targeted therapy for adrenocortical tumors ... · hecate, a 23-amino acid lytic peptide, was...

Endocrine-Related Cancer (2008) 15 635–648

Targeted therapy for adrenocortical tumorsin transgenic mice through their LH receptorby Hecate-human chorionic gonadotropin bconjugate

Susanna Vuorenoja1,2, Adolfo Rivero-Muller1, Adam J Ziecik3,Ilpo Huhtaniemi1,4, Jorma Toppari1,2 and Nafis A Rahman1

1Department of Physiology, University of Turku, Kiinamyllynkatu 10, 20520 Turku, Finland2Department of Pediatrics, University of Turku, 20520 Turku, Finland3Institute of Animal Reproduction and Food Research, Polish Academy of Sciences, 10-714 Olsztyn, Poland4Department of Reproductive Biology, Imperial College London, London W12 ONN, UK

(Correspondence should be addressed to N A Rahman; Email: [email protected])

Abstract

Novel strategies are needed for the treatment of adrenocortical tumors that are usually resistantto chemotherapy. Hecate, a 23-amino acid lytic peptide, was conjugated to the 15-amino acid(81–95) fragment of the human chorionic gonadotropin b (CGb) chain, which would selectivelykill cancer cells expressing the LH receptor (LHR) sparing the normal ones with LHR. To provethe principle that Hecate-CGb conjugate may eradicate tumors ectopically expressing plasmamembrane receptors, transgenic (TG) inhibin a-subunit promoter (inha)/Simian Virus 40 T-antigen mice, expressing LHR in their adrenal gland tumors, were used as the experimentalmodel. Wild-type control littermates and TG mice with adrenal tumors were treated with eitherHecate or Hecate-CGb conjugate at the age of 6.5 months for 3 weeks and killed 7 days afterthe last treatment. The Hecate-CGb conjugate reduced the adrenal tumor burden significantly inTG male but not in female mice, in comparison with Hecate-treated mice. Hecate-CGb

conjugate treatment did not affect normal adrenocortical function as the serum corticosteronelevel between Hecate and Hecate-CGb conjugate groups were similar. The mRNA and proteinexpressions of GATA-4 and LHR colocalized only in tumor area, and a significantdownregulation of gene expression was found after the Hecate-CGb conjugate in comparisonwith Hecate- and/or non-treated adrenal tumors by western blotting. This finding providesevidence for a selective destruction of the tumor cells by the Hecate-CGb conjugate. Hereby,our findings support the principle that Hecate-CGb conjugate is able to specifically destroytumor cells that ectopically express LHR.


Introduction

Adrenocortical carcinomas (ACCs) are rare and aggres-

sive types of humanmalignancies with poor prognosis as

they often are diagnosed late and the survival rate

remains quite low (Schulick & Brennan 1999b). ACCs

tend to occur in the first and fifth decades of life and are

relativelymore common inwomen thanmen (Schulick&

Brennan 1999a,b). There are still no efficient forms of

therapy for adrenal tumors. The only effective treatment


1351–0088/08/015–635 q 2008 Society for Endocrinology Printed in Great

at this moment is complete adrenalectomy since

with partial adrenalectomies the survival rate remains

quite poor. However, even in patients undergoing

complete surgical resection, recurrent and metastatic

disease is common (Reincke et al. 1994, Bornstein et al.

1999). Chemotherapy with mitotane (o,p0-DDD) or

cisplatin is indicated for advanced stages of disease,

although the outcome remains often poor with plenty of

side effects including adrenal insufficiency (Ahlman

et al. 2001). Thus, there is a great need for new curative

Britain

DOI: 10.1677/ERC-08-0015

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S Vuorenoja et al.: Hecate-CGb conjugate and adrenocortical tumor

methods as well as novel markers for early detection

of these rare ACCs. Besides the normal gonadal

expression of luteinizing hormone receptor (LHR),

there are reports about malignant androgen-producing

LH/human chorionic gonadotropin (hCG)-dependent

adrenal tumors (de Lange et al. 1980, Leinonen et al.

1991, Lacroix et al. 1999). The LHR is expressed at low

levels in normal human adrenal cortex (Pabon et al.

1996), and hCG stimulates dehydroepiandrosterone

sulfate synthesis of human fetal adrenals (Jaffe et al.

1981). LHRexpression has been shown in different kinds

of human adrenal pathologies i.e., virilizing or Cushing’s

syndrome (CS)-related adrenocorticotropin-independent

macronodular adrenal hyperplasias (AIMAH; Lacroix

et al. 1999, Bourdeau et al. 2001, Miyamura et al. 2002,

Feelders et al. 2003, Goodarzi et al. 2003,Mijnhout et al.

2004). Pregnancy-associated CS is also a known

condition (Sheeler 1994). LHR expression has also

been shown in aldosterone- (Saner-Amigh et al. 2006) or

androgen-producing LH-dependent adrenal adenomas

(Werk et al. 1973, Givens et al. 1975, Larson et al. 1976,

Smith et al. 1978, Takahashi et al. 1978, de Lange et al.

1980, Leinonen et al. 1991) and inACCs (Wy et al. 2002,

Barbosa et al. 2004). Thus, this abundant/upregulated

LHR expression in the adrenal hyperplasia/adenomas

and adenocarcinomas makes them a susceptible target to

ligandas a strategy for tumor eradication throughHecate-

human chorionic gonadotropin b (CGb) conjugate.During the last few years, a novel targeted

treatment approach for hormonally active, LHR-

bearing tumors has been developed using the

Hecate-CGb conjugate (Leuschner et al. 2001).

Hecate is a synthetic lytic peptide based on the

structure of melittin, the principal toxin of honeybee

venom. It rapidly destroys the outer membrane shell

of only negatively charged cells such as bacteria and

cancer cells by making small pores to the membrane

(Leuschner & Hansel 2004, Rivero-Muller et al.

2007). Cancer cells have been shown to be negatively

charged due to the changed distribution of phospha-

tidylserines in the outer cell membrane and thus, in

comparison with the healthy cells, only negatively

charged cells are destroyed by this lytic peptide

(Utsugi et al. 1991). To target the action of this lytic

peptide directly to the cancer cells through a

membrane receptor, Hecate can be conjugated to a

ligand or its receptor-binding domain, such as, in our

case, to the 15-amino acid fragment (81–95) of the

human CGb chain (Hecate-CGb conjugate). This

allows the Hecate-CGb conjugate to bind specifically

and disrupt cells bearing the LHR. The same principle

should apply to the plasma membrane receptors

ectopically expressing in cancer cells. The mechanism

636

of destruction is necrosis without activation of

apoptosis (Bodek et al. 2005b) and Hecate-CGbconjugate appears to selectively kill only cancer

cells with LHR and spare all other gonadal/non-

gonadal healthy cells (even with LHR) due to

negative/positive charge changes in their membrane

potential (Leuschner & Hansel 2004, Bodek et al.

2005b). Hecate-CGb conjugate has previously been

studied in specific cancer cell lines and xenografts of

LHR-bearing carcinomas of prostate (Leuschner et al.

2001, 2003b, Bodek et al. 2005a), mammary gland

(Bodek et al. 2003, Leuschner et al. 2003a), and ovary

(Gawronska et al. 2002). In vivo studies have so

far been carried out in transgenic (TG) mice bearing

ovarian and testicular tumors (Bodek et al. 2005b).

TG mice expressing the inhibin a promoter/Simian

virus 40 (SV40) T-antigen (inha/Tag) were

originally found to produce gonadal tumors with

100% penetrance at the age of 6 months, and if

gonadectomized prepubertally, they produced dis-

cernible adrenal tumors at the same age, but never in

intact mice (Kananen et al. 1996a, Rilianawati et al.

1998, Rahman et al. 2004). The adrenal tumors and a

tumor-derived cell line (Ca1) express very high

levels of LHR (Kananen et al. 1996a, Rilianawati

et al. 1998, Rahman et al. 2004), and the growth and

steroidogenesis of the adrenal tumors were shown to

be dependent on LH stimulation (Mikola et al. 2003).

The post-castration elevation of LH levels apparently

induced the ectopic LHR expression, which together

with the potent oncogene Tag expression triggered

adrenocortical tumorigenesis (Mikola et al. 2003).

LH dependence of the tumors was proven by findings

that they failed to appear if the post-castration

increase in gonadotropins was blocked by either

treating the mice with a gonadotropin-releasing

hormone antagonist or crossbreeding them to the

gonadotropin-deficient hpg (hypergonadotropic)

genetic background (Cattanach et al. 1977, Kananen

et al. 1997). The adrenocortical tumors tend to grow

in prepubertally gonadectomized mice by hyperpla-

sia–adenoma–carcinoma sequence, as hyperplasia of

the adrenal cortex/adrenal adenoma could be

observed at the age of 4 months, whereas discernible

gonadal tumors were seen only at 6 months with low

metastatic incidence (Rahman et al. 2004). In this

study, we took the advantage of this inha/Tag TG

adrenocortical tumor model in order to prove the

principle that adrenocortical tumor cells expressing

ectopically a hormone receptor are sensitive to the

Hecate-CGb conjugate treatment in vivo, sparing

simultaneously healthy cells.

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Materials and methods

Experimental animals

In order to induce adrenal tumors, we gonadectomized

inha/Tag TG mice prepubertally as described pre-

viously (Kananen et al. 1996a). As these mice have

earlier been extensively characterized (Kananen et al.

1996a, Rilianawati et al. 1998, 2000, Kero et al. 2000,

Rahman et al. 2001, 2004), the discernible adrenocor-

tical tumors appear at the age of 6 months with 100%

penetrance. We started all the treatments at the age of

6.5 months, in order to make sure that any treatment

effect will be only due to the anti-tumoral effect, but

not by prevention of the tumor development. Gona-

dectomy was performed under Avertin anesthesia and

postoperative buprenorphine analgesia was adminis-

tered on a routine basis. Seven to ten mice per

treatment group were selected for the experiments.

Wild-type (WT) control littermate mice (C57Bl/6N)

were used as controls. For routine genotyping, PCR

analysis was carried out using DNA extracts from ear

biopsies, as previously described (Kananen et al.

1995). After weaning at the age of 21 days, the mice

were housed two to four per cage, females and males in

separate cages, in a room of controlled light (12 h

light:12 h darkness) and temperature (21G1 8C). They

were fed with mouse chow SDS RM-3 (Whitham,

Essex, UK) and tap water ad libitum. The mice were

kept in a specific pathogen-free surrounding and were

routinely screened for common mouse pathogens. The

University of Turku Ethics Committee on Use and

Care of Animals approved the animal experiments.

Preparation of drugs

Hecate and the Hecate-CGb conjugate were syn-

thesized and purified in the Peptide and Protein

Laboratory,Department ofVirology,Hartman Institute,

University of Helsinki, as described previously

(Bodek et al. 2003).

Hecate and Hecate-CGb conjugate treatments

Male and female mice (inha/Tag or WT C57Bl control

littermates; nZ7–10 per group) were treated at the age

of 6.5 months with either Hecate-CGb conjugate

(12 mg/kg b.w.) or Hecate (12 mg/kg b.w.) by i.p.

injections. Since no substantial weight changes could

be observed between Hecate and Hecate-CGbconjugate by adding hCG to Hecate, both Hecate and

Hecate-CGb conjugate were given in the same dose

(Leuschner et al. 2001). The mice were injected once

per week for three consecutive weeks, according to an

earlier protocol for in vivo treatment for nude mice


xenografts (Leuschner et al. 2001) and TG mice with

gonadal tumors (Bodek et al. 2005b). Seven days

following the last injection, mice were killed by

cervical dislocation and blood was collected by cardiac

puncture. Weights of body, tumor, and different organs

were recorded. Tissues were either snap-frozen in

liquid nitrogen, or fixed in Bouin’s solution or 4%

paraformaldehyde and embedded in paraffin. Paraffin

sections of 5 mm thickness were stained for further

histological analysis with hematoxylin–eosin or used

for immunohistological analysis. For each tissue, at

least five independent specimens were examined from

each group.

Northern hybridization analysis

Total RNAwas isolated from cells using the single-step

guanidinium thiocyanate–phenol–chloroform extrac-

tion method (Chomczynski & Sacchi 1987). Twenty

micrograms of RNA per lane were resolved on 1.2%

denaturing agarose gel and transferred ontoHybond-XL

nylon membranes (Amersham, Amersham Inter-

national). Membranes were prehybridized overnight at

65 8C in a solution containing 5! sodium chloride/

sodium citrate (SSC), 5! Denhardt’s solution, 0.5%

SDS, 50% formamide, and 5 g/l of denatured calf

thymus DNA. A complementary RNA probe for the rat

LHR generated from a fragment of the LHR cDNA,

spanning nucleotides 441–849 of its extracellular

domain, subcloned into the pGEM-4Z plasmid was

used for hybridization (LaPolt et al. 1990). The

[32P]-dUTP (800 Ci/mmol, Amersham) labeled probe

was generated using a Riboprobe system II kit

(Promega). The probes were purified with NickCol-

umns (Pharmacia Biotech). Hybridization was carried

out at 65 8C for 20 h in the same prehybridization

solution after an addition of labeled probe. After

hybridization, the membranes were washed twice in

2! SSC with 0.1% SDS at room temperature for

10 min, followed by two washes in 0.1! SSC with

0.1% SDS at 65 8C to remove most of the background.

For GATA-4, the membranes were prehybridized

overnight at 42 8C in a solution containing 5! SSC,

5! Denhardt’s, 0.5% SDS, 50% formamide, and 5 g/l

denatured calf thymus DNA. The GATA-4 cDNA

probes were cut out from pMT2-GATA4 (Arceci et al.

1993, Ketola et al. 1999), using EcoRI/PstI and SmaI

restriction enzymes respectively labeled with Prime-

a-Gene kit (Pharmacia) using [a-P32]-dCTP for 4 h at

37 8C and purified with NickColumns. Hybridization

was carried out at 42 8C for 20 h in the same

prehybridization solution after an addition of the

denatured labeled cDNA probe. After hybridization,

637



themembraneswerewashed twice in 2!SSC and 0.1%

SDS at room temperature for 10 min, followed by two

washes in 0.1! SSC and 0.1% SDS at 42 8C to remove

most of the background. Finally, the membranes were

exposed to KodakX-ray films (Kodak XAR-5; Eastman

Kodak) at K70 8C for 4–7 days or to phosphor-imager

(Fujifilm BAS-5000, Fujifilm IaI, Tokyo, Japan) for

4–24 h. The intensities of specific bandswere quantified

using the Tina software (Raytest, Stranbenhardt,

Germany) which came as an integrated program with

the phosphor-imager (Fujifilm BAS-500; El-Hefnawy

& Huhtaniemi 1998, El-Hefnawy et al. 2000, Manna

et al. 2001, Strassburg et al. 2002, Moran et al. 2006),

and related to those of the 28S rRNA in the gel stained

with ethidium bromide. The molecular sizes of the

mRNA species were estimated by comparison with the

mobility of the 18S and 28S rRNAs.

Hormone measurements

Serum levels of LH were measured by immunofluoro-

metric assay for rat (Delfia; Wallac, Turku, Finland) as

described previously (Haavisto et al. 1993). Cortico-

sterone levelsweremeasured fromdiethyl ether extracts

of the sera by RIA using a kit for rats and mice (MP

Biomedicals, Orangeburg, NY, USA) and progesterone

was measured by Delfia Progesterone Kit (Wallac). The

approximate assay sensitivities for LH, progesterone,

and corticosterone are 0.75 pg/tube, 50 fmol/tube, and

30 fmol/tube respectively. The intra- and inter-assay

coefficients of variations for these assays were below

10–15% respectively.

Immunoblotting analysis for GATA-4 and LHR

Western blotting was used to determine the concen-

tration changes in GATA-4 and LHR due to the

different treatments (Hecate or Hecate-CGb conju-

gate). Tissue samples were subjected to homogeni-

zation and protein concentration was measured using

the bicinchoninic acid (BCA) protein assay kit (Pierce,

Rockford, IL, USA). Electrophoresis was carried out

through a 12.5% SDS-PAGE gel (1 mg protein/well).

Dual Color Precision Plus Protein Standards (Bio-Rad)

were used as standards. After electrophoresis, proteins

were electrotransferred to Hybond-P PVDF Membrane

(Amersham Biosciences) using Trans-Blot SD cell

(Bio-Rad) by 20 V for 60 min. After blocking the

membrane with TBS containing 2% fat-free milk

powder C0.05 M Tween, the incubation with the

primary antibody was performed at C4 8C overnight.

Anti-GATA-4 (goat polyclonal antibody, dilution

1:100; C-20, sc-1237; Santa Cruz Biotechnology,

Santa Cruz, CA, USA) or anti-LHR (rabbit polyclonal

638

antibody, dilution 1:4000; Acris Antibodies, Hidden-

hausen, Germany) were used separately. As secondary

antibody, either bovine anti-goat IgG-HRP (sc-2350,

Santa Cruz Biotechnology) or ECL Anti-rabbit IgG,

HRP-linked whole AB (Amersham Biosciences) was

used in dilution 1:10 000. Signals were visualized

using ECL Plus Western blotting detection reagents

(Amersham Pharmacia Biotech) and finally exposed to

Fuji X-ray film (Super RX, Fuji Photo Film Ltd,

Bedford, UK). The intensities of specific bands were

quantified using the Tina Software (Raytest).

Laser pressure catapulting (LPC)

LPC was performed by Laser MicrobeamMicrodissec-

tion (PALM Microlaser Technologies, Bernried,

Germany) using a protocol described previously

(Westphal et al. 2002). Briefly, for laser microdissec-

tion, cryosections (5 mm) were prepared from three

independent specimens. Subsequent dehydration was

achieved using graded alcohol and xylene treatment as

follows: 95% ethanol for 30 s (twice), 100% ethanol

for 30 s (twice), and xylene for 5 min (twice). The

slides were dried under laminary flow for 10 min and

then stained with hematoxylin using RNase-free

conditions and kept on dry ice until LPC. LPC was

performed using a Zeiss inverted microscope PALM

Laser Micro-Beam System u.v. laser at 337 nm, linked

to a PC with the required software programs. The

tumor and non-tumor areas to be dissected were

selected by means of high-precision microcuts (the

specimens may range from as little as 1 to 1000 mm in

diameter). LPC dissection was performed using a few

shots each of 100 mm in diameter from both tumor and

non-tumor areas and from each slide. After catapulting

similar amounts of tumor and non-tumor area, the

material was removed from the caps for further

analysis. Total RNA was extracted from tumor tissues

using RNeasy kit (Qiagen).

RT-PCR and Southern hybridization

One microgram of total RNA was reverse transcribed

using 20 IU of avian myeloma virus reverse tran-

scriptase (RT), 50 IU RNase inhibitor, 25 pmol random

primers, and 10 mmol of each dNTP (all from

Promega), in 25 ml final volume for 1 h at room

temperature. For amplification of LHR and GATA-4

cDNA, specific primers were used as described

previously (Morrisey et al. 1997, Kero et al. 2000).

PCR was performed in a 25 ml final volume, 4 ml RTsolutions were mixed with 2 IU of the DyNAzyme II

DNA polymerase (Finnzymes Oy, Espoo, Finland),

10 pmol of each primer, and 10 mmol of each dNTP




(Zhang et al. 1994). The RT and PCRs were carried out

sequentially in the same assay tube. First, the RT

reaction was carried out (50 8C for 10 min), followed

by a denaturation period of 3 min at 97 8C. Thereafter,

a PCR with 40 cycles (96 8C for 1.5 min, 57 8C for

1.5 min, and 72 8C for 3 min, with a final extension

period of 10 min at 72 8C) was performed. The

sense primer for LHR corresponded to nucleotides

176–195 (5 0-CTCTCACCTATCTCCCTGTC-3 0) and

the antisense primer to nucleotides 878–858

(5 0-TCTTTCTTCGGCAAATTCCTG-3 0) of the

mouse 700 bp fragment of the LHR cDNA. The

sense primer for GATA-4 corresponded to sense

nucleotides 1527–1550 (5 0-AAACGGAAGCCCAA-

GAACCTGAAT-3 0) and the antisense primer to

1935–1953 (5 0-GGCCCCCACGTCCCAAGTC-3 0)

(expected size: 427 bp) of mouse GATA-4 cDNA

(Morrisey et al. 1997). As a control for RNA quality, a

395 bp fragment of the L19 ribosomal protein gene was

co-amplified with each sample (sense primer, 5 0-

GAAATCGCCAATGCCAACTC-3 0; antisense pri-

mer, 5 0-TCTTAGACCTGCGAGCCTCA-3 0). Kidney

RNA was used as a negative control. After RT-PCR,

20 ml aliquots of the reaction mixtures were loaded on

1% agarose gel containing ethidium bromide (0.4 mg/

l), to identify the amplified DNA fragments. Southern

hybridization was used, according to standard tech-

niques, to confirm the specificity of these PCR

products. Hybridization was carried out with the 5 0-

end labeled oligonucleotide 5 0-TGGAGAAGATGCA-

CAGTGGA-3 0, corresponding to nucleotides 641–660

of LHR cDNA and for GATA-4 with the 5 0-

AGTGGCACGTAGACGGGCGAGGAC-3 0, corre-

sponding to nucleotides 1676–1699. The membranes

were washed according to the manufacturer’s instruc-

tions and then exposed to Kodak X-ray film (XAR 5;

Eastman Kodak).

Immunohistochemistry (IHC)

Paraformaldehyde-fixed paraffin sections (5 mm) of

mouse adrenal tumors were deparaffinized and rehy-

drated. For immunostaining, 3% H202 in water was

used to block endogenous peroxidases and all sections

were boiled by microwave treatment for 15 min in

10 mM citric acid (pH 6.0) for antigen retrieval. Two

washes were done after each step with 0.05 M Tris and

150 mM NaCl (TBS) with 0.1% Tween (TBS-T).

Serial sections of adrenal gland and testes as a positive

control were subjected to immunohistochemistry with

an anti-LHR antibody (dilution 1:1000, a rabbit

polyclonal antiserum directed against human peptide

sequence, KKLPSRETFVNLLEA; a gift of Dr A


Fazleabbas) and/or commercial rabbit polyclonal

anti-GATA-4 IgG (dilution 1:800, Santa Cruz Bio-

technology). IHC for anti-steroidogenic factor (SF)-1

(goat polyclonal antibody, 1:400, Santa Cruz Bio-

technology) was also carried out. The slides were

incubated with primary antibody at 4 8C overnight, and

after incubation and washings with TBS–Tween buffer

the slides were incubated with the anti-rabbit (Vector

Laboratories, Inc., Burlingame, CA, USA) or anti-goat

(Santa Cruz Biotechnology) secondary antibody. The

avidin–biotin immunoperoxidase system was used to

visualize bound antibody (Vectastain Elite ABC Kit,

Vector Laboratories) with 3.3 0-diaminobenzidine

(Sigma) as a substrate. As a control for antibodies,

adjacent sections were incubated with either 1%

normal goat serum in PBS or rabbit IgG as a primary

antibody to differentiate unspecific staining from

specific staining.

Statistical analysis

Statistical analyses were carried out by ANOVA and/or

the non-parametrical Mann–Whitney Rank test using

SAS Enterprise Quide 3.0 program (SAS Institute Inc.,

Cary, NC, USA). Non-parametrical tests were used

when the variances were not equal. ANOVA was used

in other cases. P values !0.05 were regarded as

statistically significant. All the values are presented as

meanGS.E.M.

Results

Hecate-CGb conjugate treatment reduced

adrenal tumor volume

Hecate-CGb conjugate had an antineoplastic effect on

the adrenal tumors of inha/TagTGmice.Wehave earlier

characterized the ontogeny of adrenocortical tumorigen-

esis in these mice and observed tumors weighing 20- to

30-fold more than normal adrenals (290G69 mg) at

6–6.5 months of age following pre-pubertal gonadect-

omy (Rahman et al. 2004). Following a 1-month

treatment with Hecate-CGb conjugate, adrenal tumor

weight was reduced by an average of 59% in males

compared with Hecate treatment (364G147 vs 150G130 mg;Hecate versusHecate-CGb conjugate;P!0.01;

Fig. 1A). In females, the differencewas only 18% (292G156 vs 240G225 mg; Hecate versus Hecate-CGbconjugate; PO0.05; Fig. 1B). As the age-related

individual tumor progression rate was variable between

inha/Tag TGmice and has been observed already earlier

in inha/Tag andTagmice (Hanahan 1989,Kananen et al.

1995, 1996b, Rahman et al. 1998), we also analyzed the

639


Figure 1 Total adrenal weights of (A) male (nZ10) and(B) female (nZ10) inha/Tag transgenic (TG) and wild-type(WT) (WT male, nZ7 and female, nZ8) mice after 3 weeks ofHecate-CGb conjugate or Hecate treatment. Tumor burden,tumor weight/body weight in grams. The values aremeanGS.E.M. *P!0.01; Hecate-CGb conjugate versus Hecate.


tumor burden (tumor weight/body weight). This para-

meter also decreased after Hecate-CGb conjugate

treatment significantly in males in comparison with

Hecate treatment. In females, the treatment effect

remained non-significant (Fig. 1A and B). We did not

observe any statistically significant differences between

the total body weights of the mice between the treatment

groups (data not shown). No statistically significant

640

differences in tumor weights/burden between the sexes

could be observed in the present study, which was in line

with the earlier observations (Rahman et al. 2004). The

adrenal tumors metastasized in 10% (nZ10 for both

male and/or female inha/Tag mice) cases in the Hecate-

treated group to liver (proven by histopathological

analysis, data not shown), but never in Hecate-CGbconjugate-treated groups. According to earlier publi-

cations and observations, these tumorsmetastasized only

occasionally (around 10% cases) to liver, more

commonly in females thanmales (Kananen et al. 1996a).

LHR expression is higher in the adrenal tumors of

male mice

To find out why Hecate-CGb conjugate treatment was

less effective in female adrenal tumors, we quantified

LHR and GATA-4 mRNA expressions by northern

hybridization in male and female non-treated tumors

(Fig. 2). GATA-4 was included in the study due to its

close association with LHR expression and adreno-

cortical tumorigenesis (Kiiveri et al. 1999, Rahman et al.

2004).We alsomeasuredLHRandGATA-4 expressions

in the established Ca1 cell line derived from inha/Tagadrenocortical tumor (Kananen et al. 1996a, Rilianawati

et al. 1998) and from non-treated adrenocortical tumors.

A significantly higher level of LHR mRNA expression

was found in the male than in the female in adrenal non-

treated tumors (P!0.01; Fig. 2). No significant

differences inGATA-4mRNAlevels between the female

and male adrenocortical tumors could be observed.

Endocrine consequences of the treatments

As sham-treated group (castrated TG mice treated with

vehicle) shows identical hormonal values with the

Hecate-treated group, which has been demonstrated

earlier for testicular and ovarian tumors (Bodek et al.

2005b), ovarian xenograft model (Gawronska et al.

2002), and also rat mammary gland tumor model

(Zaleska et al. 2004), in this study the sham-treated

group was not taken into account in order to concentrate

on the differences between the Hecate and Hecate-CGbconjugate groups. In the WT groups, gonadectomy-

induced elevated levels of LH were clear due to the lack

of negative feedback from the gonads. After 1 month of

treatment with Hecate-CGb conjugate, no statistically

significant differences could be seen in Hecate and

Hecate-CGb conjugate treatment groups in the LH,

corticosterone, or progesterone levels in either sex

(Fig. 3), although progesterone levels were twofold

lower, but not statistically significant in Hecate-CGbconjugate-treated males in comparison with Hecate



Figure 2 Northern hybridization analysis of (A) LHR mRNA and(B) western blot analysis of the protein expression in inha/TagTG adrenal tumors of male and female mice and from tumor-derived Ca1 cells. Here, in three independent experiments,each time two different tumors for both sexes were used (nZ6for both males and females). RNA was extracted from one-halfof the tumor and protein from the other half of the tumor.(A) Each lane contains 20 mg total RNA. The migration of the28S and 18S rRNAs is shown on the left side of the LHR panel.The sizes of the different LHR mRNA splice variants (in kb) arepresented on the right. Two lanes for each type of sample aredepicted. The upper panel shows, on the top, the ethidiumbromide (EtBr) staining of the 28S rRNA for RNA loadingcontrol. The middle and lower panels show northern hybrid-ization for GATA-4 and LHR mRNA respectively. The bottompanel shows densitometric quantification of the longest (7.0 kb)LHR mRNA splice variant (filled bars) and GATA-4 (open bars)in arbitrary densitometric units (the mean of Ca1 tumor cells forGATA-4 or LHR was designated as 100%) corrected forintensity of the 28S rRNA band. Each bar represents themeanGS.E.M. of three independent experiments, each time twodifferent tumors for both sexes and from two different cellextracts. Densitometric quantification of the six different bandsis represented by one bar on the densitometric analysis. Eachband shows a different tumor/mouse. **P!0.01, *P!0.05(P!0.01 and P!0.05; male versus female for LHR, maleand/or female versus Ca1 cells for LHR). Ca1, adrenaltumor cell line derived from a transgenic mice expressing(inha)/Simian Virus 40 T-antigen (Tag) adrenal tumor; tu, tumor.



group (in females nodifferences inhormonal values seen,

data not shown), due to the high individual variations

between the serum progesterone values (Fig. 3). Signi-

ficant change in LH levels was seen between gonadecto-

mized WT mice and the TG mice for both Hecate and

Hecate-CGb conjugate-treated groups (P!0.05; Fig. 3).

Serum progesterone levels were three- to four-fold

higher in the TG mice than in WT mice, which might

explain the lower level of LH in adrenal tumor-bearing

TG mice (P!0.05; Fig. 3). This finding is in agreement

with the earlier findings in adrenal tumors and adrenal

tumor-derived Ca1 cells (Kananen et al. 1996a)

indicating their steroidogenic activity, e.g., progesterone

secretion.

Hecate-CGb conjugate selectively destroyed the

LHR possessing adrenal tumor cells

To assess whether the Hecate-CGb conjugate treatment

can eradicate specifically the LHR-expressing adreno-

cortical tumor cells, wemeasured the LHR and GATA-4

protein expression levels in Hecate-CGb conjugate-

treated adrenals in comparison with the Hecate-treated

and non-treated adrenocortical tumor LHR protein

levels. Western blotting analysis revealed that there

was a concomitant significant decrease in LHR and

GATA-4 in the Hecate-CGb conjugate-treated adrenal

tumors (P!0.01 Hecate-CGb conjugate versus non-

treated or Hecate-treated adrenal tumors; Fig. 4),

indicating selective destruction of LHR and GATA-4

positive adrenocortical tumor cells. Higher GATA-4

expression compared with LHR after Hecate-CGbconjugate might reflect the previously described

phenomenon that these adrenocortical cells might

express GATA-4 without LHR, but not vice versa and

their expression patternwas higher/broader (Kiiveri et al.

1999, 2002). The LHR or GATA-4 protein expression in

WT control mouse adrenals could not be detected.

Histopathological analysis demonstrating anti-

tumoral effects of Hecate-CGb conjugate

treatment

Histopathological analysis revealed that Hecate-CGbconjugate treatment of TG adrenocortical tumor mice

(B) The upper panel shows GATA-4, middle panel LHR, andlower panel b-actin protein expressions in western blottinganalysis. Each lane represents three independent experiments,each time three different tumors for both sexes and fromthree different cell extracts. Each band shows a differenttumor/mouse. Ca1, adrenal tumor cell line derived from atransgenic mice expressing (inha)/Simian Virus 40 T-antigen(Tag) adrenal tumor; tu, tumor; on the right side, the molecularweights of the proteins in kilodaltons are indicated.

641


Figure 4 Western blot analysis for LHR and GATA-4. Proteinextracts from male inha/Tag TG mice treated with Hecate orHecate-CGb conjugate or from non-treated tumor sampleswere analyzed by western blotting. The upper panel showsLHR, GATA-4, and b-actin expressions and the lower paneltheir densitometric analysis. The representative band datashown are from one of the four different adrenal tumors/mouseper group/independent experiments (nZ4). Each bar indensitometric quantification shows the quantification of fourseparate independent tumor samples/experiments. Each barrepresents the meanGS.E.M. of four independent experiments,each time different tumor sample. Hecate-CGb conjugatedownregulated significantly both LHR and GATA-4 expressionscompared with non-treated control adrenal tumor or Hecate-treated tumor (**P!0.001). No detectable LHR or GATA-4 wasdetected in WT C57Bl mouse adrenals. On the right side, themolecular weights of the proteins in kilodaltons are indicated.

Figure 3 Serum (A) LH, (B) progesterone, and (C) corticoster-one concentrations in transgenic inha/Tag male mice aftera 3-week treatment with Hecate-CGb conjugate or Hecate.WT, wild-type or TG, inha/Tag mice treated with Hecate-CGbconjugate or Hecate. The values are meanGS.E.M.; nZ5,*P!0.05; Hecate-CGb conjugate-treated TG mice versusHecate or Hecate-CGb conjugate-treated WT mice.


induced a definite reduction of the adrenocortical tumor

mass where residual tumor tissue could be observed in a

reduced area of the zona glomerulosa in hematoxylin–

eosin-stained sections (Fig. 5B). This finding in the

representative sample was reproducible as the same

phenomenon was observed in all stained samples of the

same treatment groups (nZ5 from each group). The

adrenal tumors metastasized in 10% (nZ10 for both

males and/or female inha/Tag mice) cases in the

Hecate-treated group to liver (proven by histopatholo-

gical analysis, data not shown), but never in Hecate-

CGb conjugate-treated groups. According to earlier

publications and observations, these tumors metasta-

sized only occasionally (around 10% cases) to liver,

more commonly in females than males (Kananen

et al. 1996a).

642

LHR and GATA-4 expressions are colocalized in

the tumors

We next demonstrated that LHR and GATA-4 are

co-localized in the same adrenocortical tumor region.

The data obtained by LPC of the tumorous and non-

tumorous tissues from the same slice demonstrated the

co-localization of GATA-4 and LHR mRNA messages

in adrenal tumor tissue and their absence in normal

adrenal cortex (Fig. 6). The finding in the representa-

tive sample was reproducible as the same phenomenon

was observed in all other three samples.

We then investigated whether the expression

patterns of these proteins were affected by the Hecate

or Hecate-CGb conjugate treatment. GATA-4

expression was found to directly localize to the



Figure 5 Hematoxylin–eosin staining of adrenal tumors aftertreatments with (A and C) Hecate or (B and D) Hecate-CGbconjugate. (A and B, 20! magnification, C and D, 10!magnification). The picture from (C) Hecate-treated tissue istaken from the contralateral side of the big tumor to show theexpression of the tumorous tissue in the whole adrenal gland.

Figure 6 RT-PCR/Southern hybridization of the PALM micro-dissected samples. (A and B) Hematoxylin-stained specimensof cryosections of the tumorous adrenal tissue. (A) The tumor


H–E-stained tumor area that was in line with our

previous findings (Rahman et al. 2004; Fig. 7A and C).

In Hecate-CGb conjugate-treated sections, no LHR

staining could be detected even in the same H–E-

stained, marginalized, and reduced tumorous area

(Fig. 7A and D). We also found SF-1 expression in

the same tumor areas where GATA-4 expression could

be observed (Fig. 7B and C).
sections (dark-stained cells) and (B) non-tumor tissues (light-stained area in the middle) were dissected out and catapulted.(C) The catapulted tumor (WT, wild-type; adr tum, adrenaltumor) and (D) normal non-tumorous tissues in the cap of anEppendorf tube (magnification 63!; adr non-tu, non-tumorusadrenal tissue).
Discussion

The lack of a suitable animal model for adrenocortical

tumorigenesis has been a major obstacle for unraveling

the molecular mechanisms involved in their patho-

genesis, as well as for improving their diagnostic and

therapeutic strategies. Thus, establishment of suitable

animal models is of extremely high importance in order

to study human adrenal tumorigenesis. Chronically

elevated LH, ectopic/upregulated LHR expression,

hyperplasia–adenoma–adenocarcinoma sequence tumor

formation, slow tumor growth, late discernible tumor

incidence (analogous to human postmenopausal/andro-

pausal age), low metastases frequency (5–10%): these

characteristics make the inha/Tag TG murine model a

good tumor relevance model for human ACCs studies.

Our inha/Tag TG mouse is at the moment one of the

rare and unique murine models in which adrenocortical

tumorigenesis and its molecular mechanisms or any

treatment strategies can be investigated. Although the

physiologic significances of extragonadal LHR

expression in reproductive function is questioned

(Pakarainen et al. 2005, 2007), some recent studies


(Rao Ch et al. 2004, Alevizaki et al. 2006, Vuorenoja

et al. 2007) suggest that the adrenal cortex expresses low

levels of LHR (Pabon et al. 1996) whose function

becomes significantwhenLH levels are elevated, as after

gonadectomyorpostmenopausally. In light of the present

study, we could postulate that the upregulated LHR

expression in adrenal tumors is functionally relevant and

can be used to target the tumors by the cytotoxic Hecate-

CGb conjugate. This model serves as an example

demonstrating how ectopically expressed receptors in

tumors can be used as therapeutic target.

We used in the present study an established inha/TagTG mouse model for post-gonadectomy adrenocortical

tumorigenesis, where the pathophysiological and

endocrine responses induced by tumorigenesis are

well characterized. We found that Hecate-CGb con-

jugate treatment reduced the total adrenocortical tumor

weights without detectable side effects, e.g., adrenal

643


Figure 7 Hematoxylin–eosin staining (A) and immunohisto-chemistry for (B) SF-1, (C) GATA-4 and (D) LHR in Hecate-CGbconjugate-treated adrenal tumor sections.


insufficiency. Curiously, the treatment was, for

unknown reason, ineffective in female with adrenocor-

tical tumors. The adrenal response to elevated LH in

inha/Tag mice was probably more sensitive in females

andwe believe it was alsomore aggressive, which could

explainwhy immortalization of Ca1 cells from a female

inha/Tag founder mouse tumor was possible (Kananen

et al. 1996a, Rilianawati et al. 1998). It has been shown

that the castrated male mice developed adrenocortical

tumors more slowly than gonadectomized females

along with the ectopic LHR expression (Bielinska

et al. 2003). Moreover, the elevated LH levels induced

abundant ectopic LHR expression and acted as a tumor

promoter selectively only in the intact female double-

positive TG mice, where bLHb-CTP mice producing

constitutively elevated levels of LH (Risma et al. 1995)

were crossbred with inha/Tag TG mice (Mikola et al.

2003). This never happened in the male double-positive

mice (Mikola et al. 2003). In humans, ACCs are also

more common in postmenopausal women than in aging

males (Schulick & Brennan 1999a,b). Therefore, it

could be an established phenomenon that female

adrenals respond more readily to high LH and

tumorigenesis might be more aggressive in them,

which might imply that the suppression of LH action

needed to block tumorigenesis in females should be

more complete/stronger, whereas male tumorigenesis

can be blocked with less-effective suppression. It is

likely that this mechanism occurred in our Hecate-CGbconjugate-treated inha/Tag mice where, with the

applied dose of the drug, we achieved much a better

response in the males than in the females. Probably, this

phenomenon was additionally helped by the higher

concentration of LHR in the tumor mass in the male

644

tumors attracting larger amounts of Hecate-CGbconjugate, exerting consequently a more severe lytic

effect. The reason for the higher LHR expression in

male adrenal tumors remains unknown. The putative

difference in tumor cell membrane charges between the

male and females could also be one of the potential

mechanistic reasons behind this differential treatment

effects, which needs to be further evaluated in future

studies.

As we did not find significant changes in the serum

endocrine profiles of the Hecate- or Hecate-CGbconjugate-treated mice, the treatment outcome could

not be monitored by serum hormone measurements.

The progesterone levels are decreased in Hecate-CGbconjugatemale in comparisonwithHecate group but the

high individual variations made them non-significant.

LH levels were higher in gonadectomized WT than in

TGmales, which indicated increased endocrine activity

of the tumors. The hormone with increased production

by the tumors exerting a negative feedback action of LH

secretion is probably progesterone. However, the

adrenal tumors can also produce sex steroids (androgens

or estrogens; Leinonen et al. 1991, Kananen et al.

1996a, Lacroix et al. 1999, Bielinska et al. 2005). No

effect of the treatment could be seen on corticosterone

levels between Hecate and Hecate-CGb conjugate

treatment groups, which indicates that the Hecate-

CGb conjugate treatment does not destroy normal

adrenal tissue or induce adrenocortical hypofunction.

This additionally supports the principle that this peptide

attacks only the negatively charged tumor cells and

spares the healthy ones.

We have shown earlier a precise co-initiation of

GATA-4 along with the LHR expression during

adrenocortical tumorigenesis ontogeny (Rahman et al.

2004). GATA-4 has been proposed to be a potential

marker for adrenocortical tumorigenesis (Kiiveri et al.

1999), along with LHR (Rahman et al. 2004). Further

proof is needed to determine whether GATA-4works as

a specific co-activator for LHR (or even vice versa) in

adrenocortical tumorigenesis. Thus, we wanted to see

whether GATA-4 expression would also diminish due

to the Hecate-CGb conjugate treatment, which could

lead us to further mechanistic studies in the future. As

there are some difficulties with the availability of good

LHR antibodies, GATA-4 immunohistochemistry

could also additionally serve as a marker for treatment

efficacy. We found that both LHR and GATA-4 protein

expressions decreased significantly after Hecate-

CGb conjugate treatment compared with Hecate. By

immunohistochemistry, we could detect GATA-4 in

residual tumor area indicating that some tumor

steroidogenic cells are still left, as GATA-4 is expressed




only in tumor adrenocortical steroidogenic cells

(Kiiveri et al. 1999, 2002, Peterson et al. 2004), but

LHR staining was no longer detectable in the Hecate-

CGb conjugate-treated samples. These results indicated

that Hecate-CGb conjugate killed specifically the tumor

cells expressing LHR. This finding is well in line with

the adrenal tumor weight results. Even after the Hecate-

CGb conjugate treatment, the adrenal has not returned

to totally normal size. However, GATA-4 and SF-1

were colocalized in the same tumor area. SF-1 is known

to be another factor activating adrenocortical steroido-

genesis and it is shown to transactivate both GATA-4

and LHR activities (Tremblay & Viger 1999). The

expression pattern of SF-1 may implicate its role as a

co-activator of GATA-4 and LHR in adrenocortical

tumorigenesis of the inha/Tag TG mice.

Human adrenal LH/hCG responsive tumors have been

well described (Leinonen et al. 1991, Lacroix et al.

1999). Higher basal levels of corticosteroids in post-

menopausal women are also associated with higher

circulating LH and LHR expressions in adrenals (Pabon

et al. 1996, Alevizaki et al. 2006). A recent publication

confers that ectopic LHR may induce adrenal hyper-

plasia- and Cushing’s syndrome-like symptoms as well

as hCG-mediated steroidogenic activity (Mazzuco et al.

2006). Ectopic expression of LHR in the adrenal gland

has been shown to be associated with not only

adrenocorticotropin-independent Cushing’s syndrome

but also benign aldosterone-producing adenomas

(Saner-Amigh et al. 2006). There are also reports about

malignant androgen-producing LH-dependent adrenal

tumors (de Lange et al. 1980, Leinonen et al. 1991).

Hence, LH/hCG-dependent mechanisms of adrenal

pathogenesis do exist and they may be clinically

important in circumstances with elevated LH levels,

e.g., in connectionwith polycystic ovarian syndrome and

after menopause in women.

Taken together, the present data provide novel

evidence that targeted destruction of adrenocortical

tumors expressing ectopically LHR by the Hecate-CGbconjugate is possible. In addition to the currently usedTG

mouse model, our findings prove the more general

principle that receptors expressed ectopically in malig-

nant cells can be exploited in targeted cytotoxic therapies

without affecting the normal healthy cells.

Acknowledgements

Thisworkwas supported by grants fromFinnishCultural

Foundation at Varsinais-Suomi, the Finnish Cancer

Society, the Moikoinen Cancer Research Foundation,

the Academy of Finland, and Turku University Hospital.

We thank Dr A Fazleabbas for the kind donation of the


LHR antibody and Dr Sonia Bourguiba for fruitful

discussions. The authors also thank Ms Johanna

Lahtinen, Ms Tarja Laiho and Ms Heli Niittymaki for

their technical assistance and the personnel of Turku

University Animal Facility for taking good care of the

mice. This project was supported by grants from the

Finnish Cultural Foundation at Varsinais-Suomi (S V),

the Moikoinen Cancer Research Foundation (N R), the

Academy of Finland (I H, J T, N R), and the Turku

University Hospital (J T). We declare that there is no

conflict of interest that would prejudice thismanuscript’s

impartiality.

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