Inhibition of endometrial cancer by n-3 polyunsaturated fatty acids (PUFAs… · 2014. 5. 24. · 1...

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Inhibition of endometrial cancer by n-3 polyunsaturated fatty acids

(PUFAs) in preclinical models

Hang Zheng1,5, Hongjun Tang1,5, Miao Liu1,5, Minhong He1, Pinglin Lai2, Heling Dong2, Jun Lin2,

Chunhong Jia2, Mei Zhong3, Yifan Dai4 Xiaochun Bai2 and Liping Wang3

1Department of Oncology, Nanfang Hospital, Southern Medical University, Guangzhou 510515, China;

2Department of Cell Biology, School of Basic Medical Sciences, Southern Medical University, Guangzhou

510515, China; 3Department of Obstetrics and Gynecology, Nanfang Hospital, Southern Medical

University, Guangzhou 510515, China and 4State Key Laboratory of Reproductive Medicine and Jiangsu

Key Laboratory of Xenotransplantation, Nanjing Medical University, Nanjing 210029, China

5These authors contributed equally to this manuscript

Running title: Inhibition of endometrial cancer by n-3 PUFAs

Keywords: endometrial cancer; n-3 polyunsaturated fatty acids (n-3 PUFAs); mTORC1; mTORC2; fat-1;

endometrial hyperplasia

Grant support:

The State Key Development Program for Basic Research of China (2013CB945203), National Natural

Sciences Foundation of China (81270088 and 81372136) and Program for Changjiang Scholars and

Innovative Research Team in University (IRT1142)

Corresponding author

Liping Wang, Department of Obstetrics and Gynecology, Nanfang Hospital, Southern Medical University,

Guangzhou 510515, China (E-mail: [email protected]); Xiaochun Bai, Department of Cell Biology,

School of Basic Medical sciences, Southern Medical University, Guangzhou, 510515, China (T:

+86-20-61648724; F: +86-20-61648208; E-mail: [email protected]).

Word count: 4981 words

Total number of tables and figures: 6

Disclosure of Potential Conflicts of Interest:

No potential conflicts of interest were disclosed.

for Cancer Research. on September 10, 2021. © 2014 American Associationcancerpreventionresearch.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on May 27, 2014; DOI: 10.1158/1940-6207.CAPR-13-0378-T

http://cancerpreventionresearch.aacrjournals.org/

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Abstract

Although preclinical and epidemiological studies have shown the importance of n-3 polyunsaturated

fatty acids (PUFAs) in the prevention of hormone-responsive cancers such as breast cancer, evidence of the

association between n-3 PUFAs and endometrial cancer risk is limited and no previous study has examined

the effect of n-3 PUFAs on endometrial cancer in cellular and animal models. In this study, we

demonstrated that docosahexenoic acid (DHA) dose-and time-dependently inhibited endometrial cancer

cell proliferation, colony formation and migration, and promoted apoptosis. Dietary n-3 PUFAs efficiently

prevented endometrial cancer cell growth in xenograft models. Moreover, ectopic expression of fat-1, a

desaturase, catalyzed the conversion of n-6 to n-3 PUFAs and produced n-3 PUFAs endogenously, also

suppressed endometrial tumor cell growth and migration, and potentiated apoptosis in endometrial cancer

cell lines. Interestingly, implanted endometrial cancer cells were unable to grow in fat-1 transgenic SCID

(severe combined immune deficiency) mice. Further study revealed that mammalian target of rapamycin

(mTOR) signaling, which plays an essential role in cell proliferation and endometrial tumorigenesis, is a

target of n-3 PUFAs. Exogenous or endogenous n-3 PUFAs efficiently suppressed both mTOR complex 1

(mTORC1) and mTORC2 in vitro and in vivo. Moreover, both dietary n-3 PUFAs and transgenic

expression of fat-1 in mice effectively repressed mTORC1/2 signaling and endometrial growth elicited by

unopposed oestrogen. Taken together, our findings provide comprehensive preclinical evidences that n-3

PUFAs efficiently prevent endometrial cancer and establish mTORC1/2 as a target of n-3 PUFAs.




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Introduction

Endometrial cancer is the most common gynecologic malignancy and a major cause of morbidity and

mortality in women worldwide, with nearly 200 000 cases diagnosed every year and a rising incidence in

postmenopausal women (1-4). Although the precise cause of endometrial cancer is unknown, various

associated risk factors for the disease have been identified. The main risk factors for the development of

endometrial carcinoma are obesity and chronic unopposed estrogen stimulation of the endometrium (5-7).

Previous publications demonstrated that parity, oral contraception, body mass index (BMI), physical

activity and diet may explain up to 80% of the risk of endometrial cancer, emphasizing the importance of

lifestyle modification for prevention of this disease (8). On the other hand, although it is highly treatable by

surgery when diagnosed at an early stage and grade, therapies for advanced and recurrent disease are rarely

curative (1, 9, 10). Currently, the treatment of metastatic or recurrent disease is based on conventional

chemotherapy combination regimens (11). Advances in the understanding of the molecular pathology of

endometrial carcinoma have lead to the development and testing of targeted therapies (12, 13). Of the

potential therapeutic targets identified to date, the mechanistic target of rapamycin (mTOR) signaling

pathway is a major target for treatment of this disease (14).

mTOR is a highly conserved Ser/Thr kinase which integrates diverse signals including nutrients, growth

factors, energy and stresses to control cell growth, proliferation, survival, and metabolism (15-18). mTOR

elicits its pleiotropic functions in the context of two functionally distinct signaling complexes, termed

mTOR complex 1 (mTORC1) and complex 2 (mTORC2). mTORC1 plays a key role in translation

initiation by directly phosphorylating p70 S6 kinase 1 (S6K1) and 4E-BP1, and is sensitive to rapamycin.

mTORC2 is not susceptible to acute rapamycin inhibition (17, 18). The function of mTORC2 is less clear,

but it has been shown to phosphorylate Akt (S473) and to regulate cell survival and cell motility (15, 18).




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As a critical drug target, mTOR signaling is up-regulated in endometrial cancer and plays key roles in the

carcinogenesis and progression of the disease (19). However, clinical trials have shown that responses to

indirect (allosteric) mTORC1 inhibitors are modest, which in part, relate to positive feedback from

mTORC2 on the Akt pathway that can continue following inhibition of mTORC1 (20). Therefore,

second-generation catalytic mTORC1/2 inhibitors that can act directly on mTOR are being developed and

have entered clinical trials (21, 22).

Omega-3 (n-3) and omega-6 (n-6) polyunsaturated fatty acids (PUFAs) are essential fatty acids necessary

for human health. Laboratory and animal studies have shown that the long-chain n-3 PUFAs,

eicosapentenoic acid (EPA) and docosahexenoic acid (DHA), inhibit tumorigenesis at various cancer sites

(23). n-6 PUFAs such as arachidonic acid (AA), on the other hand, have been shown to promote tumor

growth and progression (24). Total fat intake and the ratio of n-6 to n-3 PUFAs in the Western diet have

increased significantly since the Industrial Revolution, which is thought to contribute to obesity,

inflammation and cancer (24). Studies in human populations have linked high consumption of fish or fish

oil (n-3 PUFAs) to a reduced risk of colon, prostate, and breast cancer, and combined with the

demonstrated effects of n-3 PUFAs on cancer from preclinical (animal and in vitro) models, has motivated

the development of clinical interventions using n-3 PUFAs in the prevention and treatment of these cancers

(25, 26).

Although the role of dietary PUFAs in various cancers has received significant attention in the literature,

and the preventive effect of n-3 PUFAs on obesity has been established (27), evidence on the association

between n-3 PUFAs and endometrial cancer is limited. A recent epidemiological study suggests that higher

dietary intake of EPA and DHA in food and supplements were associated with lower risk of endometrial

cancer (28). No previous study has examined the effect of n-3 PUFAs on endometrial cancer cell and




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animal models. We previously developed a transgenic mouse model that expresses fat-1 (29), a desaturase,

that catalyzes the conversion of n-6 to n-3 PUFAs and produces n-3 PUFAs endogenously, which enables

investigation of the biological properties of n-3 PUFAs without having to incorporate n-3 PUFAs such as

DHA in the diet. Our recent work also identified a critical role for the n-6 PUFA (AA) and n-3 PUFAs in

the mTORC1/2 signaling in breast carcinogenesis and angiogenesis (30, 31). This study aimed to identify

the effects of dietary n-3 PUFAs and transgenic fat-1 expression on mTORC1/2 signaling and the

prevention of endometrial cancer in preclinical models.

Materials and Methods

Materials

All cell culture reagents were obtained from Gibco BRL Technology. Arachidonic acid (AA) and

docosahexenoic acid (DHA) were obtained from Cayman Chemical. Tamoxifen citrate, cisplatin (DDP),

antibodies against �-actin and HRP-conjugated anti-mouse and anti-rabbit IgG were from Sigma. Primary

antibodies against phospho-S6 (S235/236) and poly (ADP-ribose) polymerase (PARP) were from Cell

Signaling Technology. Anti-S6, phospho-Akt (S473) and Akt antibodies were bought from Santa Cruz

Biotechnology, Inc. Recombinant adenovirus with or without the fat-1 gene was produced by cloning fat-1

cDNA into the RAPAd® CMV Adenoviral Expression System (Cell Biolabs).

Cell culture

Endometrial cancer cell lines, HEC-1-A, HEC-1-B and RL95-2 were obtained from American Type

Culture Collection (ATCC). Cell lines were frozen in bulk when received and maintained in McCOY's 5A

(HEC-1-A) or MEM (HEC-1-B) or D-MEM/F-12 (RL95-2) supplemented with 10% fetal bovine serum




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(FBS) at 37°C, 5% CO2, and 95% humidity. They had been passed for less than 6 months in culture when

the experiments were carried out. Cell lines were authenticated using single tandem repeat analysis (STR).

Wound healing assay

HEC-1-A and HEC-1-B cells were seeded in 12-well plates and DHA was added or the cells were

transfected with recombinant adenovirus, and grown until 80% confluent. The cells were then pretreated

with 1 �g/ml of mitomycin C for 24 h in a minimum medium (containing 0.5 % FBS). After making a

straight scratch using a pipette tip, the cells were incubated in medium containing 0.5% FBS in a 37°C

humidified incubator for 48 h and the wound distances were measured under a microscope.

Transwell assay

The cell culture inserts (Corning, NY) were placed in a 24-well plate. Before use, the cell culture inserts

were rehydrated with 200 �l warm McCOY's 5A or MEM for 30 min. HEC-1-A and HEC-1-B cells were

plated in the upper chamber with 500 �l McCOY's 5A or MEM containing various concentrations of DHA.

The lower chamber was filled with 800 �l McCOY's 5A or MEM containing 10% FBS. The cells were then

incubated at 37°C for 24 h. After swabbing of non-invaded cells in the upper chambers, cells that migrated

to the lower chambers were fixed with formaldehyde and stained with crystal violet. For quantification, the

cells that had migrated to the lower surface were counted under a light microscope.

Tumor xenograft models

Four-week-old female BALB/c nude mice were purchased from Guangdong Medical Experiment Animal

Centre (Guangzhou, China). Each animal was injected with 1×106/0.1ml of HEC-1-A or RL95-2 cell




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suspension into the flanks. The mice were randomized into four groups (n = 6) and administered a normal

(low) or high n-3 PUFAs diet (Supplementary Table S1). For cisplatin (DDP) combination therapy,

cisplatin (50 μg per mouse) in 0.25 ml of saline were injected intraperitoneally once per day. Bidimensional

tumor measurements were taken every three days. At the end of the experiment, the mice were killed and

tumors were removed, weighed, and the proteins extracted for analysis. Tumor volume was measured along

two major axes using calipers. Tumor volume (mm3) was calculated as follows: V = 1/2LW2 (L: length, W:

width).

Homozygous severe combined immune deficiency (SCID) mice (Jax Number: 001131) (Balb/c

background) were bred with fat-1 transgenic mice (originally on the C57BL/6 background) produced

previously (29) to generate fat-1-SCID double-hybrid mice. These mice were backcrossed for 10 generation

onto Balb/c. Female littermates lacking the fat-1 transgenic gene were used as controls. DNA extractions

from the tail tips of offspring were subjected to PCR for genotyping in accordance with the protocol on the

Jackson Laboratory webpage, using primers listed in Supplementary Table S2. Four-week-old female

homozygous SCID mice (n = 6) with or without the fat-1 gene were injected with 100 �l (1×105) of RL95-2

cell suspension. Subsequent bidimensional tumor measurement and tumor sample analysis were performed

as described above.

Tamoxifen-elicited endometrial growth

The mouse model of endometrial growth induced by unopposed estrogenic stimulation was established

as described previously (32). Briefly, ten-week-old Balb/c mice underwent midline laparotomy and

bilateral oophorectomy and were randomly assigned (n = 8) to a normal (low), high n-3 PUFAs or high n-6

PUFAs (6 g/kg/d AA) diet. One week after recovery, the mice received saline or 1 or 3 mg/kg/d tamoxifen




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citrate with the normal, high n-3 PUFAs or high n-6 PUFAs (6 g/kg/d AA) diet. All drugs were

administered by oral gavage. The animals were sacrificed by decapitation after the end of treatment, and

their uteri were removed by hysterectomy for histopathological examination. Morphometric analysis was

performed on midhorn uterine cross-sections for all animals (n = 8 per treatment group). Luminal epithelial

cell height was quantified for each slide using 40× magnification. In another set, wild type or fat-1

transgenic mice (n = 8) underwent bilateral oophorectomy and were treated with 1mg/kg/d tamoxifen

citrate for 3 d, subsequently sacrificed and subjected to histology and morphometric analysis as described

above.

Statistical analysis

All animal procedures were carried out in the mouse facility using protocols approved by the Animal

Care and Use Committee of Southern Medical University. Data are presented as mean ± SD of at least three

independent experiments. Differences between groups were analyzed using Student’s t test (SPSS 13.0),

and a level of p<0.05 was considered statistically significant.

Methods for cell proliferation assay, colony formation assays, cell viability assay, western blot analysis

and gas chromatography analysis of fatty acids compositions were described in Supplementary data.

Results

Exogenous n-3 PUFAs inhibit endometrial cancer cell growth in vitro and in xenograft models

To investigate the potential protective role of n-3 PUFAs against endometrial cancer, we first examined

the effect of DHA on the proliferation of cultured endometrial carcinoma HEC-1-A, HEC-1-B and RL95-2

cell lines. We found that DHA dose- and time-dependently inhibited cell proliferation in these cells (Fig.




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1A, B and C). On the contrary, n-6 PUFA (AA) stimulated cell proliferation in these endometrial cancer cell

lines (Supplementary Fig. S1), which is in consistent with our previous results in breast cancer cells [29].

The colony formation assay further confirmed that DHA prevented colony formation of these cell lines in a

dose-dependent manner (Fig. 1D, E and F; Supplementary Fig. S2). These results demonstrate that DHA

effectively inhibits endometrial cancer cell growth in vitro.

To further identify the effect of n-3 PUFAs on endometrial cancer cell growth in vivo, we established

endometrial tumor xenograft models by subcutaneously implanting HEC-1-A and RL95-2 cells into nude

mice. We found that High n-3 PUFAs diet efficiently prevented tumor growth and reduced the average

tumor weight and tumor volume (Figure 1G and H). Fatty acid composition analysis identified a

significantly increased ratio of n-3/n-6 PUFAs in the tumors of mice with high n-3 PUFAs diet compared

with that of mice with normal diet (Supplementary Table S3). Interestingly, high n-3 PUFAs diet could also

enhance the inhibitory effect of cisplatin, a cytotoxic anti-endometrial cancer drug (Supplementary Fig. S3).

These results indicate that dietary n-3 PUFAs suppress endometrial cancer cell growth in vivo.

Together, these findings demonstrate that DHA or dietary n-3 PUFAs effectively inhibit endometrial

cancer cell growth in animal and cell culture models.

Endogenously produced n-3 PUFAs inhibit endometrial cancer cell growth in vitro and in xenograft

models

Diet or nutritional supplements contain many nutrients and other components that may interact, which

adds a layer of complexity to their evaluation. Transgenic expression of fat-1 is capable of converting n-6

to n-3 PUFAs, leading to an increase in n-3 PUFAs and a decrease in the n-6/n-3 ratio, and allows

well-controlled studies to be performed in the absence of restricted diets (33). We further established




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endometrial cancer cell culture and tumor xenograft models producing endogenous n-3 PUFAs. Firstly,

proliferation and colony formation of HEC-1-A, HEC-1-B and RL95-2 cells transfected with recombinant

adenovirus with or without the fat-1 gene (Ad-fat-1 or Ad) were assessed. As expected, both proliferation

rate (Fig. 2A, B and C; Supplementary Fig. S4) and colony formation (Fig. 2D, E and F; Supplementary

Fig. S5) of fat-1 expressing cells were significantly decreased compared with the control cells in all tested

endometrial cancer cell lines.

Next, we established fat-1 transgenic SCID mice. Fatty acid composition analysis identified a

significantly increased ratio of n-3/n-6 PUFAs in these mice compared with wild type (WT) SCID mice

lacking fat-1 expression (Supplementary Table S4). Interestingly, although xenograft tumors with an

average volume of 223 mm3 were observed within 3 weeks in control SCID mice, we failed to observe

tumor growth in any of the fat-1 SCID animals (Fig. 2G and H). We suggest that endometrial tumor cells

are unable to proliferate or survive in the presence of high levels of endogenously-produced n-3 PUFAs in

vivo. Taken together, these data unequivocally demonstrate that endogenously produced n-3 PUFAs

suppress endometrial cancer cell growth in vitro and in vivo.

n-3 PUFAs prevent endometrial cancer cell migration

Metastasis is the major cause of mortality and morbidity in endometrial cancer patients. Invasion of

cancer cells into surrounding tissue and the vasculature is an initial step in tumor metastasis. This requires

migration of cancer cells. To investigate the potential role of n-3 PUFAs in endometrial cancer cell

migration, the effects of DHA on cell migration were examined using a wound healing assay in serum-free

medium., We found that 25 μM of DHA-treated HEC-1-A and HEC-1-B cells filled the gap more slowly

than vehicle control cells, suggesting that DHA prevented endometrial cancer cell migration (Fig. 3A and B;




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Supplementary Fig. S6). We further confirmed these results by the cell migration transwell assay. Cells

were treated with mitomycin C to inhibit proliferation thus facilitating cell motility analysis and cells which

had migrated to the lower chamber were quantified 48 h after incubation with DHA. The results showed

that DHA significantly prevented HEC-1-A (Fig. 3C) and HEC-1-B (Fig. 3D) cell migration as measured

by crystal violet staining. Moreover, fat-1 expressing HEC-1-A (Fig. 3E) and HEC-1-B (Fig. 3F) cells

migrated more slowly than vehicle control cells, indicating that endogenous n-3 PUFAs inhibit endometrial

cancer cell migration.

n-3 PUFAs promote endometrial cancer cell apoptosis in vivo and in vitro

n-3 PUFAs have been shown to promote apoptosis in a variety of cancer cells (26). We next examined if

n-3 PUFAs potentiate endometrial cancer cell apoptosis in vitro and in xenografts. It was found that DHA

dose-dependently increased the number of dead cells in serum-starved HEC-1-A, HEC-1-B and RL95-2

cell lines (Fig. 4A, B and C; Supplementary Fig. S7). Cleavage of PARP was enhanced by DHA in a dose-

and time-dependent manner (Fig. 4D). DHA could also enhance the pro-apoptotic effect of cisplatin on

HEC-1-A and RL95-2 cells (Supplementary Fig. S8). These results suggest that DHA promotes endometrial

cancer cell apoptosis in vitro. The effect of n-3 PUFAs on apoptosis was further examined in xenograft

models. The results revealed that administration of n-3 PUFAs promoted xenografted endometrial tumor

cell apoptosis as manifested by the increased apoptotic cell numbers and enhanced cleavage of PARP in

tumors in mice with high n-3 PUFAs diet (Fig. 4E). We conclude that n-3-PUFAs promote endometrial

cancer cell apoptosis in vivo and in vitro.

n-3 PUFAs inhibit mTORC1/2 signaling in endometrial cancer cell lines and xenograft models




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Previous studies have shown that activation of mTOR and phosphorylation of 4E-BP1 occur frequently

in advanced-stage and high-grade endometrial tumors, respectively, and are associated with cancer

progression and reduced survival (34). mTOR signaling plays important roles in tumorigenesis and

progression of endometrial cancer and have revealed a clinical advantage in targeting this pathway (12, 14).

We recently reported a critical role for n-6 PUFA-activated mTORC1/2 signaling in mammary

tumorigenesis and angiogenesis (30). We then determined if n-6 PUFAs stimulate mTORC1/2 activity in

endometrial cancer cells. As expected, AA (n-6 PUFA) acutely stimulated mTORC1-directed

phosphorylation of S6 (S235/235) and mTORC2-directed phosphorylation of Akt at position Ser 473 (Fig.

5A). We next examined whether DHA inhibits mTORC1/2 in endometrial cancer cell lines. In HEC-1-A

cells, DHA rapidly and dose-dependently suppressed AA-stimulated phosphorylation of S6 (S235/235) and

Akt (S473) (Fig. 5B). We further examined the role of endogenously produced n-3 PUFAs on mTORC1/2

signaling. In HEC-1-A cells transfected with fat-1 cDNA, the phosphorylation of S6 (S235/235) and Akt

(S473) were significantly reduced compared with cells transfected with the control vector (Fig. 5C). These

results were further repeated in HEC-1-B cells (Supplementary Fig. S9). It is suggested that both mTORC1

and mTORC2 signaling pathways are targets of exogenous and endogenous n-3 PUFAs in endometrial

cancer cells.

We next determined if n-3 PUFAs suppress mTORC1/2 in vivo. Interestingly, high dietary n-3 PUFAs

repressed both mTORC1 and mTORC2 activities in the endometrial tumor xenograft model, as manifest by

decreased phosphorylation levels of S6 (S235/236) and Akt (S473) in mice with a high n-3/n-6 PUFAs diet

(Fig. 5D). Moreover, levels of phosphorylated S6 (S235/236) and Akt (S473) were lower in the livers of

fat-1 SCID mice compared to wild type (WT) mice (Fig. 5E). Importantly, PP242, an mTORC1/2 inhibitor

did not enhance the inhibitory effects of n-3 PUFAs on endometrial cancer cells (Fig. 5F and G). We




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suggest that mTORC1 and mTORC2 signaling are targets of n-3 PUFAs in vivo and the suppression of

mTORC1/2 signaling by n-3 PUFAs may contribute to their inhibitory effects on endometrial tumor

growth.

n-3 PUFAs repress the endometrial growth elicited by unopposed oestrogenic stimulation in mouse

model

Many known endometrial cancer risk factors are associated with unopposed oestrogenic stimulation of

the endometrium (35, 36). To elucidate the effect of PUFAs on the growth of endometrium in response to

estrogen exposure, a mouse model of unopposed tamoxifen-elicited uterotrophy was established. Mice

treated with tamoxifen had a significantly increase in uterine wet weights, luminal epithelial cell heights

(p<0.05) and phosphorylation of S6 (S235/236) and Akt (S473) in the endometrium compared to the

vehicle-only group (Fig. 6A, B, C and D; Supplementary Fig. S10A). The high n-3 PUFAs diet decreased

the uterine wet weights, epithelial cell heights and the levels of P-S6 (S235/236) and P-Akt (S473) (Fig. 6A,

B, C and D; Supplementary Table S5 and Fig. S10A), while the high n-6 PUFAs (AA) diet significantly

increased the uterine grwoth in response to tamoxifen (Supplementary Fig. S10B and C). Furthermore, the

prevention of endometrial growth and inhibition of mTORC1/2 by n-3 PUFAs was reproducible in fat-1

transgenic mice (Fig. 6E, F and G; Supplementary Fig. S10D). These data indicate that both dietary and

endogenous n-3 PUFAs inhibit mTORC1/2 and prevent unopposed estrogen-elicited endometrial growth.

Discussion

Although n-3 PUFAs have been shown to prevent carcinogenesis and progression of

hormone-responsive cancers such as breast cancer in vitro and in animal experiments, and epidemiological




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studies suggest that higher dietary intake of n-3 PUFAs in food and supplements lowers the risk of these

cancers (23-26), evidence of the association between n-3 PUFAs and endometrial cancer risk remains

sparse (28). Multiple case-control studies and cohort studies reported no association between total fish or

polyunsaturated fats intake and endometrial cancer risk, but did not separately analyze n-3 and n-6 fatty

acids (37-40). A nationwide case-control study in Sweden showed that the major dietary source of

long-chain n-3 PUFAs, fatty fish consumption, lowers the risk of endometrial cancer (41). Another recent

case-control study suggested that total fish intake is not associated with risk, but higher intakes of EPA and

DHA or fish oil supplement use are significantly associated with reduced risk of endometrial cancer (28).

This study, for the first time, investigates the roles and mechanisms of n-3 PUFAs in the pathogenesis and

progression of endometrial cancer using in vitro and animal models and provides comprehensive preclinical

evidence that both dietary and endogenously produced n-3 PUFAs efficiently inhibit endometrial cancer by

preventing cell growth, migration and promotion of apoptosis.

Although increasing evidence from animal and in vitro studies indicate that n-3 PUFAs present in fatty

fish and fish oils inhibit the carcinogenesis of many tumors, inconsistencies remain (42, 43). Several factors

may account for these inconsistent results, namely: 1) wide variations in the amount and source (the type

and even the bioavailability) of n-3 PUFAs consumed in each study; 2) the ratio of n-6 to n-3 may be more

important than the absolute amount of n-3 PUFA, as suggested by animal and human studies. Since

transgenic expression of fat-1 enables the host to produce n-3 PUFAs endogenously while concomitantly

reducing the levels of n-6 PUFAs, the fat-1 transgenic mouse is capable of increasing n-3 content with a

balanced n-6/n-3 PUFAs ratio in all tissues and allows carefully controlled studies to be performed in the

absence of restricted diets (29, 33). Using fat-1 mouse and fat-1 transgenic SCID mouse models, combined

with a conventional dietary approach, our results unequivocally demonstrate that n-3 PUFAs efficiently




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inhibit endometrial carcinogenesis and tumor progression. Because the fat-1 mouse produces a mixture of

different n-3 PUFAs, the in vivo function of individual n-3 PUFA in suppression of endometrial cancer

need to be further defined. Previous study has shown that DHA is a more potent inhibitor of breast cancer

than EPA (44). The high ratio of DHA in total n-3 PUFAs of fat-1 mice (Supplementary Table S4 and S5)

and our in vitro data that DHA effectively represses endometrial cancer cell growth, proliferation and

migration, and promotes apoptosis also implicate that DHA is the more bioactive component.

Endometrial cancer is a heterogeneous disease with distinct molecular characteristics. The most frequent

aberration is activation of the PI3-K/mTOR pathway, however, the first generation mTOR inhibitors have

been tested in clinical trials as single agents with only modest results (12, 14, 45). A significant problem in

targeting mTOR with rapalogs is that these agents show partial inhibitory activity, which only block

mTORC1 and have little effect on mTORC2, thereby increasing Akt activity. The development of

mTORC1/2 kinase inhibitors that block mTORC1 and mTORC2 and consequent upregulation of Akt

activity may obviate this issue. Our findings that n-3 PUFAs target to both mTORC1 and mTORC2

pathways may explain their strong inhibitory effects on endometrial cancer in vitro and in vivo. n-3 PUFAs

such as DHA are able to target multiple intracellular signaling pathways (26) and numerous upstream

signaling regulators and mechanisms involved in the regulation of mTOR (16), leading to the complexity of

the regulation of mTOR by n-3 PUFAs. The mechanisms by which n-3 PUFAs inhibit mTORC1/2 remain

to be identified.

Because unopposed estrogen exposure significantly increases endometrial hyperplasia and cancer risk

(35) , we examined the effect of n-3 PUFAs on unopposed estrogenic stimulation of endometrial growth.

To our knowledge, no research studies have evaluated the efficacy of dietary changes and n-3 PUFAs in

reversing the estrogen-elicited uterine growth. We found that both dietary and endogenous n-3 PUFAs




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effectively repressed the estrogen-stimulated endometrial growth in a mouse model. Investigators have

found that treatment with rapalogs slowed the progression of endometrial hyperplasia and reduced tumor

burden in animal models (46). Our results showing that mTOR is a potential target of n-3 PUFAs in this

model further support the above finding. Future study of clinical interventions using n-3 PUFAs is

therefore warranted.

In conclusion, this study provides comprehensive preclinical evidence that n-3 PUFAs efficiently inhibit

endometrial cancer and implicates that n-3 PUFAs are anti-carcinogenic nutrients of potential benefit in

endometrial cancer. n-3 PUFAs may work through inhibition of mTORC1/2 signaling to decrease tumor

cell proliferation, migration and enhance tumor cell apoptosis. These evidences could have public health

implications with regard to prevention of endometrial through dietary and lifestyle interventions such as

fish oil (FO) supplementation and encourage public health authorities to design primary prevention

campaigns promoting long chain n-3 PUFA consumption in populations at risk. However, studies are

needed to investigate the potential negative effects before making any recommendations regarding the use

of n-3 PUFAs or their dosage in endometrial cancer prevention. Future research will also have to answer

the question of individual n-3 PUFA efficacy in endometrial cancer prevention and the exact mechanisms

through which n-3 PUFAs inhibit endometrial cancer.

Acknowledgements

This work was supported by The State Key Development Program for Basic Research of China

(2013CB945203), National Natural Sciences Foundation of China (81202072, 81270088) and Program for

Changjiang Scholars and Innovative Research Team in University (IRT1142).




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Reference

1. Wright JD, Barrena Medel NI, Sehouli J, Fujiwara K, Herzog TJ. Contemporary management of

endometrial cancer. Lancet. 2012;379:1352-60.

2. Barakat RR. Contemporary issues in the management of endometrial cancer. CA Cancer J Clin.

1998;48:299-314.

3. Parkin DM, Bray F, Ferlay J, Pisani P. Global cancer statistics, 2002. CA Cancer J Clin. 2005;55:74-108.

4. Amant F, Moerman P, Neven P, Timmerman D, Van Limbergen E, Vergote I. Endometrial cancer. Lancet.

2005;366:491-505.

5. Weiderpass E, Adami HO, Baron JA, Magnusson C, Bergstrom R, Lindgren A, et al. Risk of endometrial

cancer following estrogen replacement with and without progestins. J Natl Cancer Inst. 1999;91:1131-7.

6. Schapira DV, Kumar NB, Lyman GH, Cavanagh D, Roberts WS, LaPolla J. Upper-body fat distribution and

endometrial cancer risk. JAMA. 1991;266:1808-11.

7. Renehan AG, Tyson M, Egger M, Heller RF, Zwahlen M. Body-mass index and incidence of cancer: a

systematic review and meta-analysis of prospective observational studies. Lancet. 2008;371:569-78.

8. Terry P, Baron JA, Weiderpass E, Yuen J, Lichtenstein P, Nyren O. Lifestyle and endometrial cancer risk: a

cohort study from the Swedish Twin Registry. Int J Cancer. 1999;82:38-42.

9. Tangjitgamol S, Anderson BO, See HT, Lertbutsayanukul C, Sirisabya N, Manchana T, et al. Management

of endometrial cancer in Asia: consensus statement from the Asian Oncology Summit 2009. Lancet Oncol.

2009;10:1119-27.

10. Salvesen HB, Haldorsen IS, Trovik J. Markers for individualised therapy in endometrial carcinoma. Lancet

Oncol. 2012;13:e353-61.

11. Tangjitgamol S, See HT, Kavanagh J. Adjuvant chemotherapy for endometrial cancer. Int J Gynecol Cancer.




18

2011;21:885-95.

12. Dedes KJ, Wetterskog D, Ashworth A, Kaye SB, Reis-Filho JS. Emerging therapeutic targets in endometrial

cancer. Nat Rev Clin Oncol. 2011;8:261-71.

13. Banno K, Kisu I, Yanokura M, Masuda K, Ueki A, Kobayashi Y, et al. Epigenetics and genetics in

endometrial cancer: new carcinogenic mechanisms and relationship with clinical practice. Epigenomics.

2012;4:147-62.

14. Korets SB, Czok S, Blank SV, Curtin JP, Schneider RJ. Targeting the mTOR/4E-BP pathway in endometrial

cancer. Clin Cancer Res. 2011;17:7518-28.

15. Zoncu R, Efeyan A, Sabatini DM. mTOR: from growth signal integration to cancer, diabetes and ageing.

Nat Rev Mol Cell Biol. 2011;12:21-35.

16. Laplante M, Sabatini DM. mTOR signaling at a glance. J Cell Sci. 2009;122:3589-94.

17. Bai X, Jiang Y. Key factors in mTOR regulation. Cell Mol Life Sci. 2010;67:239-53.

18. Laplante M, Sabatini DM. mTOR signaling in growth control and disease. Cell. 2012;149:274-93.

19. Efeyan A, Sabatini DM. mTOR and cancer: many loops in one pathway. Curr Opin Cell Biol.

2010;22:169-76.

20. Carew JS, Kelly KR, Nawrocki ST. Mechanisms of mTOR inhibitor resistance in cancer therapy. Target

Oncol. 2011;6:17-27.

21. Janes MR, Limon JJ, So L, Chen J, Lim RJ, Chavez MA, et al. Effective and selective targeting of leukemia

cells using a TORC1/2 kinase inhibitor. Nat Med. 2010;16:205-13.

22. Li H, Lin J, Wang X, Yao G, Wang L, Zheng H, et al. Targeting of mTORC2 prevents cell migration and

promotes apoptosis in breast cancer. Breast Cancer Res Treat. 2012;134:1057-66.

23. MacLean CH, Newberry SJ, Mojica WA, Khanna P, Issa AM, Suttorp MJ, et al. Effects of omega-3 fatty




19

acids on cancer risk: a systematic review. JAMA. 2006;295:403-15.

24. Larsson SC, Kumlin M, Ingelman-Sundberg M, Wolk A. Dietary long-chain n-3 fatty acids for the

prevention of cancer: a review of potential mechanisms. Am J Clin Nutr. 2004;79:935-45.

25. Gleissman H, Johnsen JI, Kogner P. Omega-3 fatty acids in cancer, the protectors of good and the killers of

evil? Exp Cell Res. 2010;316:1365-73.

26. Berquin IM, Edwards IJ, Chen YQ. Multi-targeted therapy of cancer by omega-3 fatty acids. Cancer Lett.

2008;269:363-77.

27. Buckley JD, Howe PR. Anti-obesity effects of long-chain omega-3 polyunsaturated fatty acids. Obes Rev.

2009;10:648-59.

28. Arem H, Neuhouser ML, Irwin ML, Cartmel B, Lu L, Risch H, et al. Omega-3 and omega-6 fatty acid

intakes and endometrial cancer risk in a population-based case-control study. Eur J Nutr. 2013;52:1251-60.

29. Wei D, Li J, Shen M, Jia W, Chen N, Chen T, et al. Cellular production of n-3 PUFAs and reduction of

n-6-to-n-3 ratios in the pancreatic beta-cells and islets enhance insulin secretion and confer protection against

cytokine-induced cell death. Diabetes. 2010;59:471-8.

30. Wen ZH, Su YC, Lai PL, Zhang Y, Xu YF, Zhao A, et al. Critical role of arachidonic acid-activated mTOR

signaling in breast carcinogenesis and angiogenesis. Oncogene. 2013;32:160-70.

31. Chen Z, Zhang Y, Jia C, Wang Y, Lai P, Zhou X, et al. mTORC1/2 targeted by n-3 polyunsaturated fatty

acids in the prevention of mammary tumorigenesis and tumor progression. Oncogene. 2013.

32. Erdemoglu E, Guney M, Giray SG, Take G, Mungan T. Effects of metformin on mammalian target of

rapamycin in a mouse model of endometrial hyperplasia. Eur J Obstet Gynecol Reprod Biol. 2009;145:195-9.

33. Kang JX. A transgenic mouse model for gene-nutrient interactions. J Nutrigenet Nutrigenomics.

2008;1:172-7.




20

34. Darb-Esfahani S, Faggad A, Noske A, Weichert W, Buckendahl AC, Muller B, et al. Phospho-mTOR and

phospho-4EBP1 in endometrial adenocarcinoma: association with stage and grade in vivo and link with response

to rapamycin treatment in vitro. J Cancer Res Clin Oncol. 2009;135:933-41.

35. Arora V, Quinn MA. Endometrial cancer. Best Pract Res Clin Obstet Gynaecol. 2012;26:311-24.

36. Lacey JV, Jr., Brinton LA, Lubin JH, Sherman ME, Schatzkin A, Schairer C. Endometrial carcinoma risks

among menopausal estrogen plus progestin and unopposed estrogen users in a cohort of postmenopausal women.

Cancer Epidemiol Biomarkers Prev. 2005;14:1724-31.

37. Tzonou A, Lipworth L, Kalandidi A, Trichopoulou A, Gamatsi I, Hsieh CC, et al. Dietary factors and the

risk of endometrial cancer: a case--control study in Greece. Br J Cancer. 1996;73:1284-90.

38. Goodman MT, Hankin JH, Wilkens LR, Lyu LC, McDuffie K, Liu LQ, et al. Diet, body size, physical

activity, and the risk of endometrial cancer. Cancer Res. 1997;57:5077-85.

39. McCann SE, Freudenheim JL, Marshall JR, Brasure JR, Swanson MK, Graham S. Diet in the epidemiology

of endometrial cancer in western New York (United States). Cancer Causes Control. 2000;11:965-74.

40. Fernandez E, Chatenoud L, La Vecchia C, Negri E, Franceschi S. Fish consumption and cancer risk. Am J

Clin Nutr. 1999;70:85-90.

41. Terry P, Wolk A, Vainio H, Weiderpass E. Fatty fish consumption lowers the risk of endometrial cancer: a

nationwide case-control study in Sweden. Cancer Epidemiol Biomarkers Prev. 2002;11:143-5.

42. Signori C, El-Bayoumy K, Russo J, Thompson HJ, Richie JP, Hartman TJ, et al. Chemoprevention of breast

cancer by fish oil in preclinical models: trials and tribulations. Cancer Res. 2011;71:6091-6.

43. Olivo SE, Hilakivi-Clarke L. Opposing effects of prepubertal low- and high-fat n-3 polyunsaturated fatty

acid diets on rat mammary tumorigenesis. Carcinogenesis. 2005;26:1563-72.

44. Rahman MM, Veigas JM, Williams PJ, Fernandes G. DHA is a more potent inhibitor of breast cancer




21

metastasis to bone and related osteolysis than EPA. Breast Cancer Res Treat. 2013;141:341-52.

45. Colombo N, McMeekin DS, Schwartz PE, Sessa C, Gehrig PA, Holloway R, et al. Ridaforolimus as a

single agent in advanced endometrial cancer: results of a single-arm, phase 2 trial. Br J Cancer.

2013;108:1021-6.

46. Contreras CM, Akbay EA, Gallardo TD, Haynie JM, Sharma S, Tagao O, et al. Lkb1 inactivation is

sufficient to drive endometrial cancers that are aggressive yet highly responsive to mTOR inhibitor monotherapy.

Dis Model Mech. 2010;3:181-93.

Figure legends

Figure 1 DHA and dietary n-3 PUFAs inhibit endometrial cancer cell growth in vitro and in xenograft

models. A, HEC-1-A, B, HEC-1-B and C, RL95-2 cells plated in 24-well plates were treated with serial

concentrations of DHA (12.5-50 �M) for 96 h and total viable cells were counted at indicated times. *, P <

0.05 compared with respective controls. D, HEC-1-A, E, HEC-1-B and F, RL95-2 cells in 6-well plates

were incubated with 12.5-50 �M of DHA and subjected to the colony formation assay. G, four-week-old

female nude mice were randomized into four groups (n = 6) and injected with a suspension of HEC-1-A, or

H, RL95-2 cells. The mice were then fed with normal (Con) or high dietary ratios of n-3/n-6 PUFAs. The

tumor growth curve and a comparison of average tumor weight on the final day between the two groups are

shown. Bars indicate mean ± SD for three independent experiments. *, P values compared with controls.




22

Figure 2 Endogenously produced n-3 PUFAs inhibit endometrial cancer cell growth in vitro and in vivo. A,

HEC-1-A, B, HEC-1-B and C, RL95-2 cells plated in 24-well plates were transfected with recombinant

adenovirus with (fat-1) or without (Con) the fat-1 gene and total viable cells were counted at indicated

times. *, p < 0.05, compared with respective controls. D, HEC-1-A, E, HEC-1-B and F, RL95-2 cells in

6-well plates were transfected with recombinant adenovirus with or without the fat-1 gene and subjected to

the colony formation assay. G, fat-1/SCID or wild type (WT) SCID mice (n = 6) were injected with a

suspension of RL95-2 cells. The tumor growth curve and H a comparison of average tumor weight on the

final day between the two groups are shown. Bars indicate mean ± SD for three independent experiments. *,

P = 0.00 compared with respective controls.

Figure 3 n-3 PUFAs prevent endometrial cancer cell migration. A, HEC-1-A, and B, HEC-1-B cells were

incubated with or without 25 μM DHA and subjected to the wound healing assay. C, HEC-1-A, and D,

HEC-1-B cells were treated with or without 25 μM DHA and subjected to the transwell assay. Scale bars =

50 �m. E, HEC-1-A, and F, HEC-1-B cells were transfected with recombinant adenovirus with (fat-1) or

without (Con) the fat-1 gene and subjected to the wound healing assay. Bars indicate mean ± SD for three

independent experiments. *, p < 0.05, compared with the control.

Figure 4 n-3 PUFAs promote endometrial cancer cell apoptosis in vitro and in vivo. A, HEC-1-A, B,

HEC-1-B and C, RL95-2 cells were incubated with the indicated concentration of DHA in serum-free

medium for 8 h and cell viability was assessed. D, endometrial cancer cells were treated as in A and cell

lysates were subjected to western blot to assess cleavage of PARP. E, four-week-old female nude mice were

injected with a suspension of HEC-1-A cells. The mice were then fed with normal (Con) or high dietary n-3




23

PUFAs for 31 days. Tumors were stained with TUNEL apoptosis detection kit and the percent of apoptotic

cells was calculated under a microscope. Scale bars = 50 �m. F, Tumors were lysed and subjected to

western blot analysis to assess cleavage of PARP. Bars indicate mean ± SD for three independent

experiments. *, p < 0.05, compared with respective controls.

Figure 5 n-3 PUFAs inhibit mTORC1/2 signaling pathways in vitro and in vivo. A, HEC-1-A cells were

serum-starved for 12 h, followed by treatment with the indicated concentrations of AA for 30 min. Levels

of P-S6 (S235/236) and P-Akt (S473) were detected by immunoblotting. B, HEC-1-A cells were

serum-starved for 12 h, followed by treatment with 50 μM of AA and the indicated concentrations of DHA

for 30 min. Levels of P-S6 (S235/236) and P-Akt (S473) were examined by immunoblotting. C, HEC-1-A

cells were transfected with recombinant adenovirus with or without the fat-1 gene for 72 h, cell lysates

were extracted for western blot analysis. D, four-week-old female nude mice were injected with a

suspension of HEC-1-A cells. The mice were then administered a normal (Con) or high n-3 PUFAs diet for

31 days. Levels of P-S6 (S235/236) and P-Akt (S473) in the tumors were determine by western blot. E,

fat-1/SCID or wild type SCID mice were injected with a suspension of RL95-2 cells. 31 days after injection,

liver lysates were subjected to western blot analysis. F, HEC-1-A, and G, RL95-2 cells plated in 24-well

plates were treated with 50 �M of DHA and/or 100 μM of DDP for 96 h and total viable cells were counted

at indicated times.

Figure 6 n-3 PUFAs prevent tamoxifen-stimulated endometrial growth and inhibit mTORC1/2 in the

mouse model. Balb/c mice (n = 8) which underwent bilateral oophorectomy were administered a normal

(Con) or high n-3 PUFAs diet for 7 d, and subsequently received saline or 1 mg/kg/d tamoxifen citrate for




24

another 3 d. The uteri were removed for A, weighting and B, HE staining. C, luminal epithelial cell heights

were quantified. D, proteins were extracted from the uteri for western blot analysis. E, wild type or fat-1

mice (n = 8) underwent bilateral oophorectomy and 7 d later were administered 1 mg/kg/d tamoxifen citrate

for 3 d. The uteri were removed for HE staining. F, epithelial cell heights were quantified. G, uteri lysates

were subjected to western blot analysis to determine levels of P-S6 (S235/236) and P-Akt (S473). OVX,

ovariectomized; TAM, tamoxifen. Scale bars = 400 �m (10×) or 100 �m (40×).




Published OnlineFirst May 27, 2014.Cancer Prev Res Hang Zheng, Hongjun Tang, Miao Liu, et al. acids (PUFAs) in preclinical modelsInhibition of endometrial cancer by n-3 polyunsaturated fatty

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