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Small Molecule Therapeutics

Discovery of a Novel Small-Molecule Inhibitor thatTargets PP2A–b-Catenin Signaling and RestrictsTumor Growth and MetastasisShrankhla Maheshwari1,2, Srinivasa R. Avula3, Akhilesh Singh1, L. Ravithej Singh3,Gopala R. Palnati3, Rakesh K. Arya1, Srikanth H. Cheruvu4, Sudhir Shahi4, Tanuj Sharma5,Sanjeev Meena1, Anup K. Singh1, Ruchir Kant5, Mohammed Riyazuddin4,HimangsuK. Bora6, Mohammad I. Siddiqi2,5, Jiaur R.Gayen2,4, Koneni V. Sashidhara2,3, andDipak Datta1,2

Abstract

Molecular hybridization of different pharmacophores to tackleboth tumor growth andmetastasis by a singlemolecular entity canbe very effective and unique if the hybrid product shows drug-likeproperties. Here, we report synthesis and discovery of a novelsmall-molecule inhibitor of PP2A–b-catenin signaling that limitsboth in vivo tumor growth and metastasis. Our molecular hybrid-ization approach resulted in cancer cell selectivity and improveddrug-like properties of the molecule. Inhibiting PP2A and b-cate-nin interaction by selectively engaging PR55a-binding site, our

most potent small-molecule inhibitor diminished the expressionof active b-catenin and its target proteins c-Myc and Cyclin D1.Furthermore, it promotes robust E-cadherin upregulation on thecell surface and increases b-catenin–E-Cadherin association,which may prevent dissemination of metastatic cells. Altogether,we report synthesis and mechanistic insight of a novel drug-likemolecule to differentially target b-catenin functionality viainteracting with a particular subunit of PP2A. Mol Cancer Ther;16(9); 1791–805. �2017 AACR.

IntroductionCancer is now the second major killer of human population as

per WHO estimates for 2012, and by 2030, the global cancerburden is expected to rise to 21.7 million new cancer cases and13 million cancer-related deaths (1). Cancer metastasis is themajor reason of cancer-related morbidity and mortality. Lossof epithelial properties as well as acquisition of mesenchymalcharacteristics is one of the key features of earlymetastatic process(2). E-Cadherin and b-catenin together play a crucial role in initialdissemination of metastatic cells. Nuclear displacement of activeb-catenin and its recruitment to the cell surface along with E-cadherin firms cell-to-cell adhesion and prevents an early releaseof prometastatic cells (3, 4). In the absence of E-cadherin, b-cate-nin is phosphorylated in the cytoplasm in amultiprotein destruc-

tion complex consisting of adenomatous polyposis coli (APC),Axin, glycogen synthase kinase 3b (GSK3b), casein kinase 1a(CK1a), and protein phosphatase 2A (PP2A), which ultimatelyleads to proteasomal degradation via E3 ubiquitin ligase SCFb-TRCP (5–7). Therefore, nonphosphorylated form at Ser33/37/Thr41

is an active form of b-catenin, and it regulates transcriptionalactivation ofmultiple protumorigenic target genes like c-MYC andCCND1 (8, 9) as well as forms adherence junctions with E-cadherin (10, 11). Kinases like CK1a and GSK3b are well knownto control b-catenin phosphorylation, but a recent study by Zhangand colleagues clearly indicated that PR55a, a regulatory subunitof PP2A, specifically modulates PP2A-mediated b-catenindephosphorylation and thus regulates its oncogenic function(12). So, diminution of active b-catenin (non-phospho form)from nucleus and cytoplasm but its recruitment to the cell surfacecould be a wonderful therapeutic window to harness its full-blown effect in regulating tumor growth and metastasis.

Novel drugs for controlling cancer metastasis are of greatimportance in current cancer therapeutics. Molecular hybridiza-tion is one of the emerging strategies for rational drug designing,where multiple targets can be engaged with a combination of twoor more active groups to tackle both the tumor growth andmetastasis (13). In addition, hybridization not only alwaysincreases the efficacy of the molecule but also plays a major rolein controlling solubility and other drug-like properties of themolecule. In the continuation of our relentless efforts toward theidentification of small molecules as promising anticancer thera-peutics, previously we described a series of coumarin–monastrol,coumarin–chalcone and carboline–chalcone hybrids as potentialantitumor agents (14–16). Recently, by applying the molecularhybridization approach, we discovered a novel dual-targetingMDM2 small-molecule inhibitor, which not only inhibited

1Biochemistry Division, Council of Scientific & Industrial Research (CSIR), CentralDrug Research Institute (CDRI), Lucknow, India. 2Academy of Scientific andInnovative Research, New Delhi, India. 3Medicinal and Process Chemistry Divi-sion, CSIR-CDRI, Lucknow, India. 4Pharmacokinetics and Metabolism Division,CSIR-CDRI, Lucknow, India. 5Molecular and Structural Biology Division, CSIR-CDRI, Lucknow, India. 6Laboratory Animal Division, CSIR-CDRI, Lucknow, India.

Note: Supplementary data for this article are available at Molecular CancerTherapeutics Online (http://mct.aacrjournals.org/).

S. Maheshwari, S.R. Avula, and A. Singh contributed equally to this article.

Corresponding Authors: D. Datta, CSIR-Central Drug Research Institute, Sector10, Jankipuram Extension, Sitapur Road, Lucknow 226031, Uttar Pradesh, India.Phone: 91-522-277-2450, ext. 4347/48; Fax: 91-522-277-1941; E-mail:[email protected]; and Koneni V. Sashidhara, [email protected]

doi: 10.1158/1535-7163.MCT-16-0584

�2017 American Association for Cancer Research.

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MDM2–p53 interaction but also promoted MDM2 degradation(17). In our current endeavor, we have synthesized a series ofsemicarbazone and chalcone hybrid and put forward strongevidence for not only its antiproliferative tumor cell–selectiveeffect but also provided insight into its unique antimetastaticpotential via mitigating PP2A–b-catenin signaling pathways.

Materials and MethodsReagents and antibodies

DAPI, doxorubicin, and anti-b-actin (cat# A3854) antibody,GST protein tag, Crystal violet dye, Hoechst 33342, Meyerhematoxylin solution, and DPX were obtained from SigmaAldrich. ImmEdge pen (hydrophobic barrier pen), Bloxallblocking solution, DAB Peroxidase Substrate Kit, VECTASTAINABC Kit were purchased from Vector Laboratories, Inc. Fluo-rochrome-conjugated secondary antibodies, Annexin-V AlexaFluor 488, F(ab')2-goat anti-rabbit IgG (HþL) secondary anti-body Qdot 655, and Verso cDNA Synthesis Kit as well as SYBRGreen Real-Time PCR Master Mix were purchased from Molec-ular Probes-Invitrogen. b-Catenin (cat# 8480), active b-catenin(cat# 8814), p-GSK3bser-9 (cat# 5558), E-cadherin (cat# 3195),Cyclin D1 (cat# 2978), c-Myc (cat# 5605), DR5 (cat# 8074),cleaved PARP (cat# 5625), caspase-8 (cat# 9746), cleavedcaspase-8 (cat# 9496), caspase-9 (cat# 9502), cleaved cas-pase-9 (cat# 7237), Ki-67 (cat# 12202), and GAPDH (cat#2118) antibodies, as well as PP2A Sampler Kit (cat# 9780) wereprocured from Cell Signaling Technology, Inc. Anti-GSK3btotal (cat# 612313) antibody was purchased from BD Bios-ciences. XIAP (cat# sc-55551) and horseradish peroxidase(HRP)-conjugated secondary antibodies and normal rabbit IgG(cat# sc-2027) were obtained from Santa Cruz Biotechnology.Human recombinant GST-tagged PR55a protein (cat#H00005520- P01) was purchased from Abnova. XenoLightD-Luciferin - Kþ Salt bioluminescent substrate was boughtfrom PerkinElmer. All chemicals and antibodies were obtainedfrom Sigma unless specified otherwise.

Procurement and culture of cell linesVarious human cancer cell lines including DLD-1, SW620

(colorectal adenocarcinoma), FaDu (pharynx squamous cell car-cinoma), A549, NCI-H446 (lung carcinoma), PLC/PRF/5 (hep-atoma), DU145, PC-3 (prostate carcinoma and adenocarcino-ma), SKOV3 (ovarian adenocarcinoma), and human breast can-cer cell lines MCF-7, MDA-MB-468, ZR-75-1 SK-BR-3, BT-549,mice breast cancer cell line 4T1 (metastatic breast adenocarcino-ma), and human immortalized non-transformed breast epithelialcells MCF-10A were obtained from ATCC, USA. 4T1-luc2-GFPcells were purchased from Caliper Life Sciences (PerkinElmer).Early passage cells were resuscitated from liquid nitrogen vaporstocks as needed and cultured according to the manufacturer'sinstructions. Cells were routinely inspected microscopically forstable phenotype, and all experiments were performed withinearly passages (within 10) of individual cells. Cell line authen-tication (STR profiling) was done for SW620, as it was used formost of the important in vitro and especially in vivo experiments.

Evaluation of in vitro anticancer activity of synthetichybrid compounds

Rational and detailed synthesis of semicarbazone–chalconehybrid compounds and their characterization are given in

Supplementary Figs. S1 and S2A–S2C, Supplementary TableS1, and Supplementary Methods S1 and S2. Efficacy of thesehybrids as anticancer agents on various cancer cell lines wasassayed using standard sulforhodamine B (SRB) cytotoxicityassay (18, 19). Absorbance was measured on a plate reader(Epoch Microplate Reader, Biotek) at 510 nm to calculate thepercent inhibition in cell growth by using the formula: [100�(absorbance of compound treated cells/absorbance of untreat-ed cells)] � 100.

Clonogenic assayClonogenic assay was done following standard procedure. In

brief, cells (MCF-7 and MCF-10A) were seeded at 400 cells perwell in 6-well plates and allowed to adhere. After 24 hours, cellswere treatedwith either different doses (0, 0.125, 0.5, and 1mmol/L) of CS-11 or vehicle. After 7 days, media were removed and cellswerewashedwith PBS and fixedwith ice-coldmethanol, followedby staining with 0.5% crystal violet in methanol for 30 minutes.Excess stain was removed by washing with water thoroughly, andplates were allowed to dry. Representative images were taken tomonitor single-cell colony formation efficiency of MCF-7 andMCF-10A cells.

RNA isolation and qRT-PCRmRNA were isolated from cells by using the Pure Link RNA

Mini Kit (Ambion). cDNA was prepared by using Verso cDNASynthesis Kit (Invitrogen) following the manufacturer's proto-cols. Each cDNA sample was amplified using SYBR Green inStepOnePlus Real-Time PCR System (Applied Biosystems). Thereaction mixture contained 1 mL of cDNA and 0.1 mmol/L ofprimers in a final volume of 10 mL supermix. The experimentswere performed in triplicate. As an internal control, GAPDHmRNA was amplified and analyzed under identical conditions.Ct value (the cycle number at which emitted fluorescenceexceeded an automatically determined threshold) for gene ofinterest was corrected by the Ct value for GAPDH and expressedas DCt. Data were measured as fold changes of mRNA amount,which was calculated as follows: fold changes ¼ 2X (where X ¼DCt for control group � DCt for experimental group). The oligo-nucleotide primers used are as follows: for human GAPDH:forward: 50-GTCAGTGGTGGACCTGACCT-30, reverse: 50-AGGG-GAGATTCAGTGTGGTG-30, product size 395 bp; for humanCyclin D1, forward: 50-GCATGTTCGTGGCCTCTAAG-30, reverse:50-CGTGTTTGCGGATGATCTGT-30, product size 228 bp; and forhuman c-Myc: forward: 50-GATCCAGACTCTGACCTTTTGC-30,reverse: 50-CACCAGCAGCGACTCTGA -30, product size 102 bp.

Western blottingTreated or untreated cells were lysed in RIPA buffer supple-

mented with phosphatase and protease inhibitors cocktail.Equal amounts of protein in SDS-PAGE sample buffer wereloaded in 12% SDS polyacrylamide gels and transferred toPVDF membranes. After appropriate primary and secondaryantibody incubation (20, 21), immunoreactivity was detectedby enhanced chemiluminescence solution (Immobilon West-ern ECL, Millipore) and scanned by gel documentation system(Bio-Rad ChemiDoc XRSþ). Densitometric analyses of Westernblots were done by normalizing the pixel intensity of desiredprotein to their respective loading control by using Image LabSoftware (Bio-Rad).

Maheshwari et al.

Mol Cancer Ther; 16(9) September 2017 Molecular Cancer Therapeutics1792




Immunoprecipitation studiesFor immunoprecipitation (IP) studies, equal amounts (400mg)

of total protein of treated and untreated groups were incubatedovernight at 4�C with either anti-human PP2A-B (specific forPR55a) or anti-b-catenin antibody. The immune complexes wereprecipitated with protein A/G PLUS Agarose beads (Millipore)and subjected to SDS-PAGE along with 10% input control (22),followed by Western blot analysis using primary antibodies andcorresponding kappa light chain–specific HRP-conjugated sec-ondary antibodies (to avoid heavy chain IgG bands), Then, blotswere developed by enhanced chemiluminescence substrate. Den-sitometric analysis of IP blots was done by normalizing the pixelintensity of desired proteins to either input control or pull downprotein by using Image Lab Software (Bio-Rad). Ratio betweennormalized desired protein versus pulldown protein was repre-sented in percent change considering vehicle as the standard unit(100%).

Confocal microscopyCells were grown on cover slips and treated as designated. After

intermediate washes with cold PBS, cells were fixed with 4%paraformaldehyde, permeabilized by 0.1% NP40, followed byblocking with 2% BSA. After overnight incubation with primaryantibodies at 4�C, cells were washed and incubated with fluores-cent-conjugated appropriate secondary antibodies followed byDAPI staining. After washing, cells were mounted on slides andanalyzed under inverted Zeiss confocal laser scanningmicroscope(Zeiss Meta 510 LSM; Carl Zeiss).

In vitro scratch assayIn brief, 4T1 cells were seeded in 6-well plates; next day, when

cells were near 90% confluent, they were rinsed with PBS. Ascratch was made in each well with the help of a sterile 200-mLpipette tip. Treatment was given and pictures were taken at 0, 12,24 hours using phase-contrast microscope at �10 magnification(Primovert; ref. 23). The percentage of open scratch area wasquantified by TScratch Software (ETH Zurich).

In vivo studies in xenograft and syngeneic tumor modelsAll animal studies were conducted in accordance with the

principles and standard procedures approved by the InstitutionalAnimal Ethics Committee (IAEC) CSIR-Central Drug ResearchInstitute (Lucknow, India). SW620 (human colon cancer) xeno-graft and orthotopic 4T1-luc2-GFP models have recently beenestablished in the laboratory by following standard protocol.Briefly, early passage 98% viable 2 � 106 SW620 cells or 1 �106 4T1-luc2-GFP cells in 100 mL PBS were either subcutaneouslyinjected in the flanks of the right hind leg or mammary fat pad onright flank of each 4- to 6-week-old nude Crl: CD1-Foxn1nu mice.When tumor volume reached 50 to 100 mm3, mice were ran-domized and divided into two experimental groups. Tumor-bearing mice were administered intratumoral injection once aweek with either vehicle alone (DMSO and serum-free media) orcompound (5mg/kg) dissolved in 50 mL vehicle until sacrifice. Incase of SW620 xenograft, tumor size was measured using anelectronic digital caliper at regular intervals. Tumor volume wasestimated by standard formula V¼P/6� a2� b, wherein a is theshort and b is the long tumor axis (19). In the case of 4T1-luc2-GFPmodels, at day 25, mice were injected subcutaneously with D-luciferin (150 mg/kg body weight), anesthetized with ketamine,and whole mice were imaged up to 1 hour at various time points

using a bioluminescent imaging system (IVIS Spectrum, Caliper).Regions of interest from displayed images were identified on thetumor sites and quantified as photons per second (p/s) usingLiving Image software.

Apoptosis antibody arrayMCF-7 cells were treated with either vehicle or CS-11

(10 mmol/L) for 24 hours. Cells were harvested and lysed in lysisbuffer 17 followed by protein concentration estimation. Apopto-sis array analysis was performed using the Proteome ProfilerHuman Apoptosis Array Kit (ARY009) from R&D Systems as perthe manufacturer's instructions (24). Array images were capturedby gel documentation system (Bio-Rad ChemiDoc XRSþ) andwere analyzed using the Image Lab Software (Bio-Rad). Heatmapwas prepared by using Plotly software.

Flow cytometryTo measure intracellular cleaved caspase-8 and 9, cells were

harvested by mild treatment of TrypLE (Invitrogen), washed, andfirst fixed with 4% paraformaldehyde for 10 minutes and per-meabilized with 0.1% NP-40 in PBS for 10 minutes. Permeabi-lized cells were stained with either cleaved caspase-8 or cleavedcaspase-9 or isotype control antibody for 30 minutes at 4�C,followed by washing and staining with Qdot 655 IgG secondaryantibody for 30minutess at 4�C. The stained cellswere acquired ina FACSCalibur (Becton Dickinson) and analyzed by FlowJosoftware (Tree Star).

IHCIHC was done following standard procedure established in

the lab (19). Briefly, tumor tissues were fixed in 4% parafor-maldehyde for 48 hours and embedded in paraffin wax. Forstaining, tissue sections were deparaffinized, rehydrated, andquenched for endogenous peroxidase. Antigen retrieval wasperformed by heating of slides in 10 mmol/L sodium citratebuffer (pH 6) for 30 minutes. Slides were then rinsed in PBS,and endogenous peroxide activity was neutralized by incubat-ing with Bloxall (blocking solution) for 25 minutes. Tissuesections were then incubated with primary antibodies in 2%BSA against active b-catenin (1:800), E-cadherin (1:400), Ki-67(1:400), and BSA only for negative control overnight at 4�C.Next day, slides were rinsed with PBS and incubated withbiotinylated secondary antibody at room temperature for 1hour. Followed by PBS washing and incubation with ABCreagent (Vector Laboratories) at room temperature for 1 hour,slides were incubated with 30-30-diamino-benzidine (DAB) andcounterstained with hematoxylin. Tissue sections were thendehydrated and mounted by using DPX. Stained sections wereobserved under �40 magnification of microscope (Leica).

Pharmacokinetic studiesDetailed methodologies for different pharmacokinetic studies

are described in Supplementary Methods S3.

Docking studiesInitially binding pockets were identified on protein surface

using Discovery Studio 4.1 (25). Protein–protein docking studieswere performed using ZDOCK module of Discovery Studio-4(26) to identify possible binding residue of b-catenin with PP2Aenzyme. Least energy conformation of each cluster out of 2,000generated poses was selected and further analyzed for its binding

PP2A/b-Catenin Interaction Inhibitor Limits Tumor Metastasis

www.aacrjournals.org Mol Cancer Ther; 16(9) September 2017 1793




affinity and scoring. Structure of compound CS-11 was built andminimized using Marvin Sketch version 6.1.2 from Chem Axon(https://www.chemaxon.com/). Docking studies of CS-11 withPP2Aproteinwere performed onbinding pockets identified usingAuto Dock 4.2 tool (27), followed by molecular dynamic studiesby using Gromacs version 5.04 (28) to check the temporal

stability of the compound to its target protein. CHARMM forcefield was used and parameters for compound were generatedusing Swiss Param tool (29). Protein–compound complex wassolvated in a cubic box with TIP3P water molecules at 10 Åmarginal radiuses. At physiologic pH, the structure was foundto have a negative charge; thus, to make the system electrically

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Figure 1.

CS-11 induces breast cancer cell–selective cytotoxic effects and promotes apoptosis.A, Cytotoxicity of CS-11 was compared with parent molecule 8e at five differentdoses (1.25, 2.5, 5, 10, and 20mmol/L) for 48hours, and IC50 valuewas presented in bar graphbyutilizing SRBassay.B,Tumor cell–selective cytotoxic function of CS-11was comparedwith standard FDA-approved drug doxorubicin. MCF-7 andMCF-10A cells were treatedwith increasing concentrations (1.25, 2.5, and 5 mmol/L) of CS-11 and doxorubicin for 72 hours and cell viability was determined; left, representative photomicrographs of SRB-stained cells are shown in plate; right, MCF-7and MCF-10A cells were treated with CS-11 and doxorubicin, and cytotoxicity was assessed by SRB assay in the form of relative cell viability. Columns, average ofquadruplet readings of samples shown in the left panel; error bars,�SD. � ,P <0.01, comparedwith treatedMCF-10A cells. Results shown are representative of at leastthree independent experiments. C and D, MCF-7 and MCF-10A cells were treated with vehicle, CS-11, or parent compound 8e at a dose of 10 mmol/L for24 hours; representative of at least three independent experiments. C, Photomicrographs of MCF-7 and MCF-10A cells are shown. D, Cells were stained withAnnexin-V Alexa Fluor 488, Hoechst and analyzed under fluorescence microscope. E, Immunofluorescence staining was performed for cleaved PARP and DAPI inMCF-7 cells following CS-11 (5 and 10 mmol/L) or vehicle treatment for 24 hours and analyzed by confocal microscopy. Scale bar, 10 mm. Right, percent ofcleaved PARP-positive cells over total (DAPI stained) number of cells was calculated in five different fields of both treated and untreated groups represented and inbar diagram; bars, �SD of control and treated groups (� , P < 0.01). F, Crystal violet staining in MCF-7 and MCF-10A cells after 7 days of treatment of CS-11.Right, percentage of colony-forming efficiency in vehicle- or CS-11–treated MCF-7 and MCF-10A cells was calculated from two different experiments andrepresented in bar diagram; bars, �SD of control and treated groups (� , P < 0.01).

Maheshwari et al.




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neutral, sodium ions were added into the simulation box. Wholeprotein–compound systemwas subjected to energyminimizationby conjugant gradient minimization algorithm. Isothermal–iso-choric equilibration was performed using PME method for100,000 steps. Isotropic pressure coupling was performed on the

previously equilibrated systemusing Parrinello–Rahmanmethodfor 100,000 steps. Finally, protein–compound complex was sub-jected to molecular dynamic simulation for 20 nanoseconds.UCSF Chimera (30) was used for the model visualization andimage generations.

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CS-11 activates both extrinsic and intrinsic apoptotic pathways in breast cancer.A–C,MCF-7 cellswere treatedwith either CS-11 (10mmol/L) or vehicle for 24 hours andharvested for analysis of apoptosis-related protein expression by exploiting proteome profiler apoptosis array or individual Western blot analysis. A,Chemiluminescent image of hybridized array blots (vehicle and CS-11–treated cells) showing the expression of 35 proteins involved in apoptosis with positiveand negative controls in duplicates (left) and spot coordinates of array (top right).B,Heatmap illustrating the expression of differentially regulated proteins inMCF-7before and after CS-11 treatment. C, Enlarged photomicrographs of selected altered proteins from apoptosis array belonging to both intrinsic (XIAP; D21, D22)and extrinsic (DR5; C3, C4) apoptosis pathway are shown. D, Individual Western blots of altered proteins DR5 and XIAP in vehicle and CS-11–treated (5 and10 mmol/L for 24 hours) cells are shown. E, Immunoblot images for the protein expression of pro-caspase-8 and pro-caspase-9 along with endogenous controls ofvehicle- and CS-11–treated (5 and 10 mmol/L for 24 hours) cells are shown. F, FACS histogram overlays for the expression of cleaved caspase-8 and 9 in MCF-7 cellsfollowing vehicle and CS-11 treatment (10 mmol/L) for 24 hours. Results shown from D–F are representative of at least three independent experiments.






Internal fluorescence quenching assayFluorescence measurements were performed using spectroflu-

orometer of PerkinElmer Life Sciences LS 50B as described pre-viously (17). Briefly, the fluorescence spectra were obtained at 25

� 0.1�C with a 1-cm path length Quartz cell. The intrinsicfluorescence was measured using excitation wavelength for excit-ing protein at 280 nm, and emission spectra were recorded in therange of 300 to 420 nm. The titration curve was obtained by

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Solubility and pharmacokinetic evaluation of CS-11. A, In vitro available concentration of CS-11 in 4T1 cell culture on increasing concentration of CS-11 in cell lysateis represented in line graph. B, Table demonstrating solubility profiles of CS-11 and parent compounds 8e (chalcone), 10a (semicarbazone) over a period of24 hours in buffers of different pH. C, Stability profile of CS-11 (10 mg/mL) in SGF and SIF in the presence of enzyme (pepsin for SGF and pancreatin for SIF) isrepresented as percent availability at different time interval. D, Stability profile of CS-11 obtained in rat plasma. The values represent the mean � SEM of threeindependent experiments. Statistical analysis was carried out by the paired Student t test (P < 0.05). E, Metabolic stability of compound CS-11 (2 and 10 mg/mL)in rat liver microsomes (0.5 mg protein/mL) in the presence and absence of NADPH at different time (minute) points with percent of remaining drug andalso in phosphate buffer (10 mg/mL). F, The plasma protein binding of CS-11 at different concentrations of 2 and 10 mg/mLwas represented and found to be 78.5%�0.076% and 73.04% � 1.54%, respectively.

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adding the appropriate compound to the protein PR55a (humanrecombinant GST-tagged PR55a cat# H00005520- P01 fromAbnova) solution (40 mg of protein/mL in 50 mmol/L Tris-HCL,10 mmol/L reduced glutathione, pH 8.0). The stock compoundconcentration was 1mol/L, and the pH was adjusted to 8.0 in thesame buffer. Stock solutions were prepared freshly before eachtitration. For fluorescence quenching experiment, the concentra-tion range of 10 pmol/L to 1 mmol/L of compound was usedagainst PR55a protein, to the final volume. Background GST/buffer fluorescence was observed to be minimal and subtractedfrom experimental groups. Dissociation equilibrium constant(Kd) value was determined from the data fitted to a singleexponential hyperbolic equation, by using the PRISM3nonlinearregression tool (GraphPad).

Statistical analysisMost of the results are representative of at least three inde-

pendent experiments. Student t test and two-tailed distribu-tions were used to determine the statistical significance; P

values �0.05 between groups were considered statisticallysignificant.

ResultsChalcone-semicarbazone hybrid molecules promote tumorcell–selective cytotoxic effects and potentially induceapoptosis in cancer cells

Utilizing our molecular hybridization approach, we havesynthesized a series of chalcone–semicarbazone hybrids con-taining 17 compounds (including parent chalcone-8e andsemicarbazone-10a), and details of their synthesis and differentqualitative and quantitative analyses are provided in Supple-mentary Figs. S1 and S2A–S2C and Supplementary Methods S1.To test the anticancer potential of our synthesized novel mole-cules, we performed anticancer screening in seven differentcancer cell lines representing seven cancer types at a singledose of 10 mmol/L. Representative cell lines from differentcancer types are MCF-7 (breast adenocarcinoma), DLD-1

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CS-11 inhibits colon tumor growth andmitigates breast cancermetastasis in vivo.A–D, SW620 cells (2� 106) or 4T1-Luc2-GFP cells (1� 106) in 100 mL PBSwere eithersubcutaneously injected in the flanks of the right hind leg or mammary fat pad on right flank of each 4- to 6-week-old nude Crl: CD1-Foxn1nu mice. Whentumor volume reached 50 to 100mm3,micewere randomizedanddivided into experimental groups (n¼5). Tumor-bearingmicewere subjected to have intratumoralinjection with either vehicle alone (DMSO and serum-free media) or CS-11 (5 mg/kg/week) dissolved in 50 mL vehicle until sacrifice. A, Growth curve is shown forvehicle- and CS-11–treated mice; points are indicative of average value of tumor volume; bars, � SD. The CS-11–treated group had significantly lower averagetumor volumes from the vehicle-treated group (� , P < 0.05). Top corner photograph shows tumor-bearing mice of treated and control groups. B, Top,averageweight of harvested tumors bars,�SDof vehicle and treated groups (� ,P <0.05) depicted by bar graph. Bottom, representative tumor images of vehicle andCS-11–treated mice are shown. C, Average body weights of vehicle and treated mice is represented as line graph. D, Left, magnified bioluminescent image ofvehicle and CS-11–treated 4T1-luc2-GFP tumor-bearing mice. Right, regions of interest from displayed images were identified and quantified as photons per second(p/s) in each mouse and represented in bar diagram.






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(colorectal adenocarcinoma), FaDu (pharynx squamous cellcarcinoma), DU145 (prostate carcinoma), A549 (lung carcino-ma), SKOV-3 (ovarian adenocarcinoma), and PLC/PRF/5 (liverhepatoma). Detailed results of these investigations in terms ofcell death (percentage of control) at 10 mmol/L of each com-pound are given in Supplementary Table S2A. Overall, weobserved cytotoxicity of most of the molecules against differentcancer types. Interestingly, we observed that our hybridizationresulted in the induction of cytotoxic effects in breast and lungcancers compared with active parent chalcone (8e). To furthervalidate this observation, we extended our studies in fivedifferent breast cancer cell lines (MDA-MB-468, BT-549,SK-BR-3, ZR-75-1, and 4T1) as well as human immortalizednontransformed/nontumorigenic breast epithelial cell line(MCF-10A) and tested the cytotoxic potential of whole seriesof molecules against these cells at a 10 mmol/L dose. Thoroughanalysis of the data represented in Supplementary Table S2Brevealed that CS-11 is the molecule in which our hybridizationapproach worked best, as it showed maximum cytotoxicity tomost of the breast cancer cells but less toxic effect on non-tumorigenic breast epithelial MCF-10A cells. As in vitro anti-cancer efficacy of CS-11 as well as its cancer cell selectivity isbetter than parental chalcone (8e), we tested the comparativedose response of cytotoxic effects of these two in five differentbreast cancer cell lines as well as in MCF-10A cells and deter-mined the IC50 values for CS-11 and 8e. As demonstrated in Fig.1A and Supplementary Fig. S3, IC50 values of CS-11 are lowerthan 8e in all the breast cancer cell lines tested with maximumcytotoxic efficacy in MCF-7 cells. On the other hand, the inverseresult was obtained in the case of MCF-10A cells where CS-11was found to be less cytotoxic than parent chalcones 8e,indicating better tumor cell selectivity of CS-11 over parentchalcone. To compare tumor cell–selective effect of our hybridmolecule with standard marketed breast cancer drug, we nextexamined the effect of CS-11 and the FDA-approved cancerdrug doxorubicin, in multiple doses on nontumorigenic breastepithelial cells MCF-10A versus tumorigenic breast epithelialMCF-7 cells. Most strikingly, we observed (Fig. 1B) that theFDA-approved drug doxorubicin was equally cytotoxic in MCF-10A cells and MCF-7 cells. In contrast, CS-11 was found to besignificantly (P � 0.01) more cytotoxic to MCF-7 cells com-pared with MCF-10A cells. This is a clear indication of bettercancer cell selectivity of CS-11 over standard breast cancer drugdoxorubicin in terms of inducing in vitro cytotoxic effects. Tostudy the comparative cytotoxic efficacy of CS-11 and parent 8ein terms of inducing apoptosis in transformed tumorigenic cells

versus nontumorigenic cells, we treated MCF-7 and MCF-10Acells with CS-11 and 8e at a dose of 10 mmol/L for 24 hours andperformed Annexin-V staining. Results shown in Fig. 1C and Dclearly indicate that our lead molecule CS-11 has selectiveapoptotic effect in MCF-7 over MCF-10A cells in comparisonwith 8e. Apoptotic ability of CS-11 was further validated bycleaved PARP staining of vehicle and CS-11–treated cells, wherewe observed significant (P � 0.01) induction of cleaved PARP–positive cells in treated cells as compared with vehicle control(Fig. 1E, right and left). In addition, we also found that CS-11 at0.5 and 1 mmol/L doses significantly (P � 0.01) inhibitedclonogenic efficiency in MCF-7 cells but not in MCF-10A cells(Fig. 1F, right and left). Altogether, our results suggest thatCS-11 has better tumor cell selectivity and cytotoxic efficacyover standard drug as well as our parent molecule from where itwas originally derived.

CS-11 promotes cell death via regulating proteins of bothextrinsic and intrinsic apoptotic pathways

Furthermore, to gain mechanistic insight of CS-11–mediatedbreast cancer cell apoptosis, we made use of apoptosis antibodyarray (ARY009, R&D Systems) to simultaneously assess theexpression of 35 apoptosis-related proteins spanning both extrin-sic and intrinsic apoptosis pathways. MCF-7 cells were treatedwith either vehicle or 10 mmol/L of CS-11 for 24 hours, andharvested proteins were incubated with the array as per themanufacturer's protocol. Array immunoblots and/or heatmapimage along with spot coordinates of array blots (Figs. 2A–C)clearly indicated the differential expression pattern of multipleapoptotic and anti-apoptotic proteins following CS-11 treatmentcompared with vehicle control. For validation of array data, weselected two proteins (DR5 and XIAP) that were found to bereversely regulated upon CS-11 treatment as well as members oftwo different (DR5-extrinsic, XIAP-intrinsic) apoptotic pathwayshaving potent biological significance, although there were somechanges in other proteins too. Consistent with array results, CS-11treatment significantly (P � 0.05) upregulates the expression ofproapoptotic DR5 as well as inhibits the expression of antiapop-totic XIAP protein as compared with vehicle-treated cells (Fig. 2D;Supplementary Fig. S4). These upstream death signals ultimatelylead to cell death via activation of different prototype extrinsic(caspase-8) and intrinsic (caspase-9) caspases. To check the acti-vation and involvement of caspase-8 and 9 in CS-11–mediatedapoptosis, we performed Western blot and FACS analysis ofvehicle and treated cells and observed downregulation of pro-caspase-8 and 9 (Fig. 2E) as well as upregulation of active cleaved

Figure 5.CS-11 inhibits active b-catenin expression but promotes the expression of E-cadherin inmetastatic cancer cells.A,Metastatic cell lines fromdifferent cancers (SW620,4T1, PC-3, FaDu, andNCI-H446)were treatedwith either vehicle or two different doses of CS-11 (5 and 10 mmol/L) for 24 hours, harvested for total protein extraction,and SDS-PAGE was performed. Immunoblot analysis was performed using antibodies against active b-catenin, total b-catenin, and its downstream directtarget proteins c-Myc and Cyclin D1. GAPDH and b-actinwere used as internal protein loading controls. B, Fold changes in mRNA expression of Cyclin D1 (left) and c-Myc (right) followingCS-11 treatmentweremeasured by real-timePCR. Data reflect three independent experiments. Columns are averageof triplicate readings of thesample; bars, �SD. � , P < 0.05 versus vehicle-treated cells. C, SW620 and FaDu cells were treated with or without CS-11 (5 and 10 mmol/L) for 24 hours andharvested for total protein extraction. Immunoblotting was performed for E-cadherin and GAPDH was used as a control. D, SW620 cells were treated with eithervehicle or CS-11 (5 and 10 mmol/L) for 24 hours, and protein was extracted. Total b-catenin was immunoprecipitated from the whole-cell lysate treated witheither vehicle or CS-11 and immunoblotted with an anti-E-cadherin antibody. The levels of b-catenin and E-cadherin in the whole-cell lysates were analyzed as inputcontrol, while b-actin and IgG light chain–specific secondary antibody was used as IP control. Densitometric analysis of different IP immunoblots isrepresented in bar diagram (right), and details are described in theMethods section. E, SW620 cells were grown on coverslips and treatedwith either vehicle or CS-11for 24 hours and subjected to immunofluorescence staining for active b-catenin and E-cadherin and analyzed by confocal microscope. Inset photomicrographsrepresent the magnified area of the box. Scale bar, 10 mm. F, Representative IHC photomicrographs of SW620 xenograft tumor tissue sections from CS-11or vehicle-treated mice for active b-catenin, E-cadherin, and Ki-67 staining at �400 magnification.






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caspase-8 and caspase-9 following CS-11 treatment (Fig. 2F) ascompared with vehicle. Together, the results indicate the involve-ment of both extrinsic and intrinsic pathways in CS-11 inducedapoptosis.

Preclinical pharmacokinetic evaluation of CS-11 indicates itsdrug-like properties

Before going to test the in vivo efficacy of the active molecule(CS-11), we performed initial pharmacokinetic studies. First,we determined the concentration of CS-11 in our cell culturecondition where we detected pure CS-11 in the treated celllysate, and it was found to be enhanced with increasing thedose, indicating its in vitro stability in the media (Fig. 3A). Next,we determined the solubility of CS-11 at various pH to mimicdifferent gastrointestinal conditions and also compared itssolubility with parent molecules (8e and 10a). Interestingly,here our hybridization worked better to increase the solubilityof CS-11 as compared with phenotypically active parent 8e at aneutral pH, indicating the probability of better absorption ofCS-11 in the intestine (Fig. 3B). Stability of CS-11 in simulatedgastric fluid (SGF) and simulated intestinal fluid (SIF) is one ofthe important parameters of any drug-like molecule, and asshown in Fig. 3C, percent drug (CS-11) remaining in SGF andSIF were found to be 93.16 and 87.87, respectively, up to thedesired time point, suggesting its drug-like properties. For adrug to be effective, adequate active form must reach the targetto obtain the desired effect. Plasma stability of CS-11 at 2 and10 mg/mL in rat plasma was found to be 82.17% � 1.52% and62.24% � 1.76%, respectively (Fig. 3D). The compound wasfound to be stable in plasma after 2 hours at room temperaturefor the duration of typical sample handling and processing. Thein vitromicrosomal metabolic stability of CS-11 at two differentconcentrations of 2 and 10 mg/mL was also performed. Percentdrug remaining for 2 and 10 mg/mL was found to be 92.53% �4.40% and 92.34% � 0.57%, respectively (Fig. 3E) in rat livermicrosomes (RLM). Compound was stable when the experi-ment was conducted in the absence of NADPH (negativecontrol) and also thermally stable at 37�C (100 mmol/L phos-phate buffer). Testosterone (2 mg/mL, positive control) half-lifein the RLM was within the acceptable in-house limits. In vitrosystems with low metabolic capacity evaluate the cytotoxiceffects of parent compound 8e. To reduce misinterpretationof these data, it is important to determine metabolic stability.The plasma protein binding of CS-11 at different concentra-tions of 2 and 10 mg/mL was found to be 78.5% � 0.076% and73.04% � 1.54%, respectively (Fig. 3F). Ka and Kd at 2 and 10mg/mL were found to be (61.62 � 14.35) � 10�3, (40.67 �15.10)� 10�4 and (46.60� 11. 51)� 10�3, (79.02 � 42.90)�10�4/min; per mg/mL, respectively. Increased CS-11 binding

with plasma proteins accelerates the rate of elimination(t1/2 decreases) because it makes more drug available to thesite of metabolism. There may be less possibilities of drug–druginteraction due to <90% protein binding.

CS-11 markedly inhibits tumor growth and metastasis in vivoTo evaluate the in vivo efficacy of CS-11, we utilized SW620

subcutaneous xenograft model to study tumor growth, asSW620 has been widely used in in vitro mechanistic studies.To understand its antimetastatic potential, we utilized Luc2-GFP–tagged 4T1 syngeneic orthotopic breast tumor model.Before proceeding to SW620 xenograft model, we first deter-mined the IC50 value of CS-11 and parent 8e in SW620 cells,where we found the IC50 value of CS-11 is 4.06 � 0.11 mmol/Lcompared with 10.01 � 0.66 mmol/L in case of 8e (Supple-mentary Fig. S5A). In SW620 xenograft model, CS-11 (5 mg/kg/week) intratumor administration showed significant (P � 0.05)inhibition of tumor growth compared with vehicle as observedin Fig. 4A. We also observed significant (P � 0.05) difference inthe weight of harvested tumors at the end of the experimentbetween CS-11–treated and vehicle group (Fig. 4B). Important-ly, we did not observe any significant body weight differencebetween vehicle and treated group during the full-course treat-ment, suggesting its nontoxic nature in in vivo condition as well(Fig. 4C). Next, we evaluated the effect of CS-11 on breastcancer metastasis through live animal imaging. As observedin Fig. 4D (left and right) compared with vehicle-treated group,CS-11 treatment (5 mg/kg/week) reduced overall spread ofbreast tumor growth as well as inhibited lung metastasis in4T1-luc2-GFP orthotopic model. Therefore, CS-11 does notonly inhibit tumor growth but also have potential to alleviatemetastasis of breast cancer to the secondary organs. Afteranalyzing in vivo antimetastatic property of CS-11, we exploredits antimigratory efficacy. Here, we performed an in vitro scratchassay in the presence or absence of CS-11 in a time- and dose-dependent manner and found significantly (P � 0.05) lessmigration in CS-11–treated 4T1 cells as compared with vehi-cle-treated cells, which covered the scratch area within 24 hours(Supplementary Fig. S5B).

CS-11 reduces active b-catenin but promotes E-cadherinexpression in metastatic cancer cells

CS-11–mediated inhibition of lung metastasis inspired us topursue further its in-depth mechanistic insight. Published lit-erature in patient samples as well as animal data indicatethat metastasis of breast cancer, especially to the lungs, largelyrelies on functional activation of b-catenin signaling (31–34).To test this possibility in our case, we first selected five differentmetastatic cell lines (4T1, SW620, PC-3, FaDu, and NCI-H446)

Figure 6.In silico and biological evidence of CS-11 and PR55a interaction. A, 4T1, SW620, and FaDu cells were treated with vehicle or CS-11 (5 and 10 mmol/L) for 24 hours andharvested for protein extraction. Western blot analyses were performed for total GSK3b and GSK3bSer9. B, Residues of PR55a, which are responsible forinteraction with CS-11 at its binding pocket, are shown (3D and surface view models). C, RMSD plot of CS-11 for the 20-ns simulation run with 2.5 Å RMSD, indicatinghigh stability of CS-11 in PR55a binding groove. D, Left, fluorescence emission spectra of PR55a protein in the absence or presence of increasing concentrations (10pmol/L–200 nmol/L) of CS-11. Right, standard Scatchard exponential hyperbolic binding curve of direct physical binding of CS-11 with recombinant proteinPR55a with dissociation equilibrium constant (Kd) value. E, SW620 cells were treated with vehicle or CS-11 (5 and 10 mmol/L) for 24 hours and then harvested forprotein extraction. Cleared lysate volumes were normalized and subjected to IP with anti-PP2A-B specific for PR55a. Ten percent input volumes and 50% IPsamples were analyzed by Western blotting for non-phospho b-catenin (active), PR55a (PP2A-B), and PP2A-C. Densitometric analysis of IP immunoblots isrepresented in bar diagram (bottom), and details are described in the Methods section. F, Flowchart for the mechanism of inhibition of b-catenin signaling by acompetitive binding site of b-catenin and CS-11 at a binding pocket of PR55a of PP2A is shown.






and treated with two doses (5 and 10 mmol/L) of CS-11for 24hours and analyzed the expression of active b-catenin and itstwo predominant target proteins (c-Myc and Cyclin D1). IC50

of CS-11 is around 5 mmol/L at 48 hours, and to observe directeffect of CS-11, we opted for 5 and 10 mmol/L doses at 24hours to study the mode of action of CS-11. As shown in Fig.5A (left and right) and Supplementary Fig. S6A, in most of themetastatic cell lines, CS-11 inhibited the expression of b-cate-nin and its target c-Myc and Cyclin D1. There is a variation ofCS-11 dose response among different cell lines with respect totarget protein expression, which might be due to their differenttissue origin and genetic background of the particular cell line.To check whether the downregulation of c-Myc and Cyclin D1protein is transcriptionally regulated by the b-catenin inresponse to CS-11, we performed real-time PCR analysis inSW620 cells treated with vehicle and CS-11. Interestingly, weobserved significant (P � 0.05) dose-dependent decrease inthe mRNA expression of c-MYC and CCND1 genes followingCS-11 treatment compared with vehicle-treated cells (Fig. 5B).As b-catenin signaling is indispensably related to E-cadherinfunctionality, we next assessed the status of E-cadherin inSW620 and FaDu cells after CS-11 treatment. Strikingly, inboth, we observed significant (P � 0.05) upregulation of E-cadherin expression following CS-11 treatment compared withvehicle-treated cells (Fig. 5C; Supplementary Fig. S6B). Asso-ciation of b-catenin with E-cadherin firms the adherence junc-tion of a cell-to-cell contact, resulting in metastasis inhibitionor early dissemination of tumor cells from the primary site(35). To test this, we checked the association of b-catenin withE-cadherin before and after CS-11 treatment by IP experi-ments. Although the active b-catenin was found to be down-regulated in SW620 cells following CS-11 treatment, but toour great surprise, we observed significant (P � 0.05) increaseof b-catenin–E-cadherin association after CS-11 treatmentcompared with vehicle treatment (Fig. 5D). To find out thepossible localization of reversely regulated active b-cateninand E-cadherin expression, we performed confocal microscopyin vehicle and CS-11–treated SW620 cells. Interestingly, weobserved that cytoplasmic and nuclear active b-catenin expres-sion got decreased after CS-11 treatment compared with con-trol, but the expression of active b-catenin in the cell surfaceremained same or even more in the treated cells. On the otherhand, compared with vehicle treatment, CS-11 treatmentrobustly increased cell surface E-cadherin expression (Fig.5E). Because of antibody incompatibility, we were not ableto perform dual staining in confocal studies. So, IP resultsalong with confocal analysis clearly indicate that CS-11–induced E-cadherin is strongly associated with cell surfaceb-catenin and may lead to the inhibition of early release ofmetastatic cells from the primary tumor site. Furthermore, tovalidate our major in vitro finding of active b-catenin down-regulation and E-cadherin upregulation as well as proliferationinhibition upon CS-11 treatment, we assessed their expressionin vivo in harvested tumors via IHC. As observed in Fig. 5F,compared with vehicle control, CS-11 treatment resulted indownregulation of active b-catenin with concomitant increasein E-cadherin, suggesting the in vivo alignment of in vitrofindings. Expression of Ki-67 is a hallmark feature to deter-mine tumor cell proliferation in vivo, and here, we observedreduction of nuclear Ki-67 expression in CS-11–treated colontumors compared with vehicle control.

CS-11 interacts with PR55a subunit of PP2A and disruptsPP2A–b-catenin association preventing b-catenindephosphorylation

To address the probable target of CS-11 to destabilize theb-catenin in the cytoplasm and nucleus, we concentrated ourfocus on key regulators of b-catenin protein. The vast majorityof literature suggests that GSK3b is the major regulator ofb-catenin signaling (7). GSK3b phosphorylates b-catenin, lead-ing to its degradation, but PP2A prevents its degradation bydephosphorylating b-catenin (12, 36–38). To test the role ofGSK3b in CS-11–mediated decrease in active b-catenin expres-sion, we treated 4T1, SW620, and FaDu cells with CS-11 (5 and10 mmol/L) for 24 hours and assessed the status of total andinactive form of GSK3b (p-GSK3bser-9). However, in all threecell lines, we did not observe any significant change of eitherinactive (p-GSK3bser-9) or total form of GSK3b following CS-11treatment compared with vehicle-treated cells (Fig. 6A; Sup-plementary Fig. S7), suggesting no involvement of GSK3b inthis process. Next, we focus on another potential b-cateninregulator, PP2A, which has three different subunits, namely,PP2A-A (structural), PP2A-B (regulatory), and PP2A-C (cata-lytic). PR55a, an isoform of PP2A-B, is known to directlyinteract with b-catenin and promotes its dephosphorylation(12). To test the prospective binding of CS-11 with PP2A, weperformed molecular docking. To decipher the binding modeof b-catenin with the PP2A holoenzyme consisting of PR55a B-subunit (PDB id - 3DW8; ref. 39) protein–protein dockingstudies were carried out. The ZDOCK analysis indicated thatthe highest ranked pose has a cluster size of 48, ZDOCK score30.4, and ZRANK score -120.489. The highest ranked pose wasbound to the interface of B-subunit and C-subunit of PP2Aholoenzyme. Next, we performed docking of CS-11 with indi-vidual subunits as well as PP2A holoenzyme. Evidently, wefound the same binding pocket of PR55a for b-catenin and CS-11, indicating a probable competitive binding of b-catenin andCS-11 on PR55a.

Docking studies of top three binding grooves of PP2A pro-tein resulted binding energy of -7.12 Kcal/mol with CS-11having cluster size of 28 conformations. Residues of PR55a,which were found to be important for interaction with CS-11,were Lys95, Asn97, Lys98, Asn181, Asp340, Phe343, Asp344,Lys345, Leu420, and His421. Strong hydrogen bonds of 2.00 Åand 2.26 Å were formed by CS-11 with Phe245 and Asn97,respectively (Fig. 6B). These interactions involve hydrogenbonds, Pi–Pi, Pi–cation, salt bridges within a cut-off radius of2.5 Å. Molecular dynamic simulation on the CS-11–PP2Acomplex with lowest energy was preceded to analyze thestability of CS-11 with PP2A holoenzyme. Very low RMSD of2.5 Å was recorded for the long simulation run of 20 ns,indicating that the CS-11 molecule was highly stable with PP2A(Fig. 6C). To validate in silico results, we performed well-established internal fluorescence quenching measurementassay (17) to determine real biophysical association betweenPR55a and CS-11. In our binding assay, we found robustassociation between CS-11 and PR55a as it started to showfluorescence quenching even at 10 pmol/L dose, and finally, weobtained a Kd value of 45 pmol/L (Fig. 6D, left and right).Although fluorescence intensity of PR55a decreased regularlywith the addition of increasing concentration of CS-11, it didnot affect the basic peak appearance of PR55a, suggestingperfect binding capabilities of CS-11 (Fig. 6D, left). In an

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additional support of our binding data, we performed PP2A-B(PR55a) pull down experiments, where we analyzed the asso-ciation of PR55a with b-catenin before and after CS-11 treat-ment. Interestingly, in our pull down experiments, we observedsignificantly (P � 0.05) reduced association of PR55a withb-catenin after CS-11 treatment compared with vehicle-treatedcells, whereas an association between PP2A-B (PR55a) andPP2A-C remained unchanged. Also, we did not see any changeof basal protein expression of PP2A-B and PP2A-C after CS-11treatment. To avoid the interference of huge IgG band in pulldown experiments, we used light chain–specific secondaryantibody (Fig. 6E). Together, in silico, physical binding as wellas biological data confirmed that CS-11 occupies the samebinding pocket of PR55a where b-catenin binds and maypromote b-catenin dissociation from PR55a (Fig. 6F).

DiscussionTargeting simultaneously tumor growth and metastasis with a

single chemical entity is a daunting task in cancer drug discovery.Molecular hybridization of different pharmacophores offers thepossibility of engagement of different target proteins together tocontrol both tumor growth and metastasis. Our hybridizationapproach did not remarkably accelerate in vitro cytotoxic activityin multiple cancers, but it resulted in induced efficacy in selectivecancers like breast and lung and most strikingly increased tumorcell selective cytotoxic action. Moreover, hybridization hasincreased solubility of CS-11 at neutral pH, which may result inbetter pharmacokinetic profile. Albeit, our continuous effort isstill going on to have even better molecules around thispharmacophore.

b-Catenin is found to be an unique dual function proteinthat can control metastasis by regulating cell-to-cell adhesionvia interacting with E-cadherin, and as a transcription factor, itregulates the expression of key genes that are indispensable forcell growth and proliferation (40). A series of compellingstudies suggest that b-catenin was found to be upregulated/activated in most of the malignant tumors, including breast,colon, lung, prostate, pancreatic, and renal (34, 41–46). Mul-tiple studies have shown that b-catenin signaling dictates cellfate decisions and has the capability to transform normal cellsinto tumorigenic ones (47, 48). Thus, any disruption of b-cate-nin signaling represents a great opportunity to develop noveldrugs for selective cancer chemoprevention and therapy. How-ever, therapeutic diminution of direct b-catenin transcriptionalactivation by small-molecule inhibitors would withdraw itsantimetastatic effect, as it would result in loosening of adher-ence junction via less E-cadherin association. Therefore, idealtarget engagement situation would be indirect so that we coulddiminish b-catenin transcriptional activity as well as promoteits cell surface recruitment. Our novel chalcone–semicarbazonehybrid molecule CS-11 mimics the ideal situation, where itattenuates transcriptional activity of b-catenin via inhibiting theexpression of target genes (CCND1 and c-MYC) by directlyinteracting with PR55a as well as promotes cell surface asso-ciation of b-catenin with E-cadherin to enhance cell-to-celladherence junction. The substrate specificity of PP2A is con-sidered to be determined by its B subunit (49). It has beenshown that PR55a, a regulatory subunit of PP2A but not thecatalytic subunit, PP2A-C, directly interacts with b-catenin andis essential in PP2A-mediated b-catenin dephosphorylation

(12). Our in silico, direct physical binding assay and IP experi-ments confirmed that CS-11 selectively interacts with PR55abut not with other subunits of PP2A to finally decrease b-cate-nin nuclear function through its reduced dephosphorylation.Another important aspect is CS-11 mediated strong upregula-tion of E-cadherin on the cell surface, which helps furtherrecruitment of leftover b-catenin to the cell surface that ulti-mately leads to the nonavailability of active b-catenin in thenucleus as well as strengthens cellular adherence junction thatmay prevent early dissemination of metastatic cancer cells.Inhibition of b-catenin transcriptional targets like c-Myc andCyclin D1 is one of the consequences of CS-11–mediatedantiproliferative and proapoptotic effects where death receptorupregulation could be a major player. It has also been reportedthat overexpressed E-cadherin can also promote apoptosis incancer cells via death receptor upregulation (50), and in ourcase, CS-11–induced E-cadherin may also result in death recep-tor upregulation in treated cells. Together, our synthesizednovel small-molecule inhibitor has a unique capability ofdiminishing transcriptional activation of b-catenin target geneson one hand, but on the other hand, it enhances cell membranefunction of b-catenin along with E-cadherin, which may even-tually lead to target both tumor growth and metastasis.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

DisclaimerPart of this work has been filed for provisional Indian patent vide application

no. 65NF-DEL2014.

Authors' ContributionsConception and design: S. Maheshwari, S.R. Avula, L.R. Singh, G.R. Palnati,M.I. Siddiqi, K.V. Sashidhara, D. DattaDevelopment ofmethodology: S. Maheshwari, S.R. Avula, A. Singh, L.R. Singh,G.R. Palnati, S. Meena, M. Riyazuddin, M.I. SiddiqiAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): S. Maheshwari, A. Singh, R.K. Arya, S.H. Cheruvu,R. Kant, M. Riyazuddin, J.R. GayenAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): S. Maheshwari, S.R. Avula, A. Singh, L.R. Singh,G.R. Palnati, S.H. Cheruvu, S. Shahi, T. Sharma, R. Kant, M. Riyazuddin,H.K. Bora, M.I. Siddiqi, J.R. Gayen, D. DattaWriting, review, and/or revision of themanuscript: S.Maheshwari, S.R. Avula,A. Singh, L.R. Singh, G.R. Palnati, S.H. Cheruvu, S. Shahi, T. Sharma, A.K. Singh,M. Riyazuddin, M.I. Siddiqi, J.R. Gayen, D. DattaAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): S. Maheshwari, L.R. Singh, R.K. Arya, S. Shahi,J.R. GayenStudy supervision: K.V. Sashidhara (Chemistry), M.I. Siddiqi (Bioinformatics),J.R. Gayen (Pharmacokinetics), D. Datta (Biology)Other (designed and performed all in vivo experiments): A. SinghOther (provided help in in vivo work and confocal and fluorescence micro-copy): R.K. AryaOther (pharmacokinetic study): S. ShahiOther (anticancer screening and IC50 determination of molecules): S. MeenaOther (chemical synthesis of lead compound): K.V. Sashidhara

AcknowledgmentsWe sincerely acknowledge the excellent technical help of Drs. S. P. Singh,

Kavita Singh, and Kalyan Mitra of Electron Microscopy unit for ConfocalImaging and A.L. Vishwakarma of SAIF for the Flow Cytometry studies. Weare grateful to Dr. Sudhir Kumar Singh, Amit Kumar Tripathi, and AmitDeshmukh for their generous help in performing binding assays. We thankDr. Tejender S. Thakur of Molecular and Structural Biology Division,






CSIR-CDRI, for supervising the X-ray data collection and structure determina-tion of our compound reported in this article. Institutional (CSIR-CDRI)communication number for this article is 9493.

Grant SupportResearch of all the authors' laboratories was supported by CSIR-CDRI

Institutional Fund, Network Project BSC0106, and Fellowship grants fromCSIR and UGC.

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received September 1, 2016; revised March 8, 2017; accepted May 4, 2017;published OnlineFirst May 12, 2017.

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2017;16:1791-1805. Published OnlineFirst May 12, 2017.Mol Cancer Ther Shrankhla Maheshwari, Srinivasa R. Avula, Akhilesh Singh, et al. -Catenin Signaling and Restricts Tumor Growth and Metastasis

β−Discovery of a Novel Small-Molecule Inhibitor that Targets PP2A

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