N. Yamamoto1 J. Imai2
M. Watanabe2
N. Hiroi1 S. Sugano2
G. Yoshino1
Restoration of Transforming Growth Factor-B Type II Receptor Reduces Tumorigenicity in the Human Adrenocortical Carcinoma SW-13 Cell Line
Abstract
Transforming growth factor-ß (TGF-ß) is a potent growth sup- pressor. Acquisition of TGF-ß resistance has been reported in many tumors, and has been associated with reduced TGF-ß re- ceptor expression. In this study, we examined TGF-ß 1, TGF-B type I receptor (TBRI) and TGF-ß type II receptor (TBRII) expres- sion in SW-13 adrenocortical carcinoma cells by Northern and Western blot analysis. SW-13 cells did not express TØRII mRNA or protein. We have investigated the role of TØRII in modulating tumorigenic potential using stably transfected SW-13 cells with TØRII expression plasmid. TØRII-positive SW-13 cell growth was inhibited by exogenous human TGF-ß1 (hTGF-ß1) in a dose-de-
pendent manner. In contrast, SW-13 cells and control clones transfected with empty vector remained hTGF-ß1-insensitive. Xenograft examination in athymic nude mice demonstrated that TØRII-positive SW-13 cells reduced tumor-forming activity. Reconstructing the TBRII can lead to reversion of the malignant phenotype of TBRII-negative human adrenocortical carcinoma, which contains SW-13 cells. Reduced TØRII expression may play a critical role in determining the malignant phenotype of human adrenocortical carcinoma.
Key words
Transforming growth factor-ß (TGF-B) · TGF-ß insensitivity · TGF- B type II receptor . adrenocortical carcinoma . cell proliferation
Introduction
Transforming growth factor-ß1 (TGF-ß1), a 25 kDa homodimeric polypeptide, is a potent growth suppressor for normal and tumor cells [1-4]. Signal transduction by TGF-ß1 involves direct bind- ing to the TGF-B type II receptor (TØRII) as well as formation of a heterodimeric cell-surface complex between the ligand-bound TØRII and TGF-B type I receptor (TBRI) on the cell surface [5]. The TGF-B1 molecule binding to TBRII causes serine-threonine kinase phosphorylation, which leads to TØRI activation and phos- phorylation of intracellular SMAD protein serine residues [6-8]. Activated SMAD complexes are translocated into the nucleus and promote transcriptional activation. SMADs induce CDK inhibi-
tors that lead to TGF-ß-dependent cell growth arrest [9,10]. On the other hand, SMADs are also associated with TGF-ß-mediated apoptosis [3].
Human adrenocortical carcinomas are among the most lethal en- docrine neoplasms. They are highly aggressive and frequently metastasize; prognosis is very poor. We need to improve our un- derstanding of the mechanism of adrenocortical tumorigenesis since effective therapies have not emerged in recent decades [11]. So far, some candidate genes in human adrenocortical carci- nomas have been reported, such as point mutation of TP53 [12] and overexpression of IGF2 [13]. However, further studies would be needed to reveal the molecular and genetic basis of adreno-
Affiliation
1 Division of Diabetes, Metabolism and Endocrinology, Department of Medicine, Toho University School of Medicine
2 Laboratory of Genome Structure Analysis, Human Genome Center, Institute of Medical Science, The University of Tokyo, Tokyo, Japan
Correspondence
Natsuko Yamamoto, M. D., Ph. D. · Division of Diabetes, Metabolism and Endocrinology · Department of Medicine . Toho University School of Medicine . 6-11-1 Omorinishi . Ota-ku . Tokyo . 143-8541 Japan .
Phone: +81 (3) 3762-4151/6565 · Fax: +81 (3) 3765-6488 . E-Mail: n-yamamoto@med.toho-u.ac.jp
Bibliography
Horm Metab Res 2006; 38: 159-166 @ Georg Thieme Verlag KG Stuttgart . New York . DOI 10.1055/s-2006-925185 . ISSN 0018-5043
cortical carcinomas. The effect of TGF-ß signaling has been viewed as an autocrine or paracrine regulator of adrenocortical steroidogenesis [14,15], and as a growth regulator in fetal adre- nal cells [16,17]. Therefore, locally secreted TGF-ß1 may partici- pate in the regulation of various aspects of human adrenocortical physiology and pathology. Various studies have implicated ab- normalities in the TGF-ß signaling pathway in tumorigenesis. Especially disruptions in the signaling pathway caused by a re- duction or defect in TØRII may contribute to tumor progression, invasion, and metastasis [2-4]. Loss of TØRII expression has been reported in several tumors affecting organs including the colon [18], stomach [19,20], pancreas [21], liver [21,22], and breast [21,23]. We also previously demonstrated TØRII mRNA downregulation in human papillary thyroid carcinoma tissues [24].
In the present study, we used SW-13 cells as an appropriate adre- nocortical carcinoma model. We found that the TBRII mRNA and protein expression are decreased in SW-13 cells. We have also demonstrated the tumor-suppressing role of TØRII through TBRII expression in SW-13 cells.
Materials and Methods
Cell culture
Human adrenocortical carcinoma SW-13 cells (American Type Culture Collection; ATCC, Manassas, VA, USA) were cultured in Leibovitz’s L-15 medium (Invitrogen, Rockville, MD, USA) sup- plemented with 10% fetal bovine serum (FBS) and 60 µg/ml ka- namycin. The human hepatocellular carcinoma HepG2 cell line (ATCC) was used as a positive control for TGF-ß1, TBRI and TBRII mRNA expression. HepG2 cells were cultured in Dulbecco’s mod- ified Eagle’s medium (Invitrogen) supplemented with 10% FBS, 2 mM L-glutamine, 0.15% sodium bicarbonate, and 60 µg/ml ka- namycin. Cells were maintained at 37 ℃ in a humidified atmo- sphere with 5% CO2.
Cell growth study
SW-13 and HepG2 cells were plated at 1.5 x 104 cells/well in 24- well plates. After 24 h, various concentrations (0, 10-3, 10-2, 10-1, 1, 10 ng/ml) of recombinant human TGF-ß1 (hTGF-ß1) (R&D Sys- tems, Minneapolis, MN, USA) were added to the culture medium. After four days of incubation, the culture medium was changed and various concentrations of hTGF-B1 were added again. Cell numbers in triplicate wells were determined using a hemocyt- ometer after trypsinization after seven days of incubation.
Northern blot analysis
Total RNA was isolated from culture cells using TRIzol reagent (Invitrogen). 10 µg total RNA was separated by 1.0% formalde- hyde agarose gel electrophoresis in MOPS buffer and transferred to Hybond-N membranes (Amersham Pharmacia Biotech, Buck- inghamshire, UK), and cross-linked. The probes for human TGF- B1, TØRI and TØRII were obtained by polymerase chain reaction (PCR) using gene-specific primers (Table 1). The probes were la- beled with [o .- 32P] dCTP (Amersham Pharmacia Biotech) using a random primer DNA labeling kit (Takara, Tokyo, Japan). After 1 h of prehybridization, hybridization was performed with a @-32P- labeled probe at 42℃ for 18 h. The membranes were washed in
| Primer set | Sequence(5-3') | Product size (bp) | Accession No. |
|---|---|---|---|
| TGF-ß1-F | GGA GGG GAA ATT GAG GGC TT | 530 | NM 000660 |
| TGF-ß1-R | CAG GAG CGC ACG ATC ATG TT | ||
| TØRI-F | CGA GGC GAG GTT TGC TGG GGT GAG GC | 1653 | NM 004612 |
| TØRI-R | GAC CTC CCA AAT TAA AAC CCA GGA GC | ||
| TØRII-F | GTC CGC TGG GGG CTC GGT CTA TG | 1743 | NM 003 242 |
| TØRII-R | TTT GGT AGT GTT TAG GGA GCC G | ||
| GAPDH-F | TGA AGG TCG GAG TCA ACG GAT TTG | 982 | NM 002 046 |
| GAPDH-R | CAT GTG GGC CAT GAG GTC CAC CAC |
2XSSC/0.1%SDS (w/v) once at room temperature and in 1XSSC/ 0.1%SDS (w/v) twice at 65℃ for 20 min. The membranes were exposed to an imaging plate, and signals on the plate were visu- alized using a BAS2500 bio-imaging analyzer (FUJI FILM, Tokyo, Japan). The probe for glyceraldehyde-3-phosphate dehydrogen- ase (GAPDH) was also hybridized to the same membranes as the internal control.
Western blot analysis
Cells were grown to near confluence and washed three times in ice-cold phosphate-buffered saline, and lysed in TNE buffer (20 mM Tris-HCI, 1% Tween 20, 250 mM NaCl, 5 mM EDTA, and 5 µg/ml aprotinin, 1 µg/ml leupeptin, 1 µg/ml Pepstatin A). Pro- tein concentration in whole-cell lysate was measured using a BCA Protein Assay Kit (PIERCE, Rockford, IL, USA). 20 µg of lysate protein were separated by 10% SDS-PAGE and transferred to a Hybond-P membrane (Amersham Pharmacia Biotech) using the TRANS-BLOT SD CELL semi-dry blotting system (BIO-RAD, Her- cules, CA, USA). The membrane was incubated in rabbit polyclo- nal antibody against the C-terminal peptide of human TØRII (Santa Cruz Biotechnology, Santa Cruz, CA, USA) in rabbit mono- clonal antibody against the residues surrounding the cleavage site of human caspase-3 (Cell Signaling Technology, MA, USA), or in mouse monoclonal antibody against PCNA (BD-Transduc- tion, Lexington, KY, USA) for 2 h at room temperature under gen- tle agitation. The membranes were washed in Tris-buffered sa- line (TBS) with Tween 20 (TBST) three times for 5 min and incu- bated in 1: 1000 diluted alkaline phosphatase-conjugated don- key anti-rabbit IgG (H+L) or goat anti-mouse IgG (H+L) (Promega, Madison, WI, USA) for 1 h. The membrane was washed in TBST and TBS, and incubated in stabilized substrate for alkaline phos- phatase. Western Blue (Promega). Tubulin (Neomarker, CA, USA) was used as an internal control.
TØRII expression plasmid, pCE-TBRII
To construct an expression plasmid for TØRII, the entire open reading frame for human TØRII cDNA (accession number: NM 003 242) was amplified by PCR using the primers (TBRII-F: 5’- GCG CGG ATC CTG CCA TGG GTC GGG GGC TGC TCA G-3’ and TØRII-R: 5’-GCG CGC GGC CGC GCC CAG CCT GCC CCA TAA GAG CTA-3’) and a cDNA template obtained from HepG2. The PCR product was digested with BamHI and NotI. pCE-GFP, an expres- sion plasmid with a CMV-IE enhancer and a neomycin-resistance
gene [25] was also digested with BamHI and Notl, and entire cod- ing region of GFP was removed. After that, the fragment of TØRII was cloned into BamH I and Not I sites of pCE-GFP, and this plas- mid designated as pCE-TBRII. The sequence of the subcloned TØRII fragment was determined by restriction enzyme analysis, and DNA sequencing was performed using an ABI PRISM 377 DNA sequencer (Applied Biosystems, Foster City, CA, USA).
Generation of a cell line with stable TØRII expression; cell growth analysis
The TØRII expression plasmid, pCE-TBRII (4 µg), was transfected into SW-13 cells plated in 10 cm dishes using 20 ul of PLUS Re- agent and 30 ul of LIPOFECTAMINE Reagent (Invitrogen) follow- ing the recommended protocol. After 48 h, transfectants were se- lected with 800 µg/ml geneticin. After 6 weeks, antibiotic-resis- tant clones were ring-cloned and expanded to screen for TØRII expression by Northern and Western blot analysis. Clones ex- pressing high levels of TØRII were defined as SW-13-TØRII cells. As a transfection control, 4 µg of pCE-GFP plasmid containing the neomycin-resistant gene was also transfected alone into SW-13 cells and ring-cloned. These cells were designated as SW-13-Neo. SW-13, SW-13-Neo and SW-13-TBRII cells were plated in 6-well plates at a density of 5 × 104 cells/well. After 24h, various concentrations (0, 10, 20, 100 ng/ml) of TGF-ß1 were added to the culture medium. After six days of incubation, the culture medium was changed and various concentrations of hTGF-B1 were added again. Cell numbers of triplicate wells were counted after ten days of incubation.
Tumor formation in athymic mice
SW-13, SW-13-Neo and SW-13-TØRII cells were harvested with trypsin and resuspended in 200 ul of cell suspension containing 1.0 × 107 cells, then injected subcutaneously into the back of 6- week-old male KSN nude SLC strain athymic mice (Nippon SLC, Hamamatsu, Japan). The size of the tumors was measured using calipers, and the volume (V) was determined from the equation: V (mm3)=(Lx W2)×0.5, where Lis length (mm) and W is width (mm) of the tumor. After forty-seven days of inoculation, the mice underwent euthanasia, the masses were removed, and their weights were measured. Transplanted mass specimens were fixed in 10% neutral buffered formalin, embedded in O.C.T. com- pound (SAKURA Finetechnical Co. Ltd., Tokyo, Japan), and cut at 5 um using CM1900 freezing microtome (Leica, Germany). The sections were re-fixed with 10% neutral buffered formalin, and stained with hematoxylin and eosin.
Immunohistochemical analysis
Immunohistochemistry was performed using the DAKO ENVI- SION+ kit/HRP (DakoCytomation CA, USA) following the manu- facturer’s recommendations. Briefly, sections were pretreated in an autoclave for 5 min at 121 ℃; endogenous peroxidase activity was abolished by peroxidase block (DakoCytomation). Tissue samples reacted with the primary antibody against PCNA (BD- Transduction, Lexington, KY, USA) at room temperature for 60 min, and incubated with labeled polymer-HRP anti-mouse (DakoCytomation) at room temperature for 30 min. Sections were visualized with DAB/hydrogen peroxide solution (DakoCy- tomation).
Statistic analysis
Results were analyzed by one-way analysis of variance (ANOVA) followed by Fisher’s least significant difference. Differences were considered significant at values of p < 0.05.
Results
No expression of TØRII in SW-13 human adrenocortical cells
TGF-B1, TØRI and TØRII mRNA expression in the adrenocortical carcinoma SW-13 cell line was examined by Northern blot anal- ysis. HepG2 cells, used as positive control for expression of TGF- ß1, TØRI and TØRII mRNA, showed distinct signals, whereas SW- 13 cells only expressed TGF-ß1 and TBRI mRNA but not TBRII mRNA (Fig. 1a [a]). Western blot analysis with anti-TØRII anti- body also confirmed that HepG2 cells expressed the 70-kDa TØRII proteins, while SW-13 cells did not (Fig. 1 b [b]).
Resistance to growth inhibition by hTGF-ß1 in SW-13 cells
To investigate the response of TGF-ß1 treatment, SW-13 and HepG2 cells were cultured in the absence or presence of various concentrations of hTGF-ß1; the effects on cell growth were as- sessed by cell count. The relative cell number of HepG2 cells cul- tured with 10-3, 10-2, 10-1, 1, 10 ng/ml of hTGF-ß1 was 0.97 ±0.03 (p=0.36), 0.88 ±0.05 (p=0.007), 0.34±0.04 (p<0.0001), 0.18±0.02 (p<0.0001), 0.09±0.03 (p<0.0001), respectively. Exogenous hTGF-ß1 inhibited HepG2 cell growth in a dose-de- pendent manner, but not SW-13 cell growth at any concentra- tion. Relative SW-13 cell numbers cultured with 10-3, 10-2, 10-1, 1, 10 ng/ml of hTGF-ß1 were 0.97±0.05 (p=0.66), 0.98±0.04 (p=0.79), 1.01 ±0.09 (p=0.93), 0.99±0.09 (p=0.93), 1.01 ±0.08 (p=0.72), respectively (Fig.1). Actually, 10 ng/ml of exogenous hTGF-ß1 inhibited HepG2 cell growth by approximately 90% (Fig. 1c [a, b]), whereas hTGF-ß1-treated SW-13 cells grew to confluence as untreated cells did (Fig. 1 c [c, d]).
Restoration of sensitivity to hTGF-ß1 in SW-13 cells with TØRII expression
SW-13 cells with stable TØRII expression (SW-13-TØRII) were generated. One clone that expressed high level of TØRII mRNA (Fig. 2a [a]) and protein (Fig. 2b [b]), SW-13-TØRII-B, was select- ed for this examination. We examined the response to hTGF-ß1 treatment using non-transfected SW-13, transfection control SW-13-Neo, and stable TØRII-expressing SW-13-TBRII-B cells. After ten days of incubation, the cell numbers of SW-13, SW-13- Neo, and SW-13-TBRII-B cultured without hTGF-ß1 was 166.5 ±7.6x104 cells/well, 172.0±8.9x104 cells/well (p=0.51 vs. SW-13), 154.8±7.1×104 cells/well (p=0.18 vs. SW-13, p = 0.072 vs. SW-13-Neo), respectively. These cell numbers were used as controls to calculate the relative cell number, and were not significantly different. Relative SW 13 cell numbers cultured with 10, 20, 100 ng/ml of hTGF-ß1 were 0.94±0.02 (p=0.23), 1.02 ±0.02 (p=0.58), 1.01 ± 0.03 (p=0.83), respectively, while re- lative SW-13-Neo cell numbers cultured with 10, 20, 100 ng/ml of hTGF-ß1 were 1.00±0.03 (p=0.23), 1.01 ±0.02 (p=0.58), 0.99 ±0.03 (p=0.83), respectively. As expected, SW-13 and SW- 13-Neo cells were insensitive to hTGF-ß1 (Fig. 2 b). In contrast, proliferation of SW-13-TØRII-B cells was significantly inhibited by hTGF-ß1 in a dose-dependent manner. Relative SW-13-TØRII- B cell numbers cultured with 10, 20, 100 ng/ml of hTGF-ß1 were
HepG2
SW-13
(a)
TGFB-1
TØRI
TØRII
GAPDH
(b)
TBRII
1,2
Relative cell number
1
0,8
SW-13
#
HepG2
0,6
+
0,4
+
0,2
+
0
1
1
I
1
1
1
0
10-3
10-2
10-1
1
10
a
b
hTGFß-1 concentration (ng/ml)
(a)
(b)
(c)
(d)
3
c
HepG2
SW-13
SW-13-Neo
SW-13-TBRII -B
(a) TBRII
GAPDH
(b) TØRII
a
1.2
1
Relative cell number
0.8
SW-13
SW-13-Neo
SW-13-TBRII-B
0.6
+
+
0.4
+
0.2
0
10
20
100
b hTGF-ß1 concentration (ng/ml)
(a)
SW-13 SW-13
(b)
SW-13
-Neo
-TBRII-B
hTGF-ß1
+
+
+
PCNA
(c)
(d)
Caspase-3
C
d
Tubulin
+
0.55±0.04 (p<0.0001), 0.41 +0.02 (p<0.0001), 0.32±0.04 (<0.0001), respectively (Fig. 2 b). Thus, recovery of TØRII expres- sion in SW-13 cells was sufficient to restore growth-inhibitory response to exogenous hTGF-ß1. In cell morphology, we observed morphological changes in SW-13-TØRII-B cells induced by TØRII restoration. The cytoplasm of TGF-ß1 treated SW-13-TBRII-B cells became more extensive and cytoplasmic/nuclear ratio in- creased, indicating that the treated cells had similar morphologic features to epithelial cells (Fig. 2 c). Western blot analysis with PCNA antibody to reveal the inhibitory effect of cell proliferation showed that the PCNA protein expression was reduced in TGF-ß- treated SW-13-TØRII-B cells. Moreover, Western blot analysis with fragmentation of caspase-3 to assess the apoptotic activity detected cleaved caspase-3 in TGF-ß treated SW-13-TBRII-B cells (Fig. 2 d).
Tumor formation in athymic mice
The reduced growth capacity of SW-13-TBRII cells indicates the possibility that TBRII expression restoration may render these cells less tumorigenic. To investigate this hypothesis, we per- formed an examination on tumor formation in athymic nude mice. SW-13-TØRII cells were inoculated into athymic mice at a dose of 1.0 × 107 cells per site, and the mice were monitored for progression of xenograft formation. In tumor growth evaluation, xenograft growth of SW-13-TØRII-B cells was significantly de- creased compared to control cells twenty-five days after inocula- tion. Cumulative tumor volumes of SW-13, SW-13-Neo and SW- 13-TØRII-B cells were 98.4± 17.0 mm3, 122 + 29.2 mm3 (p=0.37, vs. SW-13), 26 ±9.4 mm3 (p=0.02, vs. SW-13) on the 25th day, respectively; 207.2 + 53.3 mm3, 246.0± 81.5 mm3 (p=0.90, vs. SW-13), 44.1 +24.5 mm3 (p=0.04, vs. SW-13) on the 33th day, respectively; 357.8 + 77.6 mm3, 485.6± 99.5 mm3 (p=0.18, vs. SW-13), 77.4±33.0mm3 (p=0.01, vs. SW-13) on the 40th day, respectively; and 577.8 ± 126.3 mm3, 604.7 ± 122.1 mm3 (p=0.86, vs. SW-13), 141.1 ±40.3 mm3 (p=0.02, vs. SW-13) on the 47th day, respectively (Fig. 3a). The weight of the SW-13, SW-13-Neo and SW-13-TBRII-B cell mass was 1.11 ±0.47 g, 1.24±0.58 g(p=0.57, vs. SW-13), 0.28 ±0.21 g (p=0.02, vs. SW- 13, p =0.002, vs. SW-13-Neo). The weight of the SW-13-TBRII-B cell mass was significantly lighter, almost less than 25% of that of SW-13 cells on the 47th day after cell inoculation (Fig. 3b). A representative mouse of either group and a macroscopic view of a dissected tumor are shown in Fig. 4a. These control cells, like SW-13 and SW-13-Neo cells, formed palpable solid tumors in 9 and 8 of the 10 injected mice, respectively (Fig. 4a [a, b]). In con- trast, five mice injected with SW-13-TØRII-B cells developed masses containing blood-rich fluid with the appearance of a he- matoma (Fig. 4a [c]). The mass formation was quite different from these solid tumors observed with SW-13 and SW-13-Neo.
Histologically, the tumor in SW-13 masses was composed of ovoid, spindle-shaped, and polyhedral cells, and the tumor cells had slightly basophilic cytoplasm and round-to-oval nuclei. The tumor cells formed net-like structure, and necrotic foci were ob- served (Fig. 4b [a, d]). We found similar histology in SW-13-Neo masses (Fig. 4b [b, e]). However, SW-13-TØRII-B masses showed cysts formed in various sizes, and the inner wall of the cyst was composed of fibroblast, fibrovascular cells and poorly developed smooth muscle fiber. The cysts contained a lot of erythrocytes and small epithelium-like cells within the cyst (Fig. 4b [c, f]). Im-
munohistochemically, we observed many positive reacted cells with antibody for PCNA in the SW-13 and SW-13-Neo-derived masses (Fig. 4b[g,h]), although we found only a few positive PCNA-reactive cells in SW-13-TØRII-B cell-derived xenografts (Fig. 4 b[i]). Although we examined apoptotic assay using TUNEL methods, we could not observe any distinct apoptosis cells among any of the transplanted samples (data not shown). These results might suggest that TØRII expression induces loss of tu- morigenesis in human SW-13 adrenocortical carcinoma cells.
Discussion
In this study, we have demonstrated that human SW-13 adreno- cortical carcinoma cells do not express either TØRII mRNA or pro- tein. Decreased TØRII led to an increase in resistance to hTGF-ß1- induced cell growth inhibition in SW-13 cells. TØRII expression restored the sensitivity of SW-13 cells to exogenous hTGF-ß1, and reduced cell growth via TGF-ß mediated antiproliferative and apoptotic effects. Furthermore, transplantation of SW-13- TØRII cells into athymic mice resulted in a remarkable reduction in tumor formation with reduced proliferation, suggesting that increased malignancy in adrenocortical tumors might be impli- cated in disruptions of TGF-ß signaling pathway and partly relat- ed to the reduction in TØRII expression.
TGF-ß has been viewed as a regulator of the adrenocortical ste- roidogenesis [14,15] and growth regulator in fetal adrenal cells [16,17]. Therefore, locally secreted TGF-ß1 may participate in the various aspects of human adrenocortical physiology and pa- thology. Among the other members of TGF-ß superfamily, activin and inhibin have also been shown to play a role as a regulator of cell growth and differentiation in the adrenal gland. Matzuk et al. and Beuschlein et al. have demonstrated increasing adrenal tu- mor formation in inhibin-null (INH-/-) mouse, eliminating endo- genous activin production by gonadectomy [26,27].
Regarding TGF-ß1, several studies have indicated relationships between disruption of TGF-ß signaling pathway and adrenocorti- cal tumorigenesis. Arnaldi et al. examined twelve adrenocortical carcinoma and sixteen adenomas, and reported a significant de- crease in TGF-ß1mRNA expression in adrenocortical carcinomas compared to adenomas [28]. The study by Boccuzzi et al. immu- nohistochemically evaluated TGF-ß1 expression in eleven adre- nocortical carcinomas as well as five functional and six non- functional adenomas. TGF-ß1-positive cells were observed in functional adenomas, whereas there were few positive cells in non-functional adenomas or adrenocortical carcinomas [29]. In contrast, we observed a distinct expression of TGF-ß1 in adreno- cortical carcinoma SW-13 cells. The difference in results may be caused by the small sample volume. It would be important to in- vestigate the large-scale expression analysis of TGF-ß related molecules using clinical samples of adrenal tumors.
Concerning the relationship between the abnormality of TØRII and the adrenal tumorigenesis, mouse adrenal Y1 cell line trans- fected with dominant negative TØRII showed decreasing TGF-ß1 sensitivity and increasing tumorigenicity [30]. Since this inter- esting study demonstrated the relationship between dysfunc- tional TØRII and adrenal tumorigenicity, it might be conceivable
800
2
SW-13
I
Cumulative tumor volume (mm3)
700
SW-13-Neo
600
SW-13-TBRII-B
1.5
Tumor weight (g)
500
İ
400
1
300
#
+
200
0.5
£
#
100
#
L
#
#
0
1
t
t
0
0
12
18
25
33
40
47
Incidence
9/10
8/10
5/10
a
Time (days)
b
Clone name
SW-13
SW-13-Neo SW-13-TBRII-B
(a)
10mm
(b)
(c)
a
SW-13
SW-13-Neo
SW-13-TBRII-B
*
-
*
*
*
a)
(b)
(c)
*
(d)
*
(e)
(f)
6
b
(g)
2
(h)
(i)
that the study supports our present study. Thus, our finding that reduced TØRII expression and reduction in tumor formation by the restoration of TØRII expression in human SW-13 adrenocorti- cal cells is novel.
Several studies have indicated that restoration of TØRII expres- sion by gene transfection restores TGF-ß sensitivity and reduced malignancy in breast [31] and gastric cancer cell lines [32], as well as K-ras-transformed thyroid cells [33]. In our study, SW- 13 cells with stable TØRII expression demonstrate that TBRII ex- pression recovery increases SW-13 cell sensitivity to the growth- inhibiting effects of TGF-ß1. In vitro experiments showed slightly slower cell growth in TBRII transfected-SW-13 cells compared to
control cells when incubated without hTGF-ß1; although there was no statistically significant difference among cell numbers, this may be due to autocrine or paracrine effects of TGF-ß1. In the tumor-forming test, stable SW-13-TBRII transfectants into athymic mice resulted in markedly reduced tumor formation compared to control cells. In addition, half of the mice injected with the SW-13-TBRII-B cells showed small cystic masses con- taining blood-rich fluid. These SW-13-TØRII-B masses were quite different in appearance from the masses observed in SW-13 and SW-13-Neo cells. Although reasons for this different mass for- mation in SW-13-TBRII-B cells are not clear, the decreased and varying tumor growth may be due to the effect of locally secreted abundant TGF-ß1 from surrounding tissues, and, possibly, from
autocrine action [3]. Although further studies would be needed to explain this phenomenon, TØRII expression may play a role in tumor formation.
Since human adrenocortical carcinomas remain among the most lethal neoplasms, significant improvement is needed in both ear- ly diagnosis and effective therapy. In recent years, the Weiss In- dex has been used in the pathological diagnosis of adrenocortical tumors. This is the most commonly used prognostic indicator of the disease, including recurrence and metastasis [34,35]. How- ever, only a few useful markers have been discovered for human adrenocortical carcinomas [35,36]. Since loss of TGF-ß receptor is a poor prognostic factor in other malignant tumors [37,38], it would be interesting to explore the correlation between loss of TØRII expression and adrenocortical malignancy in prospective clinical studies.
Abnormalities in transcriptional regulation of TØRII have also been reported in squamous cell carcinoma cells [39] and Ewing sarcomas [40]. Our previous study did not demonstrate any ab- normal rearrangement in the genomic region of TRII gene or de- crease in TØRII promoter activity in SW-13 cells [41]. The ab- normalities in transcriptional regulation of TBRII could be loca- ted in another element of the TØRII promoter or in the intron of the TØRII gene. Finding the intrinsic mechanism of TØRII downre- gulation in adrenocortical carcinogenesis will be an important task for the future.
With respect to therapy for adrenocortical carcinomas, complete initial surgical resection appears to offer the best hope for dur- able control and/or cure. When surgery is unsuccessful or insuf- ficient, mitotane seems modestly effective in some patients [42,43]. Chemotherapy alone or combination with mitotane or surgery remains an important area for further investigation. Fi- nally, as effective therapies for adrenocortical carcinoma have not emerged in recent decades [11], the results from the present study suggest a possibility of TB RII gene therapy in human adre- nocortical carcinomas in cases of low or undetectable TBRII mRNA levels.
In conclusion, our data suggest that abnormalities in the TGF-B signaling pathway and partly decreased expression of TØRII may be subject to the malignant transformation process in human adrenocortical tumors.
Acknowledgements
Dr. Yukitaka Miyachi, the professor of our department, passed away on 23rd January 2003. His sudden death came as a great shock, but the memory of the late Dr. Yukitaka Miyachi will re- main with us forever.
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