Direct interaction of metastasis-inducing S100P protein with tubulin causes enhanced cell migration without changes in cell adhesion

Min Du,Guozheng Wang,Igor L Barsukov,Stephane R Gross,Philip S Rudland,Richard Smith

doi:10.1042/bcj20190644

Abstract

Overexpression of S100P promotes breast cancer metastasis in animals and elevated levels in primary breast cancers are associated with poor patient outcomes. S100P can differentially interact with nonmuscle myosin (NM) isoforms (IIA > IIC > IIB) leading to the redistribution of actomyosin filaments to enhance cell migration. Using COS-7 cells which do not naturally express NMIIA, S100P is now shown to interact directly with α,β-tubulin in vitro and in vivo with an equilibrium Kd of 2–3 × 10−7 M. The overexpressed S100P is located mainly in nuclei and microtubule organising centres (MTOC) and it significantly reduces their number, slows down tubulin polymerisation and enhances cell migration in S100P-induced COS-7 or HeLa cells. It fails, however, to significantly reduce cell adhesion, in contrast with NMIIA-containing S100P-inducible HeLa cells. When taxol is used to stabilise MTs or colchicine to dissociate MTs, S100P's stimulation of migration is abolished. Affinity-chromatography of tryptic digests of α and β-tubulin on S100P-bound beads identifies multiple S100P-binding sites consistent with S100P binding to all four half molecules in gel-overlay assays. When screened by NMR and ITC for interacting with S100P, four chemically synthesised peptides show interactions with low micromolar dissociation constants. The two highest affinity peptides significantly inhibit binding of S100P to α,β-tubulin and, when tagged for cellular entry, also inhibit S100P-induced reduction in tubulin polymerisation and S100P-enhancement of COS-7 or HeLa cell migration. A third peptide incapable of interacting with S100P also fails in this respect. Thus S100P can interact directly with two different cytoskeletal filaments to independently enhance cell migration, the most important step in the metastatic cascade.

Highlights

The major cause of death from the commonly occurring cancers is due to metastasis, the process of tumour dissemination from its primary site to distant organs [1]
When 1μg/ml doxycycline was added to COS-7 S10 cells, the increase in S100P peaked at 24h and remained constant for at least 72h without effect on the expression of tubulin and non-expression of nonmuscle myosin IIA (NMIIA) (Figure 1C, D)
These results indicate that S100P can enhance cell migration through mechanisms other than via NMIIA and cell adhesion in the COS-7 cells

Summary

Introduction

The major cause of death from the commonly occurring cancers is due to metastasis, the process of tumour dissemination from its primary site to distant organs [1]. Multiple steps are required for the metastatic process, certain gene products, called metastasis-inducing proteins [4] can induce metastasis when expressed in rodents [5], providing they can act on a cell which is already neoplastic [6]. One such group includes some members of the S100 family of small, calcium-binding regulatory proteins [5]; one of the best studied is S100P [7, 8]. This interaction between the cytoskeleton and cell motility can occur in noncancerous cells such as trophoblasts [13]

Methods

Results

Conclusion