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Myocardin-related transcription factors and SRF are required for cytoskeletal dynamics and experimental metastasis

Abstract

Rho GTPases control cytoskeletal dynamics through cytoplasmic effectors and regulate transcriptional activation through myocardin-related transcription factors (MRTFs), which are co-activators for serum response factor (SRF). We used RNA interference to investigate the contribution of the MRTF–SRF pathway to cytoskeletal dynamics in MDA-MB-231 breast carcinoma and B16F2 melanoma cells, in which basal MRTF–SRF activity is Rho-dependent. Depletion of MRTFs or SRF reduced cell adhesion, spreading, invasion and motility in culture, without affecting proliferation or inducing apoptosis. MRTF-depleted tumour cell xenografts showed reduced cell motility but proliferated normally. Tumour cells depleted of MRTF or SRF failed to colonize the lung from the bloodstream, being unable to persist after their arrival in the lung. Only a few genes show MRTF-dependent expression in both cell lines. Two of these, MYH9 (NMHCIIa) and MYL9 (MLC2), are also required for invasion and lung colonization. Conversely, expression of activated MAL/MRTF-A increases lung colonization by poorly metastatic B16F0 cells. Actin-based cell behaviour and experimental metastasis thus require Rho-dependent nuclear signalling through the MRTF–SRF network.

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Figure 1: MRTFs and SRF activity is dependent on Rho–actin signalling in MDA-MB-231 and B16F2 cells.
Figure 2: MRTF activity is required for cell adhesion and spreading.
Figure 3: MRTFs and SRF are required for cell motility in culture.
Figure 4: MRTF–SRF signalling is required for invasion and tumour cell motility in vivo.
Figure 5: MRTF–SRF signalling is required for an early step in experimental metastasis.
Figure 6: MRTF target genes contribute to invasion and metastasis in MDA-MB-231 and B16F2 cells.
Figure 7: MRTF-dependent gene expression is sufficient to promote experimental metastasis (a) Activation of the SRF reporter gene in B16F0 cells by co-expression of the activated MRTF-A mutant MAL-xxx11,12.

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Acknowledgements

We thank Hans Clevers for pTER, Richard Hynes for B16F2 and B16F0 cells. We thank Rob Nicolas for helpful discussions and continuous support, and other members of the Treisman and Sahai laboratories for technical help and discussions. We thank the Cancer Research UK Affymetrix Facility at the Paterson Institute for Cancer Research, in particular Yvonne Hey, and Gavin Kelly, Phill East, Richard Mitter at the CRUK Biostatistics Laboratory for the microarray processing and data analysis. We thank Clare Watkins, Emma Murray and Emma Nye (LRI Experimental Pathology laboratory), Daniel Zicha and Colin Gray (LRI light microscopy), and Ayad Eddaoudi and Derek Davies (LRI FACS laboratory) for expert and efficient technical support. S.M. was funded in part by a fellowship from the FRM (Fondation pour la Recherche Medicale). This work was funded by Cancer Research UK.

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S.M. designed the siRNA and ShRNAs and characterized the shRNA cell lines. S.M. designed, conducted and interpreted the wound-healing and adhesion experiments, reporter analyses, transcriptome analyses, the experimental metastasis experiments and the studies with activated MRTF-A; C.P-S. established the stable cell lines and conducted preliminary adhesion, motility and metastasis experiments; E.S. conducted the intravital microscopy, and advised on design of the experimental metastasis experiments; C.G. and S.M. conducted the organotypic invasion experiments. R.T. conceived the project, designed experiments and interpreted data. R.T. and S.M. wrote the manuscript, with additional input from E.S.

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Correspondence to Richard Treisman.

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Medjkane, S., Perez-Sanchez, C., Gaggioli, C. et al. Myocardin-related transcription factors and SRF are required for cytoskeletal dynamics and experimental metastasis. Nat Cell Biol 11, 257–268 (2009). https://doi.org/10.1038/ncb1833

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