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

Gubbels JA, Felder M, Horibata S, Belisle JA, Kapur A, Holden H, Petrie S, Migneault M, Rancourt C, Connor JP, Patankar MS - Mol. Cancer (2010)

Bottom Line: Our results indicate that MUC16-mediated inhibition of immune synapse formation is an effective mechanism employed by ovarian tumors to evade immune recognition.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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Obstetrics and Gynecology, University of Wisconsin-Madison, Madison, WI, USA.

ABSTRACT

Background: Cancer 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.

Results: Expression 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 MUC16(low) 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.

Conclusion: MUC16, 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.

Show MeSH

Related in: MedlinePlus

Expression of mucins and NK cell activating and inhibitory molecules on csMUC16neg-OVC and csMUC16pos-OVC. MUC1 expression on both sublines is similar and MUC4 expression is absent. Expression of DNAM-1 and NKG2D ligands on the sublines was determined by staining with Fc chimeras (15 μg/mL) of these two receptors followed by labeling with fluorophore conjugated anti-Fc secondary antibody. HLA class I expression on the sublines was determined by labeling with FITC conjugated w6/32. Histograms of live single events are shown in all plots. Data is representative of three independent experiments. Shaded peaks show control cells incubated with only the fluorescently-tagged secondary antibodies. Un-shaded histograms are for cells incubated with the primary antibodies or Fc chimeras and the fluorescently labeled secondary antibodies.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2818693&req=5

Figure 3: Expression of mucins and NK cell activating and inhibitory molecules on csMUC16neg-OVC and csMUC16pos-OVC. MUC1 expression on both sublines is similar and MUC4 expression is absent. Expression of DNAM-1 and NKG2D ligands on the sublines was determined by staining with Fc chimeras (15 μg/mL) of these two receptors followed by labeling with fluorophore conjugated anti-Fc secondary antibody. HLA class I expression on the sublines was determined by labeling with FITC conjugated w6/32. Histograms of live single events are shown in all plots. Data is representative of three independent experiments. Shaded peaks show control cells incubated with only the fluorescently-tagged secondary antibodies. Un-shaded histograms are for cells incubated with the primary antibodies or Fc chimeras and the fluorescently labeled secondary antibodies.

Mentions: The significantly reduced ability of NK cells to bind to csMUC16high OVCAR-3 cells suggested a potential role for this mucin in protecting ovarian cancer cells from immune recognition. To investigate this immuno-protective role, we employed MUC16 knock-down cells that were successfully utilized in our recent studies [35,37]. The subline csMUC16neg-OVC expresses an endoplasmic reticulum localized scFv fragment of the anti-MUC16 antibody VK-8 that prevents expression of csMUC16 and sMUC16. A control subline designated csMUC16pos-OVC that expresses an scFv fragment of an irrelevant murine IgG has no effect on the expression of csMUC16 and sMUC16 [37]. Expression of csMUC16 on both sublines is shown in Figure 3. The scFv-MUC16 complex is rapidly degraded in the proteasomes and the mucin does not accumulate in the cytoplasm of the tumor cells (see additional file 1). The csMUC16pos-OVC and csMUC16neg-OVC were negative for MUC4 expression and did not exhibit any major difference in the levels of MUC1 (Figure 3). This is important to note as it has previously been shown that both MUC1 and MUC4 exert anti-adhesive effects on immune synapse formation [38,39].


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

Gubbels JA, Felder M, Horibata S, Belisle JA, Kapur A, Holden H, Petrie S, Migneault M, Rancourt C, Connor JP, Patankar MS - Mol. Cancer (2010)

Expression of mucins and NK cell activating and inhibitory molecules on csMUC16neg-OVC and csMUC16pos-OVC. MUC1 expression on both sublines is similar and MUC4 expression is absent. Expression of DNAM-1 and NKG2D ligands on the sublines was determined by staining with Fc chimeras (15 μg/mL) of these two receptors followed by labeling with fluorophore conjugated anti-Fc secondary antibody. HLA class I expression on the sublines was determined by labeling with FITC conjugated w6/32. Histograms of live single events are shown in all plots. Data is representative of three independent experiments. Shaded peaks show control cells incubated with only the fluorescently-tagged secondary antibodies. Un-shaded histograms are for cells incubated with the primary antibodies or Fc chimeras and the fluorescently labeled secondary antibodies.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2818693&req=5

Figure 3: Expression of mucins and NK cell activating and inhibitory molecules on csMUC16neg-OVC and csMUC16pos-OVC. MUC1 expression on both sublines is similar and MUC4 expression is absent. Expression of DNAM-1 and NKG2D ligands on the sublines was determined by staining with Fc chimeras (15 μg/mL) of these two receptors followed by labeling with fluorophore conjugated anti-Fc secondary antibody. HLA class I expression on the sublines was determined by labeling with FITC conjugated w6/32. Histograms of live single events are shown in all plots. Data is representative of three independent experiments. Shaded peaks show control cells incubated with only the fluorescently-tagged secondary antibodies. Un-shaded histograms are for cells incubated with the primary antibodies or Fc chimeras and the fluorescently labeled secondary antibodies.
Mentions: The significantly reduced ability of NK cells to bind to csMUC16high OVCAR-3 cells suggested a potential role for this mucin in protecting ovarian cancer cells from immune recognition. To investigate this immuno-protective role, we employed MUC16 knock-down cells that were successfully utilized in our recent studies [35,37]. The subline csMUC16neg-OVC expresses an endoplasmic reticulum localized scFv fragment of the anti-MUC16 antibody VK-8 that prevents expression of csMUC16 and sMUC16. A control subline designated csMUC16pos-OVC that expresses an scFv fragment of an irrelevant murine IgG has no effect on the expression of csMUC16 and sMUC16 [37]. Expression of csMUC16 on both sublines is shown in Figure 3. The scFv-MUC16 complex is rapidly degraded in the proteasomes and the mucin does not accumulate in the cytoplasm of the tumor cells (see additional file 1). The csMUC16pos-OVC and csMUC16neg-OVC were negative for MUC4 expression and did not exhibit any major difference in the levels of MUC1 (Figure 3). This is important to note as it has previously been shown that both MUC1 and MUC4 exert anti-adhesive effects on immune synapse formation [38,39].

Bottom Line: Our results indicate that MUC16-mediated inhibition of immune synapse formation is an effective mechanism employed by ovarian tumors to evade immune recognition.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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Obstetrics and Gynecology, University of Wisconsin-Madison, Madison, WI, USA.

ABSTRACT

Background: Cancer 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.

Results: Expression 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 MUC16(low) 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.

Conclusion: MUC16, 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.

Show MeSH
Related in: MedlinePlus