Angiotensin-(1 - 7) and Human Chorionic Gonadotropin (hCG) Modulate the Nuclear Transcription Factors or Nuclear Receptors Genes in the Tumorigenic Undifferentiated Breast Cancer Cell Line SKBR3

doi:10.4236/jct.2013.47A011

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Journal of Cancer Therapy, 2013, 4, 70-74

http://dx.doi.org/10.4236/jct.2013.47A011 Published Online August 2013 (http://www.scirp.org/journal/jct)

Angiotensin-(1 - 7) and Human Chorionic Gonadotropin

(hCG) Modulate the Nuclear Transcription Factors or

Nuclear Receptors Genes in the Tumorigenic

Undifferentiated Breast Cancer Cell Line SKBR3*

Isidoro Binda Neto1,2, Samuel Marcos Ribeiro de Noronha3,

Silvana Aparecida Alves Correa de Noronha3#, Maria Del Carmen Garcia Molina Wolgien1,

Alexandre Jesus Barros4, Clovis Ryiuchi Nakaie4, Suma Imura Shimuta4, Gil Facina1,

Ismael Dale Cotrim Guerreiro da Silva1

1Laboratório de Ginecologia Molecular, Universidade Federal de São Paulo, São Paulo, Brazil; 2Universidade Federal do Estado do

Rio de Janeiro, Rio de Janeiro, Brazil; 3Laboratorio de Cirurgia Translacional, Departamento de Cirurgia, UNIFESP/EPM, São Paulo,

Brazil; 4Departamento de Biofísica, Universidade Federal de São Paulo, São Paulo, Brazil.

Email: [email protected]om.br, [email protected], [email protected], #[email protected], [email protected],

[email protected], mastologista@hotmail.com, [email protected]

Received May 19th, 2013; revised June 20th, 2013; accepted June 29th, 2013

cense, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

ABSTRACT

Breast cancer is the most common cancer among women. Angiotensin-(1 - 7) [Ang-(1 - 7)] has been correlated with

cancer antiproliferative and apoptotic effects, similar properties of the human Chorionic Gonadotrofin (hCG). The aims

of this work are to evaluate the role of Ang-(1 - 7) and of hCG in modulating the expression of Nuclear Receptors and

Coregulators related genes in the tumorigenic breast cell line SK-BR3. Three experimental groups were created: control,

hCG and hCG + Ang-(1 - 7). Cells were treated for 11 days and then had their RNA extracted. Samples were loaded

into PCR Array plates containing 84 genes relate to Nuclear Receptors and Coregulators pathways. Gene expression

data were used to construct canonical pathways (MetacoreTM). hCG and hCG + Ang-(1 - 7) treatments markedly modu-

late the expression of Nuclear Receptors and Coregulators related genes. hCG differentially expressed 17% of the genes,

being 29% upregulated and 71% downregulated. Meanwhile, hCG + Ang-(1 - 7) changed the expression of 30% of the

genes on the plate, among these genes 56% were upregulated and 44% downregulated. Among these differentially ex-

pressed genes, we highlight Esr1, Nr2f2, and Nr2f1, Esr1, Hdac5, and Nr4A1 (>4 fold). Finally MetaCore analysis

based on Gene Ontology (GO) generated six networks for hCG and ten networks for the combined treatment. All gen-

erated networks are related to regulation of apoptosis or to Programmed Cell Death processes. In summary, our results

herein demonstrate that the modulation of sexual hormones and of other nuclear factor genes expression might underlie

the tumorigenic protection effect and the induction of cell differentiation caused by the hormones hCG and Ang-(1 - 7),

especially in Cancer Stem Cells.

Keywords: Breast Stem Cancer Cells; SK-BR3; hCG; Angiotensin-(1 - 7)

1. Introduction

Breast cancer is the most common cancer among women,

accounting for thousands of deaths annually. In 2012, the

estimated number of new breast cancer cases is above

two-hundred and twenty-nine thousand [1]. Among the

various mediators that act in the carcinogenic process, the

components of the renin-angiotensin system (RAS) have

assumed an important role [2-5]. Angiotensin II (Ang II),

better known peptide obtained from the cascade of events

of RAS, has vasoconstrictive, angiogenic, hyperplastic,

proliferative and metastatic properties [6,7].

Moreover, it has also been demonstrated an associa-

tion between genetic polymorphisms of some RAS

components with breast cancer [8-10]. Many are the evi-

*The work was supported by Grant number 2011/10516-0 and 2008/

54383-0 from the Sao Paulo Research Foundation (FAPESP)-Brazil.

#Corresponding author.

Angiotensin-(1 - 7) and Human Chorionic Gonadotrifin (hCG) Modulate the Nuclear Transcription Factors or Nuclear

Receptors Genes in the Tumorigenic Undifferentiated Breast Cancer Cell Line SKBR3

dences that the RAS is related to neoplasia of the breast

tissue and also that its disruption may be involved in one

or more steps that lead to carcinogenesis [11].

On the other hand, angiotensin-(1 - 7) [Ang-(1 - 7)]

another peptide component of the RAS, has been exten-

sively studied lately, for its vasodilator, antiproliferative

and apoptotic effects, opposite effects generated by Ang II

[12,13].

New breast cancer approaches have identified a small

population of highly tumorigenic cells with stem cell

properties in the human breast and in other solid tumors.

These cells have been considered the source of tumor

initiation and of its maintenance. These highly proli-

ferative cells are referred to as cancer stem cells (CSCs)

[14].

Altogether, the aims of this work are to evaluate the role

of Ang-(1 - 7) and of hCG in modulating the expression of

Nuclear Receptors and Coregulators related genes in the

tumorigenic breast cell line SK-BR3 in order to better

understand the molecular mechanisms underlying the

effects triggered by these compounds in CSCs.

2. Methods

2.1. Cell Culture and Treatments

The SKBR3 cell line was grown in DMEM supplemented

with 10% FBS, 2 mM glutamine, 100 U/ml penicillin and

100 μg/ml streptomycin. Three experimental groups were

created: control, hCG and hCG + Ang-(1 - 7). Cells were

treated for 11 days.

2.2. RNA Extraction

Pelleted cells were homogenized in Trizol reagent (Invi-

trogen) according to the manufacturer’s protocol. Total

RNA was purified with Qiagen RNeasy Mini Kit and

subjected to treatment with DNase A. The quantity and

quality of extracted RNA were measured by espectro-

photometer (Nanodrop Technologies Inc., Rockland, DE).

2.3. Real Time PCR Array

According to the manufacturer’s (Qiagen) methodology,

reverse transcriptase (RT) was carried out for the synthe-

sis of cDNA. For each sample we used as a template a

PCR array plate containing 84 different pairs of primers

for studying genes related to Nuclear Receptors and Co-

regulators pathways (RT2 ProfilerTM PCR Array; SABio-

sciences).

2.4. Anal ysis o f Relev ant Biol ogical Proces ses and

Networks by MetaCore

The MetaCore software (GeneGo, St. Joseph, MI) is a

computational resource that uses logic operations for

identifying altered biological processes based upon gene

expression changes. Genes with altered expression were

mapped to Gene Ontology (GO) using MetaCore algo-

rithm. GO annotations were used as indicators of bio-

logical functions. GO describes gene products in terms of

their associated biological processes, cellular compo-

nents, and molecular functions. The GO entries are hier-

archically linked, thus allowing construction of cluster

genes of crossed pathways.

2.5. Statistical Analysis

These results were analyzed by descriptive statistics

(means and standard deviation) and inferential statistics

through the student’s t-test, with significance level of 5%

(p < 0.05). Real-time PCR array reactions were process-

ed through the online software RT2 ProfilerTM PCR Ar-

ray Data Analysis (SABiosciences).

3. Results

hCG and hCG + Ang-(1 - 7) treatments markedly modu-

lates the expression of Nuclear Receptors and Coregula-

tors related genes (Figures 1 and 2). hCG differentially

expressed 17% of the genes, being 29% upregulated and

71% downregulated. Meanwhile, hCG + Ang-(1 - 7)

changed the expression of 30% of the genes on the plate,

among these 56% were upregulated and 44% downregu-

lated. In general, the combined treatment generates a more

downregulated expression profile of these genes than hCG

itself (Figures 1 and 2).

Among these differentially expressed genes, we high-

light Esr1, Nr2f2, and Nr2f1, Esr1, Hdac5, and Nr4A1 (>4

fold) (Figure 3). Finally MetaCore analysis based on

Gene Ontology (GO) generated six networks for hCG

treatment and ten networks for the combined treatment.

All generated networks are related to regulation of apoptosis

or to Programmed Cell Death processes (Figure 4).

4. Discussion

The search for possible new molecular targets to treat or to

early detect breast cancer is of paramount importance.

Nowadays many researchers are focusing on the actions

elicited by the hormone hCG, which has been shown to

decrease proliferation of mammary tumor cells [15,16].

The results here presented clearly demonstrate that

hCG alters the expression profile of many genes encoding

for proteins that act as nuclear transcription factors or as

nuclear receptors.

Surprisingly both treatments restored the expression of

the estrogen receptor 1 (ESR1) gene in this originally

estrogen receptor negative cell line. The ESR1 is a

ligand-activated transcription factor composed of several

domains important for hormone binding, DNA binding, and

Angiotensin-(1 - 7) and Human Chorionic Gonadotrifin (hCG) Modulate the Nuclear Transcription Factors or Nuclear

Receptors Genes in the Tumorigenic Undifferentiated Breast Cancer Cell Line SKBR3

Figure 1. Heat map of SKBR3 cells gene expression after treatment.

Figure 2. Gene expression analysis presented by scatter plot graphs.

Figure 3. Most differentially expressed genes caused by both

treatments in SKBR3 cells. Control cells were used as the

calibrator sample.

activation of transcription. Estrogen receptors are also di-

rectly involved in different pathological processes including

breast cancer, endometrial cancer, and osteoporosis [17].

Prolonged exposure to estrogens is a significant risk

factor for the development of breast cancer. Estrogens

exert carcinogenic effects by stimulating cell proliferation

or through oxidative metabolism that forms DNA-da-

maging species. In SKBR3 cells, all of these estrogen-

forming enzymes were expressed, although the lack of

ESR1 and the low levels of ESR2 expression suggest that

hCG and Ang-(1 - 7) modulate the expression of sexual

hormone genes [18].

Nuclear Receptor Subfamily 2 (NR2F2) encodes a

member of the steroid thyroid hormone superfamily of

nuclear receptors. The encoded protein is a ligand induc-

ible transcription factor involved in regulation of many

different genes (pubmed). Members of this family inhibit

cell differentiation and increase cell growth. Inhibition of

COUP-TFII (Nr2f2) may offer a novel therapeutic ap-

proach to breast cancer [19]. In the present study hCG

downregulated COUP-TFII, which might partially explain

the breast cancer protection brought about by hCG. Be-

sides that, hCG restores ESR1 gene expression, which

Angiotensin-(1 - 7) and Human Chorionic Gonadotrifin (hCG) Modulate the Nuclear Transcription Factors or Nuclear

Receptors Genes in the Tumorigenic Undifferentiated Breast Cancer Cell Line SKBR3

Figure 4. Top scored (by number of pathways) network

generated by the active experiments. Thick cyan lines indi-

cate the fragments of canonical pathways. Up-regulated

genes are marked with red circles; do wn-regulated with blue

circles. The checkerboard’ color indicates mixed expression

for the gene between files or between multiple tags for the

same gene.

might be beneficial when considering the antineoplastic

drugs available to treat breast cancer. At the same time,

NR2F2 downregulation indicates that hCG seems to in

duce cell differentiation in SKBR3 cells [19].

hCG also caused downregulation of the Nuclear Re-

ceptor Subfamily 2 (NR2F1), which may partially explain

the anti-proliferative effects of this hormone [20].

Another important action of hCG was to increase the

expression of the NR4A1 gene, which has an antimi-gra-

tion effect on normal cells.

Histone Deacetylase 5 (HDAC5) is an enzyme respon-

sible for maintenance/assembly of the heterochromatin

structure. As previously demonstrated its specific inhibi-

tion might contribute to increase the efficacy of DNA

alteration-based cancer therapies in clinic [21]. hCG in-

hibits expression of HDAC5, reducing cancer progres-

sion and cell survival.

In summary, our results herein demonstrate that the

modulation of sexual hormones and of other nuclear fac-

tor genes expression might underlie the tumorigenic pro-

tection effect and the induction of cell differentiation

caused by the hormones hCG and Ang-(1 - 7) [22], espe-

cially in CSCs [15,23].

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