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Master's Thesis

in the Department of Biomedical Sciences

Therapy Induced Senescent Tumor Cells Increase Cancer Stemness.

Ajou University Graduate School

Cancer Biology

Young Sam Kim

Therapy Induced Senescent Tumor Cells Increase Cancer Stemness.

Tae Jun Park, Advisor

I submit this thesis as the

Master's thesis in the Department of Biomedical Sciences

February, 2021

Ajou University Graduate School

Cancer Biology Major

Young Sam Kim

i

i -Abstract-

Therapy Induced Senescent Tumor Cells Increase Cancer Stemness.

Cellular senescence is defined as permanent cell cycle arrest (Alessio et al., 2019). Under the stressful condition such as physical and chemical insult, cell eventually turns into senescence (Kong et al., 2019). Therapy induced senescence is a type of cellular senescence induced by irradiation or chemotherapy (Joyner et al., 2006). Several previous studies showed increased expression of stem cell related properties with cellular senescence (Tsolou et al., 2019). Here, we proceeded research to prove cellular senescence induced by doxorubicin increases cancer cell stemness in human breast cancer. Senescence associated β-galactosidase assay and quantitative polymerase chain reaction analysis revealed BT474, a breast cancer cell line, showed senescence phenotype after treatment of doxorubicin expressing p16^INK4A (Loh et al.). Following qPCR and western blot analysis also revealed a stem cell marker CD133 was increased with the senescence marker p16^INK4A after treatment of doxorubicin (Dai et al., 2018) Flow cytometry showed the proportion of CD133 and p16^INK4A double- positive cells was increased after doxorubicin treatment. In this regard, senescent tumor cells caused by chemotherapy increase cancer cell stemness which is closely related to cancer relapse (Dou and Berger, 2018). It is anticipated that targeting senescent tumor cells is a new target to prevent cancer relapse.

Key words: cellular senescence, breast cancer, stemness, doxorubicin, relapse.

ii

Table of Contents

ABSTRACT ‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧i TABLE OF CONTENTS ‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧ ii LIST OF FIGURES ‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧ iii

Ⅰ. INTRODUCTION ‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧ 1

Ⅱ. MATERIALS & METHODS ‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧4 1. Cell culture ‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧4 2. Senescence Associated Beta Galactosidase assay‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧4 3. Western blot analysis‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧4 4. Reverse transcriptional Polymerase Chain Reaction ‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧5 5. Flow Cytometry ‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧6 6. Transfection ‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧6 7. Statistical analysis ‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧6

Ⅲ. RESULT

1. Screening of SA-β-Gal in Breast Cancer Cell Lines. ‧‧‧‧‧‧‧‧‧‧‧‧‧‧^{‧‧‧‧‧‧‧‧}7 2. Doxorubicin Induces Cellular Senescence in BT474. ‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧10 3. Dox Induces Increased Expression of Both Senescence and Stem Cell Markers

in BT474 ‧‧‧‧‧‧‧‧‧‧‧‧‧‧ ‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧ 13 4. Dox Induces Increased Expression of Both Senescence and Stem Cell Markers

after GFP-p16^INK4A Vector Infection. ‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧16

Ⅳ. DISCUSSION ‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧19

Ⅴ. REFERENCES ‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧ 21

iii

국문요약 ‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧ 24

LIST OF FIGURES

▶ Figure 1. Screening of SA-β-Gal in Breast Cancer Cell Lines. ‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧ 8

▶ Figure 2. Quantification of SA-β-Gal in Breast Cancer Cell Lines ‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧9

▶ Figure 3. SA-β-Gal of BT474 after Doxorubicin treatment‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧11

▶ Figure 4. qPCR and western blot analysis for senescent markers in BT474 after Dox‧‧12

▶ Figure 5. qPCR and western blot analysis of BT474 after Dox treatment‧‧‧‧‧‧‧‧‧‧‧‧‧14

▶ Figure 6. Flow cytometry analysis after Dox treatment in BT474‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧ 15

▶ Figure 7. GFP-p16^INK4A was introduced to BT474 by lentivirus infection‧‧‧‧‧‧‧‧‧‧‧‧ 17

▶ Figure 8. Western blot analysis for p16^INK4A-GFP stable cell line‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧18

▶ Figure 9. Schematic image of relationship between cellular senescence and cancer stemness in cancer relapse‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧20

１

Ⅰ. Introduction

Breast Cancer

Breast cancer ranks the most common cancer in women and the fourth in both sexes in South Korea (Ministry of Health and Welfare, 2019). Although survival rates have increased from past few years, its high incidence and their heterogeneity make it hardly be cured (Chakrabarty et al., 2019). The most profound characteristic of breast cancer is the expression of specific receptors on their cell surface: estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2 (HER-2) (McDermott et al., 2019). According to their positivity in immunohistochemistry analysis, treatment option can be changed (Ikeda et al., 2000). Doxorubicin (Dox) is commonly used chemotherapeutic drug to treat breast cancer patients with triple-negative to these receptors (Ikeda et al., 2000). Dox is a type of anthracyclines which is made from the bacterium streptomyces (Goldhirsch et al., 2001). It inhibits topoisomerase-2 after entering cancer cells and be inserted into the DNA (Linassier et al., 2000).

Cellular Senescence

Cellular senescence implies permanent cell cycle arrest (Dou and Berger, 2018). When a normal cell turns into a senescent cell, the size of both cell and nucleus increase (Kim and Park, 2019). As the size of a cell grows, the granular contents of a cell increase (Fernandez- Duran et al., 2019). Together with the increased number of lysosomes, a part of granular contents of the cell, the number of lysosomal β-galactosidase increases in a senescent cell (Debacq-Chainiaux et al., 2009). Therefore, the most well-known method to detect senescent cells is senescence associated-β-galactosidase assay (SA-β-Gal) which stains lysosomal β- galactosidase (Debacq-Chainiaux et al., 2009). Another major feature of senescent cells is the up regulation of SASP genes (Martinez et al., 2015). These include several cytokines, chemokines, and growth factors (Buhl et al., 2019). Cells adjacent to a senescent cell affected

２

by SASPs and even can be fallen into senescence (Fernandez-Duran et al., 2019). There are several types of cellular senescence. Firstly, replicative senescence is due to the shortening of the length of a telomere (Hares et al., 2018). As a person is getting older, exonal region can be damaged due to shortening of telomere and finally cell became senescent (Herbig et al., 2004). Secondly, DNA damaged by chemical factors such as reactive oxygen species can activates cell cycle related genes such as p53, p16^INK4A, and p21 (Capparelli et al., 2012).

Thirdly, oncogene induced senescence (OIS) is induced by activation of oncogenes (Fernandez-Duran et al., 2019). Oncogenes are cancer-causing genes which are closely related to cell cycle control such as c-Myc, Raf, and Ras (Chakrabarty et al., 2019). Lastly, therapy induced senescence (TIS) is the type of cellular senescence induced by irradiation or chemotherapy (Joyner et al., 2006).

Cellular Senescence in Cancer and Stemness

In our previous study, a mechanism of senescent tumor cells and the role of SASPs promoting local invasion in thyroid cancer was introduced (Kim et al., 2017). Senescent tumor cells in thyroid cancer increases invasiveness and lymph node metastasis. On the other hand, senescence has long been believed that it acted as a protector against cancer progression (Munoz-Galvan et al., 2019). Senescent cells are often detected in adenomatous lesion and the early stage of cancer as a protective manner against massive proliferation (McDermott et al., 2019). Therefore, cellular senescence has dual role as protective effect against cancer development, at the same time, as the part of cancer progression (Zhang et al., 2018). Cancer stemness stands for the capacity of a cell to keep its lineage, to give rise to stemness cells, and to interact with its environment to keep a balance between quiescence, cell growth, and regeneration (Dou and Berger, 2018). While adult stem cells display these feature when participating in tissue homeostasis, cancer stem cells (CSCs) conduct as their malignant equivalents (Dai et al., 2018). Therefore, CSCs reveal stemness in various condition, including the maintenance of tumor growth, and the interaction with their environment in search for main survival factors (Pustavoitau et al., 2016). These findings have magnificent

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effect for cancer therapy providing new mechanistic insights into the plasticity of cancer cells (Ritschka et al., 2017). There are several previous studies which showed the relationship between cellular senescence and cancer stemness (Munoz-Galvan et al., 2019). From this background, we tried to investigate the relationship between senescence and stemness in breast cancer. Moreover, we tried to elucidate the difference of tumor initiating ability between cells with stem cell marker and with both senescence and stem cell markers.

Understanding the development and acquirementof senescence and cancer stem cell property in cancer cells may provide opportunity for more useful therapies (Seo et al., 2018).

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Ⅱ. Materials & Methods

Cell Culture

Human breast cancer cell, MCF7, MDA-MB231, T-47D, BT-474 was purchased from ATCC (Rockville, MD) and cultured in RPMI 1640 medium (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (Invitrogen) and antibiotics (Invitrogen) at 37℃

in a humidified incubator with 5% CO2. To generate doxorubicin induced senescent cells, cells were treated with doxorubicin (Adriamycin PFS, D06001, Ildong Pharmaceutical Co., Ltd., Seoul, Korea) for 2days at indicated concentration, then recovered in normal medium for 3days.

SA- β-Gal staining

The cells were fixed with 4% paraformaldehyde for 15 min and then incubated with SA-β- Gal solution (X-gal, 1 mg/mL; citric acid/sodium phosphate, pH 5.8, 40 mM; potassium ferrocyanide, 5 mM; potassium ferricyanide, 5 mM; NaCl, 150 mM; MgCl2, 2 mM) for overnight at 37 °C. After PBS washing, SA-β-Gal-positive cells were analyzed under light microscopy. By counting the numbers of the blue-colored and total cells under using Image J software (NIH, Bethesda, MA, USA), percentage of the blue stained cells was estimated to compare the degree of senescence-associated cells.

Western Blot Analysis

Cells were lysed in RIPA buffer (20 mM Tris-HCl (pH7.4), 150mM NaCl, 1% Triton X- 100, 0.1% SDS, 1 mM EDTA) contained protease inhibitor cocktail (Catalog no. K272, Bio vision, Milpitas, USA) and phosphatase inhibitor cocktail (Catalog no. K282, Bio vision, USA). The cell lysates were centrifuged for 15 min at 13,000 rpm and supernatant solution proceed with protein quantification. Protein quantification was carried out using Bradford

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assay (Catalog no. 5000006, Bio Rad, Carlsbad, USA). The boiled protein was separated by SDS- polyacrylamide gel and transferred to the PVDF membrane (Millipore, Billerica, MA).

and blocked using 5% BSA solution for 1h at room temperature. The membranes were then incubated overnight at 4°C with the following primary antibodies. Antibodies for p16 (Catalog no. ab54210) and CD133 (Catalog no. ab19898) were purchased from Abcam (Cambridge, UK). Antibodies against p21 (Catalog no. SC-6246), GFP (Catalog no. 33-2600) and β- actin (Catalog no. MA1-140) were obtained from Santa Cruz (Dallas, TX), Thermo Fisher (Waltham, USA) and Invitrogen (Carlsbad, USA), respectively. The membranes were then incubated with horseradish peroxidase-conjugated anti-mouse (Catalog no. 170-6510, Bio Rad, Carlsbad, USA) or anti-rabbit (Catalog no. 170-6515, Bio Rad, Carlsbad, USA) antibody for 1h. Immunoreactive proteins were visualized using enhanced chemiluminescent (ECL) system (Catalog no. 1705061, Bio Rad, Carlsbad, USA) and X-Ray film (Catalog no.

PCG0810, Agfa, Mortsel, Belgium).

Real-time Polymerase Chain Reaction

Total RNA was isolated using the NucleoSpin^TM RNA kit (Macherey-Nagel GmbH & Co.

KG, USA), according to the instruction provided. Equal amounts of RNA were subjected to reverse transcription using Super Script IV Reverse Transcriptase (Invitrogen, Waltham, MA) according to the recommendations of the manufacturer. PCR reaction was performed using IQ SYBR Green Super Mix (Bio-Rad, Hercules, CA) and primer sets for target genes.

The PCR primer sets were produced by Cosmo Gene Tech (Seoul, Korea) as follows:

CD133 (5’- ACC AGG TAA GAA CCC GGA TCA A -3’, 5’- CAA GAA TTC CGC CTC CTA GCA CT -3’), CD44 (5’-CCA GAA GGA ACA GTG GTT TGG C -3’, 5’-ACT GTC CTC TGG GCT TGG TGT T -3’), p16 (5’- CCC AAC GCA CCG AAT AGT TA -3’, 5’- TCC TGC GTG TCC AGG AAG -3’), β-actin (5’-CCC TGG CAC CCA GCA C -3’, 5’-GCC GAT CCA CAC GGA GTA C -3’).

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Flow Cytometry

The cells were fixed with 70% ethanol a day before the experiment. A sample was prepared to proceed with the experiment. The primary antibodies used for p16 and CD133, and the secondary antibodies Alexa-488, anti-Rabbit (Catalog no. A11304, Invitrogen), Dylight650, anti-Mouse (Catalog no. #8455, Thermo Fisher) were used. Antibodies used in 3% bovine serum albumin (Catalog no. IC10503380, MP). Divide them into Eppendorf tube enough to check the fixed cells and put them on a centrifuge for one minute at 13,000 rpm. Supernatant was removed and the primary antibodies 100 μl each were introduced. After 1 hour of cooling, the samples were centrifuged in the 13,000 rpm 1minute and the supernatant was removed.

After cleaning, the secondary antibody was introduced in the refrigeration for 30 minutes.

After 30 minutes, the samples were washed. The samples were analyzed by flow cytometry machine (BD Science).

Production of Recombinant Lentiviruses and Generation of Stably Expressing Cell Clones

To generate the lentiviruses containing p16 GFP vector, GFP was inserted downstream of the promoter of a INK4A gene. HEK-293TN cells were transfected with plasmid DNA (pGag- pol, pVSV-G, and p16 GFP vector) using lipofectamine (Invitrogen, Carlsbad, USA). Viral supernatant was collected after 48 h and purified by filtration and transduced into BT474.

After cells were infected with the lentiviruses, stably p16 GFP expressed cells were selected with Puromycin 100 nM (Sigma aldrich, Saint Louis, USA).

Statistical Analysis

The data are values expressed as average values. In addition, an error bar and a standard deviation were displayed along with the mean value. t-test was applied to confirm statistical significance between groups. If the p value is less than or equal to 0.05, * denotes a statistical significance of *, p<0.05, **, p<0.01.

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Ⅲ. Result

1. Screening of SA-β-Gal in Breast Cancer Cell Lines

To induce TIS in human breast cancer cell lines, Dox was introduced (Figure 1A). Since too low concentration of doxorubicin could not induce senescence, and too high concentration lead to cell death, 100 nM concentration were chosen (Data not shown) (Joyner et al., 2006).

After treating doxorubicin, SA-β-Gal was introduced to confirm their senescence phenotype (Figure 1B). All cell lines showed SA-β-Gal positivity around 30-40 % out of the total cell number (Figure 2). However, MCF7, MDA-MB231 and T47D were not able to be used in following experiments because p16^INK4A was not detected in qPCR analysis (data not shown). Previous study already revealed that these breast cancer cell lines’ p16 region is already deleted or methylated (Gonzalez et al., 1997).

８

A.

B.

Figure 1. Screening of SA-β-Gal in Breast Cancer Cell Lines

(A) Experimental scheme (B) SA-β-gal staining in breast cancer cell lines MCF7, MDA- MB231, and T47D. Blue-stained cells are SA-β-Gal positive. Quantification of SA-β-Gal.

Scale bars indicate 200 μm.

Cell seeding

Dox treatment

Media change

SA-β-Gal staining&

Cell harvest

Realtime PCR &

Western blot

& FACS

1day 2days 3days

Con

Con Dox 100nM

MCF7MDA-MB231 T47D

Con Dox 100nM

Dox 100nM

９

Figure 2. Quantification of SA-β-Gal in Breast Cancer Cell Lines

All breast cancer cell lines showed SA-β-Gal positivity after treating Dox. The highest proportion of senescent cell was 37% in MDA-MB231 after Dox treatment. All cell lines constituted from at least 30% senescent cells after treating Dox.

*

１０

2. Doxorubicin Induces Cellular Senescence in BT474

As other breast cancer cell lines, 100nM and additional 150nM of Dox was introduced to induce senescence in BT474. SA-β-Gal result revealed the presence of lysosomal β- galactosidase in BT474 after treating Dox (Figure 3A). Quantification of SA-β-Gal showed positive cells increased as the concentration of Dox increased (Figure 3B). Subsequent qPCR analysis showed senescent BT474 expressed increased amount of p16 mRNA (Figure 4A).

Furthermore, western blot analysis revealed senescent BT474 expresses increased p16 and p21 proteins (Figure 4B). All experiments considered, Dox effectively induced cellular senescence in BT474.

A.

Control Dox 100nM

200um 200um 200um

Dox 150nM

200um 200um 200um

１１ Figure 3. SA-β-Gal of BT474 after Dox treatment

(A) SA-β-Gal of control BT474, Dox 100nM, and 150nM (B) Quantification of SA-β-Gal

B.

１２

Figure 4. qPCR and western blot analysis for senescent markers in BT474 after Dox treatment

(A) mRNA expression of p16^INK4A and p21 increased after 100 nM and 150 nM of Dox treatment (B) Western blot analysis shows increased protein level of p16^INK4A and p21 after treating Dox.

B.

A.

p16^INK4A (16kDa)

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3. Dox Induces Increased Expression of Both Senescence and Stem Cell Markers in BT474

mRNA level for senescent and stem cell markers of BT474 was examined through qPCR analysis. Primers for senescence marker p16^INK4A and stem cell markers, CD44 and CD133, were used (Capparelli et al., 2012; Kong et al., 2019). It revealed that Dox treatment increases intracellular mRNA level of both senescence marker and stem cell markers (Figure 5A).

Western blot analysis also revealed that Dox treatment increases protein levels of p16^INK4A, p21, and CD133 in BT474 (Figure 5B) (Pustavoitau et al., 2016). Based on the findings, we conducted a flow cytometry to determine the proportion of the cells with senescence marker and stem cell marker after Dox treatment (Figure 6). It was revealed that the proportion of the cells with having both senescence and stem cell marker increase after Dox treatment (0.98%

vs 28.98% vs 43.08%). Moreover, cells with senescence marker only increased as Dox concentration increased (0.39% vs 3.98% vs 5.73%). Likewise, cells with stem cell marker only also increased after Dox treatment (0.22% vs 2.3% vs 2.97%). Therefore, this result implies stem cell feature increases with the senescence trait after chemotherapy in breast cancer.

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p16 (16kDa)

CD133 (110kDa) Beta-actin (42kDa)

p21 (21kDa)

A

Figure 5. qPCR and western blot analysis of BT474 after Dox treatment

(A) mRNA expression level of p16^INK4A (senescence marker) and CD44 & CD133 (stem cell markers) were analyzed by qPCR analysis (B) Protein expression level of p16^INK4A and p21 (senescence markers) and CD133 (stem cell marker) were increased simultaneously after treatment of Dox

0.0 0.5 1.0 1.5 2.0 2.5

Con Dox

100nM Dox 150nM p16Ink4a mRNA expression (Fold induction)

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0

Con Dox

100nM Dox 150nM CD44 mRNA expression (Fold induction)

0.0 0.5 1.0 1.5 2.0 2.5 3.0

Con Dox

100nM Dox 150nM CD133 mRNA expression (Fold induction)

(*p<0.05 vs Control)

* *

(*p<0.05 vs Control)

(*p<0.05 vs Control)

*

*

*

*

*

*

*

B

p16^INK4A (16kDa)

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Figure 6. Flow cytometry analysis after Dox treatment in BT474

Flow cytometry result of (A) Control BT474 (B) BT474 with 100nM of Dox (C) BT474 with 150nM of Dox. The number indicates; 1: p16^INK4A positive and CD133 negative, 2: p16^INK4A positive and CD133 positive, 3: p16^INK4A negative and CD133 negative, and 4: p16^INK4A negative and CD133 positive.

2

3 4

1

0.39 % 0.98 %

0.22 % Control

CD133 p16^INK4A

2

3 4

1

3.98 % 28.68 %

2.3 % Dox 100nM

CD133 p16^INK4A

2

3 4

1

5.73 % 43.08 %

2.97 % Dox 150nM

CD133 p16^INK4A

A B

C

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4. Dox Induces Increased Expression of Both Senescence and Stem Cell Markers after GFP-p16

^INK4A

Introduction

To easily track cancer cells for in vivo experiment, GFP tagging to p16 ^INK4A promoter was introduced (Figure 7A). Fluorescence microscopic image for GFP revealed that successful GFP-tagging was applied in BT474 after lentivirus infection (Figure 7B). To confirm whether GFP-tagging system interrupted the expression of p16^INK4A and CD133, western blot analysis was conducted (Figure 8). It showed there is still stable expression of p16^INK4A, p21 and CD133 along with the GFP after Dox treatment. It implies that GFP-tagging system did not interrupt expression of senescence and stem cell phenotypes in BT474.

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A

B

Figure 7. GFP-p16^INK4A was introduced to BT474 by lentivirus infection

(A) Experimental scheme of p16^INK4A-GFP stable cell line introduction (B) Fluorescence microscopic image for GFP. Dox increases GFP signal intensity in BT474 p16^INK4A- GFP stable cell line.

BT474 - p16

^INK4A

GFP

Cell seeding

GFP infection

Media change

Puromycin selection

Doxorubicin treatment

qPCR &

Western blot

& FACS 1day 1day 2days selection 2days treatment 3days release

Media change

Media change

Dox 100nM Dox 150nM

200um

Control

200um 200um

200um 200um 200um

１８

Figure 8. Western blot analysis for p16^INK4A-GFP stable cell line

Senescence markers, p16^INK4A and p21, were still expressed after Dox treatment. Likewise, stem cell marker CD133 increased after treating Dox. GFP tagged with p16^INK4A promoter also increased with treating Dox.

p16^INK4A (16kDa)

β-actin (42kDa) GFP (30kDa)

CD133 (110kDa) p21 (21kDa)

１９

Ⅳ. Discussion

The several preceding papers demonstrated that stemness increases as cellular senescence is induced (Kim et al., 2017; Ritschka et al., 2017). Therefore, it is already known that senescence and stemness were very closely related. Likewise, Milanovic and his colleagues revealed that the relapse can be induced by the senescence and stemness (Milanovic et al., 2018). From this background, chemotherapy paradoxically increases stemness of survived cancer cells (Figure 7). Our ultimate goal is to compare tumor initiating ability of survived cancer cell groups from chemotherapy: (1) a group of cells having both senescence and stem cell trait and (2) the other group of cells with only stem cell trait.

First of all, we demonstrated that in BT474 senescence can be induced by Dox. SA-β-Gal, qPCR, and western blot analysis confirmed that senescence of BT474 can be induced by chemotherapy. Consistent with preceding papers, expression of stem cell marker CD133 increased with senescence markers, p16^INK4A and p21 after Dox treatment in western blot analysis (Capparelli et al., 2012). Flow cytometry also revealed that proportion of p16^INK4A and CD133 positive cells increased with Dox treatment. For successful in vivo experiment, fluorescence-positive cancer cells are needed. Therefore, we subsequently introduced GFP tagged p16^INK4A-promoter using lentivirus to BT474. Following experiments revealed that GFP system introduction did not interrupt the expression of senescence and stem cell markers.

Further study will be cell sorting cells through flow cytometry according to p16^INK4A and CD133 positivity. After sorting p16INK4A and CD133 double-positive and CD133 single- positive cells, cells will be injected to nude mouse to compare tumor initiating ability of two groups.

In conclusion, Dox successfully induced cellular senescence in BT474. Also, senescent BT474 showed increased stem cell property. Therefore, it is expected that targeting senescent tumor cells might be a key treatment to prevent relapse of breast cancer.

２０

Figure 9. Schematic image of relationship between cellular senescence and cancer stemness in cancer relapse

２１

V. REFERENCES

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25. Pustavoitau, A., Barodka, V., Sharpless, N.E., Torrice, C., Nyhan, D., Berkowitz, D.E., Shah, A.S., Bandeen Roche, K.J., and Walston, J.D. (2016). Role of senescence marker p16 INK4a measured in peripheral blood T-lymphocytes in predicting length of hospital stay after coronary artery bypass surgery in older adults. Exp Gerontol 74, 29-36.

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２４ -국문 요약-

치료에 의해 유도된 노화 종양세포는

암 줄기세포능을 증가시킨다.

세포 노화는 정상 세포가 더 이상 분열하지 못하는 상태를 의미한다. 즉, 정상 세포가 물리적 화학적 자극을 받아 유전자의 손상이 오면 노화 신호를 따라 노화 세포로의 변화가 일어난다. 노화된 세포는 세포 성장을 조절하는 세포 주기가 억제되어 세포 분열이 더 이상 일어나지 않는다. 노화가 진행되면 특정 물질들을 내뿜게 되는데 이것을 노화 관련분비 표현형 (senescence associated secretory phenotypes, SASPs) 이라 한다.

노화 관련 분비 표현형에는 염증관련 사이토카인과 성장인자들이 포함되며, 이들은 노화세포 주변의 미세환경 조성에 굉장히 중요한 역할을 한다.

세포 노화는 기본적으로 암과 매우 밀접한 관련이 있다. 암유전자는 암을 유발하는 인자로서, 이 유전자에 변형이 일어나게 되면 암에 대한 방어기전으로 인해 노화에 빠질 수 있다. 본 연구팀은 앞선 연구에서 노화가 유도된 노화종양세포는 갑상선암의 전이를 촉진한다는 것을 밝혔다. 따라서, 암에 대한 방어기전으로 이해되었던 세포노화가

오히려 암의 진행을 촉진시킬 수 있는 가능성이 제기되었다. 이러한 배경지식을

바탕으로, 본 연구에서는 항암치료로 유도된 노화종양세포가 암 줄기세포능을 증가시켜

암의 재발과 관련되어 있다는 가정 하에 실험을 진행하였다.

독소루비신은 유방암의 항암요법으로 흔히 선택되는 약제로서, 본 연구에서는

독소루비신을 사용하여 유방암세포주에 노화를 유도하였다. 여러 유방암세포주 중에서,

p16^INK4A에 대한 중합효소연쇄반응 검출이 가능한 BT474 유방암세포주를 최종적으로

선정하였다. 이어 진행한 실험을 통해, BT474가 안정적으로 노화가 되며 노화가 진행됨에 따라 줄기세포 마커인 CD133도 함께 증가함을 밝혔다. 이후 진행할 동물 실험에서 세포추적에 사용하기 위해 BT474의 p16^INK4A의 프로모터 부위에 GFP를 tagging하였고, 이 세포주에서도 독소루비신 처리에 따라 노화세포 마커와 줄기세포 마커가 함께 증가함을 증명하였다. 추후에는, 줄기세포 마커만 있는 세포와 줄기세포

２５

마커와 노화 마커가 모두 존재하는 세포군을 분류하고 마우스에 주입하여

종양형성능(tumor initiation ability, TIA)를 분석할 계획이다.

결론적으로, 이번 연구에서는 항암제로 인해 유방암세포가 노화가 일어날 수 있음을 밝혔다. 또한 노화가 일어남에 따라 줄기세포 특징도 함께 증가함을 밝혔다. 따라서 노화 마커와 줄기세포 마커를 가진 세포의 종양형성능에 대한 분석을 진행함으로써, 노화세포가 유방암의 재발을 막기위한 치료의 타겟이 될 수 있을 것으로 기대하고 있다.