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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

β-catenin/TCF4 as well as c-Myc regulates p68 promoter. (a) Schematic representation of human and mouse p68 promoters. (b) Activity of human p68 promoter (pGL3-hp68) was determined by luciferase assay in HEK293T, MCF7 and HCT116 cells after 36 h of transfection. Values represent the firefly luciferase activities normalised to renilla luciferase activities. (c) Similarly, pGL3-hp68 activity was determined in MCF7 cells treated with 20 mM lithium chloride (LiCl) for 2 h and 8 h. (d) Luciferase activities were determined in MCF7 cells transfected with human wild-type pGL3-hp68, or mutated transcription factor 4 (TCF4) constructs like pGL3-hp68-M1, pGL3-hp68-M2, pGL3-hp68-M3 and pGL3-hp68-M4 independently, along with pEGFP-β-catenin. (e) Mouse p68 promoter (pGL3-mp68) activities were determined in MCF7 cells transfected with either wild-type pGL3-mp68 or mutated TCF4 sites containing constructs pGL3-mp68-M1 and pGL3-mp68-M2 independently along with pEGFP-β-catenin. (f) Cross-linked chromatin fragments of MCF7, MDA-MB 231 and HCT116 (human) as well as 4T1 (mouse) cells were immunoprecipitated with respective antibodies as depicted in the figure. DNAs were isolated and PCR-amplified using primer sets designed from the promoter regions of p68 and cyclin D1 genes. Densitometry values are given below the images. (g) MCF7 cells were treated with either Wnt3a-CM or empty vector (EV)-CM (control). DNAs were isolated from cross-linked chromatins immunoprecipitated with the indicated antibodies and PCR-amplified using primer sets designed from the promoter regions of p68 and RPL30 genes. Densitometry values are given below the images. (h) β-catenin was knocked down in HCT116 and 4T1 cells using small interfering RNA (siRNA). Control siRNA (si ctrl)-transfected cells were kept as control. DNA fragments were isolated from cross-linked chromatins immunoprecipitated with the indicated antibodies and PCR-amplified using primer sets designed from the promoter regions of p68 and cyclin D1 genes. Input: 2.5% (f and g) or 5% (h) of total DNA isolated from cross-linked chromatin without immunoprecipitation. Densitometry values are given below the images.
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Fig5: β-catenin/TCF4 as well as c-Myc regulates p68 promoter. (a) Schematic representation of human and mouse p68 promoters. (b) Activity of human p68 promoter (pGL3-hp68) was determined by luciferase assay in HEK293T, MCF7 and HCT116 cells after 36 h of transfection. Values represent the firefly luciferase activities normalised to renilla luciferase activities. (c) Similarly, pGL3-hp68 activity was determined in MCF7 cells treated with 20 mM lithium chloride (LiCl) for 2 h and 8 h. (d) Luciferase activities were determined in MCF7 cells transfected with human wild-type pGL3-hp68, or mutated transcription factor 4 (TCF4) constructs like pGL3-hp68-M1, pGL3-hp68-M2, pGL3-hp68-M3 and pGL3-hp68-M4 independently, along with pEGFP-β-catenin. (e) Mouse p68 promoter (pGL3-mp68) activities were determined in MCF7 cells transfected with either wild-type pGL3-mp68 or mutated TCF4 sites containing constructs pGL3-mp68-M1 and pGL3-mp68-M2 independently along with pEGFP-β-catenin. (f) Cross-linked chromatin fragments of MCF7, MDA-MB 231 and HCT116 (human) as well as 4T1 (mouse) cells were immunoprecipitated with respective antibodies as depicted in the figure. DNAs were isolated and PCR-amplified using primer sets designed from the promoter regions of p68 and cyclin D1 genes. Densitometry values are given below the images. (g) MCF7 cells were treated with either Wnt3a-CM or empty vector (EV)-CM (control). DNAs were isolated from cross-linked chromatins immunoprecipitated with the indicated antibodies and PCR-amplified using primer sets designed from the promoter regions of p68 and RPL30 genes. Densitometry values are given below the images. (h) β-catenin was knocked down in HCT116 and 4T1 cells using small interfering RNA (siRNA). Control siRNA (si ctrl)-transfected cells were kept as control. DNA fragments were isolated from cross-linked chromatins immunoprecipitated with the indicated antibodies and PCR-amplified using primer sets designed from the promoter regions of p68 and cyclin D1 genes. Input: 2.5% (f and g) or 5% (h) of total DNA isolated from cross-linked chromatin without immunoprecipitation. Densitometry values are given below the images.

Mentions: It has been well established that nuclear β-catenin is engaged in TCF/LEF protein complexes on its target genes [52]. To further elucidate the molecular mechanism(s) for regulation of p68 gene expression we have cloned and analysed the human and mouse p68 promoter sequences. The human p68 promoter contains three putative TCF4 binding elements corresponding to the core consensus binding sequence (AC/GA/TTCAAAG) at (324-CTTTGGA-317), (554-CGTCAAAG-547) and (706-AACCAAAG-699) and one c-Myc site (380-CACGTGA-374) upstream of the transcription start site. We have mutated two of these three TCF4 sites and the c-Myc site in both human and mouse p68 promoters, represented by schematic diagrams (Figure 5a). The activity of the wild-type p68 promoter was tested in various human cell lines, with differential endogenous β-catenin levels, by luciferase assay. Results indicate that the promoter activity is differentially regulated (Figure 5b), correlating with β-catenin levels (shown in Figure 1a). This regulation is also supported by increased promoter activity due to enhanced β-catenin stabilization in LiCl-treated MCF7 cells (Figure 5c). But all the mutated p68 promoters pGL3-hp68-M1 (putative tcf4 site1), pGL3-hp68-M2 (putative tcf4 site2), pGL3-hp68-M3 (c-myc site) and pGL3-hp68-M4 (all three sites) showed reduced activity, even after β-catenin overexpression, when compared to the wild-type (Figure 5d). The activity of these mutated promoters was found to reduce 2.9-, 5.9-, 3.0- and 16-fold respectively. The mouse p68 promoter consists of one putative tcf4 site (1149-GACAAAG-1143) and one c-Myc site (478-CACGTGA-472; 100% conserved with human sequence) and the activity of the mutated promoters was found to reduce 3.8- and 7.2-fold respectively for c-Myc alone, or when both sites were mutated (Figure 5e).Figure 5


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)

β-catenin/TCF4 as well as c-Myc regulates p68 promoter. (a) Schematic representation of human and mouse p68 promoters. (b) Activity of human p68 promoter (pGL3-hp68) was determined by luciferase assay in HEK293T, MCF7 and HCT116 cells after 36 h of transfection. Values represent the firefly luciferase activities normalised to renilla luciferase activities. (c) Similarly, pGL3-hp68 activity was determined in MCF7 cells treated with 20 mM lithium chloride (LiCl) for 2 h and 8 h. (d) Luciferase activities were determined in MCF7 cells transfected with human wild-type pGL3-hp68, or mutated transcription factor 4 (TCF4) constructs like pGL3-hp68-M1, pGL3-hp68-M2, pGL3-hp68-M3 and pGL3-hp68-M4 independently, along with pEGFP-β-catenin. (e) Mouse p68 promoter (pGL3-mp68) activities were determined in MCF7 cells transfected with either wild-type pGL3-mp68 or mutated TCF4 sites containing constructs pGL3-mp68-M1 and pGL3-mp68-M2 independently along with pEGFP-β-catenin. (f) Cross-linked chromatin fragments of MCF7, MDA-MB 231 and HCT116 (human) as well as 4T1 (mouse) cells were immunoprecipitated with respective antibodies as depicted in the figure. DNAs were isolated and PCR-amplified using primer sets designed from the promoter regions of p68 and cyclin D1 genes. Densitometry values are given below the images. (g) MCF7 cells were treated with either Wnt3a-CM or empty vector (EV)-CM (control). DNAs were isolated from cross-linked chromatins immunoprecipitated with the indicated antibodies and PCR-amplified using primer sets designed from the promoter regions of p68 and RPL30 genes. Densitometry values are given below the images. (h) β-catenin was knocked down in HCT116 and 4T1 cells using small interfering RNA (siRNA). Control siRNA (si ctrl)-transfected cells were kept as control. DNA fragments were isolated from cross-linked chromatins immunoprecipitated with the indicated antibodies and PCR-amplified using primer sets designed from the promoter regions of p68 and cyclin D1 genes. Input: 2.5% (f and g) or 5% (h) of total DNA isolated from cross-linked chromatin without immunoprecipitation. Densitometry values are given below the images.
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Fig5: β-catenin/TCF4 as well as c-Myc regulates p68 promoter. (a) Schematic representation of human and mouse p68 promoters. (b) Activity of human p68 promoter (pGL3-hp68) was determined by luciferase assay in HEK293T, MCF7 and HCT116 cells after 36 h of transfection. Values represent the firefly luciferase activities normalised to renilla luciferase activities. (c) Similarly, pGL3-hp68 activity was determined in MCF7 cells treated with 20 mM lithium chloride (LiCl) for 2 h and 8 h. (d) Luciferase activities were determined in MCF7 cells transfected with human wild-type pGL3-hp68, or mutated transcription factor 4 (TCF4) constructs like pGL3-hp68-M1, pGL3-hp68-M2, pGL3-hp68-M3 and pGL3-hp68-M4 independently, along with pEGFP-β-catenin. (e) Mouse p68 promoter (pGL3-mp68) activities were determined in MCF7 cells transfected with either wild-type pGL3-mp68 or mutated TCF4 sites containing constructs pGL3-mp68-M1 and pGL3-mp68-M2 independently along with pEGFP-β-catenin. (f) Cross-linked chromatin fragments of MCF7, MDA-MB 231 and HCT116 (human) as well as 4T1 (mouse) cells were immunoprecipitated with respective antibodies as depicted in the figure. DNAs were isolated and PCR-amplified using primer sets designed from the promoter regions of p68 and cyclin D1 genes. Densitometry values are given below the images. (g) MCF7 cells were treated with either Wnt3a-CM or empty vector (EV)-CM (control). DNAs were isolated from cross-linked chromatins immunoprecipitated with the indicated antibodies and PCR-amplified using primer sets designed from the promoter regions of p68 and RPL30 genes. Densitometry values are given below the images. (h) β-catenin was knocked down in HCT116 and 4T1 cells using small interfering RNA (siRNA). Control siRNA (si ctrl)-transfected cells were kept as control. DNA fragments were isolated from cross-linked chromatins immunoprecipitated with the indicated antibodies and PCR-amplified using primer sets designed from the promoter regions of p68 and cyclin D1 genes. Input: 2.5% (f and g) or 5% (h) of total DNA isolated from cross-linked chromatin without immunoprecipitation. Densitometry values are given below the images.
Mentions: It has been well established that nuclear β-catenin is engaged in TCF/LEF protein complexes on its target genes [52]. To further elucidate the molecular mechanism(s) for regulation of p68 gene expression we have cloned and analysed the human and mouse p68 promoter sequences. The human p68 promoter contains three putative TCF4 binding elements corresponding to the core consensus binding sequence (AC/GA/TTCAAAG) at (324-CTTTGGA-317), (554-CGTCAAAG-547) and (706-AACCAAAG-699) and one c-Myc site (380-CACGTGA-374) upstream of the transcription start site. We have mutated two of these three TCF4 sites and the c-Myc site in both human and mouse p68 promoters, represented by schematic diagrams (Figure 5a). The activity of the wild-type p68 promoter was tested in various human cell lines, with differential endogenous β-catenin levels, by luciferase assay. Results indicate that the promoter activity is differentially regulated (Figure 5b), correlating with β-catenin levels (shown in Figure 1a). This regulation is also supported by increased promoter activity due to enhanced β-catenin stabilization in LiCl-treated MCF7 cells (Figure 5c). But all the mutated p68 promoters pGL3-hp68-M1 (putative tcf4 site1), pGL3-hp68-M2 (putative tcf4 site2), pGL3-hp68-M3 (c-myc site) and pGL3-hp68-M4 (all three sites) showed reduced activity, even after β-catenin overexpression, when compared to the wild-type (Figure 5d). The activity of these mutated promoters was found to reduce 2.9-, 5.9-, 3.0- and 16-fold respectively. The mouse p68 promoter consists of one putative tcf4 site (1149-GACAAAG-1143) and one c-Myc site (478-CACGTGA-472; 100% conserved with human sequence) and the activity of the mutated promoters was found to reduce 3.8- and 7.2-fold respectively for c-Myc alone, or when both sites were mutated (Figure 5e).Figure 5

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