Cooperative participation of CagA and NFATc1 in the pathogenesis of antibiotics-responsive gastric MALT lymphoma

Abstract

Background: This study aimed to explore whether cytotoxin-associated gene A (CagA) can inhibit cell cycle progression by activating nuclear factor of activated T cells (NFAT) in lymphoma B cells and contribute to Helicobacter pylori eradication (HPE) responsiveness (complete remission [CR] after HPE) in gastric mucosa-associated lymphoid tissue (MALT) lymphoma.

Materials and methods: We co-cultured three B-lymphoma cell lines (MA-1, OCI-Ly3, and OCI-Ly7) with HP strains (derived from HPE-responsive gastric MALT lymphoma) and evaluated the expression patterns of CagA, phosphorylated (p)-CagA (CagA^P-Tyr), and CagA-signaling molecules, cell-cycle inhibitors, p-NFATc1 (Ser¹⁷²), and NFATc1 using western blotting. Furthermore, we evaluated the association between nuclear NFATc1 expression in the tumor cells of 91 patients who received first-line HPE (59 patients with HPE responsiveness and 32 without HPE responsiveness) and HPE responsiveness and CagA expression in tumor cells.

Results: In HP strains co-cultured with B cell lymphoma cell lines, CagA was translocated to the nucleus through tyrosine phosphorylation (CagA^P-Tyr) and simultaneously dephosphorylated NFATc1, subsequently causing nuclear NFATc1 translocation and stimulating the expression of p-SHP-2/p-ERK/Bcl-xL. Activated NFATc1 causes G1 cell cycle retardation in both MA-1 and OCI-Ly3 cells by triggering p21 and p27 production. Nuclear NFATc1 localization was significantly associated with the presence of CagA in gastric MALT lymphomas (80% [41/51] vs. 33% [13/40]; p < 0.001) and with HPE responsiveness (73% [43/59] vs. 25% [8/32]; p < 0.001). Patients exhibiting both the presence of CagA and nuclear NFATc1 localization responded more rapidly to HPE than those without (median interval to CR, 4.00 vs. 6.00 months, p = 0.003).

Conclusions: Our findings indicated that CagA and NFATc1 cooperatively participate in the lymphomagenesis of HPE-responsive gastric MALT lymphoma.

Keywords: Helicobacter pylori; CagA; MALT lymphoma; NFATc1; Stomach.

Fig. 1

CagA, tyrosine-phosphorylated CagA, and NFATc1 can be translocated to the nucleus in HP-co-cultured DLBCL cells. A Immunofluorescence showed that NFATc1 translocated to the nucleus in HP (HM#2)-co-cultured MA-1#46 cells (upper panel) (scale bar = 40 μm). Similarly, NFATc1 translocated to the nucleus in HP (HM#2)-co-cultured OCI-Ly3 cells (ABC-DLBCL) (middle panel) (scale bar = 15.9 μm). In HP (HM#2)-co-cultured OCI-Ly7 cells (GCB-DLBCL) (lower panel), NFATc1 also translocated to the nucleus (scale bar = 15.9 μm). B In HP (HM#2)-co-cultured MA-1 cells, nuclear CagA expression was observed at 1, 3, 6, and 24 h, whereas phosphorylated (p)-CagA (CagA^P−Tyr) expression was observed at 1 and 3 h after HP stimulation. However, the effect decreased at 6 h and eventually disappeared at 24 h. Simultaneously, nuclear NFATc1 expression was observed at 1, 3, and 6 h after HP stimulation and continued until 24 h in HP (HM#2)-co-cultured MA-1 cells. Compared with that noted in the control MA-1 cells, the expression of cytoplasmic p-NFATc1 decreased at 1 and 3 h, and the decreased effect was significantly noted at 6 and 24 h in HP (HM#2)-co-cultured MA-1 cells. The p-NFATc1 expression was not detected in the nucleus of MA-1 cells. C CagA and p-CagA translocated to the nucleus of the HP (HM#2)-co-cultured OCI-Ly3 cells at 1 and 6 h. Simultaneously, nuclear expression of NFATc1 was detected at 1 and 6 h after HP stimulation. In addition, the cytoplasmic expression of p-NFATc1 was significantly diminished at 1 and 6 h in HP (HM#2)-co-cultured OCI-Ly3 cells. After the treatment of clarithromycin (CAM, 0.25 mg/L) in HP (HM#2)-co-cultured OCI-Ly3 cells, CagA and p-CagA expression decreased at 6 h, and nuclear NFATc1 expression decreased simultaneously at 6 h, whereas cytoplasmic expression of p-NFATc1 was reversed at 1 and 6 h

Fig. 2

CagA, tyrosine-phosphorylated CagA, and NFATc1 can be translocated to the nucleus in HP-co-cultured GCB-DLBCL and gastric epithelial cells, CagA-related signaling molecules in HP-co-cultured DLBCL cell lines, and HP-co-cultured MA-1 cells and OCI-Ly3 cells exhibit G1 cell-cycle retardation. A In HP (HM#2)-co-cultured OCI-Ly7 cells, nuclear CagA expression was detected 1 and 3 h after HP stimulation; however, the effect decreased gradually at 6 h and decreased predominantly at 24 h. The expression of p-CagA in the nucleus was detected at 1 and 3 h; however, its signal intensity decreased at 6 h and eventually disappeared at 24 h. Simultaneously, the nuclear expression of NFATc1 was detected at 1, 3, 6, and 24 h after HP stimulation. B In HP (HS235)-co-cultured AGS cells, CagA and p-CagA translocated to the nucleus at 0.5 and 1 h. However, the signal intensity of both CagA and p-CagA decreased at 3 and 6 h. Simultaneously, nuclear expression of NFATc1 was detected at 0.5 and 1 h after HP stimulation; however, its signal intensity decreased at 3 and 6 h. C Compared with non-HP-co-cultured MA-1 cells, G1 arrest was predominantly detected at 24 h in HP (HM#2)-co-cultured MA-1 cells. However, G1 phase arrest was reversed after administration of CsA in HP (HM#2)-co-cultured MA-1 cells. The results were expressed in triplicate for each treatment group and measured by flow cytometry analysis (error bar means standard error). D Compared with that noted in non-HP-co-cultured OCI-Ly3 cells, G1 arrest was predominantly detected at 24 h in HP (HM#2)-co-cultured OCI-Ly3 cells. After the administration of CsA to HP (HM#2)-co-cultured OCI-Ly3 cells, the G1 arrest at 24 h was reversed. E Immunoblotting showed that HP induced the expression of CagA-related signaling molecules, including p-CagA, p-SHP2, p-ERK1/2, p21, p27, and Bcl-xL in HP (HM#2)-co-cultured MA-1 cells at 6 and 12 h. Quantification of western blotting in (E) showed that the expression levels of p-CagA, p-SHP2, p21, and p27 were higher at 6 than at 24 h, whereas levels of p-ERK1/2, and Bcl-xL were higher at 24 than at 6 h. F In HP (HM#2)-co-cultured OCI-Ly3 cells, HP provoked the expression of p-CagA, p-SHP2, p-ERK1/2, p21, p27, and Bcl-xL at 6 and at 12 h. Quantification of western blotting in (F) showed that the expression levels of p-CagA, p-SHP2, p-ERK1/2, p21, p27, and Bcl-xL were higher at 6 than at 24 h

Fig. 3

Nuclear expression pattern of NFATc1 in lymphoma cells and time to CR of patients with HPE-responsive gastric MALT lymphoma A Moderate nuclear localization of NFATc1 was found in the lymphoma cells of gastric mucosa in an HPE-responsive case (time to CR: 6.00 months); right upper inset, × 1000 B Strong nuclear localization of NFATc1 was found in the lymphoma cells of gastric mucosa in an HPE-responsive case (time to CR: 3.00 months); right upper inset, × 1000 C No nuclear localization of NFATc1 was found in lymphoma cells of gastric mucosa in an HPE-irresponsive case; right upper inset, × 1000 D A high baseline expression of nuclear localization of NFATc1 in lymphoma cells of an HPE-responsive case before HPE (case #2); right upper inset, × 1000 E Decreased expression of nuclear localization of NFATc1 in remitting tumor cells (case #2) 1.00 month after completion of HPE; left lower inset, × 1000 F No expression of nuclear localization of NFATc1 in remitting tumor cells (case #2) 4.00 months after completion of HPE (case #2, the time to CR: 7.00 month); left lower inset, × 1000 G Confocal laser-scanning microscopy showing that most CagA-positive cells (green fluorescence) expressed nuclear localization of NFATc1 (red fluorescence); Representative images of nucleus (stained with DAPI, blue) (right upper panel) and merged image of CagA and NFATc1 expression (right lower panel). H Time to CR was calculated from the completion of HPE to first evidence of CR using Kaplan–Meier analysis (CagA( +)/NFATc1( +) [both expression of CagA and nuclear NFATc1 localization in lymphoma cells] vs. CagA(−)/NFATc1(−) [either CagA or nuclear NFATc1localization, or absence expression of CagA and nuclear NFATc1 localization in lymphoma cells]; two-sided log-rank test; p = 0.003). I In the stomach, persistent HP infection can result in the translocation of HP-encoded CagA into B lymphocytes and trigger the tyrosine phosphorylation-dependent signaling pathway, including SHP-2 and its ERK, p38 MAPK, Bcl-2, and Bcl-xL. This CagA-regulated signaling pathway further promotes the proliferation and impedes the apoptosis of lymphoma B cells. Simultaneously, CagA stimulates the production of NFATc1, which further upregulates the expression of p21 and p27, restricting cell cycle progression to the G1 phase and limiting the growth of lymphoma B cells. N number, MT median time, CR complete remission