Novel small molecules disrupting Hec1/Nek2 interaction ablate tumor progression by triggering Nek2 degradation through a death-trap mechanism.

Hec1 (highly expressed in cancer 1) or Nek2 (NIMA-related kinase 2) is often overexpressed in cancers with poor prognosis. Both are critical mitotic regulators, and phosphorylation of Hec1 S165 by Nek2 is required for proper chromosome segregation. Therefore, inactivation of Hec1 and Nek2 by targeting their interaction with small molecules represents an ideal strategy for tackling these types of cancers. Here we showed that new derivatives of INH (inhibitor for Nek2 and Hec1 binding) bind to Hec1 at amino acids 394-408 on W395, L399 and K400 residues, effectively blocking Hec1 phosphorylation on S165 by Nek2, and killing cancer cells at the nanomolar range. Mechanistically, the D-box (destruction-box) region of Nek2 specifically binds to Hec1 at amino acids 408-422, immediately adjacent to the INH binding motif. Subsequent binding of Nek2 to INH-bound Hec1 triggered proteasome-mediated Nek2 degradation, whereas the Hec1 binding defective Nek2 mutant, Nek2 R361L, resisted INH-induced Nek2 degradation. This finding unveils a novel drug-action mechanism where the binding of INHs to Hec1 forms a virtual death-trap to trigger Nek2 degradation and eventually cell death. Furthermore, analysis of the gene expression profiles of breast cancer patient samples revealed that co-elevated expressions of Hec1 and Nek2 correlated with the shortest survival. Treatment of mice with this kind of tumor with INHs significantly suppressed tumor growth without obvious toxicity. Taken together, the new INH derivatives are suitable for translation into clinical application.

Isolation and culture of neuroendocrine cells from fetal rabbit lung using immunomagnetic techniques.

We describe a novel method for the isolation and subsequent culture of pulmonary neuroendocrine cells (PNEC) from normal fetal rabbit lung using immunomagnetic techniques with a monoclonal antibody, MOC-1. This surface antigen has originally been identified on small cell carcinoma of the lung. Our immunohistochemical studies have shown that MOC-1 cross-reacts with PNEC of human and rabbit fetal lungs on frozen sections, and in fixed cultures of rabbit fetal lung. Using a combination of mechanical and enzymatic disaggregation, a single-cell suspension of fetal rabbit lung was obtained. These cells were incubated with MOC-1 conjugated to magnetic beads. PNEC were selectively removed from the heterogeneous mixture using a magnet, giving up to 2-fold enrichment compared with our previously reported method. These cells were maintained in culture in a functional state for up to 7 days. The ability to prepare PNEC from rabbit fetal lung offers an opportunity to develop in vitro models to investigate the physiologic and biochemical properties of these cells, and ultimately it may lead to a better understanding of their function in health and disease.