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Sci Rep ; 6: 28532, 2016 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-27334688


Sox2 is a pluripotency transcription factor that as an oncogene can also regulate cell proliferation. Therefore, genes implicated in several different aspects of cell proliferation, such as the VRK1 chromatin-kinase, are candidates to be targets of Sox2. Sox 2 and VRK1 colocalize in nuclei of proliferating cells forming a stable complex. Sox2 knockdown abrogates VRK1 gene expression. Depletion of either Sox2 or VRK1 caused a reduction of cell proliferation. Sox2 up-regulates VRK1 expression and both proteins cooperate in the activation of CCND1. The accumulation of VRK1 protein downregulates SOX2 expression and both proteins are lost in terminally differentiated cells. Induction of neural differentiation with retinoic acid resulted in downregulation of Sox2 and VRK1 that inversely correlated with the expression of differentiation markers such as N-cadherin, Pax6, mH2A1.2 and mH2A2. Differentiation-associated macro histones mH2A1.2and mH2A2 inhibit CCND1 and VRK1 expression and also block the activation of the VRK1 promoter by Sox2. VRK1 is a downstream target of Sox2 and both form an autoregulatory loop in epithelial cell differentiation.

Ciclo Celular/genética , Diferenciação Celular/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Oncogenes/genética , Proteínas Serina-Treonina Quinases/genética , Fatores de Transcrição SOXB1/genética , Biomarcadores/metabolismo , Caderinas/genética , Carcinogênese/genética , Linhagem Celular , Linhagem Celular Tumoral , Proliferação de Células/genética , Ciclina D1/genética , Regulação para Baixo/genética , Epitélio/metabolismo , Células HEK293 , Células HeLa , Humanos , Células MCF-7 , Fator de Transcrição PAX6/genética , Regiões Promotoras Genéticas/genética , Regulação para Cima/genética
Oncotarget ; 5(7): 1770-8, 2014 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-24731990


Vaccinia-related kinase 1 (VRK1) belongs to a group of sixteen kinases associated to a poorer prognosis in human breast carcinomas, particularly in estrogen receptor positive cases based on gene expression arrays. In this work we have studied the potential molecular mechanism by which the VRK1 protein can contribute to a poorer prognosis in this disease. For this aim it was first analyzed by immunohistochemistry the VRK1 protein level in normal breast and in one hundred and thirty six cases of human breast cancer. The effect of VRK1 to protect against DNA damage was determined by studying the effect of its knockdown on the formation of DNA repair foci assembled on 53BP1 in response to treatment with ionizing radiation or doxorubicin in two breast cancer cell lines. VRK1 protein was detected in normal breast and in breast carcinomas at high levels in ER and PR positive tumors. VRK1 protein level was significantly lower in ERBB2 positive cases. Next, to identify a mechanism that can link VRK1 to poorer prognosis, VRK1 was knocked-down in two breast cancer cell lines that were treated with ionizing radiation or doxorubicin, both inducing DNA damage. Loss of VRK1 resulted in reduced formation of DNA-damage repair foci complexes assembled on the 53BP1 scaffold protein, and this effect was independent of damaging agent or cell type. This observation is consistent with detection of high VRK1 protein levels in ER and PR positive breast cancers. We conclude that VRK1 can contribute to make these tumors more resistant to DNA damage-based therapies, such as ionizing radiation or doxorubicin, which is consistent with its association to a poor prognosis in ER positive breast cancer. VRK1 is potential target kinase for development of new specific inhibitors which can facilitate sensitization to other treatments in combination therapies; or alternatively be used as a new cancer drugs.

Neoplasias da Mama/enzimologia , Carcinoma/enzimologia , Dano ao DNA , Reparo do DNA , Peptídeos e Proteínas de Sinalização Intracelular/análise , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Serina-Treonina Quinases/análise , Proteínas Serina-Treonina Quinases/metabolismo , Mama/enzimologia , Neoplasias da Mama/química , Carcinoma/química , Linhagem Celular Tumoral , Reparo do DNA/genética , Doxorrubicina , Feminino , Técnicas de Silenciamento de Genes , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Prognóstico , Proteínas Serina-Treonina Quinases/genética , Radiação Ionizante , Receptor ErbB-2/análise , Receptores Estrogênicos/análise , Receptores de Progesterona/análise , Proteína 1 de Ligação à Proteína Supressora de Tumor p53
BMC Clin Pathol ; 13(1): 23, 2013 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-24079673


BACKGROUND: Malignant astrocytomas are the most common primary brain tumors and one of the most lethal among human cancers despite optimal treatment. Therefore, the characterization of molecular alterations underlying the aggressive behavior of these tumors and the identification of new markers are thus an important step towards a better patient stratification and management. METHODS AND RESULTS: VRK1 and VRK2 (Vaccinia-related kinase-1, -2) expression, as well as proliferation markers, were determined in a tissue microarray containing 105 primary astrocytoma biopsies. Kaplan Meier and Cox models were used to find clinical and/or molecular parameters related to overall survival. The effects of VRK protein levels on proliferation were determined in astrocytoma cell lines. High levels of both protein kinases, VRK1 or VRK2, correlated with proliferation markers, p63 or ki67. There was no correlation with p53, reflecting the disruption of the VRK-p53-DRAM autoregulatory loop as a consequence of p53 mutations. High VRK2 protein levels identified a subgroup of astrocytomas that had a significant improvement in survival. The potential effect of VRK2 was studied by analyzing the growth characteristics of astrocytoma cell lines with different EGFR/VRK2 protein ratios. CONCLUSION: High levels of VRK2 resulted in a lower growth rate suggesting these cells are more indolent. In high-grade astrocytomas, VRK2 expression constitutes a good prognostic marker for patient survival.

Cell Mol Life Sci ; 69(22): 3881-93, 2012 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-22752157


The spatial and temporal regulation of intracellular signaling is determined by the spatial and temporal organization of complexes assembled on scaffold proteins, which can be modulated by their interactions with additional proteins as well as subcellular localization. The scaffold KSR1 protein interacts with MAPK forming a complex that conveys a differential signaling in response to growth factors. The aim of this work is to determine the unknown mechanism by which VRK2A downregulates MAPK signaling. We have characterized the multiprotein complex formed by KSR1 and the Ser-Thr kinase VRK2A. VRK2A is a protein bound to the endoplasmic reticulum (ER) and retains a fraction of KSR1 complexes on the surface of this organelle. Both proteins, VRK2A and KSR1, directly interact by their respective C-terminal regions. In addition, MEK1 is also incorporated in the basal complex. MEK1 independently interacts with the CA5 region of KSR1 and with the N-terminus of VRK2A. Thus, VRK2A can form a high molecular size (600-1,000 kDa) stable complex with both MEK1 and KSR1. Knockdown of VRK2A resulted in disassembly of these high molecular size complexes. Overexpression of VRK2A increased the amount of KSR1 in the particulate fraction and prevented the incorporation of ERK1/2 into the complex after stimulation with EGF. Neither VRK2A nor KSR1 interact with the VHR, MKP1, MKP2, or MKP3 phosphatases. The KSR1 complex assembled and retained by VRK2A in the ER can have a modulatory effect on the signal mediated by MAPK, thus locally affecting the magnitude of its responses, and can explain differential responses depending on cell type.

Retículo Endoplasmático/metabolismo , MAP Quinase Quinase 1/metabolismo , Sistema de Sinalização das MAP Quinases , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Sítios de Ligação , Linhagem Celular Tumoral , Regulação para Baixo , Células HEK293 , Células HeLa , Humanos , Células MCF-7 , Complexos Multiproteicos/metabolismo , Ligação Proteica , Proteínas Quinases/genética , Estrutura Quaternária de Proteína , Proteínas Serina-Treonina Quinases/genética , Interferência de RNA , RNA Interferente Pequeno
Mol Cell Biol ; 30(19): 4687-97, 2010 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-20679487


The epidermal growth factor (EGF)-ErbB-mitogen-activated protein kinase (MAPK) transcription signaling pathway is altered in many types of carcinomas, and this pathway can be regulated by new protein-protein interactions. Vaccinia-related kinase (VRK) proteins are Ser-Thr kinases that regulate several signal transduction pathways. In this work, we study the effect of VRK2 on MAPK signaling using breast cancer as a model. High levels of VRK2 inhibit EGF and ErbB2 activation of transcription by the serum response element (SRE). This effect is also detected in response to H-Ras(G12V) or B-Raf(V600E) oncogenes and is accompanied by a reduction in phosphorylated extracellular signal-regulated kinase (ERK) levels, p90RSK levels, and SRE-dependent transcription. Furthermore, VRK2 knockdown has the opposite effect, increasing the transcriptional response to stimulation with EGF and leading to increased levels of ERK phosphorylation. The molecular mechanism lies between MAPK/ERK kinase (MEK) and ERK, since MEK remains phosphorylated while ERK phosphorylation is blocked by VRK2A. This inhibition of the ERK signaling pathway is a consequence of a direct protein-protein interaction between VRK2A, MEK, and kinase suppressor of Ras 1 (KSR1). Identification of new correlations in human cancer can lead to a better understanding of the biology of individual tumors. ErbB2 and VRK2 protein levels were inversely correlated in 136 cases of human breast carcinoma. In ErbB2(+) tumors, there is a significant reduction in the VRK2 level, suggesting a role for VRK2A in ErbB2-MAPK signaling. Thus, VRK2 downregulation in carcinomas permits signal transmission through the MEK-ERK pathway without affecting AKT signaling, causing a signal imbalance among pathways that contributes to the phenotype of breast cancer.

Neoplasias da Mama/metabolismo , Sistema de Sinalização das MAP Quinases , Proteínas Serina-Treonina Quinases/metabolismo , Receptor ErbB-2/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Linhagem Celular , Linhagem Celular Tumoral , Fator de Crescimento Epidérmico/farmacologia , Células HeLa , Humanos , Immunoblotting , Microscopia Confocal , Proteínas Quinases Ativadas por Mitógeno/genética , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Mutação , Fosforilação , Ligação Proteica , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas B-raf/metabolismo , Interferência de RNA , Receptor ErbB-2/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transcrição Genética/efeitos dos fármacos , Proteínas ras/genética , Proteínas ras/metabolismo