Cell Cycle-Dependent Tumor Engraftment and Migration Are Enabled by Aurora-A.

Cell-cycle progression and the acquisition of a migratory phenotype are hallmarks of human carcinoma cells that are perceived as independent processes but may be interconnected by molecular pathways that control microtubule nucleation at centrosomes. Here, cell-cycle progression dramatically impacts the engraftment kinetics of 4T1-luciferase2 breast cancer cells in immunocompetent BALB/c or immunocompromised NOD-SCID gamma (NSG) mice. Multiparameter imaging of wound closure assays was used to track cell-cycle progression, cell migration, and associated phenotypes in epithelial cells or carcinoma cells expressing a fluorescence ubiquitin cell-cycle indicator. Cell migration occurred with an elevated velocity and directionality during the S-G2-phase of the cell cycle, and cells in this phase possess front-polarized centrosomes with augmented microtubule nucleation capacity. Inhibition of Aurora kinase-A (AURKA/Aurora-A) dampens these phenotypes without altering cell-cycle progression. During G2-phase, the level of phosphorylated Aurora-A at centrosomes is reduced in hyaluronan-mediated motility receptor (HMMR)-silenced cells as is the nuclear transport of TPX2, an Aurora-A-activating protein. TPX2 nuclear transport depends upon HMMR-T703, which releases TPX2 from a complex with importin-α (KPNA2) at the nuclear envelope. Finally, the abundance of phosphorylated HMMR-T703, a substrate for Aurora-A, predicts breast cancer-specific survival and relapse-free survival in patients with estrogen receptor (ER)-negative (n = 941), triple-negative (TNBC) phenotype (n = 538), or basal-like subtype (n = 293) breast cancers, but not in those patients with ER-positive breast cancer (n = 2,218). Together, these data demonstrate an Aurora-A/TPX2/HMMR molecular axis that intersects cell-cycle progression and cell migration.Implications: Tumor cell engraftment, migration, and cell-cycle progression share common regulation of the microtubule cytoskeleton through the Aurora-A/TPX2/HMMR axis, which has the potential to influence the survival of patients with ER-negative breast tumors. Mol Cancer Res; 16(1); 16-31. ©2017 AACR.

Halofuginone Synergistically Enhances Anti-Proliferation of Rapamycin in T Cells and Reduces Cytotoxicity of Cyclosporine in Cultured Renal Tubular Epithelial Cells.

Both rapamycin (RAPA) and cyclosporin A (CsA) are commonly used for immunosuppression, however their adverse side effects limit their application. Thus, it is of interest to develop novel means to enhance or preserve the immunosuppressive activity of RAPA or CsA while reducing their toxicity. Halofuginone (HF) has been recently tested as a potential immunosuppressant. This study investigated the interaction of HF with RAPA or with CsA in cell cultures. Cell proliferation in cultures was determined using methylthiazol tetrazolium assay, and cell apoptosis assessed by flow cytometric analysis and Western blot. The drug-drug interaction was determined according to Loewe's equation or Bliss independence. Here, we showed that addition of HF to anti-CD 3 antibody-stimulated splenocyte cultures induced synergistic suppression of T cell proliferation in the presence of RAPA, indicated by an interaction index (γ) value of < 1.0 between HF and RAPA, but not in those with CsA. The synergistic interaction of RAPA with HF in the suppression of T cell proliferation was also seen in a mixed lymphocyte reaction and Jurkat T cell growth, and was positively correlated with an increase in cell apoptosis, but not with proline depletion. In cultured kidney tubular epithelial cells, HF attenuated the cytotoxicity of CsA. In conclusion, these data indicate that HF synergistically enhances anti-T cell proliferation of RAPA and reduces the nephrotoxicity of CsA in vitro, suggesting the potential use of HF for enhancing anti-T cell proliferation of RAPA and reducing CsA-mediated nephrotoxicity.

Halofuginone suppresses T cell proliferation by blocking proline uptake and inducing cell apoptosis.

Inactivation of T cells is a widely used strategy for immunosuppression. Halofuginone (HF) is an antiprotozoal agent for treating parasites in veterinary medicine, and has been demonstrated to inhibit collagen type 1 synthesis, T helper 17 cell differentiation and cytokine production in activated T cells. The present study was designed to examine the biological effects of HF against T cell receptor and interleukin (IL)-2 stimulated T cell proliferation. T cell proliferation in cultured murine splenocytes was determined by methylthiazol tetrazolium assay. Cell apoptosis was mainly determined by fluorescence-activated cell sorting with Annexin-V and 7-aminoactinomycin D staining. Here, we showed that HF significantly suppressed T cell proliferation in naïve splenocyte cultures in response to alloantigen or anti-CD3 antibody (IC50, 2-2.5 nM; P<0.0001), or in activated T cell cultures in response to IL-2 (IC50, 16 nM; P<0.0001) in a dose-dependent manner. HF did neither attenuate IL-2 production in anti-CD3 antibody activated T cells nor disrupt STAT5 signaling in IL-2-stimulated T cells, but its anti-T cell proliferation was correlated with an increase in cell apoptosis and a decrease in proline uptake in culture medium. Further experiments showed that proline supplement in cell culture medium significantly prevented HF-mediated suppression of T cell proliferation and cell apoptosis. In conclusion, these data suggest that HF interferes with proline incorporation or uptake, resulting in apoptosis via amino acid starvation response in T cells in the response to antigen/mitogen or IL-2 stimulation.