TG2 and NF-κB Signaling Coordinates the Survival of Mantle Cell Lymphoma Cells via IL6-Mediated Autophagy

Abstract

Expression of the transglutaminase TG2 has been linked to constitutive activation of NF-κB and chemotherapy resistance in mantle cell lymphoma (MCL) cells. TG2 forms complexes with NF-κB components, but mechanistic insights that could be used to leverage therapeutic responses has been lacking. In the current study, we address this issue with the discovery of an unexpected role for TG2 in triggering autophagy in drug-resistant MCL cells through induction of IL6. CRISPR-mediated silencing of TG2 delayed apoptosis while overexpressing TG2 enhanced tumor progression. Under stress, TG2 and IL6 mediate enhanced autophagy formation to promote MCL cell survival. Interestingly, the autophagy product ATG5 involved in autophagosome elongation positively regulated TG2/NF-κB/IL6 signaling, suggesting a positive feedback loop. Our results uncover an interconnected network of TG2/NF-κB and IL6/STAT3 signaling with autophagy regulation in MCL cells, the disruption of which may offer a promising therapeutic strategy. Cancer Res; 76(21); 6410-23. ©2016 AACR.

Figure 1. IL-6/STAT3 signaling is dependent on TG2/NF-kB signaling axis in MCL

(A) Cell proliferation of TG2^KO and TG2^OE cells were determined using Trypan Blue and MTT assays. The controls were mock-transfected cells. (B) The DNA binding activities of p50 and p65 were measured using nuclear extracts from TG2^KO or control (TG2^KO-Con) cells. The colorimetric values in TG2^KO cells were normalized to the controls. (C) STAT3 and p-STAT3 (Tyr705) were measured using immunoblotting with GAPDH as a loading control. (D) The DNA binding activities of p50 and p65 were measured using nuclear extract from TG2^OE or control (TG2^OE-Con) cells. The colorimetric values in TG2^OE cells were normalized to the controls. (E) STAT3 and p-STAT3 were measured using immunoblotting with GAPDH as a loading control. (F) ELISA analysis of IL-6 using conditioned media from TG2^KO and TG2^OE cells under normal conditions (10% FBS) or low-serum conditions (2% FBS). (G) TG2 levels were analyzed using immunoblotting and the relative intensity of the bands is shown compared with cells in normal conditions. The data are shown as the mean ± S.D. **p < 0.01.

Figure 2. IL-6 regulates colony formation in MCL cells mediated by TG2/NF-kB signaling

(A) The p50 and p65 levels were analyzed using immunoblotting with a scrambled shRNA plasmid (Scr) as a negative control. (B) ELISA analysis of IL-6 using conditioned media from p50^KD or p65^KD Jeko cells under low-serum conditions. The colorimetric values of p50^KD or p65^KD cells were normalized to the controls. (C) TG2^KO and control cells were treated with each drug at IC₅₀ values with or without rhIL-6 or HS5-CM for 24 h. Cell viability was determined using MTT assays, which normalized to non-treated control cells. (D) TG2^KO and TG2^OE cells were co-cultured with HS5 cells, HS5-CM, and HS5-CM plus IL-6 neutralizing antibody (α-IL-6). The average number of colonies in SP53 or Jeko cells from three parallel assays in each group is shown in the diagram. The data are shown as the mean ± S.D. *p < 0.05; **p < 0.01.

Figure 3. Interactions between TG2/NF-kB signaling and autophagy in MCL cells

(A) TG2 was measured using immunoblotting with GAPDH as a loading control. TG2 enzymatic activities in ATG5^KO cells were normalized to controls. (B) The DNA-binding activities of p50 and p65 in the nuclear extracts of ATG5^KO cells were measured using ELISA. (C) Cells were treated with the autophagy inducer pp242 (1 μM) with or without lysosomal inhibitor CQ (20 μM) for 24 h. The indicated values of LC3-II under each lane were normalized to GAPDH. (D) p62 was measured using immunoblotting with or without pp242 for 24 h. The normalized values (p62/GAPDH) in each lane are indicated. (E) Autolysosome and autophagosome formation were measured upon TG2 overexpression with normalized values (LC3-II/GAPDH) indicated under each lane. (F) p62 was measured in TG2^OE MCL cells with or without pp242 treatment. The p62/GAPDH values in each lane are indicated. (G) Knockout efficiency of ATG5 in TG2^OE cells (TG2^OE/ATG5^KO) was validated. The controls were mock-transfected cells (TG2^OE/ATG5^KO-Con). Cell proliferation was determined using Trypan Blue. (H) Enhanced TG2 levels in ATG5^KO cells (ATG5^KO/TG2^OE) were validated. Mock-transfected cells (ATG5^KO/TG2^OE-Con) were used as controls. Cell proliferation was determined using Trypan Blue. The data are shown as the mean ± S.D. **p < 0.01.

Figure 4. Autocrine and paracrine IL-6 stimulates autophagy in MCL cells

(A) ELISA analysis of IL-6 using conditioned media from ATG5^KO SP53 cells under normal conditions. The data were normalized to the control. (B) IL-6 levels were measured using conditioned media from MCL cells under 2% FBS or 10% FBS (control) for 12 h and 36 h, respectively. (C) LC3-II levels were analyzed in SP53 and Jeko cells using immunoblotting with α-IL-6 (1 μg/ml) or IgG1 antibody (control). The LC3-II/GAPDH values are indicated. (D) Cells were cultured in 2% FBS with α-IL-6 or IgG1. Autophagic flux was determined with or without CQ treatment (20 μM). (E) MCL cells were cultured in 2% FBS with α-IL-6 or IgG1. p62 was measured with normalized values under each lane. (F) Cells were treated with rhIL-6 (50 ng/ml) or DMSO with or without CQ. The LC3-II/GAPDH values in each lane are indicated. (G) MCL cells were treated with rhIL-6 or DMSO. p62 was measured with normalized values under each lane. (H) Jeko cells were cultured in HS5-CM or HS27a-CM with or without CQ. The LC3-II/GAPDH values are indicated. (I) Jeko cells were cultured in HS5-CM with α-IL-6 or IgG1 in the presence or absence of CQ.

Figure 5. TG2 overexpression enhances tumor progression in xenograft mice

(A) TG2^OE or TG2^OE-Con MCL cells were subcutaneously injected into NOD/SCID mice (n=5). Xenograft mice were sacrificed 4 weeks post injection. Dotted circles represent area of subcutaneous tumors. Tumors and spleens were isolated and photographed against a centimeter ruler. (B) The size of tumors and spleens were measured. (C) CD45⁺ human cells were isolated from spleens and bone marrows using CD45-MicroBeads, and the numbers of CD45⁺ cells were counted using Trypan Blue. (D) Immunoblots of tumor samples for TG2, p-STAT3, and STAT3 with GAPDH as a loading control. (E) The DNA binding activities of p50 and p65 in the nuclear extracts of xenograft tumors. The colorimetric values in TG2^OE xenografts were normalized to the controls. (F) Xenograft tumor cells were cultured in complete RPMI1640 medium for 48 h. IL-6 expression was measured using ELISA. The data were normalized to the controls. (G) Xenograft tumor cells were cultured in complete RPMI1640 medium with or without CQ treatment (20 μM) for 24 h. The LC3-II/GAPDH values in each lane are indicated. The data are shown as the mean ± S.D. **p < 0.01.

Figure 6. Expression patterns of TG2 and NF-kB in MCL patients

(A) TG2 levels were measured in MCL patients (n=5) and normal B (NB) cells (n=1). The intensity of the bands in MCL patients (Figure 6A) and cell lines (Figure 1E) were analyzed using ImageJ software and normalized to GAPDH. The values compared to NB cells are indicated. (B) Nuclear extracts from MCL patients (n=5) and NB cells (n=1) were subjected to ELISA assays to evaluate p50 and p65 DNA-binding activities. The colorimetric values in MCL samples were normalized to the NB cells. (C) TGM2 mRNA levels were measured in CD19⁺ B cells isolated from MCL blastoid patients (n=12) or non-blastoid patients (n=12). (D) TGM2 mRNA levels of MCL patients with multi-site infiltrations besides BM involvement (n=8) were compared to those with solely BM involvement (n=13). (E) Overall survival of MCL patients (n=24) (p=0.05, Mantel-Cox curve analysis). TGM2^high and TGM2^low refer to the upper and lower 50% of the TGM2 levels in MCL patients, respectively. The results are shown as the mean ± S.D. **p < 0.01.

Figure 7. Calcium blockers suppressed IL-6/STAT3 signaling and autophagy formation in MCL patients

(A) ELISA analysis of IL-6 using conditioned media from MCL patients cells (n=5) cultured in complete RPMI1640 medium for 48 h. The values compared to NB cells are indicated. **p < 0.01. (B) Linear regression analysis of relative TG2 expression (Figure 6A) and relative IL-6 expression (Figure 7A) based on densitometry analyses of immunoblots. R-square (R²) value is 0.8247. (C) Primary MCL cells were treated with or without BAPTA/AM (60 μM) for 24 h for p-STAT3 immunoblotting and 48 h for IL-6 analysis. (D) Primary MCL cells and NB cells were treated with or without CQ for 24 h. The LC3-II/GAPDH values in each lane are indicated. (E) Autolysosome and autophagosome formation were measured in MCL patients upon BAPTA/AM treatment with or without of CQ for 24 h. (F) p62 was measured in MCL patients with or without BAPTA/AM treatment. (G) Interactions among TG2/NF-kB, IL-6/STAT3 signaling and autophagy in MCL cells. TG2 affects NF-kB activity and STAT3 signaling. IL-6, the upstream activator of STAT3, is stimulated by TG2/NF-kB signaling. Both TG2/NF-kB signaling and IL-6 contribute to the progression of autophagy, which in turn regulates TG2/NF-kB signaling and IL-6 secretion, suggesting a potential feedback loop underlying the survival mechanism in MCL cells.