Expression and therapeutic implications of cyclin-dependent kinase 4 (CDK4) in osteosarcoma

Accepted Manuscript Expression and therapeutic implications of cyclin-dependent kinase 4 (CDK4) in osteosarcoma

Yubing Zhou, Jacson K. Shen, Zujiang Yu, Francis J. Hornicek, Quancheng Kan, Zhenfeng Duan PII: DOI: Reference:

S0925-4439(18)30047-4 doi:10.1016/j.bbadis.2018.02.004 BBADIS 65052

To appear in: Received date: Revised date: Accepted date:

7 August 2017 24 January 2018 9 February 2018

Please cite this article as: Yubing Zhou, Jacson K. Shen, Zujiang Yu, Francis J. Hornicek, Quancheng Kan, Zhenfeng Duan , Expression and therapeutic implications of cyclindependent kinase 4 (CDK4) in osteosarcoma. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Bbadis(2018), doi:10.1016/j.bbadis.2018.02.004

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ACCEPTED MANUSCRIPT REVISED Manuscript (text Unmarked) Expression and therapeutic implications of cyclin-dependent kinase 4 (CDK4) in osteosarcoma

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Yubing Zhoua,b, Jacson K. Shenb, Zujiang Yua, Francis J. Hornicekb, Quancheng Kana* and

Department of Pharmacy, The First Affiliated Hospital of Zhengzhou University, Zhengzhou,

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a

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Zhenfeng Duana,b#

Henan 450052, People’s Republic of China Sarcoma Biology Laboratory, Center for Sarcoma and Connective Tissue Oncology,

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b

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Massachusetts General Hospital and Harvard Medical School, Boston, MA 02114, USA

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Corresponding author: *Quancheng Kan, Department of Pharmacy, The First Affiliated Hospital of Zhengzhou University, 1 Jianshe East Road, Zhengzhou 450052, China. Phone: 86-371-

#

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66913114; Fax: 86-371-66970906. E-mail address: [email protected] Zhenfeng Duan, Sarcoma Biology Laboratory, Center for Sarcoma and Connective Tissue

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Oncology, Massachusetts General Hospital, 100 Blossom St, Jackson 1115, Boston, MA 02114,

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USA. Phone: 617-724-3144; Fax: 617-726-3883. E-mail address: [email protected]

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ACCEPTED MANUSCRIPT Abstract Overexpression and/or hyperactivation of cyclin-dependent kinase 4 (CDK4) has been found in many types of human cancers, and a CDK4 specific inhibitor, palbociclib, has been recently approved by the FDA for the treatment of breast cancer. However, the expression and the

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therapeutic potential of CDK4 in osteosarcoma remain unclear. In the present study, CDK4 was

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found to be highly expressed in human osteosarcoma tissues and cell lines as compared with normal human osteoblasts. Elevated CDK4 expression correlated with metastasis potential and

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poor prognosis in osteosarcoma patients as determined by immunohistochemical analysis in a human osteosarcoma tissue microarray (TMA). CDK4 inhibition by either palbociclib or specific

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small interference RNA (siRNA) exhibited dose-dependent inhibition of osteosarcoma cell

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proliferation and growth, accompanied by suppression of the CDK4/6-cyclinD-Rb signaling pathway. Flow cytometry analysis showed that CDK4 knockdown arrested osteosarcoma cells in

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the G1 phase of the cell cycle and induced cell apoptosis. Furthermore, inhibition of CDK4

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significantly decreased osteosarcoma cell migration in vitro determined by the wound healing assay. These data highlight that CDK4 may be a potential promising therapeutic target in the

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treatment of human osteosarcoma.

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Key words: Osteosarcoma, Cyclin-dependent kinase 4, Palbociclib, Rb, Cell cycle.

Abbreviation Lists: CDK4, cyclin-dependent kinase 4; RNAi, RNA interference; siRNA, small interfering RNA; Rb: retinoblastoma; TMA: tissues microarray.

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ACCEPTED MANUSCRIPT 1. Introduction The current osteosarcoma treatment regimen consists of the combination of surgery and intensive multi-agent chemotherapy, which has improved the five-year survival rate of osteosarcoma patients to 60%-75%[1, 2]. However, 30%-40% of osteosarcoma patients develop

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pulmonary metastasis and relapse, which has a significantly poor prognosis with an overall five-

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year survival rate of about 20%[3]. Hence, development of new therapeutic strategies for osteosarcoma treatment remains an important but unmet clinical need.

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Uncontrolled cell proliferation and growth is defined to be one of the hallmarks of cancer[4].

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Cyclin-dependent kinases (CDKs) are a large family of serine/threonine (Ser/Thr) protein kinases that play crucial roles in the regulation of cell cycle progression[5, 6]. CDKs are often

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overexpressed and/or overactive in human cancers owing to various genetic and epigenetic

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events that affect their regulatory pathways, bringing about loss of checkpoint integrity, and ultimately resulting in uncontrolled cell proliferation and malignant transformation[7-9]. Among

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them, CDK4 regulates the G1-S phase of the cell cycle by deactivating the tumor suppressor

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retinoblastoma protein (Rb) in cancer cells as well as dividing cells[10]. Specifically, in response to pro-proliferative stimuli, CDK4 complexes with cyclin D1 to induce the phosphorylation of

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Rb (pRb) and switch off the tumor suppressing function of Rb. Consequently, once phosphorylated, pRb is not able to bind with transcription factor E2F, thus allowing cancer cell cycle progression through transcription of various cell-cycle and anti-apoptotic genes[11, 12]. In osteosarcoma, abnormalities of the Rb and p53 genes are common phenomena[13]. Rb family proteins (including pRb/p105, pRb2/p130and p107) have three functionally distinct binding domains and interact with critical regulatory proteins including the E2F family of transcription factors[14]. Until now, six members of the E2F family have been identified, and each E2F 3

ACCEPTED MANUSCRIPT subunit has a DNA binding and a dimerization domain. E2F-1 to E2F-5 activate transcription. E2F-1 to E2F-3 bind pRb, and E2F-4 and E2F-5 bind p107 or p130, and these interactions are under cell cycle control[15, 16]. Rb mutations are detected in approximately 70% of all adolescent osteosarcomas[17]. Loss of Rb and p53 in mesenchymal stem/progenitor cells has

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been shown to transform these cells and initiate osteosarcoma formation in vivo[18].

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Overexpression and activation of the CDK4/Cyclin D1/Rb pathway has been shown to correlate

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with unrestricted tumor cell growth and proliferation, and thus is a hallmark of various types of malignancies, including sarcoma[19]. Several studies have demonstrated that CDK4, as well as

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MDM2, was highly expressed in osteosarcoma, especially in low-grade subtype, and CDK4

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expression correlated to chemotherapy responses[20, 21]. More recent investigations have

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proven that CDK4 inhibition sensitized cancer cells to targeted therapy[22, 23]. CDKs are attractive targets for the development of anticancer therapeutics, and inhibition of

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CDKs in malignant cells provides a promising approach in the defense against cancer[24-27].

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Recently, many selective CDK inhibitors targeting specific CDKs have been developed, which represent promising anti-cancer agents due to their strong anti-proliferative efficacy combined

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with a relative low direct cytotoxicity[28-31]. Notably, palbociclib (IBRANCE®), a dual

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CDK4/6 inhibitor, recently received accelerated approval by the Food and Drug Administration (FDA) for breast cancer treatment due to its potent and selective inhibitory effect on estrogen receptor (ER) positive/human epidermal growth factor receptor 2 (HER2) negative breast cancer[32-34]. Furthermore, palbociclib has also been used in phase II clinical trial for liposarcoma therapy[35, 36]. However, the expression and clinical significance of CDK4, and the potentials of targeting CDK4 as a putative therapeutic strategy in osteosarcoma are unclear.

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ACCEPTED MANUSCRIPT In the present study, we evaluate the expression of CDK4 in human osteosarcoma and the therapeutic applications. Our findings show that CDK4 was highly expressed in human osteosarcoma tissues and cells, and high CDK4 expression correlated with poor prognosis for patients with osteosarcoma. Inhibition of CDK4 decreased cell proliferation and migration, and

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induced cell cycle arrest and apoptosis. These results indicate that CDK4 plays an important role

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in the development and progression of human osteosarcoma, and therefore warrants further

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evaluation as a therapeutic target of osteosarcoma.

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2. Materials and Methods

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2.1. Human osteoblast and osteosarcoma cell lines and cell culture Human osteoblast cell line HOB-c was purchased from PromoCell GmbH (Heidelberg,

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Germany), and NHOst was purchased from Lonza Walkersville Inc. (Walkersville, MD, USA).

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Osteoblast cell lines were cultured in osteoblast growth medium (PromoCell) with a supplement mix. Human osteosarcoma cell lines U2OS, MNNG/HOS, 143B, and MG-63 were obtained from

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the American Type Culture Collection (Rockville, Maryland, USA). KHOS was kindly provided

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by Dr. Efstathios Gonos (Institute of Biological Research & Biotechnology, Athens, Greece). All osteosarcoma cell lines were cultured at 37°C in a humidified 5% CO2 atmosphere in RPMI

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1640 (Life Technologies, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (GIBCO, Grand Island, NY, USA), 100 units/ml penicillin G, and 100 μg/ml streptomycin. 2.2. Human osteosarcoma tissues Eight human osteosarcoma tissues were obtained from the Massachusetts General Hospital Sarcoma Tissue Bank and used in accordance with the policies of the institutional review boards of the hospital (IRB protocol number: 2007P-002464). All tissue diagnoses were confirmed 5

ACCEPTED MANUSCRIPT histologically. Written informed consent was obtained from all patients whose specimens and clinical information were used for this research study. 2.3. Human osteosarcoma tissues microarray (TMA) construction A total of 72 formalin-fixed, paraffin-embedded tumor specimens including primary,

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recurrent and metastatic specimens from 54 individual osteosarcoma patients were selected to

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construct the TMA in the Tissue Microarray Core at the Dana-Farber/Harvard Cancer Center. Representative triplicate 0.5-mm-diameter core biopsies of each tissue block were obtained

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through the pathology reports and reading of corresponding HE (haematoxylin and eosin)stained slides by a pathologist. The enrolled patients were diagnosed as having osteosarcoma and

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received treatment between 1992 and 2010 at the Massachusetts General Hospital. Clinical

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information of all the subjects was collected and managed from the archives, including age, gender, tumor location, pathologic stage, histologic subtype, recurrence and metastasis status,

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metastasis location, whether the patient received pre-operative chemotherapy or not, as well as

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the follow-up time and patient outcome (Supplementary Table S1). All the experimental protocols were approved by the Partners Human Research Committee (IRB protocol number:

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2007P-002464). The methods described in this study were carried out in accordance with the

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approved guidelines. Written informed consent was obtained from all patients whose specimens and clinical information were used for this research study. 2.4 Immunohistochemistry analysis of tissue microarray The expression of CDK4 in the tissue microarray was determined by immunohistochemistry assay according to the manufacturer’s instructions (Cell Signaling Technology, Beverly, MA, USA). Briefly, the paraffin-embedded slide was baked for 1 h at 60˚C, deparaffinized in xylene,

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ACCEPTED MANUSCRIPT and then transferred through graded ethanol (100% and 95%) for rehydration. After heat-induced epitope retrieval, the endogenous peroxidase was quenched by 3% hydrogen peroxide. Following blocking by normal goat serum for 1 h, the slide was incubated with rabbit polyclonal antibody to human CDK4 (#12790, 1:500 dilution, Cell Signaling Technology, Beverly, MA, USA) at

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4°C overnight in a humidified chamber. Subsequently, bound antibody on the array was detected

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by SignalStain® Boost Detection Reagent (Cell Signaling Technology, Beverly, MA, USA) and

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SignalStain® DAB (Cell Signaling Technology, Beverly, MA, USA). The nuclei of osteosarcoma cells were counterstained with hematoxylin QS (Vector Laboratories, Burlingame,

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CA) to obtain better images. Finally, the section was mounted with VectaMount AQ (Vector

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Laboratories) for long-term preservation. The TMA slides were also stained in the absence of CDK4 antibody to evaluate nonspecific secondary antibody reactions. The slide was imaged

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using an Olympus microscope (BX51, Olympus, PA, USA).

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Immunostaining of the whole slide area was viewed and scored separately by three

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independent pathologists who were blinded to tumor characteristics and patient details of the samples. According to the percentage of cells with positive nuclear staining, CDK4 expression

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was divided into 6 groups: 0, no nuclear staining; 1+, <10% of positive cells; 2+, 10%-25% of

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positive cells; 3+, 26%-50% of positive cells; 4+, 51%-75% of positive cells; 5+, >75% of positive cells. Tumors with a staining score of ≥3 were designated as high CDK4 expression and ≤2 were designated as low CDK4 expression. 2.5. Protein preparation and Western blot Protein lysates were extracted from cells or tissues with 1× RIPA lysis buffer (Upstate Biotechnology, Charlottesville, VA) supplemented with complete protease inhibitor cocktail

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ACCEPTED MANUSCRIPT tablets (Roche Applied Science, IN, USA). The concentrations of the protein lysates were determined by protein assay reagents (Sigma-Aldrich, MO, USA) with a Beckman spectrophotometer (Beckman Instruments, Inc., IN, USA). Western blot was performed as previously described. Briefly, denatured proteins were run on an SDS-PAGE gel and then

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transferred to nitrocellulose membranes. After blocking in 5% nonfat milk for 1 h, the

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membranes were incubated with rabbit monoclonal antibodies to human CDK4, CDK6,

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pRB(Ser780), and Survivin (1:1000 dilution, Cell Signaling Technology, Beverly, MA, USA), and mouse monoclonal antibodies to human Rb (1:1000 dilution, Cell Signaling Technology,

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Beverly, MA, USA) and β-actin (1:2000 dilution, Santa Cruz Biotechnology, TX, USA) at 4°C

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overnight. Following primary antibody incubation, the membranes were washed with TBST, and Goat anti-rabbit IRDye 800CW (926-32211, 1:5000 dilution) or Goat anti-mouse IRDye 680LT

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secondary antibody (926-68020, 1:10000 dilution) (Li-COR Biosciences, NE, USA) was added,

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respectively. After incubation at room temperature for 2 h, the bands were detected using Odyssey Infrared Fluorescent Western Blots Imaging System from Li-COR Bioscience (Lincoln,

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NE, USA). Quantification of Western blot results was analyzed with Odyssey software 3.0.

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2.6. Immunofluorescence assay

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For immunostaining of cultured osteosarcoma cells, U2OS and KHOS cells were grown in 6well plates for three days and fixed with 4% paraformaldehyde for 15 min, followed by permeabilization with ice-cold methanol and blocked in 1% BSA. The cells were then incubated with the CDK4 primary antibody (1:200 dilution, Cell Signaling Technology, Beverly, MA, USA) or β-Actin (sc-47778, Santa Cruz Biotechnology, 1:200 dilution) at 4°C overnight, followed by incubation with Alexa Fluor 488 (Green) conjugated goat anti-rabbit antibody or Alexa Fluor 594 (red) goat anti-mouse antibody (Invitrogen, NY, USA) for 1 h. Finally, cells 8

ACCEPTED MANUSCRIPT were imaged on a Nikon Eclipse Ti-U fluorescence microscope (Diagnostic Instruments Inc., NY, USA) equipped with a SPOT RT™ digital camera. 2.7. siRNA transfection and drug treatment

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CDK4 inhibition in osteosarcoma cells was performed by both CDK4 specific siRNA

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transfection and palbociclib treatment. For siRNA transfection, U2OS and KHOS cells were

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seeded into 96-well plates at a density of 4×103 cells per well or into 6-well plates at a density of 4×105 cells per well and transfected with increasing concentrations (0, 20, 40, 60 nM) of

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synthesized CDK4 siRNA (5’-CUCUUAUCUACAUAAGGAU-3’) (Sigma-Aldrich, MO, USA)

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by using Lipofectamine RNAiMax reagent (Invitrogen, CA, USA) according to the manufacturer’s instructions. The nonspecific siRNA (60nM) was used as a negative control. For

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drug treatment, NHOst, U2OS and KHOS cells were seeded into 96-well plates at a density of

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4×103 cells per well or into 6-well plates at a density of 6×105 cells per well and incubated with increasing concentrations (0, 0.04, 0.16, 0.625, 2.5, 10 µM) of palbociclib (Selleck Chemicals,

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Houston, TX, USA) for 2, 4, and 6 days, followed by subsequent experiments.

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2.8. Cell proliferation assay

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Three days after CDK4 siRNA transfection, and 2, 4, and 6 days after palbociclib treatment, the cell viability of NHOst, U2OS and KHOS cells was determined using the MTT assay. Briefly, at the end of cell treatment, 20 μL of MTT (5 mg/mL, Sigma-Aldrich) was added to each well and the 96-well plates were incubated at 37°C in a humidified 5% CO2 atmosphere for 4 h. Finally, the resulting formazan product was dissolved with 100 μL of acid isopropanol and the absorbance at a wavelength of 490 nm was measured on a SpectraMax Microplate® Spectrophotometer (Molecular Devices LLC, Sunnyvale, CA). Meanwhile, the morphological 9

ACCEPTED MANUSCRIPT changes of U2OS and KHOS cells were observed with a Nikon microscope (Diagnostic Instruments Inc., NY, USA) after 4 days of palbociclib treatment. 2.9. Cell migration assay

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Cell migration activity was detected by the wound healing assay. In brief, U2OS and KHOS

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cells were seeded into 6-well plates at a density of 4×105 cells per well and incubated overnight.

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Then the adherent cell layer was scraped in two parallel lines with a sterile 10 μL tip, and 1 µM of palbociclib was added into the cell medium immediately for an additional 72 h of starved

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incubation with low-serum medium containing 2% FBS. The wounds were photographed under a

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Nikon microscope (Diagnostic Instruments Inc., NY, USA) equipped with a Zen Imaging software after 0, 24, 48, and 72 h of palbociclib treatment. The wound width was evaluated by

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measuring the distance between the two edges of the scratch at five sites in each image. Cell

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migration distance was determined using the following formula: (wound width at the 0 h time

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point - wound width at the observed time point) / 2. 2.10. Flow cytometry analysis

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Cell cycle and apoptosis were analyzed by flow cytometry after 24 and 48 hours of

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palbociclib treatment, respectively. The experiment was performed in two different osteosarcoma cell lines, U2OS and KHOS. For cell cycle analysis, the cells were collected and fixed in 70% ethanol at 4°C overnight, followed by incubation in RNase A (Thermo Scientific, NY, USA) at 37°C for 30 min and dyed with Propidium Iodide (Sigma-Aldrich, MO, USA) for an additional 30 min. The DNA content was determined by flow cytometry (FACSCanto II, BD, NJ, USA) and the population of cells in each cell cycle phase was analyzed by the equipped ModFIT software (Verity Software House, ME, USA). For cell apoptosis analysis, the cells were 10

ACCEPTED MANUSCRIPT collected by trypsinization and resuspended in annexin-binding buffer, followed by staining with Annexin V-FITC and Propidium Iodide (Invitrogen, NY, USA) for 30 min, and then subjected to flow cytometry analysis.

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2.11. Statistical analysis

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Statistical analysis was performed using the GraphPad PRISM 5 software (GraphPad

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Software, San Diego, CA, USA). Data are expressed as mean ± SD. Student’s t-test was used to determine the statistical significance of differences between groups. Survival analysis was

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performed using the Kaplan-Meier method, and significance was determined by the log-rank test.

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A P value of ≤ 0.05 was considered as statistically significant.

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3. Results

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3.1. CDK4 was highly expressed in human osteosarcoma tissues and cell lines To explore the potential role of CDK4 in human osteosarcoma cell growth, we first

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determined the expression of CDK4 in human osteosarcoma cell lines. As demonstrated by

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Western blot, all five human osteosarcoma cell lines, with diverse histological staining characteristics (Supplementary Table S2), exhibited high levels of CDK4 expression, whereas

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the expression of CDK4 was tightly regulated in the normal osteoblast cell line (Fig. 1A). To further confirm the expression of CDK4 and determine its subcellular localization in osteosarcoma cells, immunofluorescence was performed in U2OS and KHOS cells. As shown in Fig. 1B, CDK4 protein was mainly localized in the nucleus of osteosarcoma cells with some expression in the cytoplasm. To preclude the possibility that CDK4 expression is an artifact induced by in vitro propagation, we also examined eight freshly isolated primary osteosarcoma specimens. The results demonstrated that CDK4 was highly expressed in most of the tested 11

ACCEPTED MANUSCRIPT osteosarcomas, with six out of eight osteosarcoma tissues showing dramatic CDK4 expression (Fig. 1C). Due to the fact that CDK6 is structurally similar to CDK4, and that Rb/pRb is involved in the CDK4/6 signaling pathway, we also determined these proteins’ levels in the tested osteosarcoma specimens. We observed that most of the osteosarcoma cell lines and tissues

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expressing CDK4 simultaneously expressed CDK6 and Rb, with high phosphorylation of Rb

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(pRb) (Fig.1 A and C).

3.2. CDK4 expression levels correlated with the clinicopathological characteristics of

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osteosarcoma patients

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CDK4 is highly expressed in both osteosarcoma cell lines and patient tissues, implying that CDK4 may play roles in the initiation and progression of osteosarcoma. To further validate the

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clinical significance of CDK4 expression in patients with osteosarcoma, we determined CDK4

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levels in a human osteosarcoma tissue microarray by immunohistochemistry, and evaluated the correlation between CDK4 expression and the pathological characteristics, as well as clinical

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prognosis of osteosarcoma patients. Consistent with the immunofluorescence assay that showed

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that CDK4 is mainly expressed in the nucleus of in vitro osteosarcoma cells, the immunohistochemistry demonstrated that CDK4 immunoreactivity was found in the nucleus of

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osteosarcoma tissue cells. Among the determined 72 tissues, 65 (90.3%) tissues positively expressed CDK4. Based on data from up to 252 months of follow-up, CDK4 expression levels in samples from non-survivors were significantly higher than those from survivors (Fig. 2A). More importantly, Kaplan-Meier survival analysis showed that the outcome for patients in the CDK4 high-staining (≥3) group was worse than for those in the CDK4 low-staining (≤2) group (Fig. 2B). Furthermore, we found that CDK4 expression correlated with the metastasis status of osteosarcoma. As shown in Fig. 2C and 2D, CDK4 expression was significantly higher in the 12

ACCEPTED MANUSCRIPT osteosarcoma tissues from metastasis patients than that from non-metastasis patients, and CDK4 expression was significantly higher in the metastasis tissues than that in the primary tissues. However, we did not find significant correlations between CDK4 expression and the recurrence status, pathologic grades, and chemotherapy of patients (data not shown). The 2 test further

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confirmed that CDK4 higher expression significantly correlated with metastasis and the outcome

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of osteosarcoma patients (Supplementary Table S3).

3.3. CDK4 inhibition by palbociclib decreased human osteosarcoma cell proliferation

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After validating the expression and clinical significance of CDK4 in patients with

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osteosarcoma, we further assessed the functional roles of CDK4 expression in osteosarcoma cell proliferation and growth in vitro. We inhibited CDK4 activity in osteosarcoma cells using a

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recently approved CDK4 selective inhibitor palbociclib and determined cell viability by MTT

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assay. As shown in Fig. 3A and 3B, after exposure to increasing concentrations of palbociclib for 4 and 6 days, the cell viability was decreased in dose-dependent manners in both U2OS and

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KHOS cells, whereas the cell growth was not dramatically influenced after 2 days of treatment

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with palbociclib. We also employed CDK4-lower expressed NHOst cell line as the control group to evaluate palbociclib on the proliferation of osteosarcoma cell lines with lower CDK4 levels.

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Expectedly, our data showed that palbociclib exhibited no activity on CDK4-lower expressed NHOst cell proliferation, only exerted mild proliferation inhibition after 10 uM of palbociclib exposure for 4 or 6 days (Fig. 3C). The morphologic changes of U2OS and KHOS were also observed after 4 days of palbociclib exposure (Fig. 3D). To investigate the alteration of the CDK4/6-Rb-apoptosis pathway following CDK4 inhibition, we determined the levels of several respective proteins after palbociclib treatment. As shown by

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ACCEPTED MANUSCRIPT the Western blot, after 3 days of palbociclib exposure, the phosphorylation levels of Rb (pRb) were reduced in dose-dependent manners in both U2OS and KHOS cells, whereas the upstream Rb expression was not significantly altered. Dose-dependent decrease in the expression of the cell survival protein survivin was also observed after CDK4 inhibition. To be noted, due to

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palbociclib only inhibiting CDK4/6 activity, but not the production, the Western blot showed

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that palbociclib had no influence on the expression of both CDK4 and CDK6 (Fig. 3E, F). 3.4. CDK4 inhibition by siRNA decreased human osteosarcoma cell proliferation

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Palbociclib inhibits the activity of both CDK4 and CDK6, making the efficacy of palbociclib

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treatment and CDK4 inhibition on osteosarcoma cell proliferation decrease not fully equivalent. To further validate the role of CDK4 in osteosarcoma cell proliferation and growth, we knocked

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down CDK4 expression using CDK4 specific siRNA and investigated the alteration of

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osteosarcoma cell viability, as well as the CDK4/6-Rb-apoptosis pathway. As shown by the MTT, after transfection with increasing concentrations of CDK4 siRNA for three days, the cell

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viability was dose-dependently inhibited in both U2OS and KHOS cells, which was not observed

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in the nonspecific siRNA transfected cells (Fig. 4A and 4B). The Western blot showed that CDK4 siRNA transfection significantly inhibited CDK4 expression, whereas the expression of

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CDK6 was not influenced. Consistent with palbociclib treatment, CDK4 siRNA transfection inhibited the CDK4/6-Rb-apoptosis pathway in dose-dependent manners, which demonstrated that both pRb and survivin levels were decreased, whereas Rb expression was not dramatically altered (Fig. 4C and 4D). 3.5. CDK4 inhibition induced human osteosarcoma cell cycle arrest and apoptosis

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ACCEPTED MANUSCRIPT To explore the underlying mechanisms that decrease osteosarcoma cell proliferation and growth by CDK4 inhibition, we examined the alterations of cell cycle and apoptosis by flow cytometry after palbociclib treatment. Due to high concentrations of palbociclib resulting in pronounced cell death, we employed a compound dose of 1 µM for cell cycle and apoptosis

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determination. The cell cycle analysis showed that after 1 µM of palbociclib treatment for 24

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hours, a significant G1 cell cycle arrest accompanied by reductions in the fraction of cells in S

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phase were observed in both U2OS and KHOS cells (Fig. 5A), suggesting that CDK4 inhibition was able to induce osteosarcoma cell cycle arrest in G1 phase and inhibit DNA synthesis. The

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cell apoptosis analysis demonstrated significantly increased apoptosis rates in both U2OS and

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KHOS cells after palbociclib exposure for 48 hours, as compared with the control group (Fig. 5B). Collectively, these results indicated that reduced osteosarcoma cell proliferation by CDK4

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inhibit is associated with cell apoptosis induction and cell cycle arrest.

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3.6. CDK4 inhibition reduced human osteosarcoma cell migration in vitro

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The major cause of mortality in the treatment of osteosarcoma is metastasis from the sites of

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primary tumors to other organs. Tumor cell motility is crucial for cancer metastasis. As the TMA results showed that CDK4 expression significantly correlated with the metastasis status of

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osteosarcoma patients, we further evaluated the role of CDK4 in human osteosarcoma cell migration in vitro. The wound healing assay was performed in osteosarcoma cells after palbociclib treatment. As illustrated in Fig. 6, after exposure to 1 µM of palbociclib for 24, 48, and 72 hours, the cell migration activities were time-dependently repressed in both U2OS and KHOS cells, as compared with the palbociclib-free control groups (P < 0.01). 4. Discussion

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ACCEPTED MANUSCRIPT CDK4 has been identified recently as a potential therapeutic target in human breast cancer, liposarcoma, melanoma, and glioblastoma[37-39]. Due to the importance of CDK4 activity in cancer cells, CDK4 inhibitors have emerged as promising candidates for the treatment of many cancer types[11]. In the current study, we aimed to explore the expression and the therapeutic

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potentials of CDK4 in osteosarcoma.

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We first determined CDK4 expression in both osteosarcoma tissues and cell lines and found CDK4 was highly expressed in most of the tested osteosarcoma tissue samples and in all the

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human osteosarcoma cell lines. These results suggest that CDK4 may be a critical player in osteosarcoma growth and proliferation. We further explored the relationship between CDK4

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expression and the clinicopathological characteristics of osteosarcoma by using TMA containing

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72 tissue samples from 54 osteosarcoma patients with complete clinical information, as well as up to 252 months of follow-up data. Our results showed that CDK4 expression significantly

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correlated with the metastasis status and clinical prognosis of osteosarcoma patients. Previously,

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CDK4 amplification has been found associated with a poor prognosis in liposarcoma patients[40]. Ewing sarcoma cells also require CDK4 for survival and anchorage-independent growth.

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Knockdown of CDK4 abrogated proliferation and transformation of fusion-gene positive

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rhabdomyosarcoma cells via G1 phase cell cycle arrest[41]. More recently, overexpression and activation of the CDK4/6 and Rb pathways have been found in chordoma, another rare type of sarcoma[42]. These findings together support that CDK4 plays important role in the development and progression in different sarcomas including osteosarcoma. Subsequently, we explored the functional roles of CDK4 in osteosarcoma cell proliferation and growth in vitro. We first inhibited CDK4 in osteosarcoma cells by the clinically approved CDK4 inhibitor palbociclib and investigated the cellular phenotypic alternations. Our findings 16

ACCEPTED MANUSCRIPT demonstrated that CDK4 inhibition by palbociclib decreased osteosarcoma cell proliferation and growth in a dose-dependently manner, but exhibited only mild inhibition on CDK4-lower expressed NHOst cells. We further specifically knocked down CDK4 expression using CDK4 specific siRNA and determined osteosarcoma cell viability. Consistently, CDK4 inhibition by

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siRNA reduced osteosarcoma cell growth dose-dependently. These studies collectively suggest

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that CDK4 plays a crucial role in osteosarcoma proliferation and growth.

As an important cell division modulator, CDK4 exerts its functional role mainly through

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phosphorylation of Rb and promotion of the transition from G1 to S phase of the cell cycle[43]. In response to mitogenic signals, CDK4/6 combines with cyclin D and transfers into the nucleus.

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Then, the cyclin D-CDK4/6 complex phosphorylates Rb into pRb and releases the E2F

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transcription factor, which activates downstream target genes and promotes cell cycle progression, and cell proliferation and growth[30]. In human cancer, the cyclin D-CDK4/6-Rb

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pathway is universally disrupted[44, 45]. Thus, we determined the alternations of respective

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protein expression after CDK4 inhibition by palbociclib. We found that both palbociclib and CDK4 siRNA dose-dependently inhibited the downstream Rb phosphorylation and decreased

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cell survival protein expression. This finding implies that pRb and survivin act as positive

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regulators of cell proliferation and growth via the same signaling pathway, which has been observed in other studies[46-48]. To be noted, due to palbociclib mainly inhibiting CDK4 activity, but not its expression, the Western blot showed that palbociclib had no influence on CDK4 amount, but effectively inhibited CDK4 downstream Rb phosphorylation and downregulated survivin expression. Regarding survivin repression by palbociclib, it might be mediated by E2F. Several previous studies have shown E2F downregulating survivin via direct binding to the survivin promoter and inducing survivin transcription[49, 50]. 17

ACCEPTED MANUSCRIPT To elucidate the potential mechanism underlying osteosarcoma cell growth decrease by CDK4 inhibition, flow cytometry analysis was used to determine cell cycle and apoptosis in human osteosarcoma cells after palbociclib treatment. The results showed that osteosarcoma cells were arrested in G1 phase of the cell cycle after CDK4 inhibition by palbociclib. Cell apoptosis

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determination simultaneously revealed that CDK4 inhibition dramatically induced cell apoptosis

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in osteosarcoma cells. Hence, we propose that CDK4 inhibition by palbociclib decreases

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osteosarcoma cell proliferation and growth through induction of cell apoptosis via arresting the

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cell cycle in G1 phase.

TMA analysis revealed that CDK4 expression positively correlated with metastasis in

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osteosarcoma patients. We further assessed the influence of CDK4 inhibition on osteosarcoma

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cell migration in vitro. Treatment of osteosarcoma cells with lower doses of palbociclib illustrated that CDK4 inhibition reduced osteosarcoma cell migration, suggesting that CDK4

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may be a contributor to osteosarcoma metastasis, which is the main obstacle in the treatment of

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osteosarcoma.

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Taken together, our current study demonstrates that CDK4 is highly expressed in osteosarcoma, and elevated CDK4 expression correlates with metastasis and poor outcome in osteosarcoma

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patients. CDK4 inhibition decreases osteosarcoma cell proliferation and growth through apoptosis induction via cell cycle arrest. These findings, together with the potency of palbociclib, highlight CDK4 as a potential therapeutic target and palbociclib as a promising candidate agent for osteosarcoma treatment. Acknowledgements

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ACCEPTED MANUSCRIPT This work was supported by a Joint Research Fund devoted to clinical pharmacy and precision medicine (Z.D and Q.K). Support has also been provided by the National Natural Science Foundation of China (No.: 81402266, 81372875, 31670895), the Gattegno and Wechsler funds, Medical Science and Technology Research Projects of Henan Province (No.: 201403074,

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201501002, 2015009) and Henan Scientific and Technological Research Projects (No.:

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162102410057, 161100310100). Dr. Zhou is supported by a scholarship from the China

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Scholarship Council. Conflict of Interest

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The authors declare no conflict of interest.

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Figure legends

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Fig. 1. CDK4 was highly expressed in human osteosarcoma cell lines and tissues. (A) Levels of

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CDK4 and downstream proteins expression in two normal human osteoblast cell line HOB-c and NHOst and five osteosarcoma cell lines U2OS, KHOS, MNNG/HOS, 143B, and MG-63 were

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detected using Western blot. (B) Expression of CDK4 in U2OS and KHOS cells was assessed by

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immunofluorescence with antibodies to CDK4 and actin. Cells were visualized under a fluorescence microscope after incubation with secondary fluorescent conjugated antibodies Alexa Fluor 488 goat anti-rabbit IgG (green) or Alexa Fluor 594 goat anti-mouse IgG (red). (C) Eight human osteosarcoma tissues were lysed and immunoblotted to determine CDK4 and downstream proteins expression, with actin as an internal reference. Fig. 2. CDK4 expression levels correlated with clinicopathological characteristics of osteosarcoma patients. CDK4 levels in human osteosarcoma tissue microarray were determined 22

ACCEPTED MANUSCRIPT by immunohistochemistry, and the correlation of CDK4 expression with clinicopathological characteristics of osteosarcoma patients was evaluated. (A) Distribution of CDK4 staining scores among survivor and non-survivor osteosarcoma tissues. (B) Kaplan-Meier survival curve of osteosarcoma patients with CDK4 low staining (≤2) or high staining (≥3). (C) Distribution of

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CDK4 staining scores in the osteosarcoma tissues from non-metastasis patients and metastasis

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patients. (D) Distribution of CDK4 staining scores in the primary tissues and the metastasis

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tissues. (E) Representative images of different immunohistochemical staining intensities of CDK4. On the basis of the percentage of cells with positive nuclear staining, CDK4 staining

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patterns were categorized into 6 groups: 0, no nuclear staining; 1+: <10% of positive cells; 2+,

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10%-25% of positive cells; 3+, 26%-50% of positive cells; 4+, 51%-75% of positive cells;

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5+, >75% of positive cells. Original magnification 400×. Fig. 3. CDK4 inhibition by palbociclib decreased human osteosarcoma cell proliferation. Human

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osteosarcoma U2OS and KHOS cells, and human osteoblast NHOst cells were treated with

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increasing concentrations of palbociclib for the indicated time, and cell proliferation and growth was determined subsequently. (A, B and C) Cell viability was determined by MTT assay after

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palbociclib exposure for 2, 4, and 6 days. (D) The morphologic changes of cells were observed

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in microscope after 4 days of palbociclib treatment (Original magnification 100×). (E and F) The expression of respective proteins in CDK4/6-Rb-apoptosis pathway in cells was examined by Western blot after 3 days of palbociclib treatment. Fig. 4. CDK4 inhibition by siRNA decreased human osteosarcoma cell proliferation. Human osteosarcoma U2OS and KHOS cells were transfected with increasing concentrations of CDK4 specific siRNA or nonspecific siRNA for 3 days, and cell proliferation and growth was determined subsequently. (A and B) Cell viability was determined by MTT assay after siRNA 23

ACCEPTED MANUSCRIPT transfection. (C and D) The respective proteins of CDK4/6-Rb-apoptosis pathway in cells were examined by Western blot after 3 days of siRNA transfection. **P < 0.01 compared with the cell only group. Fig. 5. CDK4 inhibition induced human osteosarcoma cell cycle arrest and apoptosis. After

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exposure to 1 µM of palbociclib for 24 and 48 hours, the cell cycle and apoptosis of U2OS and

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KHOS cells was assessed by flow cytometry analysis. (A) Representative images of cell cycle distribution alterations in U2OS and KHOS cells, respectively, after palbociclib treatment. Cell

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numbers in different cell cycle phases were counted. *P < 0.05, **P < 0.01 compared with the cell only group. (B) Representative images of cell apoptosis in U2OS and KHOS cells,

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respectively, after palbociclib treatment. Cell apoptosis rate was analyzed. **P < 0.01 compared

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with the cell only group.

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Fig. 6. CDK4 inhibition reduced human osteosarcoma cell migration in vitro. After exposure to 1 µM of palbociclib for the indicated time, the cell migration of U2OS and KHOS cells was

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determined by wound healing assay. (A) Representative images of U2OS cell migration after

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palbociclib treatment for 24, 48, and 72 hours (Original magnification 100×). (B) Cell migration distance of U2OS cells was measured after palbociclib treatment. (C) Representative images of

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KHOS cell migration after palbociclib treatment for 24, 48, and 72 hours. (D) Cell igration distance of KHOS cells was measured after palbociclib treatment (Original magnification 100×). **P < 0.01 compared with the cell only group.

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ACCEPTED MANUSCRIPT Highlights:

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CDK4 is highly expressed in human osteosarcoma tissues and cell lines Elevated CDK4 correlates with clinicopathological features of osteosarcoma patients CDK4 inhibition decreases osteosarcoma cell proliferation, growth and migration CDK4 inhibition induces osteosarcoma cell cycle arrest and apoptosis CDK4 may be a promising therapeutic target for osteosarcoma treatment

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