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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.

To study the signaling mechanisms which mediate ventricular hypertrophy, we utilized the induction of the ANF gene as a marker of the hypertrophic response. The induction of the atrial natriuretic factor gene (ANF) is one of the most conserved features of ventricular hypertrophy, occurring in multiple species (mouse, rat, hamster, canine, and human) in response to diverse stimuli (hormonal, mechanical, pressure/volume overload, genetic, IHSS, hypertension, etc.). The ANF gene is expressed in both the atrial and ventricular compartments during embryonic development, but shortly after birth ANF expression is down-regulated to negligible levels in the adult myocardium. Since the reactivation of ANF gene expression in the hypertrophied ventricle is a hallmark of the activation of an embryonic gene program, it has also become of interest to determine if similar mechanisms activate ANF expression during hypertrophy and the initial stages of cardiogenesis. A combination of co-transfection, microinjection, and transgenic approaches has been coupled to well characterized cultured cell systems and in vivo murine models employing normal and transgenic mice. The microinjection of oncogenic RAS proteins into living myocardial cells does not lead to the activation of cell proliferation, but activates ANF gene expression, as assessed by immunofluorescence. Co-transfection of mutant and wild-type RAS expression vectors with a ANF-luciferase fusion gene supports a direct effect of activated RAS on ANF gene transcription. Co-transfection of a dominant negative RAS vector effectively inhibits the induction of the ANF gene during α adrenergic mediated hypertrophy of ventricular muscle cells, thereby establishing that a RAS-mediated pathway is required for ANF induction. Studies in transgenic mice have suggested that the cis sequences within the ANF gene which control atrial specificity and inducibility during in vivo hypertrophy can be segregated. Transient assays in cultured ventricular cells transfected with a series of ANF-luciferase fusion genes have identified a 638 bp fragment of the ANF promoter which can confer inducibility during α adrenergic agonist and endothelin-1-mediated hypertrophy of cultured neonatal rat ventricular cells. Recent studies have systematically mutated the Egr-1; CRE; AP-1; AP-2; and A-T-rich sites within a 323 bp ANF gene fragment that mediates inducibility, to identify the cis regulatory elements and their correspondng factors that control ANF expression during myocardial cell hypertrophy. These and related studies will be discussed in the context of elucidating the molecular signals which control ANF gene expression during this important adaptive response of the myocardium.