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Overexpression of phosphorylated-STAT3 and phosphorylated-ERK protein in dermatofibrosarcoma protuberans

European Journal of Dermatology. Volume 16, Number 3, 262-5, May-June 2006, Investigative report

Summary

Author(s) : Nengxing Lin, Kazunori Urabe, Yoichi Moroi, Hiroshi Uchi, Takeshi Nakahara, Teruki Dainichi, Hisashi Kokuba, Yating Tu, Masutaka Furue , Department of Dermatology, Graduate School of Medical Sciences, Kyushu University, Maidashi 3-1-1, Higashiku, Fukuoka, 812-8582, Japan, Department of Dermatology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology.

Summary : The overexpression of phosphorylated signal transducer and activator of transcription-3 (p-STAT3) and phosphorylated extracellular signal-regulated kinase (p-ERK) have recently been shown to play an important role in the pathogenesis of various human tumors. However, the role of these two major signal transduction pathways in dermatofibrosarcoma protuberans (DFSP) remains unknown. This study was designed to investigate the significance of p-STAT3 and p-ERK expression in DFSP. The expressions of p-STAT3 and p-ERK were analyzed by immunohistochemical staining in formalin-fixed, paraffin-embedded tissue sections of human DFSP and dermatofibroma. Ten cases were positive for p-STAT3 expression in 14 cases of DFSP, however, only 5 cases were positive in 20 cases of dermatofibroma. Eleven out of 14 cases of DFSP expressed p-ERK, but only four cases were positive in 20 cases of dermatofibroma. The expressions of p-STAT3 and p-ERK were significantly higher than those in dermatofibroma (both p <\; 0.01). This study suggests that the overexpression of p-STAT3 and p-ERK may play a pivotal role in the oncogenesis of DFSP.

Keywords : DFSP, p-STAT3, p-ERK, dermatofibroma

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ARTICLE

Auteur(s) : Nengxing Lin^1,2, Kazunori Urabe¹, Yoichi Moroi¹, Hiroshi Uchi¹, Takeshi Nakahara¹, Teruki Dainichi¹, Hisashi Kokuba¹, Yating Tu², Masutaka Furue¹

¹Department of Dermatology, Graduate School of Medical Sciences, Kyushu University, Maidashi 3-1-1, Higashiku, Fukuoka, 812-8582, Japan
²Department of Dermatology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology

accepté le 25 Janvier 2006

In general, cell division and proliferation are closely correlated with large amounts of signal transduction from extracellular components to the nucleus. Janus tyrosine Kinases(JAK)/STAT3 and Raf/Mitogen-activated protein Kinase Kinase(MEK))/ERK pathways are the two main signal transduction pathways that are associated with cell proliferation, apoptosis, differentiation and malignant transduction. STAT3 and ERK are activated by phosphorylation. Overexpression of p-STAT3 and p-ERK has been shown in many human tumors and tumor cell lines. p-STAT3 and p-ERK can directly or indirectly upregulate the expression of genes that are required for uncontrolled proliferation and survival of tumor cells. These include genes that encode cell cycle-associated proteins [1, 2].DFSP is a slow-growing, locally aggressive tumor of disputed histogenesis, with a marked tendency to local recurrence, but a low tendency to metastasis [3]. The exact pathogenesis of DFSP is still unknown. Therefore, we examined whether the overexpression of p-STAT3 and p-ERK, which have recently been shown to play an important role in the pathogenesis of other human tumors, are involved in the oncogenesis of DFSP. Our study demonstrated that overexpression of p-STAT3 and p-ERK may play a pivotal role in the oncogenesis of DFSP.

Materials and methods

Tissue samples

The paraffin-embedded tissues were obtained from the archives of the Department of Dermatology in Kyushu University, Japan. They included 14 cases of DFSP, and 20 cases of dermatofibroma specimens (Table 1). Clinical and demographic data were retrieved from the patients’ files, all cases were reviewed by two dermatopathologists who were blinded to the lesion site, age and gender of the patients. The paraffin- embedded tissues were evaluated with hematoxylin and eosin staining.
Table 1 Expression of p -STAT3, p-ERK in DFSP and dermatofibroma

Groups	n	p-STAT3	p-ERK
		- + ++ +++	- + ++ +++
DFSP^#*	14	4 3 4 3	3 4 4 3
dermatofibroma^#*	20	15 3 2 0	16 2 2 0

Immunohistochemistry

For immunohistochemistry, the avidin-biotin complex (ABC) method was performed on 4-μm-thick tissue sections. Sections were deparaffinized with xylene for 15 min and rehydrated through graded ethanol concentrations, followed by blocking of endogenous peroxidase activity in H₂O₂/methanol for 12 min. Antibody-binding epitopes were retrieved by autoclaving the tissue sections in 10 mM EDTA, pH 7.0 for 2.5 min, and nonspecific binding was blocked with 10% goat serum. The sections were then incubated with antibodies against p-STAT3 (tyr705) (Cell signaling technology, Beverly, USA) or p-ERK1/2(E4)(tyr204) (Santa Cruz. Biotechnology, Santa Cruz, CA) at dilutions of 1:50 in 5% goat serum in PBS at 4°C overnight. Immunodetection was achieved by an avidin-biotin horseradish peroxidase method with 3,3-diaminobenzidine (DAB) as a chromogen, followed by light counterstaining with hematoxylin. All incubations were performed at room temperature. Washing with TBS or PBS was performed between each step according to the manufacturer’s protocols. Positive controls (using tissue samples from human breast carcinoma) and negative controls (using PBS as well as non-specific goat antibody instead of the individual primary antibodies) were stained by the same procedures.

P-STAT3 and p-ERK immunoreactivities were limited to the nucleus; cells with yellow or brown staining in the nucleus were regarded as positive cells. The degree of p-STAT3 and p-ERK immunopositivity in tumors was graded as follows according to Fromowitz [4]: Pink brown staining scored 1, yellow brown staining scored 2, dark brown staining scored 3. Positive cell rate: < 25% scored 1, 25-50% scored 2; 51-75% scored 3; > 75% scored 4. Then color score and positive cell rate score were added, and the results were graded into one of three categories: score 2-3 means weakly staining, marked (+); score 4-5 means moderate staining , marked (++); score > 5 means strongly staining, marked (+++); and completely negative staining was marked (–).

Statistical analysis

Statistical analysis was carried out with SPSS using the X² test (including Fisher’s exact test). P < 0.05 was considered statistically significant.

Results

P-STAT3 expression

P-STAT3-positive staining was limited to the nucleus. In 10 of the 14 DFSP specimens, the tumor cells were positive for p-STAT3 (figure 1A): Three were weakly stained, four moderately, and three strongly (tables 1 and 2( Table 2). In five of the 20 dermatofibroma specimens, the tumor cells were positive for p-STAT3 (figure 1B): Three were weakly stained, and two were moderately stained (tables 1 and 2). The expression of p-STAT3 in DFSP was significantly higher than that in dermatofibroma (X² = 7.201, p = 0.007).
Table 2 Expression of p -STAT3, p-ERK in DFSP

No	Years	Sex	p-STAT3	p-ERK
1	36	m	++	++
2	78	f	++	+
3	23	f	-	+
4	36	m	+	+++
5	43	f	+++	++
6	23	f	+++	-
7	39	m	++	+
8	25	m	+	+
9	26	m	++	+++
10	65	f	-	-
11	53	m	+	++
12	76	m	-	++
13	10	m	+++	-
14	11	m	-	+++
Positive	rate		10/14	11/14

P-ERK expression

P-ERK-positive staining was limited to the nucleus. In 11 of the 14 DFSP specimens, the tumor cells were positive for p-ERK: Four were weakly stained, four were moderately stained, and three were strongly stained (figure 2A) (tables 1 and 2). In four of the 20 dermatofibroma specimens, the tumors cells were positive for p-ERK (figure 2B): Two were weakly stained, and two were moderately stained (tables 1 and 2). The expression of p-ERK in DFSP was significantly higher than that in dermatofibroma (X² = 11.459, p = 0.001). It is of interest that there was no significant correlation between p-STAT3 and p-ERK expression in DFSP, suggesting that the p-STAT3 and p-ERK expressions may be differentially regulated during oncogenesis of DFSP.

Discussion

STAT3 is a cytoplasmic latent transcription factor that is activated by tyrosine phosphorylation. P-STAT3 modulates cell proliferation, apoptosis, differentiation, and many other important biological activities [5]. P-STAT3 may directly or indirectly upregulate the expression of genes that are required for uncontrolled proliferation and survival of tumor cells. These include genes that encode apoptosis inhibitors (Bcl-xL) [6] and cell cycle regulators (Cyclin D1/D2) [1]. Overexpression of p-STAT3 has been found in several tumor cell lines and samples derived from human tumors, including those of breast [7], hematopoietic origin [8], head and neck [9, 10], lung [11], prostate [12] and ovary [13].

The activated STAT3 can also induce the expression of vascular endothelial cell growth factor (VEGF) [14] and matrix metalloproteinase-2 (MMP-2) [15], which are the most widely studied and potent inducers of angiogenesis. Blocking of Stat3 activation inhibited the expression of VEGF and MMP-2 in tumor cell lines. In prostate carcinoma cells, inhibition of Src kinase activity by Ad-mda inhibited STAT3 activation, resulting in suppression of VEGF expression [16]. A recent study shows that the activated STAT3 may affect the cell adhesion and/or the cytoskeleton molecules, contributing to the invasion and metastasis of tumors [17]. In ovarian carcinomas, STAT3 was more frequently activated in high-grade ovarian carcinomas than in low-grade carcinomas and organ-confined borderline tumors [18]. In renal cell cancer, there was a strong correlation of activated Stat3 with aggressive cancers that had metastasized [19]. Cai et al. found that the overexpression of p-STAT3 was correlated with the invasion and metastasis of cutaneous squamous cell carcinoma [20]. Interestingly, blocking of Stat3 in pancreatic cancer cells by expression of a dominant-negative form inhibited tumor growth and liver metastasis in mice, whereas expression of a constitutively dimerized form of STAT3 promoted pancreatic cancer metastasis [21].

The Ras/MEK/ERK pathway is a central signal transduction pathway, which transmits signals from multiple cell surface receptors to transcription factors in the nucleus. The Ras/MEK/ERK pathway can be activated via the IRS-1 and/or Src proteins. ERK is the main physiological substrate of MEK. ERK are activated through dual phosphorylation of Thr and Tyr residues by MEK1 kinases. Phosphorylation of ERK (p-ERK) can activate its downstream targets, including p90^Rsk kinase and the CREB, c-Myc and other transcription factors [22]. Many studies have revealed that aberrant regulation of the Ras/MEK/ERK cascade is involved in malignant progression [23, 24] .Constitutively active MEK1 has been associated with malignant transformation in the human A375 malignant melanoma cell line [25]. Expression of activated ERK1/2 in melanocytic lesions appears to be related to malignant potential, so that activation of ERK1/2 may be important in melanoma progression [26]. It is well established that the Ras/MEK/ERK signaling pathway is a key regulator of cell proliferation. Overexpression of the Ras/MEK/ERK cascade has been frequently observed in human tumors, including papillary thyroid carcinoma [27], ovarian serous carcinoma [28], malignant melanoma [29], leukemia [30], carcinoid tumor [31], and lung adenocarcinoma [32].

DFSP is considered as a rare, low-grade, cutaneous sarcoma with autocrine overproduction of the platelet-derived growth factor (PDGF) β-chain from gene rearrangement as a key pathogenetic factor. Recent studies found DFSP arose from the rearrangement of chromosomes 17 and 22, with the fusion between the collagen type I1 gene (COL1A1) and the platelet-derived growth factor (PDGF) β-chain gene (PDGFB) [33]. This results in deregulation of PDGF β-chain expression and activation of PDGF receptor β (PDGFRß) protein tyrosine kinase. PDGF isoforms are consided to be the principal mediators of mesenchymal cell proliferation, incluning dermal fibroblasys [34].

In the present study, 10/14 and 11/14 specimens of DFSP were positive for p-STAT3 and p-ERK expression, respectively, suggesting that the STAT3 and ERK are significantly activated in DFSP. The activation of STAT3 and ERK in DFSP may be involved in PDGF, associated with its receptors (PDGFRß), acting through intrinsic receptor tyrosine kinases. The phosphorylation of tyrosine kinase was also involved in increased mitogenic activity of DFSP cells, furthermore, using an MEK1-specific inhibitor (PD98059) and a p38 MAPK inhibitor (SB202190), Hironobu Ihn et al. found that ERK, but neither p38 MAPK nor JNK, was involved in increased mitogenic activity of DFSP cells [35].

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