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RNAi-mediated stathmin suppression reduces lung metastasis in an orthotopic neuroblastoma mouse model

Oncogene volume 33, pages 882–890 (2014)Cite this article

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Abstract

Metastatic neuroblastoma is an aggressive childhood cancer of neural crest origin. Stathmin, a microtubule destabilizing protein, is highly expressed in neuroblastoma although its functional role in this malignancy has not been addressed. Herein, we investigate stathmin’s contribution to neuroblastoma tumor growth and metastasis. Small interfering RNA (siRNA)-mediated stathmin suppression in two independent neuroblastoma cell lines, BE(2)-C and SH-SY5Y, did not markedly influence cell proliferation, viability or anchorage-independent growth. In contrast, stathmin suppression significantly reduced cell migration and invasion in both the neuroblastoma cell lines. Stathmin suppression altered neuroblastoma cell morphology and this was associated with changes in the cytoskeleton, including increased tubulin polymer levels. Stathmin suppression also modulated phosphorylation of the actin-regulatory proteins, cofilin and myosin light chain (MLC). Treatment of stathmin-suppressed neuroblastoma cells with the ROCKI and ROCKII inhibitor, Y-27632, ablated MLC phosphorylation and returned the level of cofilin phosphorylation and cell invasion back to that of untreated control cells. ROCKII inhibition (H-1152) and siRNA suppression also reduced cofilin phosphorylation in stathmin-suppressed cells, indicating that ROCKII mediates stathmin’s regulation of cofilin phosphorylation. This data demonstrates a link between stathmin and the regulation of cofilin and MLC phosphorylation via ROCK. To examine stathmin’s role in neuroblastoma metastasis, stathmin short hairpin RNA (shRNA)\luciferase-expressing neuroblastoma cells were injected orthotopically into severe combined immunodeficiency-Beige mice, and tumor growth monitored by bioluminescent imaging. Stathmin suppression did not influence neuroblastoma cell engraftment or tumor growth. In contrast, stathmin suppression significantly reduced neuroblastoma lung metastases by 71% (P<0.008) compared with control. This is the first study to confirm a role for stathmin in hematogenous spread using a clinically relevant orthotopic cancer model, and has identified stathmin as an important contributor of cell invasion and metastasis in neuroblastoma.

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Acknowledgements

We thank Dr Vladimir Ponomarev (Memorial Sloan Kettering Cancer Center) for kindly providing the TGL reporter vector, Dr Michael Shum and Dr Toby Trahair for helpful discussions and Zillan Neiron for technical assistance. Biological Resources Imaging Laboratory and the Biomedical Imaging Facility, Mark Wainwright Analytical Center, University of New South Wales. This study was financially supported by: Children’s Cancer Institute Australia for Medical Research, which is affiliated with the University of New South Wales and the Sydney Children’s Hospital. Funding from the Anthony Rothe Memorial Trust (F Byrne, The Anthony Rothe Scholarship) and grants from the National Health and Medical Research Council (NHMRC) (M Kavallaris and J McCarroll), Cancer Council New South Wales Program Grant (M Kavallaris), Cure Cancer Australia Foundation Grant (J McCarroll), Cancer Institute New South Wales Early Career Development Fellowship (J McCarroll, J Fletcher), NHMRC Career Development Fellowship (P Phillips) and an NHMRC Senior Research Fellowship (M Kavallaris).

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Authors and Affiliations

  1. Tumour Biology and Targeting Program, Children’s Cancer Institute Australia, Lowy Cancer Research Centre, University of New South Wales, Randwick, New South Wales, Australia

    F L Byrne, L Yang, J A McCarroll & M Kavallaris

  2. Pancreatic Cancer Translational Research Group, School of Medical Sciences, University of New South Wales, Randwick, New South Wales, Australia

    P A Phillips

  3. Cell Biology Program, Hospital for Sick Children, University of Toronto, Toronto, ON, Canada

    L M Hansford

  4. Experimental Therapeutics Program, Children’s Cancer Institute Australia, Lowy Cancer Research Centre, University of New South Wales, Randwick, New South Wales, Australia

    J I Fletcher

  5. Cancer Research Program, Garvan Institute of Medical Research, Darlinghurst, New South Wales, Australia

    C J Ormandy

  6. Australian Centre for Nanomedicine, University of New South Wales, Randwick, New South Wales, Australia

    J A McCarroll & M Kavallaris

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  1. F L Byrne
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Correspondence to J A McCarroll or M Kavallaris.

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Byrne, F., Yang, L., Phillips, P. et al. RNAi-mediated stathmin suppression reduces lung metastasis in an orthotopic neuroblastoma mouse model. Oncogene 33, 882–890 (2014). https://doi.org/10.1038/onc.2013.11

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