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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

AnxA2 and MIEN1 silencing inhibit tPA dependent plasmin generation. Plasmin activity was determined at 460 nm using recombinant plasminogen, TPA and fluorogenic plasmin substrate D-VLK-AMC. Western blotting was performed to confirm depletion of MIEN1 and AnxA2 in HCC-70 (a) and MCF-7 (c) cells following siRNA knockdown. PGK served as a loading control. Total fold change in plasmin level in HCC-70 (b) and MCF-7 (d) cells was calculated by normalizing the initial rates of plasmin in untreated cells (which were assigned a value of 1). The data is presented as means ± s.d. (n = 6 for untreated controls and n = 6 for siRNA treatments)
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Fig6: AnxA2 and MIEN1 silencing inhibit tPA dependent plasmin generation. Plasmin activity was determined at 460 nm using recombinant plasminogen, TPA and fluorogenic plasmin substrate D-VLK-AMC. Western blotting was performed to confirm depletion of MIEN1 and AnxA2 in HCC-70 (a) and MCF-7 (c) cells following siRNA knockdown. PGK served as a loading control. Total fold change in plasmin level in HCC-70 (b) and MCF-7 (d) cells was calculated by normalizing the initial rates of plasmin in untreated cells (which were assigned a value of 1). The data is presented as means ± s.d. (n = 6 for untreated controls and n = 6 for siRNA treatments)

Mentions: Extracellular surface localized AnxA2 functions as a receptor for tPA and plasminogen; by binding to both enzymes, and catalytically activating plasmin generation [28, 29]. Increased levels of plasmin enhance proteolytic cleavage of ECM thus allowing cancer cells to migrate and invade to distant sites. To confirm that MIEN1 regulates AnxA2 dependent plasmin generation, we investigated the effects of MIEN1 ablation either alone or in combination with AnxA2 plasmin generation in breast cancer cells. Using basal-like HCC70 and luminal MCF7 cells (these two lines express high levels of both AnxA2 and MIEN1), we inquired whether the interaction of MIEN1 and AnxA2 had an effect on plasmin generation. We first confirmed siRNA-mediated knockdown of MIEN1 and AnxA2 in HCC-70 and MCF-7 (Fig. 6a, c) respectively. Western blotting analysis confirmed that the expression of MIEN1 or AnxA2 was dramatically reduced in the cells upon transfection with either MIEN1 or AnxA2 siRNA. Next, we examined the biochemical conversion of plasminogen to plasmin in both cell lines upon siRNA treatment. Silencing of both MIEN1 and AnxA2 led to a significant decrease in plasmin levels in both HCC-70 and MCF-7 (P < 0.05) (Fig. 6b, d). While the knockdown of AnxA2 led to a decrease in plasminogen conversion to plasmin in the initial hours as previously shown [30], the total change in plasmin levels were insignificant compared to the control siRNA treated cells. Interestingly, suppression of MIEN1 in MCF-7 cells led to approximately 1.7 fold decrease in plasmin levels but not in HCC-70; a difference which can be attributed to the expression of various regulators of the plasminogen-plasmin system. Taken together, these data indicate that co-expression of both AnxA2 and MIEN1 enhance plasmin generation and lead to an increase in breast tumor cell migration and invasion which in turn drive the metastatic process.Fig. 6


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)

AnxA2 and MIEN1 silencing inhibit tPA dependent plasmin generation. Plasmin activity was determined at 460 nm using recombinant plasminogen, TPA and fluorogenic plasmin substrate D-VLK-AMC. Western blotting was performed to confirm depletion of MIEN1 and AnxA2 in HCC-70 (a) and MCF-7 (c) cells following siRNA knockdown. PGK served as a loading control. Total fold change in plasmin level in HCC-70 (b) and MCF-7 (d) cells was calculated by normalizing the initial rates of plasmin in untreated cells (which were assigned a value of 1). The data is presented as means ± s.d. (n = 6 for untreated controls and n = 6 for siRNA treatments)
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Fig6: AnxA2 and MIEN1 silencing inhibit tPA dependent plasmin generation. Plasmin activity was determined at 460 nm using recombinant plasminogen, TPA and fluorogenic plasmin substrate D-VLK-AMC. Western blotting was performed to confirm depletion of MIEN1 and AnxA2 in HCC-70 (a) and MCF-7 (c) cells following siRNA knockdown. PGK served as a loading control. Total fold change in plasmin level in HCC-70 (b) and MCF-7 (d) cells was calculated by normalizing the initial rates of plasmin in untreated cells (which were assigned a value of 1). The data is presented as means ± s.d. (n = 6 for untreated controls and n = 6 for siRNA treatments)
Mentions: Extracellular surface localized AnxA2 functions as a receptor for tPA and plasminogen; by binding to both enzymes, and catalytically activating plasmin generation [28, 29]. Increased levels of plasmin enhance proteolytic cleavage of ECM thus allowing cancer cells to migrate and invade to distant sites. To confirm that MIEN1 regulates AnxA2 dependent plasmin generation, we investigated the effects of MIEN1 ablation either alone or in combination with AnxA2 plasmin generation in breast cancer cells. Using basal-like HCC70 and luminal MCF7 cells (these two lines express high levels of both AnxA2 and MIEN1), we inquired whether the interaction of MIEN1 and AnxA2 had an effect on plasmin generation. We first confirmed siRNA-mediated knockdown of MIEN1 and AnxA2 in HCC-70 and MCF-7 (Fig. 6a, c) respectively. Western blotting analysis confirmed that the expression of MIEN1 or AnxA2 was dramatically reduced in the cells upon transfection with either MIEN1 or AnxA2 siRNA. Next, we examined the biochemical conversion of plasminogen to plasmin in both cell lines upon siRNA treatment. Silencing of both MIEN1 and AnxA2 led to a significant decrease in plasmin levels in both HCC-70 and MCF-7 (P < 0.05) (Fig. 6b, d). While the knockdown of AnxA2 led to a decrease in plasminogen conversion to plasmin in the initial hours as previously shown [30], the total change in plasmin levels were insignificant compared to the control siRNA treated cells. Interestingly, suppression of MIEN1 in MCF-7 cells led to approximately 1.7 fold decrease in plasmin levels but not in HCC-70; a difference which can be attributed to the expression of various regulators of the plasminogen-plasmin system. Taken together, these data indicate that co-expression of both AnxA2 and MIEN1 enhance plasmin generation and lead to an increase in breast tumor cell migration and invasion which in turn drive the metastatic process.Fig. 6

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