Establishment and characterization of radiation resistant strains from squamous cell carcinoma cell lines in serum free defined culture

ABBREVIATIONS A431-HDR A431 radiation resistant cells established in high dose rate irradiation A431-LDR A431 radiation resistant cells established in low dose rate irradiation A431-WT A

Ph.D THESIS

Establishment and characterization of radiation resistant strains from squamous cell carcinoma cell lines in serum-free

defined culture

by

NGUYEN QUANG TAM

Department of Molecular Oral Medicine and Maxillofacial Surgery

Graduate School of Biomedical & Health Sciences

Hiroshima University

2018

ACKNOWLEDGEMENT

Foremost, I would like to express my deep gratitude to my Academic Supervisor Professor Tetsuji Okamoto, who has accepted me to study in the department of Molecular Oral Medicine and Maxillofacial Surgery and also in Phoenix Education Leading Program of Hiroshima University He always finds out my mistakes and teach me how to fix in a better way I have learned a lot from him that I must try my best in doing scientific research and never give up, also be creative in thinking

I would also like to thank Professor Chisa Shukunami, Professor Shinya Matsuura, Professor Satoru Endo and Professor Nobuyuki Chikudate for being my great co-advisers during my study

My deep thank is also extended to Professor Naoya Kakimoto, Professor Masaru Sugiyama and Assoc Professor Shigeaki Toratani for reviewing my thesis and their great comments

I would like to thank my instructor Dr Hamada Atsuko for teaching me all molecular techniques in my research and help me to analyze the data

I would like to thank all Associate and Assistant Professors in my lab, all my seniors,

my lab-mates for their supports in my research and other activities

My grateful thanks are also extended to Assoc Prof Tran Diep Tuan, Assoc Prof Ngo Thi Quynh Lan, and Dr Luong Van To My for introducing me to study in Hiroshima University and my colleagues, who are covering my works in Vietnam

Finally, I would like to extend my utmost gratitude to my family for their great encouragement when I study in Japan

TABLE OF CONTENTS

Summary

Chapter 1: Introduction ……….1

Chapter 2: Establishment and characterization of radiation resistant strains …………3

I Materials and methods .3

1 Cell culture & culture medium

2 Irradiation procedure for generation of radiation resistant (RR) strains

3 Colony survival assay for wild type (WT) and radiation resistant strains

4 Growth rate of WT and RR- strains

5 Sphere formation assay of WT and RR- strains

6 Expression of cancer stem cell marker CD133 in WT and RR- strains

7 Expression of pluripotent stem cell markers Nanog, Oct4, and Sox2 in WT and RR- strains by Real time-quantitative polymerase chain reaction (RT-qPCR)

8 Migration assay of WT and RR- strains

9 Tumor formation ability of WT and RR- strains in nude mice

10 Immunohistochemical examination of ki67 in nude mouse tumors derived from WT and RR- strains

11 Statistical analysis

II Results .9

1 Establishment of radiation resistant cancer cell strains and colony survival formation

2 Proliferation of the cells in serum-free monolayer culture

3 Sphere forming ability of the cells in serum-free suspension culture

4 Expression of cancer and pluripotent stem cell markers in WT and strains

RR-5 Migration ability of WT and RR- strains

6 Tumor formation ability of WT and RR-strains in nude mice

7 Immunohistochemical expression of ki67 in nude mice tumors derived from

WT and RR-strains

Chapter 3:

Identification and characterization of novel genes involved in radiation resistance … 12

I Materials and methods 12

1 DNA microarray analysis

2 Expression of IGF2 and krt13 in wild type and radiation resistant strains by

1 Results of DNA microarray analysis

2 Expression of IGF2 gene and protein in WT and RR-strains by RT-qPCR,

western-blotting and immunohistochemical staining

3 Expression of krt13 gene and protein in WT and RR-strains by RT-qPCR,

western-blotting and immunohistochemical staining

Chapter 4: Functional analysis of IGF2 and krt13 in radiation resistance 16

I Materials and methods 16

1 Effect of krt13 siRNA and IGF2 siRNA transduction on colony formation ability of RR-strains

2 Effect of radiation on IGF2-siRNA transduced-A431-LDR, -HDR,

NA-LDR and -HDR cells

3 Effect of radiation of krt13-siRNA transduced-A431-LDR and -HDR cells

4 Expression of pluripotent stem cell markers, krt13 and IGF2 in IGF2- and

krt13-siRNA transduced cells

5 Generation of stable transfectant of krt13 in A431 cells

6 Characterization of krt13-transfected A431 cell and its radiation resistant ability

II Results 19

1 Effect of various IGF2- and krt13-siRNA on silencing ability in the cells

2 Effect of radiation on colony survival formation of IGF2-siRNA transduced

A431-LDR, -HDR, NA-LDR and -HDR cells

3 Effect of radiation on colony survival formation of krt13-siRNA transduced

A431-LDR, -HDR cells

4 Expression of krt13, IGF2 and pluripotent stem cell marker genes in

IGF2-siRNA transduced A431-LDR

5 Expression of IGF2, krt13 and pluripotent stem cell marker genes in

krt13-siRNA transduced A431-LDR

6 Characterization of A431 cells overexpressing krt13 in serum-free culture

Chapter 5: Discussion 21

Chapter 6: Conclusion 27

References 29

Figure legends 36

ABBREVIATIONS

A431-HDR A431 radiation resistant cells established in high dose rate irradiation A431-LDR A431 radiation resistant cells established in low dose rate irradiation

A431-WT A431 wild type cells

CSCs cancer stem cells

DMEM Dulbecco’s modified Eagle’s medium

EDTA Ethylene Diamine Tetra Acetic acid

GAPDH Glyceraldehyde-3-phosphate dehydrogenase

IGF2 insulin-like growth factor 2

Jak/STAT Janus kinase/signal transducer of activation

krt13 keratin 13

MAPK mitogen-activated protein kinase

NA-HDR NA radiation resistant cell established in high dose rate irradiation

NA-LDR NA radiation resistant cell established in low dose rate irradiation

NA-WT NA wild type cell

OSCC oral squamous cell carcinoma

RT-qPCR Reverse Transcription-quantitative Polymerase Chain Reaction

SCC squamous cell carcinoma

siNC cells transfected with negative control siRNA

siRNA small interfering RNA

Summary Introduction

Squamous cell carcinoma (SCC), including oral squamous cell carcinoma (OSCC), has been increasing in the world and being the most common cancer in South-East Asian countries where have a betel-quid and areca-nut chewing habit It has been considered that cancer cells are functionally heterogeneous that undergo not only proliferation but also differentiation and maturation to a certain degree and contain a small population of cancer stem cells (CSCs) It seems logical that cures of cancer can be achieved only if the CSCs population is eliminated Several treatment modalities such as operation, radiation, and chemo therapy have been reported to be effective in treating many kinds of cancer including OSCC Among them, radiation therapy (RT) plays a major role in the management of OSCC Despite therapeutic and technological advances such as ionizing radiation, gamma rays and charged particles to kill cancer cells through DNA damage directly or indirectly caused by free ion radicals, some patients will have persistence of irradiated tumor or develop locoregional failure, resulting in significant morbidity and mortality

RT using high dose rate (HDR) radiation has been widely used as an effective modality

to treat human cancer by various types of modern delivering techniques On the other hand,

RT using low dose rate (LDR) radiation has been introduced in the treatment of prostate and oral cancers

Therefore, to elucidate the cellular and molecular mechanisms involved in radiation resistance of cancer cells upon radiation therapy using HDR or LDR might be worthwhile to develop effective therapies to circumvent radiation resistance In this work, mechanisms of radio-resistance to RT in squamous cell carcinomas including oral SCC and strategies used to overcome this resistance were studied To study that, it might be useful to establish radiation

resistant-cancer cells in in vitro Although there have been several reports of isolating radiation

resistant cancer cells, and their use to elucidate cellular and molecular mechanisms in radiation resistance, these radiation resistant (RR) SCC cells were isolated under serum-supplemented culture condition Serum-supplemented medium contains a lot of undefined or unknown

proteins, factors, and lipids which may exhibit unknown biological effects on cancer cells in

vitro Thus, serum-free defined medium can show exact biological characteristic of the cells

In this study, first I have tried to isolate and establish RR-SCC strains from SCC and OSCC cell lines in serum-free defined culture, and then characterized their cellular and molecular properties, and defined functional genes involved in radiation resistance

Materials and methods

Two SCC cell lines, A431 derived from vulvar SCC, and NA/HO-1-N-1 from OSCC were used in this study The cells were cultured in serum-free DF6F medium (1:1 mixture of Dulbecco’s Modified Eagle Media and Ham-F12 medium supplemented with insulin (10

g/ml), transferrin (5 g/ml), aminoethanol (10 M), sodium selenite (10 nM), mercaptoethanol (10 M), and oleic acid conjugated with fatty acid-free bovine serum albumin (9.4 g/ml))

2-All cell lines were irradiated weekly at a dose of 2.2Gy/day, 4 days/week with a low dose rate (LDR) irradiation system (RM1000, Chugai Technos, Japan), or at 5Gy/5.75 mins, twice

a week with a high dose rate (HDR) system (Gamma cell 40 Exactor, Best Theratronics, Canada) in serum-free defined culture After irradiating under 60Gy as a whole dose by LDR and HDR irradiation system, we have isolated 4 RR sub-strains from 2 SCC cell lines To confirm the radiation resistance of these RR-strains, colony survival assay was performed as follow The wild type (WT) and RR strains were irradiated at a dose of 0Gy, 2Gy, 4Gy, 6Gy, and 8Gy, respectively, and examined radiation resistance by colony survival assay After 14 days of culture, the colonies were stained with Giemsa, and counted

To clarify the biological properties of these cells, several cellular abilities such as growth

in monolayer culture, sphere formation in suspension culture, and migration in chamber method were examined in serum-free culture For the growth assay, the cells were seeded at 104cells/well in 24-well plate and counted cell numbers every day by the Coulter Counter For sphere formation assay, the cells were seeded in 35mm prime surface (low-attachment) dish at 103cells/dish, and then sphere numbers were counted on day 5 For migration assay, the cells were seeded at 5x104cells/well in 24-well collagen coated chemotaxicell well in DF medium supplemented with 0.1% BSA and stained with Giemsa for counting number of migrated cells/mm2 The ratio of CD133 positive cell in each cell line was examined by flowcytometry as cancer stem cell marker

Boyden-Total RNAs extracted and purified from all cell lines, were used for Real

Time-quantitative PCR (RT-qPCR), and DNA microarray analysis Expression of pluripotent stem

cell markers Nanog, Oct4 and Sox2 in WT and RR-strains in WT and RR-strains was examined

with RT-qPCR

To study the tumor forming ability in nude mouse, WT and RR-strains (0.25 x106 -1x106 cells/100l DF) were injected subcutaneously to the dorsal back skin of nude mice (BALB/c-nu/nu), and tumor size was measured every week Then the tumors were excised, weighted, fixed in 4% paraformaldehyde for 24hr, and embedded in paraffin for H&E and immuno-histochemical staining

DNA microarray analysis of all cells was conducted for further investigation In DNA

microarray data, IGF2 and krt13 are highly expressed in RR-strains compare to WT among

and were chosen for further investigation Theirs high expression in RR-strains in RNA level and protein level were confirmed by RT-qPCR analysis, western blot and immunohistochemical staining of nude mice tumors Silencing of those genes in RR-strains by

of the IGF2 and krt13 in RR-strains were studied by silencing with siRNA and overexpression

by generation of stable transfectant krt13-A431 cell for checking their radiation sensitivity, pluripotent stem cell marker expression, growth in monolayer and sphere forming ability

Results

By using LDR and HDR system, RR-strains from A431-WT and NA-WT cells designated A431-LDR, NA-LDR, A431-HDR, and NA-HDR, were successfully isolated in serum-free defined culture The D37 value of A431-LDR, A431-HDR, A431-WT, NA-LDR, NA-HDR, and NA-WT was 5Gy, 3.7Gy, 2.3Gy, 7.5Gy, 5.5Gy and 4.6Gy, respectively These cells exhibited higher expression of cancer stem cell marker such as CD133, higher sphere formation and higher migration abilities compare to those of WT cell lines LDR-RR cells

showed significant higher expression of pluripotent stem cell marker Nanog than in WT and

HDR-RR cells In addition, the RR cells exhibited higher tumor forming ability compared to

WT cells in nude mice xenograft

LDR radiation can generate higher radiation resistance of cancer cells, higher expression

of Nanog, higher migration ability and tumor forming ability than in HDR system

DNA microarray analysis revealed over 500 genes were overexpressed in RR-strains

compared to WT cells Among them, IGF2 and krt13 genes were high expression in RR-strains

compare to WT cells RT-qPCR analysis further confirmed that both RR-strains overexpressed

IGF2 and krt13 In addition, pathway analysis of the DNA microarray analysis revealed that

various pathways, such as MAPK, JAK/STAT signaling pathway, apoptosis pathway, TGF- signaling pathway and cytokine-cytokine receptor interaction were activated in RR-strains Gene Ontology analysis of microarray analysis also showed that the genes involved in keratinization, inflammatory response, wound healing and response to cytokine stimulus were enriched

Silencing of IGF2 by siRNA in RR-strains made them sensitive to radiation compare to

RR-strains transduced with negative control siRNA (NC) The D37 value of A431-LDR-siNC, A431-HDR-siNC and A431-LDR-siIGF2, A431-HDR-siIGF2 are averagely 5.5Gy, 5.9Gy, and 3.8Gy, 3.2Gy, respectively Further, the D37 of NA-LDR siNC siNC, NA-HDR, NA-LDR-siIGF2, NA-HDR-siIGF2 are averagely 5.5Gy, 5.6Gy, 3.8Gy, and 4.5Gy, respectively

When silencing krt13 with siRNA-krt13, A431 RR-strains showed higher radiation

sensitivity than negative control cells i.e D37 value of A431-LDR siNC, A431-HDR siNC

A431-LDR-sikrt13, and A431-HDR-sikrt13 are averagely 5.5Gy, 5Gy, 2.8Gy, and 1.9Gy,

respectively

Silencing of IGF2 in A431-LDR exhibited lower expression of krt13, Nanog and Oct4 in mRNA level WT-A431 cells overexpressed krt13 showed higher cellular ability, such as proliferation, sphere formation, migration and exhibited elevated expression of IGF2, Nanog and Oct4 in mRNA level than in control cells

Conclusion

This study clearly demonstrated that radiation resistant SCC cells can lead to cancer recurrence after radiation therapy, having higher population of cancer stem cells and high

tumorigenicity through overexpressing various genes such as IGF2 and krt13

Establishment of radiation resistant cancer cell model in serum-free defined culture and its use would be powerful tools to elucidate the mechanism of acquisition of radiation resistance and could potentially be targeted for the development of novel diagnosis and therapeutic modalities

Chapter 1: Introduction

Squamous cell carcinoma (SCC), including oral squamous cell carcinoma (OSCC), has been increasing in the world and being the most common cancer in South-East Asian countries where people have a betel-quid and areca-nut chewing habit An SCC also has a high rate of recurrence up to 40% in cancer patients depend on tumor size and stage of cancer [Te Grootenhuis et al (2018)]

Several treatment modalities such as operation, radiation, and chemo therapy have been reported to be effective in treating many kinds of cancer including oral cancer [Deorukhkar and Krishnan (2010); Krause et al (2011); Macha et al (2017); K Ogawa et al (2013)] Among them, radiation therapy (RT) plays a major role in the management of oral squamous cell carcinomas Despite therapeutic and technological advances such as ionizing radiation, gamma rays and charged particles to kill cancer cells through DNA damage directly or indirectly caused by free ion radicals, some patients will have persistence of irradiated tumor

or develop locoregional failure, resulting in significant morbidity and mortality [Blanchard et

al (2011)]

It has been considered that population of cancer stem cells (CSCs) has an important role

in not only cell proliferation but also in differentiation and maturation to a certain degree Therefore, if CSCs population can be survived after treatment, cancer and tumor can be recurred In addition, CSCs normally have different intrinsic factors compare to non-CSCs population that help them to be radiation resistance [Krause et al (2011)] It seems logical that curing of cancer can be achieved only if the CSCs population is eliminated [Krause et al (2017)]

RT using high dose rate (HDR) radiation has been widely used as an effective modality

to treat human cancer by various types of modern delivering techniques [Ogama et al (2010)]

On the other hand, RT using low dose rate (LDR) radiation has been introduced in the treatment

of prostate and oral cancers [Lazarev et al (2018); Umeda et al (2005)]

Therefore, to elucidate the cellular and molecular mechanisms involved in radiation resistance of cancer cells might be worthwhile to develop effective therapies by circumventing radiation resistance In this work, mechanisms of radio-resistance to RT in oral cancer and strategies used to overcome this resistance were studied To study that, it might be useful to establish radiation resistant-cancer cells in in vitro culture

There have been many reports in establishment of radiation resistant (RR) cancer cells

by HDR irradiation [Condon et al (2002); Park et al (2016); Shiiba et al (2013); Skinner et

al (2017); Terakado et al (2004); Y Zhao et al (2018)] However, there is no report of establishment of RR cancer cells using LDR irradiation To clarify biological effects of two

types (LDR and HDR) of irradiation on SCC cell lines in vitro and in vivo might be worthwhile

to circumvent radiation resistance for cancer

RR-SCC cells have been isolated and established under serum-supplemented culture condition This contains a lot of undefined and/or unknown proteins, factors, and lipids which

may exhibit unknown biological effects on cancer cells in vitro [Barnes and Sato (1980); J

Denry Sato et al (2002)] Thus, serum-free defined medium containing defined proteins, hormones and growth factors, can show exact biological characteristic of the cells Currently, there are no report of isolation of RR-SCC cells in serum-free defined culture, which ensures exact biological mechanisms and prevent instability of using serum

In this study, first I have tried to isolate and establish RR-SCC strains from SCC and OSCC cell lines, and then characterized their cellular and molecular properties in serum-free defined culture

Chapter 2:

Establishment and characterization of radiation resistant strains

I Materials and methods

1 Cell culture & Culture medium

The used cell lines were NA (HO-1-N-1), established from an oral cancer patient in our laboratory [Miyauchi et al (1988)] and A431, a vulvar squamous cell carcinoma cell line [Fabricant et al (1977)] All cell lines were cultured in serum-free medium designated DF6F which is composed of the basal nutrient medium (DF) supplemented with insulin (10 g/ml), transferrin (5 g/ml), 2-aminoethanol (10 M), sodium selenite (10 nM), 2-mercaptopethanol (10 M) and oleic acid conjugated with fatty acid-free serum albumin (9.4 g/ml) [Okamoto

et al (1996); J D Sato et al (1987)] The DF basal medium is a mixture of Dulbecco’s modified Eagle’s medium (DMEM) [Dulbecco and Freeman (1959)] and Ham’s F-12 medium [Ham (1965)] (DF) in a ratio of 1:1 (Merck, Darmstadt, Germany), and dissolved in mili-Q water (Direct-Q UV, Merck) added with 90 mg/L of ampicillin sodium (Meiji Seika Kaisha, Tokyo, Japan), 90 mg/L of kanamycin sulfate (Gibco, Life Technologies Corp, USA), 20 nM

of 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES, Dojindo, Kumamoto, Japan), 165mg of sodium pyruvate and 2g/L of sodium bicarbonate (Merck), adjusted to pH 7.2 and filtered with 0.2 m membrane filter (Sartorius Stedim Biotech GmbH, Germany)

Cells were cultured and incubated at 37°C in a humid atmosphere of 95% air/5% CO2 incubator (Astec, Fukuoka, Japan) until they reached at 70-80% confluency

The cells were harvested in 0.01% trypsin (Sigma-Aldrich, Merck, Missouri, USA) -0.01% ethylene diamine tetra acetic acid (EDTA; Dojindo) in Ca2+ and Mg2+-free phosphate-buffered saline (CMF-PBS) [Dulbecco and Vogt (1954)], and the trypsin was inactivated with 0.1%

soybean trypsin inhibitor (Sigma-Aldrich) in DF6F, and sub-cultured every 7 days at cell density of 105 cells/4ml/60mm dish

2 Irradiation Procedure for generation of radiation resistant strains (RR-strains)

All cell lines were seeded at density of 1x106 cells/60mm culture dish (Corning, Falcon, NY, USA) After 24-hour in culture, the cells were irradiated at a dose of 2.2Gy/day (1.52mGy/min), continuously in 4 days in a low dose rate (LDR) irradiation system (RM1000, Chugai Technos, Japan), or at 5Gy/5.75min (880mGy/min), twice a week with a high dose rate (HDR) system (Gamma cell 40 Exactor, Best Theratronics, Canada) in DF6F medium Control dishes were cultured in 0Gy incubator All cell dishes were subcultured one day after each irradiation cycle, the irradiation procedure was repeated continuously in 6 weeks with HDR system and 7 weeks with LDR system After irradiating 60Gy as a whole by LDR and HDR irradiation system, we have isolated four sub-strains from two SCC cell lines, A431-LDR and A431-HDR from A431-WT, NA-LDR and LA-HDR from NA-WT These strains were continuously irradiated every week at a dose of 0.5Gy from passage 5 To verify the radiation resistant ability of these cell strains, colony survival assay was performed

3 Colony survival assay for wild type and radiation resistant strains

To investigate their radio-sensitivities, both wild type (WT) and RR-strains were irradiated at doses of 0Gy, 2Gy, 4Gy, 6Gy, and 8Gy, then colony survival assay was performed as previously described [Franken et al (2006)] All cells were cultured in DF6F medium in 60mm dish until they reached at 70-80% confluency Then, the cells were irradiated in HDR system

at various doses of 0Gy, 2Gy, 4Gy, 6Gy, and 8Gy (880mGy/min) The cells were harvested and seeded immediately at a cell density of 300, 400, 800, 1,200 and 1,600 cells/60mm dish, respectively After 14 days in culture, cells were fixed in 99% methanol for 1 minute, then

stained with 10% Giemsa solution (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) for 30 minutes and washed with mili-Q All dishes were dried up in 24 hours before counting Colonies consist of more than 50 cells were counted as a positive colony Experiment was done in triplicate Plating efficiency (PE) and survival fraction (SF) were calculated as formulas below

PE =Number of colonies counted

Number of cells plated x 100

SF =PE of treated sample

PE of control x 100 The D37 value is defined as the dose which kill 63% of cell population or leave 37% cell population viable in radiation biology The D37 value of each cells was calculated from the survival curves of each colony forming assay

4 Growth rate of wild type and radiation resistant strains

Cells were seeded at a density of 1x104cells/well in 24-well plate (Corning) and cultured in DF6F serum-free medium Cells were harvested with trypsin/EDTA (Sigma-Aldrich) and cell numbers were counted every 24 hours with Z1 Coulter particle counter (Beckman Coulter Inc,

CA, USA) Experiment was done in triplicate

5 Sphere formation assay of wild type and radiation resistant strains

Cells were harvested and seeded at a density of 1,000 cells/35mm prime surface low attachment dish (Sumitomo Bakelite Co., Tokyo, Japan) in DF6F serum-free medium On day 5, the spheres bigger than 25 m in diameter were counted as positive sphere Experiment was done

in triplicate

6 Expression of cancer stem cell marker CD133 in wild type and radiation resistant strains Cells were cultured in DF6F serum-free medium until they reached at 80% confluency Then, cells were washed with CMF-PBS [Dulbecco and Vogt (1954)], harvested with trypsin/EDTA

(Difco) and centrifuged at 300xg for 5 minutes The cells were incubated with CD133 FITC

antibody (1:20, CD133/1-Viobright FITC, Miltenyi Biotec GmbH, Bergisch Gladbach, Germany) in FACs buffer solution (2 mM EDTA, 5% BSA in PBS) CD133-positive cells were analyzed with Cell sorter SH800 (Sony Co., Tokyo, Japan)

7 Expression of pluripotent stem cell markers Nanog, Oct4 and Sox2 in WT and RR-strains

by Real time-quantitative polymerase chain reaction (RT-qPCR)

Total RNA of all the cells was extracted by using RNease Mini kit (Qiagen, Tokyo, Japan) One g of purified RNA was reverse transcripted into cDNA by using SuperScript VILO Mastermix (Thermo Fisher Scientific)

Sequencing primers and Taqman fluorogenic probes were designed by using ProbeFinder based software (https://lifescience.roche.com/global_en/brands/universal-probe-library.html)

web-of Roche Universal Probe Library (Roche Applied Science, Nurley, USA) List web-of used primers and probes were listed in Table 1

RT-qPCR analyses were performed using the Stratagene Mx3000P system (Stratagene, Agilent Technologies, CA, USA) with GAPDH (Glyceraldehyde-3-Phosphate Dehydrogenase) as an internal control Each tube contains 10 l FastStart Universal Probe Master (Roche Diagnostics GmbH, Germany), 0.2 l of Universal Probe Library, 0.8 l of forward and reverse primers, 8

l nuclease-free water (Thermo Fisher Scientific) and 1 l of cDNA Experiment was done in triplicate Gene expression was calculated with normalization to GAPDH

8 Migration assay of WT and RR-strains

Chemotaxicell well (Chemotaxi 8, Kurabo, Japan) was coated with 100g/ml type I-collagen (Cellmatrix Type I-A, FUJIFILM Wako) in 0.1M Acetic acid (FUJIFILM Wako) and incubated in 40C overnight Cells were seeded at 5x104 cells/chemotaxicell well in DF basal nutrient medium supplemented with 0.1% bovine serum albumin (Merck) in 24-well plate in incubator After 24-hour, chemotaxicell wells were stained with Diff-Quick (Sysmex, Japan) and the membranes were fixed on glass slide (Matsunami Glass Ind, Osaka, Japan) with glue Canada Balsam (Nacalai Tesque, Kyoto, Japan) and covered with covering glass (Matsunami Glass Ind) Number of migrated cells per mm2 were counted Experiment was done in triplicate

9 Tumor formation ability of WT and RR-strains in nude mice

WT and RR-strains of A431 cells (0.25 x106-1x106cells/100l) and NA cells (1x106-5x106cells/100l) were injected subcutaneously to the dorsal back skin of nude mice (BALB/c-nu/nu), and tumor size was measured every week Four to thirteen weeks after inoculation of

106 cells, the tumors were excised, weighted, fixed in 4% paraformaldehyde in PBS (Nacalai Tesque) for 24hr, and embedded in paraffin Then the 5m slice was used for Hematoxylin and eosin (H&E) staining, and immuno-histochemical staining The tumors by 5x106 NA cells were excised 6 weeks after inoculation, and same procedure were done as described above Tumor size was calculated by using formula: tumor volume (mm3) = (length x width2) x ½

10 Immunohistochemical examination of ki67 in nude mouse tumors derived from WT and RR-strains

Paraffin blocks of nude mouse tumors were sectioned at 5m in thickness for immunohistochemical staining with ki67 antibody Sections were deparaffinized in xylene and

ethanol following standard protocol and pre-treated using heat induced epitope retrieval method before blocking with endogenous peroxidase Dako Target retrieval solution at pH 6.0 (Dako, Agilent Technologies) was used for ki67 antigen retrieval Deparaffinized sections were embedded in 1x diluted retrieval solution and heated in microwave at power of 200W for 15 minutes After Endogenous Peroxidase blocking (Dako) for 10 minutes, all sections were incubated with 10% goat serum (Histofine, Nichirei Bioscience Inc, Japan) for 30 minutes at room temperature to prevent non-specific binding and were stained overnight in 40C with ki67 antibody (1:100, mouse monoclonal, clone MIB-1, Dako) in Dako REAL antibody diluent (Dako) The sections were then put in room temperature for 1 hour and washed with PBS prior

to staining with second antibodies All sections were stained with second antibodies (Dako Envision+ System-HRP Labelled Polymer Anti-Mouse, Dako) for immuno-reactivities visualization and counterstained with Mayer’s Hematoxylin solution (FUJIFILM Wako) before permanent mounting Images were visualized and captured with Nikon DS-Ri2 camera (Nikon Corporation, Tokyo, Japan) on Nikon Eclipse E800 microscope (Nikon Corporation) After staining, each tumor section was first visually examined under x100 magnification of microscope to identify highest ki67 positive region The highest ki67 positive area of each tumor section was captured and calculated under the microscope at x400 magnification

11 Statistical analysis

Student’s t-test was performed by data analysis software in Microsoft’s Office EXCEL 2016 (Microsoft Corporation, Redmond, USA) and p-values <0.01 or <0.05 are considered significant Statistical analysis was used in all experiment

II Results

1 Establishment of radiation resistant cancer cell strains and colony survival formation Total irradiation dose for A431 and NA in LDR and HDR system is 60Gy After irradiation, from each wild-type cell line (A431-WT and NA-WT), two cell strains from each WT cell line have been derived; designated as A431-LDR, A431-HDR, NA-LDR, and NA-HDR (Figure 1)

To clarify radiation resistance of these cells, colony survival assay was performed Colony formation of the cells at irradiation dose of 0, 2, 4, 6, 8Gy revealed that survival fraction (SF)

of A431-LDR and A431-HDR is significantly higher than that of A431-WT (Fig 2A)

In addition, SF of NA-LDR and NA-HDR is significantly higher than that of NA-WT (Fig 2B) These results suggested that these cells are radiation resistant To confirm this, we analyzed D37 value of the cells The D37 value of A431-LDR, A431-HDR and A431-WT are averagely 5Gy, 3.7Gy, and 2.3Gy, respectively Further, the D37 value of NA-LDR, NA-HDR and NA-WT are averagely 7.5Gy, 5.5Gy and 4.6Gy, respectively D37 value of RR-SCC cells are significantly higher than WT cells (p<0.05, p <0.01) and D37 value of HDR-RR-SCC cells are significantly higher than LDR-RR-SCC cells (p<0.01) These results clearly confirmed that these strains are radiation resistant compared to WT cell lines

2 Proliferation of the cells in serum-free monolayer culture

Monolayer culture of WT and RR-strains revealed that RR cells exhibited lower proliferation rate compare to that of WT cells (Figure 3) Doubling times of A431-WT, A431-LDR, A431-HDR are 25.671.63 hours, 27.050.73 hours, 27.881.15 hours and NA-WT, NA-LDR, NA-HDR are 26.920.1 hours, 28.330.71 hours, 29.950.81 hours, respectively

3 Sphere forming ability of the cells in serum-free suspension culture

As a result of sphere formation assay in suspension culture using low-attachment culture dish, Sphere number of A431-LDR (25 spheres) and A431-HDR (18 spheres) was higher than that

of A431-WT (15 spheres), especially that of A431-LDR was significantly high (p<0.05) (Figure 4A) Sphere number of NA-LDR (18 spheres) and NA-HDR (16 spheres) was also high, but not significant compare to that NA-WT (14 spheres) (Figure 4B)

4 Expression of cancer and pluripotent stem cell markers in WT and RR-strains

CD133 was used as a cancer stem cell marker to verify the stem cell population in WT and RR-strains Flow cytometric analysis revealed that the existence of CD133 positive subpopulation in both WT and RR-strains CD133 subpopulation was 0.44%, 1.1%, 0.71% in A431-WT, A431-LDR, A431-HDR cell and 0.5%, 0.72%, 0.76% in NA-WT, NA-LDR, NA-HDR, respectively (Figure 5)

RT-qPCR analysis of the expression of Nanog, Oct4 and Sox2 in both WT and RR-strains revealed that Nanog and Oct4 expressions were high in both A431-LDR and NA-LDR strains compare to those of WT cells Especially Nanog expression in A431-LDR and NA-LDR was significantly higher than that of WT cells (p<0.05) (Figure 6)

5 Migration ability of wild type and radiation resistant strains

Cell migration assay by Chemotaxicell revealed that amount of migrated cells/mm2 of strains is significantly higher than that of WT, suggesting that RR-strains have higher migration ability compare to WT cells (Figure 7)

RR-6 Tumor formation ability of WT and RR-strains in nude mice

Tumors formed in nude mice by A431-LDR, and -HDR at the inoculation numbers of 1x106cells exhibited significantly higher growth rate compared to the tumors formed by A431-WT Tumors by A431-LDR and A431-HDR at an inoculation number of 1x106 cells were able to grow to the visually detectable size at the second weeks after inoculation, but A431-WT at the third weeks At a low inoculation numbers such as 0.25x106 cells, only A431-LDR was able

to form tumors (Figure 8A)

When injected with 1x106 cells/injection, Tumors by NA-LDR and NA-HDR were able to grow to the visually detectable size at the eighth week and tenth week, respectively, but no tumors were formed with NA-WT at the thirtieth weeks To verify the tumor formation ability

of NA-WT and –LDR, and HDR cells, a high inoculation number such as 5x106 cells was injected As a result, NA-LDR and NA-HDR could form tumors at the third weeks after inoculation, but no tumors were formed with NA-WT (Figure 8B)

7 Immunohistochemical expression of ki67 in nude mice tumors derived from WT and strains

RR-Expression of ki67 in nude mice tumors was examined to study the proliferation ability of the cells Percentage of ki67 positive cells in the tumors formed by A431-LDR and –HDR were high compared to that by A431-WT, but not significant (Figure 9)

Chapter 3:

Identification and Characterization of novel genes involved in radiation resistance

I Materials and methods

1 DNA microarray analysis

One g of total extracted RNA was hybridized with 3D-Gene Human Oligo Chip 25k (Toray Industries Inc, Tokyo, Japan) and performed with manufacturer’s protocol Genes expressed at least two times higher in 95% confidence interval after global normalization were listed Raw data was analyzed base on Gene Ontology terms (GO) Pathway analysis of enriched genes were performed by using online tool named The Database for Annotation, Visualization and Integrated Discovery (DAVID) v6.8 (https://david.ncifcrf.gov/)

2 Expression of IGF2 and krt13 in wild type and radiation resistant strains by RT-qPCR

Total RNA of the cells was extracted by using RNease Mini kit (Qiagen) One g of purified RNA was reverse transcribed into cDNA by using SuperScript VILO Mastermix (Thermo

Fisher Scientific) List of krt13 and IGF2 primers and probes were shown in Table 1

RT-qPCR analyses were performed using the Stratagene Mx3000P system (Stratagene, Agilent Technologies, CA, USA) with GAPDH (Glyceraldehyde-3-Phosphate Dehydrogenase) as an internal control Each tube contains 10 l FastStart Universal Probe Master (Roche Diagnostics GmbH, Germany), 0.2 l of Universal Probe Library, 0.8 l of forward and reverse primers, 8

l nuclease-free water (Thermo Fisher Scientific) and 1 l of cDNA Experiment was done in triplicate Gene expression was calculated with normalization to GAPDH

3 Western-blotting

The cells at 80% confluency were lysed in lysis buffer (0.1% SDS, 10 mM Tris, 150 mM NaCl,

5 mM EDTA, 1% Na-deoxycholate, 1% NP-40, 1% Protease inhibitor cocktail, Merck) and homogenized by sonication on ice for 25 seconds (UD-100, Tomy Seiko Co., Ltd., Japan) Protein content of each sample was quantified by Bradford method [M.Bradford (1976)] using Pierce BCA Protein assay kit (Thermo Fisher Scientific, MA, USA) Samples (20 g) were electrophoresed on 10% SDS-polyacrylamide gel (40 mA/gel) under reducing condition (96°C,

3 mins) and transferred to polyvinylidene difluoride (PVDF) membrane filters (Bio-Rad Laboratories, CA, USA) by semi-dry blotting system (90 mA/gel, Bio-Rad Laboratories) The membrane was blocked with 0.1% tween-TBS containing 5% non-fat milk for 30 minutes at room temperature then incubated with primary antibodies for 1 hour (Table 2), followed by second antibodies Immuno-reactive bands were visualized with Clarity Western ECL Substrate (Bio-Rad Laboratories) and light captured with Chemidoc Touch Imaging system (Bio-Rad Laboratories)

4 Immunohistochemical expression of IGF2, and KRT13 proteins in nude mice tumors derived from WT and RR-strains

Immunohistochemical expression of IGF2, and KRT13 proteins in nude mice tumors derived from WT and RR-strains were studied as the method described in Chapter 2-I-10 except for IGF2 antibody (1:100, rabbit polyclonal, ab9574, Abcam) and KRT13 antibody (1:200, rabbit monoclonal, ab92551, Abcam)

II Results

1 Results of DNA microarray analysis

DNA microarray results showed overexpression of 500-700 genes (SerpinB2, krt13, IGF2,

s100A8, s100A9, krt15, rad51….) in LDR- and HDR-RR-SCC cells compare to wild type

Graph was made by over eight times higher co-expression of LDR and HDR-RR-SCC cells Pathway analysis was also listed in the figure (Figure 10) Several important genes in cancer

research were increased in this DNA microarray analysis, such as: IL1A, IL1B (interleukin 1 alpha, keratinization, activation of MAPK activity), IL1R2 (interleukin a receptor 2, immune response, cytokine-mediated signaling pathway), SPRR1B, SPRR2A, SPRR2B (small proline rich protein, keratinization, keratinocyte differentiation), FGFBP1 (fibroblast growth factor binding protein 1, fibroblast growth, regulation of cell proliferation), RAD51 (RAD51 recombinase, repair of DNA), S100A6, S100A8, S100A9, S100A10 (S100 calcium binding protein, inflammatory response), KRT5, KRT13, KRT14, KRT15 (keratin, epithelial cell differentiation, response to radiation), IGF2 (insulin growth factor 2, regulation of gene expression), SERPINB2 (serpin peptidase inhibitor clade B member 2, regulation of endopeptidase activity) and FGF5 (fibroblast growth factor 5, activation of MAPKK activity)

2 Expression of IGF2 gene and protein in WT and RR-strains by RT-qPCR, western-blotting,

and immunohistochemical staining

RT-qPCR analysis revealed that expression of IGF2 mRNA was significantly higher in

A431-LDR, -HDR, and NA-A431-LDR, -HDR strains compare to that of A431-, and NA-WT cells (Figure 11A)

In addition, IGF2 protein expression by western blotting analysis showed that IGF2 protein was in A431-LDR, -HDR, and NA-LDR, -HDR were significantly overexpressed compared to that in WT cells (p<0.01) (Figure 11B) Immunohistochemical examination of IGF2

expression in nude mice tumors revealed higher expression of IGF2 in the tumors derived from A431-LDR, and –HDR compared to tumors from A431-WT In addition, tumors derived from NA-LDR and -HDR showed high expression of IGF2 (Figure 11C)

3 Expression of krt13 gene and protein in WT and RR-strains by RT-qPCR, western-blotting,

and immunohistochemical staining

RT-qPCR analysis showed that A431-LDR and A431-HDR exhibited significantly higher

expression of krt13 compare to WT cells (Fig 12A) These results were further confirmed by

western blotting analysis showing that immune-positive band corresponding to KRT13 was relatively high compared to that of WT cells (Fig 12B) In NA cells, NA-LDR showed high

expression of krt13 but not significantly high (Fig 12A) In western botting analysis, there was

no difference in KRT13 expression (Fig 12B)

Immunohistochemical examination of KRT13 revealed that nude mice tumors derived from A431-LDR and A431-HDR exhibited high expression of KRT13 compared to tumors derived from A431-WT (Fig 12C) NA-LDR and NA-HDR also showed high expression of KRT13 (Figure 12C)

Chapter 4: Functional analysis of IGF2 and krt13 in radiation resistance

I Materials and methods

1 Effect of krt13 siRNA and IGF2 siRNA transduction on colony formation ability of RR-

strains

A431- and NA-RR-SCC (LDR or HDR) cells cultured in serum-free medium were transfected with selected or customized siRNA and negative control (NC) siRNA using lipid-based siPORTTM NeoFXTM Transfection Agent (Invitrogen, Thermo Fisher Scientific) for 48 hours After 48 hours, total RNA of each transfected well was extracted and transcribed to cDNA for RT-qPCR

SiRNA for krt13 #1, #2, and #3 (Ambion, Life Technologies, CA, USA) and NC siRNA used were as follow; Krt13-siRNA primer:

Cells transduced with NC siRNA were used as a control

2 Effect of radiation on IGF2-siRNA transduced-A431-LDR, HDR, NA-LDR and -HDR cells After 48 hours of transduction, IGF2-siRNA transduced-A431-LDR, HDR, NA-LDR and -

HDR cells were irradiated at 0, 2, 4, 6, and 8Gy at 880mGy/min in HDR system and a colony forming ability was examined as described in Chapter 2-I-3

3 Effect of radiation on krt13-siRNA transduced-A431-LDR and -HDR cells

After 48 hours of transduction, krt13-siRNA transduced-A431-LDR and -HDR cells were

irradiated at 0, 2, 4, 6, and 8Gy at 880mGy/min in HDR system and a colony forming ability was examined as described in Chapter 2-I-3

4 Expression of pluripotent stem cell markers, krt13 and IGF2 in IGF2-, and krt13-siRNA

transduced cells

Cells were seeded at 3x105 cells/60mm dish and transfected with krt13 siRNA, IGF2 siRNA

and NC siRNA for 48 hours Total RNA of the cells was extracted by RNease Mini kit (Qiagen) One g of RNA was reverse transcribed into cDNA by using SuperScript VILO

Mastermix (Thermo Fisher Scientific) Primers of Nanog, Oct4, Sox2, krt13 and IGF2 used

were shown in table 1

RT-qPCR analyses were performed as described in chapter 2-I-7 Experiment was done in triplicate Gene expression was calculated with normalization to GAPDH

5 Generation of stable transfectant of krt13 in A431 cells

A431 cells were transfected with pPvKRT13-IRES2-AcGFP1 (Clontech Laboratories, CA,

USA) vector inserted krt13 sequence or pIRES2-AcGFP1 empty vector using Lipofectamine

LTX Reagent (Thermo Fisher Scientific) at 1 g/0.5ml/well following manufacturer’s instruction As pIRES2-AcGFP1 was constructed with Kanamycin/Neomycin resistant sequence, the transfected cells were selected with G418 (Geneticin, FUJIFILM Wako) from 48h after transfection at a concentration of 200 g/ml Culture medium containing G418 was changed every 3 days for 2 weeks Then G418-resistant cells were isolated as stable krt13-transfected A431 cells and empty vector-transfected cells, designated as an A431-krt13 and A431-control, respectively Then total RNA of transfected cells was extracted for RT-qPCR to

confirm overexpression of krt13 gene Experiment was done in triplicate

6 Characterization of krt13-transfected A431 cell and its radiation resistant ability

Growth assay, sphere formation assay and migration assay of A431-krt13 and A431-control

were examined in serum-free culture All assays were performed as described in chapter

2-I-4, chapter 2-I-5 and chapter 2-I-8

RT-qPCR of krt13, Nanog, Oct4, Sox2 and IGF2 was performed as described in chapter 2-I-7 except for cDNA from A431-krt13 and A431-control

Colony survival assay was also performed as described in chapter 2-I-3 except for A431-krt13

and A431-control Experiment was done in triplicate

II Results

1 Effect of various IGF2- and krt13-siRNA on silencing ability in the cells

Among #1 and #2 IGF2-siRNAs examined, #1 IGF2-siRNA exhibited highest silencing ability

by RT-qPCR analysis In addition, among #1, #2, and #3 siRNAs examined, #3 siRNA showed highest silencing ability (Figure 13) Therefore, #3 siRNA for krt13, and #1 siRNA for IGF2 were used for further study

krt132 Effect of radiation on colony survival formation of IGF2siRNA transduced A431LDR,

-HDR, NA-LDR and -HDR cells

IGF2-siRNA transduced-A431-LDR, -HDR, NA-LDR and -HDR cells exhibited highly

sensitive to radiation exposure compared to A431-LDR, -HDR, NA-LDR and -HDR cells The

D37 value of A431-LDR siNC, A431-HDR siNC, A431-LDR siIGF2 and A431-HDR siIGF2 are averagely 5.5Gy, 5.9Gy, 3.8Gy and 3.2Gy, respectively Further, the D37 values of NA-LDR-siNC, NA-HDR-siNC, NA-LDR siIGF2 and NA-HDR siIGF2 are averagely 5.5Gy, 5.6Gy, 3.8Gy and 4.5Gy, respectively (Figure 14A and B) D37 value of A431-LDR and -HDR siIGF2 are significantly lower than A431-LDR and -HDR siNC (p<0.01) D37 value of NA-LDR and -HDR siIGF2 are significantly lower than NA-LDR and -HDR siNC (p<0.01)

3 Effect of radiation on colony survival formation of krt13siRNA transduced A431LDR,

-HDR cells

krt13-siRNA transduced-A431-LDR, and -HDR exhibited highly sensitive to radiation

exposure compared to A431-LDR, and -HDR The D37 value of A431-LDR siNC, A431-HDR siNC, A431-LDR siKRT13 and A431-HDR siKRT13 are averagely 5.5Gy, 5Gy, 2.8Gy and 1.9Gy, respectively (Figure 15) D37 value of A431-LDR and -HDR siKRT13 are significantly lower than A431-LDR and -HDR siNC (p<0.01)

4 Expression of krt13, IGF2, and pluripotent stem cell marker genes in IGF2-siRNA

6 Characterization of A431 cells overexpressing krt13 (A431-krt13) in serum-free culture

A431-krt13 cells showed significantly higher growth rate compared to A431 control cell (Figure 18 A) Sphere formation assay of both cell lines revealed that A431-krt13 cells showed significantly higher sphere numbers (Figure 18 B) Migration ability of A431-krt13 was also significantly high compared to that of control cells (Figure 18 C)

By RT-qPCR analysis, expression of krt13, Oct4 and IGF2 in A431-krt13 was significantly

higher than those of control cells (Figure 18 D)

A431-krt13 cells showed significantly higher survival rate after irradiation compared to that of control cells (Figure 18 E)

Chapter 5: Discussion

In this stud y, RR-SCC strains from A431 vulvar squamous cell carcinoma cell line and

NA oral squamous cell carcinoma cell line has been successfully isolated in serum-free defined culture Two radiation treatment regimens including Low Dose Radiation (LDR) and High Dose Radiation (HDR) were used for the isolation of the RR-SCC strains LDR; total dose of 60Gy with dose per fraction of 2Gy at 1.52mGy/min per exposure: HDR; total dose of 60Gy with dose per fraction of 5Gy at 880mGy/min per exposure

Total radiation dose of 60Gy in this experiment was chosen based on common radiation dose for SCC treatment [He et al (2014)] Clinically, HDR under various techniques is widely used as common radiation therapies to treat cancer in patients but using LDR radiation therapy

is very rare

HDR irradiation in vitro model to isolate RR-SCC cells have been reported through

irradiation 2Gy/fraction in 25 fractions to reach 50Gy in 5 weeks [Condon et al (2002)], 4Gy/fraction to a total dose of 37Gy in 10 weeks [Pan et al (2017)], and 6 or 10Gy in total [Matsuoka et al (2016)] by X-ray generator or Gamma-ray generators in serum-supplemented medium However, there is no LDR irradiation model to establish and isolate RR-SCC cells Colony formation is the ability of single cell to form colony after a definite duration in culture [Fisher et al (1959)] As colony forming ability could demonstrate cell death and cell survival after irradiation, it has been used to demonstrate radio-sensitivity of cancer cells [Franken et al (2006)] A result that A431-LDR, -HDR, NA-LDR, and -HDR strains exhibited high colony forming ability and higher D37 value compared to those of WT cells clearly suggested that these isolated RR cells are radiation resistance

1-2-Sphere forming ability in suspension culture have gained a wide popularity in cancer stem cell research and for a wide range of human tumor cells [Bertolini et al (2009); Leung et

al (2010)] Under serum-free culture condition, only cancer cells with self-renewal ability are

expected to grow and maintain their spheroid morphology Sphere formation from a single cell suspension in culture medium is also a basic tumorigenic characteristic of cancer stem cells [Cao et al (2011)] RR-SCC cells exhibited higher sphere forming ability than WT cells, especially significantly high in A431-LDR, suggesting that RR-strains have maintained high population of cancer stem cells and tumor forming ability than WT cells

CD133 (Prominin-1), a cell surface marker for cancer stem cells of various cancers including SCC, can be used as a marker to identify tumor-initiating cells, and CD133-positive cells have been reported to be resistant to radiotherapy and chemotherapy [Canis et al (2012); Zhang et al (2010)] Higher sphere forming ability of RR-cells was supported by the results of flow cytometric analysis of CD133 expression that percentage of CD133-positive ratio in A431- and NA-RR-SCC cells exhibited about twice as much as that of WT cells

Nanog, Oct4, and Sox2 are transcription factors and used as pluripotent stem cell markers

for the embryonic stem cells (ESC), induced pluripotent stem cells (iPSC) [Okita and Yamanaka (2010); Tanabe et al (2014)] and cancer stem cells [A Liu et al (2013); van

Schaijik et al (2018)] It has been reported that high expression of Oct4 was related to poor

differentiation, tumor size and cancer stage in rectal cancer [You et al (2018)], and that

overexpression of Nanog was related to poor response and low survival rate of patients treated with chemotherapy [Chang et al (2017)], and Nanog, Oct4 and Sox2 were closely associated

with OSCC progression [Fu et al (2016)]

Migration of cancer cells represents their capability to avoid ionizing effect of radiation, migrate to distant sites and invasive promotion, especially after radiation therapy [Vilalta et al (2016)] Suppressing of migration and invasion ability of cancer cells are suggested to reduce metastasis [Xu and Deng (2006)] Higher migration ability of A431- and NA-RR cells are in agreement with their results

In nude mice xenograft experiments, A431-LDR, and -HDR showed significantly higher tumorigenicity compare to A431-WT In addition, low inoculation number (2.5 x 105) of A431-LDR cells formed tumors suggesting their high tumorigenic potential compared to A431-HDR and -WT cells

LDR and NA-LDR have exhibited higher resistance to radiation compare to HDR and NA-HDR In addition, A431-LDR and NA-LDR showed higher sphere formation ability, higher migration ability, also higher expression of Nanog and tumor forming ability compared to A431-HDR and NA-HDR cells These results suggested that LDR irradiation can induce highly radio-resistant phenotype through higher malignant properties such as stemness, migration ability, sphere forming ability, and tumorigenicity than HDR irradiation This is the first report finding a difference between RR-LDR cells and RR-HDR cells

A431-DNA microarray analysis revealed over 500 genes (SerpinB2, krt13, S100A8, S100A9,

RAD51, IL1R2…) were overexpressed in RR-strains compared to WT cells Among them, IGF2 gene was overexpressed in NA-LDR compare to NA-WT cells Furthermore, krt13 gene

was overexpressed in A431-LDR and A431-HDR compared to WT cells These results by DNA microarray analysis were further confirmed by RT-qPCR analysis Elevated expression

of krt13 and IGF2 genes in RR-strains strongly suggested that these genes might contribute to

radiation resistance in RR-SCC cells

In addition, pathway analysis of the DNA microarray analysis revealed that various pathways, such as MAPK signaling pathway, JAK/STAT signaling pathway, apoptosis pathway, TGF- signaling pathway and cytokine-cytokine receptor interaction were activated

in RR-strains While the exact mechanisms responsible for the activation of these signaling pathways by radiation has not yet been clearly elucidated, several signaling mechanisms have been proposed to be involved in this activation [Hein et al (2014)]

Gene Ontology analysis of microarray analysis also showed that the genes involved in keratinization, inflammatory response, wound healing and response to cytokine stimulus were enriched This result suggested that multi pathways could be activated under complicated effects of radiation for cell survival and their response to promote radiation resistance Consequently, these signaling pathways act conjointly to rescue the cells from radiation-induced injury and promote survival Thus, activated pathways and enriched multi pathways might be good targets for further investigation to verify their roles in cancer cell radiation resistance

IGF2 is one of imprinted genes in mammalian cells, by which epigenetic silencing results

in mono-allelic expression specific to parental origin In human, hepatocytes continue to secrete IGF2, but its physiological role is not so important [Harris and Westwood (2012)]

Recently, IGF2 abnormalities and/or overexpression have been demonstrated in various malignant tumors, where its overexpression is usually associated with worse prognosis [Liou

et al (2007); Lu et al (2006); Singer et al (2004)] In addition, IGF axis can play an important role in cancer stem cells renewal and its overexpression may enhance stemness, self-renewal and therapeutic resistance of cancer cells [Bodzin et al (2012)] A large cohort study also revealed that ovarian cancers with high immuno-histochemical expression of IGF2 showed

decreased progression-free survival time, and that an increase in IGF2 mRNA was associated

with worse prognosis [Huang et al (2010)] In addition, knocking down IGF2 in the resistant head and neck SCC cell lines sensitized the cells, while CDDP-sensitive cells overexpressing IGF2 became resistant [T Ogawa et al (2010)]

CDDP-Overexpression of IGF2 mRNA and protein in A431-, NA-RR cells, and in these

cell-derived nude mouse tumors may suggest that the RR cells are expressing their radiation resistant characteristic through high stemness and tumorigenicity induced by IGF2

It has been reported that IGF2 was involved in CD133-positive cancer stem cell phenotype in colon cancer, breast cancer, and prostate cancer [Liou et al (2007); X Zhao et

al (2016)] Radiation sensitivity of A431-LDR and NA-LDR cells transiently knock downed

IGF2 with IGF2-siRNA was restored to that of WT cells and exhibited a decrease of Nanog

and Oct4 expression suggesting importance of IGF2 in not only radiation resistance but also pluripotency In addition, IGF2 silencing in A431-LDR significantly decreased krt13 expression This result strongly suggested that IGF2 gene might be located in a higher hierarchy than krt13

DNA microarray and RT-qPCR analysis also revealed that krt13 was overexpressed in A431-LDR and –HDR cells Cytokeratin 13/krt13 is located in the long arm of chromosome

17 and is expressed as an intermediate filament protein in oral squamous epithelium and oral squamous cell carcinoma cells, but not abundant in other types of cancer [Ida-Yonemochi et

al (2012); Li et al (2016); S Liu et al (2016); Naganuma et al (2014)] Transfection of krt13

in prostate cancer cells induced higher proliferation, migration, invasion, and can reprogram

bone and brain metastases [Li et al (2016)], and that enrichment of krt13 in tubule-initiating

cells of prostate induced stem-like cell state and highly aggressive, promote resistance, apoptosis-resistance and lead to poor prognosis [S Liu et al (2016)] Silencing of

androgen-krt13 in RR cells by siRNA showed significantly decreased survival rate of A431-LDR and

A431-HDR in colony formation assay after radiation On the other hand, A431-WT cells

overexpressing krt13 exhibited not only high proliferation rate, high sphere forming ability, and high migration ability, but also higher expression of Nanog, Oct4 and IGF2 compare to the WT cells, in addition to higher radio-resistance suggesting importance of krt13 in radiation resistance This is a first discovery that krt13 was involved in radiation resistance of cancer cells in vitro while other several genes related to radiation resistance of cancer cells were

previously reported It is worth to mention that these previous reports were used radiation