Antitumor Effects of Interleukin-6 (IL-6)/Interleukin-6 Receptor (IL-6R) Signaling Pathway Inhibition in Clear Cell Carcinoma of the Ovary Nozomu Yanaihara,1* Yukihiro Hirata,1 Noriko Yamaguchi,1 Yukiko Noguchi,1 Misato Saito,1 Chie Nagata,1,2 Satoshi Takakura,1 Kyosuke Yamada,1 and Aikou Okamoto1 1 2

Department of Obstetrics and Gynecology, The Jikei University School of Medicine, Tokyo, Japan Department of Education for Clinical Research, National Center for Child Health and Development, Tokyo, Japan

Among epithelial ovarian cancers, clear cell carcinoma of the ovary (CCC) has unique clinical and molecular characteristics that include chemoresistance resulting in poor prognosis. It was shown that CCC recently was characterized by specific upregulation of the IL-6/IL-6R-signal transducer and activator of transcription 3 (Stat3) signaling pathway. In this study, we aim to clarify whether IL-6/IL-6R mediated signaling pathway could have clinical relations with CCC and to evaluate inhibitory effects of the pathway on CCC carcinogenesis. A total of 84 CCC cases collected from primary surgical specimens were evaluated by the immunohistochemical analysis for IL-6R and phosphorylated Stat3 (pStat3), and we found that high IL-6R expression correlated with poor patient survival both by the univariate and multivariate analyses, suggesting that IL-6/IL-6R signaling pathway could be implicated in the progression of CCC. We further investigated the effects of IL-6/IL-6R mediated signaling pathway inhibition either by IL-6R small interfering RNA (siRNA) approach or humanized anti-human IL-6R antibody (tocilizumab) in CCC. Inhibition of endogenous IL-6R including tocilizumab in CCC cells did reduce cell invasion ability and restored their response to cytotoxic reagent. These data suggest that IL-6/IL-6R signaling pathway could act on CCC cells to enhance invasion and chemoresistance and, therefore, targeting IL-6/IL-6R mediated signaling pathway could be a promising therapeutic strategy for CCC. © 2015 Wiley Periodicals, Inc.

Key words: IL-6; IL-6R; Stat3; tocilizumab; clear cell carcinoma of the ovary

INTRODUCTION Clear cell carcinoma of the ovary (CCC), differing from the other histological types of ovarian cancer in its clinical and molecular characteristics, behaves like a distinct entity [1–4]. In Japan, there were 9012 new ovarian cancer cases and 4599 cases of mortality due to ovarian cancer in 2008 (Center for Cancer Control and Information Services, National Cancer Center, Japan). The prevalence of CCC in Japan is now reaching up to approximately 25% of all cases of the cancer whereas that in western country is estimated as low as 1–12%. A recent meta-analysis showed that CCC had poorer prognosis than non-CCC, especially in advanced stages [5]. Molecular biological definition involved in the development and progression of CCC is emerging. We recently reported that CCC had a dominant Th-2 cytokine expression signature largely due to IL-6 that might be involved in the pathogenesis of this subtype, and that inhibition of IL-6 in CCC cells induced signal transducer and activator of transcription 3 (Stat3) inactivation and increased chemosensitivity [6]. Modulation of IL-6 expression or its related signaling pathway could, therefore, be suggested as a promising strategy of treatment for CCC. Combination of IL-6 signaling pathway inhibition and cytotoxic agent has been shown by small ß 2015 WILEY PERIODICALS, INC.

interfering RNA (siRNA) approach or by monoclonal anti-IL-6 antibody to disrupt progression of serous ovarian cancer in which constitutive production of IL-6 was found [7,8]. Furthermore, anti-IL-6 antibody (siltuximab) had therapeutic activities with platinumresistant ovarian cancer, including one patient with CCC, in Phase II clinical trial [9]. There is also Phase I/ II trial of siltuximab in solid tumors that shows no

Abbreviations: CCC, clear cell carcinoma of the ovary; FBS, fetal bovine serum; FIGO, International Federation of Gynecology and Obstetrics; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; gp130, glycoprotein 130; gp80, glycoprotein 80; IC50, half maximal (50%) inhibitory concentration; IL-6, interleukin-6; IL-6R, interleukin6 Receptor; MMP, matrix metalloproteinase; OS, overall survival; PCR, polymerase chain reaction; PFS, progression-free survival; pStat3, phosphorylated Stat3; SD, standard deviation; siRNA, small interfering RNA; Stat3, signal transducer and activator of transcription 3. Conflicts of interest: None. Grant sponsor: JSPS KAKENHI; Grant numbers: 25462615; 25462616; Grant sponsor: The Jikei University Research Fund *Correspondence to: Department of Obstetrics and Gynecology, The Jikei University School of Medicine, 3-25-8 Nishi-Shinbashi, Minato-ku, Tokyo 105-8461, Japan. Received 11 October 2014; Revised 6 March 2015; Accepted 16 March 2015 DOI 10.1002/mc.22325 Published online in Wiley Online Library (



clinical activity in 84 patients, of whom 29 had ovarian cancer (without histological information) [10]. In this study, we aim to clarify whether IL-6/IL-6R signaling pathway could have clinical relations with CCC and to investigate the effects of IL-6/IL-6R mediated signaling pathway inhibition in CCC. We further seek to evaluate the availability of tocilizumab, a humanized anti-human IL-6R antibody, as a therapeutic strategy for CCC. MATERIALS AND METHODS Cell Lines and Clinical Samples Eleven human CCC cell lines (JHOC-5, JHOC-7, JHOC-8, JHOC-9, HAC-2, RMG-I, RMG-II, OVTOKO, OVMANA, OVISE, and ES-2) and five human ovarian non-CCC cell lines were used in this study. RMG-I and RMG-II were maintained in Ham’s F12 medium (Sigma-Aldrich, Tokyo, Japan) with 10% fetal bovine serum (FBS). ES-2 was cultured in McCoy’s 5A (SigmaAldrich) with 10% FBS, and all other cells were cultured in RPMI-1640 (Sigma-Aldrich) containing 10% FBS. RMG-I, while RMG-II was provided by Dr. D. Aoki (Keio University, Tokyo, Japan) and HAC-2 by Dr. M. Nishida (Tsukuba University, Tsukuba, Japan). In addition, A2780 (undifferentiated carcinoma) and 2008 (serous adenocarcinoma) were provided by Dr. E. Reed (NCI, Bethesda, MD), and Dr. S.B. Howell (UCSD, San Diego, CA), respectively. JHOC-5, JHOC7, JHOC-8, and JHOC-9 were obtained from Riken Bioresource center (Tsukuba, Japan). OVTOKO, OVMANA, and OVISE were obtained from the JCRB Cell Bank (Osaka, Japan). We were able to secure SKOV3 (adenocarcinoma), MCAS (mucinous adenocarcinoma), OV-1063 (ovarian metastatic adenocarcinoma), and ES-2 from ATCC (Rockville, MD). The Jikei University School of Medicine Ethics Review Committee approved the study protocol and informed consent was obtained from each of the participating patients. Tumor specimens were surgically obtained from patients with CCC who were treated at the Department of Obstetrics and Gynecology, The Jikei University School of Medicine. Total of 84 samples were examined on hematoxylin and eosin–stained sections by pathologists and pure CCC were histologically confirmed. Tumors were staged in accordance with the International Federation of Gynecology and Obstetrics (FIGO) system (1988). FIGO stage was as follows: Stage I, 56; II, 7; III, 17; IV, 4. Quantitative Reverse Transcription-Polymerase Chain Reaction (PCR) Analysis Total RNA (3 mg), isolated with TRIzol reagent (Invitrogen, Carlsbad, CA), was submitted to complementary DNA base paring with random hexamers and SuperScript III First-Strand Synthesis kit (Invitrogen). TaqMan Gene Expression Assays (Applied Biosystems, Foster City, CA) were performed to assess Real-time Molecular Carcinogenesis

PCR Reaction for IL-6R (gp80) and gp130 using Applied Biosystems StepOnePlus Real-time PCR System (Applied Biosystems). GAPDH labeled with FAM reporter dye (Applied Biosystems) was used as an endogenous control, and a normal ovarian tissue, arbitrarily sampled for an intercomparison of cell lines, functioned as the reference [6]. Gene expression was quantified using the comparative method (2DDCT), where CT ¼ threshold cycle, DDCT ¼ (CT IL-6R  CT GAPDH)  (CT reference CT GAPDH) [11]. Reagents Tocilizumab was provided by Chugai Pharmaceutical Co., Ltd (Tokyo, Japan). Human recombinant IL-6 was purchased from PeproTech (London, UK). Antibodies against Stat3 (clone 79D7; 1:2000), phosphorylated Stat3 (pStat3) (Tyr705) (clone D3A7; 1:500), and b-actin (clone 13E5; 1:1000) were obtained from Cell Signaling Technology (Beverly, MA), and anti-IL-6Ra (clone C-20; 1:1000) from Santa Cruz Biotechnology (Santa Cruz, CA). Mouse monoclonal anti-MMP-2 (clone VB3; 1:500) and anti-MMP-9 (clone 56-2A4; 1:500) were purchased from Daiichi Fine Chemical (Toyama, Japan) and Abcam (Cambridge, UK), respectively. Western Blot Analysis Total cell lysates were prepared in 1 RIPA lysis buffer and protein concentration was prepared according to the DC Protein Assay protocol (Bio-Rad, Hercules, CA). Total protein (20–40 mg) was resolved on gradient NuPage 4–12% Bis-Tris gels (Invitrogen) and immunoblotted with specific antibodies. All blots were incubated with primary antibodies diluted in TBS with 0.1% Tween-20 and 5% bovine serum albumin for overnight at 48C with gentle agitation. Horseradish peroxidaseconjugated secondary anti-rabbit or anti-mouse antibody (Cell Signaling Technology; 1:10000) was diluted in TBS with 0.1% Tween-20 and 5% nonfat milk for 1 h at the room temperature with gentle agitation. Positive immunoreactions were detected using ImmunoStar LD chemiluminescence system (Wako, Tokyo, Japan). Small Interfering RNA (siRNA) Transfection of siRNA was performed using LipofectamineTM RNAiMAX (Invitrogen). Stealth RNAiTM siRNA for IL-6R (Invitrogen) and Stealth RNAi siRNA negative control (Invitrogen) were used for a comparative purpose and they had been transfected at the final concentration of 5 nM as per the manufacturer’s instruction. MTS Assay MTS assay was performed using the CellTiter 96 AQueous One Solution Cell Proliferation Assay kit (Promega, Madison, WI) following the manufacturer’s protocol. MTS solution was added to each of the 96-well plates and incubated for 4 h and, then, absorbance was measured at 490 nm using a microplate reader.



Cell Proliferation Assay


Two sets of assay materials were prepared, one with transfection and the other without it. After a 24-hour transfection, RMG-I cells and OVISE cells were reseeded into 96-well plates at a density of 1  104 and 1  103 cells per well, respectively, at least in triplicate. RMG-I and OVISE cells without transfection were also seeded into another set of 96-well plates at a density of 1  104 and 1  103 cells per well, respectively, with medium containing tocilizumab (1 mM) and/or IL-6 (0.1 ng/ml) reagents. MTS solution was added 4 h before each of the desired time points and cells were incubated at 378C. Data were collected as the average absorbance of the wells, and each experiment was repeated three times and values are presented as the mean  1 standard deviation (SD).

Immunohistochemical analyses for IL-6R and pStat3 expressions were performed on 3-mm paraffin sections of formalin-fixed, paraffin-embedded tissues with Ventana Discovery XT automated stainer (Ventana 20 Medical Systems, Tucson, AZ). After deparaffinization, antigen retrieval was carried out using CC1 buffer (Cell Conditioning 1; citrate buffer pH 6.0, Ventana Medical Systems). Expression levels were scored on the basis of the stain intensity and extent by two investigators (N. Y. and Y. H.). Immunohistochemical scoring was conducted entirely independently of all clinical variables. Positive staining in tumor cells was assigned using a semi-quantitative five-category grading system: 0, 60 years FIGO stage I, II III, IV Endometriosis Without With Residual tumor 1 cm >1 cm pStat3 Low High


Low (n ¼ 34)

High (n ¼ 50)

69 15

27 7

42 8


63 21

28 6

35 15


24 60

10 24

14 36


76 8

31 3

45 5


51 33

23 11

28 22



IL-6R, Interleukin-6 Receptor; FIGO, International Federation of Gynecology and Obstetrics; pStat3, phosphorylated signal transducer and activator of transcription.

(Table 2). The univariate analysis revealed that the clinicopathological determinants of reduced progression-free survival (PFS) included FIGO stage and IL-6R expression. Regarding overall survival (OS), residual tumor in addition to these two parameters were significantly related to reduced OS. Kaplan–Meier curves with P - values calculated by log rank tests according to IL-6R expression are shown in Figure 4. In the multivariate analysis, we adopted Cox proportional hazard models that included all of the variables assessed in the univariate analyses, and then, FIGO stage and IL-6R expression were identified as the key determinant of PFS and OS. On the other hand, residual tumor was found to be independent predictor for OS, but not for PFS. In addition, pStat3 expression was related to both PFS and OS with marginal statistical significance in the multivariate analysis. DISCUSSION The involvement of IL-6 in ovarian cancer, a pleiotropic cytokine that plays important roles in the immune response and inflammation, has been explored by several preclinical and clinical studies. Most of the clinical reports suggest an association between high IL-6 levels in serum and ascites, and poor prognosis of patients with ovarian cancer [14]. Earlier studies have shown that autocrine and paracrine effects of IL-6 in ovarian cancer could regulate tumorigenic properties by inducing IL-6 signaling pathway, strategies for which include Stat3 activation [14–17], angiogenesis [18], and



Table 2. Univariate and Multivariate Analyses for the Association of Clinicopathological Parameters and Gene Expression With Clinical Outcome in Patients With Clear Cell Carcinoma of the Ovary PFS Factors

Hazard ratio

Univariate Age (60 vs. >60 years) FIGO stage (I, II vs. III, IV) Endometriosis (without vs. with) Residual tumor (1 vs. >1 cm) IL-6R (low vs. high) pStat3 (low vs. high) Multivariate Age (60 vs. >60 years) FIGO stage (I, II vs. III, IV) Endometriosis (without vs. with) Residual tumor (1 vs. >1 cm) IL-6R (low vs. high) pStat3 (low vs. high)


95% CI


Hazard ratio

95% CI


1.106 6.401 1.112 4.356 2.858 1.338

0.104–3.051 0.839 4.088–24.029

interleukin-6 receptor (IL-6R) signaling pathway inhibition in clear cell carcinoma of the ovary.

Among epithelial ovarian cancers, clear cell carcinoma of the ovary (CCC) has unique clinical and molecular characteristics that include chemoresistan...
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