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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 activates translocation of AnxA2 to plasma membrane to promote breast cancer cell migration. a Control and MIEN1 transfected MDA-MB231 cells were subjected to western blot analysis with anti-MIEN1 or anti-AnxA2 antibody. PGK served as a loading control. b Down-regulation of MIEN1 and AnxA2 reduces the abilities of MDA-MB231 breast cancer cells to migrate in vitro. Confluent MDA-MB231 monolayers transfected with control GFP, AnxA2 or/and MIEN1 were wounded using a pipet tip, 60 h after transfection. Following the wound formation, plates were incubated for 12 h and the wound closure areas were visualized on an Olympus Microscope (Carl Zeiss). Representative images were acquired at 0 and 12 h. c Quantification of cell migration was achieved using Image J software. Data are presented as percentages of the recovered scratch area relative to untreated control cells. Columns are the means five replicates from two independent experiments and bars are s.d. d MDA-MB231 and MDA-MB231 cells stably transfected with control GFP or GFP-MIEN1WT were subjected to western blot analysis with anti-phosho AnxA2, anti-AnxA2 and anti-MIEN1 antibodies. GAPDH served as a loading control. The quantification of the representative blots is the densitometric average of three independent experiments analyzed using ImageJ and normalized to GAPDH. e Control and MIEN1 transfected MDA-MB231 cells were subjected to western blot analysis with anti-phosho AnxA2, AnxA2, anti-MIEN1 and actin as loading control. f Representative images, as captured using a 60X oil immersion TIRF microscope, of MDA-MB231 and MDA-MB231 cells stably transfected with GFP-MIEN1WT grown to sub-confluence on coverslips, fixed with PFA, unpermeabilized, and processed for TIRF microscopy with the specific antibodies. At least three independent fields were analyzed for confirmation of the staining pattern represented. Scale bar denotes 20 μm
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Fig5: MIEN1 activates translocation of AnxA2 to plasma membrane to promote breast cancer cell migration. a Control and MIEN1 transfected MDA-MB231 cells were subjected to western blot analysis with anti-MIEN1 or anti-AnxA2 antibody. PGK served as a loading control. b Down-regulation of MIEN1 and AnxA2 reduces the abilities of MDA-MB231 breast cancer cells to migrate in vitro. Confluent MDA-MB231 monolayers transfected with control GFP, AnxA2 or/and MIEN1 were wounded using a pipet tip, 60 h after transfection. Following the wound formation, plates were incubated for 12 h and the wound closure areas were visualized on an Olympus Microscope (Carl Zeiss). Representative images were acquired at 0 and 12 h. c Quantification of cell migration was achieved using Image J software. Data are presented as percentages of the recovered scratch area relative to untreated control cells. Columns are the means five replicates from two independent experiments and bars are s.d. d MDA-MB231 and MDA-MB231 cells stably transfected with control GFP or GFP-MIEN1WT were subjected to western blot analysis with anti-phosho AnxA2, anti-AnxA2 and anti-MIEN1 antibodies. GAPDH served as a loading control. The quantification of the representative blots is the densitometric average of three independent experiments analyzed using ImageJ and normalized to GAPDH. e Control and MIEN1 transfected MDA-MB231 cells were subjected to western blot analysis with anti-phosho AnxA2, AnxA2, anti-MIEN1 and actin as loading control. f Representative images, as captured using a 60X oil immersion TIRF microscope, of MDA-MB231 and MDA-MB231 cells stably transfected with GFP-MIEN1WT grown to sub-confluence on coverslips, fixed with PFA, unpermeabilized, and processed for TIRF microscopy with the specific antibodies. At least three independent fields were analyzed for confirmation of the staining pattern represented. Scale bar denotes 20 μm

Mentions: We investigated the effects of MIEN1 and AnxA2 interaction on cell migration, given their independent implications in cell migration and invasion (Fig. 5a-c). We silenced MIEN1 and AnxA2 alone or in combination (Fig. 5b) and examined the effects on motility of MDA-MB231 cells. In monolayer migration assays, the wound closure of cells with MIEN1 knockdown was significantly inhibited compared to siGFP (control) transfected MDA-MB231 cells (Fig. 5a, Additional file 3: Figure S3A-B). Silencing of AnxA2 resulted in impairment of cell motility at 6 and 12 h following wound creation (Fig. 5a, Additional file 3: Figure S3A-B). Using similar knockdown strategy, we observed that dual depletion of MIEN1 and AnxA2 led to a two-fold decrease compared to siGFP (Fig. 5c), indicating that MIEN1 and AnxA2 functionally cooperate to promote breast cancer cell migration. AnxA2 is a phospholipid binding protein localizing to the plasma membrane towards the cytosolic side. Previous studies proved that the phosphorylation of AnxA2 at the Y23 residue in the N-terminus of the protein causes its translocation to the extracellular surface, thereby activating extracellular proteolysis [26, 27]. To verify whether MIEN1 expression affects AnxA2 phosphorylation and subsequent translocation to the extracellular surface, we performed immunoblotting analysis followed by total internal reflection microscopy (TIRF-M). As shown in Fig. 5d, over-expression of MIEN1 enhanced AnxA2 phosphorylation at Y23. Conversely, down-regulation of MIEN1 led to a decreased phosphorylation of AnxA2 at Y23 (Fig. 5e). To further confirm this observation, we performed total internal reflection fluorescence microscopy (TIRF-M) in MDA-MB231 cells. Stable MDA-MB231 cells expressing vector control or MIEN1WT were probed with AnxA2 and phospho-AnxA2Tyr23 antibodies followed by TIRF microscopy to determine cell surface total-AnxA2 and phospho-AnxA2. MIEN1 overexpression significantly enhanced phosphorylation of AnxA2 at the tyrosine 23 residue and subsequently cell surface localized AnxA2 levels; indicating MIEN1 dependent signaling and interaction activate AnxA2 function (Fig. 5f).Fig. 5


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 activates translocation of AnxA2 to plasma membrane to promote breast cancer cell migration. a Control and MIEN1 transfected MDA-MB231 cells were subjected to western blot analysis with anti-MIEN1 or anti-AnxA2 antibody. PGK served as a loading control. b Down-regulation of MIEN1 and AnxA2 reduces the abilities of MDA-MB231 breast cancer cells to migrate in vitro. Confluent MDA-MB231 monolayers transfected with control GFP, AnxA2 or/and MIEN1 were wounded using a pipet tip, 60 h after transfection. Following the wound formation, plates were incubated for 12 h and the wound closure areas were visualized on an Olympus Microscope (Carl Zeiss). Representative images were acquired at 0 and 12 h. c Quantification of cell migration was achieved using Image J software. Data are presented as percentages of the recovered scratch area relative to untreated control cells. Columns are the means five replicates from two independent experiments and bars are s.d. d MDA-MB231 and MDA-MB231 cells stably transfected with control GFP or GFP-MIEN1WT were subjected to western blot analysis with anti-phosho AnxA2, anti-AnxA2 and anti-MIEN1 antibodies. GAPDH served as a loading control. The quantification of the representative blots is the densitometric average of three independent experiments analyzed using ImageJ and normalized to GAPDH. e Control and MIEN1 transfected MDA-MB231 cells were subjected to western blot analysis with anti-phosho AnxA2, AnxA2, anti-MIEN1 and actin as loading control. f Representative images, as captured using a 60X oil immersion TIRF microscope, of MDA-MB231 and MDA-MB231 cells stably transfected with GFP-MIEN1WT grown to sub-confluence on coverslips, fixed with PFA, unpermeabilized, and processed for TIRF microscopy with the specific antibodies. At least three independent fields were analyzed for confirmation of the staining pattern represented. Scale bar denotes 20 μm
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Fig5: MIEN1 activates translocation of AnxA2 to plasma membrane to promote breast cancer cell migration. a Control and MIEN1 transfected MDA-MB231 cells were subjected to western blot analysis with anti-MIEN1 or anti-AnxA2 antibody. PGK served as a loading control. b Down-regulation of MIEN1 and AnxA2 reduces the abilities of MDA-MB231 breast cancer cells to migrate in vitro. Confluent MDA-MB231 monolayers transfected with control GFP, AnxA2 or/and MIEN1 were wounded using a pipet tip, 60 h after transfection. Following the wound formation, plates were incubated for 12 h and the wound closure areas were visualized on an Olympus Microscope (Carl Zeiss). Representative images were acquired at 0 and 12 h. c Quantification of cell migration was achieved using Image J software. Data are presented as percentages of the recovered scratch area relative to untreated control cells. Columns are the means five replicates from two independent experiments and bars are s.d. d MDA-MB231 and MDA-MB231 cells stably transfected with control GFP or GFP-MIEN1WT were subjected to western blot analysis with anti-phosho AnxA2, anti-AnxA2 and anti-MIEN1 antibodies. GAPDH served as a loading control. The quantification of the representative blots is the densitometric average of three independent experiments analyzed using ImageJ and normalized to GAPDH. e Control and MIEN1 transfected MDA-MB231 cells were subjected to western blot analysis with anti-phosho AnxA2, AnxA2, anti-MIEN1 and actin as loading control. f Representative images, as captured using a 60X oil immersion TIRF microscope, of MDA-MB231 and MDA-MB231 cells stably transfected with GFP-MIEN1WT grown to sub-confluence on coverslips, fixed with PFA, unpermeabilized, and processed for TIRF microscopy with the specific antibodies. At least three independent fields were analyzed for confirmation of the staining pattern represented. Scale bar denotes 20 μm
Mentions: We investigated the effects of MIEN1 and AnxA2 interaction on cell migration, given their independent implications in cell migration and invasion (Fig. 5a-c). We silenced MIEN1 and AnxA2 alone or in combination (Fig. 5b) and examined the effects on motility of MDA-MB231 cells. In monolayer migration assays, the wound closure of cells with MIEN1 knockdown was significantly inhibited compared to siGFP (control) transfected MDA-MB231 cells (Fig. 5a, Additional file 3: Figure S3A-B). Silencing of AnxA2 resulted in impairment of cell motility at 6 and 12 h following wound creation (Fig. 5a, Additional file 3: Figure S3A-B). Using similar knockdown strategy, we observed that dual depletion of MIEN1 and AnxA2 led to a two-fold decrease compared to siGFP (Fig. 5c), indicating that MIEN1 and AnxA2 functionally cooperate to promote breast cancer cell migration. AnxA2 is a phospholipid binding protein localizing to the plasma membrane towards the cytosolic side. Previous studies proved that the phosphorylation of AnxA2 at the Y23 residue in the N-terminus of the protein causes its translocation to the extracellular surface, thereby activating extracellular proteolysis [26, 27]. To verify whether MIEN1 expression affects AnxA2 phosphorylation and subsequent translocation to the extracellular surface, we performed immunoblotting analysis followed by total internal reflection microscopy (TIRF-M). As shown in Fig. 5d, over-expression of MIEN1 enhanced AnxA2 phosphorylation at Y23. Conversely, down-regulation of MIEN1 led to a decreased phosphorylation of AnxA2 at Y23 (Fig. 5e). To further confirm this observation, we performed total internal reflection fluorescence microscopy (TIRF-M) in MDA-MB231 cells. Stable MDA-MB231 cells expressing vector control or MIEN1WT were probed with AnxA2 and phospho-AnxA2Tyr23 antibodies followed by TIRF microscopy to determine cell surface total-AnxA2 and phospho-AnxA2. MIEN1 overexpression significantly enhanced phosphorylation of AnxA2 at the tyrosine 23 residue and subsequently cell surface localized AnxA2 levels; indicating MIEN1 dependent signaling and interaction activate AnxA2 function (Fig. 5f).Fig. 5

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