MIEN1, a novel interactor of Annexin A2, promotes tumor cell migration by enhancing AnxA2 cell surface expression

Abstract

BackgroundMigration and invasion enhancer 1 (MIEN1) is a novel gene found to be abundantly expressed in breast tumor tissues and functions as a critical regulator of tumor cell migration and invasion to promote systemic metastases. Previous studies have identified post-translational modifications by isoprenylation at the C-terminal tail of MIEN1 to favor its translocation to the inner leaflet of plasma membrane and its function as a membrane-bound adapter molecule. However, the exact molecular events at the membrane interface activating the MIEN1-driven tumor cell motility are vaguely understood.MethodsMIEN1 was first studied using in-silico analysis on available RNA sequencing data of human breast tissues and its expression was ascertained in breast cells. We performed several assays including co-immunoprecipitation, wound healing, western blotting and immunofluorescence to decipher the molecular events involved in MIEN1-mediated tumor cell migration.ResultsClinically, MIEN1 is predominantly overexpressed in Her-2 and luminal B subtypes of breast tumors, and its increased expression correlates with poor disease free survival. Molecular studies identified a phosphorylation-dependent activation signal in the immunoreceptor tyrosine based activation motif (ITAM) of MIEN1 and the phosphorylation-deficient MIEN1-mutants (Y39F/50 F) to regulate filopodia generation, migration and invasion. We found that ITAM-phosphorylation of MIEN1 is significantly impaired in isoprenylation-deficient MIEN1 mutants indicating that prenylation of MIEN1 and membrane association is required for cross-phosphorylation of tyrosine residues. Furthermore, we identified MIEN1 as a novel interactor of Annexin A2 (AnxA2), a Ca2+ -dependent phospholipid binding protein, which serves as an extracellular proteolytic center regulating plasmin generation. Fluorescence resonance energy transfer (FRET) confirmed that MIEN1 physically interacts with AnxA2 and functional studies revealed that they mutually cooperate to accentuate tumor cell motility. Interestingly, our study identified that ectopic overexpression of MIEN1 significantly enhances Tyr23-phosphorylation on AnxA2, thereby stimulating cell surface translocation of AnxA2 and catalyzing the activation of its proteolytic activity.ConclusionOur data show that the presence and interaction of both MIEN1 and AnxA2 in breast tumors are crucial drivers of cell motility. Our study has now deciphered a novel regulatory network governing the vicious process of breast tumor cell invasion-metastasis, and findings suggest MIEN1-AnxA2 as prospective targets to counter the deadly disease.Electronic supplementary materialThe online version of this article (doi:10.1186/s12943-015-0428-8) contains supplementary material, which is available to authorized users.

MIEN1 drives breast tumor cell migration by regulating cytoskeletal-focal adhesion dynamics

“…In breast tumors, FAK mediates tumor cell adhesion, and its overexpression induces survival signals to promote breast cancer growth and metastasis [42, 50, 51]. Furthermore, our results demonstrate that depletion of MIEN1 leads to a reduction in FAK phosphorylation at Tyr-925 along with reduced phosphorylation of previously shown markers of motility such as Akt, Erk1/2 and NF- κB [14, 18, 19, 43, 52]. These results are consistent with previous findings which demonstrate that Src-mediated phosphorylation of FAK at Tyr-925 creates a binding site for Grb2 and then stimulate mitogen-activated protein kinase (MAPK) signaling via a FAK/Grb2/Sos/Ras/Erk2 pathway [43, 52].…”

Intracellular targeting of annexin A2 inhibits tumor cell adhesion, migration, and in vivo grafting

“…Preclinical studies have revealed a functional role for extracellular annexin A2 in the regulation of adhesion, migration, homing, and invasion of cancer cells 13–16 . Several annexin A2-interacting proteins, e.g.…”

Construction of C35 gene bait recombinants and T47D cell cDNA library

“…These studies indicated that the C-terminal "CVIL" motif and the ITAM domain act as the dominant functional motif of C35. Furthermore, the ITAM-phosphorylation of MIEN1 could be significantly impaired in isoprenylation-deficient MIEN1 mutants, and imply that the post-translational modification of MIEN1 was required for cross-phosphorylation of tyrosine residues (16).…”

“…In particular, this "CVIL" motif is completely conserved among all species. Additionally, the ITAM motif was identified to determine the phosphorylation-dependent activation of MIEN1, which regulated filopodia generation, migration and invasion of breast cells (16). These studies indicated that the C-terminal "CVIL" motif and the ITAM domain act as the dominant functional motif of C35.…”