casein-kinase-ii and Breast-Neoplasms

Structurally, protein kinase CK2 consists of two catalytic subunits (α and α') and two regulatory subunits (β), which play a critical role in targeting specific CK2 substrates. Compelling evidence shows the complexity of the CK2 cellular signaling network and supports the view that this enzyme is a key component of regulatory protein kinase networks that are involved in several aspects of cancer. CK2 both activates and suppresses the expression of a number of essential oncogenes and tumor suppressors, and its expression and activity are upregulated in blood tumors and virtually all solid tumors. The prognostic significance of CK2α expression in association with various clinicopathological parameters highlighted this kinase as an adverse prognostic marker in breast cancer. In addition, several recent studies reported its implication in the regulation of the epithelial-to-mesenchymal transition (EMT), an early step in cancer invasion and metastasis. In this review, we briefly overview the contribution of CK2 to several aspects of cancer and discuss how in mammary epithelial cells, the expression of its CK2β regulatory subunit plays a critical role in maintaining an epithelial phenotype through CK2-mediated control of key EMT-related transcription factors. Importantly, decreased CK2β expression in breast tumors is correlated with inefficient phosphorylation and nuclear translocation of Snail1 and Foxc2, ultimately leading to EMT induction. This review highlights the pivotal role played by CK2β in the mammary epithelial phenotype and discusses how a modest alteration in its expression may be sufficient to induce dramatic effects facilitating the early steps in tumor cell dissemination through the coordinated regulation of two key transcription factors.

Topics: Breast Neoplasms; Casein Kinase II; Epithelial-Mesenchymal Transition; Female; Forkhead Transcription Factors; Gene Expression Regulation, Neoplastic; Humans; Signal Transduction; Snail Family Transcription Factors; Transcription Factors

CK2 is upregulated in rapidly dividing cells including most human tumours. Transgenic overexpression of CK2 in lymphoid or mammary lineages predisposes to transformation. Multiple signalling and oncogene pathways could be regulated by CK2 in this process. Our studies suggest that phosphorylation of critical oncogenes by CK2, as well as by other serine-threonine kinases, regulates their stability via susceptibility to the proteasomal degradation system. Beta-catenin is a transcriptional co-factor in the Wnt signalling pathway that is regulated in this fashion. Inactivating mutations in the adenomatosis polyposis coli (APC) gene, which encodes a carrier protein for beta-catenin, or stabilizing mutations in beta-catenin itself, frequently occur in human tumours. CK2 and the monomeric serine-threonine kinase GSK3 have opposing actions on beta-catenin: GSK-3 phosphorylation of the N-terminus of beta-catenin promotes degradation; while phosphorylation by CK2 in the armadillo repeat protein interaction domain protects it. Beta-catenin is overexpressed in mammary tumours occurring in mice transgenic for CK2 or a dominant negative form of GSK3, and also in mammary tumours arising following treatment with the environmental carcinogen DMBA. Experiments are underway to determine whether expression of both CK2 and kinase inactive GSK3 further accelerates tumorigenesis. Inhibitors of GSK3 under development for treatment of diabetes could promote tumours, while CK2 inhibitors should be useful agents for treatment of cancer.

Topics: Animals; beta Catenin; Breast Neoplasms; Casein Kinase II; Female; Glycogen Synthase Kinase 3; Humans; Mutation; Phosphorylation; Protein Structure, Tertiary; Signal Transduction; Wnt Proteins

Mounting evidence demonstrates that long noncoding RNAs (lncRNAs) have critical roles in the initiation and progression of cancer. Here, we report that small nucleolar RNA host gene 3 (SNHG3) is a key regulator of breast cancer progression. We analyzed RNA sequencing data to explore abnormally expressed lncRNAs in breast cancer. The effects of SNHG3 on breast cancer were investigated via

Topics: Breast Neoplasms; Casein Kinase II; Cell Line, Tumor; Cell Proliferation; Female; Gene Expression Regulation, Neoplastic; Humans; In Situ Hybridization, Fluorescence; MicroRNAs; RNA, Long Noncoding

Casein kinase 2 (CK2) plays a critical role in the proliferation and apoptosis of cancer cells. Resveratrol is a bioactive compound with anticancer and anti-inflammatory effects. This study investigated the pro-oxidant cytotoxic effects of resveratrol in association with the inhibition of CK2 activity on human breast carcinoma cells MCF-7. We showed that resveratrol and TBB, an inhibitor of CK2, decreased cell viability in a concentration dependent manner with an IC

Topics: Antineoplastic Agents; Apoptosis; Breast Neoplasms; Casein Kinase II; Female; Humans; MCF-7 Cells; Reactive Oxygen Species; Resveratrol

Overexpression of the ubiquitous protein kinase, CK2α, has been reported in various human cancers. Here, we demonstrate that nuclear and nucleolar CK2α localization in invasive ductal carcinomas of the breast is a reliable predictor of poor prognosis. Cellular localization of CK2α in nuclei and nucleoli was analyzed immunohistochemically using surgical tissue blocks from 112 patients, who had undergone surgery without neoadjuvant chemotherapy. Clinical data collection and median follow-up period were for more than 5 y. In total, 93.8% of patients demonstrated elevated CK2α expression in nuclei and 36.6% of them displayed elevated expression predominantly in nucleoli. Clinicopathological malignancy was strongly correlated with elevated nuclear and nucleolar CK2α expression. Recurrence-free survival was significantly worse (P = .0002) in patients with positive nucleolar CK2α staining. The 5-y survival rate decreased to a roughly 50% in nucleolar CK2α-positive patients of triple-negative (P = .0069) and p Stage 3 (P = .0073) groups. In contrast, no patients relapsed or died in the triple-negative group who exhibited a lack of nucleolar CK2α staining. Evaluation of nucleolar CK2α staining showed a high secondary index with a hazard ratio of 6.629 (P = .001), following lymph node metastasis with a hazard ratio of 14.30 (P = .0008). Multivariate analysis demonstrated that nucleolar CK2α is an independent factor for recurrence-free survival. Therefore, we propose that histochemical evaluation of nucleolar CK2α-positive staining may be a new and robust prognostic indicator for patients who need further treatment. Functional consequences of nucleolar CK2 dysfunction may be a starting point to facilitate development of novel treatments for invasive breast carcinoma.

Topics: Adult; Aged; Aged, 80 and over; Biomarkers, Tumor; Breast Neoplasms; Carcinoma, Ductal, Breast; Casein Kinase II; Cell Nucleolus; Cell Nucleus; Female; Humans; MCF-7 Cells; Middle Aged; Prognosis

The complexity of intracellular signalling requires both a diversity of molecular players and the sequestration of activity to unique compartments within the cell. Recent findings on the role of liquid-liquid phase separation provide a distinct mechanism for the spatial segregation of proteins to regulate signalling pathway crosstalk. Here, we discover that DACT1 is induced by TGFβ and forms protein condensates in the cytoplasm to repress Wnt signalling. These condensates do not localize to any known organelles but, rather, exist as phase-separated proteinaceous cytoplasmic bodies. The deletion of intrinsically disordered domains within the DACT1 protein eliminates its ability to both form protein condensates and suppress Wnt signalling. Isolation and mass spectrometry analysis of these particles revealed a complex of protein machinery that sequesters casein kinase 2-a Wnt pathway activator. We further demonstrate that DACT1 condensates are maintained in vivo and that DACT1 is critical to breast and prostate cancer bone metastasis.

Topics: Adaptor Proteins, Signal Transducing; Animals; Bone Neoplasms; Breast Neoplasms; Casein Kinase II; Cell Line, Tumor; Cell Movement; Cell Proliferation; Female; Gene Expression Regulation, Neoplastic; HEK293 Cells; Humans; Male; Mice, Inbred NOD; Mice, Nude; Mice, SCID; Neoplasm Invasiveness; Nuclear Proteins; Prostatic Neoplasms; Transforming Growth Factor beta; Wnt Signaling Pathway; Wnt3A Protein

Protein kinase CK2 has been considered as an attractive drug target for anti-cancer therapy. The synthesis of

Topics: Antineoplastic Agents; Apoptosis; Benzimidazoles; Breast Neoplasms; Casein Kinase II; Cell Proliferation; Female; Humans; MCF-7 Cells; Protein Kinase Inhibitors; Structure-Activity Relationship

The combination effect of 5-fluorouracil (5-FU) with either CX-4945 or a new inhibitor of protein kinase CK2, namely 14B (4,5,6,7-tetrabromo-1-(3-bromopropyl)-2-methyl-1. Combination index (CI) values were determined using an MTT-based assay and the Chou-Talalay model. The effect of the tested drug combinations on pro-apoptotic properties and cell cycle progression was examined using flow cytometry. The activation of FAK, p38 MAPK, and ERK1/2 kinases and the expression of selected pro-apoptotic markers in MDA-MB-231 cell line after the combined treatment were evaluated by the western blot method. Confocal microscopy was used to examine actin network in MDA-MB-231.. Our results showed that a synergistic effect (CI < 1) occurred in MDA-MB-231 after treatment with both combinations of 5-FU with 14B or CX-4945, whereas the combination of 5-FU and 14B evoked an antagonistic effect in MCF-7. We conclude that the synergistic interactions (CI < 1) observed for both the combinations of 5-FU and 14B or CX-4945 in MDA-MB-231 correlated with an activation of p38 MAPK, inhibition of FAK, increased expression of apoptogenic markers, prolongation of S-phase of cell cycle, and destabilization of actin network.. The obtained results support the recent observation that CK2 inhibitors can improve 5-FU-based anticancer therapy and FAK kinase can be an attractive molecular target in breast cancer therapy.

Topics: Breast Neoplasms; Casein Kinase II; Cell Line, Tumor; Cell Survival; Down-Regulation; Drug Synergism; Female; Fluorouracil; Focal Adhesion Kinase 1; Gene Expression Regulation, Neoplastic; Humans; MCF-7 Cells; Naphthyridines; p38 Mitogen-Activated Protein Kinases; Phenazines; Protein Kinase Inhibitors

CK2 is a serine/threonine kinase that is overexpressed in breast cancer and its inhibition is associated to reduced tumor growth and disease progression. CIGB-300 is an antitumor peptide with a novel mechanism of action, since it binds to protein kinase CK2 catalytic subunit alpha and to CK2 substrates thus preventing the enzyme activity. Our aim was to evaluate the potential therapeutic benefits of CIGB-300 on breast cancer disease using experimental models with translational relevance. We demonstrated that CIGB-300 reduces breast cancer cell growth in MDA-MB-231, MCF-7 and F3II cells, exerting a pro-apoptotic action and cell cycle arrest. We also found that CIGB-300 decreased cell adhesion, migration and clonogenic capacity of malignant cells. Effect on experimental breast cancer lung metastasis was evaluated after surgical removal of primary F3II tumors or after tail vein injection of tumor cells, also we evaluated CIGB-300 effect on spontaneous lung metastasis in an orthotopic model. Systemic CIGB-300 treatment inhibited breast cancer colonization of the lung, reducing the size and number of metastatic lesions. The present preclinical study establishes for the first time the efficacy of CIGB-300 on breast cancer. These encouraging results suggest that CIGB-300 could be used for the management of breast cancer as an adjuvant therapy after surgery, limiting tumor metastatic spread and thus protecting the patient from distant recurrence.

Topics: Animals; Antineoplastic Agents; Apoptosis; Breast Neoplasms; Casein Kinase II; Cell Line, Tumor; Cell Proliferation; Female; Humans; Lung Neoplasms; MCF-7 Cells; Mice, Inbred BALB C; Neoplasm Invasiveness; Peptides, Cyclic; Protein Kinase Inhibitors

The intracellular zinc profiles of breast and prostate cancer cells are diametrically opposed, with hyper-accumulation of zinc in breast cancer, and low level in prostate cancer. This phenomenon is poorly understood. This study employs two breast and two prostate cancer cell lines to investigate the role of protein kinase CK2 in regulating zinc homeostasis. CK2 was targeted by its specific inhibitors 4,5,6,7-tetrabromobenzotriazole (TBB) and CX-4945, and by the specific siRNA against each of the three CK2 genes. The effect of zinc exposure after the above CK2 manipulation was observed by MTT [3-(4,5-dimethyliazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide] cell viability assay and confocal microscopy for intracellular zinc level. The results demonstrate that CK2 is involved in regulating zinc homeostasis in breast and prostate cancer cells as both TBB and CX-4945 substantially decreased cell viability upon zinc exposure. siRNA-mediated knockdown of the three CK2 subunits (α, α' and β) revealed their discrete roles in regulating zinc homeostasis in breast and prostate cancer cells. Knockdown of CK2α' decreased the intracellular zinc level of breast cancer cells and in turn increased the cell viability while the opposite findings were obtained for the prostate cancer cells. Knockdown of CK2β expression substantially increased the zinc level in breast cancer cell lines whilst decreased the zinc level in prostate cancer cells. Taken together, this study shows that CK2 is involved in zinc homeostasis of breast and prostate cancer cells and opens a new avenue for research on these cancers.

Topics: Antineoplastic Agents; Breast Neoplasms; Casein Kinase II; Cell Survival; Dose-Response Relationship, Drug; Drug Screening Assays, Antitumor; Female; Homeostasis; Humans; Male; MCF-7 Cells; Prostatic Neoplasms; Structure-Activity Relationship; Tumor Cells, Cultured; Zinc Sulfate

Transforming growth factor β1 (TGF β1) is a potent regulator of breast tumorigenesis. It inhibits proliferation of carcinoma cells, but the strength of its inhibitory action varies for cells from benigh, non-metastatic or metastatic tumors. The aim of this work was to generate a proteome profile of TGF β1 action on non-tumorigenic human breast epithelial cells 184A1, and validate predicted involvement of casein kinase 2α (CK2α), p53 and structure-specific recognition protein-1 (SSRP1).. Two-dimensional gel electrophoresis and mass spectrometry were used to identify TGF β1-regulated proteins in 184A1 human breast immortalized non-tumorigenic cells. 184A1 cells may serve as a model of benign breast neoplasia. These cells were obtained from normal mammary tissue, were immortalized but are not malignant, and were obtained from the American Type Culture Collection. The systemic analysis was performed by using the Cytoscape tool. Transfection of cells with CK2α construct and small interfering RNAs to CK2α and SSRP1 were used to assess an impact of CK2α and SSRP1 on phosphorylation of the p53 and cell proliferation.. Proliferation of 184A1 cells was transiently inhibited by TGF β1. We identified 100 and 47 unique proteins which changed their expression and/or. We report here the list of 147 TGF β1-regulated proteins in immortalized non-tumorigenic human breast epithelial cells, and show involvement of CK2α in the regulation of p53 Ser392 phosphorylation.

Topics: Breast; Breast Neoplasms; Casein Kinase II; Cell Line; Cell Movement; Cell Proliferation; Epithelial Cells; Female; Humans; Phosphorylation; Protein Interaction Maps; Proteomics; Signal Transduction; Transforming Growth Factor beta1; Tumor Suppressor Protein p53

Dysfunction of p53 is a key cause of cancer development, while CCDC106 can reduce p53 stability and is associated with lung cancer. However, the roles of CCDC106 in other cancer types and its upstream regulators have not been investigated.. The phosphorylation status was investigated by in vitro kinase assay and Western blotting using phosphorylation-specific antibodies. Co-immunoprecipitation assay and GST-pulldown were used to detect protein interaction. Cell viability, apoptosis, colony formation, wound-healing and invasion assays were measured for in vitro functional analyses. The in vivo effect of CCDC106 on tumor growth was investigated using a subcutaneous xenograft tumor mouse model.. We demonstrated that CCDC106 knockdown enhanced apoptosis by stabilizing p53 and suppressed cell viability, colony formation, migration and invasion in cervical cancer HeLa and breast cancer MCF7 cells with wild-type p53 (wtp53), whereas CCDC106 overexpression exerted the opposite effects in normal breast epithelial HBL100 and cervical cancer SiHa cells with wtp53. However, CCDC106 had no similar effects on p53-mutant cervical and breast cancer cells (C33A and MDA-MB-231). Further study showed that CK2 interacts with CCDC106 through its regulatory β subunit and then phosphorylates CCDC106 at Ser-130 and Ser-147. The phosphorylation of CCDC106 at Ser-130 and Ser-147 is required for its interaction with p53 and nuclear localization, respectively. Inhibiting CCDC106 phosphorylation by substituting both Ser-130 and Ser-147 with alanine or treating cells with the CK2 inhibitor CX-4945 abrogated CCDC106-induced p53 degradation and its oncogenic function in cells with wtp53. Wildtype CCDC106, but not Ser-130/- 147 mutant CCDC106, enhanced tumor growth and p53 degradation in a xenograft mouse model. Moreover, suppression of CCDC106 increased CX-4945 sensitivity of cancer cells with wtp53.. This study revealed a CK2/CCDC106/p53 signaling axis in the progression of breast and cervical cancers, which may provide a new therapeutic target for cancer treatment.

Topics: Animals; Breast Neoplasms; Carrier Proteins; Casein Kinase II; Cell Line, Tumor; Cell Proliferation; Disease Progression; Female; Gene Knockdown Techniques; HEK293 Cells; HeLa Cells; Heterografts; Humans; MCF-7 Cells; Mice; Mice, Inbred BALB C; Mice, Nude; Phosphorylation; Transfection; Tumor Suppressor Protein p53; Uterine Cervical Neoplasms

Protein kinase CK2 was recently identified as a promising therapeutic target for combination therapy. Our study aims to investigate the anticancer effect of a simultaneous inhibition of thymidylate synthase (TS) and CK2 in MCF-7 breast cancer and CCRF-CEM leukemia cells.. The type of interaction between CK2 inhibitors: CX-4945, 4,5,6,7-tetrabromo-1H-benzimidazole (TBBi), or recently obtained 4,5,6,7-tetrabromo-2-methyl-1H-benzimidazol-1-yl)acetonitrile (2b) and TS-directed anticancer drug, 5-fluorouracil (5-FU) was determined using the MTT assay and a combination index method. The influence of the combined treatment on apoptosis in leukemia cells, as well as on cell-cycle progression and the levels of TS, CK2α and P-Ser529-p65 were determined in both cell lines, using flow cytometry and western blot analysis, respectively.. The best synergistic effect was observed in CCRF-CEM cell line with the combination of 5-FU and 2b which correlated with a decrease in the endocellular CK2 activity and enhancement of the pro-apoptotic effect.. The obtained results demonstrate the ability of CK2 inhibitors to enhance the efficacy of 5-FU in anticancer treatment, indicating a different molecular mechanism of the studied CK2 inhibitors interaction with 5-FU.

Topics: Antineoplastic Agents; Apoptosis; Benzimidazoles; Breast Neoplasms; Casein Kinase II; Cell Line, Tumor; Female; Fluorouracil; Humans; Leukemia; MCF-7 Cells; Naphthyridines; Phenazines; Protein Kinase Inhibitors; Thymidylate Synthase

Protein kinase CK2α is frequently upregulated in different cancers. Alteration of CK2α expression and its activity is sufficient to induce dramatic changes in cell fate. It has been established that CK2α induces oncogenesis through modulation of both AKT and PML. CK2α has been found to be overexpressed in breast cancer. In contrary, statistical reports have shown low level of PML. However, the regulation of CK2α gene expression is not fully understood. In the current study, we found that CK2α and activated AKT positively correlate with ERα, whereas PML follows an inverse correlation in human breast cancer tissues. Modulation of ERα signalling leads to recruitment of activated ERα on the ERE sites of CK2α promoter, resulting in CK2α transactivation. Furthermore, the DMBA induced tumours in rat showed elevated level of active CK2α. Consequently it mediates enhancement of AKT activity and PML degradation, resulting in increased cellular proliferation, migration and metastasis. Syngeneic ERα overexpressing stable mouse 4T1 cells produce larger primary tumours and metastatic lung nodules in mice, corroborating our in vitro findings. Hence, our study provides a novel route of ERα dependent CK2α mediated oncogenesis that causes upregulation and consequent AKT activation along with degradation of tumour suppressor PML.

Topics: Animals; Breast Neoplasms; Carcinogenesis; Carcinogens; Casein Kinase II; Cell Line, Tumor; Estrogen Receptor alpha; Female; Gene Expression Regulation, Neoplastic; HEK293 Cells; Humans; Lung Neoplasms; Mammary Neoplasms, Experimental; MCF-7 Cells; Mice; Mice, Inbred BALB C; Nuclear Proteins; Promoter Regions, Genetic; Promyelocytic Leukemia Protein; Proto-Oncogene Proteins c-akt; Rats; Rats, Sprague-Dawley; Signal Transduction; Transcription Factors; Transcriptional Activation; Tumor Suppressor Proteins

We previously showed that protein kinase CK2 downregulation mediates senescence through the reactive oxygen species (ROS)-p53-p21(Cip1/WAF1) pathway in various human cells. In the present study, we investigated whether the FoxO3a transcription factor is associated with ROS production during CK2 downregulation-induced senescence in human colon cancer HCT116 and breast cancer MCF-7 cells. FoxO3a overexpression suppressed ROS production and p53 stabilization induced by a CK2α knockdown. CK2α downregulation induced nuclear export of FoxO3a through stimulation of AKT-mediated phosphorylation of FoxO3a and decreased transcription of its target genes (Cu/ZnSOD, MnSOD, and catalase). In contrast, CK2α overexpression inhibited AKT-mediated FoxO3a phosphorylation. This resulted in nuclear accumulation of FoxO3a, and elevated expression of its target genes. Therefore, these data indicate for the first time that CK2 downregulation stimulates ROS generation by inhibiting FoxO3a during premature senescence in human colon and breast cancer cells.

Topics: Active Transport, Cell Nucleus; Breast; Breast Neoplasms; Casein Kinase II; Cellular Senescence; Colon; Colonic Neoplasms; Down-Regulation; Female; Forkhead Box Protein O3; Gene Expression Regulation, Neoplastic; HCT116 Cells; Humans; MCF-7 Cells; Proto-Oncogene Proteins c-akt; Reactive Oxygen Species; Up-Regulation

Recently, the roles of sirtuins (SIRTs) in tumorigenesis have been of interest to oncologists, and protein kinase CK2 α1 (CSNK2A1) has been shown to be involved in tumorigenesis by phosphorylating various proteins, including SIRT1. Therefore, we evaluated the roles of CSNK2A1, SIRT6, and phosphorylated SIRT6 and their relationships in breast carcinoma. Nuclear expression of CSNK2A1 and SIRT6 predicted shorter overall survival and relapse-free survival by multivariate analysis. Inhibition of CSNK2A1 decreased the proliferative and invasive activity of cancer cells. In addition, CSNK2A1 was bound to SIRT6 and phosphorylated SIRT6; evidence for this is provided from immunofluorescence staining, co-immunoprecipitation of CSNK2A1 and SIRT6, a glutathione S-transferase pull-down assay, an in vitro kinase assay, and transfection of mutant CSNK2A1. Knockdown of SIRT6 decreased the proliferation and invasiveness of cancer cells. Overexpression of SIRT6 increased proliferation, but mutation at the Ser338 phosphorylation site of SIRT6 inhibited the proliferation of MCF7 cells. Moreover, both knockdown of SIRT6 and a mutation at the phosphorylation site of SIRT6 decreased expression of matrix metallopeptidase 9, β-catenin, cyclin D1, and NF-κB. Especially, SIRT6 expression was associated with the nuclear localization of β-catenin. This study demonstrates that CSNK2A1 and SIRT6 are indicators of poor prognosis for breast carcinomas and that CSNK2A1-mediated phosphorylation of SIRT6 might be involved in the progression of breast carcinoma.

Topics: beta Catenin; Breast Neoplasms; Casein Kinase II; Cell Proliferation; Cyclin D1; Disease Progression; Gene Expression; Gene Expression Regulation, Neoplastic; Humans; Mutation; NF-kappa B; Phosphorylation; Prognosis; Sirtuins

A series of indeno[1,2-b]indole-9,10-dione derivatives were synthesized as human casein kinase II (CK2) inhibitors. The most potent inhibitors contained a N(5)-isopropyl substituent on the C-ring. The same series of compounds was found to also inhibit the breast cancer resistance protein ABCG2 but with totally different structure-activity relationships: a N(5)-phenethyl substituent was critical, and additional hydrophobic substituents at position 7 or 8 of the D-ring or a methoxy at phenethyl position ortho or meta also contributed to inhibition. The best ABCG2 inhibitors, such as 4c, 4h, 4i, 4j, and 4k, behaved as very weak inhibitors of CK2, whereas the most potent CK2 inhibitors, such as 4a, 4p, and 4e, displayed limited interaction with ABCG2. It was therefore possible to convert, through suitable substitutions of the indeno[1,2-b]indole-9,10-dione scaffold, potent CK2 inhibitors into selective ABCG2 inhibitors and vice versa. In addition, some of the best ABCG2 inhibitors, which displayed a very low cytotoxicity, thus giving a high therapeutic ratio, and appeared not to be transported, constitute promising candidates for further investigations.

Topics: ATP Binding Cassette Transporter, Subfamily G, Member 2; ATP-Binding Cassette Transporters; Biological Transport; Breast Neoplasms; Casein Kinase II; Cell Proliferation; Cell Survival; Dose-Response Relationship, Drug; Drug Resistance, Neoplasm; Female; HEK293 Cells; Humans; Indoles; MCF-7 Cells; Mitoxantrone; Models, Chemical; Molecular Structure; Neoplasm Proteins; Protein Kinase Inhibitors

Nuclear Foxc2 is a transcriptional regulator of mesenchymal transformation during developmental epithelial-mesenchymal transition (EMT) and has been associated with EMT in malignant epithelia. Our laboratory has shown that in normal epithelial cells Foxc2 is maintained in the cytoplasm where it promotes an epithelial phenotype. The Foxc2 amino terminus has a consensus casein kinase 2 (CK2) phosphorylation site at serine 124, and we now show that CK2 associates with Foxc2 and phosphorylates this site in vitro. Knockdown or inhibition of the CK2α/α' kinase subunit in epithelial cells causes de novo accumulation of Foxc2 in the nucleus. Mutation of serine 124 to leucine promotes constitutive nuclear localization of Foxc2 and expression of mesenchymal genes, whereas an S124D phosphomimetic leads to constitutive cytoplasmic localization and epithelial maintenance. In malignant breast cancer cells, the CK2β regulatory subunit is downregulated and FOXC2 is found in the nucleus, correlating with an increase in α-smooth muscle actin (SMA) expression. Restoration of CK2β expression in these cells results in cytoplasmic localization of Foxc2, decreased α-SMA expression and reduced cell migration and invasion. In contrast, knockdown of CK2β in normal breast epithelial cells leads to FOXC2 nuclear localization, decreased E-cadherin expression, increased α-SMA and vimentin expression, and enhanced cell migration and invasion. Based on these findings, we propose that Foxc2 is functionally maintained in the cytoplasm of normal epithelial cells by CK2α/α'-mediated phosphorylation at serine 124, which is dependent on proper targeting of the holoenzyme via the CK2β regulatory subunit.

Topics: Breast; Breast Neoplasms; Cadherins; Casein Kinase II; Cell Movement; Cytoplasm; Epithelial Cells; Epithelial-Mesenchymal Transition; Female; Forkhead Transcription Factors; Gene Expression Regulation, Neoplastic; Humans; Vimentin

The heterotetrameric protein kinase CK2 has been associated with oncogenic transformation, and our previous studies have shown that it may affect estrogenic signaling. Here, we investigate the role of the protein kinase CK2 in regulating ERα (estrogen receptor α) signaling in breast cancer. We determined the correlation of CK2α expression with relapse free breast cancer patient survival utilizing Kaplan Meier Plotter (kmplot.com/analysis/) to mine breast cancer microarrays repositories. Patients were stratified according to ERα status, histological grade, and hormonal therapy. Luciferase reporter assays and flow cytometry were implemented to determine the impact of CK2 inhibition on ERE-mediated gene expression and expression of ERα protein. CK2α expression is associated with shorter relapse free survival among ERα (+) patients with grade 1 or 2 tumors, as well as among those patients receiving hormonal therapy. Biochemical inhibition of CK2 activity results in increased ER-transactivation as well as increased expression among ERα (+) and ERα (-) breast cancer cell lines. These findings suggest that CK2 may contribute to estrogen-independent cell proliferation and breast tumor progression, and may potentially serve as a biomarker and pharmacological target in breast cancer.

Topics: Biomarkers, Tumor; Breast Neoplasms; Casein Kinase II; Cell Line, Tumor; Disease-Free Survival; Estrogen Receptor alpha; Female; Humans; Kaplan-Meier Estimate; Neoplasm Recurrence, Local

Cellular senescence is an important tumor suppression process in vivo. Tamoxifen is a well-known anti-breast cancer drug; however, its molecular function is poorly understood. Here, we examined whether tamoxifen promotes senescence in breast cancer and colon cancer cells for the first time.. Human breast cancer MCF-7, T47D, and MDA-MB-435 and colorectal cancer HCT116 cells were treated with tamoxifen. Cellular senescence was measured by SA-β-gal staining and based on the protein expression of p53 and p21(Cip1/WAF1). The production of reactive oxygen species (ROS) was determined by staining with CM-H2DCFDA and dihydroethidium (DHE). CK2 activity was assessed with a specific peptide substrate.. Tamoxifen promoted senescence phenotype and ROS generation in MCF-7 and HCT116 cells. The ROS scavenger, N-acetyl-l-cysteine (NAC), and the NADPH oxidase inhibitor, apocynin, almost completely abolished this event. Tamoxifen inhibited the catalytic activity of CK2. Overexpression of CK2α antagonized senescence mediated by tamoxifen, indicating that tamoxifen induced senescence via a CK2-dependent pathway. A well-known CK2 inhibitor, 5,6-dichloro-1-β-d-ribofuranosylbenzimidazole (DRB), also stimulated ROS production and senescence in MCF-7 cells. Finally, experiments using T47D (wild-type p53) and MDA-MB-435 (mutant p53) cell lines suggested that tamoxifen induces p53-independent ROS production as well as p53-dependent senescence in breast cancer cells.. These results demonstrate that tamoxifen promotes senescence through a ROS-p53-p21(Cip1/WAF1) dependent pathway by inhibiting CK2 activity in breast cancer and colon cancer cells.

Topics: Antineoplastic Agents, Hormonal; Breast Neoplasms; Casein Kinase II; Cell Line, Tumor; Cellular Senescence; Colonic Neoplasms; Cyclin-Dependent Kinase Inhibitor p21; Female; HCT116 Cells; Humans; Reactive Oxygen Species; Tamoxifen; Tumor Suppressor Protein p53

Protein Kinase (casein kinase 2, CK2) is a pleiotropic serine-threonine kinase that is frequently dysregulated in many human tumors; microRNAs (miRNAs) are a class of small noncoding RNAs which play important roles in human cancers. This study aimed to investigate the role of CK2 and miRNA expression in breast cancer.. Casein kinase 2 in MCF-7 breast cancer cell line was inhibited by the CK2 inhibitor TBB (4,5,6,7-tetrabromobenzotriazole), then cell proliferation was studied using MTT assay, and cell cycle distribution and apoptosis were detected by flow cytometry. The alteration of microRNAs expression profile was determined by microarray technology, followed by RT-PCR confirmation.. Here, we for the first time showed that inhibition of CK2 in MCF-7 breast cancer cells causes suppressed cell growth, which was related with dysregulation of the miRNA profile and altered expression. CK2 inhibition induced the up-regulated expression of 17 miRNAs and 10 down-regulated microRNAs which contributed to the impaired growth, inhibited cell cycle progress and increased apoptosis of MCF-7 cells by a CK2 inhibitor.. These findings highlight the potential role of dysregulated miRNA expression regulated by CK2 in breast cancer.

Topics: Apoptosis; Breast Neoplasms; Casein Kinase II; Cell Cycle; Cell Proliferation; Cell Survival; Flow Cytometry; Gene Expression Regulation, Neoplastic; Humans; MCF-7 Cells; MicroRNAs; Oligonucleotide Array Sequence Analysis; Real-Time Polymerase Chain Reaction; Triazoles

Breast cancer is the most common malignancy in women worldwide and remains a major cause of mortality, thus necessitating further therapeutic advancements. In breast cancer, numerous cell signaling pathways are aberrantly activated to produce the myriad phenotypes associated with malignancy; such pathways include the PI3K/Akt/mTOR, NF-κB and JAK/STAT cascades. These pathways are highly interconnected, but one prominent lateral enhancer of each is the remarkably promiscuous kinase CK2. CK2 expression has been shown to be elevated in cancer, thus implicating it in tumorigenesis through its effects on oncogenic signaling cascades. In this study, we identify aberrant expression of CK2 subunits in human breast samples from The Cancer Genome Atlas dataset. Additionally, two specific small molecule inhibitors of CK2, CX-4945 and TBB, were used to examine the role of CK2 in two human breast cancer cell lines, MDA-MB-231 and MCF-7 cells. We show that CK2 inhibition attenuates constitutive PI3K/Akt/mTOR, NF-κB and STAT3 activation and inducible NF-κB and JAK/STAT activation and downstream transcriptional activity. CX-4945 treatment caused a range of phenotypic changes in these cell lines, including decreased viability, cell cycle arrest, apoptosis and loss of migratory capacity. Overall, these results demonstrate the tremendous potential of CK2 as a clinical target in breast cancer.

Topics: Breast Neoplasms; Casein Kinase II; Cell Line, Tumor; Cell Survival; Female; Humans; MCF-7 Cells; Naphthyridines; Phenazines; Phosphorylation; Protein Kinase Inhibitors; Signal Transduction; Tumor Cells, Cultured; Up-Regulation

Cancer is a leading cause of death worldwide. Cancer cells proliferate uncontrollably and, many cases, spread to other parts of the body. A protein historically involved in cancer is protein kinase CK2. CK2 is a serine-threonine kinase that has been involved in cell growth, cell proliferation and cell apoptosis. CK2 functions as an oncogene when overexpressed in mouse tissues, and can synergize with known oncogenes, such as ras, to induce cell transformation in cells in culture. CK2, typically the CK2α protein, is found elevated in a number of human tumors. However, we have little information on CK2α' and CK2β proteins, and scarce information on CK2 gene transcript expression. Here, we explore the expression of CK2 transcripts in primary tumor tissues using the database Oncomine in the six cancers with the highest mortality in the U.S.A. In addition, we studied the correlation between CK2 expression and overall survival using the Kaplan-Meier Plotter database in breast, ovarian, and lung cancers. We found widespread upregulation in the expression of CK2 genes in primary tumor tissues. However, we found underexpression of CK2α' transcripts in some tumors, increased CK2β transcripts in some invasive tumors, and deregulation of CK2 transcripts in some tumor precursors. There was also correlation between CK2 expression levels and patient survival. These data provides additional evidence for CK2 as a biomarker for cancer studies and as a target for cancer therapy.

Topics: Biomarkers, Tumor; Breast Neoplasms; Carcinoma; Casein Kinase II; Female; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; Male; Ovarian Neoplasms; Prognosis; RNA, Messenger

An inhibitor of the protein kinase CKII (CKII) was purified from leaves of Glycine max (L.) Merrill and was identified as coumestrol by structural analysis. Coumestrol inhibited the phosphotransferase activity of CKII toward β-casein, with an IC50 of about 5 μM. It acted as a competitive inhibitor with respect to ATP as a substrate, with an apparent Ki value of 7.67 μM. Coumestrol at 50μM resulted in 50% and 30% growth inhibition of human breast cancer MCF-7 and colorectal cancer HCT116 cells, respectively. Coumestrol promoted senescence through the p53-p21(Cip1/WAF1) pathway by inducing reactive oxygen species (ROS) production in MCF-7 and HCT116 cells. The ROS scavenger N-acetyl-l-cysteine (NAC), NADPH oxidase inhibitor apocynin and p22(phox) siRNA almost completely abolished this event. Overexpression of CKIIα antagonised cellular senescence mediated by coumestrol, indicating that this compound induced senescence via a CKII-dependent pathway. Since senescence is an important tumour suppression process in vivo, these results suggest that coumestrol can function by inhibiting oncogenic disease, at least in part, through CKII inhibition-mediated cellular senescence.

Topics: Breast Neoplasms; Casein Kinase II; Cell Line, Tumor; Cell Proliferation; Cellular Senescence; Colonic Neoplasms; Coumestrol; Down-Regulation; Female; Glycine max; HCT116 Cells; Humans; Kinetics; Male; Plant Extracts; Reactive Oxygen Species

Progesterone receptors (PR) are transcription factors relevant to breast cancer biology. Herein, we describe an N-terminal common docking (CD) domain in PR-B, a motif first described in mitogen-activated protein kinases. Binding studies revealed PR-B interacts with dual-specificity phosphatase 6 (DUSP6) via the CD domain. Mutation of the PR-B CD domain (mCD) attenuated cell cycle progression and expression of PR-B target genes (including STAT5A and Wnt1); mCD PR-B failed to undergo phosphorylation on Ser81, a ck2-dependent site required for expression of these genes. PR-B Ser81 phosphorylation was dependent on binding with DUSP6 and required for recruitment of a transcriptional complex consisting of PR-B, DUSP6 and ck2 to an enhancer region upstream of the Wnt1 promoter. STAT5 was present at this site in the absence or presence of progestin. Furthermore, phospho-Ser81 PR-B was recruited to the STAT5A gene upon progestin treatment, suggestive of a feed-forward mechanism. Inhibition of JAK/STAT-signaling blocked progestin-induced STAT5A and Wnt1 expression. Our studies show that DUSP6 serves as a scaffold for ck2-dependent PR-B Ser81 phosphorylation and subsequent PR-B-specific gene selection in coordination with STAT5. Coregulation of select target genes by PR-B and STAT5 is likely a global mechanism required for growth promoting programs relevant to mammary stem cell biology and cancer.

Topics: Breast Neoplasms; Casein Kinase II; Cell Line, Tumor; Cell Proliferation; Dual Specificity Phosphatase 6; Enhancer Elements, Genetic; Female; Gene Expression Regulation, Neoplastic; Humans; Janus Kinases; Phosphorylation; Progestins; Protein Interaction Domains and Motifs; Receptors, Progesterone; S Phase; Serine; Signal Transduction; STAT Transcription Factors; STAT5 Transcription Factor; Transcription, Genetic; Wnt1 Protein

MicroRNAs (miRNAs) play important roles in diverse biological processes and are emerging as key regulators of tumorigenesis and tumor progression. To explore the dysregulation of miRNAs in breast cancer, a genome-wide expression profiling of 939 miRNAs was performed in 50 breast cancer patients. A total of 35 miRNAs were aberrantly expressed between breast cancer tissue and adjacent normal breast tissue and several novel miRNAs were identified as potential oncogenes or tumor suppressor miRNAs in breast tumorigenesis. miR-125b exhibited the largest decrease in expression. Enforced miR-125b expression in mammary cells decreased cell proliferation by inducing G2/M cell cycle arrest and reduced anchorage-independent cell growth of cells of mammary origin. miR-125b was found to perform its tumor suppressor function via the direct targeting of the 3'-UTRs of ENPEP, CK2-α, CCNJ, and MEGF9 mRNAs. Silencing these miR-125b targets mimicked the biological effects of miR-125b overexpression, confirming that they are modulated by miR-125b. Analysis of ENPEP, CK2-α, CCNJ, and MEGF9 protein expression in breast cancer patients revealed that they were overexpressed in 56%, 40-56%, 20%, and 32% of the tumors, respectively. The expression of ENPEP and CK2-α was inversely correlated with miR-125b expression in breast tumors, indicating the relevance of these potential oncogenic proteins in breast cancer patients. Our results support a prognostic role for CK2-α, whose expression may help clinicians predict breast tumor aggressiveness. In particular, our results show that restoration of miR-125b expression or knockdown of ENPEP, CK2-α, CCNJ, or MEGF9 may provide novel approaches for the treatment of breast cancer.

Topics: 3' Untranslated Regions; Breast Neoplasms; Casein Kinase II; Cell Line; Cell Proliferation; Cell Transformation, Neoplastic; Cluster Analysis; Cyclins; Female; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Genes, Tumor Suppressor; Glutamyl Aminopeptidase; Humans; Membrane Proteins; MicroRNAs; Nerve Tissue Proteins; RNA Interference

Epithelial-to-mesenchymal transition (EMT) is closely linked to conversion of early-stage tumours into invasive malignancies. Many signalling pathways are involved in EMT, but the key regulatory kinases in this important process have not been clearly identified. Protein kinase CK2 is a multi-subunit protein kinase, which, when overexpressed, has been linked to disease progression and poor prognosis in various cancers. Specifically, overexpression of CK2α in human breast cancers is correlated with metastatic risk. In this article, we show that an imbalance of CK2 subunits reflected by a decrease in the CK2β regulatory subunit in a subset of breast tumour samples is correlated with induction of EMT-related markers. CK2β-depleted epithelial cells displayed EMT-like morphological changes, enhanced migration, and anchorage-independent growth, all of which require Snail1 induction. In epithelial cells, Snail1 stability is negatively regulated by CK2 and GSK3β through synergistic hierarchal phosphorylation. This process depends strongly on CK2β, thus confirming that CK2 functions upstream of Snail1. In primary breast tumours, CK2β underexpression also correlates strongly with expression of EMT markers, emphasizing the link between asymmetric expression of CK2 subunits and EMT in vivo. Our results therefore highlight the importance of CK2β in controlling epithelial cell plasticity. They show that CK2 holoenzyme activity is essential to suppress EMT, and that it contributes to maintaining a normal epithelial morphology. This study also suggests that unbalanced expression of CK2 subunits may drive EMT, thereby contributing to tumour progression.

Topics: Breast Neoplasms; Carcinoma; Casein Kinase II; Cells, Cultured; Epithelial-Mesenchymal Transition; Female; Gene Expression Profiling; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Humans; Isoenzymes; Microarray Analysis; Models, Biological; Protein Subunits; Snail Family Transcription Factors; Tissue Array Analysis; Transcription Factors; Up-Regulation

In the present study we identified a novel gene, Homo Sapiens Chromosome 1 ORF109 (c1orf109, GenBank ID: NM_017850.1), which encodes a substrate of CK2. We analyzed the regulation mode of the gene, the expression pattern and subcellular localization of the predicted protein in the cell, and its role involving in cell proliferation and cell cycle control.. Dual-luciferase reporter assay, chromatin immunoprecipitation and EMSA were used to analysis the basal transcriptional requirements of the predicted promoter regions. C1ORF109 expression was assessed by western blot analysis. The subcellular localization of C1ORF109 was detected by immunofluorescence and immune colloidal gold technique. Cell proliferation was evaluated using MTT assay and colony-forming assay.. We found that two cis-acting elements within the crucial region of the c1orf109 promoter, one TATA box and one CAAT box, are required for maximal transcription of the c1orf109 gene. The 5' flanking region of the c1orf109 gene could bind specific transcription factors and Sp1 may be one of them. Employing western blot analysis, we detected upregulated expression of c1orf109 in multiple cancer cell lines. The protein C1ORF109 was mainly located in the nucleus and cytoplasm. Moreover, we also found that C1ORF109 was a phosphoprotein in vivo and could be phosphorylated by the protein kinase CK2 in vitro. Exogenous expression of C1ORF109 in breast cancer Hs578T cells induced an increase in colony number and cell proliferation. A concomitant rise in levels of PCNA (proliferating cell nuclear antigen) and cyclinD1 expression was observed. Meanwhile, knockdown of c1orf109 by siRNA in breast cancer MDA-MB-231 cells confirmed the role of c1orf109 in proliferation.. Taken together, our findings suggest that C1ORF109 may be the downstream target of protein kinase CK2 and involved in the regulation of cancer cell proliferation.

Topics: Breast Neoplasms; Casein Kinase II; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Proliferation; Female; Humans; Phosphoproteins; Phosphorylation; RNA, Small Interfering; Substrate Specificity; TATA Box; Transcriptional Activation

CK2α is a signalling molecule that participates in major events in solid tumour progression. The aim of this study was to evaluate the prognostic significance of the immunohistochemical expression of CK2α in breast carcinomas.. Quantitative measurements of immunohistochemical expression of 33 biomarkers using high-throughput densitometry, assessed on digitised microscopic tissue micro-array images were correlated with clinical outcome in 1000 breast carcinomas using univariate and multivariate analyses.. In univariate analysis, CK2α was a significant prognostic indicator (p<0.001). Moreover, a multivariable model allowed the selection of the best combination of the 33 biomarkers to predict patients' outcome through logistic regression. A nine-marker signature highly predictive of metastatic risk, associating SHARP-2, STAT1, eIF4E, pmapKAPk-2, pAKT, caveolin, VEGF, FGF-1 and CK2α permitted to classify well 82.32% of patients (specificity 81.59%, sensitivity 92.55%, area under ROC curve 0.939). Importantly, in a node negative subset of patients an even more (86%) clinically relevant association of eleven markers was found predictive of poor outcome.. A strong quantitative CK2α immunohistochemical expression in breast carcinomas is individually a significant indicator of poor prognosis. Moreover, an immunohistochemical signature of 11 markers including CK2α accurately (86%) well classifies node negative patients in good and poor outcome subsets. Our results suggest that CK2α evaluation together with key downstream CK2 targets might be a useful tool to identify patients at high risk of distant metastases and that CK2 can be considered as a relevant target for potential specific therapy.

Topics: Biomarkers, Tumor; Breast Neoplasms; Casein Kinase II; Female; Humans; Immunohistochemistry; Neoplasm Metastasis; Prognosis; Risk Factors; ROC Curve; Tissue Array Analysis

Myosin II motor proteins play important roles in cell migration. Although myosin II filament assembly plays a key role in the stabilization of focal contacts at the leading edge of migrating cells, the mechanisms and signaling pathways regulating the localized assembly of lamellipodial myosin II filaments are poorly understood. We performed a proteomic analysis of myosin heavy chain (MHC) phosphorylation sites in MDA-MB 231 breast cancer cells to identify MHC phosphorylation sites that are activated during integrin engagement and lamellar extension on fibronectin. Fibronectin-activated MHC phosphorylation was identified on novel and previously recognized consensus sites for phosphorylation by protein kinase C and casein kinase II (CK-II). S1943, a CK-II consensus site, was highly phosphorylated in response to matrix engagement, and phosphoantibody staining revealed phosphorylation on myosin II assembled into leading-edge lamellae. Surprisingly, neither pharmacological reduction nor small inhibitory RNA reduction in CK-II activity reduced this stimulated S1943 phosphorylation. Our data demonstrate that S1943 phosphorylation is upregulated during lamellar protrusion, and that CK-II does not appear to be the kinase responsible for this matrix-induced phosphorylation event.

Topics: Animals; Breast Neoplasms; Casein Kinase II; Cell Line, Tumor; Cell Movement; Chlorocebus aethiops; COS Cells; Female; Fibronectins; HeLa Cells; Humans; Models, Molecular; Myosin Heavy Chains; Myosin Type II; Phosphorylation; Protein Isoforms; Proteome; Pseudopodia; Recombinant Fusion Proteins; RNA, Small Interfering

Progesterone receptors (PR) are critical mediators of mammary gland development and contribute to breast cancer progression. Progestin-induced rapid activation of cytoplasmic protein kinases leads to selective regulation of growth-promoting genes by phospho-PR species. Herein, we show that phosphorylation of PR Ser81 is ck2 dependent and progestin regulated in intact cells but also occurs in the absence of PR ligands when cells enter the G(1)/S phase of the cell cycle. T47D breast cancer cells stably expressing a PR-B mutant receptor that cannot be phosphorylated at Ser79/81 (S79/81A) formed fewer soft agar colonies. Regulation of selected genes by PR-B, but not PR-A, also required Ser79/81 phosphorylation for basal and/or progestin-regulated (BIRC3, HSD11β2, and HbEGF) expression. Additionally, wild-type (wt) PR-B, but not S79/81A mutant PR, was robustly recruited to a progesterone response element (PRE)-containing transcriptional enhancer region of BIRC3; abundant ck2 also associated with this region in cells expressing wt but not S79/81A PR. We conclude that phospho-Ser81 PR provides a platform for ck2 recruitment and regulation of selected PR-B target genes. Understanding how ligand-independent PRs function in the context of high levels of kinase activities characteristic of breast cancer is critical to understanding the basis of tumor-specific changes in gene expression and will speed the development of highly selective treatments.

Topics: 11-beta-Hydroxysteroid Dehydrogenase Type 2; Animals; Baculoviral IAP Repeat-Containing 3 Protein; Breast Neoplasms; Casein Kinase II; Cell Line, Tumor; Female; Gene Expression Regulation, Neoplastic; Heparin-binding EGF-like Growth Factor; Humans; Inhibitor of Apoptosis Proteins; Intercellular Signaling Peptides and Proteins; Mice; Phosphorylation; Promoter Regions, Genetic; Protein Isoforms; Receptors, Progesterone; Serine; Transcription, Genetic; Ubiquitin-Protein Ligases

Agents to counteract acquired resistance to hormonal therapy for breast cancer would substantially enhance the long-term benefits of hormonal therapy. In the present study, we demonstrate how resveratrol (Res) inhibits human breast cancer cell proliferation, including MCF-7 tamoxifen-resistant cells (IC(50) values for viability were in the 30-45 μM range). We show that Res, through p38(MAPK) phosphorylation, causes induction of p53, which recruits at the estrogen receptor α (ERα) proximal promoter, leading to an inhibition of ERα expression in terms of mRNA and protein content. These events appear specifically p53 dependent, since they are drastically abrogated with p53-targeting siRNA. Coimmunoprecipitation assay showed specific interaction between p53, the Sin3A corepressor, and histone deacetylase 1 (HDAC1), which was phosphorylated. The enhancement of the tripartite complex p53/Sin3A/HDAC1, together with NF-Y on Res treatment, was confirmed by chromatin immunoprecipitation analyses, with a concomitant release of Sp1 and RNA polymerase II, thereby inhibiting the cell transcriptional machinery. The persistence of such effects in MCF-7 tamoxifen-resistant cells at a higher extent than parental MCF-7 cells addresses how Res may be considered a useful pharmacological tool to be exploited in the adjuvant settings for treatment of breast cancer developing hormonal resistance.

Topics: Antineoplastic Agents, Phytogenic; Breast Neoplasms; Casein Kinase II; CCAAT-Binding Factor; Cell Line, Tumor; Estrogen Receptor alpha; Female; Gene Expression Regulation, Neoplastic; Histone Deacetylase 1; Humans; p38 Mitogen-Activated Protein Kinases; Resveratrol; Sin3 Histone Deacetylase and Corepressor Complex; Stilbenes; Tumor Suppressor Protein p53

Protein kinase CK2 is involved in cell proliferation and survival, and found overexpressed in virtually all types of human cancer, including breast cancer. We demonstrate that inhibition of CK2 with 2-dimethylamino-4,5,6,7-tetrabromo-benzimidazole (DMAT), a potent and specific CK2 inhibitor, results in caspase-mediated killing of human breast cancer cells with acquired resistance to antiestrogens, while DMAT fails to kill parental MCF-7 cells. The antiestrogen resistant breast cancer cells express reduced levels of Bcl-2 compared to MCF-7 cells. Reduced Bcl-2 protein level is also found in a tamoxifen resistant human breast tumor grown as a xenograft. We show that re-expression of Bcl-2 partially rescues antiestrogen resistant MCF-7 sublines from DMAT-induced cell death. In summary, our data suggest a novel role of CK2 in antiestrogen resistance.

Topics: Antineoplastic Agents; Apoptosis; Benzimidazoles; Breast Neoplasms; Casein Kinase II; Caspases; Cell Line, Tumor; Cell Proliferation; Dose-Response Relationship, Drug; Drug Resistance, Neoplasm; Estradiol; Estrogen Receptor Modulators; Female; Fulvestrant; Humans; Polyunsaturated Alkamides; Proto-Oncogene Proteins c-bcl-2; Tamoxifen; Time Factors; Transfection

Densely functionalised pyrazole carboxamides and carboxylic acids were synthesised in an expedient manner through saponification and transamidation, respectively, of ester-functionalised pyrazoles. This synthetic protocol allowed for three diversifying steps in which appendages on the pyrazole scaffold were adjusted to optimise inhibition of protein kinases. Thirty-five analogues were tested in CK2, AKT1, PKA, PKCalpha, and SAPK2a (p38) kinase inhibition bioassays. Blocking of these kinases may lead to effective therapies for treating inflammatory diseases and cancer. In order to investigate potential biological activity, MCF-7 human breast cancer cells were incubated with the most promising derivatives. Two analogues caused changes in MCF-7 cell growth, one of them through cell cycle arrest demonstrated by cell cycle analysis.

Topics: Amides; Breast Neoplasms; Carboxylic Acids; Casein Kinase II; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cyclic AMP-Dependent Protein Kinases; Female; Humans; Mitogen-Activated Protein Kinase 14; Protein Kinase C-alpha; Protein Kinase Inhibitors; Protein Kinases; Proto-Oncogene Proteins c-akt; Pyrazoles; Signal Transduction

Aberrant activation of nuclear factor-kappaB (NF-kappaB) transcription factors has been implicated in the pathogenesis of breast cancer. We previously showed elevated activity of IkappaB kinase alpha (IKKalpha), IKKbeta, and protein kinase CK2 in primary human breast cancer specimens and cultured cells. A novel inducible IKK protein termed IKK-i/IKKepsilon has been characterized as a potential NF-kappaB activator. Here, we provide evidence that implicates IKK-i/IKKepsilon in the pathogenesis of breast cancer. We show IKK-i/IKKepsilon expression in primary human breast cancer specimens and carcinogen-induced mouse mammary tumors. Multiple breast cancer cell lines showed higher levels of IKK-i/IKKepsilon and kinase activity compared with untransformed MCF-10F breast epithelial cells. Interestingly, IKK-i/IKKepsilon expression correlated with CK2alpha expression in mammary glands and breast tumors derived from MMTV-CK2alpha transgenic mice. Ectopic CK2 expression in untransformed cells led to increased IKK-i/IKKepsilon mRNA and protein levels. Inhibition of CK2alpha via the pharmacologic inhibitor apigenin or upon transfection of a CK2 kinase-inactive subunit reduced IKK-i/IKKepsilon levels. Expression of a kinase-inactive IKK-i/IKKepsilon mutant in breast cancer cells reduced NF-kappaB activity as judged by transfection assays of reporters driven either by NF-kappaB elements or the promoters of two NF-kappaB target genes, cyclin D1 and relB. Importantly, the kinase-inactive IKK-i/IKKepsilon mutant reduced the endogenous levels of these genes as well as the ability of breast cancer cells to grow in soft agar or form invasive colonies in Matrigel. Thus, CK2 induces functional IKK-i/IKKepsilon, which is an important mediator of the activation of NF-kappaB that plays a critical role in the pathogenesis of breast cancer.

Topics: 9,10-Dimethyl-1,2-benzanthracene; Animals; Apigenin; Breast; Breast Neoplasms; Carcinogens; Casein Kinase II; Cell Movement; Collagen; Cyclin D1; Drug Combinations; Enzyme Activation; Epithelial Cells; Female; Gene Expression Regulation, Neoplastic; Humans; I-kappa B Kinase; Laminin; Mice; Mice, Transgenic; NF-kappa B; Proteoglycans; Proto-Oncogene Proteins c-rel; Transcription Factor RelB; Transfection; Tumor Cells, Cultured

Tumor-cell death can be triggered by engagement of specific death receptors with Apo2 ligand/tumor necrosis factor-related apoptosis-inducing ligand (Apo2L/TRAIL). Apo2L/TRAIL-induced apoptosis involves caspase-8-mediated cleavage of BID. The active truncated form of BID (tBID) triggers the mitochondrial activation of caspase-9 by inducing the activation of BAK or BAX. Although a broad spectrum of human cancer cell lines express death receptors for Apo2L/TRAIL, many remain resistant to TRAIL/Apo2L-induced death. A variety of human cancers exhibit increased activity of casein kinase II (CK2). Here we demonstrate that CK2 is at the nexus of two signaling pathways that protect tumor cells from Apo2L/TRAIL-induced apoptosis. We find that CK2 inhibits Apo2L/TRAIL-induced caspase-8-mediated cleavage of BID, thereby reducing the formation of tBID. In addition, CK2 promotes nuclear factor kappa B (NF-kappa B)-mediated expression of Bcl-x(L), which sequesters tBID and curtails its ability to activate BAX. Tumor cells with constitutive activation of CK2 exhibit a high Bcl-x(L)/tBID ratio and fail to activate caspase-9 or undergo apoptosis in response to Apo2L/TRAIL. Conversely, reduction of the Bcl-x(L)/tBID ratio by inhibition of CK2 renders such cancer cells sensitive to Apo2L/TRAIL-induced activation of caspase-9 and apoptosis. Using isogenic cancer cell lines that differ only in the presence or absence of either the p53 tumor suppressor or the BAX gene, we show that the enhancement of Apo2L/TRAIL-induced tumor-cell death by CK2 inhibitors requires BAX, but not p53. The identification of CK2 as a key survival signal that protects tumor cells from death-receptor-induced apoptosis could aid the design of Apo2L/TRAIL-based combination regimens for treatment of diverse cancers.

Topics: Adenocarcinoma; Antineoplastic Agents; Apoptosis; Apoptosis Regulatory Proteins; bcl-2-Associated X Protein; bcl-X Protein; BH3 Interacting Domain Death Agonist Protein; Breast Neoplasms; Carrier Proteins; Casein Kinase II; CASP8 and FADD-Like Apoptosis Regulating Protein; Colonic Neoplasms; Drug Synergism; Humans; Intracellular Signaling Peptides and Proteins; Membrane Glycoproteins; NF-kappa B; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Recombinant Proteins; TNF-Related Apoptosis-Inducing Ligand; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha; Tumor Suppressor Protein p53

The Her-2/neu oncogene, the second member of the epidermal growth factor (EGF) receptor family, encodes a transmembrane tyrosine kinase receptor. Overexpression of Her-2/neu in approximately 30% of breast cancers is associated with poor overall survival. Recently, we have found that Her-2/neu activates nuclear factor (NF)-kappaB via a phosphatidylinositol 3 kinase (PI3-K)-Akt kinase signaling pathway in mouse mammary tumor virus (MMTV)-Her-2/neu NF639 mouse breast cancer cells. Surprisingly, the IkappaB kinase (IKK) kinase complex, implicated in proteasome-mediated degradation of IkappaB-alpha and activation of NF-kappaB via the canonical pathway, was not activated in these cells. Degradation of IkappaB-alpha was mediated via calpain, which in B cells is facilitated by phosphorylation of IkappaB-alpha by the protein kinase CK2. Here, we report that the inhibition of CK2 blocks Her-2/neu-mediated activation of NF-kappaB. NF639 breast cancer cells, stably expressing CK2alpha or CK2alpha' kinase-inactive mutants, displayed decreased NF-kappaB binding and reduced ability to grow in soft agar, as well as increased sensitivity to tumor necrosis factor (TNF)-alpha killing. Similarly, CK2 kinase-inactive subunits inhibited NF-kappaB activity in Hs578T human breast cancer cells, which also display elevated CK2 activity. In NIH 3T3 fibroblasts, which express low basal NF-kappaB and CK2 activities, overexpression of CK2 by retroviral gene delivery led to increased IkappaB-alpha turnover and the induction of classical NF-kappaB (p50/RelA). Thus, CK2 plays an important role in Her-2/neu signaling, promoting IkappaB-alpha degradation and, thereby, NF-kappaB activation. Furthermore, because ectopic CK2 activity appears sufficient to induce NF-kappaB, the elevated CK2 activity observed in many primary human breast cancers likely plays a role in aberrant activation of NF-kappaB and, therefore, represents a potential therapeutic target.

Topics: 3T3 Cells; Animals; Apoptosis; Breast Neoplasms; Casein Kinase II; Cell Survival; Cell Transformation, Neoplastic; Gene Amplification; Genes, erbB-2; Humans; I-kappa B Proteins; Kidney; Mammary Neoplasms, Experimental; Mice; NF-kappa B; NF-KappaB Inhibitor alpha; Protein Serine-Threonine Kinases; Transcriptional Activation; Transfection; Tumor Cells, Cultured

The tumor suppressor phosphatase PTEN regulates cell migration, growth, and survival by dephosphorylating phosphatidylinositol second messengers and signaling phosphoproteins. PTEN possesses a C-terminal noncatalytic regulatory domain that contains multiple putative phosphorylation sites, which could play an important role in the control of its biological activity. The protein kinase CK2 phosphorylated, in a constitutive manner, a cluster of Ser/Thr residues located at the PTEN C terminus. PTEN-phosphorylated defective mutants showed decreased stability in comparison with wild type PTEN and were more rapidly degraded by the proteasome. Inhibition of PTEN phosphorylation by the CK2 inhibitor 5,6-dichloro-1-beta-d-ribofuranosyl-benzimidazole also diminished the PTEN protein content. Our results support the notion that proper phosphorylation of PTEN by CK2 is important for PTEN protein stability to proteasome-mediated degradation.

Topics: Amino Acid Sequence; Breast Neoplasms; Casein Kinase II; Cloning, Molecular; Consensus Sequence; Cysteine Endopeptidases; Enzyme Stability; Female; Genes, Tumor Suppressor; Humans; Kinetics; Multienzyme Complexes; Mutagenesis; Phosphoproteins; Phosphoric Monoester Hydrolases; Phosphorylation; Proteasome Endopeptidase Complex; Protein Serine-Threonine Kinases; PTEN Phosphohydrolase; Recombinant Proteins; Sequence Deletion; Transfection; Tumor Suppressor Proteins

Nuclear factor-kappaB (NF-kappaB)/Rel transcription factors regulate genes that control cell proliferation, survival, and transformation. In normal breast epithelial cells, NF-kappaB/Rel proteins are mainly sequestered in the cytoplasm bound to one of the specific inhibitory IkappaB proteins, whereas in breast cancers they are activated aberrantly. Human breast tumor cell lines, carcinogen-transformed mammary epithelial cells, and the majority of primary human or rodent breast tumor tissue samples express constitutively high levels of nuclear NF-kappaB/REL: To begin to understand the mechanism of this aberrant NF-kappaB/Rel expression, in this study we measured the activity of the major kinases implicated in regulation of IkappaB stability, namely IKKalpha, IKKbeta, and protein kinase, CK2 (formerly casein kinase II). Hs578T, D3-1, and BP-1 breast cancer cell lines displayed higher levels of IKKalpha, IKKbeta, and CK2 activity than untransformed MCF-10F mammary epithelial cells. Inhibition of IKK activity upon expression of dominant negative kinases or of CK2 activity by treatment with selective inhibitors decreased NF-kappaB/Rel activity in breast cancer cells. Inactivation of the IkappaB kinase complex in Hs578T cells via expression of a dominant negative IKKgamma/NF-kappaB essential modulator/IKK-associated protein 1 reduced soft agar colony growth. Thus, the aberrant expression of CK2 or IKK kinases promotes increased nuclear levels of NF-kappaB/Rel and transformation of breast cancer cells. Furthermore, primary human breast cancer specimens that displayed aberrant constitutive expression of NF-kappaB/Rel were found to exhibit increased CK2 and/or IKK kinase activity. These observations suggest these kinases play a similar role in an intracellular signaling pathway that leads to the elevated NF-kappaB/Rel levels seen in primary human mammary tumors and, therefore, represent potential therapeutic targets in the treatment of patients with breast cancer.

Topics: Breast Neoplasms; Casein Kinase II; Cell Adhesion; Cell Division; Cell Survival; Cell Transformation, Neoplastic; Enzyme Activation; Humans; I-kappa B Kinase; NF-kappa B; Protein Serine-Threonine Kinases; Tumor Cells, Cultured

Protein kinase CK2 is a ubiquitous and evolutionarily conserved serine/threonine kinase that is upregulated in many human cancers and can serve as an oncogene in lymphocytes. Recently, we have demonstrated that CK2 potentiates Wnt/beta-catenin signaling in mammary epithelial cells. To determine whether CK2 overexpression contributes to mammary tumorigenesis, we have performed comparative studies of human and rat breast cancer specimens and we have engineered transgenic mice with dysregulated expression of CK2alpha in the mammary gland. We find that CK2 is highly expressed in human breast tumor specimens and in carcinogen-induced rat mammary tumors. Overexpression of CK2alpha in the mammary gland of transgenic mice, under control of the MMTV-LTR, causes hyperplasia and dysplasia of the female mammary gland. Thirty per cent of the female MMTV-CK2alpha transgenic mice develop mammary adenocarcinomas at a median of 23 months of age, often associated with Wnt pathway activation, as evidenced by upregulation of beta-catenin protein. NF-kappaB activation and upregulation of c-Myc also occur frequently. Thus, in mice, rats, and humans, dysregulated expression of CK2 is associated with and is capable of contributing to mammary tumorigenesis. Targeted inhibition of CK2 could be useful in the treatment of breast cancer.

Topics: Adenocarcinoma; Age of Onset; Animals; beta Catenin; Breast Neoplasms; Casein Kinase II; Cell Transformation, Neoplastic; Cytoskeletal Proteins; Female; Fibrocystic Breast Disease; Humans; Hyperplasia; Lactation; Mammary Glands, Animal; Mammary Neoplasms, Animal; Mammary Tumor Virus, Mouse; Mice; Mice, Transgenic; NF-kappa B; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-myc; Signal Transduction; Trans-Activators; Wnt Proteins; Zebrafish Proteins

Protein P1, which is a nuclear protein resembling high mobility group proteins, has been studied in human breast adenocarcinomas and compared to those from control tissue. The presence of the protein was confirmed by in vitro phosphorylation by casein kinase II and immunoblotting, using antibodies raised in rabbits against rat liver P1. The protein has been isolated by reverse phase HPLC chromatography which provides a more rapid method of purification requiring smaller amounts of material. The levels of P1 expression were investigated and it was found that there was a three-fold increase in the ratio of P1/histone H1 in normal breast tissue as compared to the neoplastic tissue. In two other malignant and non-malignant tissues studied, the level of P1 was also decreased in the malignant tissues. Thermolytic phosphopeptides of P1 from normal and malignant human breast tissues exhibited the same pattern, though when compared to the phosphopeptide pattern from rat tissue, differences were observed.

Topics: Adenocarcinoma; Amino Acids; Animals; Blotting, Western; Breast Neoplasms; Casein Kinase II; Cell Cycle Proteins; Chromatography, High Pressure Liquid; Densitometry; DNA-Binding Proteins; Electrophoresis, Polyacrylamide Gel; Female; HeLa Cells; High Mobility Group Proteins; Histones; Humans; Image Processing, Computer-Assisted; In Vitro Techniques; Minichromosome Maintenance Complex Component 3; Molecular Weight; Nuclear Proteins; Phosphopeptides; Phosphorylation; Protein Serine-Threonine Kinases; Rabbits; Transcription Factors

Breast cancer is a major cause of cancer death in women, and the genetic abnormalities leading to the common sporadic forms of the disease are still under active investigation. CK2 has been reported to be upregulated in human breast cancer, which these studies confirm; CK2 is also upregulated in rat carcinogen-induced breast tumors. Transgenic mice overexpressing CK2alpha in the mammary gland develop mammary hyperplasia, dysplasia, and eventually adenocarcinomas, demonstrating that dysregulated expression of CK2 can contribute to transformation of the mammary epithelium. These mammary tumors have evidence of activation of the Wnt and NFkappaB pathways and upregulation of c-Myc. CK2 is capable of phosphorylating the key signaling molecule in the Wnt pathway, the transcriptional cofactor beta-catenin, and regulating its turnover. CK2 is known to phosphorylate IkappaB and thereby regulate basal NFkappaB levels; in the mammary cell lines and tumors, CK2 activity correlates with NFkappaB levels and inhibition of CK2 downregulates NFkappaB. Thus, CK2 may promote breast cancer through dysregulation of key pathways of transcriptional control in the mammary epithelium, and inhibition of CK2 has a potential role in the treatment of breast and other cancers.

Topics: Adenocarcinoma; Animals; Apigenin; Blotting, Western; Breast Neoplasms; Casein Kinase II; DNA, Complementary; Dose-Response Relationship, Drug; Down-Regulation; Flavonoids; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Genes, Reporter; Humans; Immunohistochemistry; Mammary Neoplasms, Animal; Mice; Mice, Transgenic; Neoplasms, Experimental; NF-kappa B; Phosphorylation; Precipitin Tests; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-myc; Rats; Signal Transduction; Time Factors; Transcription, Genetic; Up-Regulation; Wnt Proteins; Zebrafish Proteins

The BRCA1 gene encodes a complex protein that appears to be involved in some aspects of DNA repair, transcription, or cell cycle regulation. The phosphorylation of BRCA1 is enhanced following episodes of DNA damage or during cell cycle progression, indicating that phosphorylation may be an important regulatory mechanism. Through a yeast two hybrid assay, we found that the beta-subunit of casein kinase 2 (CK2) associated with a carboxy-terminal region of BRCA1. This association was much weaker with the same fragment bearing a missense mutation (M1775R) that has been identified in breast tumors. The interaction was also evident in Sf9 cells. Subsequent studies showed that BRCA1 was phosphorylated in vitro by CK2. An analysis by site directed mutagenesis of BRCA1 showed that in vitro phosphorylation by CK2 required a serine at aa1572. These data implicate CK2 as a potential mediator of BRCA1 activity.

Topics: Alkaline Phosphatase; Amino Acid Substitution; Animals; BRCA1 Protein; Breast; Breast Neoplasms; Casein Kinase II; Cloning, Molecular; Humans; Insecta; Phosphorylation; Precipitin Tests; Protein Binding; Protein Serine-Threonine Kinases; Recombinant Fusion Proteins; Saccharomyces cerevisiae; Serine

CD44, the predominant vertebrate cell surface receptor for hyaluronan, exists in a variety of isoforms resulting from alternative splicing of a single gene. Particular spliced variants of CD44 correlate with increased cell motility, and with poor clinical prognosis in several kinds of carcinomas. Combinations of 9 variant exons that confer this enhanced motility on tumor cells are inserted into a single site in the middle of the extracellular domain of CD44. Evidence suggests that phosphorylation of 2 serine residues in the intracellular domain of CD44 are involved in controlling these events. However, evidence is lacking as to the nature of such kinases. Acidic amino acids in close proximity to these 2 serine residues suggests casein kinase II (CKII) is involved. We now show an antisense phosphorothioate oligonucleotide designed to hybridize to the AUG translation initiation codon of subunit CKII alpha' mRNA blocks in vivo phosphorylation of CD44 in MDA231 breast tumor cells, and at the protein level decreases ectopic expression of total CD44 as well as the metastatic v-7 CD44 isoform. Furthermore subplateau RT-PCR analysis demonstrated antisense transfected MDA231 tumor cells had significant down-regulated or eliminated mRNA transcripts of metastatic CD44 isoforms. CKII as a CD44-associated serine kinase therefore may serve as an important molecule in a signaling cascade that produces a variety of cellular responses in MDA231 breast cancer cells. Since the 3'-untranslated region of CD44 mRNA contain 4 dispersed AUUUA sequences which serve as signals targeting mRNA for rapid turnover, a mechanism is proposed by which CD44 phosphorylation mediates labile message stabilization, hence providing insights into the processes involved in cancer cell growth, invasion and metastasis.

Topics: Alternative Splicing; Breast; Breast Neoplasms; Casein Kinase II; Codon, Initiator; Female; Flow Cytometry; Gene Expression Regulation, Neoplastic; Humans; Hyaluronan Receptors; Oligonucleotides, Antisense; Phosphorylation; Precipitin Tests; Protein Biosynthesis; Protein Isoforms; Protein Serine-Threonine Kinases; Reverse Transcriptase Polymerase Chain Reaction; Transfection; Tumor Cells, Cultured

We determined the amino acid and radiolabel sequences of tryptic [32P]phosphopeptides of the purified human estrogen receptor (hER) from MCF-7 cells and Sf9 cells. Serine 118 was identified as a site that was phosphorylated independently of estradiol-binding in MCF-7 cells. Proline is on the carboxy terminus of serine 118, which suggests that the serine-proline may be a consensus phosphorylation site motif for either the mitogen-activated protein (MAP) kinase or p34cdc2 kinase. MAP kinase selectively phosphorylated the recombinant hER in vitro on serine 118 independent of estradiol-binding, whereas p34cdc2 did not phosphorylate the hER. We demonstrated previously that serine 167 of the hER was phosphorylated in an estradiol-dependent manner. We therefore compared the consequence of hER phosphorylation at serine 118 by MAP kinase and phosphorylation at serine 167 by casein kinase II on the receptor's affinity for specific DNA binding. The binding of the hER to an estrogen response element was not altered by phosphorylation with MAP kinase at serine 118 but was significantly increased when phosphorylated at serine 167 by casein kinase II. These data suggest that phosphorylation of the hER by MAP kinase(s) pathways may influence receptor action by a mechanism other than the estradiol-dependent phosphorylation of hER by casein kinase II.

Topics: Adenosine Triphosphate; Amino Acid Sequence; Animals; Breast Neoplasms; Calcium-Calmodulin-Dependent Protein Kinases; Casein Kinase II; Cell Line; Chymotrypsin; DNA; DNA-Binding Proteins; Female; Humans; Molecular Sequence Data; Peptide Fragments; Phosphates; Phosphopeptides; Phosphorus Radioisotopes; Phosphorylation; Protein Serine-Threonine Kinases; Receptors, Estrogen; Recombinant Proteins; Serine; Spodoptera; Substrate Specificity; Transfection; Trypsin; Tumor Cells, Cultured

Serine 167 has been identified by radiolabel and amino acid sequencing as the major estrogen-induced phosphorylation site on the human estrogen receptor (hER) from human MCF-7 mammary carcinoma cells. The phosphorylation of the hER on serine 167 was estrogen-dependent, increasing 4-fold upon estradiol treatment of MCF-7 cells and accounted for almost half of the total [32P]phosphate incorporated into the recombinant hER from Sf9 insect cells and the native hER from MCF-7 cells. Casein kinase II was found to phosphorylate the purified recombinant hER on serine 167 in vitro. In addition, estradiol binding enhanced by 2-fold the phosphorylation of the purified recombinant hER by casein kinase II in vitro. Western blot analysis and [32P]phosphate incorporation confirmed the presence of casein kinase II in Sf9 cells. These results demonstrate that the hER is phosphorylated on serine 167 by casein kinase II in a hormone-dependent manner.

Topics: Animals; Breast Neoplasms; Casein Kinase II; Cells, Cultured; Estradiol; Moths; Neoplasms, Hormone-Dependent; Nucleopolyhedroviruses; Phosphorylation; Phosphoserine; Protein Processing, Post-Translational; Protein Serine-Threonine Kinases; Receptors, Estrogen; Recombinant Proteins; Tumor Cells, Cultured