Vimentin, an intermediate filament protein induced during epithelialto-mesenchymal transition, is known to regulate cell migration and invasion. However, it is still unclear how vimentin controls such behaviors. In this study, we aimed to find a new integrin regulator by investigating the H-Ras-mediated integrin suppression mechanism. Through a proteomic screen using the integrin β3 cytoplasmic tail protein, we found that vimentin might work as an effector of H-Ras signaling. H-Ras converted filamentous vimentin into aggregates near the nucleus, where no integrin binding can occur. In addition, an increase in the amount of vimentin filaments accessible to the integrin β3 tail enhanced talin-induced integrin binding to its ligands by inducing integrin clustering. In contrast, the vimentin head domain, which was found to bind directly to the integrin β3 tail and compete with endogenous vimentin filaments for integrin binding, induced nuclear accumulation of vimentin filaments and reduced the amount of integrin-ligand binding. Finally, we found that expression of the vimentin head domain can reduce cell migration and metastasis. From these data, we suggest that filamentous vimentin underneath the plasma membrane is involved in increasing integrin adhesiveness, and thus regulation of the vimentin-integrin interaction might control cell adhesion.

Regulation of integrin affinity for its ligand is essential for cell adhesion and migration. Here, we found that direct interaction of vimentin with integrin β1 can enhance binding of integrin α5β1 to its ligand, fibronectin. Conversely, blocking the interaction reduced fibronectin binding, cell migration on a fibronectin-coated surface, and neural tube closure during Xenopus embryogenesis. We also found that withaferin A (WFA), a natural compound known to inhibit vimentin function, can suppress the vimentin-integrin interaction and abolish fibronectin binding. Finally, we identified Ser38 of vimentin as a critical residue for integrin binding. Our results suggest that phosphorylation of vimentin at Ser38 may regulate the integrin-ligand interaction, thus providing a molecular basis for antivimentin therapeutic strategies.

Background In order to develop a new immunotherapeutic agent targeting metastatic breast cancers, we chose to utilize autocatalytic feature of the membrane serine protease Prss14/ST14, a specific prognosis marker for ER negative breast cancer as a target molecule. Methods The study was conducted using three mouse breast cancer models, 4 T1 and E0771 mouse breast cancer cells into their syngeneic hosts, and an MMTV-PyMT transgenic mouse strain was used. Prss14/ST14 knockdown cells were used to test function in tumor growth and metastasis, peptides derived from the autocatalytic loop for activation were tested as preventive metastasis vaccine, and monoclonal and humanized antibodies to the same epitope were tested as new therapeutic candidates. ELISA, immunoprecipitation, Immunofluorescent staining, and flow cytometry were used to examine antigen binding. The functions of antibodies were tested in vitro for cell migration and in vivo for tumor growth and metastasis. Results Prss14/ST14 is critically involved in the metastasis of breast cancer and poor survival rather than primary tumor growth in two mouse models. The epitopes derived from the specific autocatalytic loop region of Prss14/ST14, based on structural modeling acted as efficient preventive metastasis vaccines in mice. A new specific monoclonal antibody mAb3F3 generated against the engineered loop structure could reduce cell migration, eliminate metastasis in PyMT mice, and can detect the Prss14/ST14 protein expressed in various human cancer cells. Humanized antibody huAb3F3 maintained the specificity and reduced the migration of human breast cancer cells in vitro. Conclusion Our study demonstrates that Prss14/ST14 is an important target for modulating metastasis. Our newly developed hybridoma mAbs and humanized antibody can be further developed as new promising candidates for the use in diagnosis and in immunotherapy of human metastatic breast cancer. Electronic supplementary material The online version of this article (10.1186/s13046-019-1373-y) contains supplementary material, which is available to authorized users.