No abstract

CD44 adhesion molecules are expressed in many breast cancer cells and have been demonstrated to play a key role in regulating malignant phenotypes such as growth, migration, and invasion. CD44 is an integral transmembrane protein encoded by a single 20-exon gene. The diversity of the biological functions of CD44 is the result of the various splicing variants of these exons. Previous studies suggest that exon v10 of CD44 plays a key role in promoting cancer invasion and metastasis, however, the molecular mechanisms are not clear. Given the fact that exon v10 is in the ectodomain of CD44, we hypothesized that CD44 forms a molecular complex with other cell surface molecules through exon v10 in order to promote migration of breast cancer cells. In order to test this hypothesis, we selected DNA aptamers that specifically bound to CD44 exon v10 using Systematic Evolution of Ligands by Exponential Enrichment (SELEX). We selected aptamers that inhibited migration of breast cancer cells. Co-immunoprecipitation studies demonstrated that EphA2 was co-precipitated with CD44. Pull-down studies demonstrated that recombinant CD44 exon v10 bound to EphA2 and more importantly aptamers that inhibited migration also prevented the binding of EphA2 to exon v10. These results suggest that CD44 forms a molecular complex with EphA2 on the breast cancer cell surface and this complex plays a key role in enhancing breast cancer migration. These results provide insight not only for characterizing mechanisms of breast cancer migration but also for developing target-specific therapy for breast cancers and possibly other cancer types expressing CD44 exon v10.

There is substantial evidence indicating that the WNT signaling pathway is activated in various cancer cell types including breast cancer. Previous studies reported that FH535, a small molecule inhibitor of the WNT signaling pathway, decreased growth of cancer cells but not normal fibroblasts, suggesting this pathway plays a role in tumor progression and metastasis. In this study, we tested FH535 as a potential inhibitor for malignant phenotypes of breast cancer cells including migration, invasion, and growth. FH535 significantly inhibited growth, migration, and invasion of triple negative (TN) breast cancer cell lines (MDA-MB231 and HCC38) in vitro. We demonstrate that FH535 was a potent growth inhibitor for TN breast cancer cell lines (HCC38 and MDA-MB-231) but not for other, non-TN breast cancer cell lines (MCF-7, T47D or SK-Br3) when cultured in three dimensional (3D) type I collagen gels. Western blotting analyses suggest that treatment of MDA-MB-231 cells with FH535 markedly inhibited the expression of NEDD9 but not activations of FAK, Src, or downstream targets such as p38 and Erk1/2. We demonstrated that NEDD9 was specifically associated with CSPG4 but not with β1 integrin or CD44 in MDA-MB-231 cells. Analyses of gene expression profiles in breast cancer tissues suggest that CSPG4 expression is higher in Basal-type breast cancers, many of which are TN, than any other subtypes. These results suggest not only a mechanism for migration and invasion involving the canonical WNT-signaling pathways but also novel strategies for treating patients who develop TN breast cancer.

BackgroundPrevious studies suggest that certain transition metal complexes, such as cisplatin, are efficacious for treating various cancer types, including ovarian, lung, and breast.MethodsIn order to further evaluate ruthenium (Ru) complexes as potential anti-cancer agents, we synthesized and evaluated Ru-arene complexes. Two complexes with the general formula [Ru (η6-p-cym) (N–N) Cl]+ were tested for their abilities to inhibit cancer cells.ResultsThe complex with o-phenylenediamine as the N–N ligand (o-PDA) significantly inhibited growth of breast (MDA-MB-231, MCF-7, SKBR-3, and SUM149), lymphoma (Raji), melanoma (Bowes), and osteosarcoma (HT1080); however, the complex with o-benzoquinonediimine (o-BQDI) was ineffective except for SUM149. In contrast, o-PDA failed to inhibit growth of human breast epithelial cells, MCF-10A. Treatment of MDA-MBA-231 cells with o-PDA resulted in a significant reduction of productions of PDGF-AA, GM-CSF, and VEGF-A proteins at the transcriptional levels. Finally, we demonstrated that o-PDA synergistically inhibited MDA-MB-231 cell growth with cyclophosphamide but not doxorubicin or paclitaxel.ConclusionThese results suggest that Ru-arene complexes are promising anti-cancer drugs that inhibit progression and metastasis by blocking multiple processes for breast and other types of cancer.Electronic supplementary materialThe online version of this article (doi:10.1186/s12967-016-0797-9) contains supplementary material, which is available to authorized users.

Enhanced invasion and migration into the surrounding tissues are hallmarks of the malignancy of tumor cells. To successfully metastasize, a cancer cell has to detach from the primary tumor, invade into surrounding tissues, and intravasate into blood or lymphatic vessels. These processes are composed of complex mechanisms involving tumor recognition, degradation of extracellular matrix (ECM) proteins and migration into tissue. Triple negative (TN) breast cancers are defined by a lack of expression of estrogen, progesterone, and HER2 receptors. It is widely recognized that TN breast cancers have a poorer prognosis than any other subtype of breast cancer. Given the lack of effective targeted therapies for TN breast cancer patients, understanding of the mechanisms of migration and invasion of these tumors will provide insight into developing novel approaches to lower the mortality from TN breast cancer. Previous studies demonstrated that NEDD9 plays a key role facilitating progression and metastasis of various tumor cells including breast. We previously demonstrated that NEDD9 plays a critical role in promoting migration and growth of MDA-MB-231. In order to further characterize the mechanisms of NEDD9-mediated cancer migration and growth, we established stable cell lines expressing NEDD9 using HCC38 as a parental cell line which expresses low level of endogenous NEDD9. Microarray studies demonstrated that enzymes (CHST11, CHST15, and CSGALNACT1) involved in biosynthesis of chondroitin sulfate (CS) but not heparan sulfate (HS) were markedly upregulated in HCC38(NEDD9) compared to control HCC38(Vector) cells. These results suggest that NEDD9 regulates specific structures of tumor-associated glycans such as chondroitin sulfate. Core proteins of CD44 and Serglycin were markedly upregulated in HCC38(NEDD9) cells compared to HCC38(Vector) cells, while those of Syndecan-1, Syndecan-2, and Versican were downregulated in HCC38(NEDD9). Immunofluorescence studies using specific antibody, GD3G7, confirmed the enhanced expression of CS-E subunit in HCC38(NEDD9). Immunoprecipitation and western blotting analysis demonstrated that CS-E was attached to Serglycin and CD44 core proteins. We demonstrated that removing CS by chondroitinase ABC significantly inhibited anchorage-independent growth of HCC38(NEDD9) in methylcellulose. Importantly, the fact that GD3G7 significantly inhibited colony formation of HCC38(NEDD9) cells suggest that CS-E subunit plays a key role in this process. Furthermore, treatment of HCC38(NEDD9) cells with chondroitinase ABC or GD3G7 significantly inhibited mammosphere formation. Exogenous addition of CS-E enhanced colony formation and mammosphere formation of HCC38 parental and HCC38(Vector) cells. These results suggest that NEDD9 regulates the synthesis and expression of tumor associated glycocalyx structures including CS-E, which plays a key role in promoting and regulating breast cancer progression metastasis and possibly stem cell phenotypes. The opinion and assertions contained herein are the private views of the authors and are not to be construed as official or as representing the views of the Department of the Army or the Department of Defense. Citation Format: Iida J, Dorchak J, Clancy R, Slavik J, Cutler ML, Shriver CD. Tumor-associated glycans as key molecules to promote growth of triple-negative breast cancer cells. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P2-05-16.