ABSTRACT
Although the α6ß1 integrin has been implicated in the function of breast and other cancer stem cells (CSCs), little is known about its regulation and relationship to mechanisms involved in the genesis of CSCs. We report that a CD44(high)/CD24(low) population, enriched for CSCs, is comprised of distinct epithelial and mesenchymal populations that differ in expression of the two α6 cytoplasmic domain splice variants: α6A and α6B. α6Bß1 expression defines the mesenchymal population and is necessary for CSC function, a function that cannot be executed by α6A integrins. The generation of α6Bß1 is tightly controlled and occurs as a consequence of an autocrine vascular endothelial growth factor (VEGF) signaling that culminates in the transcriptional repression of a key RNA-splicing factor. These data alter our understanding of how α6ß1 contributes to breast cancer, and they resolve ambiguities regarding the use of total α6 (CD49f) expression as a biomarker for CSCs.
Subject(s)
Integrin alpha6/metabolism , Neoplastic Stem Cells/metabolism , RNA Splicing/physiology , Breast Neoplasms/metabolism , Breast Neoplasms/pathology , CD24 Antigen/metabolism , Cell Line, Tumor , Female , Humans , Hyaluronan Receptors/metabolism , Integrin alpha6/chemistry , Integrin alpha6/genetics , Neoplastic Stem Cells/cytology , Polycomb Repressive Complex 1/metabolism , Protein Structure, Tertiary , RNA Interference , RNA, Small Interfering/metabolism , RNA-Binding Proteins/metabolism , Signal Transduction , Vascular Endothelial Growth Factor A/metabolismABSTRACT
Mutations of the neurofibromatosis 2 (NF2) tumor suppressor gene have frequently been detected not only in schwannomas and other central nervous system tumors of NF2 patients but also in their sporadic counterparts and malignant tumors unrelated to the NF2 syndrome such as malignant mesothelioma, indicating a broader role for the NF2 gene in human tumorigenesis. However, the mechanisms by which the NF2 product, merlin or schwannomin, is regulated and controls cell proliferation remain elusive. Here, we identify a novel GTP-binding protein, dubbed NGB (referring to NF2-associated GTP binding protein), which binds to merlin. NGB is highly conserved between Saccharomyces cerevisiae, Caenorhabditis elegans, and human cells, and its GTP-binding region is very similar to those found in R-ras and Rap2. However, ectopic expression of NGB inhibits cell growth, cell aggregation, and tumorigenicity in tumorigenic schwanomma cells. Down-regulation and infrequent mutation of NGB were detected in human glioma cell lines and primary tumors. The interaction of NGB with merlin impairs the turnover of merlin, yet merlin does not affect the GTPase nor GTP-binding activity of NGB. Finally, the tumor suppressor functions of NGB require merlin and are linked to its ability to suppress cyclin D1 expression. Collectively, these findings indicate that NGB is a tumor suppressor that regulates and requires merlin to suppress cell proliferation.
Subject(s)
GTP-Binding Proteins/metabolism , Neurofibromin 2/metabolism , Tumor Suppressor Proteins/metabolism , Amino Acid Sequence , Animals , Base Sequence , Cell Adhesion , Cell Movement , Cell Proliferation , Conserved Sequence , Cyclin D1/metabolism , Down-Regulation , GTP-Binding Proteins/chemistry , GTP-Binding Proteins/genetics , GTP-Binding Proteins/isolation & purification , Gene Expression Regulation, Neoplastic , Humans , Mice , Molecular Sequence Data , Neoplasms/genetics , Neoplasms/metabolism , Neoplasms/pathology , Neurofibromin 2/genetics , Protein Binding , Rats , Sequence Alignment , Tumor Suppressor Proteins/chemistry , Tumor Suppressor Proteins/genetics , Tumor Suppressor Proteins/isolation & purification , Two-Hybrid System Techniques , Ubiquitin/metabolismABSTRACT
Previous studies have demonstrated amplification of the centrosome serine/threonine kinase BTAK/Aurora-A in 10-25% of ovarian cancers. However, alterations of BTAK/Aurora-A at kinase and protein levels and its role in ovarian cancer progression have not been well documented. In this study, we examined the kinase activity and protein levels of BTAK/Aurora-A in 92 patients with primary ovarian tumors. In vitro kinase analyses revealed elevated BTAK/Aurora-A kinase activity in 44 cases (48%). Increased BTAK/Aurora-A protein levels were detected in 52 (57%) specimens. High protein levels of BTAK/Aurora-A correlated well with elevated kinase activity. Activation and overexpression of BTAK/Aurora-A were more frequently detected in early stage/low-grade ovarian tumors, although there was no statistic significance at the kinase level between early stage/low-grade and late stage/high-grade tumors. Moreover, BTAK/Aurora-A was preferentially expressed in noninvasive tumors, as revealed by immunohistochemical staining, suggesting that alterations of BTAK/Aurora-A could be an early event in human ovarian oncogenesis. To our knowledge, this is the first demonstration of recurrent activation and overexpression of BTAK/Aurora-A in human ovarian cancer, which may play a critical role in development of this malignancy.
Subject(s)
Ovarian Neoplasms/enzymology , Protein Serine-Threonine Kinases/biosynthesis , Protein Serine-Threonine Kinases/metabolism , Aurora Kinase A , Aurora Kinases , Blotting, Northern , Blotting, Southern , Blotting, Western , Cell Line , DNA, Complementary/metabolism , Female , Glutathione Transferase/metabolism , Humans , Immunohistochemistry , Ovarian Neoplasms/metabolism , Ovary/metabolism , Precipitin Tests , Recombinant Fusion Proteins/metabolism , TransfectionABSTRACT
Akt2 is a member of the Akt/PKB family, which is involved in a variety of cellular events including cell survival, proliferation, and differentiation. During skeletal muscle differentiation, the Akt2 but not Akt1 expression was significantly increased. Microinjection of anti-Akt2 but not anti-Akt1 antibody efficiently abrogated myogenesis, indicating that Akt2 plays a specific role in muscle differentiation. It has been well documented that ectopic expression of MyoD is sufficient to induce muscle differentiation in myoblasts. However, the mechanism of induction of Akt2 during muscle differentiation and the significance of Akt2 protein in MyoD-induced myogenesis are largely unknown. In this study, we provide direct evidence that Akt2 is transcriptionally regulated by MyoD and activates MyoD-myocyte enhancer binding factor-2 (MEF2) transactivation activity. The Akt2 promoter was isolated and found to contain nine putative E-boxes (CANNTG), which are putative MyoD binding sites. Electrophoretic mobility shift analyses revealed that MyoD bound to eight of the sites. The expression of MyoD significantly enhanced Akt2 promoter activity and up-regulated Akt2 mRNA and protein levels. Moreover, Akt2 but not Akt1 was activated during differentiation. The expression of Akt2 activated MyoD-MEF2 transcriptional activity and induced myogenin expression. These data indicate that there is a positive feedback regulation loop between Akt2 and MyoD-MEF2 during muscle differentiation, which is essential for MyoD-induced myogenesis.
