The CARMA3-BCL10-MALT1 (CBM) complex contributes to DNA damage-induced NF-κB activation and cell survival

Chemotherapy is one of major means for cancer treatments, and many of chemotherapeutic drugs are DNA damaging agents that reduce tumor growth through triggering cancer cell apoptosis or necrosis. Following DNA damage, ataxia telangiectasia mutated (ATM), a protein kinase, was activated and a cytosolic complex containing ATM, NEMO, RIP1 were formed (Biton and Ashkenazi, 2011). This ATM/NEMO/ RIP1 complex cooperates to activate cytoplasmic adaptor protein TRAF6 (Hinz et al., 2010) and Ser/Thr kinase, TGFβ-activated kinase 1 (TAK1) and IKK complex to induce NFκB activation through multiple signal transduction mechanisms depending on the severity of genotoxic stress and cell type (Wu et al., 2010). It has been shown that irradiation or chemotherapeutic drugs induce NF-κB activation through the functional IKK complex, but some reports suggest that UV irradiation activates NF-κB signal through IKK complexindependent but CK2-dependent manner (Kato et al., 2003). Of note, many DNA damaging agents can also activate NFκB family of transcription factors that induce the expression of anti-apoptotic genes, thereby protecting cancer cells from apoptosis and resulting in the resistance of cancer cells to the chemotherapy. Although there are many studies on the mechanism of DNA damage-induced drug resistance, the molecular mechanism by which DNA damage induces NFκB activation is not fully defined. The caspase recruitment domain (CARD) and membrane-associated guanylate kinase-like domain protein (CARMA) family of proteins has three members, CARMA1, CARMA2, and CARMA3 that are encoded by three different genes (Blonska and Lin, 2011). CARMA proteins share the same set of structural domains, but a different pattern of tissue expression profile. Upon different stimuli, all CARMA proteins can form a complex with BCL10 and MALT1, and the CARMA-BCL10-MALT1 (CBM) complex functions to activate NF-κB signaling (Pomerantz et al., 2002; Grabiner et al., 2007; Jiang et al., 2011). Besides in NF-κB signaling, CBM complex is also involved in antiviral innate immune response by suppressing IRF3-type I IFN expression through inhibiting the formation of MAVS oligomerization in mitochondrion (Jiang et al., 2016). CARMA3 was reported to be relatively overexpressed in many tumor cells and associated with the malignant behavior of cancer cells (Li et al., 2012; Pan et al., 2016). It has been shown that CARMA3 and TRAF6 form a complex to mediating GPCRand EGFRinduced NF-κB activation (Grabiner et al., 2007). Therefore, we hypothesized that the CBM complex contributes to DNA damage-induced NF-κB activation to protect genotoxic agent-induced cell death. To determine the role of CBM complex in DNA damageinduced NF-κB activation, MEF cells isolated from Malt1 Het (+/−) or knockout (−/−) mice were stimulated with doxorubicin, a genotoxic reagent inhibiting the activity of topoisomerase II. Consistent with our hypothesis, NF-κB was potently activated by doxorubicin in control cells, but completely impaired in Malt1-deficient cells (Fig. 1A). To confirm that Malt1 is responsible to DNA damage-induced NF-κB activation, MEFs (Malt1) and MALT-deficient MEFs (Malt1) were treated with two other genotoxic reagent VP16 (etoposide) (10 μmol/L) and CPT (2 μmol/L), respectively. Consistently, we found that VP16and CPT-induced NF-κB activation were defective in MALT1-deficient cells compared with controls (Fig. 1B), indicating that MALT1 is required for DNA damage-induced NFκB activation. Since MALT1 forms a complex with CARMA1 and Bcl10 in hematopoietic cells, and CARMA3 and Bcl10 in non-hematopoietic cells to activate NF-κB, we examined the impact of CARMA3 or Bcl10 on DNA damage-induced signaling (Fig. 1C). CARMA3-deficient (Carma3) and BCL10deficient (Bcl10) MEFs together with wild-type controls were treated with doxorubicin. Consistent with the result in Malt1 KO MEF cells, both CARMA3-deficient and BCL10deficient cells were completely defective for NF-κB activation upondoxorubicin treatment. Similarly, we found that genotoxic reagents-induced NF-κB activation was significantly reduced in JPM50.6 cells (Fig. S1), in which the expression of CARMA1 is defective (Wang et al., 2002). Together, these results indicate that CBM complex mediates DNA damageinduced NF-κB activation in both hematopoietic and non-hematopoietic cells. PKC family of kinases has been shown to play important roles in CBM complex-mediated NF-κB activation upon

Western Blot Analysis-The procedure from immunoblotting has been performed as described previously. Briefly, monolayer cells in 30mm dish were lysed in 100µl lysis buffer (250mM NaCl, 50mM HEPES at pH 7.4, 1mM EDTA, 1% NP-40, protease inhibitors) for 15 minutes. The lysates were centrifuged for 10min by 13000rpm at 4℃ and then denatured at 95℃ in 1XSDS loading buffer. 10 µl of the denatured protein sample was resolved on 10% SDS-PAGE gels, then transferred onto PVDF membranes (Bio-Rad) and probed with antibodies.
Transfection and Immuno-precipitation assay-For transfection, HEK-293T cells were seed in 30mm dishes at density of 2x10 6 . DNA plasmids were transfected by PEI as described previously.
Thirty-six hours later, cells were lysed in 250 µl lysis buffer (150mM NaCl, 50mM HEPES at pH 7.4, 1mM EDTA, 1% NP-40, protease inhibitors) for 15 minutes. Then the cell lysates were collected by centrifuging at 13000rpm for 10min. 40µl of the lysates were denatured for western blot analysis as input control. 200µl of the lysates were incubated with 20µl protein A/G (abmart) and indicated 2 specific antibody for 3h at 4℃. After three times washes by RIPA lysis buffer, the precipitated proteins were suspended in 40µl lysis buffer containing 1xSDS loading buffer for western blot.

Quantitative real-time PCR (qPCR)-Total RNA was isolated from cells or tissues using
TRIzol RNA isolation reagent (Invitrogen, Grand Island, NY,USA) and cDNA was generated using oligo-dT and SuperScriptII Reverse Transcriptase (Invitrogen) according to the manufacturer's protocol. Quantitative PCR was performed using SYBR Green PCR Master Mix with ROX(Genstar).
The CT values for IL-6, IFN-β and TNF-α were normalized to GAPDH mRNA levels. The amplification primers used in the studies corresponded to: 5'-CCCTATGGAGATGACGGAGA-3'

5'-GGCTGTTGTCATACTTCTCATGG-3'(reverse) for human GAPDH;
Animals and irradiation-All animal experimental protocols were approved by the Tsinghua University Animal Care Committee. Progeny homozygous for CARMA3 +/and CARMA3 -/mice with the same genetic background were bred separately for the experiments, and mice at the age of seven weeks were used. All animals were housed in modified barrier facility at the Tsinghua University Animal Center. For abdominal irradiation, mice were randomly divided into each group and were placed in a specially designed well-ventilated acrylic container and were exposed to 10 Gy of irradiation. After all mice were scarified at 3 days post irradiation, their colon samples were collected for immunochemistry staining with specific Ki67 antibody and for quantitative real-time PCR analysis of inflammatory cytokines. For survival ratio, CARMA3 +/and CARMA3 -/mice were exposed to 12 Gy of irradiation.
Statistical Analysis-At least two biological replicates were performed for all experiments unless otherwise indicated. Student's t test for paired observations was used for statistical analyses.
Statistical significance was set at a p value of 0.05 or 0.01.