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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

Canonical Wnt signaling upregulates p68 expression. (a) Whole cell lysates (WCLs) were prepared from MCF7, MDA-MB 231, 4T1, H1299, HCT116 and HEK293T cells. β-catenin, transcription factor 4 (TCF4) and p68 protein levels were analysed by immunoblotting (IB). Densitometry values are given below the blots. (b, c) Breast cancer patient samples (E-cadherin+ and E-cadherin-) were analysed by immunohistochemistry (IHC) using antibodies against β-catenin, TCF4 and p68, as well as E-cadherin, to differentiate the samples. H scores of these samples were determined. (d) Wnt3a-MCF7 and empty vector (EV)-MCF7 stable cells were immunostained with β-catenin (primary) and Alexa Fluor488 (secondary, Green) antibodies and observed under fluorescence microscope to see its localisation pattern. Images were captured along with DAPI-stained nuclei at 600X magnification (scale: 10 μm) (top). Cytoplasmic and nuclear extracts were prepared from Wnt3a-MCF7 and EV-MCF7 stable cells and were analysed for β-catenin by IB. Densitometry values are given below the blots (bottom). (e) WCLs of Wnt3a-MCF7 and EV-MCF7 stable cells were analysed for Axin-2, β-catenin, Cyclin D1 and p68 proteins by IB. Densitometry values are given below the blots. (f, g) MCF7, MDA-MB 231 and 4T1 cells were serum starved for 24 h before treatment with Wnt3a condition medium (Wnt3a-CM). WCLs and total RNAs were prepared from 24 h post-treated cells to check the expression of β-catenin, p68 and Cyclin D1 by IB and qRT-PCR.
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Fig1: Canonical Wnt signaling upregulates p68 expression. (a) Whole cell lysates (WCLs) were prepared from MCF7, MDA-MB 231, 4T1, H1299, HCT116 and HEK293T cells. β-catenin, transcription factor 4 (TCF4) and p68 protein levels were analysed by immunoblotting (IB). Densitometry values are given below the blots. (b, c) Breast cancer patient samples (E-cadherin+ and E-cadherin-) were analysed by immunohistochemistry (IHC) using antibodies against β-catenin, TCF4 and p68, as well as E-cadherin, to differentiate the samples. H scores of these samples were determined. (d) Wnt3a-MCF7 and empty vector (EV)-MCF7 stable cells were immunostained with β-catenin (primary) and Alexa Fluor488 (secondary, Green) antibodies and observed under fluorescence microscope to see its localisation pattern. Images were captured along with DAPI-stained nuclei at 600X magnification (scale: 10 μm) (top). Cytoplasmic and nuclear extracts were prepared from Wnt3a-MCF7 and EV-MCF7 stable cells and were analysed for β-catenin by IB. Densitometry values are given below the blots (bottom). (e) WCLs of Wnt3a-MCF7 and EV-MCF7 stable cells were analysed for Axin-2, β-catenin, Cyclin D1 and p68 proteins by IB. Densitometry values are given below the blots. (f, g) MCF7, MDA-MB 231 and 4T1 cells were serum starved for 24 h before treatment with Wnt3a condition medium (Wnt3a-CM). WCLs and total RNAs were prepared from 24 h post-treated cells to check the expression of β-catenin, p68 and Cyclin D1 by IB and qRT-PCR.

Mentions: To address this issue, we first examined the expression of β-catenin, TCF4 and p68 in various established cancer cell lines and HEK293T cells (Figure 1a). We have also analysed the expression pattern of these molecules by IHC analysis in human breast tumour and normal samples (Figure 1b and Figure S1 in Additional file 2) and classified based on the E-cadherin expression status [48-50]. The results indicate that there is a probable correlation for β-catenin and TCF4 with p68 in the cancer cell lines. Moreover, this positive correlation was also found in E-cadherin samples (Figures 1b and c). Our results also indicate that p68 expression is quite low in MCF-7 cells as compared to highly metastatic MDA-MB 231 and mouse 4T1 cells with reduced expression or absence of E-cadherin, which is corroborated with the previous findings in breast tumours and cell lines [35]. Since Wnt3a is known to stabilize β-catenin and increase its nuclear accumulation, we generated constitutively expressing Wnt3a stable MCF7 (Wnt3a-MCF7) cells and examined the cytoplasmic/nuclear distribution of β-catenin in these cells under serum-starved conditions. Here, we visualised the localisation pattern of β-catenin by both immunofluorescence and IB and observed an increased level of β-catenin in the nucleus of these cells when compared to the empty vector- containing control (EV-MCF7) cells (Figure 1d). Wnt3a-MCF7 cells showed a significant increase in p68 level along with β-catenin and its targets Cyclin D1 and Axin-2 (positive control) compared to EV-MCF7 (Figure 1e). To strengthen our finding, we have analysed p68 expression in various breast cancer cell lines under enhanced Wnt signaling by Wnt3a-CM treatment. We observed that Wnt3a-CM upregulates p68 expression at both protein and mRNA level (Figures 1f and g), where Cyclin D1 was kept as a positive control. We have further observed a similar effect in HEK293T and other cancer cell lines (Figure S2 in Additional file 3). Further, we observed that the stabilized β-catenin due to GSK3β inactivation in lithium chloride (LiCl)-treated serum-starved MCF7 cells, leads to increased expression of p68, where Cyclin D1 served as positive control (Figure S3 in Additional file 4). Collectively, all these results clearly indicate that p68 expression is controlled by Wnt/β-catenin signaling.Figure 1


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)

Canonical Wnt signaling upregulates p68 expression. (a) Whole cell lysates (WCLs) were prepared from MCF7, MDA-MB 231, 4T1, H1299, HCT116 and HEK293T cells. β-catenin, transcription factor 4 (TCF4) and p68 protein levels were analysed by immunoblotting (IB). Densitometry values are given below the blots. (b, c) Breast cancer patient samples (E-cadherin+ and E-cadherin-) were analysed by immunohistochemistry (IHC) using antibodies against β-catenin, TCF4 and p68, as well as E-cadherin, to differentiate the samples. H scores of these samples were determined. (d) Wnt3a-MCF7 and empty vector (EV)-MCF7 stable cells were immunostained with β-catenin (primary) and Alexa Fluor488 (secondary, Green) antibodies and observed under fluorescence microscope to see its localisation pattern. Images were captured along with DAPI-stained nuclei at 600X magnification (scale: 10 μm) (top). Cytoplasmic and nuclear extracts were prepared from Wnt3a-MCF7 and EV-MCF7 stable cells and were analysed for β-catenin by IB. Densitometry values are given below the blots (bottom). (e) WCLs of Wnt3a-MCF7 and EV-MCF7 stable cells were analysed for Axin-2, β-catenin, Cyclin D1 and p68 proteins by IB. Densitometry values are given below the blots. (f, g) MCF7, MDA-MB 231 and 4T1 cells were serum starved for 24 h before treatment with Wnt3a condition medium (Wnt3a-CM). WCLs and total RNAs were prepared from 24 h post-treated cells to check the expression of β-catenin, p68 and Cyclin D1 by IB and qRT-PCR.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4308923&req=5

Fig1: Canonical Wnt signaling upregulates p68 expression. (a) Whole cell lysates (WCLs) were prepared from MCF7, MDA-MB 231, 4T1, H1299, HCT116 and HEK293T cells. β-catenin, transcription factor 4 (TCF4) and p68 protein levels were analysed by immunoblotting (IB). Densitometry values are given below the blots. (b, c) Breast cancer patient samples (E-cadherin+ and E-cadherin-) were analysed by immunohistochemistry (IHC) using antibodies against β-catenin, TCF4 and p68, as well as E-cadherin, to differentiate the samples. H scores of these samples were determined. (d) Wnt3a-MCF7 and empty vector (EV)-MCF7 stable cells were immunostained with β-catenin (primary) and Alexa Fluor488 (secondary, Green) antibodies and observed under fluorescence microscope to see its localisation pattern. Images were captured along with DAPI-stained nuclei at 600X magnification (scale: 10 μm) (top). Cytoplasmic and nuclear extracts were prepared from Wnt3a-MCF7 and EV-MCF7 stable cells and were analysed for β-catenin by IB. Densitometry values are given below the blots (bottom). (e) WCLs of Wnt3a-MCF7 and EV-MCF7 stable cells were analysed for Axin-2, β-catenin, Cyclin D1 and p68 proteins by IB. Densitometry values are given below the blots. (f, g) MCF7, MDA-MB 231 and 4T1 cells were serum starved for 24 h before treatment with Wnt3a condition medium (Wnt3a-CM). WCLs and total RNAs were prepared from 24 h post-treated cells to check the expression of β-catenin, p68 and Cyclin D1 by IB and qRT-PCR.
Mentions: To address this issue, we first examined the expression of β-catenin, TCF4 and p68 in various established cancer cell lines and HEK293T cells (Figure 1a). We have also analysed the expression pattern of these molecules by IHC analysis in human breast tumour and normal samples (Figure 1b and Figure S1 in Additional file 2) and classified based on the E-cadherin expression status [48-50]. The results indicate that there is a probable correlation for β-catenin and TCF4 with p68 in the cancer cell lines. Moreover, this positive correlation was also found in E-cadherin samples (Figures 1b and c). Our results also indicate that p68 expression is quite low in MCF-7 cells as compared to highly metastatic MDA-MB 231 and mouse 4T1 cells with reduced expression or absence of E-cadherin, which is corroborated with the previous findings in breast tumours and cell lines [35]. Since Wnt3a is known to stabilize β-catenin and increase its nuclear accumulation, we generated constitutively expressing Wnt3a stable MCF7 (Wnt3a-MCF7) cells and examined the cytoplasmic/nuclear distribution of β-catenin in these cells under serum-starved conditions. Here, we visualised the localisation pattern of β-catenin by both immunofluorescence and IB and observed an increased level of β-catenin in the nucleus of these cells when compared to the empty vector- containing control (EV-MCF7) cells (Figure 1d). Wnt3a-MCF7 cells showed a significant increase in p68 level along with β-catenin and its targets Cyclin D1 and Axin-2 (positive control) compared to EV-MCF7 (Figure 1e). To strengthen our finding, we have analysed p68 expression in various breast cancer cell lines under enhanced Wnt signaling by Wnt3a-CM treatment. We observed that Wnt3a-CM upregulates p68 expression at both protein and mRNA level (Figures 1f and g), where Cyclin D1 was kept as a positive control. We have further observed a similar effect in HEK293T and other cancer cell lines (Figure S2 in Additional file 3). Further, we observed that the stabilized β-catenin due to GSK3β inactivation in lithium chloride (LiCl)-treated serum-starved MCF7 cells, leads to increased expression of p68, where Cyclin D1 served as positive control (Figure S3 in Additional file 4). Collectively, all these results clearly indicate that p68 expression is controlled by Wnt/β-catenin signaling.Figure 1

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