Core2 O-glycan-expressing prostate cancer cells are resistant to NK cell immunity

Abstract

Core2 β-1,6-N-acetylglucosaminyltransferase (C2GnT) forms an N-acetylglucosamine branch in the O-glycans (core2 O-glycans) of cell surface glycoproteins. We previously revealed that the expression of C2GnT is positively correlated with poor prognosis in prostate cancer patients. However, the detailed mechanisms underlying their poor prognosis remain unclear. In the current study, we report that the core2 O-glycans carried by the surface MUC1 glycoproteins of prostate cancer cells play an important role in the evasion of NK cell immunity. In C2GnT‑expressing prostate cancer cells, the MUC1 core2 O-glycans are modified with poly-N-acetyllactosamine. MUC1 glycoproteins carrying poly-N-acetyllactosamine attenuated the interaction of the cancer cells with NK cells, resulting in decreased secretion of granzyme B by the NK cells. Poly‑N‑acetyllactosamine also interfered with the ability of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) to access the cancer cell surface. These effects of poly-N-acetyllactosamine on NK cells render C2GnT-expressing prostate cancer cells resistant to NK cell cytotoxicity. By contrast, C2GnT-deficient prostate cancer cells carrying a lower amount of poly-N-acetyllactosamine than the C2GnT-expressing prostate cancer cells were significantly more susceptible to NK cell cytotoxicity. Our results strongly suggest that C2GnT-expressing prostate cancer cells evade NK cell immunity and survive longer in the host blood circulation, thereby resulting in the promotion of prostate cancer metastasis.

Figure 1

Establishment of core2 β-1,6-N-acetylglucosaminyltransferase (C2GnT)-deficient prostate cancer cells. (A) Biosynthesis pathway of O-glycan core structures, core1-4. N-acetylgalactosamine (GalNAc) is transferred to serine (Ser) or threonine (Thr) residues in a polypeptide by peptide GalNAc transferase (GalNAc-T). GalNAcα1-Ser/Thr is converted by Core1 synthase to Galβ1-3GalNAcα1-Ser/Thr (core1). Core1 is then converted by C2GnT-1 to core2. Core1 is also converted by Core3 synthase to core3. Core3 is converted by C2GnT-2 to core4. β-1,4-galactosyltransferase IV (β1-4Gal-T IV) together with β-1,3-N-acetylglucosaminyltransferase (β1-3GlcNAc-T) synthesize poly-N-acetyllactosamine in core2 branched oligosaccharides. Lycoperiscon esculentum (tomato) lectin (LEL) binds specifically to poly-N-acetyllactosamine with at least three lactosamine unit repeats. (B) Relative expression levels of C2GnT in prostate cancer cell lines were analyzed by RT-PCR. (C) Cell morphologies of PC3 and C2GnT-deficient cells. Bar is 50 μm.

Figure 2

Core2 O-glycosylation of MUC1 in core2 β-1,6-N-acetylglucosaminyltransferase (C2GnT)-expressing prostate cancer cells. Total cell lysates from PC3 and C2GnT-deficient PC3 (C2KD-1) prostate cancer cells, were analyzed by western blotting with anti-MUC1 (lanes 1 and 2), anti-lysosome-associated membrane glycoprotein 1 (anti-LAMP1) (lanes 3 and 4) and anti-actin (lanes 5 and 6) as a sample loading control. Total lysates were immunoprecipitated with Lycoperiscon esculentum lectin (LEL)-agarose followed by western blotting analysis with anti-MUC1 (lanes 7 and 8).

Figure 3

Effects of poly-N-acetyllactosamine on NK cell functions (A) Conjugate formation of NK cells with PC3 (open bar) and C2GnT-deficient PC3 (C2KD-1; closed bar) cells. (B) Granzyme B secretion by NK cells upon stimulation with the NK cell-target prostate cancer cell interaction was measured by assaying the protease activity of granzyme B in the co-culture supernatants of PC3 (open bar) and C2KD-1 (closed bar) cells. Specific granzyme B secretion was expressed as a percentage of the total cellular enzyme activity after subtracting the spontaneous release. Mean values ± SE of three independent experiments.

Figure 4

Effect of core2 β-1,6-N-acetylglucosaminyltransferase (C2GnT) expression on NK cell cytotoxicity. (A) Cytotoxicity of human NK cells against PC3 (open circles) and C2GnT-deficient PC3 (C2KD-1; closed circles) was assayed. (B) Expression of death receptor 4 (DR4), a receptor for tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in the PC3 (lane 1) and C2KD-1 (lane 2) cells was analyzed by western blotting. (C) Cell viability of PC3 (open circles) and C2KD-1 (closed circles) cells. Cells were incubated with soluble recombinant TRAIL for 24 h at the indicated concentrations. Cell viability was determined using Cell Counting Kit-8. Mean values ± SE of three independent experiments.

Figure 5

Schematic drawing of the evasion of NK cell immunity by core2 β-1,6-N-acetylglucosaminyltransferase (C2GnT)-expressing prostate cancer cells. (A) Prostate cancer cells with low or no C2GnT expression are susceptible to NK cell immunity. NK cells are activated by the interaction of the NK receptor (NKR) with its ligand (NKR-L) expressed on the surface of cancer cells. Activated NK cells secrete perforin and granzyme B to induce the apoptosis of target cancer cells. In addition, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) expressed in NK cells interacts with death receptors expressed in tumor cells such as death receptor 4 (DR4) to induce the apoptosis of target tumor cells. (B) Prostate cancer cells with high C2GnT expression levels are resistant to NK cell immunity. Poly-N-acetyllactosamine carried by MUC1 core2 O-glycans attenuates the NK cell-cancer cell interaction due to its bulkiness. The attenuated interaction results in decreased NK cell degranulation and also interferes with the access of TRAIL to DR4, impairing the TRAIL-mediated killing of target tumor cells. This shielding of MUC1 carrying core2 O-glycans and poly-N-acetyllactosamine increases the survival of the C2GnT-expressing prostate cancer cells in the host blood circulation, promoting the prostate cancer metastasis.