CK2 abrogates the inhibitory effects of PRH/HHEX on prostate cancer cell migration and invasion and acts through PRH to control cell proliferation.
ABSTRACT: PRH/HHEX (proline-rich homeodomain protein/haematopoietically expressed homeobox protein) is a transcription factor that controls cell proliferation, cell differentiation and cell migration. Our previous work has shown that in haematopoietic cells, Protein Kinase CK2-dependent phosphorylation of PRH results in the inhibition of PRH DNA-binding activity, increased cleavage of PRH by the proteasome and the misregulation of PRH target genes. Here we show that PRH and hyper-phosphorylated PRH are present in normal prostate epithelial cells, and that hyper-phosphorylated PRH levels are elevated in benign prostatic hyperplasia, prostatic adenocarcinoma, and prostate cancer cell lines. A reduction in PRH protein levels increases the motility of normal prostate epithelial cells and conversely, PRH over-expression inhibits prostate cancer cell migration and blocks the ability of these cells to invade an extracellular matrix. We show that CK2 over-expression blocks the repression of prostate cancer cell migration and invasion by PRH. In addition, we show that PRH knockdown in normal immortalised prostate cells results in an increase in the population of cells capable of colony formation in Matrigel, as well as increased cell invasion and decreased E-cadherin expression. Inhibition of CK2 reduces PRH phosphorylation and reduces prostate cell proliferation but the effects of CK2 inhibition on cell proliferation are abrogated in PRH knockdown cells. These data suggest that the increased phosphorylation of PRH in prostate cancer cells increases both cell proliferation and tumour cell migration/invasion.
Project description:Cancer cells go through a process known as epithelial-mesenchymal transition (EMT) during which they acquire the ability to migrate and invade extracellular matrix. Some cells also acquire the ability to move across a layer of endothelial cells to enter and exit the bloodstream; intra- and extravasation, respectively. The transcription factor PRH/HHEX (proline-rich homeodomain/haematopoietically expressed homeobox) controls cell proliferation and cell migration/invasion in a range of cell types. Our previous work showed that PRH activity is downregulated in prostate cancer cells owing to increased inhibitory PRH phosphorylation and that this increases cell proliferation and invasion. PRH inhibits migration and invasion by prostate and breast epithelial cells in part by activating the transcription of Endoglin, a transforming growth factor ? (TGF?) co-receptor. Here we show that depletion of PRH in immortalised prostate epithelial cells results in increased extravasation in vitro. We show that blood platelets stimulate extravasation of cells with depleted PRH and that inhibition of TGF? signalling blocks the effects of platelets on these cells. Moreover, TGF? induces changes characteristic of EMT including decreased E-Cadherin expression and increased Snail expression. We show that in prostate cells PRH regulates multiple genes involved in EMT and TGF? signalling. However, both platelets and TGF? increase PRH phosphorylation. In addition, TGF? increases binding of its effector pSMAD3 to the PRH/HHEX promoter and downregulates PRH protein and mRNA levels. Thus, TGF? signalling downregulates PRH activity by multiple mechanisms and induces an EMT that facilitates extravasation and sensitises cells to TGF?.
Project description:Protein kinase CK2 promotes cell survival and the activity of this kinase is elevated in several cancers including chronic myeloid leukaemia. We have shown previously that phosphorylation of the Proline-Rich Homeodomain protein (PRH/Hhex) by CK2 inhibits the DNA-binding activity of this transcription factor. Furthermore, PRH represses the transcription of multiple genes encoding components of the VEGF-signalling pathway and thereby influences cell survival. Here we show that the inhibitory effects of PRH on cell proliferation are abrogated by CK2 and that CK2 inhibits the binding of PRH at the Vegfr-1 promoter. Phosphorylation of PRH by CK2 also decreases the nuclear association of PRH and induces its cleavage by the proteasome. Moreover, cleavage of phosphorylated PRH produces a stable truncated cleavage product which we have termed PRH?C (Hhex?C). PRH?C acts as a transdominant negative regulator of full-length PRH by sequestering TLE proteins that function as PRH co-repressors. We show that this novel regulatory mechanism results in the alleviation of PRH-mediated repression of Vegfr-1. We suggest that the re-establishment of PRH function through inhibition of CK2 could be of value in treatment of myeloid leukaemias, as well as other tumour types in which PRH is inactivated by phosphorylation.
Project description:The proline-rich homeodomain protein (PRH/Hex) regulates transcription by binding to specific DNA sequences and regulates mRNA transport by binding to translation initiation factor eIF4E. Protein kinase CK2 plays multiple roles in the regulation of gene expression and cell proliferation. Here, we show that PRH interacts with the beta subunit of CK2 in vitro and in cells and that CK2 phosphorylates PRH. Phosphorylation of PRH by CK2 inhibits the DNA binding activity of this protein and dephosphorylation restores DNA binding indicating that this modification acts as a reversible switch. We show that phosphorylation of the homeodomain is sufficient to block DNA binding and we identify two amino acids within this the domain that are phosphorylated by CK2: S163 and S177. Site-directed mutagenesis demonstrates that mutation of either of these residues to glutamic acid partially mimics phosphorylation but is insufficient to completely block DNA binding whereas an S163E/S177E double mutation severely inhibits DNA binding. Significantly, the S163E and S177E mutations and the S163E/S177E double mutation all inhibit the ability of PRH to regulate transcription in cells. Since these amino acids are conserved between many homeodomain proteins, our results suggest that CK2 may regulate the activity of several homeodomain proteins in this manner.
Project description:Breast tumours progress from hyperplasia to ductal carcinoma in situ (DCIS) and invasive breast carcinoma (IBC). PRH/HHEX (proline-rich homeodomain/haematopoietically expressed homeobox) is a transcription factor that displays both tumour suppressor and oncogenic activity in different disease contexts; however, the role of PRH in breast cancer is poorly understood. Here we show that nuclear localization of the PRH protein is decreased in DCIS and IBC compared with normal breast. Our previous work has shown that PRH phosphorylation by protein kinase CK2 prevents PRH from binding to DNA and regulating the transcription of multiple genes encoding growth factors and growth factor receptors. Here we show that transcriptionally inactive phosphorylated PRH is elevated in DCIS and IBC compared with normal breast. To determine the consequences of PRH loss of function in breast cancer cells, we generated inducible PRH depletion in MCF-7 cells. We show that PRH depletion results in increased MCF-7 cell proliferation in part at least due to increased vascular endothelial growth factor signalling. Moreover, we demonstrate that PRH depletion increases the formation of breast cancer cells with cancer stem cell-like properties. Finally, and in keeping with these findings, we show that PRH overexpression inhibits the growth of mammary tumours in mice. Collectively, these data indicate that PRH plays a tumour suppressive role in the breast and they provide an explanation for the finding that low PRH mRNA levels are associated with a poor prognosis in breast cancer.
Project description:PRH (proline-rich homeodomain protein)/Hex is important in the control of cell proliferation and differentiation. We have shown previously that PRH contains two domains that can bring about transcriptional repression independently; the PRH homeodomain represses transcription by binding to TATA box sequences, whereas the proline-rich N-terminal domain can repress transcription by interacting with members of the Groucho/TLE (transducin-like enhancer of split) family of co-repressor proteins. The proteasome is a multi-subunit protein complex involved in the processing and degradation of proteins. Some proteasome subunits have been suggested to play a role in the regulation of transcription. In the present study, we show that PRH interacts with the HC8 subunit of the proteasome in the context of both 20 and 26 S proteasomes. Moreover, we show that PRH is associated with the proteasome in haematopoietic cells and that the proline-rich PRH N-terminal domain is responsible for this interaction. Whereas PRH can be cleaved by the proteasome, it does not appear to be degraded rapidly in vitro or in vivo, and the proteolytic activity of the proteasome is not required for transcriptional repression by PRH. However, proteasomal digestion of PRH can liberate truncated PRH proteins that retain the ability to bind to DNA. We discuss these findings in terms of the biological role of PRH in gene regulation and the control of cell proliferation.
Project description:The PRH (proline-rich homeodomain) [also known as Hex (haematopoietically expressed homeobox)] protein is a critical regulator of vertebrate development. PRH is able to regulate cell proliferation and differentiation and is required for the formation of the vertebrate body axis, the haematopoietic and vascular systems and the formation of many vital organs. PRH is a DNA-binding protein that can repress and activate the transcription of its target genes using multiple mechanisms. In addition, PRH can regulate the nuclear transport of specific mRNAs making PRH a member of a select group of proteins that control gene expression at the transcriptional and translational levels. Recent biophysical analysis of the PRH protein has shown that it forms homo-oligomeric complexes in vivo and in vitro and that the proline-rich region of PRH forms a novel dimerization interface. Here we will review the current literature on PRH and discuss the complex web of interactions centred on this multifunctional protein.
Project description:Endothelin-converting enzyme-1c (ECE-1c) is a membrane metalloprotease involved in endothelin-1 synthesis, which has been shown in vitro to have a role in breast, ovary and prostate cancer cell invasion. N-terminal end of ECE-1c displays three putative phosphorylation sites for the protein kinase CK2. We studied whether CK2 phosphorylates N-terminal end of ECE-1c as well as whether this has a role in migration and invasion of colon cancer cells. CK2 phosphorylated the N-terminal end of ECE-1c and this was precluded upon inhibition of CK2. Inhibition also led to diminished protein levels of both endogen ECE-1 or GFP-fused N-terminal end of ECE-1c in 293T embryonic and DLD-1 colon cancer cells, which highlighted the importance of this motif on UPS-dependent ECE-1c degradation. Full-length ECE-1c mutants designed either to mimic or abrogate CK2-phosphorylation displayed increased or decreased migration/invasion of colon cancer cells, respectively. Moreover, ECE-1c overexpression or its silencing with a siRNA led to increased or diminished cell migration/invasion, respectively. Altogether, these data show that CK2-increased ECE-1c protein stability is related to augmented migration and invasion of colon cancer cells, shedding light on a novel mechanism by which CK2 may promote malignant progression of this disease.
Project description:Inflammation-induced carcinogenesis is associated with increased proliferation and migration/invasion of various types of tumor cells. In this study, altered ?-catenin signaling upon TNF? exposure, and relation to loss of function of the tumor suppressor NKX3.1 was examined in prostate cancer cells. We used an in vitro prostate inflammation model to demonstrate altered sub-cellular localization of ?-catenin following increased phosphorylation of Akt(S473) and GSK3?(S9). Consistently, we observed that subsequent increase in ?-catenin transactivation enhanced c-myc, cyclin D1 and MMP2 expressions. Consequently, it was also observed that the ?-catenin-E-cadherin association at the plasma membrane was disrupted during acute cytokine exposure. Additionally, it was demonstrated that disrupting cell-cell interactions led to increased migration of LNCaP cells in real-time migration assay. Nevertheless, ectopic expression of NKX3.1, which is degraded upon proinflammatory cytokine exposure in inflammation, was found to induce the degradation of ?-catenin by inhibiting Akt(S473) phosphorylation, therefore, partially rescued the disrupted ?-catenin-E-cadherin interaction as well as the cell migration in LNCaP cells upon cytokine exposure. As, the disrupted localization of ?-catenin at the cell membrane as well as increased Akt(S308) priming phosphorylation was observed in human prostate tissues with prostatic inflammatory atrophy (PIA), high-grade prostatic intraepithelial neoplasia (H-PIN) and carcinoma lesions correlated with loss of NKX3.1 expression. Thus, the data indicate that the ?-catenin signaling; consequently sub-cellular localization is deregulated in inflammation, associates with prostatic atrophy and PIN pathology.
Project description:In order to establish a profile of gene expression of tumours overexpressing PRH we performed agilient microarray analysis on four 4T1 tumour samples engineered to overexpress PRH, and four control tumours with normal PRH expression. cancer, colorectal metastasis to the liver and matched healthy tissues. The tumours had recieved no treatement prior to resection. The expression profiles were compared by one colour microarray analysis between PRH overexpression and control tumours. Genes consistently differentially expressed between the groups were taken forward for further analysis. Preparation of the tumours: 4T1 cells were infected with Adenoviral-PRH or empty Adenovirus at MOI 50 for 24 hrs. Adenoviral constructs described in Kershaw, R.M., et al., PRH/HHex inhibits the migration of breast and prostate epithelial cells through direct transcriptional regulation of Endoglin. Oncogene, 2014. 33(49): p. 5592-600. Cells were washed 4x with PBS, trypsinised, washed and resuspended in Opti-mem (Invitrogen) at 1.25x106 cells/ml and kept on ice for 20-30 min before injection. Aliquots 200 μl / 2.5x105 of the cells were injected into the no. 3 fat pad of 6-8 week old female BALB/C mice. Total RNA was extracted from each tumour using the Qiagen miRNAeasy kit, processed into cRNA using the Agilient low input, one colour labelling kit, hybridised onto the 8 x 66k whole human genome expression microarrays. Data was normalised in R64 and comparison matrices between matched Tumour and Healthy endothelum were constructed. Genes upregualated in all the comparisons were taken forward for further analysis.
Project description:The proline-rich homeodomain protein, PRH/HEX, participates in the early development of the brain, thyroid, and liver and in the later regenerative processes of damaged liver, vascular endothelial, and hematopoietic cells. A virulent strain of lymphocytic choriomeningitis virus (LCMV-WE) that destroys hematopoietic, vascular, and liver functions also alters the transcription and subcellular localization of PRH. A related virus (LCMV-ARM) that does not cause disease in primates can infect cells without affecting PRH. Biochemical experiments demonstrated the occurrence of binding between the viral RING protein (Z) and PRH, and genetic experiments mapped the PRH-suppressing phenotype to the large (L) segment of the viral genome, which encodes the Z and polymerase genes. The Z protein is clearly involved with PRH, but other viral determinants are needed to relocate PRH and to promote disease. By down-regulating PRH, the arenavirus is able to eliminate the antiproliferative effects of PRH and to promote liver cell division. The interaction of an arenavirus with a homeodomain protein suggests a mechanism for viral teratogenic effects and for the tissue-specific manifestations of arenavirus disease.