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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 knockdown reduces breast tumour volume and expression of EMT markers in mice. (a) p68 expression level was determined in p68 knockdown mouse 4T1 stable cells. Control short hairpin RNA (shRNA)-4T1 stable cells were kept as control. (b) Images of mouse breast tumours after 16 days of injections in the mammary fat pad regions with stable cells expressing either control shRNA or p68 shRNA. Tumours obtained from the mammary fat pad regions were analysed for tumour volume (right panel). (c) Images of the hematoxylin and eosin (H&E)-stained sections of the tumours and immunohistochemical analysis of the adjacent sections were performed with the indicated antibodies as shown in the figure. Images were captured at magnifications of 200X.
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Fig7: p68 knockdown reduces breast tumour volume and expression of EMT markers in mice. (a) p68 expression level was determined in p68 knockdown mouse 4T1 stable cells. Control short hairpin RNA (shRNA)-4T1 stable cells were kept as control. (b) Images of mouse breast tumours after 16 days of injections in the mammary fat pad regions with stable cells expressing either control shRNA or p68 shRNA. Tumours obtained from the mammary fat pad regions were analysed for tumour volume (right panel). (c) Images of the hematoxylin and eosin (H&E)-stained sections of the tumours and immunohistochemical analysis of the adjacent sections were performed with the indicated antibodies as shown in the figure. Images were captured at magnifications of 200X.

Mentions: To further confirm that p68 is crucial in Wnt signaling-mediated tumourigenesis, the p68 knockdown and EV stable cells were generated using the mouse syngeneic 4T1 cell line and p68 expression was checked (Figure 7a). These cells were injected into the mouse mammary fat pad to generate tumours. p68 knockdown resulted in reduced tumour volume, indicating the involvement of p68 in tumour progression (Figure 7b). The morphological features of these tumours were ascertained by hematoxylin and eosin (H&E) staining. IHC analysis of the tumour regions confirmed the reduced expression of TCF4, β-catenin and p68 as well as PCNA in the tumours generated with p68 knockdown cells when compared to the tumours generated with EV control cells (Figure 7c). It also exhibited reduced expression of Vimentin and Snail, the EMT markers, and increased expression of E-cadherin, implying involvement of p68 in breast cancer progression and metastasis.Figure 7


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 knockdown reduces breast tumour volume and expression of EMT markers in mice. (a) p68 expression level was determined in p68 knockdown mouse 4T1 stable cells. Control short hairpin RNA (shRNA)-4T1 stable cells were kept as control. (b) Images of mouse breast tumours after 16 days of injections in the mammary fat pad regions with stable cells expressing either control shRNA or p68 shRNA. Tumours obtained from the mammary fat pad regions were analysed for tumour volume (right panel). (c) Images of the hematoxylin and eosin (H&E)-stained sections of the tumours and immunohistochemical analysis of the adjacent sections were performed with the indicated antibodies as shown in the figure. Images were captured at magnifications of 200X.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig7: p68 knockdown reduces breast tumour volume and expression of EMT markers in mice. (a) p68 expression level was determined in p68 knockdown mouse 4T1 stable cells. Control short hairpin RNA (shRNA)-4T1 stable cells were kept as control. (b) Images of mouse breast tumours after 16 days of injections in the mammary fat pad regions with stable cells expressing either control shRNA or p68 shRNA. Tumours obtained from the mammary fat pad regions were analysed for tumour volume (right panel). (c) Images of the hematoxylin and eosin (H&E)-stained sections of the tumours and immunohistochemical analysis of the adjacent sections were performed with the indicated antibodies as shown in the figure. Images were captured at magnifications of 200X.
Mentions: To further confirm that p68 is crucial in Wnt signaling-mediated tumourigenesis, the p68 knockdown and EV stable cells were generated using the mouse syngeneic 4T1 cell line and p68 expression was checked (Figure 7a). These cells were injected into the mouse mammary fat pad to generate tumours. p68 knockdown resulted in reduced tumour volume, indicating the involvement of p68 in tumour progression (Figure 7b). The morphological features of these tumours were ascertained by hematoxylin and eosin (H&E) staining. IHC analysis of the tumour regions confirmed the reduced expression of TCF4, β-catenin and p68 as well as PCNA in the tumours generated with p68 knockdown cells when compared to the tumours generated with EV control cells (Figure 7c). It also exhibited reduced expression of Vimentin and Snail, the EMT markers, and increased expression of E-cadherin, implying involvement of p68 in breast cancer progression and metastasis.Figure 7

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