Neurotensin Receptor 1 (NTSR1) Overexpression in Breast Carcinomas Is Common and Independent of ER/PR/Her2 Expression

doi:10.4236/jct.2013.47A003

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Journal of Cancer Therapy, 2013, 4, 12-17

http://dx.doi.org/10.4236/jct.2013.47A003 Published Online August 2013 (http://www.scirp.org/journal/jct)

Neurotensin Receptor 1 (NTSR1) Overexpression in Breast

Carcinomas Is Common and Independent of ER/PR/Her2

Expression

Xianyong Gui1*, Shuhong Liu1, Ziran Meng1, Zu-Hua Gao2

1Calgary Laboratory Services, Department of Pathology and Laboratory Medicine, University of Calgary, Calgary, Canada;

2Department of Pathology, McGill University, Montreal, Canada.

Email: *[email protected]

Received May 5th, 2013; revised June 6th, 2013; accepted June 14th, 2013

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

ABSTRACT

Neurotensin (NT) is a 13-amino acid peptide with trophic effects on some neoplasms. Its bioactivities are mainly medi-

ated by neurotensin receptor 1 (NTSR1). Both NT and NTSR1 were found to be upregulated in breast cancer.

NT/NTSR1 thus becomes a potential therapeutic target. We studied whether any correlation exists between the expres-

sion of NTSR1 in breast carcinomas and the expression of ER, PR, and Her2. A total 85 cases of invasive ductal (62)

and lobular (23) breast carcinomas were studied. Based on their ER/PR profiles, the ductal carcinomas (DCs) were

subcategorized into ER+/PR+ (21), ER+/PR− (20), and ER−/PR− (21). All of the lobular carcinomas (LCs) were

ER+/PR+. 21.57% of all DCs and 5.56% of LCs were Her2 positive. 77.78% of ER−/PR− DCs were also Her2 negative

(triple negative). The expression of NTSR1 was detected by immunohistochemistry and was semiquantitated (as nega-

tive, 1+, 2+, 3+). Both 2+ and 3+ were collectively defined as overexpression. The expression of NTSR1 was weak and

focal in non-neoplastic mammary epithelial cells. It is increased in 74.19% of DCs (80.95% in ER+/PR+, 75% in

ER+/PR−, and 66.67% in ER−/PR− group), and in 95.65% of LCs. The overexpression of NTSR1 is similar between

ER+ DCs and ER− DCs (75% vs 66.67%, p > 0.05) as well as between PR+ DCs and PR− DCs (80.95% in ER+/PR+

DCs vs 75% in ER+/PR− DCs, p > 0.05). And it was seen in 77.78% of Her2+ DCs, 78.38% of Her2− DCs, 94.12% of

Her2− LCs, and 78.57% of triple negative DCs. Overall, NTSR1 is commonly overexpressed in both ductal and lobular

breast carcinomas and is independent of the ER/PR/Her2 profiles of the tumors. The present data supports the potential

benefit of developing NTSR1 blockers in the adjuvant therapy of breast carcinomas, particularly for those “triple nega-

tive” tumors.

Keywords: Neurotensin; Neurotensin Receptor; NTSR1; Breast Carcinoma; Estrogen Receptor; Progesterone

Receptor; Her2

1. Introduction

Neurotensin (NT) is a 13-amino acid regulatory peptide

that was initially isolated from bovine hypothalamus by

Carraway and Leeman [1] and was subsequently found to

express highest in the small intestine [2]. Its physiologic

secretion is stimulated significantly by fat intake [3].

Over the years, NT was found to possess diverse func-

tions including trophic effects on some normal and neo-

plastic cells. Most of NT’s effects are mediated via high-

affinity neurotensin receptor 1 (NTSR1), a member of

G-protein-coupled receptor family. The activation of

NT/NTSR1 pathway involves several intracellular sig-

naling events, including increased intracellular calcium

release, activation of phosphatidylinositol 3’-kinase

(PI3K), mitogen activating protein kinase (MAPK) fam-

ily member ERK1/2, NF-κB pathway, and Rho family of

small GTPases, leading to cell proliferation. The growth-

promoting effect of NT/NTSR1 has been found in a vari-

ety of carcinomas including those of the colon, pancreas,

prostate, and lung [4]. It has also been found in breast

carcinomas that near all breast ductal carcinomas over

express NTSR1 and about a third coexpress both NT

(autocrine) and NTSR1 [5], NT exerted an inhibitory

activity on the apoptosis in breast cancer cells [6], and

the upregulated expression of NTSR1 is related to the

tumor progression [7]. All of these features make NT/

*Corresponding author.

Neurotensin Receptor 1 (NTSR1) Overexpression in Breast Carcinomas Is Common and Independent

of ER/PR/Her2 Expression

NTSR1 an ideal novel supplementary therapeutic target

in the hormonal therapy of breast cancer. Meanwhile,

many efforts on development of NTSR1 antagonists (e.g.,

SR48692, SR142948) have been undertaken [8,9]. Addi-

tionally, NT/NTSR1 activation was found to stimulate

the expression of epidermal growth factor receptor

(EGFR) as well as to transactivate EGFR [10-12]. This is

very interesting in view of that a member of the EGFR

family, Her2 (cErbB2) is currently the primary hormonal

therapeutic target for breast cancer. Moreover, a cross

talk between NT and estrogen and between NT and pro-

gesterone has been noted, at least in the physiologic

regulation of central nervous system [13-16]. In order to

better evaluate the possible application and benefits of

the anti-NTSR1 treatment, further understanding on

whether the NTSR1 expression correlates with the ex-

pression of ER/PR/Her2, as well as the histologic type

and grade of breast cancer will be important. Little data

regarding these questions is currently available. In the

present study we have found that NTSR1 overexpression

is common in breast carcinomas and it is independent of

the expression of ER, PR, or Her2.

2. Methods and Materials

2.1. Breast Carcinoma Patients and Samples

Eighty five cases of invasive breast carcinomas (age 24 -

83, mean 59.98 years old), including both ductal (62) and

lobular (23) types, resected by mastectomy or segmental

resection were retrieved from the 2010 surgical pathol-

ogy file at Calgary Laboratory Services. All tumors were

graded histologically using the modified Bloom-Rich-

ardson (MBR) system.

The study was approved by the Research Committee

of Calgary Laboratory Services. The ethics approval was

granted by Calgary Health Research Ethics Board.

2.2. Detection of NTSR1, ER, PR, and Her2

One of the most representative sections of tumor was

selected from each case for the study. The immunoreac-

tivity of NTSR1 was detected by immunohistochemistry

carried out on deparaffinized sections using the avidin-

biotin-peroxidase complex method. The formalin fixed

paraffin embedded tissue sections were pretreated in

CINTec Epitope Retrieval Solution (10 mM Tris/1mM

EDTA, pH 9.0) for 20 minutes at 95˚C - 100˚C, and then

cooled to room temperature. The NTSR1 antibody was a

rabbit polyclonal antibody (IMGENEX, California, USA)

against the third cytoplasmic domain of human NTSR1.

All slides were stained with Ventana Nexes IHC auto-

stainer at 1:40 dilution using UltraView Universal DAB

Detection (Ventana #760 - 500). The expression of

NTSR1 was semiquantitated based on the positivity and

intensity of the immunostain as negative (no stain at all),

1+ (faint or less than 10% of tumor cells stained), 2+

(moderate intensity or up to 50% of tumor cells stained),

and 3+ (strong intensity and more than 50% of tumor

cells stained). A comparison of NTSR1 expressions be-

tween these groups was analyzed. Both 2+ and 3+ were

collectively defined as increased expression (overexpres-

sion) in our results, considering that 1+ was observed in

non-neoplastic ductal epithelial cells.

ER and PR expression were routinely assessed by

immunohistochemistry (performed at the diagnostic im-

munohistochemistry laboratory of Calgary Laboratory

Services), and Her2 was routinely detected by silver

in-situ hybridization (SISH) (performed at the diagnostic

laboratory of the Tom Baker Cancer Centre). The meth-

odology of all of the lab tests and the result analysis were

validated. The results were reported routinely and in-

cluded in the clinical pathology reports.

2.3. Statistic Analysis

The rate (%) of NTSR1 overexpression in tumors of dif-

ferent groups was calculated and compared between

groups by Fisher Exact test.

3. Results

The details of the patients’ information are summarized

in Table 1.

Based on their ER/PR profiles, the ductal carcinomas

(DCs) were subcategorized into ER+/PR+ (21 cases),

ER+/PR− (20 cases), and ER−/PR− (21 cases). The 23

lobular carcinomas (LCs) were all ER+/PR+. The data of

Her2 was available in 69 cases. 11 of 51 (21.57%) DCs

and 1 of 18 LCs (5.56%) were Her2 positive. 14 of 18

(77.78%) ER−/PR− DCs were also Her2 negative (triple

negative).

The immunoreactivity of NTSR1 appeared in both

membranous and cytoplasmic patterns, as shown in Fig-

ure 1. It was weak and partial in the non-neoplastic

mammary epithelial cells, but it was stronger in both in-

situ and invasive carcinomas.

As shown in Figure 2, the overexpression of NTSR1

was detected in 46/62 (74.19%) DCs (80.95% in ER+/

PR+, 75% in ER+/PR−, and 66.67% in ER−/PR − group),

and 22/23 (95.65%) LCs. The overexpression of NTSR1

appeared to be slightly elevated in tumors with the ex-

pression of both ER and PR, although no statistically

significant difference existed (p > 0.05) between any of

these groups based on our sample size.

As shown in Figure 3, the overexpression of NTSR1

was slightly more common in ER+ DCs than in ER− DCs

(75% versus 66.67%, p > 0.05), and was similar between

PR+ DCs and PR− DCs (80.95% in ER+/PR+ DCs versus

Neurotensin Receptor 1 (NTSR1) Overexpression in Breast Carcinomas Is Common and Independent

of ER/PR/Her2 Expression

Table 1. Summary of breast cancer patients and NTSR1 immunohistochemistry.

MBR Grade NTSR1

Type ER/PR Number

Age

(mean) 1 2 3

Her2 (+/Total)Neg 1+ 2+ 3+

ER+/PR+ 21 63.67 6 11 4 4/16 0 4 13 4

ER+/PR− 20 59.12 5 8 7 7/17 0 5 4 11

Ductal

ER−/PR− 21 60.91 0 3 18 4/18 1 6 7 7

Lobular ER+/PR+ 23 60.18 2 19 2 1/18 0 1 19 3

Figure 2. NTSR1 expression (% of high expression) in dif-

ferent groups of breast carcinomas. No significant differ-

ence existed between any of the groups.

In summary, the overexpression of NTSR1 is less

common in high grade carcinomas than in low grade

ones. There seems to be a trend of higher expression of

NTSR1 in ER+/PR+ tumors than in ER−/PR− tumors.

However, there is no statistically significant difference

between ER+ and ER−, PR+ and PR−, and between

Her2+ and Her2− tumors.

Figure 1. The intensity and pattern of NTSR1 expression on

immunohistochemistry. (a) Weak (1+) expression in normal

ductal epithelial cells (×200); (b) Moderate expression (2+)

in ductal carcinoma in situ (DCIS) (×200); (c) Relatively

membranous pattern of immunostain (×400); (d) Rela-

tively cytoplasmic pattern of immunostain (×400); (e)

Strong expression (3+) in invasive ductal carcinoma (×200);

(f) Strong expression (3+) in invasive lobular carcinoma

(×200).

4. Discussion

The detection of certain biologic markers in breast cancer

has had significant clinical impact and become routine in

the clinical pathologic assessment of breast carcinomas.

These markers are related either to the aggressiveness,

invasiveness, and the extent of tumor spread, or to the

response of certain therapeutic agents. Thus they have

both prognostic and predictive values. So far, the most

useful biomarkers include estrogen receptor (ER), pro-

gesterone receptor (PR), Her2, and Ki67. Currently, the

application of ER, PR, and Her2 in the selection and pre-

diction of hormonal therapy in breast cancers is critical.

The antagonists of ER (e.g., Tamoxifen) and Her2 (e.g.,

Trastuzumab) play important roles in the management

of breast cancer patients. Several new markers have

been explored in recent years, including p53, ARF,

TBX2/3, Cyclin D1, Cyclin E, BRCA1, BRCA2, and

VEGF [17]. The utility of these emerging novel markers

are being investigated.

75% in ER+/PR− DCs, p > 0.05).

As shown in Figure 4, the overexpression of NTSR1

was seen in 7/9 (77.78%) Her2+ DCs, 29/37 (78.38%)

Her2− DCs, and in 16/17 (94.12%) Her2− LCs. In addi-

tion, the NTSR1 overexpression was seen in 11/14 (78.57%)

triple negative DCs (not shown in the figure). No statis-

tically significant difference existed (p > 0.05) between

any of these groups.

Between the tumors with different histologic grades,

as shown in Figure 5, the expression of NTSR1 seemed

to be negatively correlated with the histologic grade of

the tumors. The difference between Grade 1 and Grade 3

was statistically significant (p = 0.032), although the dif-

ference between Grade 1 and Grade 2, or between grade

2 and grade 3, was not statistically significant. NT/NTSR1 is still a relatively little-known marker

Neurotensin Receptor 1 (NTSR1) Overexpression in Breast Carcinomas Is Common and Independent

of ER/PR/Her2 Expression

(a)

(b)

Figure 3. NTSR1 expression (% of high expression) in

breast carcinomas with regards to ER and PR expression.

(a) Relationship of NTSR1 and ER expression; (b) Rela-

tionship of NTSR1 and PR expression. No significant dif-

ference existed between ER+ and ER− tumors or between

PR+ and PR− tumors.

with regards to breast cancer. Although the tumor growth-

promoting effects of NT/NTSR1 on breast cancer cells

and the overexpression of NTSR1 in breast cancer tissue

have been reported for years, the prognostic and predic-

tive values of detection of NTSR1 in breast cancer have

not been studied. In a study reported by Dupouy et al.,

the overexpression of NTSR1 seemed to be associated

with the histological grade, size of tumor, and number of

positive axillary lymph nodes, and it was also found to be

an independent prognostic factor associated with the poor

outcome [6]. In our data, however, the NTSR1 overex-

pression appeared to be negatively correlated with the

histologic grade of the tumors. Further study is required

to clarify this discrepancy.

In the present study, NTSR1 was found to be universally

expressed in both ductal and lobular breast carcinomas, and

was independent of the ER/PR/Her2-neu profiles of the

tumors. The study was not designed to further corre-

(a)

(b)

Figure 4. NTSR1 expression (% of high expression) in

breast carcinomas with regards to Her2 expression. No

significant difference existed between Her2+ and Her2-

tumors. (a) Her2+ vs Her2− ductal carcinomas; (b) Her2+

vs Her2− lobular carcinoma.

late the NTSR1 expression with the patient outcome and

many other cliniopathologic features. The data of the

potentially prognostic value of NTSR1, unfortunately, is

not available.

Our finding that NTSR1 is expressed in all kinds of

breast carcinomas further supports the potential benefits

of developing NTSR1 blockers in the adjuvant therapy of

breast carcinomas, particularly for the “triple negative”

tumors. It is reasonable to postulate that the anti-

NT/NTSR1 approach would be a supplement and/or al-

ternative to the established hormonal treatment of breast

carcinomas. In addition, in view of the transactivation of

NT/NTSR1 pathway on EGFR and the interaction be

tween NT and estrogen/progesterone, the intervention of

NT/NTSR pathway may also modulate the therapeutic

effects of ER−/Her2 antagonists.

Several experimental studies using breast cancer cell

Neurotensin Receptor 1 (NTSR1) Overexpression in Breast Carcinomas Is Common and Independent

of ER/PR/Her2 Expression

Figure 5. NTSR1 expression (% of high expression) in

breast carcinomas with regards to the histologic grade

(MBR grading system). A significant difference existed be-

tween Grade 1 and Grade 3 tumors.

lines have demonstrated the significant growth-inhibitory

effects of various means of NTSR1 blockage. Earlier

studies using SR48692, the first NTSR1 antagonist has

shown a significant reduction of cancer cell proliferation

in ex vivo cell culture or xenografted model [18,19]. In

the study of Souaze et al., small interfering RNA knock-

down of NTSR1 in xenografted MDA-MB-231 cells re-

sulted in a 70% decrease in tumor growth as compared to

wild-type cells [5]. In the study of Heakal et al, nanoli-

posomal short-chain ceramide, a chemical that can in-

hibit agonist-dependent translocation of NTSR1, was

found to significantly inhibit NTSR1-mediated MDA-

MB-231 breast cancer progression (mitogenesis, migra-

tion, and matrix metalloproteinase activity) [8]. Casti-

llo-Rodriguez et al. recently developed a neurotensin-

polyplex that transfects therapeutic genes into NTSR1-

expressing breast cancer cells (MDA-MB-231 cells) and

kills tumor cells in both in vitro and in vivo models [20].

Unfortunately, to the best knowledge of the authors, a

clinically usable NTSR1 antagonist is still not available.

The exact efficacy of the proposed anti-NTSR1 treatment

awaits validation through future study, provided a clini-

cally applicable drug becomes a reality in the near future

with the recent better understanding of the chemical

structure of NTSR1 [21].

5. Acknowledgements

This study was supported by the Calgary Laboratory

Services (CLS, RS10-534). Ms. Michelle Darago pro-

vided very helpful technical support.

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