International Seminars in Surgical Oncology BioMed Seminars in Surgical Oncology

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    International Seminars in Surgical Oncology

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    Open AcceResearchThe expression and prognostic value of the guanine nucleotide exchange factors (GEFs) Trio, Vav1 and TIAM-1 in human breast cancerJane Lane*, Tracey A Martin, Robert E Mansel and Wen G Jiang

    Address: Metastasis research Group, University Department of Surgery, Cardiff University School of Medicine, Heath Park, Cardiff, CF4 4XN, UK

    Email: Jane Lane* - lanej1@cf.ac.uk; Tracey A Martin - martinta1@cf.ac.uk; Robert E Mansel - wsrem@cf.ac.uk; Wen G Jiang - jiangw@cf.ac.uk

    * Corresponding author

    AbstractBackground: Development of metastasis in breast cancer is a multi-step process comprisingchanges in cytoskeletal structure and gene expression of tumour cells leading to changes in celladhesion and motility. The Rho GTPase proteins, which function as guanine nucleotide regulatedbinary switches, govern a variety of cellular processes including cell motility and migration, changesin cell adhesion as well as actin cytoskeletal reorganisation and gene expression/transcription. Onegroup of activators which regulate the Rho-GTPases is the guanine nucleotide exchange factors(GEFs), and this study looked at three such GEFs, Trio, Vav1 and TIAM-1. The purpose of this studywas to investigate the expression of these GEFs, in human breast cancer and assess the affect onclinical outcome.

    Methods: Specimens of fresh, frozen breast tumour tissue (n = 113) and normal background tissue(n = 30) were processed for quantitative PCR analysis. The expression and levels of expression ofTrio, Vav1 and TIAM-1 were analysed using RT-PCR and real-time Q-PCR respectively. Sectionswere also immunostained with Trio and Tiam-1 antibodies.

    Results: Tumour tissue exhibited high levels of all three Rho activators Trio, Vav1 and TIAM-1compared with normal background breast tissue, reaching a level of significance for the GEF Trio(p = 0.013). Trio levels also increased significantly in patients with a poor prognostic index (p =0.04).

    Levels of TIAM-1 were significantly higher in tumour tissue from patients who died from breastcancer compared with those who survived (p = 0.04). No significant correlation was found betweentumour grade and histology types.

    Conclusion: High expression levels of Trio, Vav1 and TIAM-1 were seen in breast tumours,especially in those with poor prognosis. This suggests that aberrant regulation of Rho familyactivities by GEFs may have an important prognostic value in breast cancer.

    Published: 16 October 2008

    International Seminars in Surgical Oncology 2008, 5:23 doi:10.1186/1477-7800-5-23

    Received: 22 August 2008Accepted: 16 October 2008

    This article is available from: http://www.issoonline.com/content/5/1/23

    2008 Lane et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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    BackgroundDuring the development of metastasis in breast cancer,tumour cells undergo numerous changes in their cytoskel-etal structure and gene expression promoting changes incell adhesion, motility and morphology leading to metas-tasis and tissue invasion. The Rho GTPases, which func-tion as guanine nucleotide regulated binary switches,control the regulation of the actin cytoskeleton, and assuch, have been implicated in promoting a variety of cel-lular processes including cell motility and migration,changes in cell adhesion as well as actin cytoskeletal reor-ganisation [1-3] and gene expression/transcription [4].Increased expression of Rho proteins has been demon-strated in a variety of tumours with raised levels of Rho-C,Rho-G and Rho-6 detected in breast tumour tissue [5], aswell as increase in the expression of the ROCK proteins,which function as downstream effectors of the RhoGTPases [6]. Therefore this study was initiated to investi-gate the expression levels of the activators of the Rho-GTPase cycle.

    In part, the Rho-GTPases are activated by guanine nucle-otide exchange factors (GEFs), a group of regulators whichfunction as modulators of the activation/inactivationcycle of the Rho family GTPases by binding to inactiveGTPases and inducing a conformational change leadingto GDP release. The GTPases then bind free cytoplasmicGTP to become reactivated.

    There are a large number of guanine nucleotide-bindingproteins requiring an equally large range of GEFs toensure signalling specificity and, as such, a number of GEFfamilies exist. A recent review of the GEFs and GAPs(GTPase activating proteins), which both function as reg-ulators of the Rho GDP/GTP cycle, has suggested thatthese proteins may be potential therapeutic targets fordeveloping drug treatments for various cancers [7]. Threesuch GEFs which regulate the Rho family of GTPases areTrio, Vav1 and TIAM-1 (T-lymphoma invasion and metas-tasis gene).

    Trio acts as a cytoskeletal modulator activating the Rhoand/or Rac pathways and has been shown to play a vitalrole in axon guidance, neuronal cell migration and cellmotility [8] as well as in the regulation of focal adhesiondynamics [9].

    The Vav family of guanine nucleotide exchange factorshave been shown to modulate activity of Rho, Rac and/orCdc42 to effect changes in cytoskeletal organisation [10].

    Vav proteins couple tyrosine kinase signals with the acti-vation of the Rho-GTPases and are likely to play an inte-gral role in the regulation of cell differentiation in manytissues. Vav1 has been shown to function as an oncogene

    involved in malignant transformation. This protein alsoacts as a growth stimulatory protein in primary pancreaticadenocarcinoma. [11].

    Studies have shown that over expression of TIAM-1 pro-tein confers an invasive phenotype in T-lymphoma cellssuggesting that increased TIAM-1 levels may lead totumour progression and invasion [12]. This GEF has alsobeen shown to interact with the cytoskeletal proteinankyrin which promotes Rac activation leading to breasttumour cell invasion and migration [13].

    To look for evidence to support their role in the motilityand invasion of breast tumour cells we have analysed theexpression of the Rho GTPase regulators Trio, Vav1 andTIAM-1 in normal breast tissue and compared this withthe expression in breast tumour tissue and with the gradeof tumour and clinical outcome.

    MethodsSurgical specimens of fresh, frozen breast tissue compris-ing breast tumours (n = 113) and normal background tis-sue (n = 30) were collected during surgery. Informationwas available on the Nottingham Prognostic Index (NPI),grade of tumour, degree of nodal involvement and clinicaloutcome for all patients with a mean follow up period of72.2 months. The expression and levels of expression ofTrio, Vav1 and TIAM-1 were analysed using RT-PCR andreal-time quantitative PCR respectively.

    RNA extraction and RT-PCRRNA was isolated from breast cancer cell and tissue linesusing a standard RNA-zol procedure, as we previouslyreported [6]. For RT-PCR, cDNA was synthesised in a 25l reaction mixture, as described in the manufacturer'sprotocol (ABgene Reverse Transcription System, ABgene,Surrey, UK). The cDNA obtained was amplified by astandard PCR mixture (as supplied in Pre-aliquotedReddy-Load Mix, Advanced Biotechnologies). Cyclingconditions for the 25 l reaction mix were 94C for 4 min,followed by 36 cycles of 94C for 15 s, 55C annealing for15 s and 72C for 30 s, followed by a final extension of 7min at 72C. The products were visualised on a 0.8% aga-rose gel following staining with ethidium bromide.

    Quantitative-PCR analysisThe Q-PCR system used the Amplifluor Uniprimer sys-tem (Intergen Company Oxford, UK) and Thermo-Start

    (ABgene, Epsom, Surrey, UK) [5,6]. Specific primers weredesigned by the authors and manufactured by Invitrogen(Invitrogen Life Technologies, Paisley, Scotland, UK).Using the Icycler IQ system (Bio-Rad, Hemel Hempstead,UK), which incorporates a gradient thermocycler and a96-channel optical unit, the plasmid standards and breastcancer cell cDNA were simultaneously assayed in dupli-

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  • International Seminars in Surgical Oncology 2008, 5:23 http://www.issoonline.com/content/5/1/23

    cated 10 l reactions as follows: Q-master mix (5 l), for-ward primer (0.3 l), probe (0.3 l), reverse primer (0.3l), plasmid, (internal standard) or specimen cDNA (4l), water (0.1 l). Q-PCR conditions were as follows:enzyme activation at 95C for 12 min, 1 cycle, followedby 60 cycles of denaturing: 95C for 15 s; annealing: 55Cfor 40 s; extension: 72C for 25 s. Using purified plasmidsas internal standards, the level of each molecule cDNA(copies/50 ng RNA) in the breast cancer samples was cal-culated. Q-PCR for -actin was also performed on thesame samples, to correct for any residual differences in theinitial level of RNA in the specimens (in addition to spec-trophotometry). The products of Q-PCR were verified onagarose gels. Primer pairs for Q-PCR were as follows: TrioF1 (5'-accgttgttcttagatgtcg); Trio ZR (5'-actgaacctgaccgta-caggagatgctgtagtgaccat; Vav1 F1 (5'-agtctctggacaccacctt);Vav1 ZR (5'-actgaacctgaccgtacaccaaaatactttgtgcttcc); TIAM-1 F1 (5'-ctttaagaagaaacgctccca); TIAM-1 ZR (5'-actgaacct-gaccgtacacttggctcagatcagagagt).

    Immunohistochemis