polo-like kinase (plk) inhibitor, ro5203280, has potent ... · #these two authors, akl cheung and...

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Polo-like kinase (PLK) inhibitor, Ro5203280, has potent anti-tumor activity in

nasopharyngeal carcinoma

Arthur Kwok Leung Cheunga#, Joseph Chok Yan Ipa#, Hong Lok Lungad, Jim Zhen Wue,

Sai Wah Tsaob,c,d, and Maria Li Lung*a,c,d

Departments of aClinical Oncology and bAnatomy, cCenter for Nasopharyngeal

Carcinoma Research, and dCenter for Cancer Research, University of Hong Kong,

Hong Kong (SAR), People’s Republic of China; eRoche Pharma Research and Early

Development, China, Shanghai, People’s Republic of China

*Corresponding author: Maria Li Lung, Department of Clinical Oncology,

University of Hong Kong, Pokfulam, Hong Kong (SAR), People’s Republic of China.

Tel: +852 28199783; Fax: +852 28195872; Email: [email protected]

#These two authors, AKL Cheung and JCY Ip, contributed equally to this manuscript.

Running Title: PLK inhibitor with anti-tumor activity in NPC

Keywords: Nasopharyngeal carcinoma, Polo-like kinase, cell cycle arrest, apoptosis,

and anti-cancer drug

Conflict of interest Drs. Maria Lung and George Tsao acknowledge receiving

research funds from Roche Pharma Research and Early Development (pRED), China.

Dr. Jim Wu is an employee of Roche pRED, China.

on January 26, 2020. © 2013 American Association for Cancer Research. mct.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 May 17, 2013; DOI: 10.1158/1535-7163.MCT-12-1219

http://mct.aacrjournals.org/

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Abstract (214 words)

Nasopharyngeal carcinoma (NPC) is a cancer with its highest prevalence among the

southern Chinese and is rare elsewhere in the world. The main treatment modalities

include chemotherapy and radiotherapy. However, tumor chemoresistance often limits

the efficacy of NPC treatment and reduces survival rates. Thus, identifying new

selective chemotherapeutic drugs for NPC treatment is needed. In this current study,

the anti-tumor efficacy of a polo-like kinase inhibitor, Ro5203280, was investigated.

Ro5203280 induces tumor suppression both in vitro and in vivo. An inhibitory effect

was observed with the highly proliferating cancer cell lines tested, but not with the

non-tumorigenic cell line. Real-time cell proliferation and FACS analysis, together

with immunohistochemical (IHC), immunofluorescence (IF), and Annexin V staining

assays were used to evaluate the impact of drug treatment on cell cycle and apoptosis.

Ro5203280 induces G2/M cell cycle arrest and apoptosis. Western blotting shows it

inhibits PLK1 phosphorylation and down-regulates the downstream signaling

molecule, Cdc25c, and up-regulates two important mitosis regulators, Wee1 and

Securin, as well as the DNA damage-related factor Chk2 in vitro and in vivo. In vivo

tumorigenicity assays with Ro5203280 intravenous injection demonstrated its potent

ability to inhibit tumor growth in mice, with no observable signs of toxicity. These




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findings suggest the potential usefulness of Ro5203280 as a chemotherapeutic

targeting drug for NPC treatment.




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Introduction

Nasopharyngeal carcinoma (NPC) is a unique malignancy with an exceptionally

high incidence in Southeast Asia, but it is rare elsewhere in the world. There are

several co-factors that are believed to contribute to NPC development, including host

genetics, Epstein-Barr virus (EBV) infection, and environmental factors. The loss of

function of tumor suppressor genes (TSGs) and loss of control of cell cycle-regulating

genes such as cyclin D1 (1) are important for NPC development. The nasopharynx is

located deep inside the skull, making surgical excision a difficult first option line of

therapy. Therefore, chemotherapy and radiotherapy are commonly used to treat NPC

patients. At present, the choices for chemotherapy in NPC are limited and

chemoresistance often contributes to treatment failures. Therefore, there is a real need

to identify alternative drugs for selectively targeting NPC.

In this current study, the drug Ro5203280 was tested and confirmed to inhibit

NPC tumor cell growth. This drug mainly targets the Polo-like kinase 1 (PLK1)

protein. PLK1 is one of the well-studied members of the PLK family, which is an

important regulator of different signaling pathways that are responsible for cell cycle

progression and regulation of mitosis (2). PLK1 expression is commonly detected in

the highly proliferating adult tissues, including the spleen and testis (3), and it serves

as a marker of cellular proliferation (4). Over-expression of PLK1 is observed in




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many different malignancies including NPC, non-small cell lung cancer (NSCLC),

breast, endometrium, colon, esophagus, ovary, pancreas, stomach, prostate,

glioblastoma, leukemia, melanoma, and also other head and neck cancers (3, 5-8). The

over-expression of PLK1 was reported to associate with the high mitotic rate found in

cancer (9).

The PLK1 protein plays various functional roles in G2/M cell cycle control. It

regulates the entry of cells into mitosis by phosphorylating the Wee1 protein and

facilitating its degradation (10) and, thereby activating the cyclin B/Cdk1 complex

activity. Cdc25c regulates Cdk1 protein activation by removing the inhibitory

phosphorylation of the Cdk1 protein (11). PLK1 also plays a role in the G2/M

checkpoint. It is inhibited as a response to DNA damage-responsive ATM and ATR

proteins (12). Moreover, PLK1 takes part in regulating the mitotic spindle formation

by controlling the recruitment of γ-tubulin to centrosomes (13). PLK1 can also

activate the anaphase-promoting complex (APC) to promote DNA segregation (14).

This suggests PLK1 plays an important role in cancer.

Based on the unique and important function of PLK1 in cancer, PLK1 has been

molecularly targeted for cancer treatment. Interestingly, injection of a PLK1-specific

antibody to transformed cells can induce mitotic arrest (13). Several PLK1 inhibitors,

including BI2536, BI6727, Volasertib, GSK461364, and HMN-214, were used in




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clinical trials for colorectal cancer, NSCLC, and other type of solid tumors (15-17).

Phase II clinical studies of BI2536 have now been completed. In a recent study, an

orally available PLK1-specific inhibitor was reported to induce cell cycle arrest and

apoptosis in cancer cell lines and xenograft tumor models (18). These earlier studies

all suggest an important potential role of chemotherapeutic PLK1 inhibitors for

targeted cancer treatment. Ro5203280 is a new PLK1 inhibitor that is now undergoing

pre-clinical drug studies. Its structure is a close analog to BI2536, but has improved

activity to PLK1. Therefore, more studies are warranted to investigate and confirm the

functions of this new drug in inhibiting tumor growth.

In NPC, the function of PLK1 is still unclear and there are no reports to date on

investigating the usefulness of PLK1 inhibitors in NPC chemotherapy. In this study,

the function of the novel PLK1 inhibitor Ro5203280 was investigated. Results

showed its efficacy in inhibiting NPC tumor cell growth in vitro and tumor formation

in vivo by inducing cell cycle arrest and apoptosis.

Materials and Methods

Cell lines

Three NPC cell lines (HONE1, HK1, and C666-1) and an immortalized

nasopharyngeal epithelial cell line (NP460) were used in this current study. HONE1

and C666-1 were established from poorly differentiated NPC tumors, while HK1 was




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from a well-differentiated NPC. C666-1 is the only Epstein-Barr Virus (EBV)-positive

NPC cell line available for studies. NP460 is a normal nasopharyngeal epithelial cell

line, which was immortalized by human telomerase. Culture conditions for these cell

lines were as described previously (19-22). All cell lines used in the current study

were tested and confirmed to be mycoplasma-negative. They were obtained from the

Hong Kong NPC AoE Cell Line Repository and have been authenticated using the

AmpFℓSTR Identifier PCR Amplification kit (Life Technologies).

Drug preparation and Ambit selectivity screen

The Ro5203280 drug was provided by Roche Pharma Research and Early

Development, China (23). The nocodazole was purchased from Sigma-Aldrich. For in

vitro studies, Ro5203280 and nocodozole were dissolved in dimethyl sulfoxide

(DMSO) solution. For the in vivo studies, the Ro5203280 was dissolved in a vehicle

consisting of PEG400/acetate buffer (pH 5.0). The Ambit selectivity screen was

performed according to the manufacturer’s protocol (Ambit Biosciences).

Real-time cell proliferation assay

The real-time cell proliferation assay was performed using the E-plate and the

xCelligence System (Roche) according to the manufacturer’s instruction. In brief, a




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total of 1 X 104 HONE1, 1 X 104 HK1, 2 X 104 C666-1, and 2 X 104 NP460 cells were

seeded onto an E-plate. Drug treatment was begun 24 hours after cell seeding. The

cell proliferation index was automatically recorded by the xCelligence system

computer program. All cell lines were treated with varying concentrations of

Ro5203280 from 25, 50, 100, 200, 500, and 1000 nM or with DMSO, which is the in

vitro vehicle control. The percentage of cell proliferation inhibition was calculated

using the formula: % inhibition= 100 - (cell index of the treated well/cell index of

untreated well) X 100% (24). All experiments were performed in duplicate at least

twice.

In vivo nude mouse tumorigenicity assay

The in vivo nude mouse tumorigenicity assay was performed as described

previously (25). All mice were kept in the Laboratory Animal Unit in the University

of Hong Kong according to the AAALAC international guidelines. In brief, a total of

1 X 107 cells of the tumorigenic NPC cell lines, HONE1 and C666-1, were injected

subcutaneously into two groups of athymic BALB/c Nu/Nu 6-8 week old nude mice.

A total of 44 and 27 mice were used for the in vivo nude mouse tumorigenicity assay

for HONE1 and C666-1 cell lines, respectively. Tumor sizes were measured weekly.

After the tumors reached a size of > 100 mm3, the drug was then injected into the




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mice intravenously. Drug concentrations of 15 mg/kg body weight (low dose) and 30

mg/kg body weight (high dose) were used for HONE1 treatment, while only the lower

dose was used for C666-1. The drug was injected into the mice following a drug

dosing cycle regimen of two days on and five days off (2+/5-).

Cell cycle and Annexin V apoptosis analysis

The cell cycle distribution of the samples and Annexin V apoptosis assay were

analyzed by FACSCantoII (BD Bioscience) flow cytometry, as previously described

(26). For the cell cycle distribution of samples, in brief, a total of 1 X 106 cells was

seeded on a T25 flask before treatment. The cell density of the untreated control

reached more than 80% confluence before being subjected to the flow analysis. The

cells were treated for 24 hours. A 200 nM drug concentration was used for HONE1,

HK1, and C666-1. A total of 10,000 stained cells was analyzed by FACS. The

Annexin V apoptosis assay was performed by utilizing the Annexin V, Alexa Fluor

647 Conjugate assay (Life Technologies) and nucleic acid staining dye,

7-Aminoactinomycin D (7-AAD) (BD Bioscience), according to the manufacturer’s

instructions.

Immunofluorescence (IF) and Immunohistochemical (IHC) staining




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The IF and IHC staining was performed as previously described (27, 28).

Antibodies specific for phosphorylated-Histone H3 and γ-tubulin were used for IHC

and IF staining, respectively (Table 1). The stained cells were observed under

fluorescence microscopy (Nikon).

Western blot analysis

Western blot analyses of the PLK, phosphorylated PLK, and different signalling

targets were performed as previously described (26, 29). The protein cell lysates from

the cell lines and from mouse tumors were extracted as previously described (24, 29).

In brief, 1 X 106 cells were seeded on a T25 flask before treatment. The cell density of

the untreated control reached more than 80% confluence. The cells were treated for 24

hours at a 200 nM drug concentration. The protein was then extracted by RIPA

solution supplemented with proteinase inhibitors. The antibodies and conditions used

in this study are listed in Table 1. The α-tubulin was used as an internal loading

control.

Statistical analysis

The real-time cell proliferation assay results represent the arithmetic mean +

range of experimental data. The standard error of mean (SEM) was used to calculate




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the standard error of the in vivo assay. The Student’s t test was used to determine the

confidence levels for group comparisons. A p-value <0.05 was considered as

statistically significant.

Results

Ro5203280 is a selective inhibitor for PLK1

Ro5203280 is a small molecular PLK1 inhibitor that is structurally similar to BI2536

(30), but with an improved activity (Fig 1A) (23). In the Ambit protein kinase

selectivity screen, a panel of 315 wild type and mutant forms of kinases was

investigated as described previously (31). R05203280 exhibited a high selectivity and

showed a very low Kd (0.09 nM) to PLK1, when compared to the 0.2 nM Kd of

BI2536. While Ro5203280 does not bind to most of protein kinases tested

(Supplementary Table 1), for eight protein kinases, it exhibits a Kd ranging from 70 to

2000 nM in the Ambit selectivity screen. The specificity of the PLK1 inhibitor

Ro5203280 is summarized in Fig 1B. Like BI2536, Ro5203280 also exhibits potent

and broad activity in suppressing a large panel of 30 cell lines with IC50 as low as 6

nM (data not shown).

Ro5203280 inhibits in vitro cell and in vivo tumor growth




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The efficacy of Ro5203280 in inhibiting in vitro cell growth was first investigated by

the real-time cell proliferation assay. Comparisons of in vitro cell growth of NPC cell

lines versus the immortalized non-tumorigenic nasopharyngeal (NP) epithelial cell

line, NP460, showed EC50 values for Ro5203280 of 78 nM for HONE1, 193 nM for

HK1, and 358 nM for C666-1, in contrast to 950 nM for NP460 (Fig 1C). The

inhibitory effect decreases dramatically at drug concentrations less than 200 nM. This

demonstrates that Ro5203280 can inhibit in vitro NPC cell growth.

The Ro5203280 is also able to inhibit in vivo tumor growth. In a previous Roche

in-house study, different dosage levels and dosing regimes of Ro5203280 were tested

to identify an effective in vivo dosage and schedule for Ro5203280 for different

cancer cell lines, including the colorectal adenocarcinoma and small cell lung cancer

(data not shown). Results suggested that the effective dosage is around 30-40 mg/kg

with a 2+/5- dosing regimen by IV injection. Both HONE1 and C666-1 cell lines are

highly tumorigenic in nude mice (Fig 2A and Table 2). For the HONE1 cell line,

significant tumor growth inhibition can be observed with Ro5203280 treatment

(p-value = 0.002) in both the low dosage (15 mg/kg) and high dosage (30 mg/kg)

groups, when compared to the vehicle control group, which was treated with

PEG400/acetate buffer (pH 5.0). For C666-1, since a low dosage of Ro5203280 was

found to induce significant tumor suppression in the in vivo tumorigenicity assay




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(p-value <0.0005), the higher dose was not tested. The Ro5203280 does not show any

acute toxicity in the mice, as assessed by mouse behavior and body conditions

including hydration and changes of body weight (Fig 2B), and gross necropsy. The

effectiveness of the Ro5203280 in in vivo levels was further confirmed by IHC

staining with p-Histone H3 antibody. P-Histone H3 is a pharmacodynamic biomarker

of PLK1 inhibition. Ro5203280-treated HONE1 and C666-1 tumors showed elevated

p-Histone H3 levels (Supplementary Fig S1). This finding further supports the potent

role of Ro5203280 in NPC tumor inhibition.

Ro5203280 induces cell cycle arrest at G2/M phase by inhibiting mitotic spindle

formation

The tumor inhibitory mechanism for Ro5203280 was investigated. FACS

analysis confirmed that 24 hours treatment of cells with 200 nM Ro5203280 could

induce cell cycle G2/M arrest, as illustrated for HONE1 and HK1 (Fig 3A). The

percentage of cells in G2/M in HONE1, HK1, and C666-1 increased from 21.9% to

52.9%, 13.7% to 39.5%, and 15.4% to 19.6%, respectively, when compared to the

vehicle control, whereas for NP460, no significant increase in the percentage of G2/M

cells before and after treatment was observed. These results suggested that

Ro5203280 has the ability to inhibit cell proliferation through G2/M arrest in HONE1




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and HK1 cells. The Ro5203280-treated HONE1 and HK1 cells formed multinucleated

cells (Figure 3A and Supplementary Figure S2).

Ro5203280 induced cell cycle arrest through inhibition of PLK1. The ability of

treatment for 24 hours of 200 nM Ro5203280 to induce mitotic spindle formation was

investigated by DAPI and IF staining. After DAPI staining, abnormal mitoses were

observed in the Ro5203280-treated cells, providing evidence of non-uniform

distributions of condensed chromatin (Supplementary Figure S3A). All three NPC cell

lines showed significantly increased populations of cells with abnormal mitoses

(Supplementary Figure S3B); this was not the case for NP460. This is consistent with

the FACS analysis. IF staining results showed that the Ro5203280-treated HONE1

cells could selectively inhibit mitotic spindle formation, when compared to the

untreated and vehicle controls (Supplementary Fig S2). A mitotic arrest inducer,

nocodazole, was used as a mitotic arrest positive control. The Ro5203280-treated

cells showed similar mitotic spindle inhibition effects as observed with nocodazole.

These results indicate the role of Ro5203280 in inhibition of the mitotic spindle

formation.

Ro5203280 induces apoptosis in NPC cells

The ability of Ro5203280 to induce apoptosis was quantified by Annexin V




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staining with FACS analysis. After 24 hours of 200 nM Ro5203280 treatment,

HONE1 cells showed a higher proportion of early apoptotic cells (20.8%), as

compared to the untreated (3.77%) and vehicle controls (7.71%) (Fig 3B). In NP460,

no significant increase in apoptotic cells could be observed in the Ro5203280-treated

cells. This further supports the anti-apoptotic ability of Ro52035280 in cancer cells.

Ro5203280 inhibits PLK1 and its downstream target activities to inhibit tumor growth

Ro5203280 is a PLK1 inhibitor. The expression levels of PLK1 in the NPC cell

lines were investigated. The PLK1 protein is up-regulated in all three NPC cell lines,

when compared to NP460 (Fig 4A). Phosphorylation levels of PLK1 were decreased

in HONE1, HK1, and C666-1 cell lines treated with 200 nM Ro5203280 (Fig 4B).

Post-drug treatment, levels of downstream proteins were determined. Wee1 and

Securin, which are important molecules negatively regulating mitosis, were

up-regulated in the NPC cell lines treated with 200 nM Ro5203280 (Fig 4B). After

treatment with 200 nM Ro5203280, there was down-regulation of the mitosis

promoter, Cdc25c, in the NPC cell lines, HONE1 and HK1, but there was no

significant change in the Cdc25c levels in C666-1 (Fig 4B). The cyclin B protein level

dropped after treatment with 200 nM Ro5203280 in HONE1 and HK1 cell lines, but

this was not observed at the other Ro5203280 treatment concentrations. The protein




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levels of total Cdk1 did not show any significant differences after Ro5203280

treatment. Interestingly, one of the DNA damage checkpoint proteins, Chk2, appeared

to have a higher phosphorylation level in NPC cells, when treated with 200 nM

Ro5203280. This is suggestive that Ro5203280 may also be involved in regulating

this signaling pathway.

After Ro5203280 treatment in vivo, tumor proteins were analyzed by Western

blot analysis. Interestingly, phosphorylation of PLK1 was reduced in both HONE1

and C666-1 cell lines. Wee1, Securin, and phosphorylated Chk2 show up-regulation

in the Ro5203280-treated HONE1 and C666-1 tumors, while the Cdc25c and Cyclin

B show down-regulation (Supplementary Figure S4). This further confirms the

important role of Ro5203280 in NPC growth inhibition.

Discussion

NPC is a deadly cancer that is usually diagnosed only at a late stage of cancer

progression. Current treatment practice utilizes only limited standard anti-cancer

drugs. With the development of drug resistance, curative treatment for late stage NPC

patients remains a clinical challenge and improved anti-cancer drugs are needed.

PLK1 over-expression is commonly observed in many different cancers (3, 5-7).

PLK1 is the best characterized member in the PLK family and increasing evidence

suggests that PLK1 serves as a good candidate for cancer drug targeting in several




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malignancies; various PLK1 inhibitors are efficacious as anti-cancer drugs (18, 32, 33).

A previous study showed inhibition of PLK1 function suppresses cell growth and

increases radiation sensitivity in medulloblastoma cell lines (34). In a diffuse large

B-cell lymphoma (DLBCL) study, a small molecule PLK1 inhibitor, MLN0905, was

confirmed to have anti-tumor effects (35). MLN0905 induces mitotic arrest and inhibits

in vivo tumorigenicity (35). In recent clinical trial studies, a PLK1 inhibitor, BI2536,

was used in a randomized phase II clinical trial of pancreatic exocrine adenocarcinoma

(36). Furthermore, another PLK1 inhibitor, BI6727, was found to maintain broad

anti-tumor activities in several solid tumors (37). However, BI2536 failed to show

sufficient efficacy in phase II clinical trials; there was a low response rate and no

significant difference in patient outcomes (36, 38). Ro5203280 is a close analog to

BI2536, but with an improved activity against PLK1. These findings provide the

rationale for evaluating the function of this novel PLK1 inhibitor in NPC. Ro5203280

holds promise as a novel molecularly targeted treatment drug for NPC.

In this current study, the effectiveness of Ro5203280 in selectively suppressing

NPC growth, while sparing inhibition of the non-tumorigenic NP460 cell line, shows

its potential to serve as a chemotherapy drug. The three NPC cell lines displayed

different EC50 values, reflective of the levels of expression of PLK1, the molecular

target for Ro5203280. For NP460, which only expresses low levels of PLK1,




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Ro5203280 has little effect on its growth. On the other hand, PLK1 is expressed in the

highly proliferative cancer cells and is regarded as a cell proliferation marker (3, 4).

These findings further support the potential efficacy of utilizing this drug for

treatment of NPC. The Ro5203280 compound does not induce any noticeable acute

toxicity in the mice. However, due to inherent limitations in the use of mouse models,

the primary hematologic toxicity seen in patients treated with other PLK1 inhibitors

could not be studied, but remains a potential side effect that warrants further future

scrutiny.

PLK1 is involved in the cell cycle G2/M transition control. The G2/M checkpoint

is one of the important checkpoints for DNA damage repair and maintenance of

genomic integrity (39). Normal cells suffering DNA damage do not undergo mitosis.

If the damaged cells are unable to be repaired, they are destined to undergo apoptosis.

On the other hand, inhibition of the mitosis can result in multinucleated cells. The

multinucleated cells will finally undergo apoptosis. Therefore, the G2/M checkpoint is

a good target for treating cancer (39). In cancer, the abnormal PLK1 activity can

interfere with the normal G2/M checkpoint and thus, damaged cells will escape from

the DNA repair mechanism and also cell cycle control. Ro5203280 inhibits PLK1

kinase activity in cancer cells and induces G2/M arrest in the NPC cell lines. This

finding helps to explain the low toxicity of Ro5203280 in the mice, as the non-tumor




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cells are spared.

PLK1 is a key regulator modulating different signaling pathways in order to

control the G2/M transition. PLK1 induces Wee1 phosphorylation and, thus, facilitates

its degradation through proteasome-dependent degradation after ubiquitination by the

E3 ubiquitin ligase (11). Wee1 is a crucial kinase in regulating the cell cycle G2/M

transition. It was confirmed to regulate DNA replication in human cells (40). It can

phosphorylate Cdk1 and inhibit its function to drive cell cycle progression (41). It can

also induce apoptosis, as observed during early xenopus embryonic development (42).

Cdc25c removes the inhibitory phosphorylation of the Cdk1 protein and activates its

functional role in mitosis (11). Early studies show Cyclin B was up-regulated in the

PLK1 knockdown NPC cell lines (8). However, in our current study, the Cyclin B

protein level decreased in the 200 nM Ro5203280-treated cells; this may be related to

cell apoptosis. At other Ro5203280 treatment concentrations, Cyclin B protein was

maintained at a high level.

In this current study, Securin was found to be up-regulated in Ro5203280-treated

NPC cells. Securin is another key regulator for monitoring chromosome segregation,

which is controlled by PLK1. PLK1 activates the APC function by degradation of

APC inhibitors such as Emi1 (43). The activated APC then degrades Securin and

releases the separase to allow chromosome segregation (44). The up-regulation of




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Securin by Ro5203280 also indicates its potential ability to cause G2/M arrest.

Furthermore, PLK1 plays a vital role for the G2/M recovery at the DNA damage

checkpoint. Up-regulation of phosphorylated Chk2 protein, which is crucial for the

DNA damage response and induces cell cycle G2/M arrest to allow DNA repair, was

observed after Ro5203280 treatment. If the cell fails to repair the damage, it will

undergo apoptosis (45). The DNA damage-induced cell cycle arrest can inactivate

PLK1 function. However, in cancer cells, PLK1 malfunction can override the G2/M

arrest induced by DNA damage (46). PLK1 inactivates Chk2 kinase activity and, thus,

arrests the DNA damage checkpoint signaling (47). The inhibition of PLK1 function

by Ro5203280 helps to maintain Chk2 function, allows the cell to undergo cell cycle

arrest, and prevents cell proliferation. Our results are further supported by a previous

PLK1 study in NPC. Knockdown of PLK1 by siRNA in C666-1 and HK1 cell lines

resulted in cell cycle G2/M arrest, apoptosis, down-regulation of Cdc25c, and

suppression of in vivo tumorigenesis (8). This further supports our findings of the

potential usefulness of the PLK1 inhibitor, Ro5203280, to serve as a candidate

chemotherapeutic drug for NPC.

Interestingly, our data show C666-1 is more sensitive to Ro5203280 in vivo

than in vitro. One possible explanation is that C666-1 grows relatively slowly in vitro,

but comparatively fast in the nude mouse. The changes in the molecular signaling




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pathways can help to explain differences observed in the efficacy of Ro52032820. In

the Western blot analysis of C666-1 mouse tumors, down-regulation of the PLK1

downstream signaling target, CDC25C, is more obvious in Ro5203280-treated mouse

tumors than in the in vitro study. Also the levels of up-regulation of the Wee1 and

Securin were much higher in the C666-1 mouse tumors than observed in vitro. This

further supports our results that the Ro5203280 has a higher efficacy on C666-1 in

vivo than in vitro.

In conclusion, the PLK1 inhibitor Ro5203280 shows a high efficacy for

suppressing NPC growth with low toxicity in mouse studies. It suppresses NPC

growth by inducing G2/M arrest and mitotic arrest and finally induces apoptosis

through inhibiting PLK1 phosphorylation. It restricts the PLK1 function, then

activates the signaling molecules that are negatively-regulated by PLK1, and

inactivates the molecules that are positively-regulated by PLK1. Thus, this novel

PLK1 inhibitor targeting multiple pathways is a promising anti-cancer drug for NPC

treatment.

Acknowledgements

We acknowledge the use of the Faculty Core Facility of the Li Ka Shing Faculty of

Medicine, HKU for the flow cytometry equipment, and Dr. Daniel Lee (Roche) for

support.




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26

Table 1: Antibody information

Antibody Host Dilution Company Catalog no.

p-PLK1-T210 Rabbit 1:1000 Cell Signaling 5472

PLK1 Rabbit 1:1000 Cell Signaling 4513

WEE1 Rabbit 1:1000 GeneTex GTX111392

Securin Rabbit 1:1000 Genetex GTX62173

Cdc25c Rabbit 1:1000 GeneTex GTX102809

Cyclin B Rabbit 1:200 GeneTex GTX100911

Cdk1 Rabbit 1:1000 GeneTex GTX108120

p-Chk2(T68) Rabbit 1:1000 Cell Signaling 2661

α-tubulin Rabbit 1:1000 GeneTex GTX108784

p-Histone H3(S10) Rabbit 1:200 Santa Cruz Sc8656

γ-tubulin mouse 1:500 Sigma-Aldrich T5326




27

Table 2: Summary of Ro5203280 in vivo tumorigenicity assays

Cell lines/Treatment description Number of residual tumors

after drug treatment/number

of injection sites)

p-value*

HONE1/Vehicle 16/16 - HONE1/15 mg/kg IV 2+/5- 10/15 0.0019

HONE1/30 mg/kg IV 2+/5- 5/13 0.0018

C666-1/Vehicle 10/10 -

C666-1/15 mg/kg IV 2+/5- 2/17 <0.0005

*p-value obtained by comparison with vehicle control




28

Figure legends

Figure 1. Inhibitory effect of Ro5203280 on NPC cell lines. (A) Chemical structure

of Ro5203280. (B) Ambit selectivity screen specificity of PLK1 inhibitor, Ro5203280.

(C) In vitro growth inhibitory effect of Ro5203280 in three NPC cell lines (HONE1,

HK1, and C666-1) and the immortalized NP cell line, NP460. The relative inhibition

rate was compared with their corresponding untreated control. The cells were treated

with different concentrations of Ro5203280 (25, 50, 100, 200, 500, and 1000 nM) for

24 hours.

Figure 2. In vivo tumor growth inhibition studies in NPC. (A) In vivo growth

inhibitory effect of Ro5203280 on NPC cell lines, HONE1 and C666-1. The curves

represent an average tumor volume of all injected animals (16 mice for

HONE1/Vehicle, 15 mice for HONE1/15 mg/kg IV 2+/5-, 13 mice for HONE1/30

mg/kg IV 2+/5, 10 mice for C666-1/Vehicle, and 17 mice for C666-1/15 mg/kg IV

2+/5-). Drug treatments were initiated, when the average tumor size reached 100 mm3.

The vehicle control forms large tumors; Ro5203280 treatment resulted in reduced

tumor sizes. (B) Changes in body weight of the experimental animals. No significant

changes in the body weights were observed in the experimental animals, indicating

low toxicity of the administered drug.

Figure 3. Cell cycle and cell death studies of the Ro5203280-treated NPC and NP cell

lines. (A) Representative images of FACS analysis in Ro5203280-treated NPC cell




29

lines, HONE1, HK1, and C666-1, and the NP cell line, NP460. The cells were

untreated (U), vehicle-treated (V), and drug-treated (T). Treatment was with 200 nM

Ro5203280 for 24 hours. (B) Representative images of the Annexin V staining assay

of untreated, vehicle, and drug-treated NPC HONE1 and NP460 cell lines. The

Ro5203280-treated HONE1 cells showed a higher level of cell death, when compared

to the Ro5203280-treated NP460.

Figure 4. Western blot analysis of changes in molecular signaling after Ro5203280

treatment. (A) PLK1 expression levels in NPC cell lines, HONE1, HK1, and C666-1,

and NP cell line, NP460. The expression levels were normalized to the internal

control, α-tubulin. (B) PLK, cell cycle control, and DNA damage-related signaling

pathways analysis of the Ro5203280-treated NPC cells. The expression levels were

normalized to the internal control, α-tubulin. The cells were untreated (U), vehicle (V),

and drug-treated (T), as indicated. The phosphorylation status of p-PLK1 in

Ro5203280-treated cells with different concentrations of Ro5203280 is shown.

Down-regulation of the Cyclin B and Cdc25c and up-regulation of the Wee1, Securin,

and pChk2 is observed in the drug-treated NPC cell lines. There is no significant

change in the levels of Cdk1 protein in the Ro5203280-treated cells. The expression

levels were normalized to the internal control, α-tubulin.




Protein Kd (nM)

ALK 230

AB

Ro5203280

ALK 230

DAPK1 100

DAPK3 70DAPK3 70

MYLK 170

PTK2B 130

C

120

PTK2B 130

RP6KA4(KinDom 2)

2000

on (%

)

80

100

RPS6KA6(KinDom 2)

560

wth

inhi

biti

40

60

TTK 51

PLK1 0.09

Gro

w

0

20

HONE1

HK1

NP460

C666-1

Concentration (nM) Figure 1

-20

-200 0 200 400 600 800 1000 1200

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Am

3 )

m3 )

HONE1 C666-1

600.0

700.0

800.0

Vehicle

15 mg/kg IV 2+/51200.0

1400.0

1600.0

Vehicle

umor

siz

e (m

m

umor

siz

e (m

m

p=0.

002

p=0.

002

p<0.

0005

300.0

400.0

500.0

15 mg/kg IV 2+/5-

30mg/kg IV 2+/5-

600.0

800.0

1000.0 15 mg/kg IV 2+/5-

T Tu

Time (Weeks) Time (Weeks)

0.0

100.0

200.0

1 2 3 4 5

0.0

200.0

400.0

1 2 3 4e ( ee s) Time (Weeks)

HONE125.0

B

C666-125.0

eigh

t (g

)

15.0

20.0

Vehicle wei

ght

(g)

15.0

20.0

Body

we

0 0

5.0

10.0Vehicle

15 mg/kg IV 2+/5-

30 mg/kg IV 2+/5-

Body

w

0 0

5.0

10.0Vehicle

15mg/kg IV 2+/5-

Time (Weeks)

0.01 2 3 4 5

Time (Weeks)

0.01 2 3 4

Figure 2

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HONE1 HK1 NP460C666-1

Untreated

AG1:42.2%G2:20.4% G1:52.5%

G2:18.4%G1:32.1%G2:16.1%

G1:49.9%G2:16.8%

VehicleEven

t

G1:46.7%G2:21.9%

G1:54.2%G2:13.7%

G1:31.2%G2:15.4%

G1:40.0%G2:19.1%

Ro5203280G1:5.22%G2:52.9%

G1:26.6%G2:39.5%

G1:28.5%G2:19 6%

G1:31.8%G2:14 6%

DNA content

G2:19.6% G2:14.6%

BUntreated Vehicle Ro5203280

HONE1HONE1

-AA

D

3.77% 7.71% 20.8%

Figure 3

NP460

Annexin V

7-

23.3% 20.8% 26.4%

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A

NE1 6-1

460

HO

N

HK1

C66 6

NP4

PLK1α-tubulin

B HONE1

UV200

nM

100

nM50

nM

25 n

M

HK1 C666-1 NP460

UV200

nM

100

nM50

nM

25 n

M

UV200

nM10

0 nM

50 n

M25

nM

UV200

nM

100

nM50

nM

25 n

M

UVT2T1T5T2

p-PLK1PLK1CyclinBCdc25c

UVT2T1T5T2 UVT2T1T5T2 UVT2T1T5T2

p-Chk2-T68SecurinWee1α-tubulin

Cdk1

Figure 4on January 26, 2020. © 2013 American Association for Cancer Research. mct.aacrjournals.org Downloaded from



Published OnlineFirst May 17, 2013.Mol Cancer Ther Arthur Kwok Leung Cheung, Joseph Chok Yan Ip, Hong Lok Lung, et al. anti-tumor activity in nasopharyngeal carcinomaPolo-like kinase (PLK) inhibitor, Ro5203280, has potent

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