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.

Mantle cell lymphoma (MCL) is an aggressive B-cell lymphoma that accounts for approximately 6%–8% of non-Hodgkin lymphomas. Despite improved therapeutic responses with first-line therapies (1, 2), MCL patients often relapse, which results in inevitable chemoresistance and poor clinical outcomes (3, 4).

TG2, encoded by the TGM2 gene, is an 80-kDa enzyme. It has multiple physiologic functions and is associated with cancer cell survival, metastatic behavior, and drug resistance (5–9). TG2 has been proposed as a promising therapeutic target in the treatment of human cancers (10, 11). Increasing evidence has demonstrated that TG2 is closely associated with constitutive NF-κB expression in cancer cells (12, 13). Our previous study has shown that TG2 forms complexes with NF-κB components, which drives the translocation of NF-κB to the nucleus and constitutive expression of NF-κB (11). Moreover, TG2 and NF-κB are highly expressed in MCL cells that are stem-like, suggesting that TG2/NF-κB signaling plays a critical role in MCL progression (11).

Signaling pathways such as NF-κB, JAK/STAT, and MAPK signaling are linked to the upregulation of cytokines, such as IL6, IL2, or IL10 (14, 15). The JAK/STAT inhibitor degrasyn inhibits MCL cell growth, and this inhibition correlates with the downregulation of constitutive NF-κB signaling and STAT3 phosphorylation (16). A major upstream activator of STAT3 is IL6, which binds its receptor and activates JAK, which in turn phosphorylates and activates STAT3. However, it remains unclear whether these events are connected to TG2 signaling and whether the drug resistance of MCL is dependent on the IL6 expression mediated by TG2/NF-κB signaling.

Autophagy is a highly conserved homoeostatic mechanism for the lysosomal degradation of cytosolic constituents (17). It can be induced by different conditions, including nutrient deprivation/starvation, oxidative stress, hypoxia, and chemotherapeutic drugs (17–20). Autophagy also plays an important role in innate and adaptive immunity and can be regulated by different cytokines, such as TGFβ or IL6 (17, 21–24). TGM2 is considered to be a stress-responsive gene, and TG2 activity is upregulated by various stressors (13, 25). Given that both autophagy and TG2 activity can be induced under cellular stress and various cytokines are involved in autophagy regulation, we hypothesized that autophagy could be regulated by either the TG2/NF-κB signaling pathway or its downstream cytokine IL6.

In the current study, we discovered that upregulated TGM2 is correlated with a poor prognosis in MCL patients; increased TG2 levels promote tumor progression in vivo. Silencing TG2 by CRISPR-Cas9 using a lentiviral system significantly affected NF-κB activities and IL6/STAT3 signaling. Notably, our data are the first to indicate an interconnected network among TG2/NF-κB, IL6/STAT3 signaling and autophagy regulation in MCL cells, and how this network regulates MCL survival under stress. Connecting this signaling network may help in the design of targeted therapies and the identification of novel therapeutic targets in the future.

Cells and cell culture

The human MCL cell line Jeko was obtained from the ATCC. The human MCL cell line SP53 was a kind gift from MD Anderson Cancer Center (MDACC, Houston, TX). HS5 and HS27a bone marrow stromal cells (BMSC) were a kind gift from Dr. B. Torok-Storb (Fred Huchinson Cancer Research Center, Seattle, WA). Cell lines were authenticated using short tandem repeats in Characterized Cell Line Core Facility at MDACC. Cells were maintained under 5% CO2 at 37°C and cultured in complete RPMI1640 medium supplemented with 10% FBS, 2 mmol/L l-glutamine, 100 ≤ μg/mL streptomycin, and 100 IU/mL penicillin.

Human MCL samples

Peripheral blood and bone marrow samples from MCL patients were obtained after informed consent, as approved by MDACC as well as by the University of Texas Health Science Center (UT-HSC) Institutional Review Boards. Mononuclear cells were isolated from MCL patient apheresis blood or bone marrow using standard Ficoll gradient separation methods. CD19+ B cells from MCL patients or healthy donors were isolated as described previously (26). Primary cells were cultured in complete RPMI1640 medium for further analysis.

Generation of lentivirus constructs and infection

The MIT CRISPR design software was used for the design of sgRNAs (http://crispr.mit.edu). To clone individual sgRNAs, 25-bp oligonucleotides containing the sgRNA sequences were synthesized (Sigma). Details for generation of lentivirus constructs and lentivirus infection can be found in Supplementary Methods.

ELISA assay

Cells were counted using Trypan blue solution before each assay. The DNA-binding activities of NF-κB components, TG2 enzymatic activity, and IL6 levels were determined by ELISA assays, as described previously (11, 26).

Cell viability assay and IC50

Cytotoxicity was assessed with fluorimetric cell viability assay using CellTiter-Blue as described previously (27). The Hill-Slope logistic model was used to calculate IC50s using CompuSyn software.

Quantitative real-time PCR

Procedures for quantitative real-time PCR (qRT-PCR) were performed as described previously (27). The involved primers are provided in Supplementary Methods. The relative expression level of each gene was normalized to the GAPDH by the method of 2−ΔΔCt.

Immunoblotting and semiquantitative analysis

The STAT3 pathway was detected as described previously (26). Total harvested cells were lysed to perform immunoblots as reported previously (27). Immunoblotting was subjected to semiquantitative analysis using ImageJ software.

MethoCult colony assay

MCL cells (5 × 103) were suspended in 1 mL of complete MethoCult medium (see Supplementary Methods for detailed components) and plated onto 35-mm petri dishes. Cells were cocultured with HS5 BMSCs, HS5 conditioned media (HS5-CM), or HS5-CM plus IL6-neutralizing antibodies (1 μg/mL). Colonies were maintained at 37°C, 5% CO2 with 95% humidity for 5 days, and were counted and photographed at day 5 using an Olympus IX70 microscope. Only colonies consisting of 50 or more cells were considered for analysis.

Tumor xenograft studies

Immunodeficient NOD/SCID mice were purchased from The Jackson Laboratory and maintained under barrier conditions. All animal procedures were approved by the UT-HSC Animal Care Committee. Manipulated SP53 MCL cells (3.5 × 106) were subcutaneously injected into NOD/SCID mice (n = 5, male) and tumor growth was monitored weekly. Mice were sacrificed four weeks postinjection and tumors, spleens, and bone marrows were isolated for further analysis. The volumes of tumors and spleens were measured as described previously (26).

TG2/NF-κB signaling axis is critical for MCL survival

Many cancer cells constitutively express NF-κB components and show elevated levels of phosphorylated STAT3 (p-STAT3) due to the upregulation of cytokines such as IL6 or IL10 (15). To determine the downstream events of TG2 and NF-κB, we silenced TG2 (TG2KO) in MCL cells using a lentiviral CRISPR-Cas9–mediated knockout system. The construct of TG2-sgRNA-LentiCRISPR plasmid was validated by DNA sequencing, and the knockout efficiency of TG2 in MCL cells was evaluated by immunoblotting, which indicated complete silencing (Supplementary Fig. S1).We then utilized TG2KO and TG2-overexpressed cells (TG2OE) to evaluate the effects of TG2 on MCL survival, respectively. The empty vector–transfected MCL cells were used as controls. The proliferation rates of TG2KO cells were largely reduced compared with controls, while TG2OE cells showed higher proliferation rate than the control (Fig. 1A), indicating a potential oncogenic role of TG2 in the pathogenesis of MCL. Notably, TG2KO cells underwent a delayed apoptosis after a 10-day incubation, suggesting that TG2 is critical in maintaining MCL survival (Supplementary Fig. S2A).

Figure 1.

IL6/STAT3 signaling is dependent on TG2/NF-κB signaling axis in MCL. A, cell proliferation of TG2KO and TG2OE 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 TG2KO or control (TG2KO-Con) cells. The colorimetric values in TG2KO 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 TG2OE or control (TG2OE-Con) cells. The colorimetric values in TG2OE 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 IL6 using conditioned media from TG2KO andTG2OE 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 ± SD. **, P < 0.01.

Figure 1.

IL6/STAT3 signaling is dependent on TG2/NF-κB signaling axis in MCL. A, cell proliferation of TG2KO and TG2OE 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 TG2KO or control (TG2KO-Con) cells. The colorimetric values in TG2KO 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 TG2OE or control (TG2OE-Con) cells. The colorimetric values in TG2OE 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 IL6 using conditioned media from TG2KO andTG2OE 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 ± SD. **, P < 0.01.

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To elucidate the mechanisms involving reduced proliferation of TG2KO cells, cell cycle and related factors were analyzed. In SP53 MCL cells, the diminished proliferation in TG2KO cells was associated with increased G0–G1 phase (Supplementary Fig. S2B). TG2KO cells contained increased p53, p21, and p27 levels and decreased cyclin genes such as CDK4, CDK6, and CDC2, indicating a cell-cycle arrest (Supplementary Fig. S2C). TG2KO cells had decreased levels of antiapoptotic genes, BCL-XL and BCL-2, and increased expression of proapoptotic genes BAX, BAK, and NOXA. Apoptotic indicators, such as cleaved caspase-3/9 and cleaved PARP were observed after one-week incubation. Collectively, these data support TG2KO cells have a delayed apoptosis (Supplementary Fig. S2D). Interestingly, silencing TG2 reduced BCL-XL and BCL-2, but not MCL-1 levels. Similar to what was reported (28–30), accumulation of MCL-1 did not prevent apoptosis caused by TG2 silencing; instead, it may slow down an apoptotic induction. As TG2KO cells have delayed apoptosis, we were able to use viable TG2KO cells for all subsequent experiments.

To investigate the effects of TG2 signaling on the downstream events, we examined NF-κB levels in MCL cells using an ELISA-based assay, which is an alternative way to measure NF-κB binding instead of a radioactivity-based assay (31). NF-κB p50 and p65 DNA-binding activities as well as NF-κB downstream target gene such as IL8 (32), were dramatically decreased in TG2KO cells (Fig. 1B and Supplementary Fig. S3A). p-STAT3 expression was also significantly reduced in TG2KO MCL cells (Fig. 1C). Correspondingly, overexpressed TG2 resulted in higher NF-κB and STAT3 signaling activities (Fig. 1D and E and Supplementary Fig. S3B).

A major upstream activator of STAT3 signaling is IL6. We then measured IL6 basal levels in both TG2KO and TG2OE SP53 cells using ELISA, because Jeko cells do not produce IL6 under normal cell culture conditions (33). IL6 levels were significantly reduced after TG2 silencing while enhanced in TG2OE cells (Fig. 1F). We further cultured cells in low-serum conditions (2% FBS), which induces IL6 secretion in both SP53 and Jeko MCL cells (33). Compared with the control cells in low serum, TG2OE cells induced more IL6, while TG2KO cells failed to increase IL6 (Fig. 1F). Interestingly, MCL cells in low-serum conditions not only increased IL6 levels but also enhanced TG2 expression (Fig. 1G), suggesting that TG2 may be a part of the stress-induced survival mechanisms for MCL.

TG2 has been proposed as a promising therapeutic target in the treatment of human cancers due to its association with drug resistance (6, 8, 10, 11). We then investigated whether TG2 expression contributes to chemotherapy resistance in MCL. Cells were treated with chemotherapeutic drugs for 24 hours. Compared with the controls, TG2 silencing exhibited sensitivity of MCL cells to chemotherapeutic drugs, while TG2-overexpressing cells showed resistant to the drugs with higher IC50 values (Supplementary Fig. S4A and S4B). Longer incubation time with drugs in TG2OE cells also showed chemoresistance compared with the controls (Supplementary Fig. S4C). These data provide strong evidence that TG2 confers drug resistance properties to MCL.

TG2/NF-κB downstream signaling molecule IL6 regulates colony formation in MCL cells

To further investigate the roles of the TG2/NF-κB downstream cytokine IL6 in MCL, we generated p50-knockdown (p50KD) and p65-knockdown (p65KD) MCL cells using shRNA-mediated lentivirus, because NF-κB knockout leads to rapid apoptosis in MCL. It showed efficient knockdown of p50 or p65 expression in GFP+ Jeko cells (Fig. 2A and Supplementary Fig. S5). We cultured Jeko under low-serum conditions, as reported, to ensure IL6 secretion (33). IL6 levels were significantly reduced in both p50KD and p65KD cells compared with the control under low-serum conditions (Fig. 2B). Together with the result shown in Fig. 1F, these findings suggest that the increased levels of IL6 in MCL cells are mediated by TG2/NF-κB signaling.

Figure 2.

IL6 regulates colony formation in MCL cells mediated by TG2/NF-κB 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 IL6 using conditioned media from p50KD or p65KD Jeko cells under low-serum conditions. The colorimetric values of p50KD or p65KD cells were normalized to the controls. C, TG2KO and control cells were treated with each drug at IC50 values with or without rhIL6 or HS5-CM for 24 hours. Cell viability was determined using MTT assays, which normalized to nontreated control cells. D, TG2KO and TG2OE cells were cocultured with HS5 cells, HS5-CM, and HS5-CM plus IL6-neutralizing antibody (α-IL6). 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 ± SD. *, P < 0.05; **, P < 0.01.

Figure 2.

IL6 regulates colony formation in MCL cells mediated by TG2/NF-κB 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 IL6 using conditioned media from p50KD or p65KD Jeko cells under low-serum conditions. The colorimetric values of p50KD or p65KD cells were normalized to the controls. C, TG2KO and control cells were treated with each drug at IC50 values with or without rhIL6 or HS5-CM for 24 hours. Cell viability was determined using MTT assays, which normalized to nontreated control cells. D, TG2KO and TG2OE cells were cocultured with HS5 cells, HS5-CM, and HS5-CM plus IL6-neutralizing antibody (α-IL6). 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 ± SD. *, P < 0.05; **, P < 0.01.

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Studies have reported the association of IL6 expression with tumor progression, poor prognosis, and chemotherapeutic resistance (34–36). Targeting IL6 has been regarded as an adjuvant therapy for human cancers (37, 38). We next investigated whether IL6 can protect MCL cells from chemotherapy-induced cell death. MCL cells were cocultured with HS5 BMSCs or HS5-CM and treated with chemotherapeutic drugs. HS5 cells have been reported to produce large amounts of IL6 (39). MCL cells cocultured with HS5 cells or HS5-CM exhibited higher cell viability compared with the cells cultured under standard conditions (Supplementary Fig. S6). Addition of human recombinant IL6 (rhIL6) or HS5-CM was able to partially reverse the effects of TG2 depletion on increased drug sensitivity (Fig. 2C), suggesting that IL6 contributes to the drug-resistant properties of TG2. Furthermore, MCL cells cocultured with HS5 cells or HS5-CM also significantly increased colony formation in MethoCult medium, while blocking IL6 using neutralizing antibodies dramatically reduced the colony-forming ability of the MCL cells (Fig. 2D and Supplementary Fig. S7; Supplementary Table S1). In addition, TG2 expression also contributes to the colony formation compared with control cells, suggesting that TG2 is involved in the oncogenic mechanism in MCL (Fig. 2D and Supplementary Fig. S7; Supplementary Table S1).

Reciprocal interactions between TG2/NF-κB signaling and autophagy in MCL cells

Autophagy plays a dual role in different stages of cancer development. At a very early stage, autophagy prevents cancer initiation by eliminating oncogenic protein substrates; once a tumor forms and develops, autophagy is activated and plays a cytoprotective role (40, 41). Many autophagy-related (ATG) genes are involved in autophagosome initiation and maturation (17, 18, 40–43). To elucidate how autophagy and TG2/NF-κB signaling are connected, we generated ATG5-knockout (ATG5KO) MCL cells by lentivirus-mediated CRISPR-Cas9 using a same schematic protocol described above. The knockout efficiency of ATG5 in MCL cells was evaluated by immunoblotting, which showed complete silencing (Supplementary Fig. S8). ATG5KO cells showed a reduced proliferation rate compared with the controls (Supplementary Fig. S9A) due to the impaired autophagosome and autolysosome formation (Supplementary Fig. S9B and S9C), suggesting autophagy may serve as an important survival mechanism in MCL cells. Interestingly, ATG5KO cells showed increased apoptosis similar to TG2KO cells (Supplementary Fig. S9D), implying the potential interactions between TG2 and autophagy.

To investigate roles of TG2/NF-κB signaling in autophagy, we examined TG2 expression and activity in ATG5KO cells using immunoblotting and ELISA assays. TG2 expression levels and enzymatic activities were decreased dramatically compared with the controls (Fig. 3A). Silencing ATG5 also remarkably inhibited NF-κB activation (Fig. 3B), indicating that TG2/NF-κB signaling pathway is involved in autophagy in MCL.

Figure 3.

Interactions between TG2/NF-κB signaling and autophagy in MCL cells. A, TG2 was measured using immunoblotting with GAPDH as a loading control. TG2 enzymatic activities in ATG5KO cells were normalized to controls. B, the DNA-binding activities of p50 and p65 in the nuclear extracts of ATG5KO cells were measured using ELISA. C, cells were treated with the autophagy inducer pp242 (1 μmol/L) with or without lysosomal inhibitor CQ (20 μmol/L) for 24 hours. 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 hours. The normalized values (p62/GAPDH) in each lane are indicated. E, autolysosome and autophagosome formation was measured upon TG2 overexpression with normalized values (LC3-II/GAPDH) indicated under each lane. F, p62 was measured in TG2OE MCL cells with or without pp242 treatment. The p62/GAPDH values in each lane are indicated. G, knockout efficiency of ATG5 in TG2OE cells (TG2OE/ATG5KO) was validated. The controls were mock-transfected cells (TG2OE/ATG5KO-Con). Cell proliferation was determined using Trypan blue. H, enhanced TG2 levels in ATG5KO cells (ATG5KO/TG2OE) were validated. Mock-transfected cells (ATG5KO/TG2OE-Con) were used as controls. Cell proliferation was determined using Trypan blue. The data are shown as the mean ± SD. **, P < 0.01.

Figure 3.

Interactions between TG2/NF-κB signaling and autophagy in MCL cells. A, TG2 was measured using immunoblotting with GAPDH as a loading control. TG2 enzymatic activities in ATG5KO cells were normalized to controls. B, the DNA-binding activities of p50 and p65 in the nuclear extracts of ATG5KO cells were measured using ELISA. C, cells were treated with the autophagy inducer pp242 (1 μmol/L) with or without lysosomal inhibitor CQ (20 μmol/L) for 24 hours. 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 hours. The normalized values (p62/GAPDH) in each lane are indicated. E, autolysosome and autophagosome formation was measured upon TG2 overexpression with normalized values (LC3-II/GAPDH) indicated under each lane. F, p62 was measured in TG2OE MCL cells with or without pp242 treatment. The p62/GAPDH values in each lane are indicated. G, knockout efficiency of ATG5 in TG2OE cells (TG2OE/ATG5KO) was validated. The controls were mock-transfected cells (TG2OE/ATG5KO-Con). Cell proliferation was determined using Trypan blue. H, enhanced TG2 levels in ATG5KO cells (ATG5KO/TG2OE) were validated. Mock-transfected cells (ATG5KO/TG2OE-Con) were used as controls. Cell proliferation was determined using Trypan blue. The data are shown as the mean ± SD. **, P < 0.01.

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To confirm whether TG2 signaling in turn regulates autophagy, we first determined the basal levels of autophagy in TG2KO and TG2OE cells. Both autophagosome and autolysosome formation were inhibited in TG2KO cells but enhanced in TG2OE cells compared with the controls (Supplementary Fig. S10A and S10B). We then treated cells with the autophagy inducer PP242. As shown in Fig. 3C, TG2KO cells exhibited decreased LC3-II formation after PP242 treatment compared with the controls. Autophagic flux assays that measure autolysosome formation also showed decreased LC3-II levels in TG2KO cells in the presence of lysosomal inhibitor chloroquine (CQ), indicating that autophagy formation was impaired upon TG2 silencing. These data were further validated using additional detection of autophagy marker p62 (Fig. 3D). Conversely, TG2OE cells showed enhanced LC3-II formation after PP242 treatment; further CQ treatment caused more LC3-II accumulation in TG2OE cells compared with that in control cells, indicating the autophagic flux was increased upon TG2 overexpression (Fig. 3E). Consistently, a significant reduction of p62 was observed in TG2OE cells compared with the controls (Fig. 3F).

To better understand the reciprocal relationship between TG2 and autophagy, we further silenced ATG5 in TG2OE MCL cells (TG2OE/ATG5KO) using a CRISPR-Cas9 system (Fig. 3G). Compared with controls, TG2OE/ATG5KO cells have reduced proliferation rate and decreased chemoresistance (Fig. 3G and Supplementary Fig. S11A). The combination of chemotherapeutic drugs with 3-MA, an autophagy inhibitor upstream of ATG5, also decreased drug resistance of TG2OE cells (Supplementary Fig. S11B), indicating that upregulated autophagy may be responsible for drug resistance in TG2OE MCL. Correspondingly, overexpression of TG2 led to a partial increase of proliferation in ATG5KO cells (Fig. 3H), suggesting that decreased TG2 levels could be a causative factor for reduced survival of ATG5KO cells.

Targeting mTOR pathway has been a promising therapy for human cancers; however, mTOR inhibition induces autophagy, which can counteract the effect of the inhibitors (44, 45). We then investigated whether there is an association between TG2 expression levels and responses to mTOR inhibitors. Similar to what was observed, TG2 deletion sensitized MCL cells to PP242, a potent mTOR inhibitor, whereas TG2OE cells displayed lower sensitivity to PP242 (Supplementary Fig. S12A). Our data support important roles of autophagy in cancer drug resistance, because autophagy inhibitors 3-MA or CQ increased cytotoxicity of MCL cells to mTOR inhibitors (Supplementary Fig. S12B). Taken together, our results support autophagy is one of the important processes involved in the therapeutic resistance and oncogenic survival mechanism in TG2OE MCL cells.

Autocrine and paracrine cytokine IL6 regulates autophagy in MCL cells

Given that TG2/NF-κB signaling axis is involved in autophagy induction, it is important to delineate the downstream signaling pathway. We first measured IL6 levels in ATG5KO cells using ELISA. IL6 levels were significantly inhibited in ATG5KO cells (Fig. 4A), indicating that impaired ATG5 expression affects IL6 signaling in MCL cells.

Figure 4.

Autocrine and paracrine IL6 stimulates autophagy in MCL cells. A, ELISA analysis of IL6 using conditioned media from ATG5KO SP53 cells under normal conditions. The data were normalized to the control. B, IL6 levels were measured using conditioned media from MCL cells under 2% FBS or 10% FBS (control) for 12 and 36 hours, respectively. C, LC3-II levels were analyzed in SP53 and Jeko cells using immunoblotting with α-IL6 (1 μg/mL) or IgG1 antibody (control). The LC3-II/GAPDH values are indicated. D, cells were cultured in 2% FBS with α-IL6 or IgG1. Autophagic flux was determined with or without CQ treatment (20 μmol/L). E, MCL cells were cultured in 2% FBS with α-IL6 or IgG1. p62 was measured with normalized values under each lane. F, cells were treated with rhIL6 (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 rhIL6 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-IL6 or IgG1 in the presence or absence of CQ.

Figure 4.

Autocrine and paracrine IL6 stimulates autophagy in MCL cells. A, ELISA analysis of IL6 using conditioned media from ATG5KO SP53 cells under normal conditions. The data were normalized to the control. B, IL6 levels were measured using conditioned media from MCL cells under 2% FBS or 10% FBS (control) for 12 and 36 hours, respectively. C, LC3-II levels were analyzed in SP53 and Jeko cells using immunoblotting with α-IL6 (1 μg/mL) or IgG1 antibody (control). The LC3-II/GAPDH values are indicated. D, cells were cultured in 2% FBS with α-IL6 or IgG1. Autophagic flux was determined with or without CQ treatment (20 μmol/L). E, MCL cells were cultured in 2% FBS with α-IL6 or IgG1. p62 was measured with normalized values under each lane. F, cells were treated with rhIL6 (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 rhIL6 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-IL6 or IgG1 in the presence or absence of CQ.

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We then analyzed whether the autocrine IL6 produced by MCL cells plays an important role in autophagy formation. MCL cells were challenged with low FBS (2%) to stimulate autocrine IL6 (Fig. 4B and Supplementary Fig. S13A and S13B). Autophagy was induced by autocrine IL6, while neutralization of IL6 resulted in largely decreased LC3-II formation (Fig. 4C). Autophagic flux assays showed a decreased LC3-II level in the cells that are blocked by neutralizing IL6 (Fig. 4D). Blocking IL6 also led to increased p62 levels (Fig. 4E). These data indicated that IL6 signaling is critical for starvation-induced autophagy formation.

Because starvation-induced IL6 can complicate the interpretation of the autophagy formation, we studied the effects of paracrine IL6 on autophagy using either rhIL6 or HS5-CM. rhIL6 resulted in enhanced autophagy formation and increased autophagic flux; rhIL6 treatment also led to a significant reduction of p62 levels compared with the controls (Fig. 4F and G). To further verify this finding, Jeko MCL cells, which are not able to secrete IL6 under normal conditions (Fig. 4B and Supplementary Fig. S13B), were cultured in HS5-CM for 36 hours, and autophagy formation was evaluated. HS27a cells, which have been reported to produce stromal cell–derived factor 1 (SDF-1) but not IL6 (39), were used as a control. The results showed that paracrine IL6 secreted by BMSCs led to an increase in LC3-II formation; further CQ treatment caused more LC3-II accumulation under HS5-CM compared with that under HS27a-CM (Fig. 4H). We then blocked IL6 in HS5-CM using neutralizing antibodies. LC3-II levels were decreased after the neutralization of IL6 in either the presence or absence of CQ (Fig. 4I), indicating that the autophagy formation was induced by paracrine IL6. Taken together, these data suggest IL6 is an important mediator for autophagy signaling in MCL.

TG2 overexpression promotes tumor progression in xenograft mice

To verify our findings using in vivo models, we subcutaneously transplanted TG2OE SP53 MCL cells or control cells. Compared with controls, TG2OE xenograft mice developed larger tumors and enlarged spleens (Fig. 5A and B). Higher numbers of CD45+ cells were found in the spleens and bone marrows from TG2OE xenograft mice, indicating increased tumor dispersal (Fig. 5C). Immunoblotting analysis using tumor samples revealed increased levels of p-STAT3 in TG2OE mice (Fig. 5D). Overexpressed TG2 in mice also resulted in enhanced NF-κB p50 and p65 DNA-binding activities and increased IL6 levels (Fig. 5E and F). Furthermore, we found increased autophagy formation and autophagic flux in tumor samples of TG2OE xenografts (Fig. 5G). These data strongly support the oncogenic ability of TG2 in MCL, and further validate an interconnected network among TG2/NF-κB/IL6 signaling and autophagy regulation in MCL.

Figure 5.

TG2 overexpression enhances tumor progression in xenograft mice. A, TG2OE or TG2OE-Con MCL cells were subcutaneously injected into NOD/SCID mice (n = 5). Xenograft mice were sacrificed 4 weeks postinjection. 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 TG2OE xenografts were normalized to the controls. F, xenograft tumor cells were cultured in complete RPMI1640 medium for 48 hours. IL6 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 μmol/L) for 24 hours. The LC3-II/GAPDH values in each lane are indicated. The data are shown as the mean ± SD. **, P < 0.01.

Figure 5.

TG2 overexpression enhances tumor progression in xenograft mice. A, TG2OE or TG2OE-Con MCL cells were subcutaneously injected into NOD/SCID mice (n = 5). Xenograft mice were sacrificed 4 weeks postinjection. 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 TG2OE xenografts were normalized to the controls. F, xenograft tumor cells were cultured in complete RPMI1640 medium for 48 hours. IL6 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 μmol/L) for 24 hours. The LC3-II/GAPDH values in each lane are indicated. The data are shown as the mean ± SD. **, P < 0.01.

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Increased TG2 levels are associated with a poor prognosis in MCL patients

To determine the clinical relevance of our findings, we first measured TG2 protein levels in MCL patient cells using immunoblots. B cells of peripheral blood from healthy donors were used as a control. Elevated TG2 levels were noted in MCL samples compared with normal B (NB) cells (Fig. 6A). MCL cells isolated from different patients also showed elevated NF-κB activity levels compared with NB cells (Fig. 6B). We then investigated the correlation of TG2 expression with patient prognosis. TGM2 gene expression levels were compared in CD19+ B cells from MCL patients or NB cells from healthy donors. TGM2 mRNA was elevated in malignant B cells compared with NB cells; interestingly, 3 of the 24 MCL cases displayed a significant elevation of TGM2 levels, with approximately 30-fold, 130-fold, and 150-fold increases compared with the NB cells (Supplementary Fig. S14). These patients are a blastoid type of MCL, which is a highly aggressive subtype form of MCL that has a worse prognosis compared with classical MCL (Supplementary Table S2). Further analysis of samples revealed that blastoid subtypes displayed significantly higher TGM2 levels than nonblastoid cases (Fig. 6C). TGM2 levels of MCL patients that have a record of multi-site infiltrations (e.g., gastrointestinal, spleen, liver, or central nervous system) besides bone marrow were higher compared with the patients that only had bone marrow involvement (Fig. 6D). The overall survival of patients with TG2high was also significantly lower than that of patients with TG2low (Fig. 6E). Our data support that TG2/NF-κB signaling is involved in not only the pathogenesis of MCL cell lines but also of MCL patients.

Figure 6.

Expression patterns of TG2 and NF-κB 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 (Fig. 6A) and cell lines (Fig. 1E) were analyzed using ImageJ software and normalized to GAPDH. The values compared with 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 nonblastoid patients (n = 12). D,TGM2 mRNA levels of MCL patients with multisite infiltrations besides bone marrow involvement (n = 8) were compared with those with solely bone marrow involvement (n = 13). E, overall survival of MCL patients (n = 24; P = 0.05, Mantel–Cox curve analysis). TGM2high and TGM2low refer to the upper and lower 50% of the TGM2 levels in MCL patients, respectively. The results are shown as the mean ± SD. **, P < 0.01.

Figure 6.

Expression patterns of TG2 and NF-κB 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 (Fig. 6A) and cell lines (Fig. 1E) were analyzed using ImageJ software and normalized to GAPDH. The values compared with 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 nonblastoid patients (n = 12). D,TGM2 mRNA levels of MCL patients with multisite infiltrations besides bone marrow involvement (n = 8) were compared with those with solely bone marrow involvement (n = 13). E, overall survival of MCL patients (n = 24; P = 0.05, Mantel–Cox curve analysis). TGM2high and TGM2low refer to the upper and lower 50% of the TGM2 levels in MCL patients, respectively. The results are shown as the mean ± SD. **, P < 0.01.

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We next investigated the roles of the TG2/NF-κB downstream IL6 in MCL patients. Cells from MCL patients demonstrated 1.8-fold to 2.9-fold increases in IL6 expression compared with NB cells (Fig. 7A), suggesting constitutive expression. Interestingly, the calculation of coefficient of determination (R2) based on relative values revealed a linear correlation between IL6 expression and TG2 expression, which further supports the possibility that IL6 is downstream of TG2/NF-κB signaling (Fig. 7B).

Figure 7.

Calcium blockers suppressed IL6/STAT3 signaling and autophagy formation in MCL patients. A, ELISA analysis of IL6 using conditioned media from MCL patients cells (n = 5) cultured in complete RPMI1640 medium for 48 hours. The values compared to NB cells are indicated. **, P < 0.01. B, linear regression analysis of relative TG2 expression (Fig. 6A) and relative IL6 expression (Fig. 7A) based on densitometry analyses of immunoblots. R2 value is 0.8247. C, primary MCL cells were treated with or without BAPTA/AM (60 μmol/L) for 24 hours for p-STAT3 immunoblotting and 48 hours for IL6 analysis. D, primary MCL cells and NB cells were treated with or without CQ for 24 hours. 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 hours. F, p62 was measured in MCL patients with or without BAPTA/AM treatment. G, interactions among TG2/NF-κB, IL6/STAT3 signaling and autophagy in MCL cells. TG2 affects NF-κB activity and STAT3 signaling. IL6, the upstream activator of STAT3, is stimulated by TG2/NF-κB signaling. Both TG2/NF-κB signaling and IL6 contribute to the progression of autophagy, which in turn regulates TG2/NF-κB signaling and IL6 secretion, suggesting a potential feedback loop underlying the survival mechanism in MCL cells.

Figure 7.

Calcium blockers suppressed IL6/STAT3 signaling and autophagy formation in MCL patients. A, ELISA analysis of IL6 using conditioned media from MCL patients cells (n = 5) cultured in complete RPMI1640 medium for 48 hours. The values compared to NB cells are indicated. **, P < 0.01. B, linear regression analysis of relative TG2 expression (Fig. 6A) and relative IL6 expression (Fig. 7A) based on densitometry analyses of immunoblots. R2 value is 0.8247. C, primary MCL cells were treated with or without BAPTA/AM (60 μmol/L) for 24 hours for p-STAT3 immunoblotting and 48 hours for IL6 analysis. D, primary MCL cells and NB cells were treated with or without CQ for 24 hours. 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 hours. F, p62 was measured in MCL patients with or without BAPTA/AM treatment. G, interactions among TG2/NF-κB, IL6/STAT3 signaling and autophagy in MCL cells. TG2 affects NF-κB activity and STAT3 signaling. IL6, the upstream activator of STAT3, is stimulated by TG2/NF-κB signaling. Both TG2/NF-κB signaling and IL6 contribute to the progression of autophagy, which in turn regulates TG2/NF-κB signaling and IL6 secretion, suggesting a potential feedback loop underlying the survival mechanism in MCL cells.

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To further determine the clinical relevance of TG2 in regulating IL6/STAT3 signaling and autophagy formation, we inhibited TG2 enzymatic activities using a calcium chelator, 1,2-bis (o-aminophenoxy) ethane-N, N, N', N'-tetraacetic acid-acetoxymethyl ester (BAPTA/AM) in MCL primary cells (11). IL6 levels and p-STAT3 expression were significantly reduced after treatment of BAPTA/AM (Fig. 7C). Compared with NB cells, primary cells from MCL patients showed upregulated autophagy formation (Fig. 7D). However, LC3 conversion and autophagic flux were decreased upon BAPTA/AM treatment in MCL primary cells (Fig. 7E), which was in line with the further detection of p62 levels (Fig. 7F). Collectively, these findings support that upregulated TGM2 is a potential prognosis marker to predict survival of advanced MCL patients; TG2 may serve as an important signaling event in MCL that utilized NF-κB/IL6/STAT3 signaling pathway and autophagy formation to promote MCL cell survival (Fig. 7G).

Although intensive chemotherapies combined with newly approved drugs have improved MCL patient survival, highly refractory and relapsed cases remain major obstacles to improving the overall survival rate (1–4). In the current study, we demonstrated that TG2 and NF-κB were uniformly expressed at high levels in MCL patient cells compared with NB cells; overexpressed TG2 contributes to chemotherapy resistance in MCL. Notably, we found that silencing TG2 displayed a lower proliferation rate and short-term survival time, which emphasizes the important biological functions of TG2 in MCL cells. Accumulated evidence has indicated that the inhibition of TG2 can induce apoptosis (9, 46), and the same is true in our results. Interestingly, we found a delayed onset of apoptosis in TG2KO cells. One possible reason may be due to the sustained expression of MCL-1, which is able to delay apoptosis (29), and plays an essential survival role in human myeloma cells rather than BCL-2 or BCL-XL (30). Although downregulation of BCL-2 and BCL-XL failed to induce rapid apoptosis in TG2KO cells, they sensitize these cells to different types of chemotherapeutic drugs, suggesting that silencing TG2 lowers the threshold for drug-induced apoptosis.

Studies have shown that the constitutive NF-κB signaling is associated with STAT3 phosphorylation in hematologic malignancies (16, 47, 48). Being a major upstream activator of STAT3, IL6 became a target of interest with regard to its role in MCL cells. In this study, we found that IL6 was highly expressed in MCL patient cells. Silencing TG2 or NF-κB components led to abolishment of both IL6 and p-STAT3 expression, which can be reversed by TG2 overexpression, indicating that IL6/STAT3 signaling is mediated by the TG2/NF-κB signaling axis. Interestingly, we found that IL6 plays an important role in regulating colony formation of MCL and contributes to drug resistance, suggesting that it may serve as a survival gene in MCL. Nevertheless, the potential survival mechanism underlying TG2/NF-κB/IL6 remains unclear. To elucidate this situation, we introduced a major survival mechanism, autophagy, into our study.

Autophagy is well-accepted as a protective mechanism for cancer cells under different conditions (17–20). In the current study, we found that silencing ATG5 led to undetectable TG2 levels and reduced NF-κB expression, whereas TG2 in turn regulated autophagy formation and flux. What's more, the protective effects of TG2 overexpression can be reversed by inhibiting autophagy, revealing a novel mechanism of TG2-mediated autophagy in MCL cells. It is noteworthy that ATG5KO cells exhibited a similar cell fate as TG2KO cells in MCL. Apart from persistent loss of autophagy, reduced TG2 levels in ATG5KO cells might be another possible reason, as TG2 overexpression could partially rescue the phenotype of ATG5KO cells. Thus, dual targeting TG2 and autophagy may be one promising strategy to reduce chemoresistance in MCL.

IL6 has been shown to regulate autophagy via activation of AMPK/mTOR pathway (24), and ATG5 depletion inhibits IL6 production (49). Here we discovered that both autocrine and paracrine IL6 can positively regulate autophagy. This finding partially explains why MCL cells display higher cell viability and colony formation in an IL6-enriched coculture setting. More interestingly, we found that autophagy, as well as being regulated by IL6, can itself influence IL6 secretion. Combined with our results that IL6 levels are enriched in MCL patient samples and bone microenvironment, we herein conclude that MCL cells could elicit IL6-mediated autophagy as an important way to survive and migrate to the bone marrow. Nevertheless, HS27a-CM can also induce autophagy based on our result, although the autophagy induction by HS27a-CM was not as “strong” as that by HS5-CM. To some extent, we believe that the MCL cells are exposed to multiple paracrine signals from BMSCs that would modify MCL cell behavior and ultimately favor cancer progression.

Taken together, our data support a model where the MCL cells exploit both TG2/NF-κB signaling and IL6 to enhance a cytoprotective autophagy response, and autophagy in turn regulates TG2/NF-κB signaling and IL6 secretion, implying a potential feedback loop underlying the survival mechanism in MCL cells. Nonetheless, there are many unanswered questions, such as whether ATG5 genes directly associate with TG2/NF-κB and whether other inducers are involved in the regulatory network of TG2/IL6/autophagy. Further studies will be needed in the future. Collectively, this study provides valuable insights into the survival mechanism of MCL cells. Disruption of TG2/IL6/autophagy network may be a promising therapeutic target in the treatment of MCL patients and will introduce novel strategies to overcome chemoresistance in MCL.

No potential conflicts of interest were disclosed.

Conception and design: R.N. Miranda, N. McCarty

Development of methodology: H. Zhang, Z. Chen, N. McCarty

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): R.N. Miranda, L.J. Medeiros, N. McCarty

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): H. Zhang, Z. Chen, L.J. Medeiros, N. McCarty

Writing, review, and/or revision of the manuscript: H. Zhang, R.N. Miranda, N. McCarty

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): N. McCarty

Study supervision: N. McCarty

This work is funded by NCI/NIH grant (5R01CA181319 to N. McCarty).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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Supplementary data