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Ancient drug curcumin impedes 26S proteasome activity by direct inhibition of dual-specificity tyrosine-regulated kinase 2

Significance Curcumin is an ancient drug derived from turmeric and has been found to exhibit potent anticancer properties albeit through controversial mechanisms of action. Using a biochemical model, mouse cancer model, and cellular models, we show that curcumin is a highly potent and selective inhibitor of dual-specificity tyrosine-regulated kinase 2 (DYRK2), a positive regulator of the 26S proteasome. Curcumin perturbs 26S proteasome activity via DYRK2 inhibition in various cancer cells and in the mouse cancer model leading to impairment of cell proliferation and reduction of cancer burden in mice. This novel mechanism of action of curcumin opens up new avenues for potential preventative or therapeutic strategies in proteasome-addicted cancers like triple-negative breast cancer and multiple myeloma.


Curcumin preparation and treatment
For all cell based and in vitro assays, curcumin was diluted in DMSO at a stock concentration of 10 mM in the dark and prepared fresh prior to each experiment and the excess solution was never stored. Thiol scavengers like DTT or β-mercaptoethanol was added to all reaction/lysis buffers. Curcumin treatment was always carried out in media containing 10% FBS unless otherwise mentioned to maintain maximum stability of curcumin (41) and final concentration was maintained between 0.1-10 µM to avoid aggregation. Treatment time varied from 0.5-24 hr in the dark depending on experiment. For xenograft mouse experiments, curcumin was mixed with 1% carboxymethycellulose (Millipore) in 0.1 M citrate buffer pH 6 and 300 mg/Kg body weight was administered via intraperitoneal injection every alternate day. Curcumin solution was prepared fresh every time in the dark. IC 50 determination IC 50 determination was carried out at The International Centre for Protein Kinase Profiling (http://www.kinase-screen.mrc.ac.uk/). Active DYRK1A, DYRK2, DYRK3, IKKβ and GSK3β were purified as reported previously (42). Curcumin IC 50 measurements were carried out against the kinases with final concentrations between 0.1 nM to 10 µM in vitro (curcumin was added to the kinase reaction prior to ATP master mix). The values were expressed as a percentage of the DMSO control. DYRK isoforms (5-20 mU diluted in 50 mM Tris pH7.5, 0.1 mM EGTA, 0.1% β-mercaptoethanol) are assayed against Woodtide (KKISGRLSPIMTEQ) in a final volume of 25.5µl containing 50 mM Tris pH 7.5, 0.1 mM EGTA, 300 µM substrate peptide, 10 mM Magnesium acetate and 0.025-2 mM [ 33 P-γATP] (0.05mM for DYRK1A and 0.005mM for DYRK3) (50-1000 cpm/pmole) and incubated for 30 min at room temperature. GSK3β (5-20 mU diluted in 20 mM MOPS pH 7.5, 1 mM EDTA, 0.01% Brij35, 5% glycerol, 0.1% βmercaptoethanol, 1 mg/ml BSA) is assayed against Phospho-GS2 peptide (YRRAAVPPSPSLSRHSSPHQS(PO4)EDEEE) in a final volume of 25.5 µl containing 8 mM MOPS pH 7.0, 0.2 mM EDTA, 20 µM Phospho GS2 peptide, 10 mM magnesium acetate and 0.005 mM [ 33 P-γATP] (50-1000 cpm/pmole) and incubated for 30 min at room temperature. IKKβ (5-20 mU diluted in 50mM Tris (pH 7.5), 0.1mM EGTA, 1mg/ml BSA, 0.1%, β-Mercaptoethanol) is assayed against substrate peptide (LDDRHDSGLDSMKDEEY) in a final volume of 25.5µl containing 50mM Tris (pH 7.5), 0.1mM EGTA, 0.1%, β-Mercaptoethanol, 300µM substrate peptide, 10 mM magnesium acetate and 0.005 mM [ 33 P-γATP] ( 500-1000 cpm/pmole) and incubated for 30 mins at room temperature. Assays are stopped by addition of 5 µl of 0.5 M (3%) orthophosphoric acid and then harvested onto P81 Unifilter plates with a wash buffer of 50 mM orthophosphoric acid. IC 50 curves were developed and IC 50 values were calculated using GraphPad Prism software.

Protein kinase profiling
Kinase inhibitor specificity profiling assays were carried out at The International Centre for Protein Kinase Profiling (http://www.kinase-screen.mrc.ac.uk/). Curcumin kinase specificity was determined against a panel of 140 protein kinases as described previously (42,43). Results are presented as a percentage of kinase activity in DMSO control reactions. Protein kinases were assayed in vitro with 1 µM final concentration of curcumin and the results are presented as an average of triplicate reactions ± SD or in the form of comparative histograms. were maintained in RPMI-1640 with 10% fetal bovine serum (FBS) and Penicillin/Streptomycin. MCF10A cells were cultured in DMEM/F-12 medium supplemented with 5% horse serum, 20 ng/ml EGF, 0.5 µg/ml hydrocortisone, 100 ng/ml cholera toxin, 10 µg/ml insulin and Penicillin/Streptomycin (Life Technologies). AHH1 cells were cultured in RPMI-1640 with 10% horse serum, 10 mM HEPES, and 1.0 mM sodium pyruvate (Life Technologies).

Cell transfection
Transient transfection of HEK293T and MDA-MB-231 cells was carried out using Lipofectamine 2000 (Life Technologies) as recommended by the manufacturer. Stable overexpression of DYRK2 and Rpn11-TBHA were carried out by Retroviral transduction followed by puromycin selection as reported previously (37).

Genome editing
Crispr/Cas9 mediated genome editing of DYRK2 loci in MDA-MB-231 cells was carried out using guide RNA: CCTGGATCTGTCCGTGAGCG as described previously (37). DYRK2 KO MDA-MB-231 clones were ascertained by DNA sequencing. HaCaT cells with DYRK2 KO were generated as reported previously (37).

Protein expression and purification
DYRK2 74-479 with an N-terminal 6xHis tag was expressed in E. coli Rosetta (DE3). For protein expression, cultures were grown at 37 °C in LB medium to an OD 600 of 0.6-0.8 before induced with 0.5 mM IPTG overnight at 18 °C. Cells were collected by centrifugation and frozen at −80 °C.
Crystallization, structure determination and alignment studies DYRK2 74-479 was incubated with 100 µM curcumin on ice before crystallization. The DYRK2-curcumin crystals were grown at 18 °C by the sitting-drop vapor diffusion method, using a reservoir solution containing 12%-20% PEG 3350 and 0.2 M ammonium citrate tribasic, pH 7.0. Crystals grew to full size in several days and were transferred to 20% PEG 3350, 0.2 M ammonium citrate tribasic, pH 7.0, and 15% ethylene glycol before frozen in liquid nitrogen.
The diffraction data were collected at Shanghai Synchrotron Radiation Facility (SSRF) beamline BL17U. The diffraction data were indexed, integrated, and scaled using HKL2000 (HKL Research). The structure was determined by molecular replacement using the published DYRK2 structure (PDB ID: 3K2L) (44) as the search model using the Phaser program (45). Curcumin was placed using LigandFit program in Phenix (46). The structural model was then adjusted in Coot (47) and refined using Phenix. The quality of the structural model was checked using the MolProbity program in Phenix.
Structural alignments were performed in PyMOL (Schrödinger) using the align command. Sequence alignments were performed in Clustal Omega (48) and manually adjusted for clarity of interpretation in the final figure.

Proteasome activity assays
Proteasome activity assays were analysed either directly on cell lysates or on human 26S proteasome affinity-purified from MDA-MB-231 cells stably expressing Rpn11-TBHA as described previously (37). Cells were treated with or without indicated concentration of curcumin and/or carfilzomib prior to lysis. Peptidase activities of purified proteasome or proteasomes in whole cell or tumor lysates were assayed using fluorogenic peptide substrates (Enzo Life Sciences) (49) with phosphatase inhibitors present in the lysis and assay buffers. The measured activity was normalized either against total protein concentration of cell lysates or against western blot of proteasome subunits. Fluorescence signal in whole cells or cell lysates were quantified using a Tecan Infinite® M200 Pro multi-well plate reader. For casein degradation, 1 µg of self-quenching bodipy-casein (EnzChek® Protease Assay Kit, green fluorescence, Life Technology) and 1 µg of purified, 26S proteasome were mixed in the 26S assay buffer with 5 mM ATP. Reaction was carried out for 40 mins at 37°C. Fluorescence was measured in a Tecan Infinite® M200 Pro multi-well plate reader.

Cell proliferation, viability and invasion assays
Cell proliferation was carried out by cell counting as reported previously (37) in the presence or absence of 1 (for 5TGM1-GFP cells) or 3 µM (for MDA-MB-231 cells) curcumin. Media containing curcumin or DMSO control was replaced every day for 5 days to avoid excess degradation of curcumin in cell culture media. Cell viability assays were carried out with or without 24 hr treatment of carfilzomib or curcumin or both using the CellTiter 96® AQueous Non-Radioactive Cell Proliferation Assay kit. Synergistic effects of drugs on cytotoxicity were analysed using combination index (CI) by CompuSyn software (ComboSyn, Inc., Paramus, NJ, USA). CI<1 is considered synergism; CI=1 is considered additive while CI>1 is accepted to be antagonism. To analyse whether cytotoxicity induced by curcumin was apoptotic, we analysed Caspase3/7 activity in cell lysate using the CellEvent™ Caspase-3/7 Green Detection Reagent (Life Technologies) using manufacturer's instructions. The Matrigel invasion assay was performed using 8 µm pore size transwells coated with Matrigel (BD Biosciences) as described previously (43). The bottom chamber contained normal growth media (DMEM with 10% FBS with or without 5 µM curcumin) as a chemoattractant. MDA-MB-231 or Hs578T cells were seeded into the upper chamber (20,000 cells/insert) in DMEM with 1% BSA with or without 5 µM curcumin. After 24 hr of culture, cells that migrated through the matrix were quantified using Cyquant following manufacturer's instructions (Life Technologies).

Tumor study
Female NSG TM (Jackson Labs) mice were housed and maintained at the University of California-San Diego (UCSD) in full compliance with policies of the Institutional Animal Core and Use Committee (IACUC). MDA-MB-231 (parental or DYRK2 KO) cells were counted and suspended in PBS containing 50% Matrigel (BD Bioscience). 500,000 cells (100 µl) were injected subcutaneously into the neck of each mouse, 10 mice for parental and 5 mice for DYRK2 KO cells. When tumor became palpable, mice bearing parental MDA-MB-231 cells were randomized into 2 groups with 5 mice per group. Curcumin 300 mg/Kg body weight was administered to one group of n=5 mice bearing parental 231 cells. Vehicle 1% carboxymethylcellulose in 0.1 M citrate buffer pH 6 was injected into the control group of n=5 mice bearing parental 231 cells. Tumor dimensions were measured twice per week using a digital caliper and tumor volume was calculated as (length × width 2 )/2. Mice were euthanized 42 days after injection and tumors were excised and weighed. For immunohistochemistry, tumor tissue was fixed in 10% neutral buffered formalin over night at room temperature, rinsed in 70% ethanol and placed in histology cassettes for paraffin embedment. Hematoxylin and eosin (H&E) staining and anti-Ki-67 staining were performed by the UCSD Tissue Technology core. For immunoblotting and proteasome assays, tumors were lysed in proteasome assay buffer using Bullet Blender Navy Bead Lysis Kit (Next Advance Inc., NY, USA) using manufacturer's instructions.

Statistics and data presentation
Most experiments were repeated 3 times with multiple technical replicates to be eligible for the indicated statistical analyses, and representative image has been shown. All results are presented as mean ± SD unless otherwise mentioned. For animal studies, five mice per group is the standard sample size for tumor xenograft experiments, and no statistical power analysis was used to predetermine sample size. Mice bearing parental MDA-MB-231 derived tumors were randomized into two groups of n=5 prior to vehicle or drug treatment. The investigators were not blinded to allocation during experiments and outcome assessment. Data were analysed using Graphpad Prism statistical package.     The dataset was collected from a single crystal. Values in parentheses are for highestresolution shell.