Integrin Inhibitors as a Therapeutic Agent for Ovarian Cancer

Ovarian cancer is a deadly disease, with a cure rate of only 30%. Despite aggressive treatments, relapse remains almost inevitable in patients with advanced-stage disease. In recent years, great progress has been made towards targeting integrins in cancer treatment, and clinical studies with various integrin inhibitors have demonstrated their effectiveness in blocking cancer progression. Given that the initial critical step of ovarian cancer metastasis is the attachment of cancer cells onto the peritoneum or omentum, in addition to the proven positive clinical results of anti-angiogenic therapy, targeting integrins is likely to be one of the most feasible approaches. This paper summarizes the current understanding of the integrin biology in ovarian cancer metastasis and the various therapeutic approaches attempted with integrin inhibitors. Although no integrin inhibitors have shown favorable results so far, integrin-targeted therapies continue to be a promising approach to be explored for further clinical investigation

Interleukin 6 receptor is an independent prognostic factor and a potential therapeutic target of ovarian cancer.

Ovarian cancer remains the most lethal gynecologic cancer and new targeted molecular therapies against this miserable disease continue to be challenging. In this study, we analyzed the expressional patterns of Interleukin-6 (IL-6) and its receptor (IL-6R) expression in ovarian cancer tissues, evaluated the impact of these expressions on clinical outcomes of patients, and found that a high-level of IL-6R expression but not IL-6 expression in cancer cells is an independent prognostic factor. In in vitro analyses using ovarian cell lines, while six (RMUG-S, RMG-1, OVISE, A2780, SKOV3ip1 and OVCAR-3) of seven overexpressed IL-6R compared with a primary normal ovarian surface epithelium, only two (RMG-1, OVISE) of seven cell lines overexpressed IL-6, suggesting that IL-6/IL-6R signaling exerts in a paracrine manner in certain types of ovarian cancer cells. Ovarian cancer ascites were collected from patients, and we found that primary CD11b+CD14+ cells, which were predominantly M2-polarized macrophages, are the major source of IL-6 production in an ovarian cancer microenvironment. When CD11b+CD14+ cells were co-cultured with cancer cells, both the invasion and the proliferation of cancer cells were robustly promoted and these promotions were almost completely inhibited by pretreatment with anti-IL-6R antibody (tocilizumab). The data presented herein suggest a rationale for anti-IL-6/IL-6R therapy to suppress the peritoneal spread of ovarian cancer, and represent evidence of the therapeutic potential of anti-IL-6R therapy for ovarian cancer treatment

IL-6 receptor expression but not IL-6 expression correlates with poor prognosis in patients with ovarian cancer.

<p>(A) Immunohistochemical staining of tissue microarrays with malignant ovarian tissue sections. Representative areas of four different ovarian cancers stained using an anti-human IL-6R antibody and scored as “Low” or “High”. Placentae complicated with chorioamnionitis were used as a positive control. Arrows indicate membranous staining in trophoblasts. A negative control is nonimmune sera. (B) IL-6 receptor expression correlates with poor prognosis in patients with ovarian cancer. Kaplan-Meier curves of progression free survival (<i>left</i>) and overall survival (<i>right</i>) of ovarian cancer patients treated at Gifu University Hospital (n = 94). (C) Representative areas of four different ovarian cancers stained using an anti-human IL-6 antibody and scored as “Low” or “High”. Placentae complicated with chorioamnionitis were used as a positive control. Arrows indicate cytoplasmic staining in villous mesenchymal cells. A negative control is nonimmune sera. (D) IL-6 expression did not affect the prognosis in patients with ovarian cancer. Kaplan-Meier curves of progression free survival (<i>left</i>) and overall survival (<i>right</i>) of patients. Original magnification, ×200. Scale bar in each panel represents 50 μm.</p

Proposed paracrine mechanism.

<p>CD11b⁺CD14⁺ cells, which are predominantly macrophages, are one of the major sources of IL-6 production in an ovarian tumor microenvironment and promoted ovarian cancer invasion, proliferation and angiogenesis.</p

CD11b⁺CD14⁺ cells from ovarian cancer ascites promote ovarian cancer cell invasion and proliferation <i>via</i> producing IL-6.

<p>(A) The protocol of isolation of CD11b^-, CD11b⁺CD14^- and CD11b⁺CD14⁺ cells using magnetic-activated cell sorting (MACS) technology (Miltenyi Biotech). ELISA assay of IL-6 (B) and sIL-6R (C). 1 x 10⁵ of SKOV3ip1 cells and primary cells indicated in the figure were plated onto 6-well plates and cultured with 2 ml of serum-free medium for 72 h. Conditioned media were collected and the concentrations of human IL-6 (B) as well as sIL-6R (C) were measured by ELISA. Experiments were repeated three times and values are means (±SD) of triplicates. (D) Matrigel invasion assay. 1 x 10⁵ SKOV3ip1 cells were placed on the upper chamber with the same number of primary cells indicated in the figure seeded on the bottom chamber as a chemoattractant, and were allowed to invade for 72 h. The relative number of invading cells when no cells were plated on the bottom chamber was set as 1.0. (E) Anti-IL-6R antibody inhibited ovarian cancer cell invasion induced by CD11b⁺CD14⁺ cells. In this experiment, the co-culture experiment in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118080#pone.0118080.g004" target="_blank">Fig. 4D</a> was repeated with the addition of the 10 μg/ml of anti-IL-6R antibody or non-immune IgG in the bottom chamber. Representative pictures of transwells are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118080#pone.0118080.g004" target="_blank">Fig. 4D</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118080#pone.0118080.g004" target="_blank">4E</a> (<i>bottom</i>). (F) In vitro cell proliferation assay. 1 x 10⁴ SKOV3ip1 cells were plated in 24-well plates. Thereafter, polycarbonate filters with 1-μm pores were placed onto 24-well plates and the same number of primary cells indicated in the figure were seeded as a stimulant and cells were cultured for 72 h. Cell proliferation was expressed as the ratio of the number of viable cells. (G) Anti-IL-6R antibody inhibited ovarian cancer cell proliferation induced by CD11b⁺CD14⁺ cells. In this experiment, the co-culture experiment in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118080#pone.0118080.g004" target="_blank">Fig. 4F</a> was repeated with the addition of the 10 μg/μl of anti-IL-6R antibody or non-immune IgG in the upper chamber. Experiments were repeated three times and values are means ± SD of triplicates. n.s.; not significant, n.d.; not detected, *; P < 0.05, **; P < 0.01.</p

Characteristics of the patients (n = 94).

<p>Characteristics of the patients (n = 94).</p

Multivariate analysis for progression free survival of the patients.

<p>HR, hazard ratio;</p><p>95% CI, 95% confidence interval.</p><p>Multivariate analysis for progression free survival of the patients.</p

Immunohistochemical analyses of IL-6 in high-grade serous ovarian cancer tissues.

<p>Serial sections of stage III high-grade serous ovarian cancer tissues were immunostained with anti-IL-6 antibody (A, C) and anti-CD-68 antibody (B, D). IL-6 was strongly expressed in stroma, while cancer cells little expressed IL-6. (A, B) Sections from a 56 year-old female with stage IIIC high-grade serous ovarian cancer. (C, D) sections from a 63 year-old female with stage IIIC high-grade serous ovarian cancer. CD68 staining identified macrophages. Arrows indicate macrophages. Arrowheads indicate ovarian cancer cells. Original magnification, x100 (upper panels), and x400 (bottom panels). Black bar; 200 μm, red bar; 50 μm.</p

Exogenous treatment of IL-6 promotes ovarian cancer cell proliferation, invasion and VEGF production.

<p>(A) A matrigel invasion assay was done using a modified Boyden chamber system. 1 x 10⁵ of SKOV3ip1 (<i>left</i>) or RMUS-S (<i>right</i>) cells were placed on the top chamber in serum-free medium and allowed to invade for 72 h. Various concentrations of IL-6 (1–100 ng/ml) or 60 ng/ml of sIL-6R were applied in the bottom chamber as a chemoattractant. 10 μg/ml of anti-IL-6R antibody or non-immune IgG was co-treated. Non-invading cells were removed using a cotton swab, and invading cells on the underside of the filter were enumerated. Relative numbers of invading cells with respect to the control (no IL-6 treatment) are shown. (B) <i>In vitro</i> cell proliferation assay. 1 x 10⁴ cells of SKOV3ip1 (<i>left</i>) or RMG1 (<i>right</i>) cells were plated in 24-well plates in 10% FBS/DMEM for 24 h and then incubated in serum-free medium in the presence or absence of various concentrations of IL-6 (1–100 ng/ml) with or without anti-IL-6R antibody or non-immune IgG as control for 72 h. Cell proliferation was evaluated by a modified MTS assay. Cell proliferation was expressed as the ratio of the number of viable cells. (C) ELISA assay of VEGF-A. 1 x 10⁵ SKOV3ip1 cells were plated onto 6-well plates and cultured with 2 ml of serum-free medium in the presence or absence of 100 ng/ml of IL-6 for 72 h. Anti-IL-6R antibody or control IgG was co-treated. Conditioned media were collected and the concentration of human VEGF-A was measured by ELISA. Experiments were repeated three times and values are means ± SD of triplicates. n.s.; not significant, *; P < 0.05, **; P < 0.01.</p