DEAD-box protein p68 is regulated by β-catenin/transcription factor 4 to maintain a positive feedback loop in control of breast cancer progression
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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.
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.
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.
Our findings indicate that Wnt/β-catenin signaling plays an important role in breast cancer progression through p68 upregulation.
KeywordsAdenomatous Polyposis Coli Gene TCF4 Expression TCF4 Complex Tumourigenic Potential Respective siRNAs
adenomatous polyposis coli
bovine serum albumin
catenin (cadherin-associated protein) beta 1
epithelial to mesenchymal transition
hematoxylin and eosin
low-density lipoprotein-related protein
proliferating cell nuclear antigen
platelet-derived growth factor
secreted Frizzled-related proteins
small interfering RNAs
transcription factor 4
whole cell lysates
Wnt inhibitory factor-1
Compelling evidences indicate that the Wnt/β-catenin signaling is implicated in different stages of mammary gland development and is also important for mammary oncogenesis when aberrantly activated -. Genetic mutations in adenomatous polyposis coli (APC) and catenin (cadherin-associated protein) beta 1 (CTNNB1), the components of the Wnt/β-catenin signaling pathway, are the major contributors of colorectal cancer although they are typically not the key factors associated with breast cancer. It has been demonstrated that only 6% of breast tumours contain mutations in the APC gene but no mutations were detected in CTNNB1 ,. However, Wnt proteins (1, 3a, 4, 5a, 7b, 10b and 14) - and multiple Frizzled receptors (Fzd4/7) are reported to be overexpressed in human breast cancer cell lines and primary tumours ,. Recently, it has been documented that low-density lipoprotein-related protein (LRP)6 but not LRP5 is frequently upregulated in a subset of human breast carcinomas and downregulation of LRP6 is sufficient to inhibit breast cancer tumourigenesis . Moreover, Dishevelled 1 (DVL1), a central regulator of Wnt signaling is found to be upregulated in breast cancer . In addition to this, epigenetic silencing of the Wnt antagonists secreted Frizzled-related proteins (sFRPs) and Wnt inhibitory factor-1 (WIF-1) leads to aberrant regulation of Wnt/β-catenin signaling in both primary breast tumours and cell lines -. Again, approximately 60% of primary breast tumours show cytoplasmic or nuclear accumulation of β-catenin rather than its membrane localization, and this is correlated with poor prognosis .
p68 was first discovered through its immunological cross-reactivity with the anti-SV40 large T monoclonal antibody . Molecular similarity of p68 (an ATP-dependent RNA helicase) with both the large T antigen and eIF-4A (an ATP-dependent DNA helicase) implied that p68 may function as both RNA and DNA helicase . Moreover, p68 knockout mice are embryonically lethal (E11.5), indicating its importance in the development process . p68 was shown to bind, unwind and rearrange RNA secondary structures and it is also a crucial factor in the processing, alternate splicing and degradation of mRNA -. Subsequently, p68 has been implicated in a wide range of biological processes, and early studies of this protein indicate its possible involvement in the regulation of proliferation and organ differentiation . Recently, p68 has been demonstrated to act as a potent transcriptional co-activator of estrogen receptor ,, androgen receptor , tumour suppressor p53 , MyoD  and β-catenin . p68’s activation as a result of its phosphorylation at Tyr593 by platelet-derived growth factor (PDGF) was shown to be associated with cellular transformation and epithelial to mesenchymal transition (EMT) in colon cancer by promoting nuclear translocation of β-catenin, and upregulation of its target genes like cyclin D1 and c-myc ,. In addition to this, modification of p68 by the small ubiquitin-like modifier SUMO-2 was found to modulate its activity as a transcriptional regulator, favouring repression . It has been found that p68 is constitutively overexpressed in various cancers like colon , breast , prostate , head and neck as well as cutaneous squamous cell carcinoma . Serum-induced p68 expression in Swiss 3 T3 fibroblasts is involved in cellular proliferation and also connected to organ differentiation and maturation of the foetus .
p68 regulates the expression of several oncogenes through co-activation of β-catenin-mediated transcription, and controls tumour growth and metastasis. Although, there is an expansive evidence of literature deciphering the central role of p68 with respect to β-catenin in the architecture of intracellular signaling networks, little is known about its transcriptional regulation that may contribute to cancer development. Moreover, cellular consequences due to the modulation of its expression are not yet completely understood. Such knowledge might provide invaluable insights into the molecular mechanisms with respect to p68 in the context of oncogenesis.
Cell culture, transfection and drug treatments
HEK293T (human embryonic kidney); MCF7, MDA-MB 231 (human breast cancer); 4T1 (mouse breast cancer); H1299 (lung adenocarcinoma) and HCT116 (colon cancer) cell lines used in this study were obtained from the American Type Culture Collection (ATCC). Cell culture and transfections were performed using standard procedure as described previously . Small interfering RNAs (siRNAs) were purchased from Sigma-Aldrich (St Louis, MO, USA) (β-catenin), Cell Signaling Technology (Beverly, MA, USA) (p68) and Santa Cruz Biotechnology (Santa Cruz, CA, USA) (tcf4 and c-myc). G418 (2 mg/ml)-resistant MCF7 stable cells were selected by homogeneous colony formation as described earlier .
Wnt3A conditioned medium
Wnt3a-L cells were split at the ratio of 1:10 in 10 ml medium using standard procedure. Medium was collected from two consecutive batches of cells cultured for four days. Wnt-3a conditioned medium (Wnt3a-CM) was either freshly used with serum-free media (1:1) or stored at 4°C until further use.
The human wnt3a gene was cloned in pcDNA4/TO and further sub-cloned into pcDNA3.1-myc-his. Cloning of c-myc in pcDNA3.1-myc-his was done previously . pEGFP-c1-β-catenin and pEGFP-c1-p68 were sub-cloned from pGZdx-β-catenin and pGS5-p68 respectively. Human and mouse p68 promoters (-1200 to +200 and -1200 to +175 respectively) were cloned in pGL3-basic plasmid. Point mutations in these p68 promoters were generated by Site-Directed Mutagenesis using QuikChange XL kit (Stratagene, San Diego, CA, USA). Transcription factor 4 (TCF4) promoter (-1001 to +303) was cloned into pGL3-basic plasmid. All the constructs were verified by restriction digestions and confirmed by sequencing. Sequences of all the primers used are available in Table S1 in Additional file 1.
For ICC, cells were processed as described earlier . Briefly, after permeabilization, cells were incubated with primary antibodies (1:200) overnight at 4°C followed by incubation with Alexa Fluor (488 or 594)-tagged secondary antibodies (1:500) (Molecular Probes, Eugene, OR, USA). Primary antibodies (β-catenin and TCF4) were purchased from Cell Signaling Technology. Images were taken with BX61 upright fluorescence microscope (Olympus, Center Valley, PA, USA) using Image-Pro Plus software (Media Cybernetics, Silver Spring, MD, USA).
Preparation of whole cell lysates (WCL), cytoplasmic and nuclear extracts and IB were performed as described previously . Either GAPDH or β-actin was used as a loading control. Primary antibodies were purchased from Abcam (Cambridge, UK) (p68), Cell Signaling Technology (β-catenin, Cyclin D1, c-Myc, TCF4, Myc-Tag, E-cadherin, N-cadherin and Vimentin) and Santa Cruz Biotechnology (GAPDH, α-Tubulin, LaminB, β-actin and TCF4). Horseradish peroxidase (HRP)-tagged anti-rabbit and anti-goat secondary antibodies used were from Cell Signaling Technology and Sigma-Aldrich respectively.
Quantitative RT-PCR (qRT-PCR)
Total RNA was extracted and converted to cDNA, which was subsequently used for qRT-PCR analysis  using Power SYBR Green Master Mix on a 7500 Fast Real-Time PCR system (Applied Biosystems, Carlsbad, CA, USA). 18S rRNA served as the internal control. Sequences of all the primers used in qRT-PCR are given in Table S1 in Additional file 1. Standard deviation (SD) calculations are based on three technical replicates of two independent biological repeats.
Cells were transiently transfected with pGL3-p68 promoter luciferase reporter construct(s) along with Renilla luciferase plasmid (pRL-TK) and subjected to various treatments as indicated in the relevant figure(s). Luciferase activity was determined by luminometry using the GLOMAX 20/20 luminometer (Promega, Madison, WI, USA) by the dual-luciferase assay system (Promega), as specified by the manufacturer. Quantification was based on three technical replicates over at least two independent biological repeats.
Chromatin immunoprecipitation (ChIP)
Sonicated cross-linked chromatin fragments were prepared from cells treated with formaldehyde as described earlier ,. Briefly, equal amounts of all pre-cleared chromatin fragments (250 μg) were incubated with primary antibodies for 12 h at 4°C followed by pull down with 3% bovine serum albumin (BSA)-blocked protein G sepharose beads. Primary antibodies against β-catenin, TCF4, c-Myc and normal rabbit immunoglobulin G (IgG) were purchased from Cell Signaling Technology. Purified DNAs from immunoprecipitated chromatin fragments were used in PCR reactions with a standard programme using Qiagen’s Top Taq master mix (Qiagen, Venlo, Netherlands). The PCR products were analysed in 2% agarose gel. Quantitative RT-PCR (qRT-PCR) reactions were performed using SYBR Green master mix. All the primers used are listed in Table S1 in Additional file 1.
Soft agar colony formation assay and invasion assay
Colony formation assays in soft agar were performed in triplicate as described earlier . Images were captured by Olympus IX81 microscope using Image-Pro Plus software (Olympus) at 100× optical magnification and colony-forming efficiency was quantified. Matrigel invasion assay was performed as described previously . Images were captured by Olympus IX81 microscope at 40X magnification. Statistical analysis was performed by Student’s t test using GraphPad software with level of significance P <0.001 (GraphPad Software, San Diego, CA, USA).
Formalin-fixed paraffin-embedded (FFPE) tissue sections derived from normal human breast (n = 10) and breast cancer samples (n = 20) were obtained from Indian patients after formal approval from ethical committee of both Council of Scientific and Industrial Research (CSIR)-Indian Institute of Chemical Biology (IICB) and Park Clinic (registered under WB Societies Act. 1961). All patients involved in the study agreed to participate and publish the research outcome. IHC was performed and H-scores were calculated as described previously . Primary antibodies were from Abcam (p68 and Snail), Cell Signaling Technology (TCF4, E-cadherin, proliferating cell nuclear antigen (PCNA) and Vimentin) and Santa Cruz Biotechnology (β-catenin).
Orthotopic syngeneic mouse model of breast cancer
All animal care and experimentation conformed to the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA) (Govt. of India) following internationally recognized guidelines. The animal ethics approval was granted by ‘IICB-Animal Ethics Committee (IICB-AEC), CSIR, Govt. of India’. To generate the tumour model, three- to four-week-old BALB/c mice were injected once with either EV or p68 knockdown 4T1 stable cells (1 × 105) in the mammary fat pad region. Growth of the tumour was observed for a period of 21 days after which the mice were sacrificed and the tumours were excised, fixed in 10% buffered formalin and embedded in paraffin for further IHC analysis.
Statistical analysis and densitometry
All statistical calculations were done using GraphPad QuickCalcs calculator, . For the analysis of statistical significance of the H scores, U test or Student’s t test (unpaired) was used. In all the experiments a value of P <0.05 was considered as statistically significant. Image quantification and densitometric scanning of immunoblots were done using Image J software (Bethesda, MD, USA).
Canonical Wnt signaling regulates p68 expression
Canonical Wnt signaling in human colon and breast cancer cells plays a key role for the aberrant activation of the β-catenin/TCF4 in tumour progression. Like β-catenin, p68 is an important transcriptional regulator, crucial for early growth and development and is also associated with cell proliferation ,. Overexpression of p68 is common in most of the human tumours including breast . Here, we are interested in investigating the possible connections among these intensively studied oncogenes.
β-catenin and TCF4 control p68 transcription
To further support these results we have investigated this β-catenin/TCF4-mediated p68 gene regulation in both mouse 4T1 and human HEK293T cells. Here, we observed that overexpression of either β-catenin or TCF4 leads to increased expression of p68 along with Wnt targets c-Myc and Cyclin D1 (Figures 2d and e). Conversely, p68 expression was reduced in HEK293T cells transiently overexpressing dominant negative TCF4 (DN-TCF4) (Figure 2f). We have also observed p68 mRNA upregulation in TCF4 overexpressing HEK293T cells (Figure S4 in Additional file 5).
Hence, it can be stated that the transcriptional upregulation of p68 by ectopic expression of β-catenin or TCF4 indicates a direct involvement of the β-catenin/TCF4 transcription complex on p68 gene expression.
Wnt/β-catenin target c-Myc additionally contributes to p68 gene expression
Enhanced expression of both p68 and TCF4 by Wnt/β-catenin signaling constitutes a positive feedback loop in breast cancer cells
A positive feedback loop exists to regulate TCF4 expression mediated by β-catenin/p300 in the endometrial carcinoma (Em Ca) cells . Since p68 is the co-activator of β-catenin, we sought to analyse the importance of p68 in this type of feedback mechanism involving TCF4 expression. To examine this, we have selected MCF7 cells where both TCF4 and p68 expressions are low compared to other cancer cell lines.
Recruitment of β-catenin/TCF4 on p68 promoter is important for its enhanced expression
Next, to examine whether TCF4/β-catenin complex can bind directly to the endogenous p68 promoter, ChIP assay was performed with antibody against β-catenin where RNA polymerase II (Pol II) kept as a positive control. We have found occupancy of β-catenin on the p68 promoter as well as Cyclin D1 promoter (positive control) by ChIP assay in four different cancer cell lines and relatively more in the case of HCT116 and 4T1 (Figure 5f). Similarly, in case of ChIP assay with antibody against TCF4, we found enrichment of p68 as well as c-myc (Figure S6 in Additional file 7). Furthermore, binding of β-catenin and c-Myc was enhanced in Wnt3a-induced cells, keeping RPL30 promoter as the negative control (Figure 5g). Next, β-catenin was knocked down in these cells to investigate the binding of β-catenin to the p68 promoter. It is evident from our results that the occupancy of β-catenin on this promoter depends on the level of β-catenin, thus supporting the involvement of the β-catenin/TCF4 complex in p68 promoter activation (Figure 5h and Figure S6 in Additional file 7). Therefore, it indicates that the β-catenin/TCF4 complex indeed occupies the p68 promoter and the binding is enhanced when β-catenin gets activated. We have also found reduced c-Myc binding in c-myc knockdown cells when compared to the control (Figure S7 in Additional file 8). Altogether, these results highlight that Wnt signaling enhances the β-catenin/TCF4-mediated transcriptional activation of p68 promoter.
β-catenin/TCF4 mediated upregulation of p68 in breast cancer cells leads to epithelial to mesenchymal transition (EMT)
To assess the anchorage-independent growth and tumourigenic potential of Wnt3a-MCF7 cells, we conducted colony-formation assays. The results indicate that these cells form significantly more number of colonies in comparison to EV-MCF7 cells. Also, individual knockdown of β-catenin, TCF4, or p68 in Wnt3a-MCF7 cells using respective siRNAs showed that knockdown of any of these molecules resulted in reduced number of colonies, indicating that the tumour-promoting ability of β-catenin not only depends on TCF4 but also on p68 (Figure 6c). To further ascertain the role of p68 in invasion of cancer cells, a matrigel invasion assay was performed with Wnt3a-MCF7 cells. These cells showed a significantly higher invasive property compared to control cells (Figure 6d).
p68 enhances β-catenin/TCF4-dependent breast cancer progression
All these results indicate that p68 upregulation through β-catenin/TCF4 signaling is important for enhanced transactivation of β-catenin target genes and thus attributes to the tumourigenic potential of breast cancer cells.
Wnt signaling has been found to be deregulated in most cancers including breast and it plays an important role in tumour progression by upregulating various factors. β-catenin is majorly present in the cell membrane. Wnt3a induces β-catenin stabilisation and promotes its nuclear translocation ,. Several reports state the importance of p68 overexpression in cancer progression, specifically in breast cancer, which is attributed to gene locus amplification . Here, we have tried to decipher the regulation of p68 gene expression and demonstrated that β-catenin is a critical mediator for p68 expression in cancer cells. A previous study suggested a c-Myc consensus site to be present in the p68 promoter, which is involved in modulating p68 promoter activity -. The current study confirmed that the β-catenin/TCF4 complex regulates p68 promoter, and β-catenin target c-Myc is also involved in this Wnt signaling-mediated p68 gene expression. Our study, using mouse promoter and 4T1 breast cancer cells, indicates that Wnt signaling-mediated p68 gene regulation is conserved in mice. This regulation also exists in human HEK293T and other cancer cells. Thus, β-catenin stabilization and nuclear accumulation through Wnt signaling is vital for upregulation of p68 gene expression. A recent study indicates the presence of putative tcf4 sites in the rat p68 gene and assigned as novel putative target . Here, in this study, we also found the existence of putative tcf4 binding sites in both human and mouse p68 promoters. Our results from promoter analysis and ChIP assay confirm that β-catenin occupies the p68 promoter in both human and mouse cell lines and the occupancy is further increased when β-catenin is either overexpressed or induced by Wnt signaling. Hence, we have emphasized that p68 upregulation by β-catenin/TCF4 may be one of the major contributing factors in breast cancer progression.
Furthermore, we have shown that p68 being the co-activator of β-catenin is also critically involved in the Wnt signaling-mediated expression of TCF4 in breast cancer cells. Thus, p68 appears to govern the critical aspect of β-catenin nuclear function, which is the assembly of a transcription activation complex, by regulating TCF4 expression. Therefore, p68 gene regulation represents an important mechanism for controlling canonical Wnt signaling-mediated proliferation and tumourigenesis.
Nuclear import of β-catenin is another crucial phenomenon in cells in response to Wnt signaling, and it induces transition of epithelial cells to the mesenchymal phenotype and tumour invasion ,. Our study also established that constitutively overexpressed Wnt3a-MCF7 stable cells showed more tumourigenic potential than the EV-control cells. We have also shown that β-catenin/TCF4-mediated p68 regulation plays an important function in enhanced expression of EMT marker proteins in triple-negative breast cancer cells MDA-MB 231, as well as in mouse 4T1 cells. Again, our knockdown and overexpression studies support the involvement of p68 in the EMT processes, as shown earlier . We further infer that the effect of p68 on the EMT progression is, in part, mediated by its association with β-catenin/TCF4 complex. This is consistent with the fact that many target genes of Wnt/β-catenin signaling have EMT regulatory functions -, where p68 may play an important role by inducing β-catenin mediated transcription.
Our findings not only provide an improved understanding of the molecular mechanisms in the context of β-catenin and tumourigenesis but also suggest that p68 may be a crucial target for therapeutic intervention in breast cancer. Although, our data demonstrate that p68 gene regulation by Wnt/β-catenin signaling and its implication in breast cancer, further work is required to understand the p68 upregulation with reference to cancer progression and metastasis.
We would like to thank Dr. Frances V Fuller-Pace (University of Dundee) for kindly providing us pGS5-p68 construct; Dr. B. Alman (University of Toronto, Canada) for gifting us pcDNA3-TCF4 and pcDNA3-DN-TCF4 constructs; Dr. Uttara Chatterjee (IPGMER, Kolkata) for providing the human breast tumour samples and assisting in the analysis of the related data. This work is financially supported by CSIR (EMPOWER-OLP-002, miND-BSC0115 and MEDCHEM-BSC0108) grants.
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