Differential Role of Human Choline Kinase α and β Enzymes in Lipid Metabolism: Implications in Cancer Onset and Treatment

11 pages, 6 figures, 1 table.Background The Kennedy pathway generates phosphocoline and phosphoethanolamine through its two branches. Choline Kinase (ChoK) is the first enzyme of the Kennedy branch of synthesis of 1phosphocholine, the major component of the plasma membrane. ChoK family of proteins is composed by ChoKα and ChoKβ isoforms, the first one with two different variants of splicing. Recently ChoKα has been implicated in the carcinogenic process, since it is over-expressed in a variety of human cancers. However, no evidence for a role of ChoKβ in carcinogenesis has been reported. Methodology/Principal Findings Here we compare the in vitro and in vivo properties of ChoKα1 and ChoKβ in lipid metabolism, and their potential role in carcinogenesis. Both ChoKα1 and ChoKβ showed choline and ethanolamine kinase activities when assayed in cell extracts, though with different affinity for their substrates. However, they behave differentially when overexpressed in whole cells. Whereas ChoKβ display an ethanolamine kinase role, ChoKα1 present a dual choline/ethanolamine kinase role, suggesting the involvement of each ChoK isoform in distinct biochemical pathways under in vivo conditions. In addition, while overexpression of ChoKα1 is oncogenic when overexpressed in HEK293T or MDCK cells, ChoKβ overexpression is not sufficient to induce in vitro cell transformation nor in vivo tumor growth. Furthermore, a significant upregulation of ChoKα1 mRNA levels in a panel of breast and lung cancer cell lines was found, but no changes in ChoKβ mRNA levels were observed. Finally, MN58b, a previously described potent inhibitor of ChoK with in vivo antitumoral activity, shows more than 20-fold higher efficiency towards ChoKα1 than ChoKβ. Conclusion/Significance This study represents the first evidence of the distinct metabolic role of ChoKα and ChoKβ isoforms, suggesting different physiological roles and implications in human carcinogenesis. These findings constitute a step forward in the design of an antitumoral strategy based on ChoK inhibition.This work has been supported by grants to JCL from Comunidad de Madrid (GR-SAL-0821-2004), Ministerio de Ciencia e Innovación (SAF2008-03750, RD06/0020/0016), Fundación Mutua Madrileña, and by a grant to ARM from Fundación Mutua Madrileña.Peer reviewe

Generation and characterization of monoclonal antibodies against choline kinase α and their potential use as diagnostic tools in cancer

6 pages, 3 figures, 1 table.Choline kinase α (ChoKα) is a metabolic enzyme involved in the synthesis of phosphatidylcholine, recently implicated in cancer onset since it is overexpressed in a variety of human cancers such as mammary, lung, colorectal and prostate adenocarcinomas. Furthermore, overexpression of ChoKα in human HEK293T cells confers them oncogenic properties with the induction of tumors after subcutaneous injection in nude mice. ChoKα levels in tumor samples have been analyzed using polyclonal antibodies and Western blotting. These techniques have considerable limitations and do not allow for a precise and efficient evaluation of the real significance of ChoK overexpression in human carcinogenesis. We developed a set of monoclonal antibodies with high specificity and sensitivity against ChoKα, and characterized their properties. We provide evidence that the newly generated MoAbs against ChoKα have potential use in cancer diagnosis by conventional immunohistochemistry techniques.This work was supported by Laboratorios INDAS, and by grants CAM 08.1/0047/2003, FIS C03-08 and FIS C03-10 from MSyC.Peer reviewe

Choline kinase is a novel oncogene that potentiates RhoA-induced carcinogenesis

Choline kinase is overexpressed in human breast, lung, colorectal, and prostate tumors, a finding that suggests the involvement of this enzyme in carcinogenesis. Here we show that overexpression of choline kinase induces oncogenic transformation of human embryo kidney fibroblasts and canine epithelial Madin-Darby canine kidney cells. Choline kinase lays downstream of RhoA signaling and is activated through ROCK kinase, one of the best-characterized RhoA effectors. In keeping with this, coexpression of RhoA and choline kinase potentiates both anchorage independent growth and tumorigenesis. Finally, choline kinase-mediated transformation is sensitive to MN58b, a well-characterized specific choline kinase inhibitor. These results provide the definitive evidence that choline kinase has oncogenic properties and that choline kinase inhibition constitutes a novel valid antitumor strategy. ©2005 American Association for Cancer Research.Grant support: McyT grant SAF2001-2042 and MSyC grants FIS C03-08 and FIS C03-10.Peer Reviewe

OncoChok. Validación de nuevos factores pronóstico e identificación de nuevas dianas terapéuticas en carcinogénesis humana

El consorcio OncoChok está integrado por el Laboratorio de Oncología Traslacional liderado por el Dr. Juan Carlos Lacal en el Instituto de Investigaciones Biomédicas de Madrid "Alberto Sols" (IIBM) del Consejo Superior de Investigaciones Cientificas (CSIC), y tres de los hospitales universitarios más importantes de la Comunidad de Madrid: Hospital Clínico, Hospital Doce de Octubre y Hospital La Paz y la empresa Translational Cancer Drugs Pharma (TCD Pharma).-- Más información sobre el programa en: http://www.oncochok.com/.El Programa OncoChok investiga la implicación del enzima colino quinasa alfa (ChoKa) como marcador de diagnóstico, pronóstico o predictor de respuesta al tratamiento antitumoral, así como su utilidad como una nueva diana terapéutica en distintos tipos de cáncer (pulmón, mama, páncreas, colorrectal y vejiga). También propone identificar sus posibles reguladores y efectores, su participación en el proceso de carcinogénesis y dilucidar el mecanismo de acción de sus inhibidores. El estudio se completa con la posible implicación del enzima ChoKa y sus reguladores en el desarrollo y progresión del cáncer y el análisis de su posible relación con parámetros clínico-patológicos.OncoChok es un proyecto de investigación subvencionado por la Comunidad de Madrid (CAM).Peer reviewe

Comparative expression of ChoKα1 and ChoKβ1 mRNA in a panel of cancer cell lines.

<p>Q-PCR was performed to determine the level of expression of mRNA in non-tumorogenic mammary cell lines (HMEC, MCF10A), breast cancer cell lines (MDA-MB435, MDA-MB468, T47D, MCF7), non-tumorogenic lung cells (BEC) and lung cancer cell lines (H510, H82). The data were normalized with the endogenous 18S ribosomal RNA. For the comparison between tumoral and non-tumoral cell lines, the 2^−ΔΔCt method was applied and log₁₀ RQ is represented. Note that the data are referred to the Human Mammary Epithelial Cells (HMEC) mRNA levels in breast cell lines and no significant difference in the level of both ChoK isoforms mRNA was found with the normal MCF10A cell line. The reference for lung cancer cells was the primary Bronchial Epithelial Cells (BEC).</p

Anchorage independent cell growth of ChoKα1- and β1-overexpressing cells.

<p><b>A)</b> and <b>B)</b><i>In vitro</i> ChoK activity of cell-free extracts from transfected cells at the moment of plating, determined as conversion of ¹⁴C-labeled choline to PCho. <b>C)</b> Photographs of a representative experiment of the soft agar assay. A total of 10⁵ cells were plated per 60-mm dish, and the number of colonies quantified after 5-8 weeks of incubation. <b>D)</b> and <b>E)</b> Computer based automatic quantification of the number of colonies, mean values±SEM is represented. The assay was performed 3 independent times with triplicate samples obtaining similar results. Statistical significance (p≤0.05) is marked by an asterisk.</p

Differential activation of choline kinase α1 and β1 isoforms by Ras and Rho GTPases.

<p>Choline kinase isoforms were expressed alone or in combination with the indicated Ras and Rho GTPases and the <i>in vitro</i> ChoK activity determined. <b>A)</b> Analysis of ectopic expression by Western Blot in HEK293T transfected cell extracts of ChoKα1 (52 KDa), ChoKβ1(45 KDa), RhoA-QL(22 KDa), Cdc42-QL(25 KDa) and H-rasV12 (23 KDa). Empty vectors were used as controls for the endogenous levels, and GAPDH as loading control. <b>B)</b> and <b>C)</b><i>In vitro</i> choline kinase activity of ChoKα1 or ChoKβ1 in the presence of enhanced expression of constitutive active forms of RhoA, Cdc42 or H-Ras. <b>D)</b> and <b>E)</b><i>In vitro</i> ethanolamine kinase activity of ChoKα or ChoKβ in the presence of each indicated constitutive active form of GTPase. The results are represented as fold induction of conversion to the corresponding phosphorylated metabolite determined as total cpm/µg of whole cellular extract, and normalized to the empty vector transfected cells as control. Data shown represent the mean values±SEM of 3 independent experiments, each one performed with duplicate samples. Statistical significance (p≤0.05) is marked by an asterisk comparing to the activity achieved when ChoKα1 or ChoKβ1, where appropriate, are transfected alone.</p

Overexpression of ChoKβ1 is not sufficient to induce tumor growth in athymic nude mice.

<p>Xenografts were established by s.c. injection of transfected HEK293T or MDCK cells in athymic nu/nu nude mice. <b>A)</b> and <b>B)</b> Western Blot analysis of ectopic expression of choline kinase isoforms in transfected HEK293T or MDCK cells, respectively, before mice inoculation. <b>C)</b> and <b>D)</b> Analysis of choline kinase activity in ChoKα1 or β1 transfected HEK293T or MDCK cells-free extracts before mice inoculation. <b>E)</b> and <b>F)</b> Volume of tumors generated by subcutaneous injection of 10⁶ transfected cells. Tumoral volume was calculated according to the formula: <i>Vol = [D * d²]/2</i>, where D and d are major and minor tumor diameters respectively. The data from HEK293T represents mean values±SEM from two independent experiments (n₁ = 12; n₂ = 16), the MDCK experiment correspond to an equivalent experiment with n = 12.</p

Michaelis constant (Km) of ChoKα and β isoforms for choline and ethanolamine.

1<p>Data are represented in milliMolar.</p>2<p>Referenced to the lowest Km.</p><p>Km of the different isoforms of ChoK for each substrate is indicated in each case. The results were obtained from four independent experiments using the logarithmic Michaelis-Menten formula as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007819#s4" target="_blank">Material and Methods</a>.</p

Characterization of choline and ethanolamine kinase activity of ChoKα1 and Chokβ1 in HEK293T cells.

<p>HEK293T cells were transfected with eukaryotic expression vectors of human ChoKα1 and ChoKβ1 gene. pCDNA3b empty vector was used as control. <b>A)</b> Overexpression of ChoKα1 and ChoKβ1 in HEK293T cells detected by Western Blot. GAPDH detection was used as control of expression level. <b>B, C)</b> In vitro ChoK (B) and EtnK (C) activity of choline kinase α1 and β1 isoforms in cell-free extracts of HEK293T transfected cells. Percentage of conversion of ¹⁴C-choline or ¹⁴C-ethanolamine to the phosphorylated product is represented. The experiment was performed in duplicate samples, repeated 4 times, and mean±SEM values from all experiments estimated.</p