Therapeutic Effect of Quinacrine, an Anti-protozoan Drug ...mcr.aacrjournals.org/content/molcanres/early/2018/... · and prion disease. s (5). Moreover, an . anti-tumoral effect of

1

Therapeutic Effect of Quinacrine, an Anti-protozoan Drug, by Selective Suppression of

p-CHK1/2 in p53-negative Malignant Cancers

Soyoung Park1, *, Ah-Young Oh1, *, Jung-Hyun Cho1, Min-Ho Yoon1, Tae-Guen Woo1, So-mi Kang1,

Ho-Young Lee2, Youn-Jin Jung3, Bum-Joon Park1,#

Running title: Anti-tumor Effect of QNC in p53-negative Cancers

1Department of Molecular Biology, College of Natural Science, Pusan National University, BUSAN,

Korea (republic of)

2Department of Nuclear Medicine, Seoul National University Bundang Hospital, Seongnam, Korea

(Republic of)

3College of Pharmacy, Pusan National University, BUSAN, Korea (republic of)

*These authors contributed equally at this work

# Corresponding author: BJP ([email protected])

on July 20, 2019. © 2018 American Association for Cancer Research. mcr.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on March 15, 2018; DOI: 10.1158/1541-7786.MCR-17-0511

http://mcr.aacrjournals.org/

2

Abstract

Quinacrine (QNC), anti-protozoan drug commonly used against Malaria and Giardiasis, has been

recently tried for rheumatics and prion diseases via drug repositioning. In addition, several reports

suggest anti-tumor effects of QNC through suppression of NF-κB and activation of p53. This study,

demonstrates the anti-cancer effect of QNC via a novel pathway through the elimination of check

point kinase 1/2 (Chk1/2) under p53 inactivated conditions. Inhibition of p53, by PFT-α or siRNA,

promotes QNC-induced apoptosis in normal fibroblast and p53-intact cancer cells. Considering that

Chk1/2 kinases exert an essential role in the control of cell cycle, inhibition of Chk1/2 by QNC may

induce cell death via uncontrolled cell cycle progression. Indeed, QNC reduces Chk1/2 expression

under p53-impaired cancer cells and induces cell death in the G2/M phase. QNC increases the binding

between p-Chk1/2 and β-TrCP and promotes proteasome-dependent degradation. Moreover, QNC

treatment displayed anti-tumor effects in a Villin-Cre;p53+/LSL-R172H intestinal cancer mouse model

system as well as HCT116 p53-/- xenografts.

Implications: Quinacrine has been used for the past over 70 years without obvious side-effects, as

such it is a plausible drug candidate for relapsed cancers, small-cell lung cancer, breast cancer as well

as various p53-inactivated human malignancies.




3

Introduction

Despite intensive investigation of anti-cancer drug development, cancer incidence and mortality are

continuously increased by extension of life span. In addition, cancer relapse ratio and resistance

against typical anti-cancer drugs are increased. Relapsed cancers after radiation and chemotherapy, in

general, show defects in DNA damage-induced cell death pathways, particularly, p53-dependent

pathways via genetic mutations or epigenetic alternations (1, 2). Hyper-activated ATM/ATR-related

DNA repair systems also provide resistance against DNA damaging agents (3). Thus, inhibition of

DNA repair systems is a plausible therapeutic strategy for relapsed cancers and p53 inactivated

cancers.

Quinacrine (QNC) is widely used as an anti-protozoa drug; however, new applications of QNC are

being proposed. Previous literature, has reported favorable effects of QNC on autoimmune diseases (4)

and prion diseases (5). Moreover, an anti-tumoral effect of QNC has been suggested (6). Concerning

anti-cancer effects, activation of p53 and inhibition of NF-κB are suggested (7), because QNC has an

acridine orange-like ring structure that is supposed to intercalate DNA (8, 9). However, unlike other

DNA intercalating agents, such as DAPI and EtBr, it is not considered a strong DNA intercalating

agent or carcinogen. Indeed, despite long time usage of QNC since World War II, there is little

epidemiological evidence to support an increase in cancer incidence in QNC-treated patients (10).

However, we cannot also find epidemiological study about low cancer incidence in QNC-treated

patients. These facts are very curious, because the cancer prevention effect of Rapamycin, a widely

used anti-bacterial drug, has been revealed by epidemiological follow-up (11). Indeed, RAD001 has

been developed as an anti-cancer drug based on the biological effect of Rapamycin (12). These facts

suggest two possibilities: 1) QNC is not related with human cancer incidence and prevention; and 2)

QNC could be associated with cancer under special condition. However, first possibility could be

potentially excluded because QNC-mediated anti-tumor effects have been reported (13).

Cancer cells are often depicted as trying to escape from a controlling environment and transform to




4

a single cell-like state (14). Thus, we hypothesized that the anti-protozoa drugs such as anti-malaria

drugs might show meaningful anti-cancer effects. To test this hypothesis, the effect of well-known

anti-protozoa chemicals on human cancer cell lines was evaluated. Discovery of a lead candidate

could potentially be applied to human patients, with minimal delay, as these chemicals have already

been approved by the FDA.

In summary, this study revealed that QNC has anti-tumor effects on p53-deficient malignant

cancers in vitro and in vivo. Mechanistically, QNC promoted p-Chk1/2 degradation and eliminated

minimal cell cycle check point. Thus, p53 deficient cells were easily affected by QNC-induced

cytotoxicity. These results strongly suggest that QNC would be a plausible candidate drug against

relapsed human malignancies.




5

Materials and Methods

Mice

All experimental procedures using laboratory animals were approved by the animal care committee

of Pusan National University. Athymic nude mice were maintained under temperature- and light-

controlled conditions (20–23oC, 12 h/12 h light/dark cycle) and provided autoclaved food and water

ad libitum.

In Vivo Treatment Studies

For xenografts, HCT 116 p53-/- (1 × 10^7) cells were seeded in the right and left flanks of nude

mice. After a week, randomly picked tumor-bearing mice were treated with PBS (n=3) as a control,

QNC (10 mg/kg, n=4) and (20 mg/kg, n=4) by intraperitoneal (i.p.) injection three times a week.

Tumor size and body weight were measured twice a week. To generate Villin-Cre;p53+/LSL-R172H mice,

Villin-Cre mice were crossed with Villin-Cre;p53+/LSL-R172H mice. Villin-Cre;p53+/LSL-R172H mice (4-

month-old, n=5) were treated with carrier (n=2) as a control and QNC (20 mg/kg, n=3) by

intraperitoneal (i.p.) injection three times a week. Body weights were measured twice a week.

Cell Culture and Reagents

A549, H1299, HEK 293, MCF7, MDA-MB231 and MDA-MB468 were purchased and

authenticated from the American Type Culture Collection (ATCC, Manassus, VA, USA) and

maintained in RPMI-1640 or Dulbecco's Modified Eagle's medium (DMEM) supplemented with 10%

FBS and antibiotics. ACHN were obtained and authenticated from Korea cell line bank (KCLB, Seoul,

Korea) and maintained in DMEM medium containing 10% FBS and antibiotics. HCT 116 p53+/+,

p53+/- and p53-/- cells were kindly provided by Dr. B. Vogelstein (Johns Hopkins University,

Baltimore) and maintained in McCoy's 5A (modified) medium containing 10% FBS and antibiotics.

MEF cells were isolated from 14.5 day embryos using a standard protocol and cultured in

MEM/EBSS medium containing 15% FBS and 1% antibiotics. Mycoplasm tests and DNA finger




6

printing were performed every 6 months (Mycoplasm tests) and 2 years (last test was performed

2016). Cells were passaged no longer than 2 months. QNC, SB202190, 5-Fluorouracil (5-Fu), 4,7-

Dichloroquinoline and 4-Hydroxytamoxifen (4-OHT) were purchased from Sigma (Missouri, USA).

Adriamycin, Ptfithrin-a, LY294002, U0126 and MG132 were obtained from Calbiochem (Darmstadt,

Hessen, Germany) and Rapamycin, Gefitinib and Erlotinib were provided by LC Laboratory (Woburn,

MA, USA). PD98059 was purchased from StressGen (BC, Canada) and ABT-737, SB216763 and

Imatinib were obtained from Selleckchem (TX, USA).

Immunoblot Analysis

Protein was extracted using RIPA buffer (50 mM Tris-Cl, 150 mM NaCl, 1% NP-40, 0.1% SDS and

10% sodium deoxycholate) and heated with sample buffer for inactivation (heated at 95C for 7

minutes). Protein samples were applied to Sodium dodecyl sulfate-polyacrylamide gel electrophoresis

(SDS-PAGE) and immunoblot analysis was carried out following a general protocol. In brief, the

samples were transferred onto a PVDF membrane and blocked with 3% skimmed milk before

incubation with primary antibodies. The membranes were washed three times and incubated with

HRP-conjugate secondary antibodies for 1 hr at room temperature. Protein bands were detected by

chemiluminescence with ECL kit (Advansta). Antibodies against, Actin (sc-1616), GFP (sc-9996),

Chk2 (sc-9064), His (sc-8036), GST (sc-138), NF2 (sc-331), β-catenin (sc-7963), HA (sc-7392), β-

TrCP (sc-390629), Chk1 (sc-8408), α-tubulin (sc-8035) and p53DO1 (sc-126) were purchased from

Santa Cruz (California, USA). p-Chk1 (2341), p-Chk2 (2661), p-Erk (9101) and t-p53(9282) were

obtained from Cell signaling (Massachusetts, USA). Anti-Mre11 (gtx70212) was provided by

GeneTex (California, USA) and anti-FLAG (F3165), anti-Myc (M5546) was obtained from Sigma

Aldrich (Mo, USA).

Immunofluorescence

Cells were cultured on cover slips and transfected with the indicated vectors and treated with the




7

indicated chemicals. Cells were fixed with MeOH for 30 min and the nucleus was visualized using

DAPI. After washing with PBS, cover slips were mounted with mounting solution (Vector

Laboratories (Burlingame, CA, USA), H-5501). Images were captured through fluorescence

microscopy (Zeiss, Jena, Germany).

Transfection and siRNAs

peGFP-Chk1, pcDNA3 NF2-FLAG (Isoform 1 of merlin), pcDNA3 NF2-NTERM-FLAG 1-332

AA (Xu et al., 1998), β-TrCP-Myc, β-TrCP WD2-7-Myc (β-TrCP -DN), CDC4-Myc, CDC4 R465C-

Myc (CDC4-DN) and Skp2-Myc were purchased from Addgene (Cambridge, MA, USA). HA-p53

vectors kindly provided by Dr. S. Kim (Seoul National University, Seoul, KOREA). VHL-FLAG and

Siah1-FLAG vectors were obtained from Dr. Y.J. Jung (Pusan National University, Busan, Republic

of Korea) and from Dr. Lashuel (Qatar Foundation), respectively. GFP-fused Chk2 expression vector

was obtained by Dr. N.C. Ha (Seoul National University, Seoul, KOREA). Transfections, were

performed using the jetPEI transfection agent (Polyplus Transfection, New York, NY) following the

manufacturer’s protocol. In brief, vectors (1.5 μg) were mixed with 1.5 μl of jetPEI reagent in 150

mM NaCl solution. After incubation for 20 minutes at room temperature, the mixture was added to the

cell. After 3 hours, the serum-free medium was replaced with 10% FBS containing medium. For in

vitro gene knockdown, siRNAs against indicated targets were generated by a custom service (Cosmo

Genetech). The siRNA target sequence for β-TrCP is described below: 5’-

GCGACATAGTTTACAGAGA TTCAAGAGA TCTCTGTAAACTATGTCGC-3’. For siRNA

transfections, the INTERFERin® transfection reagent (Polyplus New York, USA) was used. In brief,

1.5 pmoles (21 ng) of siRNA duplexes were mixed with 4 µl of INTERFERin® in 200 µl medium

without serum. The mixture was homogenized by vortexing and incubated for 10 min at room

temperature. After the incubation, the mixture was added to the cell and incubated at least 48 hr.

Immunoprecipitation and Pulldown




8

To determine the interaction between proteins, pulldown and immunoprecipitation (IP) assays were

performed. For pulldown assay, GST-Chk1 or Ni-β-TrCP were incubated with cell lysate in RIPA

buffer for 30 min at 4°C. For IP, cells were lysed in the RIPA buffer. Whole cell lysates were

incubated with indicated primary antibodies for 1 hr at 4°C and then with protein A/G-conjugated

agarose beads (Invitrogen, Carlsbad, CA, USA) for 1 h. After centrifugation, beads with bound

proteins were washed with RIPA buffer and precipitated proteins were detected by immunoblot.

Cell Viability

Briefly, cells were treated with varying concentrations of drug for the indicated duration. The

exposed cells were then incubated with 0.5 mg/ml of MTT (3-(4, 5-Dimethylthiazol-2-yl)-2,5-

diphenyltetrazolium bromide) solution (Calbiochem) for 4 hours at 37 °C. After removing the

supernatant, the precipitate was dissolved in Dimethyl sulfoxide (DMSO) and quantified by

measuring the absorbance on a spectrophotometer at 540nm.

Luciferase Assay

To examine NF-κB promoter activity, NF-κB-luc vectors were transfected into cells for 24 hours,

and cells were treated with the indicated chemicals. After washing with wash buffer (Promega,

Wisconsin, USA), the cells were lysed by lysis buffer (Promega, Wisconsin, USA). The luciferase

activity was determined by a luminometer (MicroDigital, Gyeonggi-do, South Korea).

Apoptosis Assay

Cells were seeded on 6 well plates and incubated 12 hours with DMSO (as control) or 5 μM QNC.

After treatment, cells were stained using the Annexin V-FITC apoptosis detection kit (B32117,

Biotool, USA). A minimum of 10,000 cells were analyzed by FACS (Beckman coulter).

Histology and Immunostaining.

Tissue specimens were fixed in 4% paraformaldehyde and embedded in paraffin. The paraffin




9

blocks were sectioned at 5 μm by Leica microtome and transferred onto adhesive-coated slides

(Marienfeld laboratory glassware, Germany). After deparaffinization and rehydration, tissue sections

were stained with haematoxylin and eosin. Immunohistochemistry was performed on tissue sections

using polyclonal rabbit anti-Ki67 or mouse monoclonal p53 (Pab1801) antibodies. In brief, sections

were gradually deparaffinized and rehydrated using xylene and alcohols and rinsed in PBS. Antigen

retrieval was performed with 10 mmol/L sodium citrate (pH 6.0) 2 times at 95°C for 10 minutes each.

Endogenous peroxidase activity was blocked with 3 % hydrogen peroxide for 10 minutes. Primary

antibodies (1:200) were incubated overnight at 4°C and HRP-conjugate secondary antibodies were

incubated for 4 hr. The visualization was performed using DAB Peroxidase (HRP) Substrate Kit

(Vector Labs).

Image Acquisition of [18

F] FDG PET/CT.

To acquire [18F] -FDG PET/CT images, each mouse was fasted at least 6 h. [18F] FDG (500±23 uCi)

was intravenously administered through the tail vein. After administration of [18F] FDG, mice were

placed in a cage with dim light for 60 min. Each mouse was maintained under anesthesia with

isoflurane (2.5% flow rate) for the duration of the scan. Animals were positioned prone in the

standard mouse bed. Limbs were positioned lateral to the body to acquire uniform CT images. Whole

brain CT images were acquired with a Micro-PET/CT scanner (nanoPET/CT, Bioscan Inc.,

Washington DC, USA). For CT image acquisition, the X-ray source was set to 200 µA and 45 kVp

with 0.5 mm. The CT images were reconstructed using cone-beam reconstruction with a Shepp filter

with the cutoff at the Nyquist frequency and a binning factor of 4, resulting in an image matrix of 480

× 480 × 632 and a voxel size of 125 µm.




10




11

RESULTS

Chemical screening

To test our hypothesis that anti-protozoa drugs (Supplementary Fig. S1A) have anti-cancer effects,

MTT assays were performed using human breast cancer cell lines, MDA-MB468 and MCF7 (Fig. 1A

and B). This initial screen revealed that QNC, compared to previously well-known anti-cancer drugs

or chemical inhibitors, suppressed the cell viability of MDA-MB468 cells (Fig. 1B), but not MCF7

(Fig. 1A). Since MDA-MB468 cells are considered a more aggressive type of breast cancer than

MCF7 cells (15), it was speculated that QNC might be effective against more aggressive types of

cancer. Indeed, QNC at two different concentrations suppressed the viability of MDA-MB468 cells

with dramatic effect (Fig. 1C), compared to MCF7 cells (Fig. 1D). Similar results were obtained by

counting viable cells using tryphan blue dye exclusion (Supplementary Fig. S1B). To address how

QNC suppresses cell viability, cells were stained with FITC-Annexin V, a well confirmed early

apoptosis marker (16), and staining intensity was measured by FACS. QNC increased the annexin V-

positive staining in MDA-MB468 cells (more than 50%; Fig. 1E), compared to MCF7 cells (Fig. 1E).

To collect more evidence about the anti-cancer effects of QNC on human malignancies, additional cell

lines were tested. Reduced cell viability in response to QNC was observed in small-cell lung cancer

cell lines (Supplementary Fig. S1C). To further extend this, more cell lines (detailed information in

Supplementary Fig. S1D) were tested: two mesothelioma cell lines (H28 and H2452), a renal cancer

cell line (ACHN), and another aggressive human breast cancer cell line (MDA-MB231) all of which

consistently displayed reduced viability with QNC treatment (Fig. 1F). However, other anti-cancer

drugs such as Her2/neu inhibitors (Iressa and Tarceva) and an mTOR inhibitor (Rapamycin) did not

suppress the viability of these cell lines (Fig. 1F). In addition, QNC did not show synergistic or

additional effects with other conventional anti-cancer drugs such as EGFR kinase inhibitors

(Supplementary Fig. S2A and B). Although autophagy has been suggested as the molecular

mechanism of QNC-induced cell death, drugs that impact autophagy such as RAD001, rapamycin,

and 3-MA (inhibitor of autophagy) (17-19) did not alter the QNC-induced cell death (Supplementary




12

Fig. S2C).

The relevance of anti-cancer effect of QNC and p53 status

To determine if p53 contributes to the QNC-induced anti-cancer effects, we compared the response

to QNC in HCT116 and its isogenic HCT116 p53-/- cell line. In contrast to previous reports that QNC

induces p53 (6, 7), p53-deficient HCT116 cells were more sensitive to QNC than parental HCT116

cells with intact p53 (Fig. 2A). Indeed, suppression of p53 expression via siRNA (Supplementary Fig.

S2D) promoted QNC-induced cell death in p53-intact A549 and MCF7 cells (Fig. 2B). PFT-a p53

transcription inhibitor, also sensitized A549 and MCF7 cells to QNC-induced cell death

(Supplementary Fig. S2E). QNC-induced cell death was also observed in normal human fibroblast by

treatment with PFT-α (Supplementary Fig. S3A). These results indicated that QNC can selectively

induce cell death under p53-inactivated condition. To confirm the effect of p53 on QNC-induced cell

death, cell viability was measured in MEF cells, obtained from conditional Ubc-Cre-ER; p53+/LSL-R172H

knock-in mice (20). LSL-p53R172H mouse cells express wild-type p53 before activation of Cre

recombinase because of a stop codon between the LoxP site and p53 mutation which when expressed

by activation of Ubc-Cre-ER acts as dominant negative (21). Thus, 4-OHT treatment induced mutant

p53 expression (Fig. 2C) and consistently reduced expression of p21 a well-known p53 target (Fig.

2C). Under p53 mutant expressed condition, QNC induced cell death (Fig. 2D). To test the in vivo

anti-tumor effects of QNC, HCT116 p53-/- cells were inoculated in athymic nude mice and injected

with QNC (10 or 20 mg/kg) three times per weeks via i.p for 8 weeks. As shown in Fig. 2E, HCT116

p53-/- tumor growth was suppressed by QNC injection without body weight loss (Supplementary Fig.

S3B). These results strongly suggest that QNC suppresses tumor growth under p53 impaired

conditions. Although suppression of NF-κB has been suggested as an important mechanism for the

anti-tumor effects of QNC (6, 7), reduced NF-κB transcription was not observed by luciferase assay at

the concentrations (i.e., 1 M and 5 M) used in the study. However, reduced NF-κB-luc activity at

high concentrations (> 10 M) was observed (Supplementary Fig. S3C). Thus, considering that QNC




13

could induce cell death at concentrations of 1-5 M (i.e, less than needed to suppress NF-κB activity),

it appears that NF-κB activity is not critical for the anti-tumoral effects of QNC. Interestingly, the

renal cell line ACHN responded to QNC, despite expressing wild-type p53. It has been reported that

in ACHN cells p53 is inactivated by NF2 deletion (22). Consistent with our previous report (23),

transfection of NF2 could induce p53 expression (Supplementary Fig. S3D). In addition, QNC-

induced cell death was also suppressed by NF2 transfection (Supplementary Fig. S3E). This result

suggests that QNC might be effective in NF2-deficient cancers such as RCC or mesothelioma. To

better understand the dynamics of QNC-induced cell death, cell viability was monitored in a time-

dependent manner. Interestingly, QNC induced cell death from 12 hr to 24 hr in MBA-MB468 cells

(Fig. 2F). Considering that cell cycle progression is generally about 12-24 hr, cell death by QNC

appears to coincide with cell cycle progression, in particular, S-to-M phase. In other cell lines (e.g.,

ACHN and MDA-MB231), cell death was detected from 32hr to 36 hr and would potentially couple

with cell cycle (2 cycle or more; Fig. 2F). However, MCF7 did not show a stair-like dropped-down

phase, but rather a gradual decrease of cell viability was detected. To confirm a potential cell cycle-

dependent cell death, cell viability was measured after cell cycle inhibition at M-phase using Taxol.

Blocking cell cycle progression in M-phase diminished the QNC-induced cell death (Supplementary

Fig. S3F). These results suggested that QNC-induced cell death is potentially regulated in a cell cycle

phase-dependent manner. Indeed, previous literature showed that QNC can promote S-G2/M cell

cycle progression in p53 intact lung cancer cell lines with alteration of cell cycle and DNA repair-

related gene expression via microarray analysis (13).

Reduction of Check point kinase 1/2 activity by QNC

The potential for QNC-induced cell death to be dependent on p53 and the cell cycle, prompted

further investigation using the TCGA database (through www.cbioportal.org) (24). In several kinds of

human cancers, up-regulation of p-Chk1/2 or total-Chk2 in p53 mutated cancers was found



http://www.cbioportal.org/


14

(Supplementary Fig. S4A). This suggested that Chk1/2 activities may be required for proper cancer

cell growth under p53 inactivated condition. Indeed, Chk1/2 are key regulators of S-G2-M phase and

are also well conserved in single cell organisms to mammalian systems (25-27). In addition, it has

been proposed that inhibition of cell cycle check point kinases can enhance the sensitivity to anti-

cancer drugs under p53 deficient condition (28-30). To explore the possibility, cell viability was

determined in p53-deficient and -proficient cells after elimination of Chk1/2. Although single

knockdown of Chk1 or Chk2 did not induce cell death, co-treatment of siRNAs against Chk1 and

Chk2 (Supplementary Fig. S4B) did induce cell death in p53-null HCT116 (Supplementary Fig. S4C).

Thus, the effect of QNC on Chk1/2 activity was determined. As shown in Fig. 3A, both p-Chk1 and 2

were suppressed by QNC in UV-treated conditions. Similar results were obtained in Adriamycin-

treated HCT116 cells (Supplementary Fig. S5A). Next, a dose-dependent reduction of activated p-

Chk1/2 was detected upon QNC-treatment (Fig. 3B). Of note, a rapid and selective reduction of p-

Chk1/2 without an obvious reduction of total Chk1/2 was seen (Fig. 3C). Reduction of p-Chk1/2 in

the human lung epithelial cell line, WI-26, transformed by Large T was also observed (Supplementary

Fig. S5B). These results indicated that QNC could reduce p-Chk1/2. Next, the engagement of p53 on

QNC-induced Chk1/2 reduction was assessed. Compared to HCT116 cells, p-Chk1 and 2 were rapidly

reduced in HCT116 p53-/- cells (Fig. 3D). To confirm this, p-Chk2 was measured the in MCF7 cells.

Although 50 M of QNC could suppress p-Chk2, reduction of p-Chk2 was detected from 10 M of

QNC only in PFT-α-treated cells (Supplementary Fig. S5C). Similarly, the rapid reduction of p-Chk2

in PFT-α-treated MCF7 cells could observed (Supplementary Fig. S5C). Finally, transfection of wild-

type p53, but not mutant p53, could block the reduction of Chk2 (Fig. 3E) and QNC-induced cell

death (Supplementary Fig. S5D). These results implied that QNC can preferably eliminate activated

Chk1/2.

QNC promoted Chk1/2 destabilization




15

To address how QNC suppresses p-Chk1/2, the expression of Chk2 was measured. QNC could

reduce Chk2 expression in p53 inactivated HEK293 cells (Fig. 3F). However, MRE11, an upstream

regulator of Chk2 (31, 32), or p18, an activator of ATM kinase (33), were not reduced by QNC (Fig.

3F). Reduction of Chk2 could also be observed via IF (Fig. 3G). However, another anti-protozoan

drug, primaquine (PQ) did not suppress Chk2 expression (Supplementary Fig. S5E), indicating that

reduction of Chk2 may be specific to QNC. To understand how Chk2 was reduced by QNC, protein

stability was evaluated. QNC-induced Chk2 turnover was inhibited by the proteasome inhibitor,

MG132 (Fig. 3H). However, GFP (control protein), RAD50, and MRE11 expression were not reduced

by QNC (Fig. 3H) suggesting that QNC might selectively reduce the protein half-life of Chk2. Indeed,

expression of MRE11 or RAD50 were not altered by QNC in the presence of PFT-α (Supplementary

Fig. S5F) and an MRE11 inhibitor did not abolish QNC-reduced cell viability (Supplementary Fig.

S5G). Chk1 expression was also reduced in a QNC dose-dependent (Supplementary Fig. S6A) and

time-dependent (Supplementary Fig. S6B) manner. To confirm the reduction of Chk1/2 via

proteasome-mediated degradation, ubiquitination assays were performed. Treatment of QNC could

promote ubiquitination of Chk1/2 (Fig. 3I), without an overall increase in total ubiquitination

(Supplementary Fig. S6C). These results suggest that QNC selectively promotes Chk1/2 degradation

via the proteasome. Under p53 deficient conditions, Chk1/2 would be rapidly phosphorylated because

of rapid cell cycle progression. Thus, QNC might induce reduction of total Chk1/2, although QNC

only suppressed p-Chk1/2.

QNC promote Chk1/2 degradation using -TrCP

To further investigate the mechanism, Chk2 expression was measured in several E3 ligase

transfected cells. Among the tested E3 ligases, -TrCP, CDC4, and Siah1 reduced Chk2 expression

(Fig. 4A). Indeed, transfection of -TrCP and CDC4 promoted Chk2 degradation in response to QNC

(Supplementary Fig. S7A). In contrast, Siah1 eliminated Chk2 expression, regardless of QNC




16

treatment (Supplementary Fig. S7A). Next, the interaction between E3 ligases and Chk1 was

evaluated via GST-pulldown. QNC as well as PQ enhanced the binding between Chk1 and -TrCP or

CDC4; while, Siah1 did not associate with Chk1 (Fig. 4B). This result indicated that Siah1 is not

responsible for the observed QNC-induced Chk1 reduction. To determine which E3 ligase is essential

for QNC-induced Chk1/2 reduction, we first blocked the CDC4 activity using a dominant negative

CDC4 (CDC4-DN). However, CDC4-DN did not completely block the QNC-induced Chk2 reduction

(Supplementary Fig. S7B), although it could increase basal GFP-Chk2 expression (Supplementary Fig.

S7A and B). Next, we knockdowned -TrCP and checked the Chk1/2 expression. Elimination of -

TrCP blocked the QNC-induced reduction of exogenous Chk2 (Fig. 4C) as well as endogenous

Chk1/2 (Supplementary Fig. S7C). DN--TrCP also blocked the QNC-induced Chk2 reduction

(Supplementary Fig. S7D), implying that -TrCP could be responsible for QNC-induced Chk1/2

reduction. To confirm this, the pulldown assay and immunoprecipitation assay were performed again

using recombinant -TrCP and -TrCP antibody. QNC increased the binding between Chk1/2 and -

TrCP (Fig. 4D and Supplementary Fig. S7E) in a dose-dependent manner (Supplementary Fig. S7F).

However, inhibition of GSK3, upstream kinase for -TrCP substrate such as -catenin, did not block

the QNC-induced Chk1/2 reduction (Supplementary Fig. S7G and H). These results imply that Chk1

and 2 are not canonical targets of -TrCP. In our previous results, it was demonstrated that PQ also

promoted the binding of Chk1 and -TrCP (Fig. 4B) without cell death (Fig. 1A-D). Thus, PQ could

be competitive inhibitor against QNC, if it binds to same site. Importantly, QNC and PQ have a very

similar chemical structure (Supplementary Fig. S1A). Consistent with this hypothesis, pre-treatment

of PQ blocked the QNC-induced p-Chk2 reduction (Fig. 4E). However, high concentrations of QNC

could overcome this PQ effect (Fig. 4F). These results suggest that QNC is very selectively anchored

to Chk1/2 and promote proteasome-dependent degradation.

Anti-tumor effect of QNC in intestinal tumor model




17

To explore the anti-tumor effects of QNC in vivo, an intestinal cancer mouse model (VP mice) was

created Villin-Cre;p53+/LSL-R172H

(Supplementary Fig. S8A). After 4 months, QNC was injected (20

mg/kg, i.p.) three times per week for 22 weeks into these VP mice (Supplementary Fig. S8A). Gross

morphology and body weight did not show detectable differences between control and QNC-injected

mice (Supplementary Fig. S8B). From the PET-CT analysis, tumors were detected in control mice

(VP13 and VP17; Fig. 5A) but disappeared in QNC-treated mice (Fig. 5A; right panel). Indeed, tumor

number, detected by PET-CT were reduced by QNC-treatment (Fig. 5B). Intestinal histology was

analyzed and tumors were observable from 3-5 sites in the control mice (Fig. 5C and Supplementary

Fig. S9). Indeed, invasive or diffused cancer cells were detected in control VP mouse tissues (Fig. 5C

and Supplementary Fig. S9A-D). In contrast, QNC-treated mice only showed over-growing intestinal

epithelial cells (Fig. 5C and Supplementary Fig. S10A-D). QNC-treatment also reduced the cell

proliferation of intestinal villi (Fig. 5D). These result strongly support the notion that QNC would be a

plausible treatment strategy for p53 negative cancers.




18

Discussion

In this study, it was found that QNC, a drug previously used to fight malaria and protozoa (10),

possesses a very interesting anti-cancer property though inhibition of Chk1/2 under p53-deficient or

inactivated conditions. As mentioned above, a new strategy for drug development is the re-purposing

of old chemicals through mining of new biological effects in other diseases (34). Our purpose is to

find new anti-cancer drugs from old drugs, which is a very economic and smart approach for drugs

already approved by FDA with reported toxicities. Importantly, pre-clinical investigation is not

generally required for application of clinical trials. With regard to QNC it has been widely used to

combat malaria since World War II and is now used for anti-Giardiasis (10). In addition, QNC has

been re-positioned for prion-related neurodiseases (5), anti-inflammation (e.g., SLE) (4) and cancer

(6).

Concerning the anti-cancer effects of QNC, previous literature suggested that activation of p53 and

inhibition of NF-κB are main pathways (6, 7). Considering the chemical structure and its intercalating

property into DNA, p53 activation by QNC is very plausible hypothesis. However, at effective

concentration (5-10 M), p53 was not induced (Fig. 3D). Rather, in our hands, QNC promoted cell

death under p53-deficient conditions (Fig. 2). In addition, QNC did not suppress NF-κB activity.

Although, while we and others have already observed the anti-cancer activity of QNC the underlying

mechanism is not clearly demonstrated.

The current results revealed that QNC induced cell death under p53-deficient conditions via rapid

degradation of Chk1/2 (Fig. 6). Chk1/2 is a well conserved cell cycle regulator from single-cell

organisms, such as yeast, to humans and is speculated to perform the basic cell cycle regulation. In

contrast, the physiological role of p53 in multicellular systems appears to be important for




19

maintaining cellular homeostasis and determination of which cell will die or survive (35). Thus, under

p53-intact conditions, Chk1/2 activity seems to be not essential. However, in cancer cells that try to

escape the systematic control and move to potentially behave more like a single-cell system, Chk1/2-

mediated minimum cell cycle regulation would be critical for cell survival. Our results suggest that

elimination of Chk1/2 will lead the cancer cells to a more chaotic cell cycle progression and end in

cell death. Which is why we believe that QNC can work on protozoa and cancer. Indeed, we did not

find triple mutant cancers (i.e., p53-;Chk1-;Chk2-) in the human cancer cell lines tested.

Since p53-mediated cell regulatory systems may be collapsed at the end-stage of cancer or in

relapsed cancers, which ATM/ATR-mediated DNA repair systems and Chk1/2 activity are elevated,

QNC-induced Chk1/2 degradation would be a useful approach for therapeutic effect.

This study, reveals that QNC induces cell death under p53-inactivated conditions via rapid

degradation of Chk1/2. Since QNC has been used clinically, after determination of proper treatment

protocol, it could be used as a cancer drug, in particular, p53-deficient end-stage or relapsed human

cancers including GBM, SCLC and NF2-deficient cancers.




20

Acknowledgments

This research was supported by Basic Science Research Program through the National Research

Foundation of Korea (NRF) funded by the Ministry of Science and ICT (NRF-2017R1A2B2007355;

B.-j. Park) and by Ministry of Education (NRF-2017R1A6A3A11035837; A.-Y. Oh).




21

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25

Figure Legend

Figure 1.

Anti-cancer effect of chloroquine relative chemicals. A and B, Chemical screening of various anti-

cancer drugs and anti-protozoa drugs by MTT assay. Among various chemicals, QNC suppressed

viability of MDA-MB468 cells. In contrast, it did not show in less aggressive type of cancer, MCF7

cells. Cells were incubated with indicated chemicals for 24 hr and viability was determined by MTT

assay. **P<0.005. C and D, QNC suppressed cell viability of MDA-MB468 but not MCF7. Treating

with another anti-protozoa drug, primaquine could not inhibit both cells' viability. After incubation

with chemicals for 24 hr the cell viability was monitored by MTT assay. **P<0.005, N.S indicated

not significance. E, Apoptotic signals from QNC treated MDA-MB468 and MCF7 cells was detected

using Annexin V-FITC staining. Comparing to MCF7 cells, MDA-MB468 cells showed increased

Annexin V-FITC stained cells by QNC treatment. Cells were treated with QNC of 5 μM for 12 hr and

assessed apoptosis by Annexin V-FITC staining (early apoptosis) and PI (late apoptosis). Numbers in

each panel indicate percentage in total cells. F, All aggressive cancer cells including mesothelioma

cell lines (H28 and H2452), RCC cell line (ACHN) and another aggressive human breast cancer cell

line (MDA-MB231) were very sensitive to QNC. Cells were incubated with chemicals for 36 hr. Then

cell viability was measured by MTT assay. *0.005<P<0.05, **P<0.005.

Figure. 2.

Anti-cancer effect of Quinacrine under p53-deficient condition. A, Cell viability was gradually

suppressed depending on QNC concentration especially in p53-deficient HCT116 cells compared to

p53-intact HCT116 cells. HCT116 cells were treated with QNC for 24 hr and cell viability was

determined with MTT assay. **P<0.005. B, Inhibition of p53 using siRNA can promote QNC-

induced cell death in p53-WT A549 and MCF7 cells. A549 and MCF7 cells were transfected with si-




26

scamble (si-con) or si-p53 for 24 hr. Subsequently, cells were treated with QNC for 24 hr and cell

viability was measured by MTT assay. *0.005<P<0.05 and **P<0.005. N.S indicates not significance.

C and D, p53 mutant MEF cells’ viability were more inhibited by QNC compared to p53 intact

condition. MEF cells obtained form UBC-ER-Cre; p53+/LSL-R172H knock in mouse were pretreated with

or without 4-OHT. Subsequently, cells were treated with QNC of indicated dose for 6 hr or 12 hr.

Then immunoblot and MTT assay were performed. *0.005<P<0.05. E, QNC reduced tumor growth of

HCT116 p53 -/- cells engrafted to nude mouse. HCT116 p53 -/- cells were inoculated subcutaneous

into athymic nude mice and two concentrations of QNC or PBS was injected three times a week via

i.p for 8 weeks. Tumor volume was monitored twice a week. F, ACHN, MDA-MB231 and MDA-

MB468 cells showed a stair-like dropped-down phase in cell death by QNC treatment. These

dropped-down points induced by QNC associated with cell cycle progression (about 12-24 hr). In

contrast, it did not show in MCF7 cells. After treatment with 5μM QNC for indicated time, cell

viability was measured by MTT assay. *0.005<P<0.05, **P<0.005.

Figure 3.

Anti-cancer effect of Quinacrine via Chk1/2. A, Among various anti-protozoa drugs, QNC notably

suppressed endogenous p-Chk1/2 protein levels although Chk1/2 was activated by UV treatment.

HCT116 p53+/- and isogenic HCT116 p53 -/- cells were pretreated with or without UV. Subsequently,

cells were treated with indicated chemicals of 10 μM for 4 hr and immunoblot was performed. Actin

was used for loading control. B, QNC induced dose-dependent reduction effect and can selectively

eliminate activated, p-Chk1/2 by Adriamycin treatment. Chk2-GFP was transfected to HCT116 cells.

Subsequently cells were pretreated with or without Adriamycin and were treated with QNC of

indicated dosage for 6 hr. Then immunoblot analysis was performed. C, QNC induced time-dependent

reduction effect and can selectively eliminate activated, p-Chk1/2 by Adriamycin treatment. HCT116

p53+/- cell was pre-treated with Adriamycin and measured the expression of p-Chk1/2 and total Chk1




27

following 5 μM QNC-incubated time. D, In response to QNC, p-Chk2 expression was more rapidly

reduced in HCT116 p53-/- than HCT116. Cells were treated with QNC of indicated dosage for 4 hr.

Then immunoblot analysis was performed. E, Unlike p53 WT, abnormal status of p53 could not block

the QNC-induced cell death. Wild-type p53 and p53 R175H and Chk2-GFP were transfected to

HCT116 P53 -/- cells and subsequently QNC were treated of indicated dosage for 4 hr. Then

immunoblot analysis was performed. F, QNC can reduce exogenous Chk2 expression but not Mre11

and p18, upstream regulator of Chk2 signaling. Indicated vectors were transfected to HEK293 cells

and QNC (10 μM) was treated for 4 hr. Only Chk2 expression was decreased by QNC. G, Chk2

reduction by treatment of QNC was detected by immunofluorescence. After transfected with Chk2-

GFP (Green), Cells were stained with DAPI (Blue) to visualize the nucleus. H, Chk2 reduction by

treatment of QNC was inhibited by pre-treatment with the proteasome inhibitor, MG132. After

transfected with Chk2-GFP, cells were pre-treated with or without MG132 and were treated with QNC

of 5 μM for indicated time. Subsequently immunoblot was performed. I, Treatment with QNC

promoted ubiquitination of Chk1/2. After transfected with ubiquitin and Chk2-GFP or Chk1-GFP,

cells were pre-treated with or without MG132 and were treated with QNC of 5 μM for 2 hr.

Subsequently immunoprecipitation with a GFP antibody was performed.

Figure 4.

Quinacrine enhance β-TrCP activity. A, Chk2 expression test in several E3 ligase transfected cells.

Among them, only β-TrCP, CDC4, and Siah1 reduced the Chk2 expression. Vectors were transfected

to HEK293 cells for 24 hr. Then immunoblot analysis was performed. B, Chk1 can bind to β-TrCP

and CDC4, but not Siah1. And both QNC and Primaquine (PQ) could enhance binding affinity of

Chk1-bead with β-TrCP and CDC4. Cell lysates transfected with indicated vectors were incubated

with GST-Chk1 bead and indicated chemicals. Bound proteins were eluted and immunoblot analysis

was performed. C, Elimination of β-TrCP by RNA interference blocked the QNC-induced reduction




28

of Chk2. Vectors and siRNA were transfected to HEK293 cells and QNC (5 μM) was treated for

indicated time. Then immunoblot analysis was performed. D, Chk1/2 binding to Ni-β-TrCP bead was

enhanced by QNC. Cell lysates transfected with indicated vectors were incubated with Ni-β-TrCP

bead and QNC. Bound proteins were eluted and immunoblot analysis was performed. E and F, Pre-

treatment of primaquine (PQ) blocked the QNC-induced p-Chk2 reduction. PQ was used as binding

competitor of QNC. E, Cells were pre-treated with Adriamycin and then were treated with PQ.

Subsequently QNC were treated of 10 μM for indicated time. Then immunoblot analysis was

performed. F, Chk2-GFP was transfected to HCT116 cells. Subsequently cells were pre-treated with

or without PQ and were treated with QNC of indicated dosage for 6 hr. Then immunoblot analysis

was performed.

Figure 5.

Anti-cancer effect of Quinacrine in p53 mutant cancer model. A, Through the PET/CT analysis, QNC

suppressed the tumor formation in the Villin-Cre;p53+/LSL-R172H mouse model. Compared to control

mice (VP13 and VP17), QNC injected mice (VP15, VP20 and VP21) did not show tumor formation.

The arrow indicates the localization of the tumor. B, the number of tumors, based on PET-CT image.

C, H&E staining of Villin-Cre;p53+/LSL-R172H mouse intestines from control and QNC-injected mice.

Compared to control mice (VP13 and VP17), QNC injected mice (VP15, VP20 and VP21) did not

show tumor regions. Cancer regions of control mice and hyperplastic regions of treated mice were

presented as enlarged images. D, Immunohistochemistry (IHC) staining for Ki-67 and hematoxylin

counterstain in Villin-Cre;p53+/LSL-R172H mouse intestines of control and QNC-injected mice. The

intestines of control group mice had more Ki-67 staining than QNC-injected mice.

Figure 6.




29

The schematic diagram illustrates proposed mode of action of QNC. QNC could reduce p-Chk1/2

protein level by enhance β-TrCP activity. Thus, especially under p53-deficient conditions, QNC can

induce cancer cell death.






















Published OnlineFirst March 15, 2018.Mol Cancer Res Soyoung Park, Ah-Young Oh, Jung-Hyun Cho, et al. CancersSelective Suppression of p-CHK1/2 in p53-negative Malignant Therapeutic Effect of Quinacrine, an Anti-protozoan Drug, by

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