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Repression of SHP-1 expression by p53 leads to trkA tyrosine phosphorylation and suppression of breast cancer cell proliferation

Oncogene volume 28, pages 3787–3800 (2009)Cite this article

Abstract

The nerve growth factor (NGF) receptor, trkA, the tumour suppressor p53 and the phosphatase SHP-1 are critical in cell proliferation and differentiation. SHP-1 is a trkA phosphatase that dephosphorylates trkA at tyrosines (Y) 674 and 675. p53 can induce trkA activation and tyrosine phosphorylation in the absence of NGF stimulation. In breast cancer tumours trkA expression is associated with increased patient survival. TrkA protein expression is higher in breast-cancer cell lines than in normal breast epithelia. In cell lines (but not in normal breast epithelia) trkA is functional and can be NGF-stimulated to promote cell proliferation. This study investigates the functional relationship between trkA, p53 and SHP-1 in breast-cancer, and reveals that in wild-type (wt) trkA expressing breast-cancer cells both endogenous wtp53, activated by therapeutic agents, and transfected wtp53 repress expression of SHP-1 through the proximal CCAAT sequence of the SHP-1-P1-promoter and the transcription factor NF-Y. In these cells trkA-Y674/Y675 phosphorylation is detected when SHP-1 protein levels decrease in a wtp53-dependent manner. Proliferation and cell-cycle assays, with cells expressing endogenous or transfected wt-trkA and a temperature-sensitive p53 grown at 32 °C (when p53 is in the wt configuration), show suppressed cell proliferation. Suppression is not detected when grown at 37 °C (when p53 is in the mutant configuration). A release from suppression is observed when these cells are transiently transfected with wt-SHP-1 and grown at 32 °C. Suppression is also detected when, as control, wt-trkA-expressing cells are transiently transfected with SHP-1-siRNA, but not when a dominant-negative (DN) mutant trkA is used to abolish wt-trkA activity. Importantly, suppression is not seen with control trkA-negative breast-cancer cells (expressing wtp53, wt-SHP-1 and undetectable trkA), transfected with Y674F/Y675F mutant-trkA. BrdU-incorporation experiments reveal lack of incorporation in cells expressing wt-trkA and wtp53, or wt-trkA and SHP-1-siRNA. However, BrdU is incorporated in the presence of Y674F/Y675F mutant trkA or DN mutant trkA. These results indicate that p53 repression of SHP-1 expression leads to trkA-Y674/Y675 phosphorylation and trkA-dependent suppression of breast-cancer cell proliferation. These data provide an explanation as to why high trkA levels are associated with favourable prognosis.

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Acknowledgements

Thanks to Tessa Crompton, Sue Outram, Anna Furmanski and Ayad Eddaoudi for helping with FACS analysis; Shanie Budham-Mahadeo for helping with luciferase assays; Ralf Kirschner, Kurt Engeland, Finn Werner and David Mann for ChIP and BrdU incorporation protocols; Ben Neel and David Kaplan for wt-SHP-1, Δ-SHP-1 and anti-trk203-antibodies; Alison Sparks for sheep anti-p53-antibodies; Reiner Jänicke for MCF7-p53-RNAi cells; Borek Vojtesek for MCF7-p53DD Derek Huntley for bioinformatics and particularly to David Bacon for technical help with photography and graphics. This research was supported by a Breast Cancer Campaign grant awarded to XM.

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    Present address: 2Current address: UCL, Institute of Child Health, London WC1N 1EH, UK.,

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  1. Division of Cell and Molecular Biology, Molecular Signalling Group, Imperial College London, London, UK

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Montano, X. Repression of SHP-1 expression by p53 leads to trkA tyrosine phosphorylation and suppression of breast cancer cell proliferation. Oncogene 28, 3787–3800 (2009). https://doi.org/10.1038/onc.2009.143

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