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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

Expression of MIEN1 in breast cancer patient specimens and cultured cell lines. a Expression of MIEN1 in different subtypes of breast cancer patients as obtained by analyzing TCGA dataset from Oncomine. Legends and number of tissues analyzed are in parentheses- 0. No value (normal) (n = 61); 1. Apocrine Breast Carcinoma (n = 1); 2. Breast Large Cell Neuroendocrine Carcinoma (n = 1); 3. Ductal Breast Carcinoma (n = 1); 4. Intraductal Cribriform Breast Adenocarcinoma (n = 3); 5. Invasive Breast Carcinoma (n = 76); 6. Invasive Cribriform Breast Carcinoma (n = 1); 7. Invasive Ductal Breast Carcinoma (n = 392); 8. Invasive Ductal and Lobular Carcinoma (n = 3); 9. Invasive Lobular Breast Carcinoma (n = 36); 10. Invasive Papillary Breast Carcinoma (n = 1); 11. Male Breast Carcinoma (n = 3); 12. Metaplastic Breast Carcinoma (n = 1); 13. Mixed Lobular and Ductal Breast Carcinoma (n = 7); 14. Mucinous Breast Carcinoma (n = 4); 15. Papillary Breast Carcinoma (n = 1); 16. Pleomorphic Breast Carcinoma (n = 1). b MIEN1 mRNA expression in different molecular subtypes of breast cancer as determined by RSSPC using bc-GenExMiner database v3.0. A. The figure and table show the patients with low, intermediate and high MIEN1 expression in each molecular subtype. c MIEN1 protein expression was analyzed by immunoblotting analysis and β-actin was used as loading control. d Kaplan Meier survival curve showing the survival percentage of breast cancer patients were significantly low in MIEN1-high expressing tumors compared to low-expressing cohort. TCGA data analyzed using UCSD cancer genome browser
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Fig1: Expression of MIEN1 in breast cancer patient specimens and cultured cell lines. a Expression of MIEN1 in different subtypes of breast cancer patients as obtained by analyzing TCGA dataset from Oncomine. Legends and number of tissues analyzed are in parentheses- 0. No value (normal) (n = 61); 1. Apocrine Breast Carcinoma (n = 1); 2. Breast Large Cell Neuroendocrine Carcinoma (n = 1); 3. Ductal Breast Carcinoma (n = 1); 4. Intraductal Cribriform Breast Adenocarcinoma (n = 3); 5. Invasive Breast Carcinoma (n = 76); 6. Invasive Cribriform Breast Carcinoma (n = 1); 7. Invasive Ductal Breast Carcinoma (n = 392); 8. Invasive Ductal and Lobular Carcinoma (n = 3); 9. Invasive Lobular Breast Carcinoma (n = 36); 10. Invasive Papillary Breast Carcinoma (n = 1); 11. Male Breast Carcinoma (n = 3); 12. Metaplastic Breast Carcinoma (n = 1); 13. Mixed Lobular and Ductal Breast Carcinoma (n = 7); 14. Mucinous Breast Carcinoma (n = 4); 15. Papillary Breast Carcinoma (n = 1); 16. Pleomorphic Breast Carcinoma (n = 1). b MIEN1 mRNA expression in different molecular subtypes of breast cancer as determined by RSSPC using bc-GenExMiner database v3.0. A. The figure and table show the patients with low, intermediate and high MIEN1 expression in each molecular subtype. c MIEN1 protein expression was analyzed by immunoblotting analysis and β-actin was used as loading control. d Kaplan Meier survival curve showing the survival percentage of breast cancer patients were significantly low in MIEN1-high expressing tumors compared to low-expressing cohort. TCGA data analyzed using UCSD cancer genome browser

Mentions: Enhanced expression of MIEN1 is reported in breast cancer compared to normal breast tissues [2]. Analysis of Cancer Genome Atlas (TCGA) data sets identified significantly elevated MIEN1 expression in different subtypes of breast carcinomas (Apocrine, Large Cell Neuroendocrine, Cribiform, Papillary, Ductal, Lobular, Mixed Ductal and Lobular, Mucinous) patients compared to normal tissues (Fig. 1a). In clinical oncology, evaluations of breast tumors are accompanied by an assessment of the molecular status of ER, PR and Her-2 oncogene. To understand the differential expression of MIEN1 in various subtypes of breast cancer, we examined the expression of MIEN1 within the molecular subtypes of breast cancer. Our findings revealed that MIEN1 is predominantly overexpressed in Her-2 positive (85 % cases with elevated MIEN1) and luminal B (63 % cases with elevated MIEN1) subtypes. However, MIEN1 expression in other subtypes basal-like and luminal A were moderate, whereas majority of the normal breast tissues had low MIEN1 (Fig. 1b). Screening of various established breast tumor lines identified increased expression of MIEN1 in majority of the breast tumor lines (MCF10AT, MCF10CA1a, MCF10CA1d, MCF10CA1h, JIMT-1, BT-474, SKBR-3, MDA-MB231, T47D, MCF-7, MDA-MB436 and HCC-70) compared to the immortalized normal mammary epithelial cell line, MCF10A (Fig. 1c). As expected most of the Her-2 amplified cell lines displayed increased expression of MIEN1 protein (CRL-2330, SKBR-3, and BT-474); but MIEN1 expression is not only restricted to Her-2 amplification indicating its distinct transcriptional and post-translational modifications contributing to its elevated expression in breast tumors. Classification of the patient cohort according to high and low MIEN1 expression using TCGA dataset, confirmed a poor survival in breast cancer patients with elevated MIEN1 expression as previously shown [21] (Fig. 1d). Altogether, these findings confirm that MIEN1 is a clinically important oncogene, and its increased expression contributes towards an aggressive disease with poor survival.Fig. 1


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)

Expression of MIEN1 in breast cancer patient specimens and cultured cell lines. a Expression of MIEN1 in different subtypes of breast cancer patients as obtained by analyzing TCGA dataset from Oncomine. Legends and number of tissues analyzed are in parentheses- 0. No value (normal) (n = 61); 1. Apocrine Breast Carcinoma (n = 1); 2. Breast Large Cell Neuroendocrine Carcinoma (n = 1); 3. Ductal Breast Carcinoma (n = 1); 4. Intraductal Cribriform Breast Adenocarcinoma (n = 3); 5. Invasive Breast Carcinoma (n = 76); 6. Invasive Cribriform Breast Carcinoma (n = 1); 7. Invasive Ductal Breast Carcinoma (n = 392); 8. Invasive Ductal and Lobular Carcinoma (n = 3); 9. Invasive Lobular Breast Carcinoma (n = 36); 10. Invasive Papillary Breast Carcinoma (n = 1); 11. Male Breast Carcinoma (n = 3); 12. Metaplastic Breast Carcinoma (n = 1); 13. Mixed Lobular and Ductal Breast Carcinoma (n = 7); 14. Mucinous Breast Carcinoma (n = 4); 15. Papillary Breast Carcinoma (n = 1); 16. Pleomorphic Breast Carcinoma (n = 1). b MIEN1 mRNA expression in different molecular subtypes of breast cancer as determined by RSSPC using bc-GenExMiner database v3.0. A. The figure and table show the patients with low, intermediate and high MIEN1 expression in each molecular subtype. c MIEN1 protein expression was analyzed by immunoblotting analysis and β-actin was used as loading control. d Kaplan Meier survival curve showing the survival percentage of breast cancer patients were significantly low in MIEN1-high expressing tumors compared to low-expressing cohort. TCGA data analyzed using UCSD cancer genome browser
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4536591&req=5

Fig1: Expression of MIEN1 in breast cancer patient specimens and cultured cell lines. a Expression of MIEN1 in different subtypes of breast cancer patients as obtained by analyzing TCGA dataset from Oncomine. Legends and number of tissues analyzed are in parentheses- 0. No value (normal) (n = 61); 1. Apocrine Breast Carcinoma (n = 1); 2. Breast Large Cell Neuroendocrine Carcinoma (n = 1); 3. Ductal Breast Carcinoma (n = 1); 4. Intraductal Cribriform Breast Adenocarcinoma (n = 3); 5. Invasive Breast Carcinoma (n = 76); 6. Invasive Cribriform Breast Carcinoma (n = 1); 7. Invasive Ductal Breast Carcinoma (n = 392); 8. Invasive Ductal and Lobular Carcinoma (n = 3); 9. Invasive Lobular Breast Carcinoma (n = 36); 10. Invasive Papillary Breast Carcinoma (n = 1); 11. Male Breast Carcinoma (n = 3); 12. Metaplastic Breast Carcinoma (n = 1); 13. Mixed Lobular and Ductal Breast Carcinoma (n = 7); 14. Mucinous Breast Carcinoma (n = 4); 15. Papillary Breast Carcinoma (n = 1); 16. Pleomorphic Breast Carcinoma (n = 1). b MIEN1 mRNA expression in different molecular subtypes of breast cancer as determined by RSSPC using bc-GenExMiner database v3.0. A. The figure and table show the patients with low, intermediate and high MIEN1 expression in each molecular subtype. c MIEN1 protein expression was analyzed by immunoblotting analysis and β-actin was used as loading control. d Kaplan Meier survival curve showing the survival percentage of breast cancer patients were significantly low in MIEN1-high expressing tumors compared to low-expressing cohort. TCGA data analyzed using UCSD cancer genome browser
Mentions: Enhanced expression of MIEN1 is reported in breast cancer compared to normal breast tissues [2]. Analysis of Cancer Genome Atlas (TCGA) data sets identified significantly elevated MIEN1 expression in different subtypes of breast carcinomas (Apocrine, Large Cell Neuroendocrine, Cribiform, Papillary, Ductal, Lobular, Mixed Ductal and Lobular, Mucinous) patients compared to normal tissues (Fig. 1a). In clinical oncology, evaluations of breast tumors are accompanied by an assessment of the molecular status of ER, PR and Her-2 oncogene. To understand the differential expression of MIEN1 in various subtypes of breast cancer, we examined the expression of MIEN1 within the molecular subtypes of breast cancer. Our findings revealed that MIEN1 is predominantly overexpressed in Her-2 positive (85 % cases with elevated MIEN1) and luminal B (63 % cases with elevated MIEN1) subtypes. However, MIEN1 expression in other subtypes basal-like and luminal A were moderate, whereas majority of the normal breast tissues had low MIEN1 (Fig. 1b). Screening of various established breast tumor lines identified increased expression of MIEN1 in majority of the breast tumor lines (MCF10AT, MCF10CA1a, MCF10CA1d, MCF10CA1h, JIMT-1, BT-474, SKBR-3, MDA-MB231, T47D, MCF-7, MDA-MB436 and HCC-70) compared to the immortalized normal mammary epithelial cell line, MCF10A (Fig. 1c). As expected most of the Her-2 amplified cell lines displayed increased expression of MIEN1 protein (CRL-2330, SKBR-3, and BT-474); but MIEN1 expression is not only restricted to Her-2 amplification indicating its distinct transcriptional and post-translational modifications contributing to its elevated expression in breast tumors. Classification of the patient cohort according to high and low MIEN1 expression using TCGA dataset, confirmed a poor survival in breast cancer patients with elevated MIEN1 expression as previously shown [21] (Fig. 1d). Altogether, these findings confirm that MIEN1 is a clinically important oncogene, and its increased expression contributes towards an aggressive disease with poor survival.Fig. 1

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