Estrogen Receptor β of Host Promotes the Progression of Lung Cancer Brain Metastasis of an Orthotopic Mouse Model

doi:10.4236/jct.2012.324046

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Journal of Cancer Therapy, 2012, 3, 352-358

http://dx.doi.org/10.4236/jct.2012.324046 Published Online September 2012 (http://www.SciRP.org/journal/jct)

Estrogen Receptor β of Host Promotes the Progres sion of

Lung Cancer Brain Metastasis of an Orthotopic Mouse

Model

Lei Xu1, Guang Gao1, Jiangong Ren1, Fei Su2, Weihua Zhang1*

1Department of Biology and Biochemistry, University of Houston, Houston, USA; 2Center for Nuclear Receptors and Cell Signaling,

College of Natural Sciences and Mathematics, University of Houston, Houston, USA.

Email: *[email protected]

Received July 19th, 2012; revised August 23rd, 2012; accepted September 8th, 2012

ABSTRACT

Estrogen receptors (ERα and ERβ) in the brain play critical roles in maintaining brain tissue homeostasis and in tissue

repair after injury. Growth of cancer metastasis in the brain is a constant damaging process. The role of ERs of the host

in the progression of cancer brain metastasis is unknown. To determine the role of ERβ of host in the progression of

lung cancer brain metastasis, we used an isogenic murine lung cancer cell line, Lewis lung carcinoma cells (3LL), to

produce orthotopic lung cancer brain metastases in wild type and ERβ knockout (ERβ−/−) mice. In the wild type mice,

we found that ERα and ERβ appeared in the tumor associated reactive astrocytes at 24 hr after injection of tumor cells,

and ERβ remained thereafter while ERα disappeared after 1 week. The metastasis bearing ERβ−/− mice survived sig-

nificantly longer than the wild type mice. To further test the role of ERβ of reactive astrocytes in the survival of cancer

cells, we knocked down ERβ in cultured actrocytes using shRNA and performed 3D co-culture with 3LL cells in the

presence/absence of chemotherapeutic agents, oxaliplatin and 5-fluorouracil. We found that loss of ERβ in astrocytes

significantly reduced the survivability of 3LL cells co-cultured with astrocytes. It is concluded that ERβ of host, espe-

cially ERβ in reactive astrocytes, promotes the progression of lung cancer brain metastasis and ERβ might be a potential

therapeutic target for lung cancer brain metastasis.

Keywords: Lung Cancer; Brain Metastasis; Reactive Astrocytes; Estrogen Receptor β

1. Introduction

The development of brain metastasis in lung cancer pa-

tients is highly fatal. In the USA, 50% - 60% of lung

cancer patients will develop brain metastasis [1]. The

median survival of these patients is 2 - 3 months and ag-

gressive radiotherapy combined with chemotherapy may

prolong it to a median of 6 - 8 months [2]. Traditionally

the resistance of brain metastasis to chemotherapy has

been attributed to the blood-brain-barrier (BBB) [3].

However, it has been found that tumor cells in brain me-

tastasis produce VEGF/VPF which renders the BBB

permeable [4]. There are two other non-mutually exclu-

sive mechanisms that may be responsible for the chemo-

resistance of brain metastases. One is that the cancer

cells that can grow in the brain environment are a se-

lected subpopulation of cells that are chemoresistant; the

other possibility is that the brain microenvironment con-

fers drug resistance to tumor cells.

In the brain parenchyma, there are three major popula-

tions of cells, vascular cells, neuronal cells and glial cells.

The growth of brain metastasis is a process of combina-

tion of tissue destruction induced by invading tumor cells

and reactive alterations occurring around the metastases,

which results in a series of pathological microenviron-

mental changes. One of the most dramatic reactions in

the peritumoral region of brain metastasis is astrocytosis

[5], an increase in the number of reactive astrocytes due

to the destruction of nearby brain tissue. The physiologi-

cal function of reactive astrocytes is to repair/rescue da-

maged tissues by providing pro-survival inputs and scar

formation [6]. A tumor, “the wound that never heals” [7],

growing in the brain induces astrocytosis [8,9] similar to

other types of physical injury [10,11]. A pro-survival role

of reactive astrocytes to brain metastasis, as they have

with regard to the neurons, has been proposed [12].

Estrogen is neuroprotective and facilitates neuron re-

covery from injury [13]. It has been found that astrocytes

are the major mediator of estrogen’s neuroprotective ef-

fect [14]. The genomic functions of estrogen are medi-

ated by its receptors, estrogen receptor α (ERα) and

*Corresponding author.

Estrogen Receptor β of Host Promotes the Progression of Lung Cancer Brain Metastasis

of an Orthotopic Mouse Model

353

(ERβ). Neither ERα nor ERβ can be detected by immu-

nohistochemistry in astrocytes of intact brain, however,

both receptors are significantly upregulated in the reac-

tive astrocytes following brain injuries [15,16], and both

receptors are required to carry out estrogen’s protective

effect [17].

In this study, we used an isogenic murine lung cancer

cell line, 3LL, to produce orthotopic brain metastases in

the brains wild type (ERβ+/+) and ERβ knockout (ERβ−/−)

male mice, and investigated the role of ERβ of host in the

progression of brain metastasis of lung cancer. In addi-

tion, we also determined the effects of ERβ of astrocytes

on the survival of co-cultured 3LL cells.

2. Materials and Methods

2.1. Antibodies

Goat polyclonal antibody against glial fibrillary acidic

protein (GFAP) was from Santa Cruz. Homemade chic-

ken anti-ERβ antibody, ERβ503, and rabbit anti-ERβ

antibody, ERβ-LBD, were described previously [18,19].

Rabbit polyclonal antibody against ERα was from San-

taCruz (#MC20). Horseradish peroxidase conjugated and

alkaline phosphatase conjutated secondary antibodies

were from Sigma-Aldrich.

2.2. Cells and Cell Culture

Murine cell line, 3LL (Cat. CRL-1642), and astrocytes

(Cat. CRL-2534) were purchased from American Type

of Cell Culture (ATCC). Cells were cultured in Dul-

becco’s modified Eagle’s medium (DMEM) containing

10% fetal bovine serum (FBS) in an incubator at 37˚C

supplied with 5% CO2. For co-culturing 3LL cells and

astrocytes, we first stably transfected green fluorescent

protein (GFP) in to the 3LL cells, which allows us to di-

fferentiate cancer cells (GFP positive) from the co-cul-

tured astrocytes (GFP negative). The 3D cell co-culture

was performed with a seeding ratio of 3LL cells/astro-

cytes at 3:1 using the n3D cell culture system from

Nano3D Biosciences (Houston, TX) according to the

protocol provided by the manufacturer.

2.3. Knocking down ERβ by shRNA

The U6 promoter-driven shRNA expression vector

(pRNAT-U6.1/Neo) from Genscript (Piscataway, NJ)

was used to construct vectors expressing ERβ shRNA or

scrambled control shRNA. The target sequence for ERβ

was GTCCGCCTCTTGGAAAGCT, and the control se-

quence was GAACAATGTTGACCAGGTGA. Before

co-culturing with 3LL cells, asctrocytes were transfected

with either control shRNA or ERβ shRNA plasmids for

48 hr using trasfection reagent Genjuice according to the

manufacturer’s instruction (Roche). Western Blot was

used to determine the efficiency of ERβ shRNA.

2.4. Western Blot Assay

Cells were lysed for 30 minutes on ice in RIPA buffer

(Sigma Aldrich, St. Louis, MO) supplemented with pro-

tease and phsophatase inhibitors. The concentrations of

the protein samples were measured using the Qubit

flourometer (Invitrogen, Carlsbad, CA) and equal amounts

of protein samples were loaded onto a 10% SDS-PAGE

gel and transferred to a PVDF membrane. Membranes

were incubated in 5% milk to block the non specific

binding sites for 30 minutes and incubated with opti-

mized concentrations of primary antibody ERβ-LBD at

1:1000 dilutions at 4˚C overnight. After washing with 3×

PBS, membranes were incubated with horseradish per-

oxidase conjugated secondary antibodies (Santa Cruz

Biotechnology, Santa Cruz, CA) at 1:3000 dilutions for 1

hour at room temperature. Luminescent signals were

detected using enhanced luminescence kit (Pierce Ther-

moScientific, Rockford, IL) and exposed to X-ray films

(VWR, Bridgeport, NJ).

2.5. Cell Survival Assay

Co-cultured 3LL cells and astrocytes were treated with

5-fluorouracil (5-FU, 30 µg/ml) or oxaliplatin (50 µg/ml)

the 24 hr after the establishment of 3D co-culture. Con-

trol cells were treated with an equal volume of vehicle.

The treatments lasted for 12 hr before cells were har-

vested for trypan blue uptake assay. The dead cancer

cells were identified by dual positivity of trypan blue and

GFP signals under an inverted fluorescent microscope.

Cell viability was estimated basing on the percentage of

GFP positive dead cells from triplicate samples of each

experimental group.

2.6. Animals and Intracarotid Artery Injection

of Tumor Cells

Wild type and heterozygous of ERβ knockout mice

(ERβ+/−) were purchased from the Jackson Laboratory

(Bar Harbor, Maine). ERβ−/− mice were produced by

breeding the ER+/− pairs at the animal facility of Univer-

sity of Houston. Male mice of wild type and ERβ−/− at the

age of 3 months were used for injection of 3LL cells via

the intracarotid artery into the brain according the proto-

col described previously [20]. In brief, Mice were anes-

thetized by injection (i.p.) of a mixture of ketamine (100

mg/kg)/xylazine (5 mg/kg), washed with 70% alcohol,

and restrained to a cork board in a back position and

placed under a dissecting microscope. The head of ani-

mal was stabilized with a rubber band placed between

Estrogen Receptor β of Host Promotes the Progression of Lung Cancer Brain Metastasis

of an Orthotopic Mouse Model

354

the teeth of the upper jaw. The hair over the trachea is

shaved, the neck washed with 70% alcohol and the skin

cut by a mediolateral incision. After blunt dissection, the

trachea was exposed and the muscles were separated to

expose the carotid artery which was then separated from

the vagal nerve. The artery was prepared for injection at

a point distal to the point of division into the internal and

external carotid arteries. A ligature of 5 - 0 silk suture

was placed and tied in a position proximal to the injec-

tion site. The artery was nicked with a pair microscissors

and a plastic cannula was inserted into the blood vessel

lumen. The cells (250,000 cells in 20 µl) were injected

slowly. The cannula was removed, and the distal ligature

was tightened and the skin was closed by a tissue clamp.

2.7. Immunohistochemistry

For immunoperoxidase staining with diaminobenzidine

(DAB) labeling, tissue sections were deparaffinized in

xylene and rehydrated in a graded series of alcohol and

PBS. Antigen retrieval was performed using heated cit-

rate buffer. Endogenous peroxidase activity was blocked

with 3% hydrogen peroxide in methanol. Samples were

incubated in a blocking solution (5% donkey serum in

PBS) for 1 hour at room temperature and then overnight

at 4˚C with the primary antibodies diluted in the blocking

solution (ERα antibody was diluted at 1:200; ERβ503

antibody was diluted at 1:50; GFAP antibody was diluted

at 1:200). After three washes in PBS, samples were in-

cubated with a biotinylated goat anti-rabbit secondary

antibody (1:500) for 1 hour at room temperature and then

washed thoroughly. An ABC staining kit was used for

chromogenesis. Slides were then briefly counterstained

with hematoxylin and mounted. For double staining us-

ing horseradish peroxidase conjugated (1:500) and alka-

line phosphatase conjutated (1:500) secondary antibodies,

slides incubated with a mixture of primary antibodies of

either ERα (MC-20, rabbit origin)/GFAP (goat origin) or

ERβ503 (chicken origin)/GFAP (goat origin) were washed

with PBS and subsequently incubated with a mixture of

respective secondary antibodies. Signal of peroxidase

was first developed, then the slides were washed with

PBS before the development of alkaline phosphatase

signal. Slides were then briefly counterstained with he-

matoxylin and mounted. Staining Images were taken

using a Nikon histology imaging microscope.

2.8. Statistical Analyses

Student t-test with two tails and equal variance was used

to compare the values of two experimental groups and a

P value less than 0.05 is considered as statistically sig-

nificance.

3. Results

3.1. Characterization of Changes of ERα and

ERβ in the Brain during the Development of

Lung Cancer Brain Metastasis

The isogenic murine lung cancer cell line, 3LL (deve-

loped spontaneously in C57/BL6 mice), allows us to

study the pathogenic progression of lung cancer brain

metastasis in immnocompetent mice of the same strain.

To gain insights into the role of ERs of the brain in the

progression of lung cancer brain metastasis, by immuno-

histochemical co-staining, we profiled the expression of

ERα and ERβ in tumor associated reactive astrocytes

(identified by immnohistochemical staining of glial fi-

brillary acidic protein, GFAP) in the brain tissues of wild

type male mice collected at a series of time points after

injection of 3LL cells into the brain via intracarotid ar-

tery injection. Tissues were collected at 24 hr, 1 wk, and

3 wk after tumor injection. As shown in Figure 1, the

tumor associated reactive astrocytes are positive for ERα

and ERβ at the 24 hr time point, ERα disappeared at 1

wk while ERβ remained. These data suggest that both

ERα and ERβ are involved in the early phase of astrocyte

activation and ERβ but not ERα is involved in sustaining

the functions of reactive astrocytes in the process of tu-

mor growth.

3.2. Astrocytic ERβ Is Critical for the

Pro-Survival Function of Astrocytes in Vitro

To further determine the pro-survival role of ERβ in as-

trocytes, we performed survival analyses on 3LL cells

co-cultured with astrocytes with/without knockdown of

ERβ by shRNA. We used Western blot and immunocy-

tochemistry to determine the efficiency of ERβ shRNA.

As shown in Figure 2(a), astrocytes transfected with

Figure 1. Immunochemical double staining of ERα/GFAP

and ERβ/GFAP of tumor associated reactive astrocytes in

the brain of wild type mice. Immunoperoxidase staining

with diaminobenzidine (DAB) labeling (brown color) was

used for ERα and ERβ, and alkaline phosphatase staining

was used for GFAP (pink color). Arrows indicate nuclei of

reactive astrocytes. Arrow heads indicate tumor cells. Bar =

20 µm.

Estrogen Receptor β of Host Promotes the Progression of Lung Cancer Brain Metastasis

of an Orthotopic Mouse Model

355

Figu re 2. Lo ss of ER β in astrocytes reduced the pro-survival function of astrocytes to tumor cells. (a) Western blot analysis of

ERβ expression in astrocytes trasnfected with control-shRNA or ERβ-shRNA. Beta actin was used as a loading control; (b)

Trypan blue uptake assay of cell viability of GFP labeled 3LL cells co-cultured with astrocytes. Image of normal light (to

visualize trypan blue staining, arrows) and image of fluorescent (to visualize GFP signal) were taken for each field of obser-

vation, and total GFP positive cells (cancer cells) and trypan blue and GFP dual positive cells (double-head arrow) were

counted. The single-head arrow indicates a dead astrocyte (trypan blue positive and GFP negative); (c) Quantification of cell

death of 3LL cells cultured alone and co-cultured with astrocytes treated with control shRNA or ERβ shRNA. Triplicate

samples were used in each group. Asterisk indicates statistically significant between the linked groups (error bars = standard

deviation, P < 0.05).

3.3. Loss of ERβ in the Host Prolonged Lives of

Mice Bearing Lung Cancer Brain Metastasis

ERβ shRNA had significantly lower levels of ERβ as

compared to the control cells. To determine the effect of

astrocytes on cancer cells’ survival, we stably expressed

green fluorescent protein (GFP) in the 3LL cells, which

allows us to distinguish 3LL cells from the co-cultured

GFP negative astrocytes. We transfected astrocytes with

either ERβ-shRNA or control-shRNA 48 hr prior to co-

culture, we then co-cultured 3LL cells with these astro-

cytes using the n3D co-culture system in the presence/

absence of 5-fluorouracil or oxalaplatin for 24 hr. After

treatments, cells were trypsinized, stained with trypan

blue, plated on tissue slides and sealed with cover slides.

Green fluorescent images of GFP signal and normal light

images of trypan blue signal were taken for each field of

observation. Dead cancer cells (positive for both GFP

and trypan blue) and live cancer cells (positive for GFP

only) (Figure 2(b)) were counted in 3 fields of each

sample (there were 3 samples in each group) under an

upright fluorescent microscope. As shown in Figure 2(c),

co-culturing with astrocytes protected cancer cells from

the toxicities of both drugs, and loss of ERβ significantly

reduced the pro-survival effects of astrocytes. These data

suggest that ERβ in the cancer cell associated astrocytes

is pro-survival to the cancer cells.

To determine the role of ERβ of host in the progression

of brain metastasis, we injected tumor cells into the

brains of age matched wild type and ERβ knockout male

(C57/BL6 background). Loss of physical balance was

used as a sign of termination of experiment. When the

survival curves of these mice were compared, it was

found that the ERβ knockout mice survived significantly

longer that the wild type, the median survival time for the

wild type mice was 22 days and for the ERβ knockout

mice was 34 days (Figure 3). These data suggest that

ERβ of the host is involved in promoting the disease

progression of lung cancer brain metastasis.

4. Discussion

Cancer brain metastasis is notoriously resistant to con-

ventional therapies. Increasing attentions have been drawn

to the contribution of brain microenvironment. Studies

have found that reactive astrocytes are pro-survival to

tumor cells [21,22]. The molecular mechanisms by which

reactive astrocytes provide tumor cells with survival

support are being revealed. It has been found that physi-

Estrogen Receptor β of Host Promotes the Progression of Lung Cancer Brain Metastasis

of an Orthotopic Mouse Model

356

Figure 3. Brain metastases bearing ERβ knockout mice

survived longer than the wild type mice. The median sur-

vival time of wild type mice (n = 4) was 22 days, and the

median survival time of the ERβ knockout mice (n = 3) was

34 days. Asterisk indicates statistically significant between

the linked groups (error bars = standard deviation, P <

0.007).

cal contact between tumor cells and reactive astrocytes is

required for astrocytes to provide survival support to

tumor cells [21,22]. Targetable mechanisms are highly

desired. Our study, for the first time, found that ERβ, a

steroid hormone nuclear receptor, plays a key role in the

pro-survival functions of tumor associated reactive as-

trocytes.

The differential upregulation of ERα and ERβ in the

tumor associated astrocytes suggest that ERα and ERβ

plays different roles in the biology of tumor associated

reactive astrocytes. The quick up-regulation of ERα and

ERβ after the inoculation of tumor cells indicates that

both receptors are involved in the initiation process of

astrocyte activation. The disappearance of ERα and con-

tinuous expression of ERβ after 1 wk of inoculation of

tumor cells suggest that reactive astrocytes undergo a

significant genetic reprogram process in response to the

invasion of cancer cells to the brain. The continuous pat-

tern of ERβ expression in the tumor associated reactive

astrocytes indicates that ERβ is involved in maintaining

the function of astrocyte at its reactive status, which is

supported by the in vitro cell co-culture data that loss of

ERβ significantly reduced the pro-survival function of

astrocytes to tumor cells (Figure 2). The chemoprotec-

tive function of astrocytes has been demonstrated in

various melanoma cells, breast cancer cells and lung can-

cer cells [23]. Establishment of gap junctions between

cancer cells and astrocytes is required for astrocytes to

provide cancer cells with survival support [22]. Although

the exact molecular mechanisms by which astrocytes

offer tumor cells with chemoresistant strength remain to

be investigated, clues can be found from what is known

about how reactive astrocytes protect neurons from in-

sults [24,25]. Studies have found three major mecha-

nisms by which reactive astrocytes provide neurons in

damage with survival support, increased release of neu-

rotrophic factors, increased release of glutathione, and

enhanced energetic supply [10]. Reactive astrocytes se-

crete fibroblast growth factor 2 [26] and nerve growth

factor [27], and glutathione promotes cell survival by

reducing cellular oxidative stress. These secreted pro-

survival factors do not depend on cell-cell contact to exe-

cute their functions. The enhanced energetic metabolism

made in the reactive astrocytes deserves more attention

from a cancer brain metastasis point of view. Reactive

astrocytes have increased activities of fatty acid β-oxi-

dation, glycolysis, pentose phosphate pathway and Krebs

cycle [28,29]. These enhanced metabolisms generate

increased amounts of intermediate metabolites that could

be channeled to the neighboring cells in danger through

gap junctions [30], which shall also increase the meta-

bolic efficiency the neighboring cells to produce suffi-

cient amount of ATP for sustaining survival in a hostile

environment. Considering the death of cancer cells is

also determined by the intracellular ATP levels [31] and

the necessity of gap junctions between tumor cells and

astrocytes to increase tumor cells’ survival, the enhanced

energetic supply from reactive astrocytes to tumor cells

might be a major pro-survival mechanism offered by

reactive astrocytes.

The orthotopic brain metastasis model used in this

study, inoculating isogenic cancer cells into immuno-

competent gene knockout animals, is novel at two folds.

First, it overcomes the disadvantage of excluding the

involvement of immune systems of immunodeficient ani-

mal models, such as SCID (severe combined immunode-

ficiency) or nude mice; second, it allows the study of the

impact of a particular host gene on tumor progression.

The ERβ knockout mice used in this study is not tissue

specific, we can’t rule out the functions of ERβ in the

non-astrocytic cells are also involved in promoting the

progression of brain metastasis. Astrocyte specific ERβ

knockout is needed to conclusively define the role of

astrocytic ERβ in the progression of lung cancer brain

metastasis. However, one of the advantages of using the

whole body ERβ knockout mice to test the impact of host

ERβ on tumor progression is that this model is more

therapeutic relevant, i.e. if ERβ was proven to be a the-

rapeutic target for cancer brain metastasis, an ERβ spe-

cific ligand that can inhibit function of ERβ in reactive

astrocytes can be administrated systemically.

5. Acknowledgements

This study was supported by a startup grant from the

University of Houston to Z. W. Z. W. is also supported

Estrogen Receptor β of Host Promotes the Progression of Lung Cancer Brain Metastasis

of an Orthotopic Mouse Model

357

by grants from the American Cancer Society and the De-

partment of Defense Prostate Cancer Research Program.

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