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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

Posttranslational modifications on MIEN1 regulate its function. NIH3T3 cells transfected with the MIEN1 constructs indicated in Fig. 2a, were subjected to scratch wound assays. a–g Confluent monolayers of cells were wounded, and healing of the wound by cell migration was monitored for 18 h. Images were taken at 0 and 18 h. h The fold change in migration was normalized to GFP (empty vector) transfected cells and expressed as the means ± s.e.m of three independent experiments. i–n NIH3T3 cells transfected with indicated GFP-tagged MIEN1 constructs were stained with rhodamine-conjugated phalloidin post wound induction to evaluate effects of ITAM and CAAX motif on filopodia formation. o NIH3T3 cells expressing the GFP vector control and GFP-MIEN1 variants were analyzed for GFP and MIEN1 contents. GAPDH served as a loading control
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Fig3: Posttranslational modifications on MIEN1 regulate its function. NIH3T3 cells transfected with the MIEN1 constructs indicated in Fig. 2a, were subjected to scratch wound assays. a–g Confluent monolayers of cells were wounded, and healing of the wound by cell migration was monitored for 18 h. Images were taken at 0 and 18 h. h The fold change in migration was normalized to GFP (empty vector) transfected cells and expressed as the means ± s.e.m of three independent experiments. i–n NIH3T3 cells transfected with indicated GFP-tagged MIEN1 constructs were stained with rhodamine-conjugated phalloidin post wound induction to evaluate effects of ITAM and CAAX motif on filopodia formation. o NIH3T3 cells expressing the GFP vector control and GFP-MIEN1 variants were analyzed for GFP and MIEN1 contents. GAPDH served as a loading control

Mentions: Next, we evaluated the functional impact of MIEN1-ITAM and isoprenyl mutants compared to the wild-type protein (MIEN1WT). For this, we stably expressed MIEN1 constructs in NIH3T3-cells with low endogenous protein. Cell migration was significantly enhanced in cells expressing MIEN1WT relative to the vector control (Fig. 3a, b), whereas MIEN1C112S failed to migrate into the wound (Fig. 3c), as reported earlier [6]. Similar to the isoprenyl-mutants, NIH3T3 expressing ITAM mutants had a reduced migratory potential (Fig. 3d–f). However, we did not observe significant differences in functional contribution between individual tyrosine mutants (MIEN1Y39F and MIEN1Y50F) compared to MIEN1Y39/50F confirming that the phosphorylation of both tyrosine residues is vital for ITAM induced functions or signaling events to mediate cell migration. The MIEN1Y39/50F/C112S cells did not show any additive effect suggesting both isoprenylation and ITAM-phosphorylation are important for MIEN1 function (Fig. 3g). To determine whether MIEN1 posttranslational modifications are also required for invasive function, we performed matrigel invasive assays with MDA-MB231 cells transfected with MIEN1 constructs. Consistent with the data from the migration assays, MIEN1WT expression increased the invasive potential of breast cancer cells whereas ITAM and isoprenyl mutants failed to show an invasive phenotype (Additional file 1: Figure S1).Fig. 3


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)

Posttranslational modifications on MIEN1 regulate its function. NIH3T3 cells transfected with the MIEN1 constructs indicated in Fig. 2a, were subjected to scratch wound assays. a–g Confluent monolayers of cells were wounded, and healing of the wound by cell migration was monitored for 18 h. Images were taken at 0 and 18 h. h The fold change in migration was normalized to GFP (empty vector) transfected cells and expressed as the means ± s.e.m of three independent experiments. i–n NIH3T3 cells transfected with indicated GFP-tagged MIEN1 constructs were stained with rhodamine-conjugated phalloidin post wound induction to evaluate effects of ITAM and CAAX motif on filopodia formation. o NIH3T3 cells expressing the GFP vector control and GFP-MIEN1 variants were analyzed for GFP and MIEN1 contents. GAPDH served as a loading control
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Related In: Results  -  Collection

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Fig3: Posttranslational modifications on MIEN1 regulate its function. NIH3T3 cells transfected with the MIEN1 constructs indicated in Fig. 2a, were subjected to scratch wound assays. a–g Confluent monolayers of cells were wounded, and healing of the wound by cell migration was monitored for 18 h. Images were taken at 0 and 18 h. h The fold change in migration was normalized to GFP (empty vector) transfected cells and expressed as the means ± s.e.m of three independent experiments. i–n NIH3T3 cells transfected with indicated GFP-tagged MIEN1 constructs were stained with rhodamine-conjugated phalloidin post wound induction to evaluate effects of ITAM and CAAX motif on filopodia formation. o NIH3T3 cells expressing the GFP vector control and GFP-MIEN1 variants were analyzed for GFP and MIEN1 contents. GAPDH served as a loading control
Mentions: Next, we evaluated the functional impact of MIEN1-ITAM and isoprenyl mutants compared to the wild-type protein (MIEN1WT). For this, we stably expressed MIEN1 constructs in NIH3T3-cells with low endogenous protein. Cell migration was significantly enhanced in cells expressing MIEN1WT relative to the vector control (Fig. 3a, b), whereas MIEN1C112S failed to migrate into the wound (Fig. 3c), as reported earlier [6]. Similar to the isoprenyl-mutants, NIH3T3 expressing ITAM mutants had a reduced migratory potential (Fig. 3d–f). However, we did not observe significant differences in functional contribution between individual tyrosine mutants (MIEN1Y39F and MIEN1Y50F) compared to MIEN1Y39/50F confirming that the phosphorylation of both tyrosine residues is vital for ITAM induced functions or signaling events to mediate cell migration. The MIEN1Y39/50F/C112S cells did not show any additive effect suggesting both isoprenylation and ITAM-phosphorylation are important for MIEN1 function (Fig. 3g). To determine whether MIEN1 posttranslational modifications are also required for invasive function, we performed matrigel invasive assays with MDA-MB231 cells transfected with MIEN1 constructs. Consistent with the data from the migration assays, MIEN1WT expression increased the invasive potential of breast cancer cells whereas ITAM and isoprenyl mutants failed to show an invasive phenotype (Additional file 1: Figure S1).Fig. 3

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