Downregulation of miR-155-5p enhances the anti-tumor effect of cetuximab on triple-negative breast cancer cells via inducing cell apoptosis and pyroptosis

Cetuximab resistance is the main obstacle for the treatment of EGFR overexpression cancer, including triple-negative breast cancer (TNBC). MicroRNA (miRNA)-155-5p is upregulated in TNBC cells; thus, the present study explored whether the downregulation of miR-155-5p enhanced the anti-tumor effect of cetuximab in TNBC cells. MDA-MB-231 and MDA-MB-468 cells were infected with lentivirus-epidermal growth factor receptor (EGFR) for 72 h to obtain EGFR-overexpressed cell lines (MDA-MB-231 and MDA-MB-468). The inhibitory effects of cetuximab on the proliferation and migration of EGFR-overexpressed MDA-MB-468 cells were enhanced following transfection with the miR-155-5p antagomir, and miR-155-5p knockdown enhanced the pro-apoptotic effect of cetuximab on EGFR-overexpressed MDA-MB-468 cells. Further, the luciferase reporter assay revealed that gasdermin E (GSDME) was the direct binding target of miR-155-5p. The combination of cetuximab with the miR-155-5p antagomir promoted pyroptosis in EGFR-overexpressed MDA-MB-468 cells via the upregulation of GSDME-N and cleaved caspase-1. Results from the in vivo experiments confirmed that the downregulation of miR-155-5p enhanced the anti-tumor effect of cetuximab in an MDA-MB-468 xenograft model and on EGFR-overexpressed TNBC cells via inducing cell apoptosis and pyroptosis. Therefore, cetuximab combination with an miR-155-5p antagomir may be a novel therapeutic strategy for the treatment of TNBC.

AGING of EGFR and suppresses the activation of a series of downstream signaling pathways [7]. Results from previous studies demonstrated that the expression of EGFR was increased in patients with TNBC [8,9]. Liao et al indicated that cetuximab treatment could induce apoptosis and inhibit growth of the EGFR-expressed TNBC cells in vitro and in vivo [10]. Therefore, cetuximab is an effective treatment for some patients with breast cancer. However, a large percentage of patients with breast cancer are resistant to anti-EGFR therapies after long period of treatment with EGFR inhibitor [11]. Therefore, novel therapies for the treatment of TNBC are needed.
MicroRNAs (miRNAs) are a class of endogenous noncoding single-stranded RNA molecules that contain 18-24 nucleotides [12]. MiRNAs regulate posttranscriptional gene expression by binding to the complementary sequences in the 3'-untranslated region (3'-UTR) of their target mRNAs [13]. Recently, miRNAs have emerged as novel biomarkers for various cancers, including breast cancer [14]. Liu et. al. [15] found that the level of miR-155-3p was up-regulated in breast cancer cells. Results from another study revealed that miR-155 promoted the proliferation of breast cancer cells and suppressed apoptosis in breast cancer cells [16].
In this study, we identified GSDME harbored a conserved miR-155-5p cognate sites using TargetScan bioinformatics tool, and predicted that GSDME was a potential target of miR-155-5p. GSDME was identified as the executioner of pyroptosis [17]. Pyroptosis is a novel form of programmed necrosis, which is triggered upon formation of caspase-1-activating inflammasomes [18]. Active caspase-1 can lead to increased production of gasdermin D and proinflammatory cytokines IL-1β and IL-18 [17]. Therefore, this study investigated whether the downregulation of miR-155-5p enhanced the anti-tumor effect of cetuximab in TNBC cells via targeting GSDME in order to provide an alternative therapeutic option for patients with TNBC.

EGFR is overexpressed in TNBC cells
First, we established TNBC cell lines (e.g., MDA-MB-231 and MDA-MB-468) with stable EGFR overexpression. As shown in Figure 1A and 1B, the fluorescent expression confirmed that the MDA-MB-231 and MDA-MB-468 cells were effectively transfected with the lentivirus after incubation for 72 h. In addition, the results from the quantitative real-time polymerase chain reaction (qRT-PCR) assay indicated that the expression of EGFR was significantly increased in MDA-MB-231 and MDA-MB-468 cells following transfection with lentivirus-EGFR ( Figure 1C-1F). These findings indicated that EGFR was
Results from immunofluorescence staining assay demonstrated that the proliferation of EGFR-overexpressed MDA-MB-231 and MDA-MB-468 cells was slightly inhibited following treatment with cetuximab or the miR-155-5p antagomir as compared with the proliferation of cells in the control group ( Figure 2E and 2F). Additionally, the proliferation of EGFR-overexpressed MDA-MB-231 and MDA-MB-468 cells was significantly inhibited in the cetuximab and miR-155-5p treatment group as compared with that in the cetuximab treatment group ( Figure 2E and 2F). These results suggested that the downregulation of miR-155-5p enhanced the anti-proliferative effect of cetuximab in EGFR-overexpressed TNBC cells.

Downregulation of miR-155-5p enhanced the cytotoxicity effect of cetuximab in TNBC cells
Next, the cytotoxicity effect of cetuximab combined with the miR-155-5p antagomir on EGFR-overexpressed MDA-MB-468 cells was measured with flow cytometry,  which suggested the forms of death including apoptosis (in Q2 and Q3 quadrant), necrosis or pyroptosis (in Q1 and Q2 quadrant). As indicated in Figure 3A and 3B, 10 nM cetuximab markedly induced the apoptosis of EGFRoverexpressed MDA-MB-468 cells, and the downregulated miR-155-5p significantly enhanced the pronecrosis or pro-pyroptosis effect in EGFR-over-expressed TNBC cells. In addition, the expression of Bax and cleaved caspase-3 were increased in EGFR-overexpressed MDA-MB-468 cells in the cetuximab and miR-155-5p antagomir treatment group as compared with that in the cetuximab treatment group. However, the level of BCl-2 was reduced in EGFR-overexpressed MDA-MB-468 cells in the cetuximab and miR-155-5p treatment group as compared with that in the cetuximab treatment group ( Figure 3C-3F). These data illustrated that the downregulation of miR-155-5p enhanced the cytotoxicity effect of cetuximab in EGFR-overexpressed MDA-MB-468 cells, however, the type of death need be identified by other assays.

Downregulation of miR-155-5p enhanced the inhibitory effect of cetuximab on the migration and invasion of TNBC cells
We performed wound healing and transwell invasion assays to determine the effect of cetuximab and the miR-155-5p antagomir on the migration and invasion of EGFRoverexpressed TNBC cells. As shown in Figure   AGING treatment group were suppressed as compared with those in the cetuximab treatment group ( Figure 4A-4D). These results indicated that the downregulation of miR-155-5p enhanced the inhibitory effect of cetuximab on the migration and invasion of EGFR-overexpressed MDA-MB-468 cells.

GSDME was a direct binding target of miR-155-5p
Although various target genes of miR-155-5p were obtained from the TargetScan database (http://www. targetscan.org/vert_71/), GSDME, a marker of pyroptosis, was used as the predicted target gene of miR-155-5p in the current study ( Figure 5A). In addition, the level of miR-155-5p was significantly upregulated following transfection with the miR-155-5p agomir ( Figure 5B). Furthermore, the results from the dual luciferase reporter assay confirmed that luciferase activity was reduced in the EGFR-overexpressed MDA-MB-468 cells following cotransfection with the WT-GSDME segment and miR-155-5p agomir as compared with that in the vector-control group ( Figure 5C). These results indicate that GSDME is a direct binding target of miR-155-5p.

Cetuximab combined with the miR-155-5p antagomir induced pyroptosis in TNBC cells
Results from previous studies indicate that the N-terminus of GSDME trans-locates to the cell membrane and induces cell pyroptosis [19]. To identify the mechanism underlying miR-155-5p-regulated pyroptosis in EGFRoverexpressed MDA-MB-468 cells, the protein levels of GSDME-N, cleaved caspase-1, and p-EGFR were detected by Western blotting assays. Results from the Wester blotting analysis revealed that cetuximab had no effect on the expression of GSDME-N and cleaved caspase-1 in EGFR-overexpressed MDA-MB-468 cells; however, the miR-155-5p antagomir significantly increased the levels of GSDME-N and cleaved caspase-1 in the EGFR-overexpressed MDA-MB-468 cells ( Figure  6A-6C). Cetuximab markedly inhibited the expression of p-EGFR in EGFR-overexpressed MDA-MB-468 cells, whereas treatment with the miR-155-5p antagomir had no effect on the expression of p-EGFR in the EGFRoverexpressed MDA-MB-468 cells ( Figure 6A and 6D). Moreover, results from the immunofluorescence staining assay confirmed that cetuximab combined with the AGING miR-155-5p antagomir increased the level of GSDME-N in EGFR-overexpressed MDA-MB-468 cells ( Figure 6E and 6F). Meanwhile, results from the RT-qPCR analysis revealed that cetuximab had no effect on the levels of IL-1β and IL-18 in EGFR-overexpressed MDA-MB-468 cells; however, the miR-155-5p antagomir markedly increased the levels of IL-1β and IL-18 in the EGFRoverexpressed MDA-MB-468 cells ( Figure 6G and 6H). Morphologically, cells in the miR-155-5p antagomir and cetuximab + miR-155-5p antagomir group exhibited the characteristic pyroptotic phenotype (e.g., cell swelling and large bubbles emerging from the plasma membrane) ( Figure 6I). These data indicated that cetuximab combined with the miR-155-5p antagomir induced pyroptosis in EGFR-overexpressed TNBC cells.

Downregulation of miR-155-5p enhanced the antitumor effect of cetuximab in TNBC cells in vivo
We further investigated the role of cetuximab combined with the miR-155-5p antagomir in a MDA-MB-468 xenograft mouse model. As shown in Figure 7A-7C, the tumor volume and tumor weight were decreased in the cetuximab + miR-155-5p antagomir treatment group as compared with those in the cetuximab treatment group. In addition, treatment with the miR-155-5p antagomir or combination treatment notably decreased the level of miR-155-5p in tumor tissues ( Figure 7D). Moreover, results from the immunohistochemistry (IHC) assay indicated that combination treatment markedly suppressed cell proliferation in tumor tissues ( Figure 7E and 7F). Meanwhile, results from the TUNEL assay revealed that combination treatment notably induced cell apoptosis in tumor tissues ( Figure  7G and 7H). Combination treatment upregulated the expression of GSDME-N and cleaved caspase-1 in tumor tissues, but downregulated the expression of p-EGFR ( Figure 8A-8D). These data indicated that the downregulation of miR-155-5p enhanced the anti-tumor effect of cetuximab in TNBC cells in vivo via inducing pyroptosis.

DISCUSSION
EGFR is overexpressed in TNBC cells; therefore it is a potential target for anticancer drugs [10]. Results from a study conducted by Nakamura et. al. [20] revealed that EGFR, which is a therapeutic target for oral cancer, was overexpressed in oral cancer cells. Cetuximab exhibits anti-tumor effects in human cancers via targeting EGFR [20][21][22]. In this study, we confirmed that cetuximab suppressed the proliferation of EGFR-overexpressed TNBC cells in vitro and in vivo via inhibiting the activation of EGFR.
Recently, miRNAs have gained attention as novel targets for the treatment of breast cancer [23]. Hong et al [24] indicated that miR-7 sensitized breast cancer cells to paclitaxel and carboplatin, while Shi et al [25] found that the overexpression of miR-129-5p enhanced the anti-tumor effect of taxol in breast cancer cells. AGING Studies have also demonstrated that miR-155 functioned as an oncogene in several cancers, such as oral squamous cell carcinoma, bladder cancer, and anaplastic thyroid cancer [26][27][28]. In this study, we found that miR-155-5p antagomir or cetuximab slightly inhibited the proliferation of EGFR-overexpressed TNBC cells. However, the complete knockdown of miR-155-5p markedly enhanced the anti-proliferative effect of cetuximab in EGFRoverexpressed TNBC cells in vitro and in vivo. Moreover, the upregulation of miR-155-5p enhanced the antiapoptotic effect of cetuximab in EGFR-overexpressed MDA-MB-468 cells via upregulating the expressions of Bax and cleaved caspase-3 and downregulating the expression of Bcl-2. These data suggest that the downregulation of miR-155-5p enhances the anti-tumor effect of cetuximab in EGFR-overexpressed TNBC cells via inducing apoptosis.
To further investigate the molecular mechanisms underlying the miR-155-5p-mediated growth of EGFRoverexpressed TNBC cells, we performed TargetScan and luciferase reporter assays to predict and confirm potential binding targets of miR-155-5p. Results from these assays indicated that GSDME was a potential binding target of miR-155-5p. GSDME is a member of the gasdermin family, which participates in the activation of pyroptosis [29]. Pyroptosis is a type of programmed cell death that is mediated by caspase-1 [30]. Specifically, activated caspase-1 cleaves GSDME into two fragments: N-and C-terminal domains [31]. The N-terminus of GSDME trans-locates into the cell membrane where it induces cell pyroptosis [19,29]. Pyroptosis is characterized by rapid plasma-membrane rupture and the release of proinflammatory intracellular contents [32]. In this study, we found that the  Apoptosis is the most widely recognized programmed process that lead to non-inflammatory cell death [32]. Meanwhile, pyroptosis (also known as caspase 1dependent programmed cell death) is another programmed cell death process, and is inherently inflammatory [33]. Our data found that combination treatment suppressed the growth of EGFRoverexpressed TNBC cells in vitro and in vivo by inducing apoptosis and pyroptosis. However, it is not clear at this point which one (apoptosis or pyroptosis) plays the leading role in triggering cell death in EGFR-overexpressed TNBC cells. Therefore, further study is needed to elucidate the exact mechanism of combination-mediated cell death in TNBC.

CONCLUSION
In this study, we found that cetuximab exerted antitumor effect in EGFR-overexpressed MDA-MB-468 cells via the inhibition of EGFR. In addition, the downregulation of miR-155-5p enhanced the antitumor effect of cetuximab in EGFR-overexpressed TNBC cells via inducing apoptosis and pyroptosis. Therefore, the combination of the miR-155-5p antagomir with cetuximab may be an alternative therapeutic approach for the treatment of TNBC.

Cell culture
The human TNBC cell lines (MDA-MB-231, MDA-MB-468) were obtained from Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). The cells were cultured in DMEM (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% heatinactivated fetal bovine serum (FBS) and antibiotics (100 U/mL penicillin and 100 mg/mL streptomycin) and incubated at 37 °C in a humidified atmosphere with 5% CO 2 .

Lentivirus production and cell infection
The EGFR sequence was synthesized by GenePharma and cloned into the lentiviral expression vector. Next, the lenti-EGFR plasmids were infected into 293T cells and incubated for 48 h at 32 °C. After incubation, the supernatant containing the virus particles were collected, and the MDA-MB-231 and MDA-MB-468 cells were seeded into 60-mm cell plates at a density of 4 x 10 5 cells/well and cultured overnight. The following day, the lenti-EGFR supernatant was added directly to the MDA-MB-231 and MDA-MB-468 cells. Finally, the cells were treated with Puromycin (2.5 μg/mL, Sigma Aldrich) for three days to select stable EGFR overexpressed cell lines (MDA-MB-231, MDA-MB-468).

Immunofluorescence staining assay
Cells were washed twice with PBS, pre-fixed with 4% paraformaldehyde at room temperature for 10 min, and post-fixed with pre-chilled methanol at -20 °C. Next, the cells were incubated with the following primary antibodies: anti-Ki67 and anti-GSDME-N at 4 °C overnight. The following day, the cells were incubated with secondary antibodies at 37 °C for 1 h. All of the antibodies were purchased from Abcam (Cambridge, MA, USA), and the cells were observed under a fluorescent microscope (Olympus BX53 Tokyo, Japan).

Flow cytometry
Cells were washed twice with pre-chilled PBS and resuspended in a binding buffer. Next, the cells were stained with 5 μL of Annexin V-FITC and 5 μL of propidium Iodide (PI, Thermo Fisher Scientific) for 30 min in the dark according to the protocol. A flow cytometer (BD FACSCanto II, BD Bioscience, Franklin Lake, NJ, USA) was used to determine the number of annexin V-FITC-positive apoptotic cells.

Wound healing assay
Cells (2 × 10 5 cells/well) were seeded into a 12-well culture plate and cultured in DMEM supplemented with 10% FBS. Once the cells reached 80 % confluency, a wound area was generated with a sterile 200 μL pipette tip, and the cells were washed with PBS to remove nonadherent cells. Next, the cells were treated with cetuximab (10 nM) and/or miR-155-5p antagomir (10 nM) for 48 h. Finally, the wound closure was photographed at 0 and 48 h in five random microscopic regions using a fluorescent microscope (Olympus).

Transwell invasion assay
A 24-well transwell (Corning New York, NY, USA) was pre-coated with Matrigel (BD Bioscience, Franklin Lake, NJ, USA). Cells (5 × 10 4 cells) that were suspended in serum-free DMEM medium were seeded into the upper chamber, and 600 μL of DMEM supplemented with 10% FBS were added to the lower chamber to induce cell invasion. After 24 h of incubation, the cells that attached to the lower surface of the chamber were stained with 0.2% crystal violet.
Finally, the cells were photographed at 0 and 48 h in five random microscopic regions using a fluorescent microscope (Olympus).

Transmission electron microscopy
The ultrastructure of the cells was observed with a transmission electron microscope (TEM, H-600IV, Hitachi Ltd., Japan). Briefly, the cells were fixed in 2.5% glutaraldehyde (GA, Sigma-Aldrich, St. Louis, MO, USA) at 4 °C overnight and dehydrated in ethanol. The samples were captured using a TEM as described previously [35].

Animal study
Four to 6-week-old BALB/c nude mice were purchased from the Hubei Provincial center for Laboratory Animal and maintained following the guidelines of the Institutional Animal Care and Use Committee. Animals were randomized into four groups: 1) Vehicle, 2) cetuximab, 3) miR-155-5p antagomir, and 4) cetuximab + miR-155-5p antagomir group. EGFR-overexpressed MDA-MB-468 cells (1 × 10 7 per mouse in 100 μL of PBS) were subcutaneously injected into the left flank of nude mice. When the tumors reached approximately 200 mm 3 , 50 nM miR-155-5p antagomir was directly injected into the tumors twice weekly, and the mice received weekly intraperitoneal injections of cetuximab (50 mg/kg). Tumor volume was measured every week using the following formula: V = (length x width 2 )/2 (width < length) The mice were euthanized at 28 days after treatment, and the tumors were isolated and weighed. All of the animal experiments were approved by the Institutional Ethical Committee of Longhua Hospital Affiliated to Shanghai University of TCM.

IHC analysis
IHC assays were performed to determine the expression level of Ki67 in tumor tissues. Tissue specimens were sectioned into 5 μm thick slices and incubated with the primary Ki67 antibody overnight at 4 °C. The following day, the tissue was incubated with biotinylated goat antirabbit IgG for 30 min at room temperature. IHC reactions were visualize using the IHC detection system (EnVision kit; Dako Japan).

Statistical analysis
All data were repeated in triplicate. Data are presented as the mean ± standard deviation (S.D.). All statistical analyses were performed using GraphPad Prism software (version 7.0, La Jolla, CA, USA). One-way analysis of variances (ANOVAs) were performed for multiple group comparisons, and pairwise comparisons were conducted with Tukey's tests when applicable. *P < 0.05 was considered to be statistically significant.

Editorial note
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AUTHOR CONTRIBUTIONS
WX and CS made major contributions to the conception, design and manuscript drafting of this study. WX, CS, XW and YL were responsible for data acquisition, data analysis and data interpretation. XB and XL were responsible for data acquisition and data analysis. JW and JL made substantial contributions to conception and design of the study and revised the manuscript. All authors agreed to be accountable for all aspects of the work. All authors read and approved the final manuscript.