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Matrix Metalloproteinase 11 Is a Potential Therapeutic Target in Lung Adenocarcinoma

Lung cancer is one of the leading causes of cancer-associated death, with the etiology largely unknown. The aim of this study was to identify key driver genes with therapeutic potentials in lung adenocarcinoma (LUAD). Transcriptome microarray data from four GEO datasets (GEO: GSE7670, GSE10072, GSE68465, and GSE43458) were jointly analyzed for differentially expressed genes (DEGs). Ontologic analysis showed that most of the upregulated DEGs enriched in collagen catabolic and fibril organization processes were regulated by matrix metalloproteinases (MMPs). Matrix metalloproteinase 11 (MMP11), the highest upregulated MMP family member in LUAD-transformed cells, acted in an autocrine manner and was significantly increased in sera of LUAD patients. MMP11 depletion severely impaired LUAD cell proliferation, migration, and invasion in vitro, in line with retarded tumor growth in xenograft models. Treatment of different human LUAD cell lines with anti-MMP11 antibody significantly retarded cell growth and migration. Administration of anti-MMP11 antibody at a dose of 1 μg/g body weight significantly suppressed tumor growth in xenograft models. These findings indicate that MMP11 is a key cancer driver gene in LUAD and is an appealing target for antibody therapy.


INTRODUCTION
Lung cancer is one of the most common malignant tumors worldwide. Surgery, radiation, and chemotherapy are three common methods for lung cancer therapy. 1 However, regional therapeutic methods, including surgery and radiotherapy, have a poor effect on patients with advanced metastasis. Chemotherapy is currently a major treatment for lung cancer with metastasis, but chemotherapeutic drugs are not specific to killing tumor cells and will cause resistance in patients receiving multiple rounds of chemotherapy. With the emerging concept of "precision medicine" in recent years, more and more molecularly targeted drugs have been effectively applied, 2 and the treatment of lung cancer has entered a new era. In the concept of precision medicine, key genes driving carcinogenesis could be used as therapeutic targets. 3 Biological reagents or drugs specifically designed to target these genes would interfere with tumor cells and their related microenvironments, so as to eliminate or suppress the occurrence and development of cancer. A variety of therapeutic targets with different biological functions, including EGFR, 4 VEGF, 5 ALK, 4 MET, 6 KRAS, 4 ROS1, 7 HER2, 8 and others, 9 have been investigated. Accordingly, therapeutic drugs targeting specific molecules in lung cancer cells have been developed, include cell growth factor receptor inhibitors, 10 angiogenesis inhibitors, 11 and signal transduction inhibitors. 12 Although clinical results have indicated that molecularly targeted therapy has an overall advantage, with the emergence of resistance to drugs, curing lung cancer still faces challenges, 13,14 greatly requiring development of new types of targeted therapy drugs.
Genome-wide profiling provides deep insights into tumorigenesis and has proved to be a robust way to identify pathogenic gene targets. 15 Furthermore, substantial amounts of gene expression data have been created and deposited in public databases, providing a great opportunity for deciphering the molecular mechanisms of tumorigenesis. 16 Two frequently used public gene expression databases, GEO (https://www.ncbi.nlm.nih.gov/geo/) and The Cancer Gene Atlas (TCGA) project (https://gdc.cancer.gov/), contain deposits of a great amount of transcriptomic data generated from microarray or high sequencing. To identify novel gene targets with therapeutic potential in lung adenocarcinoma, we investigated the gene expression pattern by integrating four independent transcriptomic microarray datasets of lung adenocarcinoma. A total of 48 common differentially expressed genes (DEGs) were identified. Collagen catabolic and fibril organization processes were the most enriched biological processes associated with 30 upregulated common DEGs. A group of four matrix metalloproteinases (MMPs) identified in this screen are functionally related to this process. After validation by TCGA data, we proposed that MMP11 was the most significantly overexpressed DEG, and it was the focus of the current study.
MMPs are a class of enzymes that degrade extracellular matrix (ECM). 17 They are important tumor microenvironment regulators and play an extremely important role in tumor invasion and metastasis. MMP11, also named stromelysin-3, is a member of the stromelysin subgroup belonging to the MMP superfamily. 18 MMP11 is secreted in an enzymatically active form and can be detected in patients' serum. We proposed that MMP11 may actively participate in the regulation of the tumor microenvironment, by promoting the occurrence and development of malignant tumors. Therapeutically targeting the tumor microenvironment may have merit for improving the prognosis of cancer patients. 19 In the current study, the role of MMP11 in the regulation of lung adenocarcinoma (LUAD) development was comprehensively studied by cellular functional assay in vitro and antibody therapy assay in vivo. Our results establish the potential of MMP11 as a therapeutic target for lung cancer.

Bioinformatics Identifies DEGs in LUAD and Non-malignant Tissues
Four gene expression datasets of LUAD in the GEO database, including GSE7670, GSE10072, GSE68465, and GSE43458, were used for identification of genes differentially expressed in lung cancer. The intersecting part of the four sets of DEGs consisted of 48 genes, which were thought to be involved in development and progression of this malignancy (Table 1). Among the 48 DEGs, 30 were upregulated in tumor tissues ( Figure 1A). Impressively, the gene ceacam5, encoding the most common serum biomarker CEA for diagnosis of lung cancer, was on the list of upregulated genes, providing convincing evidence of the efficacy of the DEG profile. Next, to gain insight into the biological roles of DEGs involved in cancer progression, we performed a Gene Ontology (GO) enrichment analysis and a protein-protein interaction (PPI) analysis. Most of the enriched GO terms belonged to the biological process category. Collagen catabolic and fibril organization processes were the two most relevant biological processes, and nearly half of the terms involved regulating collagen assembly or disassembly through proteolysis. In terms of the cellular component, the enriched GO terms were mainly related to the ECM. Of the 10 enriched GO molecular functions, 4 were asso-ciated with endopeptidase activity ( Figure 1B). PPI combined with ontologic analysis revealed common pathways or processes associated with upregulated genes in LUAD ( Figure 1C), including ECMreceptor interaction, bladder cancer, elevated chemokine-related signaling, protein digestion, and absorption ontologies. Besides the chemokine-signaling-related factors CXCL13 and CXCL14, most of the upregulated genes belong to the collagen and MMP families. We propose that the DEGs encoding MMPs or collagens found in this study may play critical roles in the development of LUAD. Large studies have demonstrated that MMPs and collagens contribute to the progression of cancer by modulating the tumor microenvironment. [20][21][22] MMPs, as enzymes with peptidase activity taking an active role in ECM remodeling, are thought to be key players in this process.

MMP11 as a Serum Biomarker of LUAD
The above results indicated that four matrix metalloproteinases, including MMP11, MMP12, MMP9, and MMP7, are commonly upregulated in tumor tissues of LUAD. To further validate the results, we analyzed their expression based on transcript counts derived from the mRNA-seq data that was downloaded from TCGA. As shown in Figure 2A, the expression level of MMP11 has a 5-fold increase in tumor tissues-highly significant compared to the other three well-studied MMPs, 23-25 MMP12, MMP9, and MMP7 (Figure S1). The roles of MMP11 in promoting tumorigenesis have been demonstrated in several malignancies, such as breast, 26 gastric, 27 and colorectal cancers, 28 but there has been little research on its connection with lung cancer. Therefore, we focused on MMP11, to examine its regulation of the progression of LUAD.
First, to evaluate the overexpression of MMP11 in LUAD, we performed immunohistochemistry in clinically collected biopsy samples. Although MMP11 was initially considered to be expressed by fibroblasts in tumor stroma, 29 further studies indicated that it may be expressed in both tumor cells and stromal cells. 18,30,31 Our results showed that strong staining for MMP11 was observed in LUADtransformed cells within the tumor cell masses, with little or no staining seen in the adjacent stroma ( Figure 2B), which provides evidence that LUAD tumor cells may release MMP11 protein in an autocrine manner to facilitate invasion of adjacent tissues ( Figure 2B), actively exerting its biological endopeptidase activity in digesting ECM. Since MMP11 is a secreted protein, the increase of its level in the circulation may reflect oncogenic occurrence. Therefore, we examined MMP11 levels by ELISA in serum from LUAD patients compared to normal healthy individuals. There was a significant increase in MMP11 protein in sera of LUAD patients ( Figure 2C), implying that MMP11 may have potential as a serum biomarker for diagnosis of LUAD.

Critical Role of MMP11 in LUAD Cell Proliferation
To characterize the oncogenic role of MMP11 in LUAD cells, we depleted MMP11 by the CRISPR/Cas9 system through a stable infection of cells with lentiviruses harboring sequence-targeting of the MMP11 gene in the A549 cell line, a widely used LUAD cell model. MMP11 knockout was efficiently achieved as almost undetectable protein of two cell clones with different sequence targeting www.moleculartherapy.org cells, which was consistent with a reduced cellular proliferation rate, as indicated above ( Figure 2F). Western blot showed that phosphorylated AKT was downregulated in MMP11-knockout cell lines (Figure S3), indicating that MMP11 may affect cell proliferation through the PI3K/AKT signaling cascades. A colony-formation assay was performed in both control and MMP11-knockout cells. As indicated, MMP11-depleted cells had a nearly 60% reduced number of formed cell colonies with relatively smaller size in comparison with control cells ( Figure 2G). The colony formation of cancer cells is an important indicator of their malignancy, reflecting their proliferative ability.
To further evaluate the oncogenic role of MMP11, another human LUAD cell line, PC9, was used for analysis. MMP11-knockout and overexpression were efficiently achieved ( Figure 3A). Compared to the control cells, MMP11 depletion significantly reduced cellular proliferation rates, which were partially rescued by transiently overex- pression of MMP11, especially significant on day 3 ( Figure 3B). Similar cell morphological change was found in PC9 cells, compared with A549 cells (Figure S2B). In addition, significant reduction of labeling of the proliferation marker Ki67 was observed in MMP11-deficient PC9 cells, inconsistent with the results shown in A549 cells ( Figure 3C). The reduction of Ki67 labeling in PC9 cells with MMP11 depletion was partially rescued by transient MMP11 overexpression ( Figure 3C). A colony-formation assay was also performed in PC9 cells with MMP11 depletion. As indicated, MMP11-depleted PC9 cells had a significantly reduced number of formed cell colonies, with relative smaller size in comparison with control cells (Figure 3D), which was partially restored by overexpression of MMP11. In summary, the above results suggest that MMP11 is critical for LUAD cell proliferation.

Exogenous Antibody against MMP11 Inhibits LUAD Cell Proliferation
The above experiments demonstrated the inhibitory effect of endogenous depletion of MMP11 on the proliferation of LUAD cells. We considered whether inhibition of MMP11 activity by exogenously applied antibody would have the same effect on cell proliferation. Cells including A549 and PC9 were treated with different concentrations of monoclonal antibody against MMP11 or control vehicle for up to 96 h. The inhibitory effect of antibody treatment on cell proliferation was shown in an antibody concentration-dependent fashion (Figure 4). With the increase of antibody concentration and the extension of the treatment period, the cells' status progressively deteriorated, and they started to shrink, with a morphological change from spindle to cobblestone similar to that shown in MMP11knockout cells (Figures 4 and S2). Significant reduction of cell proliferation was observed in cells treated with antibody concentrations as low as 1 mg/mL in both A549 ( Figure 4A) and PC9 cells ( Figure 4B). The above results suggest that exogenous inhibition of MMP11 by antibody may be an effective therapeutic intervention for LUAD.

MMP11 Depletion Impairs Cellular Motility and Invasion
Subsequently, we investigated whether morphological and proliferative changes in MMP11-depleted cells would affect cellular migratory and invasive behaviors. As shown in Figure 5, MMP11 depletion severely impaired the migratory ability of A549 and PC9 cells, as examined by wound-healing assay. The wound area closed significantly slower in the MMP11-depleted A549 cells compared to the control cells. Whereas the control cells almost closed the wound after culturing for 56 h, MMP11-knockout cells still left significant wound area unhealed ( Figure 5A). Cell invasion is an important indicator of the metastatic ability of cancer cells, which can be evaluated by transwell invasion assay. As indicated, MMP11-depleted A549 cells exhibited significantly lower (60% reduction) infiltration rates in transwell invasion experiments than control cells ( Figure 5B). To emphasize the specific role of MMP11 on LUAD cell migration and invasion, both wound-healing assay and transwell invasion assay were performed on the PC9 cell line, and similar results were obtained, indicating that MMP11 depletion severely impaired cell migration and invasion (Figures 5C and 5D). Importantly, rescue studies confirmed that transient MMP11 overexpression partially restored the migratory and invasive capacities of MMP11-deficient PC9 cells, demonstrating the specific role of MMP11 in regulation of LUAD cells' oncogenic abilities. The above results demonstrate that MMP11 plays an essential role in LUAD cell migration and invasion.

Exogenous Antibody to MMP11 Inhibits Cell Migration Ability
We next investigated if exogenous treatment of A549 cells with antibody against MMP11 would affect the migratory ability of cells. Prior  experiments had shown that application to the cells of a dose of antibody as low as 1 mg/mL could effectively inhibit cell proliferation. Herein, two different concentrations, 0.5 and 1 mg/mL, were selected for a wound-healing assay. As shown in Figure 6A, under both concentrations of antibody, the migratory ability of the A549 cells was decreased in a dose-dependent manner. In concurrence with the results in the A549 cell line, a similar phenomenon was found in the PC9 cell line ( Figure 6B). In both cell lines, considering that the wound area did not be close, even up to 56 h, and that the cells exhibited an unhealthy cobblestone morphology, 1 mg/mL was the more preferable concentration for antibody inhibition of cell migration ( Figure 6). We argue that exogenously administered MMP11 antibody may be an attractive therapy for LUAD.

In Vivo Tumorigenesis and Tumor Targeting
The above results demonstrated the oncogenic properties of MMP11 and the efficacy of MMP11 antibody treatment to inhibit oncogenic behaviors of LUAD tumor cells in vitro. We next sought to examine whether silencing MMP11 or antibody targeting of MMP11 affects tumor development in vivo. A xenograft model was established using the LUAD cell line A549. First, mice were injected subcutaneously with MMP11-depleted A549 cells and control normal A549 cells to develop solid tumors. Tumor xenografts were excised and analyzed at day 30. The mouse body weights were comparable over the course of treatment between the different groups of mice: those with subcutaneous injection of MMP11-depleted cells and those with control normal A549 cells ( Figure S4A). Immunohistochemistry assays validated the loss of expression of MMP11 in excised tumors derived from MMP11-depleted cells ( Figure S5A). Importantly, the volume of the tumor masses in mice injected with control cells was almost four times larger at day 30 than in those with the MMP11-depleted xenografts ( Figure 7A), suggesting that MMP11 affects tumor development in vivo.
To further validate the role of MMP11 on tumorigenesis in vivo, we tested the efficacy of MMP11 antibody therapy on tumorigenic inhibition in mouse xenografts. Female BALB/c nude mice were inoculated subcutaneously with A549 cells. Beginning on day 14, anti-MMP11 antibody and sterile saline control were injected via the tail vein at intervals of 4 days. After four rounds of antibody injections up to day 30, tumor pads were excised for measurement. The mouse body weights were comparable between the groups at various time points ( Figure S4B). Importantly, MMP11 antibody treatment significantly retarded LUAD tumor growth, given that tumor volumes in the antibody-treated group were two to three times smaller than those in the control group ( Figure 7B). That is, antibody treatment reduced the growth of pre-established tumors by more than 60% compared with the saline control ( Figure 7B), which was inconsistent with greatly reduced Ki67 expression ( Figure S5B). These results suggest that MMP11 antibody therapy can effectively reduce the LUAD tumor load.

DISCUSSION
In this study, we identified MMP11 as a tumor biomarker in LUAD, which poses the potential for its use as a therapeutic target for treatment of this malignancy. MMP11 has long been found to be associated with tumor progression in some types of cancer, such as breast, liver, and colon. To date, however, data are limited on the role of MMP11 in lung cancer. In this study, we first performed gene expression profiling on four independent datasets of LUAD and uncovered MMP11 as a potential driver gene associated with LUAD. We found that the expression of MMP11 increased in both tumor tissues and sera from LUAD patients. Functional studies indicated that MMP11 played a key role in promoting tumor cell proliferation, migration, and invasion. Most important, its suitability as a therapeutic target was assessed, using an anti-MMP11 antibody, in both in vitro and in vivo studies.
It is well known that almost all solid tumors are composed not only of tumor cells, but also of various non-tumor stromal cells, as well as cytokines, growth factor, proteases, and ECM. 32 Tumor cells and stromal components are tightly linked and orchestrated with each other through extracellular elements to form the tumor microenvironment, which promotes tumorigenesis by malignantly transforming normal epithelial cells or by accelerating tumor cell growth and invasion. Generally, degradation and remodeling of the ECM is a prerequisite for tumor cell invasion of the adjacent normal parenchyma and metastasis to distant tissues. 33 MMPs are a family of zinc-dependent endopeptidases with a powerful capacity for degrading ECM proteins, revealing its critical role during the process of tumorigenesis. In this study, we found that tumor cells may release MMP11 in an autocrine manner, thus actively modifying the microenvironment to adapt their malignant biological behaviors.
So far, there have been many studies on the MMPs family proteins, but the research on MMP11 has not been adequate, especially in lung cancer. Human MMP11 was first identified in the stromal cells of breast carcinoma. 34 It is upregulated in colon cancer, 31 and its downregulation inhibits tumor proliferation and metastasis in hepatocellular carcinoma. 35 In colorectal cancer, tumor metastasis can be effectively prevented by let-7-mediated inhibition of MMP11. 28 Knockdown of MMP11 also inhibits cell proliferation and metastasis in gastric cancer. 27 It is also highly expressed in laryngeal cancer. 36 In prostate cancer, downregulation of MMP11 can reduce tumor migration. 37 Downregulation of mir125a5p in osteosarcoma targets MMP11, to suppress tumor progression, 38 and MMP11 may even serve as a new prognostic factor in hormone-receptor-negative, HER2-positive breast cancers. 39  www.moleculartherapy.org proteins, MMP11 has its unique characteristics and functions. It is intracellularly processed to secrete in an active form instead of as an inactive zymogen like other MMPs. Moreover, unlike other MMPs that degrade the laminin, fibronectin, and elastin of these classic substrates, MMP11 preferentially hydrolyzes serine protease inhibitor a1-antitrypsin, insulin-like growth factor binding protein-1 (IGFBP-1), 40 and collagen VI. 41 IGF-1-mediated cell growth signaling was generally neutralized by IGFBP-1. MMP11 cleaves IGFBP-1 to release extra free IGF-1 in the tumor microenvironment, thus hyperactivating the IGF-1-mediated PI3k/AKT signaling cascade. Our work supports this hypothesis, since MMP11-deficient cells showed impaired proliferative ability and downregulation of phosphorylated AKT protein. Our results are also consistent Deng et al. 42 , which shows that knockdown of MMP11 in gastric cell lines inhibits cell growth and colony formation. It is not surprising to find that MMP11 plays roles in promoting cell migration and invasion, since MMP11 functions in ECM reconstruction. Substantial evidence supports this notion in various cell models, since the first study demonstrated that the oncogene Gli1 enhances migration and invasion via upregulation of MMP11 in breast cancer cell lines. 43 Our work demonstrated that MMP11 protein levels in serum was significantly increased in patients with LUAD compared to normal healthy individuals. Because of the importance of early diagnosis of malignant tumors in decreasing mortality, this finding suggests that MMP11 is a potential biomarker for clinical diagnosis in LUAD. A prior study proved the efficacy of serum MMP11 as a diagnostic marker for gastric cancer, with higher sensitivity compared to other traditional tumor markers, such as CEA, CA199, CA242, and its family member MMP9. 44 In addition, as a diagnostic marker, another important application for MMP11 in LUAD may be as a therapeutic target. As an important component of the cancer microenvironment, MMP11 is released by LUAD tumor cells, which may remodel ECM to facilitate tumorigenesis and progression of cancer. Our results indicate that anti-MMP11 antibody application could efficiently inhibit growth and migration of lung cancer cells in vitro. In vivo treatment of human LUAD xenografts with anti-MMP11 antibody strongly prevented tumor growth, which was inconsistent with in vitro data. In a previous study, immunotherapy using MMP11 as antigen exerted effective anti-tumor effects in a colon adenocarcinoma mouse model. 45 Combined with our current work, MMP11 holds potential as a therapeutic target in different solid tumors.
In summary, MMP11 expression was highly expressed in tissues and sera from patients with LUAD, and MMP11 antibody therapy inhibited tumor growth in both in vitro experiments and in vivo xenografts. This work demonstrates that MMP11 may serve as a potent therapeutic target through antibody therapy for LUAD. Additional studies are required to delineate in detail the exact molecular mechanism, as well as the in vivo efficacy and safety of this therapeutic strategy.

Microarray Data Mining and Gene Expression Analysis
We analyzed microarray datasets containing LUAD patient samples from the GEO  from American Type Cell Culture (ATCC) and cultured in the recommended media at 37 C and 5% CO 2 in a Thermo Fisher X50 incubator.

Collection of Serum and Tumor Tissues
The collection of tumor and adjacent normal tissues and plasma from LUAD patients was approved by the Institutional Review Board of the Cancer Hospital (Chinese Academy of Sciences, Hefei, China), in accordance with the Declaration of Helsinki.

Antibodies
Mouse monoclonal antibody against MMP11 was purchased from Boster Biological Technology. Mouse monoclonal antibodies against AKT, p-AKT, ERK, p-ERK, and Vimentin were purchased from Cell Signaling Technology. Rabbit antibodies against Ki67 and b-actin were obtained from Fuzhou Maixin Biotech (China) and Sangon Biotech (China), respectively.

Immunohistochemistry and Western Blot
Immunohistochemical staining and western blot were performed according to standard protocols. 48,49 Details are available in Supplemental Materials and Methods.

Plasmid Construction, Lentivirus Packaging, and Infection
The single guide RNAs were designed with the CRISPR Design webtool (https://zlab.bio/guide-design-resources/). The oligos were annealed and cloned into the lentiviral shuttle vector lenti-CRISPRv2 (Addgene). Lentivirus packaging and infection were performed as previously described. 50 An MMP11-overexpressing plasmid was constructed by subcloning of MMP11 cDNA into pcDNA3.1, subsequently verified by sequencing. More details are available in Supplemental Materials and Methods.

Proliferation Assay
Cells were seeded into 6-well plates in DMEM with 10% FBS culture medium, at the density indicated Figures 2E and 3B, and maintained at 37 C and 5% CO 2 in a humidified incubator. At www.moleculartherapy.org the indicated time points, cell were counted by trypan blue exclusion.

Plate Colony Formation Assay
The cells were diluted in gradients to the appropriate concentrations. For the A549 cells, 1,000 cells were seeded and uniformly distributed in 10 cm dishes with complete medium, and for the PC9 cells, 800 cells were seeded and uniformly distributed in 6-well dishes, then placed in an incubator for 2-3 weeks until single clones formed. Cell colonies were stained with crystal violet, and complete dishes were scanned with a scanner. The number of cell colonies was analyzed by Image-Pro Plus 6.0 (IPP6) software.

Wound-Healing and Invasion Assay
The wound-healing and invasion assay was performed as previously described. 48

Tumor Xenografts and Treatment
Six-to eight-week-old female BALB/c nude mice were purchased from the animal model center of Nanjing University. To obtain solid tumors, each mouse was inoculated with 1 Â 10 7 cells/mL suspended in Matrigel (1:1) via subcutaneous injection into the left or right shoulder flank. Tumor development was monitored over a period of 30 days before the mice were killed for further analysis. For antibody therapy, beginning on day 14, inoculated mice were injected via the tail vein every 4 days with 1 mg/kg anti-MMP11 antibody, or a vehicle control (sterile saline). Tumor volume (mm 3 ) is calculated by the formula V = L Â B 2 /2, where V is tumor volume (mm 3 ), L is tumor length (mm), and B is tumor breadth (mm). All animal care and handling were in compliance with the guidelines of the Institutional Animal Care and Use Committee at the Hefei Institutes of Physical Science, Chinese Academy of Sciences.

Statistical Analysis
Comparisons of data were performed with a paired two-tailed Student's t test with equal variance or one-way ANOVA with Bonferroni's post hoc test. Data are presented as means ± SD. Results were considered to be statistically significant when *p < 0.05, **p < 0.01, or ***p < 0.001, or as indicated in the figures.

CONFLICTS OF INTEREST
Ownership interest and patents: Hefei Institutes of Physical Science, Chinese Academy of Sciences.