High expression of MMP19 is associated with poor prognosis in patients with colorectal cancer

Background Matrix metalloproteinase 19 (MMP19) is a member of zinc-dependent endopeptidases, which have been involved in various physiological and pathological processes. Its expression has been demonstrated in some types of cancers, but the clinical significance of MMP19 in colorectal cancer (CRC) has not been reported. Thus, we aimed to analyze the clinical significance of MMP19 in CRC in present study. Methods The expression of MMP19 was first explored in The Cancer Genome Atlas (TCGA) cohort, and then validated in the GSE39582 cohort and our own database. Clinicopathological features and survival rate were also investigated. Results MMP19 was found to be a predictor for overall survival (OS) in both univariate (hazard ratio [HR]: 1.449, 95% confidence interval [CI]: 1.108–1.893, P = 0.007) and multivariate survival analyses (HR: 1.401, 95% CI: 1.036–1.894, P = 0.028) in the TCGA database. MMP19 was further validated as an independent factor for recurrence free survival in the GSE39582 database by both univariate analysis (HR: 2.061, 95%CI: 1.454–2.921, P < 0.001) and multivariate analysis (HR = 1.470, 95% CI: 1.025–2.215, P = 0.032). In an in-house cohort, MMP19 was significantly upregulated in CRC tissues when compared with their adjacent normal controls (P < 0.001). Ectopic MMP19 expression was positively associated with lymph node metastases (P = 0.029), intramural vascular invasion (P = 0.015) and serum carcinoembryonic antigen levels (P = 0.045). High MMP19 expression correlated with a shorter OS (HR = 5.595; 95% CI: 2.573–12.164; P < 0.001) and disease free survival (HR = 4.699; 95% CI: 2.461–8.974; P < 0.001) in multivariate cox regression analysis. Conclusions Expression of MMP19 was upregulated in CRC. High expression of MMP19 was determined to be an independent and poor prognostic factor in CRC. These results suggest that MMP19 may be a good biomarker for CRC.


Background
Colorectal cancer (CRC) is the third most common and the third leading cause of cancer-related death in the United States [1]. In China, both the incidence and mortality rate of CRC has been increasing, and CRC is ranked as the third leading cause of cancer-related deaths [2]. The development of distant metastasis is the main reason of cancer-related death regardless of effective surgical procedures and systemic chemotherapy.
Approximately 20-25% of patients are initially diagnosed with synchronous metastases, of which approximately 50% ultimately develop metachronous disease after colectomy [3,4]. The survival outcome of CRC is mainly determined by tumor stage and some other clinicopathological factors. However, the heterogeneity of this disease makes it difficult to predict patient prognosis with these traditional factors [5]. Recent genetic and molecular analyses of CRC have identified a set of predictive biomarkers, including RAS status, BRAF mutation and mismatch repair protein expression, that can aid in the identification of patients who are at high risk of disease progression or recurrence [3,[6][7][8]. Although available biomarkers are commonly used for predicting long-term outcome, some previous studies have reported that a proportion of patients are misdiagnosed [7,8]. Therefore, the identification of novel markers that can be used to screen various prognostic risk subgroups to guide individual treatment is urgently needed.
Matrix metalloproteinases (MMPs) are zinc-dependent endopeptidases that involve in a variety of physiological processes [9], and they act in concert in tumor invasion and metastasis [10]. Over the years, they have been investigated for their roles in cancer progression and metastasis [9,[11][12][13][14]. MMP14 plays an important role in CRC progression and prognosis [15]. Immunohistochemical score based on major members of MMP/TIMP profile can identify a distinct group of colorectal cancers with poor prognosis [14]. However, some MMPs, such as MMP19, have not been fully investigated in CRC. MMP19 was first isolated as an autoantigen from the synovium of a rheumatoid arthritis patient [16,17]. MMP19 contains classical MMP structural domains, including a signal peptide, pro-peptide, catalytic domain, hinge region, and C-terminal domain [17,18]. MMP19 is reportedly involved in the progression and metastases of various cancers, but its role in CRC remains unknown.
In this study, we used The Cancer Genome Atlas (TCGA) and whole-genome expression microarray (Gene Expression Omnibus, accession number GSE39582) databases to investigate the expression of MMP19 and RNA sequence. Furthermore, we explored the relationship between MMP19 expression and cancer prognosis using our data to determine whether MMP19 can serve as a valuable prognostic predictor in CRC patients.

Methods
TCGA and GSE39582 database MMP19 mRNA expression was retrieved from the TCGA portal (http://tcga-data.nci.nih.gov) and GSE39582 database (https://www.ncbi.nlm.nih.gov/geo/). We selected patients who had both RNA sequencing data and clinicopathological factors available for the correlation analysis. The inclusion criteria were as follows: pathologically diagnosed with invasive adenocarcinoma, available intact survival information, and RNA sequencing data available. A total of 359 CRC samples in the TCGA cohort and 474 cases in the GSE39582 database were selected. The relationship between MMP19 expression and the prognosis of CRC patients was explored.

Validation cohort
The study was approved by the Ethics Committee of Taizhou Municipal Hospital, Medical College of Taizhou University (Zhejiang, China). Before surgery, all patients provided written informed consents in compliance with the ethics of the World Medical Association (Declaration of Helsinki) for the donation of their tissue for the present research. All patients underwent radical colectomy, and all fresh tissues, including tumor tissues and normal controls, were frozen in liquid nitrogen immediately after resection and stored in RNA later at − 20°C. Pathological diagnoses were made by at two pathologists and restaged according to the 8th American Joint Committee on Cancer guidelines. Normal control tissue was retrieved at least 10 cm from the tumor margin.
All patients were underwent followed up strategy according to NCCN guidelines. The primary endpoint for patients were OS and DFS. The OS was defined as the time from diagnoses to death from any cause, and the DFS was defined as the time from diagnoses to the first recurrence or death [19]. The survival data was got from the medical records or contacts with patients by phone or email.

Ethics statement
This study was approved by the Taizhou Municipal Hospital Research Ethics Committee (ID: 2018-03-0039). The study was implement according to the approved guidelines. Informed consent was obtained from each patient before surgery.

Immunohistochemistry (IHC) study
Immunohistochemistry (IHC) was performed on formalin-fixed, paraffin-embedded tissue sections as previous described [15,22]. MMP19 was detected using the rabbit anti-MMP19 polyclonal antibody AP6202a (Abgent Inc.). Primary antibodies was omitted as negative control. Data were assessed by two independent single-blinded pathologists. A semi quantitative immunoreactivity scoring system was used to sort patients into high and low expression groups according to the immunoreactivity score [22,23].

Statistical analysis
OS, DFS or recurrence free survival (RFS) was used as primary endpoint for TCGA, GSE39582 and validation cohort. Survival analysis were compared using the univariate and multivariate Cox proportional hazard model among different MMP19 mRNA expression levels in the TCGA and GSE39582 database. The results were demonstrated as hazard ratios (HR) and 95% confidence intervals (CI). MMP19 was also classified into high and low expression subgroups in TCGA and GSE39582 cohorts by the X-tile program with a maximum χ 2 value and minimum P value. [24]. A one-sided P value < 0.05 was considered as statistically significance.

Results
MMP19 was an independent prognostic factor for survival in the TCGA cohort A total of 359 eligible patients were included in this study from the TCGA database, including 199 (55.4%) men and 160 (44.6%) women. The median age was 64 (range 31-90) years. The median follow-up time was 32 (range, 0-15) months and 82 patients (22.8%) died after the last follow-up.

MMP19 was correlated with inferior clinical characteristics in the validation cohort
To investigate the potential relevance of MMP19 expression in CRC tissues in terms of clinical characteristics, MMP19 mRNA expression was further examined in 198 CRC cancer tissues and paired normal controls. The results indicated that MMP19 expression was significantly up-regulated in cancer tissues than in normal controls (P < 0.05; Fig. 3a). The clinical and histopathologic characteristics classified by the median MMP19 mRNA expression level are summarized in Table 3. High MMP19 expression was significantly correlated with lymph node metastases (P = 0.029), intramural vascular invasion (P = 0.015) and serum carcinoembryonic antigen status (P = 0.045; Table 3). Genes usually exert function through their encoded proteins. Therefore, we used immunohistochemistry to detect the expression of MMP19 protein in 42 patients in the validation group, and found MMP19 mRNA expression is highly correlated with their protein levels (P < 0.001) ( Table 3).

Discussion
With recent advances in high-throughput technologies (e.g., RNA deep sequencing), the transcriptomes of many tumors have been surveyed and many novel biomarkers and therapeutic targets have been identified. To validate MMP19 as a potential novel target gene to predict survival, we designed our study in three steps. First, we found MMP19 as a potential novel biomarker in terms  of survival in the TCGA database. Second, we studied MMP19 in the GSE39582 database and confirmed it as novel biomarker for CRC. Finally, because some important clinical characteristics were missed in the TCGA and GSE39582 database, such as strategies of adjuvant therapy, and the quality of surgery,, we further validated the results from our own database. We found that MMP19 expression was significantly upregulated in cancer tissues relative to the normal controls, and that high MMP19 expression was associated with inferior clinical characteristics. Importantly, MMP19 was validated as an independent predictor for both OS and DFS for CRC after colectomy. Our results point to a crucial role for MMP19 in the evolution of CRC. MMP19 is a classical member of the MMP family, which consists of at least 23 enzymes [17]. MMP19 shares the typical structural domains of MMP, including a signal peptide, propeptide, catalytic domain, hinge region, and C-terminal domain [18]. MMP19 has been gradually recognized as an important oncogene in carcinogenesis and progression. It is associated with increased mortality and promotes metastatic behavior in non-small cell lung cancer (NSCLC) [25]. An increase in MMP19 expression indicates the progression of cutaneous melanoma and might augment melanoma growth by promoting the invasion of tumor cells [26]. MMP19 is highly expressed in astroglial tumors and promotes the invasion of glioma cells [27]. For CRC, a previous study reported that MMP19 may involve in malignant transformation, is low expressed in normal mucosa, and is upregulate during neoplastic progression [28], but its prognostic value has not been reported. Here, we provide new evidence that MMP19 plays a crucial role in CRC. High MMP19 expression was significantly correlated with lymph node metastases and intramural vascular invasion, which suggests that MMP19 may play a critical role in CRC invasion and metastases. Distant metastases and recurrence are two main reasons for cancer-related death; thus, it was not surprising that high MMP19 expression was correlated with a poor    prognosis in CRC. Similar, increased MMP19 gene expression correlates with a worse prognosis and facilitates invasion in NSCLC [25]. Although we obtained conclusions from three independent databases, there were some limitations in our study. First, we only investigated the clinical significance of MMP19 in CRC using patient samples; no in vitro study or animal models were used in this study. Second, a further study is needed to understand the mechanisms underlying the function of MMP19 in CRC progression.

Conclusion
In summary, our results demonstrate that MMP19 is upregulated in CRC and is a potential predictor of CRC, which provides additional information for predicting survival and developing a therapeutic strategy. Our results warrant further studies on the detailed mechanisms by which MMP19 facilitates tumor progression in CRC.