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p-21-Activated kinase-1, -4 and -6 and estrogen receptor expression pattern in normal placenta and gestational trophoblastic diseases
YGYNO-975189; No. of pages: 5; 4C: Gynecologic Oncology xxx (2013) xxx–xxx
Contents lists available at ScienceDirect
Gynecologic Oncology journal homepage: www.elsevier.com/locate/ygyno
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Melina Shoni a, Zoltan Nagymanyoki b, Allison F. Vitonis a, Cynthia Jimenez b, Shu-Wing Ng a, Bradley J. Quade b, Ross S. Berkowitz a,c,⁎ a b c
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p-21-Activated kinase-1, -4 and -6 and estrogen receptor expression pattern in normal placenta and gestational trophoblastic diseases
Division of Gynecologic Oncology, Department of Obstetrics and Gynecology, Brigham and Women's Hospital, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA, USA Division of Women's and Perinatal Pathology, Department of Pathology, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA Donald P Goldstein, MD Trophoblastic Tumor Registry, New England Trophoblastic Disease Center, MA, USA
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Objectives. To delineate the potential role of p21-activated kinases (PAKs) in the pathogenesis of gestational trophoblastic diseases (GTD) by defining the expression pattern of PAK-1, -4 and -6 and their potential implication in estrogen receptor (ER) regulation of normal placental tissue and GTD. Methods. We evaluated immunohistochemically 10 normal first-trimester placentas (NP), 10 partial moles (PM), 15 complete moles (CM) and 3 choriocarcinomas (CCA) for PAK-1, PAK-4, PAK-6 and ER expression intensity and localization. Staining outcomes were assessed utilizing non-parametric Kruskal–Wallis one-way analysis of variance test followed by pairwise Wilcoxon Rank Sum tests. Statistical significance was determined by twosided p-value of b 0.05. Results. In NP, PAK-6 immunoreactivity was predominantly cytoplasmic. Compared to NP, PM and CM demonstrated significant increase of cytoplasmic PAK-6 in cytotrophoblast (p = 0.012, p = 0.033 respectively), accompanied by significantly increased nuclear immunoreactivity in cytotrophoblast (p = 0.008, p = 0.045 respectively) and intermediate trophoblast (p = 0.003, p = 0.015 respectively). PAK-4 was found significantly upregulated in both cytoplasmic and nuclear compartments of cytotrophoblast and syncytiotrophoblast in PM (p = 0.004 and p = 0.002 for cytotrophoblast; p = 0.018 and p = 0.002 for syncytiotrophoblast, respectively) and CM (p = 0.001 and p = 0.001 for cytotrophoblast; p = 0.002 and p = 0.001 for syncytiotrophoblast, respectively) when compared to NP, whereas PAK-1 expression was significantly reduced in the syncytiotrophoblast of PM (p = 0.025 for cytoplasm and p = 0.008 for nucleus). Nuclear expression of ER was undetectable in all stained samples. Conclusion. Our results reveal PAK-6 upregulation in GTD compared to NP. The absence of nuclear expression of ER might stem in part from the repressive effect of PAK-6 in trophoblastic tissue. © 2013 Published by Elsevier Inc.
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Article history: Received 24 July 2013 Accepted 11 September 2013 Available online xxxx
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Keywords: Gestational trophoblastic diseases p-21-Activated Kinases PAK-6 Estrogen receptors
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• p-21-Activated Kinase-6 (PAK-6) has increased expression in gestational trophoblastic diseases and might be implicated in its pathophysiology. • p-21-Activated Kinases (PAKs) may play an important role in regulating and limiting estrogen receptor expression in gestational trophoblastic tissue.
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Introduction
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Perturbations of the balance between cellular proliferation, differentiation, migration and death lead to aberrant tissue development. Even in its healthy state, placental trophoblastic tissue exhibits a malignant-like, though tightly controlled, behavior [1] by invading with its extravillous component the endometrium and developing a rich uterine vasculature to generate an intimate connection between the fetus and the mother [2]. Failure of the underlying regulatory mechanisms seems implicated
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⁎ Corresponding author at: 75 Francis Street, Boston, MA 02115. E-mail address: [email protected] (R.S. Berkowitz).
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in the development of gestational trophoblastic diseases (GTD), as reflected on its premalignant forms — complete and partial hydatidiform mole (HM) — which have an increased potential of persistence and malignant transformation, as well as its malignant spectrum of invasive mole, choriocarcinoma (CCA), placental site trophoblastic tumor (PSTT) and epithelioid trophoblastic tumor (ETT) [3]. While there is growing understanding of the molecular biology of GTD, the precise pathways that confer such aberrations need further exploration [4]. Recent studies point to a functional role of p21-activated kinases (PAKs) in promoting related tumorogenic pathways [5,6]. PAKs comprise a well characterized group of conserved, non-receptor serine/threonine kinase isoforms, downstream effectors of Rho-family
0090-8258/$ – see front matter © 2013 Published by Elsevier Inc. http://dx.doi.org/10.1016/j.ygyno.2013.09.010
Please cite this article as: Shoni M, et al, p-21-Activated kinase-1, -4 and -6 and estrogen receptor expression pattern in normal placenta and gestational trophoblastic diseases, Gynecol Oncol (2013), http://dx.doi.org/10.1016/j.ygyno.2013.09.010
60 61 62 63 64 65 66 67 68 69 70 71
91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110
t1:1 t1:2
Table 1 Human malignancies where PAKs have been found deregulated. PAK isoform
t1:4 t1:5
Group I PAK-1
Cancer type (alteration)
brain (increased phospho-PAK-1 in cytoplasm) breast (protein overexpression and increased nuclear localization) liver and kidney (protein and gene overexpression) colon (protein overexpression) bladder (gene amplification (11q13Aq14 amplicon) lung (protein overexpression) ovarian (protein overexpression and gene amplification (11q13Aq14 amplicon) T-cell lymphoma (gene amplification) NF1 (PAK-1 deletion), NF2 (PAK1 deletion) gestational trophoblastic diseases choriocarcinoma – neuroendocrine (protein overexpression)
U
N
t1:3
t1:6 t1:7 t1:8 t1:9
t1:10 t1:11
115
Under protocol approved by the Brigham and Women's Hospital Institutional Review Board, paraffin-embedded, gestationally agematched normal placenta from elective first trimester abortions (NP) (n = 10), partial molar (PM) (n = 10) and complete molar (CP) (n =15) tissues as well as choriocarcinomas (CCA) (n = 3) were provided by the Division of Women's and Perinatal Pathology. Precisely, the mean gestational age was 8.8 weeks in normal pregnancies, 11.6 weeks in complete and 9.1 weeks in partial molar pregnancies. Persistent gestational trophoblastic neoplasia (GTN) developed in 2 of 15 CM cases only. Molar tissues were collected at the time of uterine evacuation while normal placentas were obtained at the time of elective pregnancy termination. Clinical information including gestational age, pathologic diagnosis and development of persistent gestational trophoblastic neoplasia was collected for all cases. Conventional histologic sections were made of paraffin-embedded tissues and routinely stained with hematoxylin– eosin (HE). The underlying diagnosis was based on standard histopathologic criteria as determined by experienced gynecologic pathologists.
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Placental trophoblastic tissue samples
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87 88
Materials and methods
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of PAKs in the pathophysiology of GTD by determining the expression 111 and distribution of PAK-1, PAK-4 and PAK-6 as well as the potential ef- 112 fect of PAK-1 and PAK-6 on ER regulation in normal placenta and GTD. 113
PAK-2 PAK-3 Group II PAK-4
PAK-5 PAK-6
esophagus (protein overexpression) breast (protein overexpression and increased nuclear localization) pancreas (gene amplification (19q13 amplicon), protein overexpression) colon (protein overexpression, gene amplification (19q13 amplicon) and 2 somatic mutations) choriocarcinoma – prostate (protein overexpression)
118 119 120 121 122 123 124 125 126 127 128 129 130 131 132 133
Immunolocalization of PAK-1, PAK-4, PAK-6 and estrogen receptors
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Immunohistochemistry (IHC) was performed on formalin-fixed, paraffin-embedded trophoblastic tissue slides. The corresponding blocks were sectioned at a thickness of 7 μm, mounted on Superfrost Plus microscopic slides (Fisher Scientific, Pittsburgh, PA), and dried at 50 °C for at least 24 h. Deparaffinization was achieved using xylene and rehydration with a graded ethanol series. Antigen retrieval was performed within antigen-unmasking solution (Vector Laboratories, Burlingame, CA) utilizing a pressure-cooker. Endogenous peroxidases were then blocked using 3% H202 in distilled water for 15 min. Following a 30-min blocking with the corresponding normal serum, sections were incubated with primary antibodies (rabbit anti-PAK-1 antibody, Cell Signaling Technology Inc., dilution 1:100; rabbit anti-PAK-4, Cell Signaling Inc., dilution 1:150; rabbit anti-PAK-6, Sigma Aldrich, dilution 1:200; mouse antiER, Sigma, dilution 1:80) at 4 °C overnight. The reaction was visualized using Vectastain Elite ABC Kit with diaminobenzidine chromogen as a substrate (Vector Laboratories, Burlingame, CA). Sections were lightly counterstained with hematoxylin and mounted. Immunohistochemical staining of cytotrophoblast, syncytiotrophoblast, intermediate trophoblast and decidual stromal cells was evaluated independently by two experienced pathologists using a semi-quantitative scoring system. The final histological score was obtained by multiplying the percentage of the stained area (0 when absent, 1+ when less than 25%, 2+ when 25% to 50% and 3+ when more than 50% of the total epithelial area) with the intensity of the stain (from 0 for no stain to 3 for intense stain).
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Statistical analysis
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The non-parametric Kruskal–Wallis one-way analysis of variance test was used to identify significant differences among independent variable groups. This was followed by pairwise Wilcoxon Rank Sum test to determine which group differences were statistically significant, as defined by the two-sided p-value of b0.05. All statistical analyses were performed utilizing SAS.
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79 80
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78
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76 77
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GTPases (especially Rho, Rac and Cdc42). Divided into two distinct groups based on structural and functional similarities (group I: PAK1-3 and group II: PAK4-6), these pluripotent kinases interact with a wide array of intracellular proteins exerting regulatory control over hallmark biological processes, including cell morphogenesis and survival, gene transcription, actin cytoskeletal dynamics and motility, angiogenesis, growth factor- and steroid receptor- signaling, and energy homeostasis [7,8]. PAKs overexpression or hyperactivation in various human malignancies (Table 1) indicates their role in cancer-related signaling networks [9–14]. PAKs modulate the expression of genes for both survival and metastatic potential through transcriptional modulator substrates and by their association with nuclear chromatin [9–14]. With regard to GTD, PAK-1 was found to enhance cellular proliferation and invasive phenotypes [6], whereas PAK-4 was reported to potentiate proliferation, invasion and migration in choriocarcinomas [5]. To date, less is known about PAK-6 in GTD. Findings link PAKmediated signaling to the steroid hormone receptor pathway, with PAK-6 first identified by yeast two-hybrid screening as an androgen receptor (AR)-interacting protein [15]. Since the latter pertains to the same nuclear receptor superfamily with estrogen receptors (ER), similar PAK-6/ER interactions could be anticipated. Although PAK-6 is primarily localized in the cytoplasm, in the presence of AR or ER-α and the respective ligand, PAK-6 can translocate into the nucleus as part of an AR/ER-α complex and repress AR/ER-α-mediated transcription through both kinase-dependent and -independent manner [15–17]. From this standpoint, PAK-6 inhibits prostate tumorogenesis by regulating AR homeostasis [18]. However, the inhibitory effect of PAK-6 on AR- and ER-mediated gene transcription is opposed by the ER transactivation as induced by PAK-1-mediated receptor phosphorylation [19]. Mammary tissues with active PAK-1 exhibit stimulated ER-mediated transactivation and expression of endogenous ER target genes, revealing a novel role for the PAK-1/ ER pathway in promoting hyperplasia of mammary epithelium [19]. Previous attempts for the elucidation of the role of sex hormone receptors in GTD revealed almost negative ER expression in HM and placentas from spontaneous abortions [20,21]. On the other hand, Jiang et al. [22] highlight the importance of functional ER in controlling choriocarcinoma cell proliferation, suggesting a potential role of estrogens in mediating placental trophoblastic growth. In the present study, we aimed at expanding our understanding of the potential role
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Please cite this article as: Shoni M, et al, p-21-Activated kinase-1, -4 and -6 and estrogen receptor expression pattern in normal placenta and gestational trophoblastic diseases, Gynecol Oncol (2013), http://dx.doi.org/10.1016/j.ygyno.2013.09.010
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M. Shoni et al. / Gynecologic Oncology xxx (2013) xxx–xxx
Normal
Cytotrophoblastic cytoplasm nucleus Syncytiotrophoblastic cytoplasm nucleus Intermediate trophoblast cytoplasm nucleus Decidual cell cytoplasm nucleus
CCA
n
Median (range)
n
Median (range)
n
Median (range)
n
Median (range)
KW p
Normal vs. PM
Normal vs. CM
PM vs. CM
Normal vs. CM
10 10
5 (2–6) 9 (4–9)
10 10
4.5 (3–9) 6 (6–9)
14 14
6 (2–9) 9 (4–9)
2 2
2.5(2–3) 5 (4–6)
0.160 0.358
0.871 0.538
0.224 0.920
0.371 0.410
0.167 0.226
10 10
3 (0–6) 6 (1–9)
10 10
1 (0–3) 1.5(1–4)
14 14
2.5 (0–6) 4 (0–6)
2 2
2.5(2–3) 4 (2–6)
0.023 0.011
0.025 0.008
0.574 0.100
0.012 0.040
0.588 0.592
10 10
6 (4–9) 9 (4–9)
10 10
6 (3–9) 9 (4–9)
15 15
6 (3–9) 9 (4–9)
2 2
2.5 (2–3) 5 (4–6)
0.095 0.181
0.721 0.724
0.769 0.862
0.463 0.527
0.059 0.119
9 9
3 (0–3) 2 (0–6)
9 9
1 (0–3) 2 (0–4)
15 15
3 (0–6) 6 (1–9)
2 2
1.5 (0–3) 3 (0–6)
0.102 0.020
0.513 0.334
0.174 0.137
0.027 0.004
0.999 0.903
PM: partial mole, CM: complete mole, CCA: choriocarcinoma.
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Results
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Immunohistochemical staining outcomes
169 170
Immunohistochemical staining of normal placenta for PAK-1 revealed cytoplasmic and nuclear expression in all cellular groups (cytotrophoblastic, syncytiotrophoblastic, intermediate trophoblastic and decidual stromal cells). Apart from decidual cells, nuclear expression was higher than cytoplasmic. These outcomes were retained in CM and CCA cases. In PM, although with the same cellular immunolocalization, PAK-1 exhibited decreased expression compared to normal placenta, which reached statistical significance only in syncytiotrophoblastic cells (p = 0.025 for cytoplasmic expression and p =0.008 for nuclear expression). Table 2 demonstrates in detail the immunohistochemical expression of PAK-1 in normal placenta and GTD. In normal placenta, PAK-4 immunoreactivity was predominantly confined to the cytoplasm of all cellular groups with occasional nuclear staining detected in intermediate trophoblastic cells. When CM and PM were compared to normal placenta, PAK-4 cytoplasmic expression was significantly increased in cytotrophoblastic cells (p = 0.001 and p = 0.004, respectively) and syncytiotrophoblastic cells (p = 0.002 and p = 0.018, respectively). This was also accompanied by significantly increased nuclear immunoreactivity in cytotrophoblastic cells (p = 0.001 and p = 0.002, respectively) and syncytiotrophoblastic cells (p =0.001 and p = 0.002, respectively). CCA intermediate cells showed reduced cytoplasmic and nuclear stain, which, however, did not reach statistical significance. Table 3 reviews in detail the immunohistochemical outcomes of PAK-4 staining in normal placenta and GTD. Immunohistochemical staining of normal placenta for PAK-6 demonstrated primarily cytoplasmic expression that spared only the decidual stromal cells. This staining pattern was intensified in PM tissue, reaching a statistically significant increase in the cytoplasm of cytotrophoblastic cells (p = 0.012) and a significant simultaneous PAK-6 translocation in the nucleus of cytotrophoblastic (p = 0.007) and intermediate trophoblastic cells (p = 0.003). These significant changes were also noticed in CM cases, which additionally exhibited increased cytoplasmic expression in intermediate (p = 0.011) and decidual cells (p = 0.033). Nuclear cytotrophoblastic and intermediate trophoblastic expression was also increased in CCA but statistical significance was not reached. However, the limited number of the included CCA cases might need to be considered for the interpretation of this outcome. Table 4 demonstrates in detail the immunohistochemical outcomes of PAK-6 staining in normal placenta and GTD. ER expression, both cytoplasmic and nuclear, was undetectable in normal placental tissue and this negative pattern was also retained in all of our PM and CM as well as CCA cases. However, weak ER staining
183 184 185 186 187 188 189 190 191 192 193 194 195 196 197 198 199 200 201 202 203 204 205 206 207 208 209 210
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181 182
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179 180
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was observed in the decidual cells of normal placenta and GTD while 211 endometrial stromal and glandular cells were found strongly positive 212 and thus, were used as positive internal controls. 213
N C O
175 176
U
173 174
R O
t2:17
171 172
Kruskal Wallis (KW) & Wilcoxon Rank Sum test p-values
F
t2:5 t2:6 t2:7 t2:8 t2:9 t2:10 t2:11 t2:12 t2:13 t2:14 t2:15 t2:16
CM
O
t2:4
PM
P
PAK-1
Discussion
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Trophoblastic tumor biology stems from its placental origin with aberrations in a heterogeneous population of cells, including villous cytotrophoblasts, syncytiotrophoblasts, extravillous trophoblasts and intermediate trophoblasts [3]. p21-activated kinases (PAKs) are a conserved family of six serine/threonine kinases implicated in salient pathways of aberrant tissue development [23], including but not limited to anchorage-independent growth (mainly through the canonical MAP kinase cascade of Ras/Raf/MeK/ERK), inhibition of apoptosis (through the BAD/Bcl-2 pathway) and stimulation of cellular motility (by targeting LIM kinase) [7]. In this study, we demonstrated an upregulated expression profile of PAK-6 in GTD and investigated immunohistochemically the potential role of PAK members in the regulation of ER in trophoblastic tissue. The rationale for this investigation was in part based on previously published data demonstrating a potential role for PAK-1 and PAK-4 in GTD. PAK-1, the best-characterized member of the PAK family, has been placed as a convergence point in cellular transformation induced by various small GTPases and mitogenic factors [24]. Siu et al. [6] found upregulated PAK-1 expression in hydatidiform mole (HM) and choriocarcinoma (CCA) when compared to normal placentas, a pattern that was associated with more aggressive behavior and increased potential of HM progression to gestational trophoblastic neoplasia (GTN). The expression level of PAK-1 was overall decreased in PM compared to normal placenta, reaching statistical significance only in syncytiotrophoblastic cells (p =0.025 for cytoplasmic expression and p = 0.008 for nuclear expression). CM and CCA retained the PAK1 expression pattern of normal placenta. Our immunohistochemical outcomes for PAK-1 differ from these data and this might be in part attributable to the different sample categories that have been assessed in these studies. Whereas Siu et al. [6] distinguished HM into nonpersistent and persistent HM, they did not distinguish PM versus CM, as we did. Moreover, it should be noted that the limited number of cases included in the study may contribute to a potential bias and this should also be considered when interpreting the data. Contrary to PAK-1, our findings regarding PAK-4 expression were in agreement with the outcomes of Zhang et al. [5], who reported ubiquitously expressed PAK-4 in normal placenta. According to the same study, PAK4 was found to affect cell proliferation, migration and invasion of CCA cells through deregulation of p16, CDK6 and MT1-MMP. We found that the predominantly cytoplasmic staining of PAK-4 in normal
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t2:3
Table 2 Immunohistochemical expression of PAK-1 in normal placenta and gestational trophoblastic diseases.
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t2:1 t2:2
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Please cite this article as: Shoni M, et al, p-21-Activated kinase-1, -4 and -6 and estrogen receptor expression pattern in normal placenta and gestational trophoblastic diseases, Gynecol Oncol (2013), http://dx.doi.org/10.1016/j.ygyno.2013.09.010
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t3:3
Table 3 Immunohistochemical expression of PAK-4 in normal placenta and gestational trophoblastic diseases. PAK-4
Normal
t3:4 t3:5 t3:6 t3:7 t3:8 t3:9 t3:10 t3:11 t3:12 t3:13
Cytotrophoblastic cytoplasm nucleus Syncytiotrophoblastic cytoplasm nucleus Intermediate trophoblast cytoplasm nucleus
PM
CM
CCA
n
Median (range)
n
Median (range)
n
Median (range)
n
Median (range)
KW p
Normal vs. PM
Normal vs. CM
PM vs. CM
Normal vs. CCA
10 10
3 (0–4) 0 (0–1)
10 10
6 (3–9) 3 (0–9)
10 10
6 (4–9) 5 (1–9)
0 0
– –
0.001 0.001
0.004 0.002
0.001 0.001
0.899 0.649
– –
10 10
3 (0–6) 0 (0–2)
10 10
6 (0–9) 4 (0–9)
10 10
6 (6–9) 6 (2–9)
0 0
– –
0.001 0.001
0.018 0.002
0.002 0.001
0.082 0.228
– –
2 2
3 (0–6) 2 (0–4)
8 8
6 (3–6) 2 (0–6)
13 13
3 (0–6) 1 (0–6)
3 3
0 (0–3) 0 (0–0)
0.065 0.258
0.441 0.895
0.860 0.931
0.101 0.540
0.763 0.460
PM: partial mole, CM: complete mole, CCA: choriocarcinoma.
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placenta was significantly increased in both partial and complete HM accompanied by an obvious nuclear translocation of PAK-4 in cytotrophoblastic and syncytiotrophoblastic cells (p = 0.001 and p =0.001, respectively). To further advance previous studies, we investigated the expression profile of PAK-6 and we found significant upregulation in GTD compared to normal placenta. In terms of protein localization, PAK-6 was completely cytoplasmic in normal placenta. However, PAK-6 had significantly increased immunoreactivity not only in the cytoplasm but also in the nucleus of the cytotrophoblastic and intermediate trophoblastic cells of PM and CM. This finding is in line with the theory that suggests that PAK-6 has a role in regulating steroid receptor-mediated nuclear gene transcription. The ability of PAKs to interact with and modulate nuclear receptor functions also raises the possibility that PAKs may play a role in the hormonal independence of trophoblastic tissue. PAK6 interacts with the nuclear receptor (NR) superfamily members, androgen receptor (AR) and estrogen receptor α (ER-α), which play key roles in development, hormone signaling and hormone-independent carcinomas [15–17]. It is unclear whether PAK-6 overexpression confers oncogenic or counteracting tumor suppressing capacity in GTD. In keeping with the function of PAK-6 in hormone signaling, overexpression of PAK-6 has been reported in prostate cancer [25] at a time when a study demonstrates its tumor suppressing role in prostate tumor growth [18]. Notably, increased PAK-6 and ERα interaction is also observed in tamoxifen treated breast cancer cells [26]. In order to elucidate potential relationships between PAK-6 and ER in GTD we examined the expression status of ER in our tissue samples but the nuclear immunoreactivity to ER was undetectable in all normal placentas and GTD tissues. This is
t4:1 t4:2
Table 4 Immunohistochemical expression of PAK-6 in normal placenta and gestational trophoblastic diseases.
270 271 272 273 274 275 276 277 278
Author's disclosures of potential conflict of interest
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283
t4:4 t4:5 t4:6 t4:7 t4:8 t4:9 t4:10 t4:11 t4:12 t4:13 t4:14 t4:15 t4:16 t4:17
Cytotrophoblastic cytoplasm nucleus Syncytiotrophoblastic cytoplasm nucleus Intermediate trophoblast cytoplasm nucleus Decidual cell cytoplasm nucleus
286 287 288 289 290 291 292 293 294 295 296 297 298 299 300 301
304
Acknowledgments
We would like to thank the Foundation for Education and European 305 Culture (IPEP), Greece, for the generous support on this research effort. 306
N
PAK-6
U
t4:3
284 285
303
The authors indicated no potential conflicts of interest.
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in agreement with previous studies [20,21] but since our assessment has been approached immunohistochemically, this does not exclude ER expression in immunohistochemically undetectable levels. The opposing role that PAK-1 and PAK-6 exert on ER might have also an impact on this finding. Moreover, we should not forget that besides the natural estrogen ligand, several signaling molecules including Akt, protein kinase A and pp90rsk1 have been shown to phosphorylate the AF1 domain of ER, which contributes to the transactivation function of ER [27,28]. In summary, we have defined the expression profile of PAK-6 in normal placenta and GTD. PAK-6 upregulation in GTD may provide a novel molecular pathway to investigate and functional studies are warranted to elucidate the precise role of PAK-6 on the hormonal dependence or independence of trophoblastic tumorigenesis. Because PAKs may act as multifunctional regulators in GTD and in light of the drug-discovery efforts that have already led to the development of several direct and indirect PAK inhibitors [29], functional studies focused on PAK targeting may shed more light on the therapeutic potential of this pathway in GTD.
T
268 269
C
266 267
E
264 265
R
262 263
R
260 261
O
258 259
O
t3:14
256 257
Kruskal Wallis (KW) & Wilcoxon Rank Sum test p-values
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t3:1 t3:2
M. Shoni et al. / Gynecologic Oncology xxx (2013) xxx–xxx
Normal
PM
CM
CCA
Kruskal Wallis (KW) & Wilcoxon Rank Sum test p-values
n Median (range)
n Median (range)
n Median (range)
n Median (range)
KW p
Normal vs. PM
Normal vs. CM
PM vs. CM
Normal vs. CCA
10 10
1.25(0.5–2) 0.5 (0–2)
10 10
2.5 (1–3) 2.5 (1–4)
14 14
2(0.5–3.5) 2 (0–4)
3 3
2 (1–6) 1 (1–4)
0.025 0.016
0.012 0.007
0.033 0.045
0.349 0.142
0.190 0.284
10 10
1 (0–2) 0 (0–0.5)
10 10
1(0.5–3.5) 0.5(0–0.5)
14 14
1 (0.5–3) 0 (0–0.5)
3 3
1(1–9) 0 (0–0)
0.535 0.135
0.558 0.099
0.226 0.672
0.478 0.154
0.434 0.513
10 10
1 (0–2) 0 (0–2)
10 10
1.3 (1–2) 2.5 (1–4)
14 14
3 (0.5–5) 1.5 (0–4)
3 3
1 (0.5–6) 1 (1–3)
0.030 0.003
0.088 0.003
0.011 0.015
0.094 0.167
0.606 0.082
10 10
0 (0–1) 0 (0–4)
9 9
1 (0–1.5) 1 (0–2)
15 14
1 (0–3.5) 0.8 (0–2)
1 1
0 (0–0) 0 (0–0)
0.002 0.370
0.007 0.224
0.033 0.245
0.620 0.873
0.819 0.695
PM: partial mole, CM: complete mole, CCA: choriocarcinoma.
Please cite this article as: Shoni M, et al, p-21-Activated kinase-1, -4 and -6 and estrogen receptor expression pattern in normal placenta and gestational trophoblastic diseases, Gynecol Oncol (2013), http://dx.doi.org/10.1016/j.ygyno.2013.09.010
M. Shoni et al. / Gynecologic Oncology xxx (2013) xxx–xxx
[16]
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[1] Lala PK, Graham CH. Mechanisms of trophoblast invasiveness and their control: the role of proteases and protease inhibitors. Cancer Metastasis Rev 1990;9:369–79. [2] Huppertz B, Kertschanska S, Demir AY, Frank HG, Kaufmann P. Immunohistochemistry of matrix metalloproteinases (MMP), their substrates, and their inhibitors (TIMP) during trophoblast invasion in the human placenta. Cell Tissue Res 1998;291:133–48. [3] Seckl MJ, Sebire NJ, Berkowitz RS. Gestational trophoblastic disease. Lancet 2010;376:717–29. [4] Shih Ie M. Gestational trophoblastic neoplasia–pathogenesis and potential therapeutic targets. Lancet Oncol 2007;8:642–50. [5] Zhang HJ, Siu MK, Yeung MC, Jiang LL, Mak VC, Ngan HY, et al. Overexpressed PAK4 promotes proliferation, migration and invasion of choriocarcinoma. Carcinogenesis 2011;32:765–71. [6] Siu MK, Yeung MC, Zhang H, Kong DS, Ho JW, Ngan HY, et al. P21-Activated kinase-1 promotes aggressive phenotype, cell proliferation, and invasion in gestational trophoblastic disease. Am J Pathol 2010;176:3015–22. [7] Kumar R, Gururaj AE, Barnes CJ. P21-activated kinases in cancer. Nat Rev Cancer 2006;6:459–71. [8] Jaffer ZM, Chernoff J. P21-activated kinases: three more join the Pak. Int J Biochem Cell Biol 2002;34:713–7. [9] Aoki H, Yokoyama T, Fujiwara K, Tari AM, Sawaya R, Suki D, et al. Phosphorylated Pak1 level in the cytoplasm correlates with shorter survival time in patients with glioblastoma. Clin Cancer Res 2007;13:6603–9. [10] Ching YP, Leong VY, Lee MF, Xu HT, Jin DY, Ng IO. P21-activated protein kinase is overexpressed in hepatocellular carcinoma and enhances cancer metastasis involving c-Jun NH2-terminal kinase activation and paxillin phosphorylation. Cancer Res 2007;67:3601–8. [11] Bagheri-Yarmand R, Mandal M, Taludker AH, Wang RA, Vadlamudi RK, Kung HJ, et al. Etk/Bmx tyrosine kinase activates Pak1 and regulates tumorigenicity of breast cancer cells. J Biol Chem 2001;276:29403–9. [12] Carter JH, Douglass LE, Deddens JA, Colligan BM, Bhatt TR, Pemberton JO, et al. Pak-1 expression increases with progression of colorectal carcinomas to metastasis. Clin Cancer Res 2004;10:3448–56. [13] Schraml P, Schwerdtfeger G, Burkhalter F, Raggi A, Schmidt D, Ruffalo T, et al. Combined array comparative genomic hybridization and tissue microarray analysis
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Our abstract has been accepted for an oral presentation at the 2013 World Congress XVII of the Society for the Study of Trophoblastic Diseases (ISSTD) in Chicago, IL.
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Please cite this article as: Shoni M, et al, p-21-Activated kinase-1, -4 and -6 and estrogen receptor expression pattern in normal placenta and gestational trophoblastic diseases, Gynecol Oncol (2013), http://dx.doi.org/10.1016/j.ygyno.2013.09.010
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Contents lists available at ScienceDirect
Gynecologic Oncology journal homepage: www.elsevier.com/locate/ygyno
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Melina Shoni a, Zoltan Nagymanyoki b, Allison F. Vitonis a, Cynthia Jimenez b, Shu-Wing Ng a, Bradley J. Quade b, Ross S. Berkowitz a,c,⁎ a b c
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p-21-Activated kinase-1, -4 and -6 and estrogen receptor expression pattern in normal placenta and gestational trophoblastic diseases
Division of Gynecologic Oncology, Department of Obstetrics and Gynecology, Brigham and Women's Hospital, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA, USA Division of Women's and Perinatal Pathology, Department of Pathology, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA Donald P Goldstein, MD Trophoblastic Tumor Registry, New England Trophoblastic Disease Center, MA, USA
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Objectives. To delineate the potential role of p21-activated kinases (PAKs) in the pathogenesis of gestational trophoblastic diseases (GTD) by defining the expression pattern of PAK-1, -4 and -6 and their potential implication in estrogen receptor (ER) regulation of normal placental tissue and GTD. Methods. We evaluated immunohistochemically 10 normal first-trimester placentas (NP), 10 partial moles (PM), 15 complete moles (CM) and 3 choriocarcinomas (CCA) for PAK-1, PAK-4, PAK-6 and ER expression intensity and localization. Staining outcomes were assessed utilizing non-parametric Kruskal–Wallis one-way analysis of variance test followed by pairwise Wilcoxon Rank Sum tests. Statistical significance was determined by twosided p-value of b 0.05. Results. In NP, PAK-6 immunoreactivity was predominantly cytoplasmic. Compared to NP, PM and CM demonstrated significant increase of cytoplasmic PAK-6 in cytotrophoblast (p = 0.012, p = 0.033 respectively), accompanied by significantly increased nuclear immunoreactivity in cytotrophoblast (p = 0.008, p = 0.045 respectively) and intermediate trophoblast (p = 0.003, p = 0.015 respectively). PAK-4 was found significantly upregulated in both cytoplasmic and nuclear compartments of cytotrophoblast and syncytiotrophoblast in PM (p = 0.004 and p = 0.002 for cytotrophoblast; p = 0.018 and p = 0.002 for syncytiotrophoblast, respectively) and CM (p = 0.001 and p = 0.001 for cytotrophoblast; p = 0.002 and p = 0.001 for syncytiotrophoblast, respectively) when compared to NP, whereas PAK-1 expression was significantly reduced in the syncytiotrophoblast of PM (p = 0.025 for cytoplasm and p = 0.008 for nucleus). Nuclear expression of ER was undetectable in all stained samples. Conclusion. Our results reveal PAK-6 upregulation in GTD compared to NP. The absence of nuclear expression of ER might stem in part from the repressive effect of PAK-6 in trophoblastic tissue. © 2013 Published by Elsevier Inc.
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Article history: Received 24 July 2013 Accepted 11 September 2013 Available online xxxx
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Keywords: Gestational trophoblastic diseases p-21-Activated Kinases PAK-6 Estrogen receptors
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• p-21-Activated Kinase-6 (PAK-6) has increased expression in gestational trophoblastic diseases and might be implicated in its pathophysiology. • p-21-Activated Kinases (PAKs) may play an important role in regulating and limiting estrogen receptor expression in gestational trophoblastic tissue.
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Introduction
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Perturbations of the balance between cellular proliferation, differentiation, migration and death lead to aberrant tissue development. Even in its healthy state, placental trophoblastic tissue exhibits a malignant-like, though tightly controlled, behavior [1] by invading with its extravillous component the endometrium and developing a rich uterine vasculature to generate an intimate connection between the fetus and the mother [2]. Failure of the underlying regulatory mechanisms seems implicated
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⁎ Corresponding author at: 75 Francis Street, Boston, MA 02115. E-mail address: [email protected] (R.S. Berkowitz).
28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 49 48
in the development of gestational trophoblastic diseases (GTD), as reflected on its premalignant forms — complete and partial hydatidiform mole (HM) — which have an increased potential of persistence and malignant transformation, as well as its malignant spectrum of invasive mole, choriocarcinoma (CCA), placental site trophoblastic tumor (PSTT) and epithelioid trophoblastic tumor (ETT) [3]. While there is growing understanding of the molecular biology of GTD, the precise pathways that confer such aberrations need further exploration [4]. Recent studies point to a functional role of p21-activated kinases (PAKs) in promoting related tumorogenic pathways [5,6]. PAKs comprise a well characterized group of conserved, non-receptor serine/threonine kinase isoforms, downstream effectors of Rho-family
0090-8258/$ – see front matter © 2013 Published by Elsevier Inc. http://dx.doi.org/10.1016/j.ygyno.2013.09.010
Please cite this article as: Shoni M, et al, p-21-Activated kinase-1, -4 and -6 and estrogen receptor expression pattern in normal placenta and gestational trophoblastic diseases, Gynecol Oncol (2013), http://dx.doi.org/10.1016/j.ygyno.2013.09.010
60 61 62 63 64 65 66 67 68 69 70 71
91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110
t1:1 t1:2
Table 1 Human malignancies where PAKs have been found deregulated. PAK isoform
t1:4 t1:5
Group I PAK-1
Cancer type (alteration)
brain (increased phospho-PAK-1 in cytoplasm) breast (protein overexpression and increased nuclear localization) liver and kidney (protein and gene overexpression) colon (protein overexpression) bladder (gene amplification (11q13Aq14 amplicon) lung (protein overexpression) ovarian (protein overexpression and gene amplification (11q13Aq14 amplicon) T-cell lymphoma (gene amplification) NF1 (PAK-1 deletion), NF2 (PAK1 deletion) gestational trophoblastic diseases choriocarcinoma – neuroendocrine (protein overexpression)
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t1:3
t1:6 t1:7 t1:8 t1:9
t1:10 t1:11
115
Under protocol approved by the Brigham and Women's Hospital Institutional Review Board, paraffin-embedded, gestationally agematched normal placenta from elective first trimester abortions (NP) (n = 10), partial molar (PM) (n = 10) and complete molar (CP) (n =15) tissues as well as choriocarcinomas (CCA) (n = 3) were provided by the Division of Women's and Perinatal Pathology. Precisely, the mean gestational age was 8.8 weeks in normal pregnancies, 11.6 weeks in complete and 9.1 weeks in partial molar pregnancies. Persistent gestational trophoblastic neoplasia (GTN) developed in 2 of 15 CM cases only. Molar tissues were collected at the time of uterine evacuation while normal placentas were obtained at the time of elective pregnancy termination. Clinical information including gestational age, pathologic diagnosis and development of persistent gestational trophoblastic neoplasia was collected for all cases. Conventional histologic sections were made of paraffin-embedded tissues and routinely stained with hematoxylin– eosin (HE). The underlying diagnosis was based on standard histopathologic criteria as determined by experienced gynecologic pathologists.
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Placental trophoblastic tissue samples
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Materials and methods
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of PAKs in the pathophysiology of GTD by determining the expression 111 and distribution of PAK-1, PAK-4 and PAK-6 as well as the potential ef- 112 fect of PAK-1 and PAK-6 on ER regulation in normal placenta and GTD. 113
PAK-2 PAK-3 Group II PAK-4
PAK-5 PAK-6
esophagus (protein overexpression) breast (protein overexpression and increased nuclear localization) pancreas (gene amplification (19q13 amplicon), protein overexpression) colon (protein overexpression, gene amplification (19q13 amplicon) and 2 somatic mutations) choriocarcinoma – prostate (protein overexpression)
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Immunolocalization of PAK-1, PAK-4, PAK-6 and estrogen receptors
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Immunohistochemistry (IHC) was performed on formalin-fixed, paraffin-embedded trophoblastic tissue slides. The corresponding blocks were sectioned at a thickness of 7 μm, mounted on Superfrost Plus microscopic slides (Fisher Scientific, Pittsburgh, PA), and dried at 50 °C for at least 24 h. Deparaffinization was achieved using xylene and rehydration with a graded ethanol series. Antigen retrieval was performed within antigen-unmasking solution (Vector Laboratories, Burlingame, CA) utilizing a pressure-cooker. Endogenous peroxidases were then blocked using 3% H202 in distilled water for 15 min. Following a 30-min blocking with the corresponding normal serum, sections were incubated with primary antibodies (rabbit anti-PAK-1 antibody, Cell Signaling Technology Inc., dilution 1:100; rabbit anti-PAK-4, Cell Signaling Inc., dilution 1:150; rabbit anti-PAK-6, Sigma Aldrich, dilution 1:200; mouse antiER, Sigma, dilution 1:80) at 4 °C overnight. The reaction was visualized using Vectastain Elite ABC Kit with diaminobenzidine chromogen as a substrate (Vector Laboratories, Burlingame, CA). Sections were lightly counterstained with hematoxylin and mounted. Immunohistochemical staining of cytotrophoblast, syncytiotrophoblast, intermediate trophoblast and decidual stromal cells was evaluated independently by two experienced pathologists using a semi-quantitative scoring system. The final histological score was obtained by multiplying the percentage of the stained area (0 when absent, 1+ when less than 25%, 2+ when 25% to 50% and 3+ when more than 50% of the total epithelial area) with the intensity of the stain (from 0 for no stain to 3 for intense stain).
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Statistical analysis
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The non-parametric Kruskal–Wallis one-way analysis of variance test was used to identify significant differences among independent variable groups. This was followed by pairwise Wilcoxon Rank Sum test to determine which group differences were statistically significant, as defined by the two-sided p-value of b0.05. All statistical analyses were performed utilizing SAS.
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GTPases (especially Rho, Rac and Cdc42). Divided into two distinct groups based on structural and functional similarities (group I: PAK1-3 and group II: PAK4-6), these pluripotent kinases interact with a wide array of intracellular proteins exerting regulatory control over hallmark biological processes, including cell morphogenesis and survival, gene transcription, actin cytoskeletal dynamics and motility, angiogenesis, growth factor- and steroid receptor- signaling, and energy homeostasis [7,8]. PAKs overexpression or hyperactivation in various human malignancies (Table 1) indicates their role in cancer-related signaling networks [9–14]. PAKs modulate the expression of genes for both survival and metastatic potential through transcriptional modulator substrates and by their association with nuclear chromatin [9–14]. With regard to GTD, PAK-1 was found to enhance cellular proliferation and invasive phenotypes [6], whereas PAK-4 was reported to potentiate proliferation, invasion and migration in choriocarcinomas [5]. To date, less is known about PAK-6 in GTD. Findings link PAKmediated signaling to the steroid hormone receptor pathway, with PAK-6 first identified by yeast two-hybrid screening as an androgen receptor (AR)-interacting protein [15]. Since the latter pertains to the same nuclear receptor superfamily with estrogen receptors (ER), similar PAK-6/ER interactions could be anticipated. Although PAK-6 is primarily localized in the cytoplasm, in the presence of AR or ER-α and the respective ligand, PAK-6 can translocate into the nucleus as part of an AR/ER-α complex and repress AR/ER-α-mediated transcription through both kinase-dependent and -independent manner [15–17]. From this standpoint, PAK-6 inhibits prostate tumorogenesis by regulating AR homeostasis [18]. However, the inhibitory effect of PAK-6 on AR- and ER-mediated gene transcription is opposed by the ER transactivation as induced by PAK-1-mediated receptor phosphorylation [19]. Mammary tissues with active PAK-1 exhibit stimulated ER-mediated transactivation and expression of endogenous ER target genes, revealing a novel role for the PAK-1/ ER pathway in promoting hyperplasia of mammary epithelium [19]. Previous attempts for the elucidation of the role of sex hormone receptors in GTD revealed almost negative ER expression in HM and placentas from spontaneous abortions [20,21]. On the other hand, Jiang et al. [22] highlight the importance of functional ER in controlling choriocarcinoma cell proliferation, suggesting a potential role of estrogens in mediating placental trophoblastic growth. In the present study, we aimed at expanding our understanding of the potential role
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Please cite this article as: Shoni M, et al, p-21-Activated kinase-1, -4 and -6 and estrogen receptor expression pattern in normal placenta and gestational trophoblastic diseases, Gynecol Oncol (2013), http://dx.doi.org/10.1016/j.ygyno.2013.09.010
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Normal
Cytotrophoblastic cytoplasm nucleus Syncytiotrophoblastic cytoplasm nucleus Intermediate trophoblast cytoplasm nucleus Decidual cell cytoplasm nucleus
CCA
n
Median (range)
n
Median (range)
n
Median (range)
n
Median (range)
KW p
Normal vs. PM
Normal vs. CM
PM vs. CM
Normal vs. CM
10 10
5 (2–6) 9 (4–9)
10 10
4.5 (3–9) 6 (6–9)
14 14
6 (2–9) 9 (4–9)
2 2
2.5(2–3) 5 (4–6)
0.160 0.358
0.871 0.538
0.224 0.920
0.371 0.410
0.167 0.226
10 10
3 (0–6) 6 (1–9)
10 10
1 (0–3) 1.5(1–4)
14 14
2.5 (0–6) 4 (0–6)
2 2
2.5(2–3) 4 (2–6)
0.023 0.011
0.025 0.008
0.574 0.100
0.012 0.040
0.588 0.592
10 10
6 (4–9) 9 (4–9)
10 10
6 (3–9) 9 (4–9)
15 15
6 (3–9) 9 (4–9)
2 2
2.5 (2–3) 5 (4–6)
0.095 0.181
0.721 0.724
0.769 0.862
0.463 0.527
0.059 0.119
9 9
3 (0–3) 2 (0–6)
9 9
1 (0–3) 2 (0–4)
15 15
3 (0–6) 6 (1–9)
2 2
1.5 (0–3) 3 (0–6)
0.102 0.020
0.513 0.334
0.174 0.137
0.027 0.004
0.999 0.903
PM: partial mole, CM: complete mole, CCA: choriocarcinoma.
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Results
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Immunohistochemical staining outcomes
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Immunohistochemical staining of normal placenta for PAK-1 revealed cytoplasmic and nuclear expression in all cellular groups (cytotrophoblastic, syncytiotrophoblastic, intermediate trophoblastic and decidual stromal cells). Apart from decidual cells, nuclear expression was higher than cytoplasmic. These outcomes were retained in CM and CCA cases. In PM, although with the same cellular immunolocalization, PAK-1 exhibited decreased expression compared to normal placenta, which reached statistical significance only in syncytiotrophoblastic cells (p = 0.025 for cytoplasmic expression and p =0.008 for nuclear expression). Table 2 demonstrates in detail the immunohistochemical expression of PAK-1 in normal placenta and GTD. In normal placenta, PAK-4 immunoreactivity was predominantly confined to the cytoplasm of all cellular groups with occasional nuclear staining detected in intermediate trophoblastic cells. When CM and PM were compared to normal placenta, PAK-4 cytoplasmic expression was significantly increased in cytotrophoblastic cells (p = 0.001 and p = 0.004, respectively) and syncytiotrophoblastic cells (p = 0.002 and p = 0.018, respectively). This was also accompanied by significantly increased nuclear immunoreactivity in cytotrophoblastic cells (p = 0.001 and p = 0.002, respectively) and syncytiotrophoblastic cells (p =0.001 and p = 0.002, respectively). CCA intermediate cells showed reduced cytoplasmic and nuclear stain, which, however, did not reach statistical significance. Table 3 reviews in detail the immunohistochemical outcomes of PAK-4 staining in normal placenta and GTD. Immunohistochemical staining of normal placenta for PAK-6 demonstrated primarily cytoplasmic expression that spared only the decidual stromal cells. This staining pattern was intensified in PM tissue, reaching a statistically significant increase in the cytoplasm of cytotrophoblastic cells (p = 0.012) and a significant simultaneous PAK-6 translocation in the nucleus of cytotrophoblastic (p = 0.007) and intermediate trophoblastic cells (p = 0.003). These significant changes were also noticed in CM cases, which additionally exhibited increased cytoplasmic expression in intermediate (p = 0.011) and decidual cells (p = 0.033). Nuclear cytotrophoblastic and intermediate trophoblastic expression was also increased in CCA but statistical significance was not reached. However, the limited number of the included CCA cases might need to be considered for the interpretation of this outcome. Table 4 demonstrates in detail the immunohistochemical outcomes of PAK-6 staining in normal placenta and GTD. ER expression, both cytoplasmic and nuclear, was undetectable in normal placental tissue and this negative pattern was also retained in all of our PM and CM as well as CCA cases. However, weak ER staining
183 184 185 186 187 188 189 190 191 192 193 194 195 196 197 198 199 200 201 202 203 204 205 206 207 208 209 210
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was observed in the decidual cells of normal placenta and GTD while 211 endometrial stromal and glandular cells were found strongly positive 212 and thus, were used as positive internal controls. 213
N C O
175 176
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173 174
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t2:17
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Kruskal Wallis (KW) & Wilcoxon Rank Sum test p-values
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t2:5 t2:6 t2:7 t2:8 t2:9 t2:10 t2:11 t2:12 t2:13 t2:14 t2:15 t2:16
CM
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t2:4
PM
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PAK-1
Discussion
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Trophoblastic tumor biology stems from its placental origin with aberrations in a heterogeneous population of cells, including villous cytotrophoblasts, syncytiotrophoblasts, extravillous trophoblasts and intermediate trophoblasts [3]. p21-activated kinases (PAKs) are a conserved family of six serine/threonine kinases implicated in salient pathways of aberrant tissue development [23], including but not limited to anchorage-independent growth (mainly through the canonical MAP kinase cascade of Ras/Raf/MeK/ERK), inhibition of apoptosis (through the BAD/Bcl-2 pathway) and stimulation of cellular motility (by targeting LIM kinase) [7]. In this study, we demonstrated an upregulated expression profile of PAK-6 in GTD and investigated immunohistochemically the potential role of PAK members in the regulation of ER in trophoblastic tissue. The rationale for this investigation was in part based on previously published data demonstrating a potential role for PAK-1 and PAK-4 in GTD. PAK-1, the best-characterized member of the PAK family, has been placed as a convergence point in cellular transformation induced by various small GTPases and mitogenic factors [24]. Siu et al. [6] found upregulated PAK-1 expression in hydatidiform mole (HM) and choriocarcinoma (CCA) when compared to normal placentas, a pattern that was associated with more aggressive behavior and increased potential of HM progression to gestational trophoblastic neoplasia (GTN). The expression level of PAK-1 was overall decreased in PM compared to normal placenta, reaching statistical significance only in syncytiotrophoblastic cells (p =0.025 for cytoplasmic expression and p = 0.008 for nuclear expression). CM and CCA retained the PAK1 expression pattern of normal placenta. Our immunohistochemical outcomes for PAK-1 differ from these data and this might be in part attributable to the different sample categories that have been assessed in these studies. Whereas Siu et al. [6] distinguished HM into nonpersistent and persistent HM, they did not distinguish PM versus CM, as we did. Moreover, it should be noted that the limited number of cases included in the study may contribute to a potential bias and this should also be considered when interpreting the data. Contrary to PAK-1, our findings regarding PAK-4 expression were in agreement with the outcomes of Zhang et al. [5], who reported ubiquitously expressed PAK-4 in normal placenta. According to the same study, PAK4 was found to affect cell proliferation, migration and invasion of CCA cells through deregulation of p16, CDK6 and MT1-MMP. We found that the predominantly cytoplasmic staining of PAK-4 in normal
215 216
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t2:3
Table 2 Immunohistochemical expression of PAK-1 in normal placenta and gestational trophoblastic diseases.
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t2:1 t2:2
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Please cite this article as: Shoni M, et al, p-21-Activated kinase-1, -4 and -6 and estrogen receptor expression pattern in normal placenta and gestational trophoblastic diseases, Gynecol Oncol (2013), http://dx.doi.org/10.1016/j.ygyno.2013.09.010
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Table 3 Immunohistochemical expression of PAK-4 in normal placenta and gestational trophoblastic diseases. PAK-4
Normal
t3:4 t3:5 t3:6 t3:7 t3:8 t3:9 t3:10 t3:11 t3:12 t3:13
Cytotrophoblastic cytoplasm nucleus Syncytiotrophoblastic cytoplasm nucleus Intermediate trophoblast cytoplasm nucleus
PM
CM
CCA
n
Median (range)
n
Median (range)
n
Median (range)
n
Median (range)
KW p
Normal vs. PM
Normal vs. CM
PM vs. CM
Normal vs. CCA
10 10
3 (0–4) 0 (0–1)
10 10
6 (3–9) 3 (0–9)
10 10
6 (4–9) 5 (1–9)
0 0
– –
0.001 0.001
0.004 0.002
0.001 0.001
0.899 0.649
– –
10 10
3 (0–6) 0 (0–2)
10 10
6 (0–9) 4 (0–9)
10 10
6 (6–9) 6 (2–9)
0 0
– –
0.001 0.001
0.018 0.002
0.002 0.001
0.082 0.228
– –
2 2
3 (0–6) 2 (0–4)
8 8
6 (3–6) 2 (0–6)
13 13
3 (0–6) 1 (0–6)
3 3
0 (0–3) 0 (0–0)
0.065 0.258
0.441 0.895
0.860 0.931
0.101 0.540
0.763 0.460
PM: partial mole, CM: complete mole, CCA: choriocarcinoma.
255
281 282
placenta was significantly increased in both partial and complete HM accompanied by an obvious nuclear translocation of PAK-4 in cytotrophoblastic and syncytiotrophoblastic cells (p = 0.001 and p =0.001, respectively). To further advance previous studies, we investigated the expression profile of PAK-6 and we found significant upregulation in GTD compared to normal placenta. In terms of protein localization, PAK-6 was completely cytoplasmic in normal placenta. However, PAK-6 had significantly increased immunoreactivity not only in the cytoplasm but also in the nucleus of the cytotrophoblastic and intermediate trophoblastic cells of PM and CM. This finding is in line with the theory that suggests that PAK-6 has a role in regulating steroid receptor-mediated nuclear gene transcription. The ability of PAKs to interact with and modulate nuclear receptor functions also raises the possibility that PAKs may play a role in the hormonal independence of trophoblastic tissue. PAK6 interacts with the nuclear receptor (NR) superfamily members, androgen receptor (AR) and estrogen receptor α (ER-α), which play key roles in development, hormone signaling and hormone-independent carcinomas [15–17]. It is unclear whether PAK-6 overexpression confers oncogenic or counteracting tumor suppressing capacity in GTD. In keeping with the function of PAK-6 in hormone signaling, overexpression of PAK-6 has been reported in prostate cancer [25] at a time when a study demonstrates its tumor suppressing role in prostate tumor growth [18]. Notably, increased PAK-6 and ERα interaction is also observed in tamoxifen treated breast cancer cells [26]. In order to elucidate potential relationships between PAK-6 and ER in GTD we examined the expression status of ER in our tissue samples but the nuclear immunoreactivity to ER was undetectable in all normal placentas and GTD tissues. This is
t4:1 t4:2
Table 4 Immunohistochemical expression of PAK-6 in normal placenta and gestational trophoblastic diseases.
270 271 272 273 274 275 276 277 278
Author's disclosures of potential conflict of interest
302
E
D
P
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283
t4:4 t4:5 t4:6 t4:7 t4:8 t4:9 t4:10 t4:11 t4:12 t4:13 t4:14 t4:15 t4:16 t4:17
Cytotrophoblastic cytoplasm nucleus Syncytiotrophoblastic cytoplasm nucleus Intermediate trophoblast cytoplasm nucleus Decidual cell cytoplasm nucleus
286 287 288 289 290 291 292 293 294 295 296 297 298 299 300 301
304
Acknowledgments
We would like to thank the Foundation for Education and European 305 Culture (IPEP), Greece, for the generous support on this research effort. 306
N
PAK-6
U
t4:3
284 285
303
The authors indicated no potential conflicts of interest.
C
279 280
in agreement with previous studies [20,21] but since our assessment has been approached immunohistochemically, this does not exclude ER expression in immunohistochemically undetectable levels. The opposing role that PAK-1 and PAK-6 exert on ER might have also an impact on this finding. Moreover, we should not forget that besides the natural estrogen ligand, several signaling molecules including Akt, protein kinase A and pp90rsk1 have been shown to phosphorylate the AF1 domain of ER, which contributes to the transactivation function of ER [27,28]. In summary, we have defined the expression profile of PAK-6 in normal placenta and GTD. PAK-6 upregulation in GTD may provide a novel molecular pathway to investigate and functional studies are warranted to elucidate the precise role of PAK-6 on the hormonal dependence or independence of trophoblastic tumorigenesis. Because PAKs may act as multifunctional regulators in GTD and in light of the drug-discovery efforts that have already led to the development of several direct and indirect PAK inhibitors [29], functional studies focused on PAK targeting may shed more light on the therapeutic potential of this pathway in GTD.
T
268 269
C
266 267
E
264 265
R
262 263
R
260 261
O
258 259
O
t3:14
256 257
Kruskal Wallis (KW) & Wilcoxon Rank Sum test p-values
F
t3:1 t3:2
M. Shoni et al. / Gynecologic Oncology xxx (2013) xxx–xxx
Normal
PM
CM
CCA
Kruskal Wallis (KW) & Wilcoxon Rank Sum test p-values
n Median (range)
n Median (range)
n Median (range)
n Median (range)
KW p
Normal vs. PM
Normal vs. CM
PM vs. CM
Normal vs. CCA
10 10
1.25(0.5–2) 0.5 (0–2)
10 10
2.5 (1–3) 2.5 (1–4)
14 14
2(0.5–3.5) 2 (0–4)
3 3
2 (1–6) 1 (1–4)
0.025 0.016
0.012 0.007
0.033 0.045
0.349 0.142
0.190 0.284
10 10
1 (0–2) 0 (0–0.5)
10 10
1(0.5–3.5) 0.5(0–0.5)
14 14
1 (0.5–3) 0 (0–0.5)
3 3
1(1–9) 0 (0–0)
0.535 0.135
0.558 0.099
0.226 0.672
0.478 0.154
0.434 0.513
10 10
1 (0–2) 0 (0–2)
10 10
1.3 (1–2) 2.5 (1–4)
14 14
3 (0.5–5) 1.5 (0–4)
3 3
1 (0.5–6) 1 (1–3)
0.030 0.003
0.088 0.003
0.011 0.015
0.094 0.167
0.606 0.082
10 10
0 (0–1) 0 (0–4)
9 9
1 (0–1.5) 1 (0–2)
15 14
1 (0–3.5) 0.8 (0–2)
1 1
0 (0–0) 0 (0–0)
0.002 0.370
0.007 0.224
0.033 0.245
0.620 0.873
0.819 0.695
PM: partial mole, CM: complete mole, CCA: choriocarcinoma.
Please cite this article as: Shoni M, et al, p-21-Activated kinase-1, -4 and -6 and estrogen receptor expression pattern in normal placenta and gestational trophoblastic diseases, Gynecol Oncol (2013), http://dx.doi.org/10.1016/j.ygyno.2013.09.010
M. Shoni et al. / Gynecologic Oncology xxx (2013) xxx–xxx
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Our abstract has been accepted for an oral presentation at the 2013 World Congress XVII of the Society for the Study of Trophoblastic Diseases (ISSTD) in Chicago, IL.
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Please cite this article as: Shoni M, et al, p-21-Activated kinase-1, -4 and -6 and estrogen receptor expression pattern in normal placenta and gestational trophoblastic diseases, Gynecol Oncol (2013), http://dx.doi.org/10.1016/j.ygyno.2013.09.010
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