1. IntroductionGenome integrity maintenance is essential for the appropriate functioning and survival of all organisms. It is also crucial for reasons such as the constant attack on DNA by genotoxic agents and nucleotide misincorporation during DNA replication. In humans, these types of damage induce the development of several inherited disorders, aging, and even oncogenesis. The MRE11 (Meiotic recombination 11 homolog 1)–RAD50–NBS1 (Nijmegen breakage syndrome protein 1) (MRN) is pivotal for ataxia–telangiectasia mutated (ATM) activation during DSB (double-strand DNA break) [1,2]. A previous study revealed that ATM and ATM and RAD3-related (ATR) kinase activations are intact in lung cancer cell lines and may act as lung cancer biomarkers [3]. Moreover, studies have shown that the expression level of the MRN complex is higher in radioresistant small-cell lung cancer cells than in radiosensitive cells [4]. Identifying the critical regulator controlling MRN complex integrity may provide a crucial therapeutic target for lung cancer.IL-6, a type I interferon (IFN)-activated cytokine, is secreted by T cells and macrophages [5]. Among the cytokines linked to inflammation-associated cancer, IL-6 drives many cancer hallmarks through the downstream activation of the STAT3 (Signal transducer and activator of transcription 3) signaling pathway [6]. STAT3, a critical protein in tumor metastasis, including in the metastasis of lung cancer [7,8], is a valuable biomarker for prognosis prediction and is a therapeutic target in human solid tumors [9]. While cytokine receptors are activated, STAT3 is phosphorylated at Tyr705, leading to the nuclear translocation of activated STAT3 and the induction of downstream target genes such as CCL2 that promote various cellular processes for cancer progression [10]. Persistently activated or tyrosine-phosphorylated STAT3 (pSTAT3) is found in 50% of lung adenocarcinomas [11]. Moreover, IL-6 drives many cancer hallmarks through the downstream activation of the STAT3 signaling pathway [6]. Professor Christine Watson reported that inhibitor of DNA binding 1 (ID1, a dominant negative helix–loop–helix protein, transcript variant 1), phospholipid scramblase 1 (PLSCR1), and X-box binding protein 1 (XBP1) are vital downstream genes of STAT3 [12]. Chemokine (C-C motif) ligand 2 (CCL2), formerly known as monocyte chemoattractant protein-1, can attract monocytes in vitro [13,14]. In lung cancer, the CCL2 signaling pathway is the key mechanism through which macrophages activate the growth and metastasis of lung cancer cells by triggering the bidirectional cross-talk between macrophages and these cells [15]. Therefore, blocking the STAT3/CCL2 signaling pathway may aid in the cessation of lung cancer progression.Macrophages can promote lung tumor invasion and metastasis [16,17]. Direct mixed co-culture of macrophages and cancer cells could result in stronger transcription factor-mediated activation of signaling [18,19]. Lactate dehydrogenase A (LDHA), regulated by STAT3, is crucial for the invasion and metastasis of malignancies [20,21]. VEGF (vascular endothelial growth factor) is an important signaling protein for vasculogenesis. Previous studies have shown that VEGF is regulated by the STAT3 pathway in several types of cancer, including lung cancer [22,23]. Runt-related transcription factor 2 (RUNX2) has a key role associated with the pathogenesis of some cancers and a downstream gene of the STAT3 pathway [24,25,26]. Macrophages can migrate and accumulate in distinct tumor microenvironments depending on the chemokine expression pattern [17,27,28,29]. The CCL2 signaling pathway is pivotal for the bidirectional cross-talk between macrophages and lung cancer cells during the growth and metastasis of cancer cells [15]. Moreover, CCL2 is a STAT3 downstream gene [30].

However, the mechanism of interaction between MRE11 and IL6/STAT3/CCL2 is completely unclear. In this study, we explored the novel role of MRE11 in the IL6/STAT3/CCL2 signaling pathway in lung cancer. More importantly, we believe that MRE11 might be a novel target for lung cancer diagnosis and treatment.

2. Materials and Methods 2.1. Cell Culture and Treatment

A549 cells (human lung adenocarcinoma cell line), THP-1 cells (human acute monocytic leukemia cell line), RAW 264.7 cells (mouse macrophage cell line), and 293T cells were obtained from the Bioresource Collection and Research Center, Taiwan. The A549 cells and THP-1 cells were cultured in RPMI-1640 medium (Invitrogen Corp., Carlsbad, CA, USA), supplemented with 10% fetal bovine serum at 37 °C and 5% CO2. The 293T cells and RAW 264.7 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Invitrogen Corp., Carlsbad, CA, USA), supplemented with 10% fetal bovine serum at 37 °C and 5% CO2. The indicated cells were cultured to 60–70% confluence before treatment. Then, the medium was then replaced with a fresh medium containing the indicated compounds at the indicated concentrations. The cells treated with water alone were used as untreated vehicle controls.

2.2. shRNA Knockdown Assay

For lentiviral short hairpin RNA (shRNA) infection, 293T cells were co-transfected with MRE11 or GFP control shRNA with packing plasmid (deltaVPR8.9) and envelope plasmid (VSV-G) using Lipofectamine 2000 reagent. MRE11-lentiviral shRNA (5′-TTAAAGAACGTCATCTCGAGATGACGTTCTTTAAGAAATC-3′), and control shRNA (5′-GCAAGCTGACCCTGAAGTTC-3′) were transfected with packing plasmids into 293T cells for 2 days, and virus particles containing MRE11 or control shRNA were used to infect A549 cells. All the infected cells were cultured in a medium containing 2 μg/mL puromycin for 4 days.

2.3. Cytokine Membrane Array

The secretion medium of A549 shLeu or A549 shMRE11 cell lines was cultured and collected after being cultured for 24 h. The secretion profiles of cytokines were measured using the Human Cytokine Panel A Array kit (R&D Systems, Minneapolis, Minnesota, United States), according to the manufacturer’s instructions. Cell culture supernatants were mixed with a cocktail of biotinylated detection antibodies. Nitrocellulose membranes (spotted with different cytokine antibodies) were then incubated in the sample/antibody mixture. After several washes, streptavidin-HRP and chemiluminescent detection reagents were added, which produced light at each spot proportional to the amount of cytokine bound.

2.4. Macrophage Recruitment AssayMacrophage recruitment analyses were performed as described previously [31]. The human lung cancer cells (A549 shLeu or A549 shMRE11 cells) were cultured for 24 h. The conditioned medium was collected and plated into the lower chamber of transwell plates with a 5 μm pore polycarbonate membrane insert. RAW 264.7 cells (1 × 104 cells) were plated onto the upper chamber for macrophage migration assay. After incubation for 24 h, the cells migrated into the bottom are fixed and stained using 1% toluidine blue, and the numbers of migratory cells averaged after counting six randomly selected fields. Each sample was assayed in triplicate. Each experiment was repeated at least twice. 2.5. Enzyme-Linked Immunosorbent Assay (ELISA)The ELISA was performed as described previously [32]. The RAW 264.7 cells were cultured for 24 h. Then, the conditioned medium was collected and plated into a monoculture of lung cancer cells (A549 shLeu or A549 shMRE11 cells). After 24 h, human CCL2, IL-6, or IL-8 in the medium were detected by human CCL2, IL-6, or IL-8 ELISA kits (eBioscience, San Diego, California, United States) according to the manufacturer’s instructions. 2.6. Quantitative Real-Time PCR

Total RNA was extracted from lung cancer cells using the TRIzol reagent (Invitrogen, Waltham, Massachusetts, United States, Cat. No. 15596-026) according to the manufacturer’s instructions. Reverse transcription was performed using the Superscript first strand synthesis kit (Invitrogen, Waltham, Massachusetts, United States, Number: 11904018). Quantitative real-time PCR analyses using the comparative CT method were performed on an ABI PRISM 7700 sequence detector system using the SYBR Green PCR Master Mix kit (Perkin Elmer, Applied Biosystems, Wellesley, MA, USA) according to the manufacturer’s instructions. Following initial incubation at 50 °C for 2 min and 10 min at 95 °C, amplification was performed for 40 cycles at 95 °C for 20 s, 65 °C for 20 s, and 72 °C for 30 s. The primers used were: CCL2 forward, 5′-GTC TCT GCC GCC CTT CTG TG-3′ and CCL2 reverse, 5′-GAC ACT TGC TGC TGG TGA TTC TTC-3′. XBP1 forward, 5′-CCG CAG CAC TCA GAC TAC-3′ and XBP1 reverse, 5′-TCA ATA CCG CCA GAA TCCAT-3′. ID1 forward, 5′-CAT TCC ACG TTC TTA ACT GTT CCA-3′ and ID1 reverse, 5′-ATT CTT GGC GAC TGG CTG AA-3′. PLSCR1 forward, 5′-ATG ATT GGT GCC TGT TTC CT-3′ and PLSCR1 reverse, 5′-TCC ACT ACC ACA CTC CTG ATT TT-3′. LDHA forward, 5′-ACC CAG TTT CCA CCA TGA TT-3′ and LDHA reverse, 5′-CCC AAA ATG CAA GGA ACA CT-3′. RUNX2 forward, 5′-AGG TAC CAG ATG GGA CTG TG-3′ and RUNX2 reverse, 5′-TCG TTG AAC CTT GCT ACT TGG-3′. VEGF forward, 5′-ACC TCC ACC ATG CCA AGT G-3′ and VEGF reverse, 5′-TCT CGA TTG GAT GGC AGT AG-3′. GAPDH forward, 5′-TGC ACC ACC AAC TGC TTAGC-3′ and GAPDH reverse, and 5′-GGC ATG GAC TGT GGT CATGA-3′. GAPDH was used as the housekeeping gene for data normalization.

2.7. Western Blot Analysis

For Western blotting, cellular extracts of the human lung cancer cell line (A549 shLeu or A549 shMRE11 cells) treated with indicated compounds for 24 h were prepared according to the manufacturer’s instructions. Equal amounts of protein were fractionated on a 6% or 12% SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes. The membranes were then blocked with 5% nonfat dried milk for 30 min and incubated in a primary antibody for 3 h at room temperature. The primary antibodies used were anti-phospho (tyr705)-STAT3 antibody (p-STAT3) (cell signaling, ratio: 1:1000), anti-STAT3 antibody (cell signaling, ratio: 1:1000), anti-MRE11 antibody (IP: 1:100; IB: 1:1000, Calbiochem) or anti-β-actin antibody (Santa Cruz, Dallas, Texas, United States, IB: 1:10,000). The primary antibody and secondary antibody were diluted with 1% nonfat dried milk in 1X TBST (Tris-Buffered Saline Tween-20). The blots were washed by 1X TBST and incubated in horseradish peroxidase-conjugated secondary anti-mouse or anti-rabbit antibodies (Santa Cruz, Dallas, Texas, United States, ratio: 1:5000) for one hour at room temperature. After washing with 1X TBST again, the protein signal was detected by chemiluminescence, using the Super Signal substrate (Pierce, Number: 34087).

2.8. Immunoprecipitation (IP)

After the indicated treatment, 293T cells were lysed with E1A lysis buffer (250 mM NaCl, 50 mM HEPES (pH 7.5), 0.1% NP-40, 5 mM EDTA, protease inhibitor cocktail (Roche)). Cell lysates were precleared with normal rabbit IgG (sc-2027, Santa Cruz Biotechnology, Dallas, Texas, United States) and protein A-agarose. Specific antibodies were added to the lysates (1 μg primary antibody/1 mg protein extract) and immunoprecipitated overnight at 4 °C. After washing the beads 4 times with E1A lysis buffer, the immunoprecipitated complexes were analyzed by immunoblotting.

2.9. Statistical Analyses

All values are presented as means ± standard error of the means of the replicate samples (n = 3). These experiments were repeated a minimum of three times. Differences between the two groups were assessed using the unpaired two-tailed Student’s t-test. For testing the significance of pairwise group comparisons, Tukey’s test was used. For all comparisons, p-values of < 0.05 were considered statistically significant. SPSS version 13.0 (SPSS Inc., Chicago, IL, USA) was used for all calculations.

4. DiscussionIn a previous study, mice administered anti-IL6 antibodies exhibited increased radiation-induced mortality [35]. Moreover, IL-6 silencing in human lung cancer cell lines resulted in higher DSBs after irradiation [36,37]. Further, a previous study showed that MRE11 knockdown suppressed IL-6 expression in mouse embryonic fibroblast cells [38]. In our results, MRE11-deficient A549 cells showed decreased IL-6 expression (Figure 3). In addition, MRE11 can interact with STAT3 under IL-6 treatment and can regulate STAT3 Tyr705 phosphorylation (Figure 1B). IL-6 cannot induce STAT3 activation without MRE11 (Figure 2). The MRE11 signaling pathway can also regulate CCL2 secretion (Figure 4). These data indicate that MRE11 plays a critical role in IL-6 pathways. However, the effect of other cytokines on MRE11 must be investigated.The IL-6/STAT3 pathway is hyperactivated in patients with many cancer types, including lung cancer [39]. In the tumor environment, IL-6 can activate STAT3 signaling in both cancer cells and tumor-infiltrating immune cells, including macrophages, and can promote the proliferation and metastasis of cancer cells [39]. A previous study showed that DSBs can activate the STAT3 signaling pathway [40]. Moreover, breast cancer cell lines with MRE11 overexpression can induce STAT3 phosphorylation at tyrosine-705 and serine-727 residues and promote cancer cell proliferation and migration [41]. In our study, IL-6 could not induce STAT3 Tyr705 phosphorylation in the A549 shMRE11 cells (Figure 1A). Moreover, MRE11 could interact with STAT3 in IL-6-treated cells (Figure 1B). On the other hand, a previous study showed that MRE11 interacted with dsDNA in the cytoplasm and not in the nucleus [38]. The location of the MRE11–STAT3 interaction must be investigated for a deeper understanding of the mechanism.CCL2, an important cytokine for macrophage chemotaxis and activation [42], is also produced by many types of tumor cells [43]. DSBs can result in higher CCL2 expression in cancer cells for macrophage activation and recruitment [44]. Moreover, lung cancer is a genomically unstable cancer with a high mutation rate [45]. Lung cancer patients with high CCL2 expression have a poor prognosis [46]. CCL2 signaling is the important pathway through which macrophages can activate the growth and metastasis of lung cancer cells by triggering bidirectional cross-talk between macrophages and cancer cells [15]. Our results showed that CCL2 mRNA expression and CCL2 secretion could not be activated in MRE11-knockdown lung cancer cells after macrophage activation (Figure 4). MRE11-knockdown A549 cells also exhibited a decreased ability to recruit macrophages (Figure 5). The results suggest that MRE11 could regulate the microenvironment of lung cancer through the CCL2 pathway. On the other hand, the MRN complex plays a pivotal role in DSBs [1,2]. However, the effect of NBS1 and RAD50 on CCL2 regulation remains unclear. The mechanism should be investigated further.