a). We have also overexpressed either c-Myc or β-catenin in HEK293T cells. We observed increased expression of p68 without additional signaling by wnt3a overexpression (Figure S5 in Additional file 6). We have further confirmed this regulation by knocking down β-catenin in c-Myc-overexpressed H1299 cells and analysed by IB and qRT-PCR (Figures 3b and c). Consistently, upon knockdown of either TCF4, c-Myc or p68 in mouse 4T1 cells using respective siRNAs, we have found a substantial decrease in the expression levels of p68 (Figures 3d and e). Surprisingly, here we have also observed an unexpected result in p68 knockdown cells where TCF4 was abruptly diminished indicating a relation between them. All these results suggest that both β-catenin/TCF4 and c-Myc are important factors in regulating p68 gene expression.Figure 3

Bottom Line: Protein and mRNA expressions were determined by immunoblotting and quantitative RT-PCR respectively.Promoter activity of p68 was checked using luciferase assay.Furthermore, we have also established a positive feedback regulation for the expression of TCF4 by p68.

View Article: PubMed Central - PubMed

Affiliation: Signal Transduction in Cancer and Stem Cells Laboratory, Cancer Biology and Inflammatory Disorder Division, Council of Scientific and Industrial Research-Indian Institute of Chemical Biology (CSIR-IICB), 4 Raja S C Mullick Road, Jadavpur, Kolkata, 700032, India. kirankumarndkm@gmail.com.

ABSTRACT

Introduction: Nuclear accumulation of β-catenin is important for cancer development and it is found to overlap with p68 (DDX5) immunoreactivity in most breast cancers, as indicated by both clinical investigations and studies in cell lines. In this study, we aim to investigate the regulation of p68 gene expression through β-catenin/transcription factor 4 (TCF4) signaling in breast cancer.

Methods: Formalin-fixed paraffin-embedded sections derived from normal human breast and breast cancer samples were used for immunohistochemical analysis. Protein and mRNA expressions were determined by immunoblotting and quantitative RT-PCR respectively. Promoter activity of p68 was checked using luciferase assay. Occupancy of several factors on the p68 promoter was evaluated using chromatin immunoprecipitation. Finally, a syngeneic mouse model of breast cancer was used to assess physiological significance.

Results: We demonstrated that β-catenin can directly induce transcription of p68 promoter or indirectly through regulation of c-Myc in both human and mouse breast cancer cells. Moreover, by chromatin immunoprecipitation assay, we have found that both β-catenin and TCF4 occupy the endogenous p68 promoter, which is further enhanced by Wnt signaling. Furthermore, we have also established a positive feedback regulation for the expression of TCF4 by p68. To the best of our knowledge, this is the first report on β-catenin/TCF4-mediated p68 gene regulation, which plays an important role in epithelial to mesenchymal transition, as shown in vitro in breast cancer cell lines and in vivo in an animal breast tumour model.

Conclusions: Our findings indicate that Wnt/β-catenin signaling plays an important role in breast cancer progression through p68 upregulation.

Show MeSH
Related in: MedlinePlus