Dual targeting of MDM2 with a novel small-molecule inhibitor overcomes TRAIL resistance in cancer
Carcinogenesis
Dual targeting of MDM2 with a novel small-molecule inhibitor overcomes TRAIL resistance in cancer
Anup Kumar Singh 2
Shikha S.Chauhan 1 3
Sudhir Kumar Singh 0
Ved Vrat Verma 6
Akhilesh Singh 2
Rakesh Kumar Arya 2
Shrankhla Maheshwar 2
Md. Sohail Akhta 0
Jayanta Sarka 2
Vivek M.Rangnekar 4
Prem M.S.Chauhan 1
Dipak Datta 2 5
0 Molecular and Structural Biology Division, CSIR-Central Drug Research Institute , Lucknow, Uttar Pradesh 226031, In
1 Medicinal and Process Chemistry Division
2 Biochemistry Division
3 Present address: Pennsylvania State University , University Park, PA 16801 , USA
4 Department of Radiation Medicine and Markey Cancer Center, University of Kentucky , Lexington, KY 40536 , USA
5 Academy of Scientific and Innovative Research , New Delhi 110025 , India
6 dDiae,partment of Biophysics, Delhi University , South Campus, New Delhi 110021 , India
Mouse double minute 2 (MDM2) protein functionally inactivates the tumor suppressor p53 in human cancer. Conventional MDM2 inhibitors provide limited clinical application as they interfere only with the MDM2-p53 interaction to release p53 from MDM2 sequestration but do not prevent activated p53 from transcriptionally inducing MDM2 expression. Here, we report a rationally synthesized chalcone-based pyridb]oin[dole, CPI-7c, as a unique small-molecule inhibitor of MDM2, which not only inhibited MDM2-p53 interaction but also promoted MDM2 degradation. CPI-7c bound to both RING and N-terminal domains of MDM2 to promote its ubiquitin-mediated degradation and p53 stabilization. CPI-7c-induced p53 directly recruited to the promoters DofR4 and DR5 genes and enhanced their expression, resulting in sensitization of TNFrelated apoptosis-inducing ligand (TRAIL)-resistant cancer cells toward TRAIL-induced apoptosis. Collectively, we identified CPI-7c as a novel small-molecule inhibitor of MDM2 with a unique two-prong mechanism of action that sensitized TRAILresistant cancer cells to apoptosis by modulating the MDM2-p53-DR4/DR5 pathway.
Introduction
Mouse double minute 2 (MDM2, also termed HDM2 in humans) as Nutlin-3, is that they interact only with its N-terminal and
oncogene is amplified and overexpressed in a number of human interfere with MDM2–p53 interaction but do not exert any effect
malignancies and its expression often correlates with poor on its catalytic RING domain leading to accumulation of active
survival of cancer patients1(–3). The tumor suppressor p53 is MDM2 (
9–11
). Moreover, MDM2 and p53 regulate each other
known to be the primary target of MDM2-mediated proteasomal mutually through the autoregulatory feedback loop where-sta
degradation 4(,5). Structurally, MDM2 protein has two distinct bilization and activation of p53 results in MDM2 transactivation
domains with different functional roles. N-terminal domain of and overexpression. On the other hand, MDM2 directly interacts
MDM2 primarily interacts with p536(), and its C-terminal RING with p53 to inhibit its function4(
,5,12
). Furthermore, MDM2 is
domain has E3 ubiquitin ligase activity that promotes p53 ub-iq stabilized by a closely related protein MDMX (also called MDM4)
uitination followed by proteasomal degradatio7n,8(). The major owing to dimerization mediated by the conserved C-terminal
limitation associated with conventional MDM2 inhibitors, such RING domains of both MDM2 and MDMX and this prevents
Abbreviations previously (37). Reagents were purchased from following suppliers: Pierce
Magnetic ChIP kit, antibodies for GAPDH and verso one-step reverse tr-an
ChIP chromatin immunoprecipitation scription–PCR (RT–PCR) kit from Thermo Scientific; human apoptosis
HRP horseradish peroxidase proteome profiler kit and neutralizing antibodies for TRAIL-R1 (DR4) and
MDM2 mouse double minute 2 TRAIL-R2 (DR5) from R&D systems; sulforhodamine-B (SRB), TRAIL,
crysSRB sulforhodamine-B tal violet dye and dimethyl sulfoxide were procured from Sigma–Aldrich;
TRAIL TNF-related apoptosis-inducing ligand antibodies for p53 (DO1), MDM2, horseradish peroxidase (HRP)-conjugated
WT wild type secondary antibodies from Santacruz; Agarose A/G beads and antibody for
DR4 from Millipore; antibodies for DR5, ubiquitin and chromatin immun-o
MDM2 autoubiquitination 1(
3–15
). These observations suggest pcroeecriyptithartinio-nco(nCjhuIgPa)tgerdadaentpi5b3o(7dFy5f)ofrrDomR4Caenldl DSiRg5nuasleindginTetchhenfololwogciyetso;mph-eytry
that stabilization of p53 requires a strategy that involves notand platinum human TRAIL ELISA kit from e-Biociences; DharmaFECT
only interference with the N-terminal domain of MDM2 but also transfection reagent, control and p53-specific siRNA and MGC Human
impairment of the MDMX interacting catalytic RING domain XIAP Sequence Verified cDNA from Dharmacon; and primers for DR4, DR5,
so that it can undergo autodegradation. Therefore, ideally totalGAPDH used for semiquantitative PCR and that of p53 binding sites iDnR4
stabilization of p53 should be accomplished by blocking both an (...truncated)