MMP10 involved in Radiosensitivity in Non-Small Cell Lung Cancer via DNA damage repair pathway

doi:10.21203/rs.2.24001/v1

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MMP10 involved in Radiosensitivity in Non-Small Cell Lung Cancer via DNA damage repair pathway

https://doi.org/10.21203/rs.2.24001/v1

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posted 19 Feb, 2020

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Background

Non-small cell lung cancer (NSCLC) is an aggressive subtype of lung cancer with high mortality and morbidity. Our purpose in this study is to investigate the effect of MMP10 in NSCLC during radiotherapy and its mechanism.

Materials and methods

We analyzed MMP10 expression and overall survival rate of patients via UALCAN analysis. Western blot analysis was used to test the protein expression. We applied clonogenic assay and apoptosis assay to detect radioresistance in vitro. The level of DNA damage and the DNA damage repair were disclosed via comet assay and γH2AX foci assay.

Results

Our results showed that high MMP10 expression in Lung Adenocarcinoma specimens is associated with poor outcomes of patients. Knockdown MMP10 induced radiosensitization in A549 cells via clonogenic assay and apoptosis assay. Furthermore, we found MMP10 siRNA enhanced DNA damage of NSCLC cells and MMP10 involved in DNA damage repair after IR.

Conclusion

Our data highlighted that MMP10 plays a crucial role in NSCLC after radiation via DNA damage repair pathway, and the regulating effect of MMP10 on radiosensitivity in NSCLC might confer to therapeutic implications to NSCLC radiotherapy.

Oncology

MMP10

NSCLC

Radiosensitize

DNA damage repair

Non-small cell lung cancer (NSCLC) is an aggressive subtype of lung cancer with high mortality and morbidity, which includes squamous cell carcinoma, adenocarcinoma, and large cell carcinoma[1]. Surgery, radiation and chemotherapy are three common methods for lung cancer therapy[2]. However, the development of "precision radiotherapy" in recent years, represented as Stereotactic Body Radiation Therapy (SBRT), has highlighted its non-invasive, precise, and low side effects, and has provided more cure possibilities for lung cancer radiotherapy[3]. Although a number of comprehensive radiotherapy-based comprehensive treatment methods have involved in the treatment of non-small cell lung cancer, lung cancer has gradually tolerated ionizing radiation with the progression of radiotherapy, which indicates that most patients got radiotherapy resistance more or less and seriously affects the effect of radiotherapy[4–6].

Matrix metalloproteinase-10 (MMP10) is an important constituent of matrix metalloproteinases (MMPs)[7]. MMP10 belongs to the mesenchymal lysing enzymes in the MMPs family, which can degrade the core proteins of IV, V, Ⅸ, X-type collagen, fibronectin, laminin, elastin, gelatin and proteoglycan[8]. MMP10 participates in many physiological and pathological processes, and plays an important role in embryonic development, tissue damage repair and other processes[9, 10]. Studies have found that MMPs participate in the destruction and degradation of the extracellular matrix (ECM) and major tissues of the basement membrane to promote tumor growth, invasion and metastasis, and their mediation of the extracellular matrix and basement membrane is an invasion and necessary steps for tumor transfer[11, 12]. At present, there are many research reports on matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9), but few reports on the relationship between MMP10 and tumor[13, 14]. Recent research shows that MMP10 plays a very important role in the activation of pro MMPs[15]; MMP10 is highly expressed in epithelial tumors such as gastric cancer, bladder transitional cell cancer, esophageal cancer, skin cancer and non-small cell lung cancer[16–19]. These indicate that MMP10 is closely related to the occurrence and development of tumors.

In our study, we investigated the radiosensitization effects of MMP10 in NSCLC and found MMP10 conferred to radioresistance in NSCLC through DNA damage repair pathway. The regulating effect of MMP10 on radiosensitivity in NSCLC might confer to therapeutic implications to NSCLC radiotherapy.

High MMP10 expression in Lung Adenocarcinoma specimens is associated with poor outcomes of patients.

We used UALCAN analysis to disclose the MMP10 expression in Lung Adenocarcinoma (LUAD) specimens based on TCGA samples. As shown in Fig. 1A, MMP10 expression in LUAD specimens was significantly higher than normal tissues in 515 primary Lung adenocarcinoma tissues compared to 59 normal tissues. Next we analyzed the association of MMP10 expression with the lifespan of LUAD patient via UALCAN analysis. Figure 1B showed that the overall survival rates for patients with MMP10^high LUAD were significantly lower than those with MMP10^low tumors(P < 0.05).

Effects of MMP10 siRNA on cell proliferation and apoptosis of A549 cells after IR.

To disclose the effects of MMP10 in radiotherapy, firstly we used MMP10 siRNA to inhibit MMP10 expression in A549 cells (Fig. 2A). Then we applied these cell models to clonogenic assay, the figure showed knockdown MMP10 expression significantly sensitized these cells to IR (Fig. 2B). Furthermore, it was found that knockdown MMP10 expression significantly promotes apoptosis of A549 cells after IR (Fig. 2C,2D).

MMP10 siRNA enhances DNA damage of NSCLC cells after IR.

To fully disclose the role of MMP10 in NSCLC after IR, we applied comet assay to assess the level of DNA damage via MMP10 siRNA. The images and column diagrams showed that knockdown MMP10 expression promoted the DNA damage after IR (Fig. 3A, 3B, 3C), which meant that MMP10 might play the important role in DNA damage repair pathway after IR.

MMP10 is involved in DNA damage repair pathway.

To verify our inference, we used immunofluorescence and western bolt to test the DNA damage repair pathway after IR and MMP10 siRNA treatment. The γH2AX foci assay showed that the number of foci in siMMP10 + IR group was much higher than that in IR group at 8 h after IR (Fig. 4A, 4B), which indicated knockdown MMP10 significantly impaired DNA repair in response to IR. Then we tested the phosphorylation of DNA damage repair proteins after MMP10 siRNA and radiation, the results showed that the phosphorylation of DNA damage repair proteins was inhibited after MMP10 siRNA (Fig. 5), which indicated MMP10 involved in DNA damage repair pathway after IR.

In this study, we figured out that MMP10 involved in radiosensitivity in Non-Small Cell Lung Cancer via DNA damage repair pathway. Firstly, we used UALCAN analysis to disclose the MMP10 expression in Lung Adenocarcinoma (LUAD) specimens based on TCGA samples, we found MMP10 expression in LUAD specimens was significantly higher than normal tissue. Next we analyzed the association of MMP10 expression with the lifespan of LUAD patient via UALCAN analysis, which we found that the overall survival rates for patients with MMP10^high LUAD were significantly lower than those withMMP10^low tumors(P < 0.05). Then we applied A549 to detect Effects of MMP10 on cell proliferation and apoptosis after IR, which showed that MMP10 conferred to radioresistance in NSCLC. Furthermore, we discovered knock down MMP10 enhanced DNA damage after IR via inhibiting DNA damage repair pathway, which we considered the main reason conferred to radioresistance in NSCLC.

Radiotherapy is the important tumor therapy strategy in lung cancer, however, the outcome of the radiotherapy is not quite satisfied[20]. The main research fields of radiosensitization involved in following respects: tumor cell hypoxia tolerance, DNA damage repair, apoptosis, cell cycle disorders and angiogenesis[21–23]. However, although research on radiosensitization has made some progress, it is still in the exploratory stage. The radiotherapy resistance phenomenon of lung cancer cells still exists, which gains many uncertainties in treatment[24, 25].

Matrix metalloproteinase-10 (MMP10) is an important constituent of matrix metalloproteinases (MMPs). MMP10 participates in many physiological and pathological processes, and plays an important role in embryonic development, tissue damage repair and other processes[26]. Recent research shows that MMP10 plays a significantly important role in the activation of pro MMPs[27]; MMP10 is highly expressed in epithelial tumors such as gastric cancer, bladder transitional cell cancer, esophageal cancer, skin cancer and non-small cell lung cancer[17, 28–30]. In our study, we found that knockdown MMP10 in A549 cells by MMP10 siRNA could significantly inhibit the proliferation and promoted apoptosis after IR. Then we found knockdown MMP10 could enhance the level of DNA damage after IR. Furthermore, we utilized immunofluorescence and western bolt technical means to check the DNA damage repair pathway, which we found MMP10 plays a crucial role in NSCLC after radiation via DNA damage repair pathway, and the regulating effect of MMP10 on radiosensitivity in NSCLC might confer to therapeutic implications to NSCLC radiotherapy.

Overall, this is the first time we showed that MMP10 could significantly radiosensitive NSCLC cells. For further step, we found that the regulating effect of MMP10 on radiosensitivity in NSCLC might confer to its participation in DNA damage repair pathway. These new findings could help us to discover new therapeutic implications to NSCLC radiotherapy.

UALCAN analysis

UALCAN is publicly available at http://ualcan.path.uab.edu. It is an interactive web-portal for in-depth analyses of gene expression data on cancers from TCGA. We utilized this website to analyze MMP10 expression and overall survival rate of patients.

Cell culture

Human lung adenocarcinoma cell line A549 was purchased from ATCC(USA). Cells were grown in DMEM supplemented with 10% fetal bovine serum at 37 °C in a 5% CO2 humidified chamber.

Irradiation

We used ⁶⁰Co γ-rays for cell radiation exposure. The ⁶⁰Co γ-rays sited in Radiation Center (Faculty of Naval Medicine, Second Military Medical University, Shanghai, China). Different cells were treated with specific dose at a dose rate of 1 Gy/min.

siRNA and transfections

siRNA against MMP10 were purchased from Thermo Fisher (Catalog # AM16708). Transfect MMP10 siRNA with Lipofectamine 3000 (Invitrogen) according to the manufacturer's instructions. Transfected cells were used for further experiments at different time point.

Clonogenic assay

The proliferation of A549 cells were assessed by Clonogenic assay. Cells were plated in 60 mm dishes, pretreated cells were irradiated with 0, 2, 4, 8 Gy. 10 days later, fixed the cells with paraformaldehyde and stain with 1% methylene blue. Wash dishes with PBS after 30 minute and dry dishes naturally. Then count the clone formation.

Apoptosis assay

Annexin V-fluorescein isothiocyanate (Annexin V-FITC) and Propidium Iodide (PI) in Apoptosis Detection Kit (Invitrogen, Carlsbad, California, USA) were utilized to stain A549 cells after radiation and analyzed by flow cytometry (Beckman Cytoflex) according to the manufacturer’ instructions.

Comet Assay

Level of DNA damage was assessed by Neutral comet assay. Comet assay Kit (Trevigen Inc., Gaithersburg, MD) were used to detect the DNA double-strand breaks of A549 cells at 4 h, 8 h after radiation following the manufacturer’s protocol.

Western blot analysis

At 0 h, 0.5 h, 8 h time point after irradiation, total cell lysates were prepared via using ProtectJETTM Mammalian Cell Lysis Reagent (Fermentas, Vilnius, Baltic, Lithuania) according to manufacturer’s protocol. The membranes were incubated overnight with each primary antibody [1: 1, 000] at 4 °C with gentle agitation: MMP10 (Abcam, US; 1:1000), p-DNA-PKcs (Abcam, US; 1:1000), p-ATM (Abcam, US; 1:1000), Rad51(Abcam, US; 1:1000), actin (Abcam, US; 1:1000). The secondary antibody (1:5000) was purchased from Abcam. All membranes were detected using the ECL system (Santa Cruz Biotechnology Inc.).

γH2AX foci assay

γH2AX foci is DNA double strand break marker. A549 cells were transfected with MMP10 siRNA after 2 Gy radiation, at specific time point fixed cells with ice-cold acetone/methanol (1:1) and blocked with 3% BSA in PBS for 1 h at room temperature. Then cells stained with γH2AX primary antibody(Abcam, US; 1:300) and stained with the secondary antibody (1:1000) at next step. Immunofluorescence was monitored by both conventional and confocal microscopy.

Statistical analysis

All data were obtained by at least three independent experiments and represented as mean ± standard deviation. The limit of statistical significance was set at P < 0.05.

Mean and standard error (SEM) of each treatment calculate all experimental groups. All pairwise comparison procedures, including calculations P-values are done using Student's t test.

NSCLC: Non-small cell lung cancer; SBRT: Stereotactic Body Radiation Therapy; MMP10: Matrix metalloproteinase-10; ECM: extracellular matrix; LUAD: Lung Adenocarcinoma; DDR: DNA damage repair; DSB: Double-Strand Breaks; DNA: Deoxyribonucleic Acid; RNA: Ribonucleic Acid; SEM: Mean and standard error; PBS: phosphate buffer saline; BSA: bovine serum albumin; siRNA: Small interfering RNA.

Acknowledgements

Not applicable.

Authors’ contributions

Xiao Lei designed the study, analyzed the data and made the manuscript. Yuanyuan Chen did the most experiments. Na Ma and Pei Zhang did some experiments and analyzed the data. Yanan Han participated in the research design. Baolin Qu and Lehui Du participated in the writing of paper and revision of manuscript. All authors read and approved the final manuscript.

Funding

No funding was received.

Availability of data and materials

The datasets are available under reasonable request.

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Author details

¹ The First Medical Center of PLA General Hospital, Department of Radiation Oncology, Beijing, P.R. China.² Department of Radiation Medicine, Faculty of Naval Medicine, Second Military Medical University, Shanghai, P.R. China

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MMP10 involved in Radiosensitivity in Non-Small Cell Lung Cancer via DNA damage repair pathway

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