Oncogenes and Tumor Suppressors The Nuclear Pool of Tetraspanin CD9 Contributes to Mitotic Processes in Human Breast Carcinoma

Tetraspanin-29 (CD9) is an integral membrane protein involved in several fundamental cell processes and in cancermetastasis. 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. Implications:The discovery of a nuclear pool of CD9 has prognostic and/or therapeutic potential for patients with ductal carcinoma of the breast. Mol Cancer Res; 12(12); 1840–50. 2014 AACR. Introduction Morbidity from breast cancer largely arises from the dissemination and growth of tumor cells at metastatic sites. Therefore, metastatic spread is the biggest concern in the treatment of human breast cancer, as it is for most cancers. The pleiotropic molecule CD9, reportedly involved in cell fusion, adhesion, motility, proliferation, and signaling (1), has been implicated in the metastatic process, as inhibitor of cell invasion and metastasis (2), or as prometastatic, depending on the context (3). 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 (also called EWI-2) and EWI-F (6–9), participating to the formation of structural and functional units called tetraspanin-enriched microdomains (TEM) or, more commonly, tetraspanin web (9). Within TEMs, CD9 can regulate many physiologic and pathologic processes, such as fertilization and metastasis (3, 10, 11). Here, we report for the first time that breast cancer cells (BCC) contain a nuclear CD9 pool and that the abrogation of CD9 expression results in multipolar mitoses and polynucleation. The presence of nuclear CD9 in patient-derived breast cancer material warrants future studies to assess the prognostic and therapeutic relevance of nuclear CD9. Materials and Methods Cell lines Human MDA-MB-231 (MDA) and MCF-7 BCC were obtained from the ATCC. The human MA-11 BCC line, established from bone marrow micrometastases of a patient with breast cancer (12, 13), was obtained by Dr. Fodstad, Norwegian Radium Hospital (Oslo, Norway). All cell lines were stored in aliquots in liquid nitrogen and kept in culture for less than 3months after resuscitation. All cells were tested for mycoplasma contamination every 6 months by PCR and DAPI staining and authenticated by morphology check every 2 weeks. Complete culture medium consisted of RPMI-1640 (Gibco), 10% FBS (Atlanta Biologicals, Inc.), 100 units/mL penicillin, 100 mg/mL streptomycin, and 2 mmol/L L-glutamine. Cancer Research Center, Roseman University of Health Sciences, Las Vegas, Nevada. Note: Supplementary data for this article are available at Molecular Cancer Research Online (http://mcr.aacrjournals.org/). Corresponding Author: Aurelio Lorico, Cancer Research Center, Roseman University of Health Sciences, Las Vegas, NV 89135. Phone: 7028225395; Fax: 702-9685949; E-mail [email protected] doi: 10.1158/1541-7786.MCR-14-0242 2014 American Association for Cancer Research. Molecular Cancer Research Mol Cancer Res; 12(12) December 2014 1840 on July 11, 2017. © 2014 American Association for Cancer Research. mcr.aacrjournals.org Downloaded from Published OnlineFirst August 7, 2014; DOI: 10.1158/1541-7786.MCR-14-0242