Jennifer A. A. Gubbels

Mesothelin-MUC16 binding is a high affinity, N-glycan dependent interaction that facilitates peritoneal metastasis of ovarian tumors

BackgroundThe mucin MUC16 and the glycosylphosphatidylinositol anchored glycoprotein mesothelin likely facilitate the peritoneal metastasis of ovarian tumors. The biochemical basis and the kinetics of the binding between these two glycoproteins are not clearly understood. Here we have addressed this deficit and provide further evidence supporting the role of the MUC16-mesothelin interaction in facilitating cell-cell binding under conditions that mimic the peritoneal environment.ResultsIn this study we utilize recombinant-Fc tagged human mesothelin to measure the binding kinetics of this glycoprotein to MUC16 expressed on the ovarian tumor cell line OVCAR-3. OVCAR-3 derived sublines that did not express MUC16 showed no affinity for mesothelin. In a flow cytometry-based assay mesothelin binds with very high affinity to the MUC16 on the OVCAR-3 cells with an apparent Kd of 5–10 nM. Maximum interaction occurs within 5 mins of incubation of the recombinant mesothelin with the OVCAR-3 cells and significant binding is observed even after 10 sec. A five-fold molar excess of soluble MUC16 was unable to completely inhibit the binding of mesothelin to the OVCAR-3 cells. Oxidation of the MUC16 glycans, removal of its N-linked oligosaccharides, and treatment of the mucin with wheat germ agglutinin and erythroagglutinating phytohemagglutinin abrogates its binding to mesothelin. These observations suggest that at least a subset of the MUC16-asscociated N-glycans is required for binding to mesothelin. We also demonstrate that MUC16 positive ovarian tumor cells exhibit increased adherence to A431 cells transfected with mesothelin (A431-Meso+). Only minimal adhesion is observed between MUC16 knockdown cells and A431-Meso+ cells. The binding between the MUC16 expressing ovarian tumor cells and the A431-Meso+ cells occurs even in the presence of ascites from patients with ovarian cancer.ConclusionThe strong binding kinetics of the mesothelin-MUC16 interaction and the cell adhesion between ovarian tumor cells and A431-Meso+ even in the presence of peritoneal fluid strongly support the importance of these two glycoproteins in the peritoneal metastasis of ovarian tumors. The demonstration that N-linked glycans are essential for mediating mesothlein-MUC16 binding may lead to novel therapeutic targets to control the spread of ovarian carcinoma.

MUC16 provides immune protection by inhibiting synapse formation between NK and ovarian tumor cells

BackgroundCancer cells utilize a variety of mechanisms to evade immune detection and attack. Effective immune detection largely relies on the formation of an immune synapse which requires close contact between immune cells and their targets. Here, we show that MUC16, a heavily glycosylated 3-5 million Da mucin expressed on the surface of ovarian tumor cells, inhibits the formation of immune synapses between NK cells and ovarian tumor targets. Our results indicate that MUC16-mediated inhibition of immune synapse formation is an effective mechanism employed by ovarian tumors to evade immune recognition.ResultsExpression of low levels of MUC16 strongly correlated with an increased number of conjugates and activating immune synapses between ovarian tumor cells and primary naïve NK cells. MUC16-knockdown ovarian tumor cells were more susceptible to lysis by primary NK cells than MUC16 expressing controls. This increased lysis was not due to differences in the expression levels of the ligands for the activating receptors DNAM-1 and NKG2D. The NK cell leukemia cell line (NKL), which does not express KIRs but are positive for DNAM-1 and NKG2D, also conjugated and lysed MUC16-knockdown cells more efficiently than MUC16 expressing controls. Tumor cells that survived the NKL challenge expressed higher levels of MUC16 indicating selective lysis of MUC16low targets. The higher csMUC16 levels on the NKL resistant tumor cells correlated with more protection from lysis as compared to target cells that were never exposed to the effectors.ConclusionMUC16, a carrier of the tumor marker CA125, has previously been shown to facilitate ovarian tumor metastasis and inhibits NK cell mediated lysis of tumor targets. Our data now demonstrates that MUC16 expressing ovarian cancer cells are protected from recognition by NK cells. The immune protection provided by MUC16 may lead to selective survival of ovarian cancer cells that are more efficient in metastasizing within the peritoneal cavity and also at overcoming anti-tumor innate immune responses.

Peritoneal natural killer cells from epithelial ovarian cancer patients show an altered phenotype and bind to the tumour marker MUC16 (CA125)

The ovarian tumour marker MUC16 (CA125) inhibits the cytotoxic responses of human natural killer (NK) cells and down‐regulates CD16. Here we show that approximately 10% of the peripheral blood NK cells (PBNK) from the epithelial ovarian cancer (EOC) patients are CD16– CD56br whereas 40% of the peritoneal fluid NK (PFNK) carry this phenotype, which is usually associated with NK cells from the lymph nodes or human decidua. PBNK from healthy donors exposed to PF show a significant increase in the CD16– CD56br population. This shift in phenotype is not caused by increased apoptosis of the CD16+ CD56dim cells or selective proliferation of the CD16– CD56br NK cells. Thus, the terminal differentiation of the CD16– CD56br NK cells to CD16+ CD56dim subset that occurs during normal NK cell development may actually be a reversible step. A majority of the NK cell receptors (NKp46, NKp44, NKG2D, CD244, CD226, CD158a, CD158b, and CD158e) studied were down‐regulated in the PFNK. MUC16 binds selectively to 30–40% of CD16+ CD56dim NK cells in EOC patients indicating that phenotypic alterations in these cells are mediated by tumour‐derived soluble factors. Similar to EOC, MUC16 in early pregnancy also binds to NK cells suggesting shared mechanisms of NK cell suppression in feto‐maternal tolerance and immune evasion by ovarian cancers.

The detection, treatment, and biology of epithelial ovarian cancer

Ovarian cancer is particularly insidious in nature. Its ability to go undetected until late stages coupled with its non-descript signs and symptoms make it the seventh leading cause of cancer related deaths in women. Additionally, the lack of sensitive diagnostic tools and resistance to widely accepted chemotherapy regimens make ovarian cancer devastating to patients and families and frustrating to medical practitioners and researchers. Here, we provide an in-depth review of the theories describing the origin of ovarian cancer, molecular factors that influence its growth and development, and standard methods for detection and treatment. Special emphasis is focused on interactions between ovarian tumors and the innate and adaptive immune system and attempts that are currently underway to devise novel immunotherapeutic approaches for the treatment of ovarian tumors.

Ab-IL2 fusion proteins mediate NK cell immune synapse formation by polarizing CD25 to the target cell-effector cell interface

The huKS-IL2 immunocytokine (IC) consists of IL2 fused to a mAb against EpCAM, while the hu14.18-IL2 IC recognizes the GD2 disialoganglioside. They are under evaluation for treatment of EpCAM+ (ovarian) and GD2+ (neuroblastoma and melanoma) malignancies because of their proven ability to enhance tumor cell killing by antibody-dependent cell-mediated cytotoxicity (ADCC) and by antitumor cytotoxic T cells. Here, we demonstrate that huKS-IL2 and hu14.18-IL2 bind to tumor cells via their antibody components and increase adhesion and activating immune synapse (AIS) formation with NK cells by engaging the immune cells’ IL-2 receptors (IL2R). The NK leukemia cell line, NKL (which expresses high affinity IL2Rs), shows fivefold increase in binding to tumor targets when treated with IC compared to matching controls. This increase in binding is effectively inhibited by blocking antibodies against CD25, the α-chain of the IL2R. NK cells isolated from the peritoneal environment of ovarian cancer patients, known to be impaired in mediating ADCC, bind to huKS-IL2 via CD25. The increased binding between tumor and effector cells via ICs is due to the formation of AIS that are characterized by the simultaneous polarization of LFA-1, CD2 and F-actin at the cellular interface. AIS formation of peritoneal NK and NKL cells is inhibited by anti-CD25 blocking antibody and is 50–200% higher with IC versus the parent antibody. These findings demonstrate that the IL-2 component of the IC allows IL2Rs to function not only as receptors for this cytokine but also as facilitators of peritoneal NK cell binding to IC-coated tumor cells.

Differential internalization of hu14.18‐IL2 immunocytokine by NK and tumor cell: impact on conjugation, cytotoxicity, and targeting

The hu14.18‐IL2 (EMD 273063) IC, consisting of a GD2‐specific mAb genetically engineered to two molecules of IL‐2, is in clinical trials for treatment of GD2‐expressing tumors. Anti‐tumor activity of IC in vivo and in vitro involves NK cells. We studied the kinetics of retention of IC on the surface of human CD25+CD16– NK cell lines (NKL and RL12) and GD2+ M21 melanoma after IC binding to the cells via IL‐2R and GD2, respectively. For NK cells, ∼50% of IC was internalized by 3 h and ∼90% by 24 h of cell culture. The decrease of surface IC levels on NK cells correlated with the loss of their ability to bind to tumor cells and mediate antibody‐dependent cellular cytotoxicity in vitro. Unlike NK cells, M21 cells retained ∼70% of IC on the surface following 24 h of culture and maintained the ability to become conjugated and lysed by NK cells. When NKL cells were injected into M21‐bearing SCID mice, IT delivery of IC augmented NK cell migration into the tumor. These studies demonstrate that once IC binds to the tumor, it is present on the tumor surface for a prolonged time, inducing the recruitment of NK cells to the tumor site, followed by tumor cell killing.

Characterization of the tumor marker muc16 (ca125) expressed by murine ovarian tumor cell lines and identification of a panel of cross-reactive monoclonal antibodies

ObjectivesThe ovarian tumor marker CA125 is expressed on human MUC16, a cell surface bound mucin that is also shed by proteolytic cleavage. Human MUC16 is overexpressed by ovarian cancer cells. MUC16 facilitates the binding of ovarian tumor cells to mesothelial cells lining the peritoneal cavity. Additionally, MUC16 also is a potent inhibitor of natural killer cell mediated anti-tumor cytotoxic responses. Extensive studies using human as well as murine ovarian tumor cell models are required to clearly define the function of MUC16 in the progression of ovarian tumors. The major objective of this study was to determine if the murine ovarian tumor cells, MOVCAR, express Muc16 and to characterize antibodies that recognize this mucin.MethodsRT-PCR analysis was used for detecting the Muc16 message and size exclusion column chromatography for isolating Muc16 produced by MOVCAR cells. Soluble and cell-associated murine Muc16 were analyzed, respectively, by Western blotting and flow cytometry assays using a new panel of antibodies. The presence of N-linked oligosaccharides on murine Muc16 was determined by ConA chromatography.ResultsWe demonstrate that murine Muc16 is expressed by mouse ovarian cancer cells as an ~250 kDa glycoprotein that carries both O-linked and N-linked oligosaccharides. In contrast to human MUC16, the murine ortholog is primarily released from the cells and cannot be detected on the cell surface. Since the released murine Muc16 is not detected by conventional anti-CA125 assays, we have for the first time identified a panel of anti-human MUC16 antibodies that also recognizes the murine counterpart.ConclusionThe antibodies identified in this study can be used in future purification of murine Muc16 and exhaustive study of its properties. Furthermore, the initial identification and characterization of murine Muc16 is a vital preliminary step in the development of effective murine models of human ovarian cancer. These models will aid in the further elucidation of the role that human MUC16 plays in the etiology and progression of ovarian tumors.

The mucin MUC16 (CA125) binds to NK cells and monocytes from peripheral blood of women with healthy pregnancy and preeclampsia

MUC16 (CA125) released from ovarian tumors binds to NK cells and monocytes via the inhibitory receptor Siglec‐9. Here, we investigate whether MUC16 also binds to circulating immune cells during pregnancy and in women with preeclampsia.

The immunomodulating roles of glycoproteins in epithelial ovarian cancer.

The complexity of the immune system demands an intricate defense mechanism by tumors. Ovarian and other tumors employ specific glycoproteins and the associated glycan sequences to modulate immune responses. Glycoproteins enable tumor cells that express or secrete these molecules to evade immune cell attack and induce the immune system to promote tumor growth. This review focuses first on the immune environment in ovarian cancer, and the mechanisms of activation and inhibition that immune cells undergo in order to either attack or ignore a target cell. Next we illustrate the immunomodulatory roles of ovarian cancer-associated glycans and glycoproteins in 1. preventing immune synapse formation, 2. serving as ligands of immune cell receptors, 3. scavenging cytokines and chemokines, and 4. participating in the formation of autoantibodies against the tumor. The importance of these immunomodulating strategies from the view points of understanding the tumor immunology of ovarian tumors, potential origin of such mechanisms, and specific strategies to circumvent the glycoconjugate-mediated suppression of immune responses is discussed in this review.

Abstract 301A: A novel epithelial ovarian cancer protein, SUSD2, inhibits platelet activation and binding to tumor cells

Over 30% of ovarian cancer (OvCa) patients present with thrombocytosis (elevated plasma platelet count above 450,000 per cubic millimeter) at the time of diagnosis. These patients exhibit shorter survival times and a higher likelihood of advanced stage disease. Platelets have also been shown to enhance metastasis and promote tumor cell survival by coating tumor cells which provides immune escape and protection from chemotherapeutic agents. While previous studies have shown that platelets exhibit differential OvCa cell binding and facilitate cancer cell growth, the mechanism and molecules involved have yet to be elucidated. SUSD2 (SUShi Domain containing 2), an 822 amino acid type I transmembrane protein, is highly abundant in ovarian tumors and normal endothelial cells that line the blood vessels. Endothelial cells do not normally adhere to platelets; therefore, we hypothesized that SUSD2 inhibits the binding of platelets to cancer cells, which subsequently decreases platelet activation. To investigate the role of SUSD2 in platelet binding, SUSD2 knock-down (SUSD2neg) and non-targeting (SUSD2pos) OVCAR3 cell lines were generated. SUSD2pos and SUSD2neg OVCAR3 cell lines were cultured and grown to confluency. Platelets were isolated, dyed with Calcein-AM, washed and added to the cancer cells. After a 15-minute incubation, the co-culture was washed to remove non-adherent platelets. We demonstrated that platelets were bound to the OVCAR3-SUSD2neg an average of 35% more compared to OVCAR3-SUSD2pos cells. In order to identify the receptor on the platelets mediating adhesion to the tumor cells, we used the drug eptifibatide to block GPIIb/IIIa, an integrin receptor on the platelet surface that primarily binds to fibrinogen. Using the same methods described above, platelets were incubated with the inhibitor before being co-incubated with the cancer cells. Treating the platelets with eptifibatide decreased platelet binding by an average of 35% to both cancer cell lines, indicating that platelet binding to tumor cells is partially, yet equally, mediated by GPIIb/IIIa regardless of the presence of SUSD2. The role of this receptor was further explored by performing flow cytometry on the supernatant collected from co-culturing platelets with the cancer cells. The conditioned media contains the platelets that were not bound to the cancer cells. We measured the activation of the unbound platelets using the anti-PAC-1 antibody that binds exclusively to the activated conformation of GPIIb/IIIa. Incubation with the OVCAR-3-SUSD2neg cells activated the unbound platelets three-fold more compared to the OVCAR3-SUSD2pos cells. These data indicate that SUSD2 expression may mediate the activation state of GPIIb/IIIa, and this, in turn, may affect adhesion mediated by other factors. Citation Format: Tyson Lager, Megan Thacker, Charissa Etrheim, Kristi A. Egland, Mark K. Larson, Jennifer A. A. Gubbels. A novel epithelial ovarian cancer protein, SUSD2, inhibits platelet activation and binding to tumor cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 301A. doi:10.1158/1538-7445.AM2015-301A