Abstract
Tetraspanin-29 (CD9) is an integral membrane protein involved in several fundamental cell processes and in cancer metastasis. Here, characterization of a panel of breast cancer cells revealed a nuclear pool of CD9, not present in normal human mammary epithelial cells. Antibody binding to surface CD9 of breast cancer cells resulted in increased nuclear CD9 fluorescence. CD9 was also found, along with a plasma membrane-associated pool, in the nuclei of all primary ductal breast carcinoma patient specimens analyzed. In all patients, about 40% of the total CD9 cellular fluorescence was nuclear. CD9 colocalized at the nuclear level with CEP97, a protein implicated in centrosome function, and with the IGSF8, an established CD9 partner in the plasma membrane. Co-immunoprecipitation of CEP97 and IGSF8 with CD9 was shown in nuclear extracts from breast cancer cells expressing a CD9-GFP fusion protein. However, by fluorescence resonance energy transfer (FRET) analysis, no direct binding of CD9 with either protein was observed, suggesting that CD9 is part of a larger nuclear protein complex. CD9 depletion or exposure of parental breast cancer cells to anti-CD9 mAb resulted in polynucleation and multipolar mitoses. These data indicate that the nuclear CD9 pool has an important role in the mitotic process. The discovery of a nuclear pool of CD9 has prognostic and/or therapeutic potential for patients with ductal carcinoma of the breast.
Highlights
Morbidity from breast cancer largely arises from the dissemination and growth of tumor cells at metastatic sites
Nuclear localization of CD9 in breast cancer cells (BCC) We investigated by confocal laser microscopy the cellular localization of CD9 in fixed and permeabilized MDA, MA-11, and MCF-7 cells
We have identified by confocal microscopy and immunoblotting a nuclear pool of the tetraspanin CD9 in MDA, MA-11, and MCF-7 cells under na€ve conditions and upon forced expression of a CD9–GFP fusion protein
Summary
Morbidity from breast cancer largely arises from the dissemination and growth of tumor cells at metastatic sites. CD9 belongs to the tetraspanin family, which in humans contains 33 distinct proteins, all characterized by 4 transmembrane domains and conserved motifs, in particular CCG and PXSC in the large extracellular loop [4]. It is expressed in multiple cell types, including hematopoietic, epithelial, and many cancer cells [5]. It interacts with other tetraspanins and with other proteins, including 2 members of the immunoglobulin superfamily, IgSF8 ( called EWI-2) and EWI-F [6,7,8,9], participating to Cancer Research Center, Roseman University of Health Sciences, Las Vegas, Nevada
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