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

Kpetemey M, Dasgupta S, Rajendiran S, Das S, Gibbs LD, Shetty P, Gryczynski Z, Vishwanatha JK - Mol. Cancer (2015)

Bottom Line: 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.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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Medical Genetics and Institute for Cancer Research, University of North Texas Health Science Center, 3500 Camp Bowie Blvd., Fort Worth, TX, 76107, USA. lkpeteme@live.unthsc.edu.

ABSTRACT

Background: Migration 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.

Methods: MIEN1 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.

Results: Clinically, 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 Ca(2+) -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.

Conclusion: Our 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.

No MeSH data available.


Related in: MedlinePlus

MIEN1 is a novel interactor of AnxA2. a MIEN1 interacts with AnxA2 in a yeast two-hybrid screen. Positive clones were selected on high-stringency medium (synthetic dropout medium) selection markers SD/−Ade/−His/−Leu/−Trp/X-α-gal, and only true interactors can activate the expression of β-galactosidase (blue color). The left panel shows positive interaction of MIEN1 with AnxA2, but not with p53. The right panel shows the positive control p53 − T-antigen interaction, while the AnxA2 − p53 interaction served as the negative control. b Co-immunoprecipitation of AnxA2 and MIEN1 in BT-474 cells showing the interaction of the two proteins. IgG was used as isotype control antibody and the total MIEN1 and AnxA2 used for the experiment was shown by western blotting. c Confocal microscopy showing co-localization of AnxA2 and MIEN1 in cancer cells. The interaction is predominantly observed around the membrane and cytosol excluding the nucleus. HCC-70, MCF-7 images were acquired with 40x objective with 1x zoom; MDA-MB231 images were acquired with 40x objective with 2x zoom. d FRET confirms the interaction of MIEN1 and AnxA2. Lifetime decays of the donor and donor − acceptor pair was measured to be 1.75 and 1.26 ns, respectively, and the lifetime decay histograms of the donor − acceptor pair (upper) and donor (lower) are shown
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Fig4: MIEN1 is a novel interactor of AnxA2. a MIEN1 interacts with AnxA2 in a yeast two-hybrid screen. Positive clones were selected on high-stringency medium (synthetic dropout medium) selection markers SD/−Ade/−His/−Leu/−Trp/X-α-gal, and only true interactors can activate the expression of β-galactosidase (blue color). The left panel shows positive interaction of MIEN1 with AnxA2, but not with p53. The right panel shows the positive control p53 − T-antigen interaction, while the AnxA2 − p53 interaction served as the negative control. b Co-immunoprecipitation of AnxA2 and MIEN1 in BT-474 cells showing the interaction of the two proteins. IgG was used as isotype control antibody and the total MIEN1 and AnxA2 used for the experiment was shown by western blotting. c Confocal microscopy showing co-localization of AnxA2 and MIEN1 in cancer cells. The interaction is predominantly observed around the membrane and cytosol excluding the nucleus. HCC-70, MCF-7 images were acquired with 40x objective with 1x zoom; MDA-MB231 images were acquired with 40x objective with 2x zoom. d FRET confirms the interaction of MIEN1 and AnxA2. Lifetime decays of the donor and donor − acceptor pair was measured to be 1.75 and 1.26 ns, respectively, and the lifetime decay histograms of the donor − acceptor pair (upper) and donor (lower) are shown

Mentions: We investigated the potential interacting partners of MIEN1 to define the mechanisms associated with tumor cell migration and invasion. In a yeast two-hybrid assay, we identified MIEN1 as potential interactor of AnxA2, a Ca(2+)-dependent phospholipid binding protein which translocates to the cell surface upon cellular signaling. Full-length AnxA2 cDNA cloned into GAL4 DNA-binding domain (GAL4 DNA-BD) of vector pGBKT7 was found to interact with MIEN1 in a yeast two-hybrid screen from a transformed human placental cDNA library as bait. Positive clones were selected on high-stringency medium (synthetic dropout medium) selection markers SD/−Ade/−His/−Leu/−Trp/X-α-gal, and only true interactor-MIEN1 could activate the expression of β-galactosidase (blue color). The left panel shows positive interaction of MIEN1 with AnxA2, but not with p53. The right panel shows the positive control p53 − T-antigen interaction, while the AnxA2 − p53 interaction served as the negative control (Fig. 4a). Following immunoblotting and real-time PCR analysis of breast cell lines expressing both MIEN1 and AnxA2 (Additional file 2: Figure S2A-B), we performed co-immunoprecipitation of endogenous AnxA2 with MIEN1 in BT-474 cells to confirm the yeast two-hybrid data. The reciprocal immunoprecipitation of MIEN1 also pulled down endogenous AnxA2, confirming that these two proteins indeed reside in a complex. The total input used for the immunoprecipitation confirmed equal loading and similar levels of expression of both the proteins (Fig. 4b). Colocalization experiments using confocal microscopy also confirmed interaction of endogenous AnxA2 and MIEN1 primarily in the cytosol, plasma membrane and the perinuclear area of breast cancer cells (Fig. 4c). Finally to confirm that AnxA2 and MIEN1 physically interact intracellularly, we performed FRET detection by fluorescence lifetime imaging microscopy (FLIM) assay to measure the proximity of MIEN1 and AnxA2. The lifetime decays of the donor (MIEN1) and donor-acceptor (MIEN1-AnxA2) pair were measured to be 1.75 and 1.26 ns, respectively. Substituting the lifetime values in the Förster equation, the efficiency of energy transfer was determined to be 28 %, which corresponds to a distance of 50.3 Å between the donor and acceptor pair; indicating that MIEN1 and AnxA2 indeed physically interact and reside in a very close proximity (Fig. 4d). These studies clearly validate MIEN1 as a novel interactor of AnxA2.Fig. 4


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

Kpetemey M, Dasgupta S, Rajendiran S, Das S, Gibbs LD, Shetty P, Gryczynski Z, Vishwanatha JK - Mol. Cancer (2015)

MIEN1 is a novel interactor of AnxA2. a MIEN1 interacts with AnxA2 in a yeast two-hybrid screen. Positive clones were selected on high-stringency medium (synthetic dropout medium) selection markers SD/−Ade/−His/−Leu/−Trp/X-α-gal, and only true interactors can activate the expression of β-galactosidase (blue color). The left panel shows positive interaction of MIEN1 with AnxA2, but not with p53. The right panel shows the positive control p53 − T-antigen interaction, while the AnxA2 − p53 interaction served as the negative control. b Co-immunoprecipitation of AnxA2 and MIEN1 in BT-474 cells showing the interaction of the two proteins. IgG was used as isotype control antibody and the total MIEN1 and AnxA2 used for the experiment was shown by western blotting. c Confocal microscopy showing co-localization of AnxA2 and MIEN1 in cancer cells. The interaction is predominantly observed around the membrane and cytosol excluding the nucleus. HCC-70, MCF-7 images were acquired with 40x objective with 1x zoom; MDA-MB231 images were acquired with 40x objective with 2x zoom. d FRET confirms the interaction of MIEN1 and AnxA2. Lifetime decays of the donor and donor − acceptor pair was measured to be 1.75 and 1.26 ns, respectively, and the lifetime decay histograms of the donor − acceptor pair (upper) and donor (lower) are shown
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Fig4: MIEN1 is a novel interactor of AnxA2. a MIEN1 interacts with AnxA2 in a yeast two-hybrid screen. Positive clones were selected on high-stringency medium (synthetic dropout medium) selection markers SD/−Ade/−His/−Leu/−Trp/X-α-gal, and only true interactors can activate the expression of β-galactosidase (blue color). The left panel shows positive interaction of MIEN1 with AnxA2, but not with p53. The right panel shows the positive control p53 − T-antigen interaction, while the AnxA2 − p53 interaction served as the negative control. b Co-immunoprecipitation of AnxA2 and MIEN1 in BT-474 cells showing the interaction of the two proteins. IgG was used as isotype control antibody and the total MIEN1 and AnxA2 used for the experiment was shown by western blotting. c Confocal microscopy showing co-localization of AnxA2 and MIEN1 in cancer cells. The interaction is predominantly observed around the membrane and cytosol excluding the nucleus. HCC-70, MCF-7 images were acquired with 40x objective with 1x zoom; MDA-MB231 images were acquired with 40x objective with 2x zoom. d FRET confirms the interaction of MIEN1 and AnxA2. Lifetime decays of the donor and donor − acceptor pair was measured to be 1.75 and 1.26 ns, respectively, and the lifetime decay histograms of the donor − acceptor pair (upper) and donor (lower) are shown
Mentions: We investigated the potential interacting partners of MIEN1 to define the mechanisms associated with tumor cell migration and invasion. In a yeast two-hybrid assay, we identified MIEN1 as potential interactor of AnxA2, a Ca(2+)-dependent phospholipid binding protein which translocates to the cell surface upon cellular signaling. Full-length AnxA2 cDNA cloned into GAL4 DNA-binding domain (GAL4 DNA-BD) of vector pGBKT7 was found to interact with MIEN1 in a yeast two-hybrid screen from a transformed human placental cDNA library as bait. Positive clones were selected on high-stringency medium (synthetic dropout medium) selection markers SD/−Ade/−His/−Leu/−Trp/X-α-gal, and only true interactor-MIEN1 could activate the expression of β-galactosidase (blue color). The left panel shows positive interaction of MIEN1 with AnxA2, but not with p53. The right panel shows the positive control p53 − T-antigen interaction, while the AnxA2 − p53 interaction served as the negative control (Fig. 4a). Following immunoblotting and real-time PCR analysis of breast cell lines expressing both MIEN1 and AnxA2 (Additional file 2: Figure S2A-B), we performed co-immunoprecipitation of endogenous AnxA2 with MIEN1 in BT-474 cells to confirm the yeast two-hybrid data. The reciprocal immunoprecipitation of MIEN1 also pulled down endogenous AnxA2, confirming that these two proteins indeed reside in a complex. The total input used for the immunoprecipitation confirmed equal loading and similar levels of expression of both the proteins (Fig. 4b). Colocalization experiments using confocal microscopy also confirmed interaction of endogenous AnxA2 and MIEN1 primarily in the cytosol, plasma membrane and the perinuclear area of breast cancer cells (Fig. 4c). Finally to confirm that AnxA2 and MIEN1 physically interact intracellularly, we performed FRET detection by fluorescence lifetime imaging microscopy (FLIM) assay to measure the proximity of MIEN1 and AnxA2. The lifetime decays of the donor (MIEN1) and donor-acceptor (MIEN1-AnxA2) pair were measured to be 1.75 and 1.26 ns, respectively. Substituting the lifetime values in the Förster equation, the efficiency of energy transfer was determined to be 28 %, which corresponds to a distance of 50.3 Å between the donor and acceptor pair; indicating that MIEN1 and AnxA2 indeed physically interact and reside in a very close proximity (Fig. 4d). These studies clearly validate MIEN1 as a novel interactor of AnxA2.Fig. 4

Bottom Line: 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.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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Medical Genetics and Institute for Cancer Research, University of North Texas Health Science Center, 3500 Camp Bowie Blvd., Fort Worth, TX, 76107, USA. lkpeteme@live.unthsc.edu.

ABSTRACT

Background: Migration 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.

Methods: MIEN1 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.

Results: Clinically, 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 Ca(2+) -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.

Conclusion: Our 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.

No MeSH data available.


Related in: MedlinePlus