The Role of MRE11 in the IL-6/STAT3 Pathway of Lung Cancer Cells

MRE11 is a pivotal protein for ATM activation during double-strand DNA break. ATM kinase activations may act as lung cancer biomarkers. The IL-6/STAT3 pathway plays an important role in tumor metastasis, including lung cancer. However, the mechanism between MRE11 and the IL-6/STAT3 pathway is still unclear. In this study, we discovered that MRE11 can interact with STAT3 under IL-6 treatment and regulate STAT3 Tyr705 phosphorylation. After the knockdown of MRE11 in lung cancer cells, we discovered that IL-6 or the conditional medium of THP-1 cells can induce the mRNA expression of STAT3 downstream genes, including CCL2, in the control cells, but not in MRE11-knockdown lung cancer cells. Moreover, CCL2 secretion was lower in MRE11-knockdown lung cancer cells than in control cells after treatment with the conditional medium of RAW264.7 cells. In addition, MRE11 deficiency in lung cancer cells decreases their ability to recruit RAW 264.7 cells. Furthermore, MRE11 is a potential target for lung cancer therapy.


Introduction
Genome 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 factormediated 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.

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% CO 2 . 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% CO 2 . 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.

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.

Macrophage Recruitment Assay
Macrophage 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 × 10 4 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.

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.

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).

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.

The Effect of MRE11 on Activation of STAT3 under Treatment with IL-6
Next, we observed the effect of MRE11 on the mRNA expression of these downstream genes. After the cells were treated with the indicated IL-6 concentration, total mRNA was extracted from the A549 shLeu and A549 shMRE11 cells. Our data demonstrated that IL-6 can induce the mRNA expression of STAT3 downstream genes, including PLSCR1 (Figure 2A), ID1 ( Figure 2B), and XBP1 ( Figure 2C) in the A549 shLeu cells, but not in the A549 shMRE11 cells (Figure 2A-C). Next, we observed the effect of MRE11 on the mRNA expression of these stream genes. After the cells were treated with the indicated IL-6 concentratio  Figure 2C) in the A549 shLeu cel not in the A549 shMRE11 cells (Figure 2A-C).

The Effect of MRE11 on Cytokine Secretion from Lung Cancer Cells
Tumor cells secrete some molecules, such as chemokines, during invasion an

The Effect of MRE11 on Cytokine Secretion from Lung Cancer Cells
Tumor cells secrete some molecules, such as chemokines, during invasion and migration. Next, we investigated the effect of MRE11 on cytokine secretion from lung cancer cells. The secreted cytokines from the A549 shLeu and A549 shMRE11 cells were screened using the human cytokine array. The results revealed that MRE11 deficiency can decrease the expression of the cytokines CCL2, CCL5, interleukin-1 receptor antagonist, G-CSF, IL-6, and intercellular adhesion molecule 1 ( Figure 3A). A previous study showed that hypoxia can induce IL-6 production and subsequent secretion of CCL2 [33,34]. Next, we investigated the effect of MRE11 on the IL-6/CCL2 pathway under hypoxia. In a hypoxia environment for 24 h, we also discovered that secretion of IL-6 of A549 shMRE11 cells can be induced. However, the secretion of IL-8 and CCL2 was inhibited in A549 shMRE11 cells ( Figure 3B-D). 2022, 44 6138 using the human cytokine array. The results revealed that MRE11 deficiency can decrease the expression of the cytokines CCL2, CCL5, interleukin-1 receptor antagonist, G-CSF, IL-6, and intercellular adhesion molecule 1 ( Figure 3A). A previous study showed that hypoxia can induce IL-6 production and subsequent secretion of CCL2 [33,34]. Next, we investigated the effect of MRE11 on the IL-6/CCL2 pathway under hypoxia. In a hypoxia environment for 24 h, we also discovered that secretion of IL-6 of A549 shMRE11 cells can be induced. However, the secretion of IL-8 and CCL2 was inhibited in A549 shMRE11 cells ( Figure 3B-D).

The Effect of MRE11 on STAT3 s Downstream Genes from Lung Cancer Cells under Activation of Macrophages
Next, we treated the A549 shLeu or A549 shMRE11 cells with the conditional medium of THP-1 cells to simulate a co-cultured system. The qPCR results revealed that VEGF, RUNX2, and LDHA mRNA expression could be activated in conditional medium-treated A549 shLeu cells, but not in the treated A549 shMRE11 cells ( Figure 4A-C).

The Effect of MRE11 on STAT3′s Downstream Genes from Lung Cancer Cells under vation of Macrophages
Next, we treated the A549 shLeu or A549 shMRE11 cells with the condition dium of THP-1 cells to simulate a co-cultured system. The qPCR results reveale VEGF, RUNX2, and LDHA mRNA expression could be activated in conditional me treated A549 shLeu cells, but not in the treated A549 shMRE11 cells ( Figure 4A-C).

Effects of MRE11A on RAW 264.7 Cell Recruitment In Vitro
Our results showed that CCL2 mRNA expression could be activated in a cond medium of THP-1 cells-treated A549 shLeu cells, but not in the treated A549 shM

Effects of MRE11A on RAW 264.7 Cell Recruitment In Vitro
Our results showed that CCL2 mRNA expression could be activated in a conditional medium of THP-1 cells-treated A549 shLeu cells, but not in the treated A549 shMRE11 cells ( Figure 5A). Moreover, CCL2 secretion was lower in the A549 shMRE11 cells than in the A549 shLeu cells after treatment with the conditional medium of THP-1 cells or RAW264.7 cells according to the ELISA results ( Figure 5B,C). 2022, 44 6140 cells ( Figure 5A). Moreover, CCL2 secretion was lower in the A549 shMRE11 cells than in the A549 shLeu cells after treatment with the conditional medium of THP-1 cells or RAW264.7 cells according to the ELISA results ( Figure 5B,C). We also investigated the effect of MRE11 on macrophage recruitment by lung cancer cells. The cancer cells were cultured for 24 h. The conditioned media were collected and placed in the lower chambers, and RAW 264.7 cells were placed in the upper chambers of transwell plates in a serum-free medium for a migration assay. These results indicated that MRE11 deficiency in lung cancer cells can decrease their ability to recruit RAW 264.7 cells ( Figure 5D,E).  We also investigated the effect of MRE11 on macrophage recruitment by lung cancer cells. The cancer cells were cultured for 24 h. The conditioned media were collected and placed in the lower chambers, and RAW 264.7 cells were placed in the upper chambers of transwell plates in a serum-free medium for a migration assay. These results indicated that MRE11 deficiency in lung cancer cells can decrease their ability to recruit RAW 264.7 cells (Figure 5D,E).

Discussion
In a previous study, mice administered anti-IL6 antibodies exhibited increased radiationinduced 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 MRE11knockdown 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.

Conclusions
Together, our results suggest that IL-6 can induce the MRE11-STAT3 interaction and activate STAT3. Then, the activated STAT3 signaling pathway can induce CCL2 secretion for macrophage recruitment. Furthermore, MRE11 is a potential target for lung cancer therapy. Funding: Financial support was obtained in the form of grants CMRPG6H0161, CMRPG6H0162, and CMRPG6H0163 from Chang Gung Memorial Hospital awarded to Ching Yuan Wu. The funding bodies allowed the design of the study, the collection, analysis, and interpretation of data, and the writing of the manuscript.
Institutional Review Board Statement: Not applicable.

Informed Consent Statement: Not applicable.
Data Availability Statement: All data generated or analyzed during this study are indicated in this article (with no patient data). The datasets generated during and/or analyzed during the present study are available from the corresponding author upon reasonable request.