ABSTRACT
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.
Subject(s)
Neoplasms/metabolism , Neoplasms/pathology , Nuclear Proteins/metabolism , Protein Serine-Threonine Kinases/metabolism , Animals , Benzamides/chemistry , Benzamides/pharmacology , Binding Sites , Cell Line, Tumor , Cytoskeletal Proteins , Disease Models, Animal , Disease Progression , Gene Expression , Heterografts , Humans , Indoles , Inhibitory Concentration 50 , Mitosis/drug effects , Models, Molecular , Molecular Conformation , NIMA-Related Kinases , Neoplasms/genetics , Neoplasms/mortality , Nuclear Proteins/antagonists & inhibitors , Nuclear Proteins/chemistry , Nuclear Proteins/genetics , Phosphorylation/drug effects , Prognosis , Protein Binding/drug effects , Protein Serine-Threonine Kinases/chemistry , Protein Serine-Threonine Kinases/genetics , Proteolysis/drug effects , Thiazoles/chemistry , Thiazoles/pharmacology , ValeratesABSTRACT
MicroRNAs (miRNAs) are involved in tumorigenecity by regulating specific oncogenes and tumor suppressor genes, and their roles in breast cancer stem cells (BCSCs) are becoming apparent. Distinct from the CD44(+)/CD24(-/low) sub-population, we have isolated a novel PROCR(+)/ESA(+) BCSC sub-population. To explore miRNA-regulatory mechanisms in this sub-population, we performed miRNA expression profiling and found miR-495 as the most highly upegulated miRNA in PROCR(+)/ESA(+) cells. Coincidently, high upregulation of miR-495 was also found in CD44(+)/CD24(-/low) BCSCs, reflecting its potential importance in maintaining common BCSC properties. Ectopic expression of miR-495 in breast cancer cells promoted their colony formation in vitro and tumorigenesis in mice. miR-495 directly suppressed E-cadherin expression to promote cell invasion and inhibited REDD1 expression to enhance cell proliferation in hypoxia through post-transcriptional mechanism. miR-495 expression was directly modulated by transcription factor E12/E47, which itself is highly expressed in BCSCs. These findings reveal a novel regulatory pathway centered on miR-495 that contributes to BCSC properties and hypoxia resistance.
Subject(s)
Cadherins/genetics , MicroRNAs/genetics , Neoplastic Stem Cells/metabolism , Transcription Factor 3/genetics , Transcription Factors/genetics , Animals , Base Sequence , Breast Neoplasms/genetics , Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Cadherins/metabolism , Cell Hypoxia , Cell Line , Cell Line, Tumor , Down-Regulation , Female , Gene Expression Profiling , HEK293 Cells , Humans , Immunoblotting , Mammary Neoplasms, Experimental/genetics , Mammary Neoplasms, Experimental/metabolism , Mammary Neoplasms, Experimental/pathology , Mice , Mice, Inbred NOD , Mice, SCID , Molecular Sequence Data , Promoter Regions, Genetic/genetics , Protein Binding , Reverse Transcriptase Polymerase Chain Reaction , Transcription Factor 3/metabolism , Transcription Factors/metabolism , Transplantation, HeterologousABSTRACT
Early studies of the retinoblastoma gene (RB) have uncovered its critical role as a regulator of the G(1)/S cell cycle phase progression. Surprisingly, genetic approaches in mammals and nematodes have also shown RB controls cell lineage specification and aspects of differentiation. The RB gene product accomplishes this by diverse mechanisms such as by interacting with tissue-specific transcription factors, enhancing RNA interference, and modifying chromatin structure. We review recent studies uncovering novel mechanisms by which RB works in several cell lineages and we provide perspectives on how these new findings might relate to RB tumor suppression.
