PRECLINICAL STUDY

Expression of membrane transporters and metabolicenzymes involved in estrone-3-sulphate disposition in humanbreast tumour tissues

Nilasha Banerjee • Naomi Miller • Christine Allen •

Reina Bendayan

Received: 5 March 2014 / Accepted: 30 April 2014 / Published online: 16 May 2014

� Springer Science+Business Media New York 2014

Abstract Two-thirds of newly diagnosed hormone-

dependent (HR?) breast cancers are detected in post-

menopausal patients where estrone-3-sulphate (E3S) is the

predominant source for tumour estradiol. Understanding

intra-tumoral fate of E3S would facilitate in the identifi-

cation of novel molecular targets for HR ? post-meno-

pausal breast cancer patients. Hence this study investigates

the clinical expression of (i) organic anion-transporting

polypeptides (OATPs), (ii) multidrug resistance protein

(MRP-1), breast cancer resistance proteins (BCRP), and

(iii) sulphatase (STS), 17b-hydroxysteroid dehydrogenase

(17b-HSD-1), involved in E3S uptake, efflux and metab-

olism, respectively. Fluorescent and brightfield images of

stained tumour sections (n = 40) were acquired at 49 and

209 magnification, respectively. Marker densities were

measured as the total area of positive signal divided by the

surface area of the tumour section analysed and was

reported as % area (ImageJ software). Tumour, stroma and

non-tumour tissue areas were also quantified (Inform

software), and the ratio of optical intensity per histologic

area was reported as % area/tumour, % area/stroma and

% area/non-tumour. Functional role of OATPs and STS

was further investigated in HR? (MCF-7, T47-D, ZR-75)

and HR-(MDA-MB-231) cells by transport studies con-

ducted in the presence or absence of specific inhibitors.

Amongst all the transporters and enzymes, OATPs and

STS have significantly (p \ 0.0001) higher expression in

HR? tumour sections with highest target signals obtained

from the tumour regions of the tissues. Specific OATP-

mediated E3S uptake and STS-mediated metabolism were

also observed in all HR? breast cancer cells. These

observations suggest the potential of OATPs as novel

molecular targets for HR? breast cancers.

Keywords Hormone-dependent breast cancers � Estrone-

3-sulphate � Organic anion-transporting polypeptides �Clinical tumour tissues � Molecular target

Introduction

Breast cancer is not only the most commonly diagnosed

cancer, but also the second leading cause of cancer asso-

ciated death in women [1]. While the incidence of hormone

receptor-positive (HR?) breast cancers has been declining

since 2003 [2], two-thirds of newly diagnosed breast can-

cers are HR? and demonstrate hormone (estradiol)-

dependent proliferation [3, 4]. Furthermore, 75 % of these

HR? breast cancers are detected in post-menopausal

women with very low ovarian production of estradiol (E2)

[5]. While plasma levels of E2 are 90 % reduced post-

menopause, breast tissue levels are comparable in pre- and

post-menopausal women [6, 7] due to in situ E2 production

through the aromatase and sulphatase pathways [8].

Estrone-3-sulphate (E3S) is the predominant source for

tumour tissue E2. The plasma-circulating levels of E3S are

5 to 10 times higher than androgens and other oestrogen

metabolites [9]. Sulphatase (STS) activity is 130–200 times

Electronic supplementary material The online version of thisarticle (doi:10.1007/s10549-014-2990-y) contains supplementarymaterial, which is available to authorized users.

N. Banerjee � C. Allen � R. Bendayan (&)

Department of Pharmaceutical Sciences, Leslie Dan Faculty

of Pharmacy, University of Toronto, 144 College Street,

Toronto, ON M5S 3M2, Canada

e-mail: r.bendayan@utoronto.ca

N. Miller

Department of Pathology, Toronto General Hospital, University

Health Network, Toronto, ON, Canada

123

Breast Cancer Res Treat (2014) 145:647–661

DOI 10.1007/s10549-014-2990-y

greater than aromatase activity [10], and tumour tissue:

plasma concentration ratio of E3S is 20:1 [6, 7, 11].

Therefore, characterizing the tumour cell uptake, efflux and

intra-tumoral cell metabolism of E3S is of interest as a

means of better understanding the pharmacological sig-

nificance of E3S in the proliferation of HR? tumours

detected in post-menopausal patients.

It has been established that cellular uptake of E3S is

mediated by the organic anion-transporting polypeptides

(OATPs), a family of membrane-associated uptake trans-

porters [12], with a demonstrated ten times higher E3S

transport efficiency in HR? (MCF-7), in comparison to

HR- (MDA-MB-231, MDA-MB-468, MDA435/LCC6),

breast cancer cells [13]. Amongst the seven human OATPs

that recognize E3S as a substrate [14], OATP1A2,

OATP2B1, OATP3A1 and OATP4A1 expression were

observed to be significantly higher in HR? breast cancer

cells [13]. Miki et al. previously reported tenfold higher

expression of OATP1A2 in breast cancer tissues [15, 16],

and Pizzagalli et al. [17] reported OATP2B1 localization in

luminal epithelium in invasive ductal carcinoma tissues.

Kindla et al. [18] also investigated the mRNA expression

of OATP2B1, OATP3A1 and OATP5A1 and reported high

inter-individual variability with no significant difference in

paired samples of normal and malignant breast tissue.

However, aside from these studies, there are very limited

clinical data comparing expression of OATP isoforms

between HR? and HR- human breast tumour tissues.

Hence, this current study focuses on comparing the

expression of OATP1A2, OATP2B1, OATP3A1 and

OATP4A1 in HR? and HR- breast tumour, in both axil-

lary lymph node positive (LN?) and negative (LN-)

patients.

Intra-cellularly, E3S is reported to be desulphated to

estrone by STS and converted to E2 by 17b-hydroxysteroid

dehydrogenase (17b-HSD-1) [9, 19]. Hence, expression of

STS and 17b-HSD-1 were compared between HR? and

HR- breast tumours. Additionally, expression of breast

cancer resistance protein (BCRP) and multidrug resistance

proteins-1 (MRP-1), efflux transporters belonging to the

ABC (ATP Binding Cassette) family, that have been

reported to recognize E3S as a substrate [20, 21], were

compared between HR? and HR- breast tumours to fur-

ther clarify mechanisms involved in tumour cell efflux of

E3S. To the best of our knowledge, this is the first study

comparing the clinical expression of (i) uptake transporters,

(ii) efflux transporters, and (iii) metabolizing enzymes,

involved in E3S uptake and metabolism, in both HR? and

HR- tumour tissues. Furthermore, tumour cell-specific

expression of OATPs, STS, 17b-HSD-1, BCRP and MRP-1

were assessed within each tumour section.

It would be valuable to collect functional data in the

clinical sample to compare the function of these

transporters and enzymes in HR? and HR- tumour tis-

sues. However, it was extremely challenging to obtain

these human breast cancer tissues. Hence, to better

understand the observed differences in expression of these

transporters and enzymes in the HR? and HR- clinical

tissues, the functional roles of OATP (in E3S uptake) and

STS (in E3S metabolism) were examined in HR? (MCF-7,

T47D, ZR-75) and HR- (MDA-MB-231) breast cancer cell

lines. While STS and 17b-HSD-1 are currently considered

therapeutic targets for ER? breast cancers, this study helps

to elucidate the potential of OATPs as novel molecular

targets for breast cancer and provides a better under-

standing of the intra-tumoral fate of E3S.

Methods

Immunohistochemistry and image analysis

Following Institutional Research Ethics Board Approval

(University Health Network REB Approval #11-0820-T),

40 tumours were selected from the pathology records on

the basis of HR and LN status as follows: 10 HR? /LN-,

10 HR? /LN?, 10 HR-/LN-, 10 HR-/LN?. A single

formalin-fixed paraffin-embedded tumour block was

selected from each case which contained both invasive

carcinoma and benign breast cancer tissue.

Immunohistochemical staining of tumour sections was

performed by the pathology research program (PRP)

[University Health Network, Toronto, ON] on 4-lm-thick

tumour sections. Sections were incubated with primary

antibodies: rabbit polyclonal for anti-OATP1A2(1:1,200),

anti-OATP2B1(1:200), anti-STS(1:100) and anti-17bHSD1 (1:100) (Sigma Aldrich, CA), goat polyclonal for

anti-OATP3A1 (1:100) and anti-OATP4A1 (1:100) (Santa

Cruz, CA), rat monoclonal for anti-MRP-1 (1:300) (Ab-

cam, CA) and mouse monoclonal for anti-BCRP (1:100)

(Abcam, CA). These sections were then incubated with

Alexa Fluor 488 labelled corresponding secondary

(Molecular Probes, Burlington, ON) or diaminobenzidine

(DAB) (chromophore) for fluorescent and bright field

images, respectively. Positive and negative controls were

performed for all markers (data not shown).

Fluorescent images were acquired at 49 magnification

(Olympus BX50 microscope). Images of complete tumour

sections were produced by stitching tiled images using

MetaMorph software. Marker densities were measured as

the total area of positive signal divided by the surface area

of the tumour section analysed using the ‘area fraction’ tool

of ImageJ software. Immunohistochemical expression was

reported as % area [22].

Bright field images were acquired using the ScanScope

XT (Aperio technologies, CA, USA) at 209 magnification

648 Breast Cancer Res Treat (2014) 145:647–661

123

and were analysed as previously described [23–25], using

inform pattern recognition-based image analysis software.

Algorithms were developed to quantify histologic tumour,

stroma and non-tumour tissue areas in tumour sections

(The non-tumour tissue comprised glandular and adipose

tissue). Target signals were then quantified within each of

the selected tissue compartments. The ratio of positive

signal per histologic area was reported as % area/tumour,

% area/stroma and % area/non-tumour using inform and

ImageJ software. Representative 16 blocks (4 HR?/LN-,

4HR?LN?, 4HR-/LN-, 4HR-,LN?) were selected

from the 40 tumour tissue blocks, and sections from each of

these blocks were quantified for tumour, stroma and non-

tumour tissue areas for each of the markers, and the %

tumour, % stroma and % non-tumour obtained from the

16 sections were extrapolated for all 40 sections. Optical

intensity for each marker was then determined in the

tumour, stroma and non-tumour regions and reported as %

area/tumour, % area/stroma and % area/non-tumour,

respectively. The outcomes of tissue segmentation and

mapping for each image were assessed by pathologist, Dr.

Naomi Miller.

Cell culture

MCF-7, T47-D, ZR-75 and MDA-MB-231 cells were

purchased from ATCC. Human embryonic kidney (HEK)

293 cells stably expressing OATP3A1 (HEK/OATP3A1)

and OATP4A1 (HEK/OATP4A1) were kindly donated by

Dr. Martin F. Fromm (Friedrich-Alexander-Universitat

Erlangen-Nurnberg, Erlangen, Germany). Human placental

tissue lysate was kindly donated by Dr. Micheline Piquette-

Miller (University of Toronto, Canada). Tissue culture

reagents were obtained from Invitrogen (Carlsbad, CA)

unless indicated otherwise.

The MCF7, MDA-MB-231, HEK/OATP3A1 and HEK/

OATP4A1 cells were grown in Dulbecco’s modified

Eagle’s medium while the ZR-75 and T47-D cells were

cultured in RPMI1640 medium. Bovine insulin (0.2 %)

(Sigma Aldrich, CA) was added to T47-D cell medium.

HEK/OATP3A1 and HEK/OATP4A1 media were supple-

mented with 800 lg/ml G-418 [26]. For transport experi-

ments, cells were seeded into 24-well plates with a cell

density of 25 9 103 cells/cm2.

Transient transfection of OATP1A2 cDNA

OATP1A2 cDNA was transiently transfected as previously

described [13]. Briefly, the pEF/Amp-OATP1A2 vector,

kindly provided by Dr. Richard Kim (University of Wes-

tern Ontario, Canada), encoding the full-length OATP was

used to generate recombinant constructs [27, 28]. Purified

plasmids were then transfected into HEK293 cells by using

lipofectamine as directed by the suppliers (Invitrogen).

After 48 h of transfection, whole cell lysates were pre-

pared, and protein overexpression was verified by immu-

noblotting using antibodies specific to each transporter.

Immunoblotting

Western blot analysis was performed as described previ-

ously by our group [13, 29, 30] with minor modifications.

10 lg (human placental tissue expressing STS) or 50 lg

[HEK-293 cells overexpressing OATP1A2, OATP3A1 and

OATP4A1 (both stably and transiently transfected cells)]

of cell/tissue lysates was loaded as positive controls, and

50 lg was loaded for MCF-7, T47-D, ZR-75 and MDA-

MB-231 cell lysates. Blots were incubated with primary

antibodies for anti-OATP1A2 (1:1,000), anti-OATP3A1

(1:600), anti-OATP4A1 (1:600) and anti-STS (1:1,200).

All blots were also incubated with primary mouse anti-

actin (C4) antibody (1:2,000) as a loading control (Santa

Cruz Biotechnology, Inc., Santa Cruz, CA). The blots were

then incubated for 1.5 h in anti-rabbit (1:15,000), anti-goat

(1:10,000) or anti-mouse (1:2,000) secondary antibody.

Densitometric analysis was performed by using Alpha-

DigiDoc RT2 software to quantify relative protein

expression.

Transport experiments

Transport experiments were conducted as described pre-

viously by our group [13] with minor modifications. Time-

course studies were performed on confluent cell mono-

layers incubated (for different times) with transport buffer

containing 20 nM E3S (spiked with 0.3 lCi/ml [3H]-E3S)

[PerkinElmer Life and Analytical Sciences (Waltham,

MA)]. The specificity of OATP-mediated uptake and STS-

mediated metabolism were demonstrated by the use of a

specific transport and enzyme inhibitors bromosulphoph-

thalein (BSP) (Bromosulphophthalein) (100 lM) and

STX64 (also known as BN83495) (20 lM), respectively

[31]. All buffers, inhibitors and Triton X-100 were pur-

chased from Sigma-Aldrich, Canada.

Data analysis

All experiments were repeated at least three times in cells

from three different passages. Within an individual

experiment, each data point represents triplicate trials.

Results are presented as mean ± SD or mean ± S.E.M as

appropriate. All statistical analyses were performed with

Graphpad InStat version 3.0 software (GraphPad Software,

Inc., San Diego, CA). Statistical significance was assessed

by two-tailed Student’s t test for unpaired experimental

values or one-way analysis of variance (ANOVA) for

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650 Breast Cancer Res Treat (2014) 145:647–661

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analysis of repeated measures, as appropriate, p \ 0.05 is

considered statistically significant.

Results

OATP1A2, OATP2B1, OATP3A1 and OATP4A1

expression in tumour tissues

Figure 1a–d shows representative breast tumour sections

stained for OATP1A2, OATP2B1, OATP3A1 and

OATP4A1, respectively. Total tumour expression (Fig. 1e) of

OATPs (reported as % area) was determined in all 40 tumour

sections. Significantly (p \ 0.001) higher OATP expression

for all four isoforms were observed in HR? as compared to

HR- tumour sections. OATP1A2 expression was 2.4 times

greater in HR? (LN-: 8.06 ± 0.45 % area; LN?:

7.18 ± 0.5 % area) in comparison to HR- (LN-: 3.5 ±

0.62 % area; LN?: 3.0 ± 0.76 % area) sections. Similar to

OATP1A2, OATP4A1 has 3.6 times greater expression in

HR? (LN-: 11.49 ± 0.56 % area; LN?: 11.13 ± 0.53 %

area) compared to HR- (LN-: 3.26 ± 0.65 % area; LN?:

3.01 ± 0.54 % area) sections. OATP3A1 has high expression

in all tumour sections with a 1.4 times higher expression in

HR? (LN-:12.86 ± 0.7 % area; LN?: 12.42 ± 0.9 %

area) compared to HR- (LN-: 9.06 ± 0.6 % area; LN?:

8.77 ± 0.8 % area) sections. OATP2B1 has the lowest total

expression with 1.5 times greater expression in HR? (LN-:

3.99 ± 0.52 % area; LN?: 3.69 ± 0.81 % area) compared

to HR- (LN-: 2.85 ± 0.45 % area; LN?: 2.4 ± 0.6 %

area) tumour sections.

Figure 2a–d shows representative images of tumour tissue

montages, wherein tissues were stained for OATP1A2,

OATP2B1, OATP3A1 and OATP4A1, respectively, to

determine % area/tumour, % area/stroma and % area/non-

tumour. For the four OATP isoforms, it was found that the

highest target signals were obtained within the tumour regions

as opposed to the stroma or non-tumour regions for HR?

tumour sections (Table 1). Amongst the four OATPS, target

signal from within the tumour region (% of total signal) was

highest for OATP3A1 (HR?/LN-: 61.88 ± 1.2 %; HR?/

Fig. 1 Immunohistochemical staining for OATP1A2 (a), OATP2B1

(b), OATP3A1 (c) and OATP4A1 (d) in HR?/LN- (i), HR?/LN?

(ii),HR-/LN- (iii) and HR-/LN? (iv) breast tumour tissues. Total

tumour expression e of OATP isoforms (reported as % area) was

determined in 40 tumour sections. Each tumour section was stained

with OATP (first row) and DAPI as a nuclear marker (second row).

An overlay of OATP and DAPI staining (third row) allowed better

understanding of the intensity of the OATP staining with respect to

the DAPI staining. Scatter plots labelled with different letters indicate

significant difference in expression, p \ 0.05

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LN?: 65.32 ± 0.98 %), followed by OATP4A1 (HR?/LN-:

57.24 ± 2.4 %; HR?/LN?: 55.86 ± 2.9 %), OATP2B1

(HR ?/LN-: 50.27 ± 1.8 %; HR?/LN?: 44.32 ± 5.2 %)

and OATP1A2 (HR?/LN-: 46.38 ± 0.9 %; HR?/LN?:

45.12 ± 2.6 %). For HR- tumour sections, the data show a

trend that target signals for OATP1A2, OATP2B1 and

OATP4A1 (for HR-/LN? sections) within the tumour

region were lower than the target signals within the stroma or

non-tumour regions, although statistical significance was not

reached.

Fig. 2 Tumour tissue montage (Inform software) used to evaluate or

estimate % tumour, % stroma and % non-tumour areas within a

tumour section. Optical intensity of OATP1A2 (a), OATP2B1 (b),

OATP3A1 (c) and OATP4A1 (d) signal from each histopathological

area was determined and reported as % area/tumour, % area/stroma

and % area/non-tumour. Regions showing red, green and blue

represent tumour, stroma and non-tumour, respectively

652 Breast Cancer Res Treat (2014) 145:647–661

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Expression of metabolic enzymes, STS and 17b-HSD-1

in tumour tissues

Figure 3a, b shows representative breast tumour sections

stained with STS and 17b-HSD-1, respectively. Similar to

previously reported data [32, 33], STS expression was

observed to be 1.4 times higher (p \ 0.001) in HR? (LN-:

7.12 ± 0.38 % area; LN?: 7.51 ± 0.48 % area), as com-

pared to HR- (LN-: 5 ± 0.27 % area; LN?: 5.47 ±

0.49 % area) tumour tissues. However, unlike STS, 17b-

Fig. 3 Immunohistochemical staining for STS (a) and 17b-HSD-1

(b) in HR?/LN- (i), HR?/LN? (ii), HR-/LN- (iii) and HR-/LN?

(iv) breast tumour tissues. Total tumour expression (c) of STS and

17b-HSD-1 (reported as %Area) was determined in 40 tumour

sections. Each tumour section was stained with STS or 17b-HSD-1

(first row) and DAPI as a nuclear marker (second row). An overlay of

STS/17b-HSD-1 and DAPI staining (third row) allowed better

understanding of the intensity of the staining with respect to the

DAPI staining. Scatter plots labelled with different letters indicate

significant difference in expression, p \ 0.05

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HSD-1 expression was ubiquitously high in all 40 tumour

sections with no significant difference between the HR ?

(LN-: 6.19 ± 0.73 % area; LN?: 6.1 ± 0.66 % area) and

HR- (LN-: 6.22 ± 0.69 % area; LN?: 6.51 ± 0.56 %

area) tumour tissues.

Figure 4a,b shows representative images of tumour tissue

montages for STS and 17b-HSD-1, respectively. For STS

expression, the highest target signals (% of total signal) were

obtained from the tumour region (HR?/LN-:

73.03 ± 8.63 %; HR?/LN?: 59.92 ± 3.2 %; HR-/LN-:

Table 1 Immunohistochemical expression in tumour, stroma and non-tumour regions of the tumour sections

Total expression

(% area) ± SEM

Expression in tumour

(% area) ± SEM

Expression in

stroma (% area)

± SEM

Expression in

non-tumour

(% area) ± SEM

OATP1A2

HR?/LN- 7.33 ± 1.4 3.4 ± 0.4 2.01 ± 0.7 1.92 ± 0.7

HR?/LN? 7.18 ± 1.6 3.24 ± 0.6 2.71 ± 1.1 1.23 ± 0.6

HR-/LN- 3.5 ± 0.8 0.58 ± 0.02 1.6 ± 0.4 1.32 ± 0.8

HR-/LN? 2.9 ± 0.9 0.6 ± 0.01 1.33 ± 0.8 0.97 ± 0.2

OATP2B1

HR?/LN- 3.7 ± 1.1 1.86 ± 0.4 0.82 ± 0.04 1.02 ± 0.2

HR?/LN? 3.7 ± 0.8 1.64 ± 0.4 1.02 ± 0.1 1.04 ± 0.08

HR-/LN- 2.8 ± 0.7 1.05 ± 0.2 0.7 ± 0.04 1.05 ± 0.3

HR-/LN? 2.4 ± 0.8 0.57 ± 0.07 1.03 ± 0.1 0.8 ± 0.07

OATP3A1

HR?/LN- 11.7 ± 3.2 7.24 ± 2.4 3.1 ± 1.1 1.36 ± 0.7

HR?/LN? 12.4 ± 2.7 8.1 ± 2.2 2.7 ± 0.4 1.6 ± 0.6

HR-/LN- 9.06 ± 1.8 5.3 ± 1.1 3.6 ± 1.2 0.16 ± 0.08

HR-/LN? 8.8 ± 3.1 4.6 ± 1.3 2.9 ± 0.8 1.4 ± 0.09

OATP4A1

HR?/LN- 10.5 ± 4 6.01 ± 2.3 2.2 ± 0.8 2.29 ± 0.7

HR?/LN? 11.1 ± 3.4 6.2 ± 1.8 3.1 ± 1.2 1.8 ± 0.8

HR-/LN- 3.3 ± 1.6 1.2 ± 0.7 1.03 ± 0.4 1.07 ± 0.2

HR-/LN? 3.01 ± 1.2 0.8 ± 0.08 1.2 ± 0.3 1.01 ± 0.2

BCRP

HR?/LN- 3.53 ± 1.2 1.31 ± 0.7 1.2 ± 0.4 1 ± 0.7

HR?/LN? 5.41 ± 1.8 2.2 ± 0.8 1.96 ± 0.8 0.94 ± 0.1

HR-/LN- 9.47 ± 3.1 6.2 ± 1.4 1.7 ± 0.4 1.8 ± 0.4

HR-/LN? 12.86 ± 4.2 6.4 ± 2.4 3.5 ± 1.1 2.96 ± 0.8

MRP-1

HR?/LN- 11.25 ± 2.2 7.2 ± 1.2 2.4 ± 1 1.6 ± 0.7

HR?/LN? 12.57 ± 3.4 7.8 ± 2.4 3 ± 1.4 1.77 ± 0.6

HR-/LN- 11.4 ± 2.4 6.5 ± 2 2.8 ± 0.8 2.1 ± 0.9

HR-/LN? 12.4 ± 3.2 6.9 ± 2.2 3.4 ± 1.1 2.1 ± 0.7

STS

HR?/LN- 7.12 ± 2.1 5.2 ± 1.2 1 ± 0.4 0.93 ± 0.3

HR?/LN? 7.51 ± 2.2 4.5 ± 1.1 1.8 ± 0.7 1.2 ± 0.2

HR-/LN- 5 ± 1.8 2.8 ± 0.7 1.2 ± 0.5 1 ± 0.1

HR-/LN? 5.47 ± 1 2.84 ± 0.8 1.7 ± 0.7 0.93 ± 0.1

HSD-1

HR?/LN- 6.19 ± 2.1 4 ± 1.4 1.5 ± 0.4 0.69 ± 0.04

HR?/LN? 6.1 ± 2 3.1 ± 1.1 1.8 ± 0.4 1.2 ± 0.2

HR-/LN- 6.22 ± 2.4 3.6 ± 1.2 1.5 ± 0.5 1.12 ± 0.2

HR-/LN? 6.51 ± 1.2 4 ± 1.1 1.5 ± 0.2 1 ± 0.3

654 Breast Cancer Res Treat (2014) 145:647–661

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56.00 ± 5.24 % and HR-/LN?: 51.92 ± 2.81 %) as

opposed to stroma or non-tumour, for both HR?and HR-

tumour sections (Table 1). Similar to STS, 17b-HSD-1 tar-

get signals were highest in the tumour region (HR?/LN-:

64.62 ± 7.56 %; HR?/LN? : 50.82 ± 6.11 %; HR-/LN-:

57.88 ± 2.72 % and HR-/LN?: 60.70 ± 7.42 %).

Expression of efflux transporters BCRP and MRP-1

in tumour tissues

Figure 5a,b shows representative breast tumour sections

stained for BCRP and MRP-1, respectively. Unlike OATP

and STS expression, total tumour expression of BCRP was

significantly greater (p \ 0.001) in HR- compared to

HR? tumour sections. Furthermore, within each category,

LN? tumours had significantly higher expression

(p \ 0.001 for HR? and HR- tumours) compared to LN-

tumours. These observations are in agreement with previ-

ously reported data suggesting that hormone receptor

expression is inversely related to BCRP expression [34,

35]. MRP-1 tumour expression was significantly higher

(p \ 0.01) in LN? tumours compared to LN- tumours,

which is in agreement with previously reported data [36,

37]. MRP-1 expression was independent of HR status.

However, similar to OATP1A2, OATP2B1, OATP4A1,

STS and 17b-HSD-1, the highest target signals for both

BCRP (HR?/LN-: 37.11 ± 4.32 %; HR?/LN?: 40.67 ±

2.82 %; HR-/LN-: 65.47 ± 5.43 % and HR-/LN?:

49.77 ± 3.02 %) and MRP-1 (HR?/LN-: 64.00 ± 3.66 %;

HR?/LN?: 62.05 ± 4.43 %; HR-/LN-: 57.02 ± 1.8 %

and HR-/LN?: 55.65 ± 3.24 %) were obtained in the

tumour regions (Table 1). Figure 6a, b shows representative

images of tumour tissue montages for BCRP and MRP-1,

respectively.

OATP1A2, OATP3A1, OATP4A1 and STS protein

expression in HR± breast cancer cells

OATP1A2, OATP3A1, OATP4A1 and STS protein expression

determined by Western blot analysis were compared between

HR? (i.e. MCF-7, T47-D and ZR-75) and HR- (i.e. MDA-MB-

231) breast cancer cell lines. OATP1A2 expression was

observed in all cell lines with significantly lower expression in

MDA-MB-231 compared to MCF-7 (p \0.05) or T47-D

(p\ 0.05) (Fig. 7a). OATP3A1 expression in MDA-MB-231

was significantly lower than that in MCF-7 (p \ 0.01), T47-D

(p\ 0.05) and ZR-75 (p \0.05) (Fig. 7b). Similar to

OATP3A1, OATP4A1 expression was also significantly lower

in MDA-MB-231 compared to MCF-7 (p\ 0.001), T47-D

(p\ 0.01) and ZR-75 (p\ 0.05) (Fig. 7c). STS expression

was significantly lower in MDA-MB-231 compared to MCF-7

(p\ 0.001) and T47-D (p\ 0.05) (Fig. 7d). Amongst the

HR? breast cancer cell lines, MCF-7 had the highest expres-

sion of OATP1A2, OATP3A1 and OATP4A1 as well as STS.

Fig. 4 Tumour tissue montage (Inform software) used to evaluate %

tumour, % stroma and % non-tumour areas within a tumour section.

Optical intensity of STS (a) and 17b-HSD-1 (b) signal from each

histopathological area was determined and reported as % area/

tumour, % area/stroma and % area/non-tumour. Regions showing

red, green and blue represent tumour, stroma and non-tumour,

respectively

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123

Specificity of OATP-mediated E3S uptake in breast

cancer cells

Specificity of OATP-mediated E3S uptake was investi-

gated in MCF-7 (Fig. 8a), T47-D (Fig. 8b) and ZR-75

(Fig. 8c) cells lines. Time-course experiments were

performed with transport buffer containing 20 nM E3S, in

the presence or absence of specific OATP inhibitor i.e.

100 lM-BSP. Significant differences observed between the

total and non-specific uptake in MCF-7 (Fig. 8a), T47-D

(Fig. 8b) and ZR-75 (Fig. 8c) suggest a specific carrier-

mediated process contributes to the intracellular

Fig. 5 Immunohistochemical staining for BCRP (a) and MRP-1

(b) in HR?/LN- (i), HR?/LN? (ii), HR-/LN- (iii) and HR-/LN?

(iv) breast tumour tissues. Total tumour expression (c) of BCRP and

MRP-1 (reported as % area) was determined in 40 tumour sections.

Each tumour section was stained with BCRP or MRP-1 (first row) and

DAPI as a nuclear marker (second row). An overlay of BCRP/MRP-1

and DAPI staining (third row) allowed better understanding of the

intensity of the staining with respect to the DAPI staining. Scatter

plots labelled with different letters indicate significant difference in

expression, p \ 0.05

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accumulation of E3S in HR? breast cancer cells. Although

significant differences were observed in the intracellular

accumulation of E3S in HR- breast cancer cells (MDA-

MB-231) (Fig. 8d) [13], the total intracellular accumula-

tion was much lower in these cells compared to that

observed in HR? cells. These data are in agreement with

our recent study that evaluated the tumour accumulation of

exogenous E3S in animal models of hormone-dependent

(MCF-7) and independent (MDA-MB-231) breast cancer

[38].

Specific STS enzyme activity in hormone-dependent

breast cancer cells

Similar time-course experiments were performed with

transport buffer containing 20 nM E3S, in the presence or

absence of specific enzyme (STS) inhibitors i.e. 20 lM-

STX-64. Significantly, higher intracellular E3S accumula-

tion was observed in the presence of STX-64 in MCF-7

(Fig. 8a), T47-D (Fig. 8b) and ZR-75 (at 5 and 10 min

time points only) (Fig. 8c) cells, suggesting the presence of

specific STS-mediated intracellular metabolism of E3S in

hormone-dependent breast cancer. There was no significant

difference between the accumulation of intracellular E3S in

the presence or absence of STX-64 in hormone-indepen-

dent (MDA-MB-231) breast cancer cells (Fig. 8d).

Discussion

Understanding uptake, metabolism and efflux of E3S

would allow evaluation of novel molecular targets (such as

OATPs) for HR? breast cancer patients, including, but not

limited to, patients who are resistant to endocrine therapies.

Expression of four OATP isoforms (i.e. OATP1A2,

OATP2B1, OATP3A1 and OATP4A1) was compared

between HR? and HR- human breast tumour tissues from

both LN? and LN- subcategories. The OATP isoforms

were chosen based on our previous data [13] and other

studies [13, 39–41] reporting their expression and func-

tional role in cellular uptake of E3S in in vitro breast

cancer cells. OATP1A2, OATP2B1, OATP3A1 and

OATP4A1 all show significantly higher expression in HR?

as compared to HR- tumour sections (Fig. 1a–d).

OATP1A2 and OATP4A1 have the greatest difference in

expression between the HR? and HR- tumour tissues,

followed by OATP3A1 and OATP2B1. These data are in

agreement with previously reported gene expression of

OATPs in breast tumour tissues [42]. To further understand

the potential of OATPs as a novel molecular target for

HR? breast cancer, expression in histological tumour

regions (i.e. tumour, stroma and non-tumour) was deter-

mined. Highest target signals were observed in tumour

regions of the HR? tumour sections (Table 1). Tumour

Fig. 6 Tumour tissue montage (Inform software) used to estimate %

tumour, % stroma and % non-tumour areas within a tumour section.

Optical intensity of BCRP (a) and MRP-1 (b) signal from each

histopathological area was determined and reported as % area/

tumour, % area/stroma and % area/non-tumour. Regions showing

red, green and blue represent tumour, stroma and non-tumour,

respectively

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123

region-specific OATP expression strongly suggests the

potential of these transporters as novel molecular targets

for HR? tumours.

To further understand the intra-tumoral fate of E3S, total

tumour and region-specific expression of metabolizing

enzymes STS and 17b-HSD-1 were determined in the

clinical tumour tissues (Fig. 3a, b). STS expression was

significantly higher in HR? tumour tissues (1.4 times) as

compared to HR- tumour tissues with highest target sig-

nals in the tumour regions of both HR? and HR- tumour

tissues (Table 1). As STS is currently considered a novel

target for treatment of HR? breast cancers, our data are in

agreement with previously published clinical data [32, 33,

43]. Moreover, given that OATP expression is higher

Fig. 7 Immunoblot and densitometric analysis of OATP transporters

and STS enzyme in HR? and HR- breast cancer cells. Protein

expression of OATP1A2 (a), OATP3A1 (b), OATP4A1 (c) and STS

(d) was determined in HR? (i.e. MCF-7, T47D, ZR-75) and HR-

(i.e.MD-MB-231) breast cancer cells applying standard western blot

analysis as described in the Materials and methods section. Results of

the densitometric analysis are expressed as mean ± SD of three

separate experiments (1: positive control; 2: MDA-MB-231; 3: MCF-

7; 4: ZR-75; 5: T47-D). ***p \ 0.001, **p \ 0.01 and *p \ 0.05 are

considered to be statistically significant

658 Breast Cancer Res Treat (2014) 145:647–661

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specifically in the tumour regions of HR? tumours (com-

pared to stroma or non-tumour regions), while STS

expression is higher in the tumour regions of both HR?

and HR- tumours; this further suggests that OATPs may

be a promising molecular target for HR? tumours. How-

ever, unlike STS, no significant differences were observed

in 17b-HSD-1 expression between HR? and HR- tumour

tissues. Contrary to OATP and STS expression, total

tumour expression of BCRP was significantly greater in

HR- compared to HR? tumour sections while MRP-1

expression had no correlation with HR status. Interestingly,

both BCRP and MRP-1 expression were significantly

higher in LN? tumours (Table 1) [34, 35]. Similar to

OATPs, STS and 17b-HSD-1, the target signals for BCRP

and MRP-1 expression were highest in the tumour regions.

These data collectively suggest that (i) the eight markers

investigated are potential tumour targets as their expression

is highest in the tumour region; (ii) OATPs are potential

novel molecular targets for HR? tumours. We acknowl-

edge that our data are semi-quantitative and only establish

a trend in the expression of each marker in the four sub-

categories of tumour tissues. Due to limited access to

clinical tumour tissues, gene and protein expression of the

markers could not be quantified.

The functional role of the transporters and enzymes was

further investigated in in vitro models of HR? (MCF-7,

T47-D, ZR-75) and HR- (MDA-MB-231) breast cancer cell

lines to better understand the fate of E3S and the relevance

of the differences in expression of OATP and STS

observed in HR? and HR- tumours. Similar to the human

breast tumour tissues, the protein expression of OATP1A2,

OATP3A1, OATP4A1 and STS was significantly

(p \ 0.05) higher in HR? than HR- breast cancer cells

(Fig. 7). These data support the use of these in vitro cell

lines as models for HR? and HR- breast cancer. Specific

OATP-mediated E3S uptake and STS-mediated metabo-

lism were observed in all HR? (Fig. 8a–c) breast cancer

cells. We acknowledge that the tumoral fate of E3S could

be better understood if the functional role of the efflux

transporters could also be established. However, cross

Fig. 8 Time course of [3H] E3S uptake by HR ?/- breast cancer

cells. Total uptake (closed circles) of E3S by the cells was evaluated

over 30 min at pH 7.4 and 37 �C. The non-specific uptake (closed

squares) and the non- specific metabolism (closed diamonds) were

calculated by determining uptake in the presence of an excess

concentration of transport inhibitor (BSP 100 lM) and enzyme

inhibitor (STX64 20 mM) as described in the Materials and Methods

section. a MCF7, b T47D, c ZR75 and d MDA-MB-231 cells.

*p \ 0.05 is considered to be statistically significant

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reactivity between the inhibitors for BCRP, MRP-1 and

OATPs makes it technically challenging to ensure OATP-

mediated cellular uptake in the presence of BCRP and

MRP-1 transport inhibitors.

Overall, the significantly higher expression of OATPs

and STS observed in human HR? tumour tissues with

specifically high target signals obtained from tumour cells

suggests the potential of these markers as novel tumour

targets. Current limitations of endocrine therapies are

acquired or de novo resistance caused primarily by muta-

tion or loss of HR [44, 45]. While selective oestrogen-

receptor modulators, such as tamoxifen and raloxifene,

aromatase inhibitors, and GnRH agonists, constitute the

first line therapy for HR? breast tumours [46, 47], the

efficacy of these agents is dependent upon ER status.

Developing OATPs as a novel molecular target could

potentially expand diagnostic and treatment options for

patients with primary hormone-dependent tumours which

have lost HR functional expression.

Acknowledgment The authors acknowledge Dr. Md. Tozammel

Hoque for his help with the cell line transient transfection studies and

Dr. Fei–Fei Liu for her excellent scientific advice and for serving as

the Principal Investigator on the Research Ethics Board (REB) Tissue

application. This research was supported by Internal University of

Toronto funds allocated to Dr. Reina Bendayan and Dr. Christine

Allen. Nilasha Banerjee is a recipient of the CIHR- Bio-Therapeutics

Strategic Training doctoral fellowship and the Canadian Breast

Cancer Foundation-Ontario region doctoral fellowship.

Conflict of interest No conflicts of interest were identified.

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