Radiation-induced Myofibroblasts Promote Tumor Growth via ......Jul 24, 2018 · Normal human...

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Radiation-induced Myofibroblasts Promote Tumor Growth via Mitochondrial

ROS-activated TGFβ Signaling

Tsutomu Shimura1*

, Megumi Sasatani2, Hidehiko Kawai

2, Kenji Kamiya

2, Junya

Kobayashi3, Kenshi Komatsu

3, Naoki Kunugita

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1 Department of Environmental Health, National Institute of Public Health 2-3-6

Minami, Wako, Saitama, Japan 351-0197

2 Department of Experimental Oncology, Research Center for Radiation Genome

Medicine, Research Institute for Radiation Biology and Medicine (RIRBM), Hiroshima

University, Hiroshima, Japan 734-8553

3 Department of Genome Dynamics, Radiation Biology Center, Kyoto University;

Kyoto, Japan 606-8501

Running title: TGFβ Pathway-mediated Acquisition of Myofibroblasts

Keywords: myofibroblast, mitochondria, ROS, TGF- signaling, tumor

microenvironment

Financial support: This research was supported by a grant from the JSPS KAKENHI

Grant Number 18H03377, 17K19944, Industrial Disease Clinical Research Grants from

the Japanese Ministry of Health, Labour, and Welfare and in part by NIFS Collaborative

Research Program (NIFS13KOBA028).

on April 8, 2021. © 2018 American Association for Cancer Research. mcr.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on July 24, 2018; DOI: 10.1158/1541-7786.MCR-18-0321

http://mcr.aacrjournals.org/

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*Corresponding author: Tsutomu Shimura, PhD

Department of Environmental Health, National Institute of Public Health, 2-3-6 Minami,

Wako, Saitama 351-0197, Japan

Tel. +81-48-458-6261; Fax +81-48-458-6270; Email: [email protected]

Disclosure of Potential Conflict of interests

The authors declare no competing financial interests.




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Abstract

Fibroblasts are a key stromal cell in the tumor microenvironment (TME) and promote

tumor growth via release of various growth factors. Stromal fibroblasts in cancer, called

cancer-associated fibroblasts (CAFs), are related to myofibroblasts, an activated form of

fibroblast. While investigating the role of stroma fibroblasts on radiation-related

carcinogenesis, it was observed following long-term fractionated radiation (FR) that the

morphology of human diploid fibroblasts changed from smaller spindle shapes to larger

flat shapes. These cells expressed smooth muscle actin (α-SMA) and platelet-derived

growth factor receptors (PDGFRs), markers of myofibroblasts and CAFs, respectively.

Long-term FR induces progressive damage to the fibroblast nucleus and mitochondria

via increases in mitochondrial reactive oxygen species (ROS) levels. Here it is

demonstrated that long-term FR-induced α-SMA positive cells have decreased

mitochondrial membrane potential and activated oxidative stress responses. Antioxidant

N-acetyl cysteine suppressed radiation-induced mitochondrial damage and generation of

myofibroblasts. These results indicate that mitochondrial ROS are associated with the

acquisition of myofibroblasts after long-term FR. Mechanistically, mitochondrial ROS

activated TGFβ signaling which in turn mediated the expression of α-SMA in

radiation-induced myofibroblasts. Finally, in vivo tumor growth analysis in a human




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tumor xenograft model system revealed that long-term FR-induced myofibroblasts

promote tumor growth by enhancing angiogenesis.

Implications: Radiation affects malignant cancer cells directly and indirectly via

molecular alterations in stromal fibroblasts such as activation of TGFβ and angiogenic

signaling pathways.




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Introduction

Ionizing radiation offers numerous clinical benefits for diagnosis and therapy.

Radiotherapy (RT) is one of the most widely used therapies for cancer treatment. In

patients being treated with RT, the radiation dose is limited by the tolerance of healthy

tissue. To preserve normal tissue function, treatment is delivered in multiple small

fractions of radiation (fractionated radiation; FR) with dose of around 2 Gy for 5 to 7

weeks (1). Technological advances have significantly improved clinical outcomes of RT

by enabling increased radiation doses directed very specifically at a tumor. However,

the late effects of cancer treatment such as radiation-induced second malignancies are a

serious public health issue (1).

Ionizing radiation is a carcinogen that causes leukemia and solid cancers in humans.

Cancer risk following exposure to radiation was estimated in a life span study of atomic

bomb survivors from Hiroshima and Nagasaki (2,3). This epidemiological research

showed an increased in leukemia and solid cancer risk among atomic bomb survivors.

DNA double-strand breaks are a major cause of the detrimental biological effects of

radiation. DNA mutations associated with radiation-related carcinogenesis, the so-called

“radiation signature,” have been intensively investigated to elucidate the risk of

radiation-induced cancer (4,5). Currently, radiation signatures remain a matter of debate




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in a variety of cancers because the development of cancer is a multistep process that

occurs over long periods of time. Basic research on radiation-related carcinogenesis is

indispensable for obtaining a rigorous body of scientific evidence on the estimated risk

of cancer following ionizing radiation.

Mitochondria govern many cellular metabolic processes and are the energy power

house in healthy cells (6,7). Ionizing radiation damages mitochondria and impairs

mitochondrial respiration, leading to the overproduction of mitochondrial reactive

oxygen species (ROS). Mitochondria are the sites of intracellular ROS generation

through mitochondrial oxidative phosphorylation (OXPHOS) and are more vulnerable

to ROS toxicity than the nucleus (8,9). We recently discovered that long-term

FR-induced enhanced mitochondrial activity is evident by elevated mitochondrial

membrane potential and elevated activity of mitochondrial complex IV (cytochrome c

oxidase), which fills the energy demands for the chronic DNA damage response in

irradiated human fibroblasts (8,9). Subsequent reduction of the intercellular antioxidant

glutathione (GSH) via continuous activation of mitochondrial oxidative phosphorylation

(OXPHOS) causes oxidative stress and genomic instability in irradiated cells.

Mitochondrial dysfunction may be critical in facilitating radiation-related

carcinogenesis. Tumor cells preferentially metabolize glucose into lactate through




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glycolysis than using mitochondrial oxidative phosphorylation for energy production

because of their inability to respire (10). Aerobic glycolysis is a metabolic hallmark of

cancer, known as the Warburg effect (11,12). Metabolic alterations associated with

mitochondrial dysfunction increase tumorigenesis. Thus, cancer metabolism is strongly

related to cancer development and resistance to anti-cancer medication and radiation

(13,14). To control the quality of mitochondria, the parkin E3 ubiquitin ligase is

recruited to abnormal mitochondria with low membrane potential (ΔΨm) and promotes

their clearance by selective degradation (mitophagy) (15,16). ATM loss leads to

defective mitophagy and increased frequency of abnormal mitochondria with decreased

mitochondrial membrane potential. ATM-deficient cells showed highly radiosensitive

phenotypes when subjected to low-dose long-term FR, which induced in these cells

severe mitochondrial damage, as assessed by mitochondrial fragmentation, and cell

death proved to occur through mitochondria-mediated apoptosis (17). Furthermore, we

previously reported that mitochondrial radiation responses such as increase in

mitochondrial biogenesis and OXPHOS were not observed after low-dose, long-term

FR in ATM-deficient cells in contrast to ATM-complemented cells. ATM-dependent

signaling pathways have an essential role in the crosstalk between the nucleus and

mitochondria in irradiated cells (8,17).




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Fibroblasts are cells in connective tissue that contribute to the synthesis and deposition

of extracellular matrix (ECM) to induce changes in the tissue microenvironment. The

tumor microenvironment plays an important role in tumor initiation, progression, and

metastasis (18-20). Cancer-associated fibroblasts (CAFs), which are related to

myofibroblasts, are an activated from of fibroblasts in tumor tissues, are distinguishable

from their normal counterparts, and promote tumor growth by inducing angiogenesis

and recruiting bone marrow-derived endothelial progenitor cells (21). Expression of

α-SMA characterizes fibroblast-to-myofibroblast differentiation (22). CAF produce a

variety of growth factors, chemokines, and matrix-degrading proteases to promote

tumor growth and cancer cell aggressiveness, such as tumor angiogenesis, extracellular

matrix remodeling, tumor proliferation, invasion, and metastasis (18-20). CAF-derived

stromal-cell-derived factor 1 (SDF1) regulates the proliferation of breast cancer cells

(23). CAF modulates extracellular matrix (ECM) remodeling ability via producing

ECM-degrading proteases such as the MMPs and collagen (24,25). CAF also interacts

with the microvasculature by secreting VEGF (26). However, the role of tumor

microenvironments in radiation-induced carcinogenesis is not well understood.

We here investigated the association between mitochondrial oxidative stress and the

formation of tumor microenvironments during the development of radiation-related




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tumors. We examined whether ionizing radiation promotes myofibroblast differentiation

to facilitate tumor growth.

Materials and Methods

Cell culture conditions and drugs

Normal human diploid lung fibroblasts (MRC-5 and TIG-3) were purchased from the

Health Science Research Resources Bank and grown in minimum essential medium

(Nacalai Tesque, Kyoto, Japan) supplemented with 10% heat-inactivated fetal calf

serum. HeLa, a cervical cancer cell line, was obtained from the Cell Resource Center

for Biomedical Research, IDAC, Tohoku University. The cells were grown in

RPMI1640 medium (Nacalai Tesque, Kyoto, Japan) supplemented with 5%

heat-inactivated fetal calf serum. LY361949 and NAC were purchased from Sigma (San

Diego, CA, USA). NAC was continuously added to the media at a final concentration of

1 mM during exposure to long-term FR. Media was changed at 2- or 3-day intervals.

Cells were treated with LY361949 (3 µM) for 1 week during FR exposure (from 25 to

31 days).

Irradiation experiments




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Cells were irradiated using a 150-kVp X-ray generator (Model MBR-1505R2, Hitachi)

with 0.5-mm Cu and 0.1-mm Al filters. We previously reported that human fibroblasts

no longer proliferate under FR exposure for 31 days when cells were exposed to ≥0.5

Gy of X-rays per fraction. In contrast, cells exposed to 0.01 or 0.05 Gy FR continue to

grow exponentially for 31 day (27). Therefore, X-ray fractions (0.01 or 0.05 Gy) were

administered twice a day for 5 days/week in this study. The total doses delivered over

31 days were 0.46 and 2.3 Gy for cells exposed to FR of 0.01 and 0.05 Gy, respectively.

Cells continued to grow for 31 days following exposure to 0.01 or 0.05 Gy FR. When

cells reached 80% confluency, they were subcultured in a new flask.

Immunofluorescence

Immunofluorescence staining was performed as previously described (27,28). Cells

were seeded onto 18 × 18-mm cover slips, which were placed in 35-mm tissue culture

dishes and cultured overnight. The cells on cover slips were fixed with 4%

formaldehyde for 10 min and permeabilized with 0.5% Triton X-100 for 5 min. For

double staining with -SMA (Sigma) and parkin, cells were fixed with 4%

formaldehyde at 37˚C for 15 min and permeabilized with 0.2% Triton X-100 for 10 min.

Antibodies against -SMA (Sigma), parkin (Santa Cruz Biotechnology, Santa Cruz, CA,




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USA), PDGF (Abcam), Nrf2 (Nichirei Bioscience, Tokyo, Japan), Phospho-TGF-

receptor serine 165 (Thermo Fisher), and secondary antibodies conjugated with Alexa

Fluor 488 (Molecular Probes, Eugene, OR, USA) or Cy-3 (Jackson ImmunoResearch

Laboratories, West Grove, PA, USA) were used. Cells were counterstained for DNA

with Hoechst 33258 (4 µg/mL in Vectashield mounting medium; Vector Laboratories,

Burlingame, CA, USA). Images were captured using a CCD camera attached to a

fluorescence microscope (Keyence). For each data point, we counted more than 50 cells

from at least three independent samples.

Measurement of TGF-β, bFGF, and VEGF

TGF-β, bFGF, and VEGF were measured using an assay kit (Sigma) according to the

manufacturer’s instructions. The activity was reported as units/mg of cell extract.

Animal experiments

The study design was approved by the Animal Ethical Committee of the National

Institute of Public Health. Male BALB/c nu/nu mice at 5 weeks of age were used. All

mice were maintained in our animal facility on a 12-h light/dark cycle at a controlled

temperature (23˚C ± 2˚C). For transplantation, 5 × 106 HeLa cells and 1.5 × 10

7 normal




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fibroblasts cells in 100 l of saline were injected in the dorsal area of mice. Tumor size

was measured with calipers. The tumor volume (V) was estimated by V = 0.5×(length ×

width2). We calculated the average of tumor size for each data point from at least three

mouse.

Immunohistochemistry

Tumor tissues were fixed in 10% buffered formalin. Immunohistochemistry staining

was performed as previously described (29). Image acquisition and evaluation were

conducted with a Keyence BZ-X700 fluorescence microscope with the Hybrid Cell

Count software (BZ-II Analyzer, Keyence, Osaka, Japan). Three or four images were

used for the image analysis in each slide. We calculated the a-SMA positive area ratio in

HeLa tumors or in small intestine of Apc Min/+ mouse from at least two independent

samples.

Statistical analysis

Error bars represent standard deviations. All experiments were repeated at least three

times using independent samples. Following one-way ANOVA, the Dunnett test was

used to detect significant differences among the means of three or more independent




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groups. Student’s t-test was used for the tumor growth analysis. Double and single

asterisks indicate significant differences with p-values of <0.01 and <0.05, respectively.

Results

Myofibroblast induction by long-term FR

We investigated the effect of exposure to FR for 31 days (long-term FR) in human lung

fibroblasts, TIG-3, and MRC-5. Fibroblasts are elongated and spindle-shaped.

Following long-term FR, there are pronounced differences in the morphology of some

irradiated fibroblasts, which appear flat with a leaf-like shape. Cells with altered

morphology expressed a myofibroblast marker, α smooth muscle actin (α-SMA) (Figure

1A). Approximately 20% of TIG-3 and MRC-5 cells exposed to long-term FR were

positive for -SMA staining (Figure 1A, right panel). Cells exposed to <5 Gy acute

single radiation (SR) did not induce -SMA expression, although -SMA-positive cells

appeared after exposure to 10 Gy SR (Supplemental Figure 1). The platelet-derived

growth factor receptor (PDGFR) is expressed in CAFs (30,31). Immunofluorescent

staining (Figure 1B) or FACS analysis using a phycoerythrin (PE)-conjugated

anti-PDGFR antibody (PE-140a) (Figure 1C) showed an increased number of

PDGFR-expressing cells after long-term FR. According to the previous reports, only a




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small fraction of cells is co-expressed with PDGFR and alpha-SMA (21). We previously

reported that long-term FR of TIG-3 and MRC-5 cells induced mitochondrial oxidative

stress via decreased glutathione (GSH) levels and the accumulation of mitochondrial

ROS (8,9). This can be mitigated by the antioxidant N-acetyl-cysteine (NAC), a GSH

precursor, which increases intracellular GSH levels. We therefore examined the role of

mitochondrial oxidative stress on long-term FR-induced acquisition of myofibroblasts.

Statistical comparisons revealed that treatment of long-term FR cells with NAC

suppressed the induction of -SMA or PDGFR in TIG-3 and MRC-5 cells (Figure 1A,

1B, and 1C).

Mitochondrial stress in myofibroblast

To clarify the association between myofibroblast differentiation and mitochondrial

dysfunction, we examined -SMA-positive myofibroblasts for mitochondrial oxidative

damage. Cells were immunostained with antibodies specific for the E3 ubiquitin ligase,

parkin, which recognizes damaged mitochondria with low ΔΨm. Myofibroblasts

positive for -SMA had parkin foci in the mitochondria of cells exposed to long-term

FR (Figure 2A). The number of single and double positive for each marker, and total

cells was shown in the table. We observed a statistically significant increase in the




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number of TIG-3 and MRC-5 cells double-positive for -SMA and parkin foci

following long-term FR compared with non-irradiated cells (Figure 2A, right graph).

We previously reported that mitochondrial damage is attributable to increased

mitochondrial ROS levels in cells exposed to long-term FR (8). We therefore examined

the activation of the oxidative stress response in long-term FR-induced myofibroblasts.

The transcription factor Nrf2 relocates from the cytoplasm to the nucleus upon

activation and regulates the expression of oxidative stress-inducible genes that defend

against oxidative stress (32). In cells exposed to long-term FR, -SMA-positive

myofibroblasts showed the nuclear accumulation of Nrf2 (Figure 2B). We further

investigated the mechanism of Nrf2 regulation following long-term FR. NAC treatment

suppressed nuclear accumulation of Nrf2 in -SMA-positive myofibroblasts (Figure

2B). These results indicated that mitochondrial oxidative stress was likely to be induced

in myofibroblasts after long-term FR.

TGF- activation in myofibroblast

In healthy tissue, ionizing radiation induces the expression of TGF- as part of the

damage recovery mechanism (33). TGF- signaling plays a key role in

fibroblast-to-myofibroblast differentiation by inducing the expression of -SMA (22).




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To determine the role of TGF- signaling in long-term FR-induced acquisition of

myofibroblasts, we measured the concentration of secreted TGF- in the culture

medium of long-term FR cells (Figure 3A). TGF-levels were significantly increased

by long-term FR compared with non-irradiated cells. NAC eliminated the increased

TGF- concentration in long-term FR cells (Figure 3A). TGF- binds to its type 2

receptor (TGFR2), which then phosphorylates the type 1 receptor (TGFR1). The active

receptor complex phosphorylates Smad2/Smad3 (34). Elevated levels of phosphorylated

TGFR1 following long-term FR in MRC-5 cells indicated that the TGF- signaling

pathway was activated (Figure 3B). We used an inhibitor of TGF- signaling,

LY-364947, to test the role of TGF-signaling and NAC in long-term FR-induced

myofibroblast differentiation. Treatment with LY-364947 prevented TGF- signaling

following long-term FR. Similarly, NAC suppressed long-term FR-induced activation of

TGF- signaling (Figure 3B). The phosphorylation of TGFR1 was detected in

-SMA-positive myofibroblasts (Figure 3C). Treatment with LY-364947 abrogated

long-term FR-induced myofibroblast differentiation by the inhibition of the TGF-

signaling pathway (Figure 3B, 3C right panel, 3D).

Long-term FR cells enhance in vivo tumor growth




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To test the in vivo interaction between cancer cells and long-term FR-induced

myofibroblasts, we made a human tumor xenograft of HeLa cells, HeLa +

non-irradiated fibroblasts (HeLa + 0FR), HeLa + long-term FR fibroblasts (HeLa +

0.05FR), HeLa + NAC-treated fibroblasts (HeLa + NAC 0FR), and HeLa +

NAC-treated long-term FR fibroblasts (HeLa + NAC 0.05FR) in a 3:1 ratio in nude

mice. Individual tumor measurements data for each mouse at each time point were

shown in supplemental figure 2. We determined the average slope of tumor growth

curves for each tumor type. HeLa cells with long-term FR irradiated fibroblasts

generated tumors of greater volume than HeLa cells with non-irradiated fibroblasts

(Figure 4A). HeLa + NAC 0FR and HeLa + NAC 0.05FR tumors did not show a growth

advantage in nude mice compared with HeLa + 0FR tumors (Figure 4B).

Immunohistochemistry with an anti--SMA antibody showed that HeLa + 0.05FR

tumors comprised a higher number of -SMA-positive myofibroblasts than HeLa and

HeLa + 0FR tumors (Figure 5A). The adenomatous polyposis coli (Apc) Min/+ mouse

model is widely used to investigate the radiation-induced formation of intestinal

adenoma. Using anti--SMA antibodies, we observed that radiation-induced tumors had

many -SMA-positive fibroblasts surrounding tumor cells (Figure 5B). Quantification

data of -SMA positive area ratio in HeLa tumors or in small intestine of Apc Min/+




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mouse were shown in Figure 5C.

Myofibroblasts promote angiogenesis

To characterize long-term FR-induced myofibroblasts, we measured levels of

extracellular factor released from long-term FR cells. Compared with non-irradiated

control cells, levels of basic fibroblast growth factor (bFGF) and vascular endothelial

growth factor (VEGF) were higher in the culture medium of long-term FR cells (Figure

6A, 6B). Treatment with NAC eliminated the stimulation of bFGF and VEGF in

long-term FR cells.

We examined in vivo tumor angiogenesis in human tumor xenografts. CD31+

endothelial progenitor cells proportion was increased in tumor xenografts containing

long-term FR cells compared with those containing non-irradiated fibroblasts (Figure

6C).

Discussion

We investigated the harmful effects of long-term FR exposure in relation to medical

exposure in normal cells. Our data demonstrated that long-term FR-induced

myofibroblasts that express α-SMA and PDGFR have an abnormal, flat, large




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morphology. For FR, a total radiation dose of 0.46 Gy (0.01 Gy/fractions, twice a day, 5

days/week for 31days) is sufficient to induce the acquisition of myofibroblasts.

However, a radiation dose of 10 Gy is required to induce -SMA-expressing

myofibroblasts in acute SR. Thus, the acquisition of myofibroblasts is influenced by the

dose and method of radiation exposure. The induction of myofibroblasts following

long-term FR is associated with mitochondrial dysfunction and oxidative stress, as

shown by the formation of parkin foci and nuclear accumulation of Nrf2 in

-SMA-expressing cells, respectively. We recently showed that long-term FR exposure

elevates mitochondrial OXPHOS in response to the persistent energy requirement of the

DNA damage response (8). The activation of mitochondrial OXPHOS leads to the

continuous mitochondrial ROS generation and a decrease in antioxidant GSH levels

(35). Alterations in GSH levels are not observed following SR (9). Long-term FR, but

not SR, causes mitochondrial oxidative stress in human fibroblasts by perturbing

mitochondrial redox. Treatment with antioxidant NAC restored GSH levels and

decreased radiation-induced mitochondrial ROS levels (9). NAC suppressed

myofibroblast appearance after long-term FR. We provided the first demonstration that

persistent mitochondrial oxidative stress induced by long-term FR stimulates the

differentiation of myofibroblasts from normal fibroblasts.




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Myofibroblasts are an activated form of fibroblasts that play important roles in wound

healing (36). TGF- signaling is a master switch for radiation-induced fibrosis (37).

Plasma TGF- levels can be used to predict the risk of radiation-induced lung injury.

TGF- plays an essential role in ECM remodeling, cell mobility, and immune function

modulation. TGF- regulates the formation and breakdown of connective tissue (34,37).

We demonstrated that long-term FR stimulates TGF-signaling in irradiated cells via

mitochondrial ROS-mediated activation of fibroblasts by TGF--signaling (Figure 7).

We showed that ROS are involved in the activation of TGF-. The pro-TGF-complex

is a large molecule containing TGF- and the latency association protein (LAP). ROS

directly oxidizes LAP and indirectly activates MMP2, which in turn cleaves LAP to

release active TGF-. TGF- then suppresses the expression of several

antioxidant enzymes including glutaredoxin, catalase, superoxide dismutase, and

glutathione peroxidase and decreases the concentration of GSH (39). Receptor

stimulation for TGF- results in the activation of a signaling pathway that increases the

transcription of the gene encoding smooth muscle actin to induce myofibroblast

differentiation. Consequently, mitochondria-mediated oxidative stress is associated with

the induction of TGF-signaling.

During skin would healing, recruited fibroblasts and differentiated myofibroblasts




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proliferate and deposit ECM to form granulation tissue (36). Myofibroblasts are

responsible for excessive ECM production, leading to fibrosis (22). Therefore, at the

later stages of wound healing, myofibroblasts are driven into senescence to control

fibrogenesis. These senescent cells promote tissue remodeling and clearance of

myofibroblasts (40). Cellular senescence is a potent tumor-suppressive mechanism to

prevent malignant transformation by arresting the growth of abnormal cells. Senescent

fibroblasts release senescence-associated secretory phenotype factors, including

interleukins, chemokines, growth factors, secreted protease, and ECM components, that

modify the tissue microenvironment (41,42). Therefore, cellular senescence is both

beneficial and deleterious for tumorigenesis.

We demonstrated that -SMA-expressing fibroblasts existed within tumors in small

intestine of Apc Min/+ mouse induced by ionizing radiation. Alterations in fibroblasts

play a key role in tumor bed effects on radiation-related tumorigenesis (19,20). Stromal

fibroblasts stimulate tumorigenesis by releasing factors that act on tumor cell growth.

Our data showed that long-term FR-induced myofibroblasts release bFGF and TGF-

which are key mediators of fibroblast activation. This study contains a limitation of not

using fibroblasts and tumor cells of similar histology, because we used lung-derived

fibroblasts with a cervical cancer cell line. In vivo animal experiments showed that




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long-term FR-induced myofibroblasts promote tumor growth of HeLa cells in nude

mice. Tumor progression is clearly dependent on angiogenesis. Long-term FR-induced

VEGF appears to play a major role in angiogenesis in tumor tissues.

In conclusion, long-term FR-induced mitochondrial stress causes the acquisition of

activated fibroblasts (myofibroblasts). Radiation-induced myofibroblasts promote tumor

growth in vivo. Alterations in stromal cells can contribute to radiation-related tumors.

Radiation affects malignant cancer cells directly and indirectly via the tumor stroma.

Author Contributions

Conception and design: T. Shimura

Development of methodology: T. Shimura

Acquisition of data (provided animals, acquired and managed patients,

provided facilities, etc.): M. Sasatani, H. Kawai, J. Kobayashi, K. Komatsu

Analysis and interpretation of data (e.g., statistical analysis, biostatistics,

computational analysis): T. Shimura

Writing, review, and/or revision of the manuscript: T. Shimura, M. Sasatani,

H. Kawai, J. Kobayashi

Administrative, technical, or material support (i.e., reporting or organizing




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data, constructing databases): M. Sasatani, H. Kawai, J. Kobayashi, K. Komatsu, K.

Kamiya

Study supervision: N. Kunugita

Acknowledgments

We thank Dr. Shinkai Youichi for the measurement of TGF-β. This research was

supported by a grant from the JSPS KAKENHI Grant Number 18H03377, 17K19944,

Industrial Disease Clinical Research Grants from the Japanese Ministry of Health,

Labour, and Welfare and in part by NIFS Collaborative Research Program

(NIFS13KOBA028). This work was performed at the Joint Usage/Research Center

(Radiation Biology Center), Kyoto University, and the Program of the network-type

joint Usage/Research Center for Radiation Disaster Medical Science of Hiroshima

University, Nagasaki University, and Fukushima Medical University.

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Figure Legends

Figure 1. Induction of -SMA and PDGFR expression by long-term FR.

(A) Images of -SMA staining in non-irradiated control cells (0FR) and TIG-3




27

cells exposed to 0.01 FR for 31 days (0.01FR) with/without NAC treatment. Cells were

stained 24 h after the last FR. Scale bar = 50 µm. The percentage of TIG-3 and MRC-5

cells with -SMA staining for each irradiation method is shown in the graph on the right.

The total number of cells observed in this analysis was shown in table. (B) Images of

PDGFR staining in 0FR cells and 0.01FR cells of TIG-3. The percentage of TIG-3 and

MRC-5 cells with PDGFR staining for each irradiation method is shown in the graph on

the right. The total number of cells observed in this analysis was shown in table. (C)

FACS analysis for PDGFR (PE-140a) in non-irradiated control (0FR; dotted black lines)

and indicated cells (solid lines) in TIG-3 cells. The percentage of TIG-3 and MRC-5

cells with PDGFR staining for each irradiation method are shown in the graph on the

right.

Figure 2. Mitochondrial dysfunction and oxidative damage in long-term FR-induced

myofibroblasts.

(A) Images of -SMA and parkin staining and (B) Images of -SMA and Nrf2 staining

in 0FR and 0.05FR cells of TIG-3. Scale bar = 50 µm. The number of single and double

positive for each marker, and total cells was shown in the table (percentage in

parenthesis). For each data point, more than 50 cells were counted from at least four




28

independent samples. The percentage of double-positive TIG-3 and MRC-5 cells for

each irradiation method was shown in the graph on the right.

Figure 3. Activation of TGF- signaling for myofibroblast differentiation.

(A) TGF- levels in TIG-3 and MRC-5 cells 72 h after the last FR. We calculated the

average of TGF-levels for each data point from at least five independent samples.

Asterisks indicate a significant difference in the levels of TGF- in irradiated cells

compared with non-irradiated cells. (B) Western blots for Phospho-TGFR1, TGFR1,

and actin in 0FR and 31FR cells with/without NAC or LY364947 treatment. (C) Images

of -SMA and phospho-TGFR1 staining in 0FR and 0.05FR cells of TIG-3 cells. Scale

bar = 50 µm. Cells were stained 24 h after the last FR. The number of single and double

positive for each marker, and total cells was shown in the table (percentage in

parenthesis). For each data point, more than 50 cells were counted from at least three

independent samples. (D) Western blots for -SMA and actin in 0FR and 31FR cells

with/without NAC or LY364947 treatment.

Figure 4. The role of radiation-induced myofibroblast for in vivo tumor growth

(A) In vivo tumor growth of HeLa cells, HeLa + non-irradiated fibroblasts (HeLa +




29

0FR), HeLa + long-term FR fibroblasts (HeLa + 0.05FR). (B) In vivo tumor growth of

HeLa cells, HeLa + NAC-treated fibroblasts (HeLa + NAC 0FR), and HeLa +

NAC-treated long-term FR fibroblasts (HeLa + NAC 0.05FR). We calculated the

average of tumor size for each data point from at least three independent samples. Black

and red asterisks indicate the significance change of tumor volume in HeLa +

non-irradiated fibroblasts samples with/without NAC treatment compared to that of

HeLa cells and HeLa + long-term FR fibroblasts samples with/without NAC treatment

compared to that of HeLa cells, respectively.

Figure 5. Expression of -SMA in human tumor xenografts and Apc Min/+ mice.

(A) Histological sections of human tumor xenografts on day 31 were immunostained

with -SMA, hematoxylin, and eosin. Scale bar = 100 µm. (B) Immunostaining with

-SMA, hematoxylin, and eosin in histological sections of intestinal tumors from Apc

Min/+ mice. Scale bar = 100 µm. Magnified images for -SMA staining were inserted.

(C) Quantification data of immunostaining with -SMA in HeLa tumors and in small

intestine of Apc Min/+ mouse.

Figure 6. The role of long-term FR-induced myofibroblasts in angiogenesis.




30

Levels of bFGF (A) and VEGF (B) in TIG-3 and MRC-5 cells 72 h after the last FR.

Asterisks indicate significant differences in the amount of bFGF or VEGF in irradiated

cells compared with non-irradiated cells. We calculated the average of the amount of

bFGF or VEGF for each data point from at least four independent samples. (C)

Quantification of blood vessel density in HeLa tumors. Blood vessels in five fields of

three tumors were quantified. Image of cd31 staining in HeLa + non-irradiated

fibroblasts (HeLa + 0FR) and HeLa + long-term FR fibroblasts (HeLa + 0.05FR). DNA

was stained with Hoechst. Scale bar = 100 µm.

Figure 7. Schematic representation of mitochondrial ROS-mediated myoblast

differentiation via TGF--signaling.




Published OnlineFirst July 24, 2018.Mol Cancer Res Tsutomu Shimura, Megumi Sasatani, Hidehiko Kawai, et al.

SignalingβMitochondrial ROS-activated TGFRadiation-induced Myofibroblasts Promote Tumor Growth via

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