MMP12 is a Potential Predictive and Prognostic Biomarker of Various Cancers Including Lung Adenocarcinoma

Cancer Control

journals.sagepub.com/home/ccx S Sage

Guo-Sheng Li’, Yu-Xing Tang2, Wei Zhang2, Jian-Di Li2, He-Qing Huang?, Jun Liu’, , Zong-Wang Fu’, Rong-Quan He4, Jin-Liang Kong5, Hua-Fu Zhou’, and Gang Chen2

Abstract

Objective: This study sought to explore the clinical value of matrix metalloproteinases 12 (MMP12) in multiple cancers, including lung adenocarcinoma (LUAD).

Methods: Using >10,000 samples, this retrospective study demonstrated the first pan-cancer analysis of MMP12. The ex- pression of MMP12 between cancer groups and their control groups was analyzed using Wilcoxon rank-sum tests. The clinical significance of MMP/2 expression in multiple cancers was assessed using receiver operating characteristic curves, Kaplan-Meier curves, and univariate Cox analysis. A further LUAD-related analysis based on 4565 multi-center and in-house samples was performed to verify the findings regarding MMP12 in pan-cancer analysis partly.

Results: MMP12 mRNA is highly expressed in 13 cancers compared to their controls, and the MMP12 protein level is elevated in some of these cancers (e.g., colon adenocarcinoma) (P < . 05). MMP12 expression makes it feasible to distinguish 21 cancer tissues from normal tissues (AUC = 0.86). A high MMP12 expression is a prognosis risk factor in eight cancers, such as adrenocortical carcinoma (hazard ratio >1, P < .05). The elevated MMP12 expression is also a prognosis protective factor in breast-invasive carcinoma and colon adenocarcinoma (hazard ratio <1, P < .05). Some pan-cancer findings regarding MMP12 are verified in LUAD-MMP12 expression is upregulated in LUAD at both the mRNA and protein levels (P <. 05), has the potential to distinguish LUAD with considerable accuracy (AUC = . 91), and plays a risk prognosis factor for patients with the disease (P < . 05).

Conclusions: MMP 12 is highly expressed in most cancers and may serve as a novel biomarker for the prediction and prognosis of numerous cancers.

Keywords

expression, prognosis, immune microenvironment, standardized mean difference, area under the curve

Received July 27, 2023. Received revised December 1, 2023. Accepted for publication January 9, 2024.

“Department of Cardiothoracic Surgery, The First Affiliated Hospital of Guangxi Medical University, Nanning, P. R. China

2Department of Pathology, The First Affiliated Hospital of Guangxi Medical University, Nanning, P. R. China

3Department of Radiotherapy, The First Affiliated Hospital of Guangxi Medical University, Nanning, P. R. China

4Department of Medical Oncology, The First Affiliated Hospital of Guangxi Medical University, Nanning, P. R. China

5Department of Respiratory and Critical Care Medicine, The First Affiliated Hospital of Guangxi Medical University, Nanning, P. R. China

Corresponding Author:

Gang Chen, Department of Pathology, The First Affiliated Hospital of Guangxi Medical University, No. 6 Shuangyong Road, Nanning, 530021, P. R. China. Email: chengang@gxmu.edu.cn

Creative Commons Non Commercial CC BY-NC: This article is distributed under the terms of the Creative Commons CC Attribution-NonCommercial 4.0 License (https://creativecommons.org/licenses/by-nc/4.0/) which permits non-commercial use, BY NC reproduction and distribution of the work without further permission provided the original work is attributed as specified on the SAGE and Open Access pages (https://us.sagepub.com/en-us/nam/open-access-at-sage).

Introduction

Cancer has gradually become one of the leading causes of human illness and mortality. International cancer statistics in- dicate that there were approximately 19 million new cancer cases worldwide and more than 10 million cancer deaths in 2020.1 Targeted therapy offers greater promise than traditional approaches (e.g., surgery) in treating several cancers.2 Imatinib provides a noteworthy example of its effectiveness in targeting the BCR-ABL fusion gene, which has led to a significant improvement in the 10-year survival rate for patients with chronic myeloid leukemia (from less than 50% to approxi- mately 80%).3 Cancer cells expressing PD-L1 can circumvent immune response inhibition by activating the interaction be- tween PD-1/PD-L1, which promotes immune evasion; PD-1 inhibitors, such as nivolumab, can effectively block this in- teraction and suppress immune evasion and provide benefits to patients with non-small-cell lung cancer.4 These examples highlight the immense potential value of targeted therapy for treating cancers. However, the presence of drug resistance and the occurrence of side effects associated with current medi- cations underscore the need for researchers to prioritize the exploration of novel cancer markers.5,6 Furthermore, a lack of valuable markers for many cancers also limits the development of targeted therapy. Therefore, it is crucial to explore markers that potentially play vital roles in multiple cancers.

Among numerous potential tumor markers, matrix met- alloproteinase 12 (MMP12) has garnered increasing attention from researchers. MMP12, a metalloproteinase secreted by macrophages, has been found to be aberrantly expressed in various tumors, affecting tumor progression and prognosis through multiple mechanisms.7-9 In liver hepatocellular car- cinoma (LIHC), upregulation of MMP12 has been associated with tumor growth and progression by promoting angio- genesis, ultimately resulting in a poorer prognosis for pa- tients.1º In lung adenocarcinoma (LUAD), MMP12 protein expression levels are significantly higher in tumor tissues than control lung tissues. Knocking down MMP12 or inhibiting its expression can suppress the proliferation and invasion of LUAD cells, possibly by affecting the expression of vascular endothelial growth factor and the epithelial-mesenchymal transition.11,12 In renal cell carcinoma and esophageal squa- mous cell carcinoma, patients with increased MMP12 ex- pression have a significantly worse prognosis compared to those with low MMP12 expression.13,14 Furthermore, MMP12 has been proposed as a target for tumor immunosuppression and immune checkpoints.15 These studies shed light on the pro-tumorigenic role of MMP12 and its potential therapeutic value. However, some studies have also suggested anti-tumor effects of MMP12 in colorectal cancer: knocking out MMP12 leads to the accumulation of M2 macrophages (which predominantly exhibit pro-cancer effects) in the tumor mi- croenvironment, thereby promoting the growth of colorectal tumors.16 In ovarian cancer, high levels of MMP12 mRNA have been associated with better overall survival (OS).17

Therefore, previous research indicates that MMP12 plays an important role in various tumors, but its clinical signifi- cance may not be consistent across different types of cancer. Closing this research gap is needed to identify the pan-cancer clinical significance of MMP12.

We analyzed MMP12’s cancer cell effects, mRNA and protein expression, immune effects, clinical significance, and potential mechanisms of MMP12 in 33 cancer tissues and 21 normal tissues by collecting data from the DepMap Portal, Xena database, and Clinical Proteomic Tumor Analysis Consortium database. Additionally, we used data from The Cancer Genome Atlas (TCGA), Genotype-Tissue Expression, ArrayExpress, and Gene Expression Omnibus to analyze MMP12’s expression in LUAD and combined those data with our in-house data to explore the role of MMP12 in LUAD as well as its important pan-cancer role, thereby providing a potential target of cancer immunotherapy.

Materials and Methods

This retrospective study was approved by the medical ethics review committee of the First Affiliated Hospital of Guangxi Medical University (No. 6 Shuangyong Road, Nanning, China) on October 26, 2021, with the approval number 2021(KY-E-246). Informed consent was signed by all patients involved in the in-house data. The personal identification information of the patients included in this study has been removed, and it is not possible to ascertain the identity of the patients through the information provided in this article. The reporting of the study adheres to REMARK guidelines.1

The DepMap Portal includes data on numerous cell types. We collected data on RNA interference (n of samples = 494) to analyze the essential roles of MMP12 in multiple cancer cells. An RNA interference score of less than 0 indicates that MMP12 is essential for a specific cancer cell.

The Xena database collects data on tumors and their normal samples from various datasets such as TCGA. TCGA data were extracted from the Xena database to analyze MMP12’s mRNA expression among 33 tumor tissue types (n of samples = 8305) and 21 normal tissue types (n of samples = 671). The Clinical Proteomic Tumor Analysis Consortium database contains MMP12 protein level data in various cancers from the Proteomic Data Commons. The protein level data from this database were collected to detect the difference in MMP12 protein levels between breast-invasive carcinoma (BRCA), colon ade- nocarcinoma (COAD), head and neck squamous cell car- cinoma (HNSCC), and uterine corpus endometrial carcinoma (UCEC) and their control samples. 19-21

Clinical parameters were collected from the Xena database, including patients’ ages, genders, and cancer stages as defined by the American Joint Committee on Cancer. The cancer patients’ prognosis data were also acquired from the Xena database, including OS, disease-specific survival, progression-free interval, and disease-free interval. The define details of clinical endpoints can be seen in previous research.22

The Prediction Effect and Prognosis Value of MMP12 for Cancers

The area under the curve (AUC) size of receiver operating characteristic (ROC) curves was applied to determine the ability of MMP12 expression to differentiate tumor tissues and normal tissues. We plotted the summary ROC (sROC) to evaluate the overall ability to discriminate between tumor tissues and normal tissues. Univariate Cox regression and Kaplan-Meier curves revealed differences in prognosis of patients with various expression levels of MMP12. The high- MMP12 and low-MMP12 groups were identified using a cutoff value determined by the “survminer” software package.

We also examined the role of MMP12 expression in the immune environment and signaling pathways. Three types of data, including neoantigen count,23 tumor mutational burden (TMB), and microsatellite instability (MSI), were acquired from Sanger Box (v3.0).24 We also revealed the regulation of MMP12 expression on six types of patient’s immune cells, including B cells, CD4+ T cells, CD8+ T cells, neutrophils, macrophages, and dendritic cells; the infiltration level data of these cells were obtained from TIMER.25 Immune environ- ment data based on the ESTIMATE algorithm 26 were ac- quired from Sanger Box (v3.0); they contained three types of scores, including immune, stromal, and ESTIMATE scores. Using the clusterProfiler package,27 Kyoto Encyclopedia of Genes and Genomes28 signaling pathways that MMP12 may participate in multiple cancers were determined with gene set enrichment analysis; those signaling pathways with the P-value <. 05 were included.

Validation of MMP12 mRNA Expression in LUAD

To validate the MMP12 expression in LUAD at mRNA levels, LUAD-related public datasets were collected from several public databases: TCGA, Genotype-Tissue Expression, Ar- rayExpress, and Gene Expression Omnibus. The data retrieval strategy for datasets was “(mRNA or gene) AND lung AND (adenocarcinoma OR [non-small cell]).” The inclusion criteria for datasets and their samples were as follows: (1) the samples were sourced from humans; (2) samples from the LUAD group were obtained from pathologically diagnosed LUAD tissues or cells; (3) samples from the control group were obtained from pathologically diagnosed normal lung tissues or cells; (4) the dataset had complete mRNA expression data. The exclusion criteria were as follows: (1) incomplete mRNA expression data;

(2) duplicate samples in various datasets. Ultimately, 59 data- sets were included in this study and merged to 29 datasets based on the same platform (e.g., GPL10558). Details of the 59 da- tasets are listed in Supplementary Material 1.

Validation of MMP12 Protein Expression in LUAD

To verify MMP12 expression in LUAD at the level of pro- teins, in-house tissue microarrays (LUC1021, LUC1502, and LUC481) were purchased from Fanpu Biotech (Guilin, China), including 64 LUAD samples and 24 non-LUAD control samples. The inclusion of samples was as follows: (1) samples were sourced from human LUAD tissues and non- LUAD control lung tissues; (2) samples were pathologically identified; (3) samples were collected from the patients who signed the informed consent. These samples were used in an immunohistochemistry experiment.

We conducted the immunohistochemistry experiment following the instructions of the reagent manufacturer. We used a .01 M citrate buffer solution (pH = 6.0) to wash the dewax and repaired tissue slides to extract the antigen, and we used 3% H2O2 to deactivate the endogenous peroxidase. We used the rabbit anti-human MMP12 monoclonal anti- body (ab137444, Abcam, UK) in a 1:100 dilution to in- cubate the prepared tissue slides at 37℃ for 30 min. In contrast, we incubated the negative control slides in phosphate buffer overnight. We added a secondary anti- body, horseradish peroxidase (D-3004-15, Changdao Biotechnology Co. Ltd., Shanghai, China), to the tissue slides, which were then kept at room temperature (ap- proximately 25°℃) for 25 minutes and finally stained with 3,3’-diaminobenzidine for 10 minutes.

The dehydrated and sealed slides were used to assess the degree of MMP12 protein expression under microscopy. Positive and negative anti-MMP12 antibody staining showed diverse colors (brown granules in the nucleus and/or cytoplasm for the positive staining and blue particles for the negative). All specimens of the tissue microarrays were evaluated for positive cells in five randomly selected regions. In the visual field, the anti-MMP12 body staining intensity score was indicated by integers from 0 through 3, representing no staining, light staining, moderate staining, and strong staining. For positive cells, integers from 0 through 4 represented <5%, 5%-25%, 26%-50%, 51%-75%, and >75%, respectively, in the visual field. The final score (i.e., the product of the intensity score and the positive cells score) represents the MMP12 protein level in the LUAD and control tissues.

Validation of the Potential Clinical Value of MMP12 in LUAD

The AUC values of the ROC curves and an sROC curve were applied to determine the ability of MMP12 expression to differentiate LUAD samples and control specimens. We used

the Kaplan-Meier curve to evaluate the prognosis differences between LUAD individuals with a high MMP12 and those with a low MMP12 expression. The “survminer” package was employed to determine the high-MMP12 and low-MMP12 groups.

Statistical Analysis

We conducted Wilcoxon rank-sum tests to explore the differ- ences in MMP12 expression between cancer groups and their control groups (e.g., LUAD vs control). The method was also

used to detect MMP12 expression differences in patients with various clinical parameters. Using the “meta” package, dif- ferences in MMP12 expression level between the LUAD group and the non-LUAD group were evaluated with a standardized mean difference (SMD). We used Begg’s test to evaluate publication bias, and the P-value of less than .1 indicated significant publication bias.29 All correlation analyses in this study were done with Spearman’s rank correlation coefficient. The sROC curve was produced using Stata (v15.0), and the remaining calculations were conducted in R (v4.1.0). Figure 1 shows the overall framework design of this research.

Figure 1. Research overflow of this study.

BRCA

The essential role and mRNA expression of MMP12 in cancers

MMP12 Protein Level

-2

Non-Tumor Tumor Sample (n= 149)

ESCA (n = 13’Vs 181)

KIRC

PAAD

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The prediction effect and prognosis value of MMP12 for cancers

Survival probability

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READ (n= 81)

TGCT (n = 147)

“MMP 12 Cog2(TPM+1)

p = 0.23, p = 0.041

The association of MMP12 with immune and signaling pathways

MMP12 Log2(TPM+1)

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0.55, p = 3.1e-13

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Log2(neoantigen count + 1)

B_cell level

In-house TMA

MMP12 Protein Level

A validation of MMP12 expression in lung adenocarcinoma

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Sample (n= 88)

ĞSE1987

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A validation of potential clinical value of MMP12 in lung adenocarcinoma

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MMP12 is a Potential Predictive and Prognostic Biomarker of Various Cancers Including Lung Adenocarcinoma

Figure 2. Essential role of MMP 12, MMP 12 mRNA expression, and MMP12 protein levels in cancers. Panel A: Identification of essential roles of MMP 12 for multiple cancers. Panel B: The differential expression of MMP12 mRNA between cancers and controls; P-value was based on the Wilcoxon rank-sum test with a false discovery rate. Panel C: The differential levels of MMP12 protein between cancers and controls. nsp ≥ .05; * P < . 05; ** P < . 01; *** P < . 001; **** P < . 0001.

MMP12 Gene Effect

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Bladder Cancer

Bone Cancer

Brain Cancer

Breast Cancer

Cervical Cancer

Colon/Colorectal Cancer

Endometrial/Uterine Cancer

Esophageal Cancer

Gastric Cancer

Head and Neck Cancer

Kidney Cancer

Leukemia

Liver Cancer

Lung Cancer

Lymphoma

Myeloma

Ovarian Cancer

Pancreatic Cancer

Prostate Cancer

Rhabdoid

Sarcoma

Skin Cancer

MMP12 mRNA Expression [log2(TPM+1)]

MMP12 expression between cancers and their controls

Group ns

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BRCA

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BRCA

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Sample (n= 174)

Sample (n= 144)

Results

Pan-Cancer MMP12 Expression Level

MMP12 is essential for various cancers, particularly esophageal cancer (ESCA) and liver cancer (Figure 2A). MMP12 mRNA overexpression compared to normal tissues was observed in the cancer tissues of 13 cancer types (P < .05), including BRCA, COAD, ESCA, HNSCC, LIHC, LUAD, UCEC, cervical squamous cell carcinoma and en- docervical adenocarcinoma (CESC), cholangiocarcinoma (CHOL), lung squamous cell carcinoma (LUSC), rectum adenocarcinoma (READ), stomach adenocarcinoma (STAD), and thyroid carcinoma (THCA) (Figure 2B). Fur- thermore, at the protein level, MMP12 expression was higher in COAD, HNSCC, and UCEC than in normal tissues, while the opposite result was found in BRCA (P <. 05) (Figure 2C).

Clinical Significance of Pan-Cancer MMP12 Expression

We drew ROC curves to identify the pan-cancer clinical significance of MMP12. The results show that 13 of 21 cancer types have an AUC value above .7 (Figure 3A), indicating that MMP12 has a conspicuous ability to dis- tinguish these cancer tissues from normal tissues. In ad- dition, the AUC in sROC was .86, indicating that MMP12 has a good ability to distinguish 21 cancer tissues from normal tissues (Figure 3B).

Univariate Cox analysis showed that high MMP12 ex- pression predicts a poor OS (hazard ratio [HR]> 1, P <. 05) in adrenocortical carcinoma (ACC), kidney renal clear cell carcinoma (KIRC), and pancreatic adenocarcinoma (PAAD) and a favorable OS in BRCA (HR < 1, P <. 05) (Table 1). For disease-specific survival, high MMP12 expres- sion was associated with poor clinical outcomes in ACC, ESCA,

Figure 3. Ability of MMP12 to differentiate the tumor tissue from the control tissue. Panel A: MMP12 can accurately distinguish cancer tissues from control tissues in some cancers. Panel B: MMP12 can well distinguish cancers from controls in 21 cancer types.

CESC (n = 3 vs 304)

CHOL (n = 9 vs 36)

COAD (n = 41 vs 288)

ESCA (n = 13 vs 181)

0.8

Sensitivity

0.8

Sensitivity

0.4

AUC: 0.961

0.4

AUC: 0.898

0.4

AUC: 0.731

0.4

AUC: 0.938

0.0

0.4

0.8

0.0

0.4

0.8

0.0

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HNSCC (n = 44 vs 518)

LIHC (n = 50 vs 369)

LUAD (n = 59 vs 513)

LUSC (n = 50 vs 498)

0.8

Sensitivity

0.4

AUC: 0.939

0.4

AUC: 0.769

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AUC: 0.885

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AUC: 0.984

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0.0

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PAAD (n = 4 vs 178)

PCPG (n = 3 vs 177)

READ (n = 10 vs 92)

STAD (n = 36 vs 414)

0.8

Sensitivity

0.8

Sensitivity

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AUC: 0.721

0.4

AUC: 0.891

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AUC: 0.768

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AUC: 0.791

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UCEC (n = 23 vs 180)

1.0

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Sensitivity

0.4

AUC: 0.895

Sensitivity

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Observed Data

Summary Operating Point

1 - Specificity

SENS = 0.72 [0.62 - 0.80]

SPEC = 0.85 [0.77 - 0.91]

SROC Curve

AUC = 0.86 [0.83 - 0.89]

- 95% Confidence Contour

95% Prediction Contour

0.0

1.0

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Specificity

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kidney chromophobe (KICH), KIRC, and PAAD (HR > 1, P < .05) (Table 1). In addition, high expression of MMP12 was associated with a poor progression-free interval in ESCA, KIRC, PAAD, sarcoma (SARC), and thymoma (THYM) (HR > 1, P< .05) and favorable progression-free interval in STAD (HR < 1, P < . 05) (Table 2). Upregulation of MMP12 in LUAD, PAAD, and SARC was associated with a decreased disease-free interval (HR > 1, P < . 05), while elevated expression of MMP12 was relevant to an increased disease-free interval in COAD and STAD (HR < 1, P < . 05) (Table 2). Finally, we used Kaplan-

Meier curves to test MMP12 expression and prognosis in cancer patients, which validated the above results (P < . 05) (Figure 4).

MMP12 expressed variously among the cancer staging levels of 21 cancer types. MMP12 expression was at high levels in the advanced stages of several cancers, including ACC, ESCA, KIRC, LIHC, and THCA (P <. 05) (Figure 5A). In contrast, it expressed at low levels in terminal cancers, including BRCA and COAD (P < . 05) (Figure 5A). Other cancers, including bladder urothelial carcinoma (BLCA), CHOL, HNSCC, KICH, kidney renal papillary cell carcinoma

Table 1. Relation of MMP12 Expression With Overall Survival (OS) and Disease-specific Survival (DSS) of Cancer Patients.
Cancer (sample)	OS HRª	P-value	Cancer (sample)	DSS HR	P-value
ACC (41)	1.555	.002	ACC (39)	6.396	<. 001
BLCA (416)	.975	.455	BLCA (401)	.972	.496
BRCA (1143)	.879	.031	BRCA (1115)	.926	.324
CESC (273)	1.072	.253	CESC (272)	1.062	.380
CHOL (40)	.988	.949	CHOL (38)	1.045	.809
COAD (317)	.920	.184	COAD (302)	.920	.339
DLBC (44)	1.035	.865	DLBC (44)	1.210	.488
ESCA (188)	1.099	.119	ESCA (185)	1.217	.007
GBM (114)	.888	.373	GBM (106)	.895	.429
HNSCC (554)	.974	.437	HNSCC (526)	.959	.347
KICH (77)	2.440	.055	KICH (77)	3.341	.017
KIRC (500)	1.282	<. 001	KIRC (486)	1.303	<. 001
KIRP (240)	1.007	.979	KIRP (238)	1.147	.576
LGG (205)	1.118	.721	LGG (201)	1.175	.606
LIHC (326)	1.121	.086	LIHC (317)	1.078	.390
LUAD (549)	1.042	.230	LUAD (515)	1.005	.913
LUSC (515)	1.000	.989	LUSC (457)	.957	.396
MESO (67)	1.001	.993	MESO (49)	.913	.528
OV (382)	.947	.337	OV (354)	.946	.369
PAAD (176)	1.146	.034	PAAD (170)	1.159	.037
PCPG (93)	1.408	.425	PCPG (93)	1.240	.718
PRAD (518)	.615	.410	PRAD (516)	.961	.951
READ (101)	.939	.664	READ (95)	.885	.596
SARC (219)	1.063	.336	SARC (214)	1.082	.240
SKCM (94)	1.118	.521	SKCM (94)	1.212	.317
STAD (406)	.944	.111	STAD (384)	.915	.065
TGCT (127)	1.647	.123	TGCT (127)	1.670	.129
THCA (503)	1.273	.226	THCA (497)	.255	.127
THYM (106)	1.180	.842	THYM (106)	2.488	.301
UCEC (178)	.915	.472	UCEC (176)	1.066	.657
UCS (54)	1.225	.106	UCS (52)	1.203	.164
UVM (33)	1.417	.082	UVM (33)	1.480	.054

ªNotes: hazard ratio.

(KIRP), LUAD, LUSC, mesothelioma (MESO), PAAD, READ, skin cutaneous melanoma (SKCM), STAD, testicular germ cell tumor (TGCT), and uveal melanoma (UVM), showed little difference in MMP12 expression between var- ious stages (Figure 5A). Furthermore, we observed that MMP12 expression did not differ significantly by age and gender in most cancers (Supplementary Materials 2 and 3).

Relationship Between the Expression of Immune Gene MMP12 and Genomic Heterogeneity

TMB is related to the number of tumor cell mutations; a high level of TMB can induce the body to produce neoantigens and cause more immune cells to play a role in immune recognition.30 MMP12 expression was positively correlated with TMB (P < .05) in STAD, ACC, uterine carcinosarcoma (UCS), UCEC, SARC, BRCA, COAD, LUAD, and CESC (Figure 5B).

Microsatellites, because they are short-chain repetitive DNA sequences, are prone to MSI when they are affected by mismatch repair.31 The expression of MMP12 was positively correlated with COAD and STAD but negatively correlated with prostate adenocarcinoma, HNSCC, LUSC, and TGCT (P < . 05) (Figure 5C).

DNA damage increases neoantigens on the surface of tumor cells, which benefits immune cells in recognizing and killing tumor cells.32 Our research found a weak correlation between MMP12 expression and cancer neoantigens in READ and COAD (P < . 05) (Figure 5D).

Correlation Assessment of MMP12 Expression and the Immune Microenvironment

MMP12 expression was correlated with the degree of six types of immune cell infiltration in various cancers (Figure 6A and Supplementary Material 4). Notably, MMP12 expression was

Table 2. Relation of MMP12 Expression With Progression-free Interval (PFI) and Disease-free Interval (DFI) of Cancer Patients.
Cancer (sample)	PFI HRª	P-value	Cancer (sample)	DFI HR	P-value
ACC (40)	1.008	.965	ACC (20)	165.846	.223
BLCA (415)	1.003	.928	BLCA (189)	.999	.995
BRCA (1142)	.973	.632	BRCA (975)	.988	.873
CESC (276)	.960	.507	CESC (173)	.858	.147
CHOL (40)	.884	.450	CHOL (29)	1.019	.910
COAD (314)	.934	.247	COAD (121)	.684	.020
DLBC (43)	.930	.697	DLBC (26)	.366	.119
ESCA (185)	1.189	.002	ESCA (91)	1.219	.055
GBM (113)	.991	.947	HNSCC (134)	1.202	.072
HNSCC (553)	.993	.839	KICH (36)	.027	.538
KICH (77)	2.011	.089	KIRC (112)	.764	.582
KIRC (489)	1.202	.009	KIRP (133)	1.279	.463
KIRP (237)	1.048	.810	LGG (52)	1.083	.894
LGG (204)	.660	.207	LIHC (274)	1.056	.532
LIHC (326)	1.061	.328	LUAD (331)	1.160	.003
LUAD (545)	1.037	.259	LUSC (316)	.950	.423
LUSC (515)	.981	.638	MESO (11)	1.987	.085
MESO (65)	.966	.783	OV (191)	.967	.632
OV (382)	.916	.087	PAAD (71)	1.382	.001
PAAD (175)	1.127	.047	PCPG (77)	2.092	.469
PCPG (92)	1.444	.084	PRAD (367)	.534	.125
PRAD (518)	1.155	.330	READ (32)	1.185	.620
READ (100)	.962	.760	SARC (129)	1.223	.002
SARC (216)	1.160	.003	STAD (252)	.864	.033
SKCM (93)	1.125	.455	TGCT (101)	1.004	.967
STAD (409)	.903	.009	THCA (357)	1.409	.110
TGCT (125)	.992	.923	UCEC (126)	.729	.077
THCA (502)	1.086	.575	UCS (26)	.894	.766
THYM (106)	2.175	.029
UCEC (178)	.899	.313
UCS (54)	1.211	.103
UVM (32)	1.186	.389

ªNotes: hazard ratio.

significantly associated with the infiltration levels of B cells and dendritic cells in CHOL, TGCT, and READ (P < . 05) (Figure 6A). Moreover, the expression levels of MMP12 were relevant to the immune microenvironment (Figure 6B and Supplementary Material 5). Among THCA, COAD, READ, and UVM, MMP12 expression had the strongest relationship with the stromal score in COAD, and the relationship of MMP12 expression with immune and ESTIMATE scores was conspicuous in READ and COAD (Figure 6B).

MMP12 Expression and Potential Signaling Pathways

The results of gene enrichment analysis show that MMP12 may participate in 31 potential molecular mechanisms in 33 cancers. The analysis results of 12 cancers (BLCA, BRCA, CHOL, COAD, HNSCC, KIRP, LIHC, LUSC, MESO, pheochromocytoma and

paraganglioma, READ, and THCA) suggest that MMP12 is associated with at least five signaling pathways, in- dicating that MMP12 is likely to regulate the occurrence and development of cancer through these pathways, such as the cytokine-cytokine receptor interaction and the chemokine signaling pathway (Figure 7 and Supplementary Material 6).

Overall Expression Level of MMP12 in LUAD

To further explore the findings regarding MMP12 in pan- cancer analysis, we examined the comprehensive expression level of MMP12 in LUAD. Among the 29 collected datasets, MMP12 mRNA was upregulated in LUAD (SMD = 1.35; 95% CI [1.04-1.66]) (Figure 8A), and no significant publication bias was found using Begg’s test (P > .1, Supplementary Material 7). A Wilcoxon rank-sum test also revealed

Figure 4. Relation of MMP12 expression with overall survival (A), disease-specific survival (B), progression-free interval (C), and disease-free interval (D) of cancer patients. The red and blue curves represent the high-MMP12 expression group and low-MMP12 expression group, respectively.

ACC

BRCA

KIRC

PAAD

Survival probability

1.00

Survival probability

1.00

Survival probability

1.00

Survival probability

1.00

0.75

0.50

Q. 0.50

0.50

0.25

0.0001

0.25

p = 0.0047

0.25

p < 0.0001

0.25

p = 0.0014+L

0.00

2.5

7.5

12.5

2.5

5 7.5 10 12.5

Time (Years)

ACC

ESCA

KICH

KIRC

PAAD

Survival probability

1.00

Survival probability

1.00

Survival probability

1.00

Survival probability

1.00

Survival probability

1.00

0.75

0.50

0.25

0.0001

0.25

p = 0.0011

0.25

p = 0.00028

0.25

p < 0.0001

0.25

p = 0.0016

0.00

2.5

7.5

12.5

2.5

7.5

2.5

7.5

2.5

7.5

12.5

Time (Years)

12.5

Time (Years)

ESCA

KIRC

PAAD

SARC

STAD

THYM

Survival probability

1.00

Survival probability

1.00

Survival probability

1.00

Survival probability

1.00

Survival probability

1.00

Survival probability

1.00

0.75

0.50

0.25

p = 0.00014

0.25

< 0.0001

0.25

p = 0.0047

0.25

p = 0.042

0.25

0.00014

0.25

= 0.0019

0.00

0.00-

0.00

0.00-

2.5

7.5

2.5

7.5

12.5

2.5

7.5

2.5

7.5

Time (Years)

12.5

COAD

LUAD

PAAD

SARC

STAD

Survival probability

1.00

Survival probability

1.00

Survival probability

1.00

Survival probability

1.00

Survival probability

1.00

0.75

0.50

0.25

p = 0.0062

0.25

p < 0.0001

0.25

p = 0.00099

0.25

0.001

0.25

p = 0.0011

0.00

2.5

7.5

12.5

2.5

7.5

Time (Years)

overexpression of MMP12 mRNA in LUAD (P < . 05) (Figure 8B). In addition, using in-house tissue microarrays, no positive MMP12 protein staging was found in the alveoli and bronchi of the control tissue (Figures 9A and C). However, a high level of MMP12 protein was observed in the LUAD tissue (Figures 9B and D), which was confirmed by the further Wilcoxon rank-sum test (P < . 05) (Figure 8C).

Clinical Value of MMP12 in LUAD

Among the 29 included datasets, ROC curves showed that MMP12 mRNA expression in LUAD exceeded moderate accuracy in 19 datasets (AUC >.75) (Figure 10A). The sROC analysis revealed that MMP12 mRNA expression accurately distinguished LUAD from non-LUAD (sensitivity = . 83, specificity = . 85; AUC = . 91) (Figure 10B). Furthermore, using OS curves, a lower MMP12 expression in LUAD pa- tients tended to predict a good prognosis (P = . 022) (Figure 10C).

Discussion

Cancer seriously threatens human health as one of the leading causes of death, so it is meaningful to explore novel markers for identifying the disease status and prognosis of cancer patients.33 As an immune gene, MMP12 can regulate in- flammatory responses and play an essential role in specific cancers.34 However, there was a dearth of a comprehensive pan-cancer analysis of MMP12.

This study used numerous multi-center samples and multiple approaches to explore the pan-cancer expression, clinicopathology, potential mechanisms, and clinical signifi- cance of MMP12. MMP12 was differently expressed in various cancers, and its expression level was related to TMB, MSI, neoantigen counts, and immune microenvironments in some cancers. The relationship between MMP12 expression and the potential molecular mechanism, prognosis, and clinical significance was also investigated in various cancers. Furthermore, we analyzed the expression of MMP12 in LUAD to support pan-cancer research by using public database

Figure 5. Relation of MMP12 expression with tumor stages (panel A), tumor mutational burden (panel B), microsatellite instability (panel C), and neoantigen (panel D) of cancer patients. * P < . 05; * P< . 01; * P < . 001.

ACC

BLCA

BRCA

CHOL

COAD

ESCA

7.5

MMP12 expression

10.0

MMP12 expression

7.5

5.0

2.5

0.0

Stage | Stage II Stage III Stage IV AJCC_stage (n = 75)

Stage | Stage II Stage II Stage IV AJCC_stage (n = 405 )

Stage | Stage II Stage III Stage IV AJCC_stage (n = 1067)

Stage | Stage II Stage III Stage IV AJCC_stage (n = 36 )

Stage | Stage II Stage III Stage IV AJCC_stage (n = 276 )

Stage | Stage II Stage III Stage IV AJCC_stage (n = 158 )

HNSCC

KICH

KIRC

KIRP

LIHC

LUAD

COM

7.5

MMP12 expression

€

5.0

3 3

2.5

0.0

Stage | Stage Il Stage III Stage IV AJCC_stage (n = 443 )

Stage | Stage II Stage Ill Stage IV AJCC_stage (n = 66 )

Stage | Stage Il Stage III Stage IV AJCC_stage (n = 527 )

Stage | Stage II Stage III Stage IV AJCC_stage (n = 258 )

Stage | Stage II Stage III Stage IV AJCC_stage (n = 345 )

Stage | Stage Il Stage III Stage IV AJCC_stage (n = 505 )

LUSC

MESO

PAAD

READ

SKCM

STAD

าร

10.0

nis

MMP12 expression

n$ ns

MMP12 expression

7.5

MMP12 expression

5.0

2.5

0.0

Stage | Stage II Stage III Stage IV AJCC_stage (n = 494 )

Stage

Stage II Stage III Stage IV AJCC_stage (n = 87 )

Stage | Stage II Stage III Stage IV AJCC_stage (n = 175)

Stage

Stage II Stage III Stage IV AJCC_stage (n = 82)

Stage

Stage II Stage III Stage IV AJCC_stage (n = 97 )

Stage

Stage Il Stage III Stage IV AJCC_stage (n = 389 )

TGCT

THCA

UVM

10.0

MMP12 expression

7.5

MMP12 expression

5.0

2.5

0.0

Stage I

Stage II

AJCC_stage (n = 79 )

Stage III

Stage | Stage II Stage III Stage IV AJCC_stage (n = 502 )

Stage II

Stage III

AJCC_stage (n = 78 )

Stage IV

GBM (119)

DLBC (37)STAD (409) ***

LUSC (490)-TOCT (147)” UVM (35)

SKCM (98)

ACC (41)*

CHOL (36)

COAD (285) ***

LIHC (309)

0.4

UCS (55)*

PAAD (176)

0.3

STAD (412) ***

LUSC (486)

0.2

UCEC (175) **

KICH (56)

0.15

UCEC (180)

KIRC (276)

SARC (199) **

MESO (65)

READ (89)

BLCA (406)

HNSCC (500)*

-0.

BRCA (968) ***

-0.15

PCPG (96)

LGG (212)

-0.4

COAD (282) ***

PRAD (474)*

-0.3

SKCM (98)

HNSCC (498)

LUAD (509) ***

GBM (120)

SARC (216)

CHOL (35)

KICH (56)

DLBC (47)

CESC (302)

MESO (64)

THYM (105)

BLCA (406)

KIRP (219)

TGCT (142)

UVM (35)

ACC (41)

BRCA (1025)

THICA (443)

CESC (286)*

OV (285)

LIHC (318)

ESCA (180)

OV (283)

UCS (55)

LUAD (511)

KIRP (215)

READ (90)

LGG (216)

KIRC (278)

PAAD (171)PRAD (471) PCPG (96)

ESCA (180) THCA (446) THYM (105)

READ (n = 81)

COAD (n = 255)

HNSCC (n = 446)

MMP12 Log2(TPM+1)

p = 0.23, p = 0.041

MMP12 Log2(TPM+1)

p = 0.21, p = 0.00055

10.0

p = 0.025

7.5

…

MMP12 Log2(TPM+1)

7.5

5.0

2.5

0.0

Log2(neoantigen count + 1)

Figure 6. Relation of MMP 12 expression and infiltration levels of immune cells. Panel A: TIMER algorithm; B: ESTIMATE algorithm.

A CHOL (n = 36)

CHOL (n= 36)

CHOL (n = 36)

CHOL (n=36)

MMP12 Log2(TPM+1)

p= 0.52, p = 0.0012

.4, p = 0.015

00.39, p =0.02

p =0.49, p = 0.0024

p=048, p= 0.0032

0.62, p= 0.000047

0.15 0.20 0.25 0.30 0.35

0.14 0.16 0.18 0.20 0.22

CD4_Tcell level

0.17

0.18

0.19

CD8_Tcell level

0.20

0.075 0.080 0.085 0.090

0.54

0.56

B_cell level

Neutrophil level

0.035 0.040 0.045 0.050 Macrophage level

Dendritic level

0.58

TGCT (n = 147)

MMP12 Log2(TPM+1)

7.5

5.0

2.5

0.0

0.55

3.1e

0.0

= 0.33, p = 0.000052

0.0

p -0.41, p = 2.7e-07

0.0

p = 1.8e-12

0.0

:p = 0.049

0.0

0.76, p < 2.2e-16

0.0 0.1 0.2 0.3 0.4

0.1 0.2 0.3 0.4 0.5

0.1 0.2 0.3 0.4

CD4_Tcell level

CD8_Tcell level

0.10 0.15 0.20 0.25

0.00 0.05 0.10 0.15 0.20

Dendritic level READ (n = 92)

0.5

1.0

B_cell level

Neutrophil level

Macrophage level

READ (n = 92)

MMP12 Log2(TPM+1)

7.5

5.0

2.5

0.0

0.19

,p= 0.065

0.0

19,

p = 0.074

0.0

0.5

56 3

p = 1.3e-08

0.0

72, 0

< 2.2e-16

0.0

p= 0.00033

0.0

p <2.2e-

0.1

.1 0.2 0.3 0.4

0.12

0.16

0.20

0.15 0.20 0.25 0.30

0.08

0.12

0.16

0.20

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

B_cell level

CD4_Tcell level

CD8_Tcell level

Neutrophil level

Macrophage level

Dendritic level

COAD (n = 282)

READ (n = 91)

MMP12 Log2(TPM+1)

7.5

5.0

2.5

0.0

P = 0.54, p < 2.20-16

0.0

p =0.69, p < 2.2e-16

0.0

0.65, p < 2,2e-16

‘S

0.0

2.5e-0

0.0

.71, p < 2.28-16

0.0

O .63

p <2.2e-16

-2000-1000 0 1000

-1000

Stromal_score

1000 2000 3000

Immune_score

-2000 0 2000 4000

ESTIMATE_score

-2000 -1000

1000

2000

-2000

Stromal_score

Immune_score

2000

UVM (n = 79)

ESTIMATE_score THCA (n = 503)

UVM (n = 79)

THCA (n = 503)

MMP12 Log2(TPM+1)

-0.4

0.015

= 0.56

00044

0.54, p =0.00075

0.59

2e-16

-1500 -1000 -500

-1000

1000

2000

-3000-2000-1000

1000 2000

-2000

-1000

1000

-1000

-2000

Stromal_score

2000

4000

Immune_score

ESTIMATE_score

Stromal_score

1000 2000 3000

Immune_score

ESTIMATE_score

datasets and in-house tissue microarrays to comprehensively determine that MMP12 mRNA and protein expression are upregulated in LUAD.

MMP12 is highly expressed in various cancers and plays a critical role in these diseases. Studies report that elevated MMP12 expression represents a poor prognosis for BRCA and that the gene was positively correlated with neutrophils and dendritic cells.35 In terms of COAD, overexpressed MMP12 promotes tumors and is associated with a poor OS of pa- tients.36 In HNSCC, increasing MMP12 expression promotes cancer cell proliferation, migration, invasion, and the me- tastasis of cancer cells.37 Overexpressed MMP12 mRNA may

promote the progression and metastasis of ESCA, LIHC, and LUSC and is associated with a poor prognosis in cancer patients.25,38,39 Additionally, a high expression of MMP12 mRNA in LUAD promotes lymph node metastasis.12 Simi- larly, our study detected that MMP12 is differently expressed in various cancers and that MMP12 is associated with patients’ cancer status and prognosis in several cancers. Briefly, in regard to expression between cancer and normal tissues, MMP12 mRNA is overexpressed in 13 cancers (CESC, etc.), and higher MMP12 protein levels are observed in COAD, HNSCC, and UCEC. Some of these findings have not pre- viously been reported. For instance, our study for the first time

Figure 7. Potential signaling pathways of MMP12 may affect multiple cancers.

BLCA

KEGG_CHEMOKINE_SIGNALING_PATHWAY

BRCA

KEGG_CHEMOKINE_SIGNALING_PATHWAY

CHOL

KEGG_CALCIUM_SIGNALING_PATHWAY

1.00

KEGG_CYTOKINE_CYTOKINE_RECEPTOR_INTERACTION

1.0

KEGG_NATURAL_KILLER_CELL_MEDIATED_CYTOTOXICITY

1.0

Running Enrichment Score

KEGG_CELL_ADHESION_MOLECULES_CAMS

0.75

KEGG_JAK_STAT_SIGNALING_PATHWAY

0.5

KEGG_OLFACTORY_TRANSDUCTION

0.5

KEGG_CYTOKINE_CYTOKINE_RECEPTOR_INTERACTION

KEGO_NEUROACTIVE_LIGAND_RECEPTOR_INTERACTION

KEGO_T_CELL_RECEPTOR_SIGNALING_PATHWAY

KEGO_HEMATOPOIETIC_CELL_LINEAGE

.50

KEGG_OLFACTORY_TRANSDUCTION

0.0

KEGG_TOLL_LIKE_RECEPTOR_SIGNALING_PATHWAY

KEGG_RIBOSOME

0.25

-0.5

0.00

-1.0

Ranked List Metric

-10

10000

20000

30000

40000

50000

10000

20000

30000

40000

50000

-20

10000

20000

30000

40000

Rank in Ordered Dataset

50000

COAD

KEGG_CELL_ADHESION_MOLECULES_CAMS

HNSCC

KEGG_CELL_ADHESION_MOLECULES_CAMS

KIRP

KEGG_CHEMOKINE_SIGNALING_PATHWAY

1.00

Running Enrichment Score

KEGG_CHEMOKINE_SIGNALING_PATHWAY

1.0-

1.00

Running Enrichment Score

KEGG_CYTOKINE_CYTOKINE_RECEPTOR_INTERACTION

Running Enrichment Score

KEGG_CYTOKINE_CYTOKINE_RECEPTOR_INTERACTION

.75

KEGG_CYTOKINE_CYTOKINE_RECEPTOR_INTERACTION

0.5

KEGG_NEUROACTIVE_LIGAND_RECEPTOR_INTERACTION

0.75

KEGG_HEMATOPOIETIC_CELL_LINEAGE

KEGG_OLFACTORY TRANSDUCTION

KEGG_NEUROACTIVE_LIGAND_RECEPTOR_INTERACTION

9.50

KEGG_OLFACTORY_TRANSDUCTION

0.0

KEGG_RIBOSOME

0.50

KEGG_TASTE_TRANSDUCTION

0.25

-0.5

0.25

0.00

=1.0

0.00

III

Ranked List Metric

-10

10000

20000

30000

Rank in Ordered Dataset

40000

50000

10000

20000

30000

50000

10000

20000

30000

40000

Rank in Ordered Dataset

40000

Rank in Ordered Dataset

50000

LIHC

KEGG_CELL_ADHESION_MOLECULES_CAMS

LUSC

KEGG_CELL_ADHESION_MOLECULES_CAMS

MESO

KEGG_AUTOIMMUNE_THYROID_DISEASE

KEGG_CHEMOKINE_SIGNALING_PATHWAY

1.00 1

KEGG_CHEMOKINE_SIGNALING_PATHWAY

1.0

Running Enrichment Score

KEGG_CYTOKINE_CYTOKINE_RECEPTOR_INTERACTION

Running Enrichment Score

KEGG_HEMATOPOIETIC_CELL_LINEAGE

Running Enrichment Score

KEOG_DRUG_METABOLISM_CYTOCHROME_P450

0.5

0.75

0.5

KEGG_NATURAL_KILLER_CELL_MEDIATED_CYTOTOXICITY

KEGG_OLFACTORY TRANSDUCTION

KEGG_NEUROACTIVE_LIGAND_RECEPTOR_INTERACTION

KEGO_OLFACTORY_TRANSDUCTION

0.0

KEGG_RETINOL_METABOLISM

0.50

KEGG_T_CELL_RECEPTOR_SIGNALING_PATHWAY

0.0

KEOG_TASTE_TRANSDUCTION

0.5

0.25

-1.0

0.00

-1.0

Ranked List Metric

-10

10000

20000

30000

40000

50000

10000

20000

30000

Rank in Ordered Dataset

40000

50000

10000

20000

30000

Rank in Ordered Dataset

40000

50000

PCPG

KEGG_HEMATOPOIETIC_CELL_LINEAGE

READ

KEGG_CELL_ADHESION_MOLECULES_CAMS

THCA

KEGG_CHEMOKINE_SIGNALING_PATHWAY

1.00-

Running Enrichment Score

KEGG_NATURAL_KILLER_CELL_MEDIATED_CYTOTOXICITY

Running Enrichment Score

KEGG_CHEMOKINE_SIGNALING_PATHWAY

1.01

9.75

KEGG_OLFACTORY_TRANSDUCTION

0.75

KEGG_CYTOKINE_CYTOKINE_RECEPTOR_INTERACTION

Running Enrichment Score

KEGG_CYTOKINE_CYTOKINE_RECEPTOR_INTERACTION

0.5

KEGG_HEMATOPOIETIC_CELL_LINEAGE

KEGG_RIBOSOME

KEGG_HEMATOPOIETIC_CELL_LINEAGE

KEGO_JAK_STAT SIGNALING_PATHWAY

.50

WEGG_TOLL_LIKE_RECEPTOR_SIGNALING_PATHWAY

0 50

KEGG_OLFACTORY_TRANSDUCTION

0.0

KEGG_OLFACTORY_TRANSDUCTION

.25

0.25

0.00

0 00

Ranked List Metric

-10

10000

20000

30000

Rank in Ordered Dataset

40000

50000

10000

20000

Rank in Ordered Dataset

40000

50000

10000

20000

Rank in Ordered Dataset

40000

50000

describes an elevated MMP12 expression in UCEC, including mRNA and protein levels, indicating the novelty of our findings. Notably, our study found that, in contrast to MMP12 mRNA expression, MMP12 protein levels are lower in BRCA than in control tissues; this may be related to various factors, such as translation-level regulation and post-translational modification regulation,40-42 which requires further

experimental verification. Such a phenomenon also indicates that investigations of gene expression should at least focus on both mRNA and protein levels rather than on only one of them. Regarding cancer status prediction, our study indicates that MMP12 makes it feasible to distinguish 21 cancer tissues from normal tissues. For prognosis, MMP12 expression is a prognosis risk factor in multiple cancers, including ACC,

A	Experimental					Control
Study	Total	Mean	SD	Total	Mean	SD
ArrayExpress_Agilent	97	4.35	1.7612	120	2.28	1.7760
GPL10558	14	7.09	1.8947	49	6.05	1.0663
GPL13497	17	7.87	3.1886	50	5.02	1.5731
GPL14951	37	2.76	1.5143	32	3.88	1.2668
GPL17586	8	5.97	1.0032	66	3.33	0.5403
GPL20115	11	4.19	2.0403	16	5.20	3.8723
GPL21290	6	6.43	1.3560	15	3.70	3.4272
GPL570	547	8.62	5.5514	351	3.89	4.3839
GPL6244	148	6.12	1.2699	239	4.32	1.2309
GPL6883	125	2.51	0.5225	124	2.61	0.5218
GPL6884	212	6.10	1.2884	200	5.22	0.7832
GPL96	120	8.38	1.4957	80	6.60	1.0105
GPL962	20	0.34	0.0371	30	0.29	0.0083
GSE103512	27	4.35	1.3116	3	3.26	0.1222
GSE11117	13	5.45	3.6880	15	2.73	1.8020
GSE11969	94	0.21	0.1592	5	0.05	0.0159
GSE1987	8	9.26	1.6526	9	6.04	0.4060
GSE21933	11	7.58	2.4891	11	3.62	1.1701
GSE32665	87	10.02	1.7092	92	7.48	0.5166
GSE37759	6	0.01	0.0835	6	-0.09	0.1166
GSE40275	11	5.62	1.0956	43	3.60	0.2734
GSE51852	4	8.06	2.2251	4	2.13	0.4766
GSE52248	12	2.51	1.9573	6	0.89	1.0861
GSE6044	10	7.02	1.8666	5	5.87	2.1635
GSE62949	28	6.59	0.1711	28	6.24	0.1194
GSE68571	86	7.91	2.2586	10	-0.80	4.7976
GSE83227	200	5.36	1.1198	17	4.26	0.5479
GSE85716	6	3.75	1.8911	6	2.27	0.2959
TCGA-GTEx	533	4.23	1.7879	347	1.19	1.5555

Figure 8. MMP12 mRNA and protein levels in LUAD. Panel A: MMP12 mRNA expression forest plot in LUAD and control tissues. Panel B: Violin plots of MMP12 mRNA expression in each dataset. Panel C: The violin plot of MMP12 protein levels. The P-value is calculated based on the Wilcoxon rank-sum test. NSP ≥ .05; * P < . 05; ** P< . 01; *** P < . 001.

Standardised Mean Difference

SMD

95%-CI Weight

1.17

[ 0.88; 1.46]

4.3%

0.80

[ 0.18; 1.41]

3.8%

1.35

[0.75; 1.95]

3.8%

-0.78

[-1.28; - 0.29]

4.0%

4.35

[ 3.32; 5.37]

3.0%

-0.30

[-1.07; 0.47]

3.5%

0.87

[-0.12; 1.86]

3.0%

0.92

[ 0.78; 1.06]

4.4%

1.45

[ 1.22; 1.68]

4.3%

-0.20

[-0.45; 0.05]

4.3%

0.82

[ 0.62; 1.02]

4.4%

1.34

[ 1.03; 1.65]

4.3%

2.02

[ 1.32; 2.72]

3.6%

0.84

[-0.38; 2.05]

2.6%

0.93

[ 0.14; 1.72]

3.4%

1.01

[ 0.10; 1.92]

3.2%

2.62

[ 1.23; 4.00]

2.3%

1.96

[ 0.91; 3.01]

2.9%

2.03

[ 1.67; 2.39]

4.2%

0.88

[-0.33; 2.09]

2.6%

3.68

[ 2.70; 4.66]

3.1%

3.20

[ 0.60; 5.81]

1.1%

0.89

[-0.15; 1.92]

3.0%

0.55

[-0.54; 1.65]

2.8%

2.31

[ 1.62; 2.99]

3.6%

3.31

[ 2.50; 4.12]

3.4%

1.01

[ 0.50; 1.51]

4.0%

1.01

[-0.22; 2.24]

2.6%

1.79

[ 1.63; 1.95]

4.4%

Random effects model 2498

1979

Heterogeneity: /2 = 93%, +2 = 0.5515, p < 0.01

1.35 [ 1.04; 1.66] 100.0%

-2

ArrayExpress_Agilent

GPL10558

GPL13497

GPL14951

GPL17586

GPL20115

MMP12 mRNA Level

12.5

MMP12 mRNA Level

10.0

NS.

7.5

5.0

2.5

-5

Non-Tumor

Tumor

Sample (n= 217)

Non-Tumor Sample (n= 63)

Tumor

Non-Tumor

Tumor

Sample (n= 67)

Non-Tumor

Tumor

Sample (n= 69)

Non-Tumor

Tumor

Sample (n= 74) GPL6884

Non-Tumor

Tumor

Sample (n= 27)

GPL21290

GPL570

GPL6244

GPL6883

GPL96

MMP12 mRNA Level

100

MMP12 mRNA Level

10.0

7.5

5.0

-5

Non-Tumor

Tumor

Sample (n= 21)

Non-Tumor

Tumor

Sample (n= 898)

Non-Tumor Sample (n= 387) GSE11117

Tumor

Non-Tumor

Tumor

Sample (n= 249) GSE11969

Non-Tumor

Tumor

Sample (n= 412)

Non-Tumor Sample (n= 200) Tumor GSE21933

GPL962

GSE103512

GSE1987

MMP12 mRNA Level

1.00

MMP12 mRNA Level

0.4

7.5

0.75

5.0

0.50

0.3

0.25

2.5

-5

0.00

Non-Tumor

Tumor

Sample (n= 50)

Non-Tumor Sample (n= 30)

Tumor

Non-Tumor Sample (n= 28)

Tumor

Non-Tumor

Tumor

Sample (n= 99)

Non-Tumor

Tumor

Sample (n= 17)

Non-Tumor

Tumor

Sample (n= 22) GSE6044

GSE32665

GSE37759

GSE40275

GSE51852

GSE52248

MMP12 mRNA Level

0.25

MMP12 mRNA Level

15.0

MMP12 mRNA Level

NS.

12.5

0.00

NS.

10.0

-0.25

7.5

5.0

0.50

-3

Non-Tumor Sample (n= 179)

Tumor

Non-Tumor

Tumor

Non-Tumor Sample (n= 54)

Tumor

Non-Tumor

Sample (n= 12)

Tumor

Sample (n= 8)

Non-Tumor

Tumor

Sample (n= 18)

Non-Tumor Sample (n= 15)

Tumor

GSE62949

GSE68571

GSE83227

GSE85716

TCGA-GTEx

In-house TMA

MMP12 mRNA Level

6.9

MMP12 mRNA Level

10.0

MMP12 mRNA Level

10.0

MMP12 Protein Level

7.5

6.6

NS.

5.0

2.5

6.3

…

2.5

6.0

0.0

Non-Tumor

Tumor

Non-Tumor Sample (n= 96)

Tumor

Non-Tumor Tumor Sample (n= 217)

Non-Tumor Tumor Sample (n= 12)

Non-Tumor

Tumor

Sample (n= 56)

Sample (n= 880)

Non-Tumor Sample (n= 88)

Tumor

Figure 9. Microscopic images of MMP12 protein levels in control tissues (panels A and C) and LUAD tissues (panels B and D). The numerical value in the bottom left corner of each image represents the magnification of the microscope. The white numerical value in the lower right corner of each image represents the scale.

Alveolus

100x

200x

400x

LUAD

100x

200x

400x

25 pm

Bronchio

100x

200x

400x

25 um

LUAD

100x

200x

400x

ESCA, KICH, KIRC, LUAD, PAAD, SARC, and THYM. Notably, MMP12 expression may be a prognosis protective factor for patients with BRCA and STAD; this may be at- tributed to the beneficial effects of MMP12 on macrophage development and suppression of angiogenesis in these two types of cancer, leading to its anti-tumor function.16,43 This indicates that the clinical significance may not be consistent across different types of cancer. Altogether, elevated MMP12 expression may serve as a potential predictive and prognostic biomarker for various cancers.

MMP12 may act as a potential target gene for immu- notherapy in cancers. Previously, highly expressed MMP12 has been identified as an immune gene promoting the proliferation of immune cells (e.g., B cells and dendritic cells), and the gene stimulates the host immune system to cause immune responsiveness.44,45 TMB and MSI are considered predictive biomarkers for immunotherapy, as they may contribute to the generation of neoantigens and thus stimulate the host immune system to recognize and clear neoantigens in the immune microenvironment.32,46,47 In our study, MMP12 expression is related to TMB, MSI, and neoantigen numbers in various cancers (e.g., STAD and COAD), implying its participation in the immune micro- environment. Such a conclusion is also supported by the

correlation (mainly positive) of MMP12 expression with several immune cells (e.g., B cells and dendritic cells) and immune scores (e.g., ESTIMATE algorithm scores). Moreover, MMP12 may participate in the occurrence and development of cancers through molecular signaling pathways, such as the cytokine-cytokine receptor inter- action and the chemokine signaling pathway as has been verified in colorectal cancer and breast cancer.48,49 The abovementioned findings suggest that MMP12 may have the potential to act as a tumor marker in tumor immunotherapy.

Some pan-cancer findings regarding MMP12 are verified in LUAD. With relatively small samples (n = 52), Lv et al. determined that MMP12 protein levels are higher in LUAD tissues than in normal tissues, and they report that MMP12 can promote the proliferation and growth of cancer cells and increase their invasiveness.12 Employing a large sample (n = 4565) from multiple centers and in house, we identified the upregulation of MMP12 expression in LUAD at both the mRNA and protein levels. We also show for the first time that MMP12 mRNA expression has the potential to distinguish LUAD with considerable accuracy. Fur- thermore, MMP12 expression serves as a risk prognosis factor for patients with LUAD. Thus, MMP12 may play an

Figure 10. Clinical value of MMP12 in LUAD. Panels A-B: MMP12 can well distinguish LUAD from controls. Panel C: Kaplan-Meier curves of the relation of MMP12 expression with overall survival of LUAD patients.

ArrayExpress_Agilent

GPL10558

GPL13497

GPL14951

GPL17586

GPL20115

Sensitivity

0.8

Sensitivity

0.8

Sensitivity

0.8

Sensitivity

0.8

Sensitivity

0.8

0.4

AUC: 0.782

0.4

AUC: 0.636

0.4

AUC: 0.760

0.4

AUC: 0.715

0.4

AUC: 0.987

0.4

AUC: 0.545

0.0

0.4

0.8

0.0

0.4

0.8

0.0

0.4

0.8

0.0

0.4

0.8

0.0

0.4

0.8

0.0

0.4

0.8

1 - Specificity

GPL21290

GPL570

GPL6244

GPL6883

GPL6884

GPL96

Sensitivity

0.8

Sensitivity

0.8

Sensitivity

0.8

Sensitivity

0.8

Sensitivity

0.8

Sensitivity

0.8

0.4

AUC: 0.722

0.4

AUC: 0.778

0.4

AUC: 0.876

0.4

AUC: 0.626

0.4

AUC: 0.695

0.4

AUC: 0.849

0.0

0.4

0.8

0.0

0.4

0.8

0.0

0.4

0.8

0.0

0.4

0.8

0.0

0.4

0.8

0.0

0.4

0.8

1 - Specificity

GPL962

GSE103512

GSE11117

GSE11969

GSE1987

GSE21933

Sensitivity

0.8

Sensitivity

0.8

Sensitivity

0.8

Sensitivity

0.8

Sensitivity

0.8

Sensitivity

0.8

0.4

AUC: 0.933

0.4

AUC: 0.716

0.4

AUC: 0.703

0.4

AUC: 0.930

0.4

AUC: 1.000

0.4

AUC: 0.909

0.0

0.4

0.8

0.0

0.4

0.8

0.0

0.4

0.8

0.0

0.4

0.8

0.0

0.4

0.8

0.0

0.4

0.8

1 - Specificity

GSE32665

GSE37759

GSE40275

GSE51852

GSE52248

GSE6044

Sensitivity

0.8

Sensitivity

0.8

Sensitivity

0.8

Sensitivity

0.8

Sensitivity

0.8

Sensitivity

0.8

0.4

AUC: 0.944

0.4

AUC: 0.639

0.4

AUC: 0.979

0.4

AUC: 1.000

0.4

AUC: 0.764

0.4

AUC: 0.740

0.0

0.4

0.8

0.0

0.4

0.8

0.0

0.4

0.8

0.0

0.4

0.8

0.0

0.4

0.8

0.0

0.4

0.8

1 - Specificity

GSE62949

GSE68571

GSE83227

GSE85716

TCGA-GTEx

Sensitivity

0.8

Sensitivity

0.8

Sensitivity

0.8

Sensitivity

0.8

Sensitivity

0.8

0.4

AUC: 0.938

0.4

AUC: 0.934

0.4

AUC: 0.868

0.4

AUC: 0.806

0.4

AUC: 0.908

0.0

0.4

0.8

0.0

0.4

0.8

0.0

0.4

0.8

0.0

0.4

0.8

0.0

0.4

0.8

1 - Specificity

Overall Survival

1.0

1.00

Survival probability

0.75

0.50

Sensitivity

0.5

0.25

p = 0.022

Observed Data

Summary Operating Point

SENS = 0.83 [0.77 - 0.88]

0.00

SPEC = 0.85 [0.79 - 0.90]

SROC Curve

AUC = 0.91 [0.88 - 0.93]

Time (Years)

95% Confidence Contour

95% Prediction Contour

Number at risk

0.0

1.0

0.5

Specificity

0.0

MMP12= Low

MMP12=High

422

important role in LUAD as a predictive and prognostic biomarker. Based on this, a promising application of MMP12 in LUAD involves detecting MMP12 mRNA ex- pression levels during pathological diagnosis of potential patients with LUAD; this approach may contribute to the evaluation of patient prognosis.

This study has a few limitations. For example, for various cancers, we need to collect more samples to determine MMP12 expression at the protein level. We also need to include more clinicopathological parameters to explore whether other clinicopathological variables may impact our results. More in vivo and in vitro experiments are needed to investigate the pan-cancer mechanisms of MMP12. In addi- tion, the pan-cancer examination of the relationship between prognosis and MMP12 expression was based on retrospective data; thus, prospective studies are needed for further validation.

Conclusion

This study comprehensively explores MMP12 in multiple cancers. MMP12 is highly expressed in most cancers. The gene may serve as a novel biomarker for the prediction and prognosis of numerous cancers.

Appendix

Abbreviations

ACC adrenocortical carcinoma

AUC area under the curve

BLCA

bladder urothelial carcinoma

BRCA breast-invasive carcinoma

CESC cervical squamous cell carcinoma and endocervical adenocarcinoma

CHOL

cholangiocarcinoma

COAD

colon adenocarcinoma

DLBC

lymphoid neoplasm diffuse large b-cell lymphoma esophageal carcinoma

ESCA

GBM glioblastoma multiforme

HNSCC

head and neck squamous cell carcinoma

hazard ratio

KEGG Kyoto Encyclopedia of Genes and Genomes

KICH

kidney chromophobe

KIRC

kidney renal clear cell carcinoma

KIRP

kidney renal papillary cell carcinoma

LGG

brain lower grade glioma

LIHC

liver hepatocellular carcinoma

LUAD

lung adenocarcinoma

LUSC

lung squamous cell carcinoma mesothelioma

MESO MMP12

matrix metalloproteinase 12

OS overall survival

ovarian serous cystadenocarcinoma

PAAD

pancreatic adenocarcinoma

PCPG PRAD

pheochromocytoma and paraganglioma

prostate adenocarcinoma

rectum adenocarcinoma

receiver operating characteristic

SARC

sarcoma skin cutaneous melanoma

SKCM sROC summary receiver operating characteristic standardized mean difference

STAD stomach adenocarcinoma

OS TCGA

overall survival The Cancer Genome Atlas

TGCT

THCA

THYM

TMB

testicular germ cell tumors thyroid carcinoma thymoma tumor mutation burden microsatellite instability

MSI

UCEC uterine corpus endometrial carcinoma

UCS

UVM

uterine carcinosarcoma uveal melanoma.

Acknowledgments

The authors thank Guangxi Key Laboratory of Medical Pathology for its technical support in computational and clinical pathology. The results shown in the study are in part based upon data generated by the DepMap Portal (https://depmap.org/portal/), Xena database (https:// xena.ucsc.edu/), Proteomic Data Commons (https://pdc.cancer.gov), Gene Expression Omnibus (https://www.ncbi.nlm.nih.gov/gds/), and TCGA Research Network (www.cancer.gov/tcga).

Authors’ Contributions

Study design and draft writing: Guo-Sheng Li, Yu-Xing Tang, Jin- Liang Kong, Hua-Fu Zhou, and Gang Chen. Data acquisition: Guo- Sheng Li, Yu-Xing Tang, Wei Zhang, Jian-Di Li, and He-Qing Huang. Data analysis and interpretation: Guo-Sheng Li, Yu-Xing Tang, Wei Zhang, Jian-Di Li, He-Qing Huang, Jun Liu, Zong-Wang Fu, and Rong-Quan He. All authors were involved in reading and revising the draft and approved the final version for publication.

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This research was funded by Guangxi Medical High-level Key Talents Training “139” Program (2020), Guangxi Zhuang Autonomous Region Medical Health Appropriate Technology Development and Application Promotion Project (S2020031), Guangxi Higher Edu- cation Undergraduate Teaching Reform Project (2022JGA146 and 2021JGA142), Guangxi Educational Science Planning Key Project (2021B167), and Guangxi Medical University Key Textbook Con- struction Project (Gxmuzdjc2223).

READ ROC

SMD

Ethical Statement

Ethical Approval

This study was approved by the medical ethics review committee of the First Affiliated Hospital of Guangxi Medical University (No. 6 Shuangyong Road, Nanning, China) on October 26, 2021, with the approval number 2021(KY-E-246). Informed consent was signed by all patients involved in the in-house data.

ORCID iD

Gang Chen ® https://orcid.org/0000-0003-2402-2987

Data Availability Statement

The data that support the findings of pan-cancer analyses are available in public databases, including DepMap Portal (https://depmap.org/ portal/), Xena database (https://xenabrowser.net/datapages), Proteo- mic Data Commons (https://pdc.cancer.gov), Gene Expression Omnibus (https://www.ncbi.nlm.nih.gov/gds/), and TCGA Research Network (www.cancer.gov/tcga). Data on in-house tissue samples used during the current study are available from the corresponding author upon reasonable request.

Supplemental Material

Supplemental material for this article is available online.

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Quartz 4

Explorer

38410859

MMP12 is a Potential Predictive and Prognostic Biomarker of Various Cancers Including Lung Adenocarcinoma

Abstract

Keywords

Corresponding Author:

Introduction

Materials and Methods

The Prediction Effect and Prognosis Value of MMP12 for Cancers

Validation of MMP12 mRNA Expression in LUAD

Validation of MMP12 Protein Expression in LUAD

Validation of the Potential Clinical Value of MMP12 in LUAD

Statistical Analysis

Results

Pan-Cancer MMP12 Expression Level

Clinical Significance of Pan-Cancer MMP12 Expression

Relationship Between the Expression of Immune Gene MMP12 and Genomic Heterogeneity

Correlation Assessment of MMP12 Expression and the Immune Microenvironment

MMP12 Expression and Potential Signaling Pathways

Overall Expression Level of MMP12 in LUAD

Clinical Value of MMP12 in LUAD

Discussion

Conclusion

Appendix

Abbreviations

Acknowledgments

Authors’ Contributions

Declaration of Conflicting Interests

Funding

Ethical Statement

Ethical Approval

ORCID iD

Data Availability Statement

Supplemental Material

References

Graph View

Table of Contents

Backlinks

Quartz 4

Explorer

38410859

MMP12 is a Potential Predictive and Prognostic Biomarker of Various Cancers Including Lung Adenocarcinoma

Abstract

Keywords

Corresponding Author:

Introduction

Materials and Methods

Collecting MMP12-related Expression and Prognosis Data Across Cancers

The Prediction Effect and Prognosis Value of MMP12 for Cancers

Validation of MMP12 mRNA Expression in LUAD

Validation of MMP12 Protein Expression in LUAD

Validation of the Potential Clinical Value of MMP12 in LUAD

Statistical Analysis

Results

Pan-Cancer MMP12 Expression Level

Clinical Significance of Pan-Cancer MMP12 Expression

Relationship Between the Expression of Immune Gene MMP12 and Genomic Heterogeneity

Correlation Assessment of MMP12 Expression and the Immune Microenvironment

MMP12 Expression and Potential Signaling Pathways

Overall Expression Level of MMP12 in LUAD

Clinical Value of MMP12 in LUAD

Discussion

Conclusion

Appendix

Abbreviations

Acknowledgments

Authors’ Contributions

Declaration of Conflicting Interests

Funding

Ethical Statement

Ethical Approval

ORCID iD

Data Availability Statement

Supplemental Material

References

Graph View

Table of Contents

Backlinks