Matrix Metalloproteinase Expressions Play Important role in Prediction of Ovarian Cancer Outcome

Ovarian cancer has a high death rate and is often not detected until late stages. While some studies found high expressions of MMPs correlated with cancer invasion, metastasis, and poor prognosis, however, several other studies indicated MMPs might inhibit cancer rather than promote cancer in certain situations. Thus, the role of different MMPs in different cancer types needs a systematic re-evaluation. In this study, we used RNA-Seq and corresponding clinical data downloaded from TCGA and analyzed the correlations between MMP expressions and the clinicopathologic characteristics and outcome in ovarian serous cystadenocarcinoma (OSC) patients. Among the MMPs investigated, MMP-3 was significantly increased in high-grade and high-stage tumors compared with low-grade and low-stage ones. Using univariate analysis and multivariate Cox model, high expressions of MMP-19 and -20 were found to associate with poor overall survival independent of clinicopathologic characteristics. Moreover, using in vitro studies, we found ovarian cancer cell lines with higher MMP-19 and -20 protein expressing levels were associated with anti-cancer drugs resistance, while knockdown of MMP-19 or -20 increased ovarian cancer cell sensitivities to several clinical using chemotherapy agents. Finally, knockdown of MMP-19 or -20 also decreased the invasion abilities of several ovarian cancer cell lines. These in vitro studies provided potential mechanisms of high MMP-19 and -20 expressions in the poor prognosis of ovarian cancer.

extracellular proteins, some MMPs regulate tumor invasion through other mechanisms, for example, MMP-2 as well as MMP-9 could activate TGF-β to promote tumor invasion 16 .
On another hand, although most studies have found poor prognosis correlated with the high expressions of MMPs 15,[17][18][19][20][21][22] , a few other studies have found certain MMPs could inhibit cancer growth rather than promote it under certain situations [23][24][25] , suggesting that different MMPs have different roles. Moreover, while several MMP inhibitors have been developed 26,27 , however, there are still no effective MMP inhibitors to treat cancer today. More importantly, studies have also suggested that some MMP inhibitors might promote cancer progression instead of inhibition 27 , which limited the usage of MMP inhibitors in the clinic. Therefore, to evaluate the significance of MMP expressions in the prediction of cancer prognosis, systematic studies of the relationships between each MMP and certain cancers were needed.
In this study, we used the RNA expression data from The Cancer Genome Atlas (TCGA) to conduct a systematic investigation of the relationships between each MMP members and the clinical characteristics in ovarian serous cystadenocarcinoma (OSC). Our study not only found some MMPs were correlated with the clinicopathologic characteristics in OSC patients, but also found high expressions of MMP-19 as well as MMP-20 predicted poor outcome. Further studies found MMP-19 and MMP-20 high expressions could cause drug resistance and cancer invasion, providing potential molecular mechanisms for the poor prognosis caused by high MMP-19 and MMP-20 expressions.
Results patient clinical characteristics. 293 ovarian cancer patients from TCGA with clinical and gene expression data were analyzed in this study. The clinical characteristics were shown in Table 1. The median age of the patient cohort was 58 years (range 30-87 years). Among these patients, about 89% were Caucasian, 6% were African or African American, and 4% were Asian. The proportion of American Indian, Alaska Native, or Native Hawaiian and other Pacific Islander was less than 1%. The proportions of Stage I, II, III, and IV of these patients were 0%, 6%, 82%, and 12%, respectively. The overall grades for these patients were: 11% at Grade 1 and 2, 89% at Grade 3, and less than 1% at Grade 4. Right, left, and bilateral for anatomic neoplasm subdivisions were 13%, 12% and 75%, respectively. The median time to last follow up was 28.0 months with the range of 0-183 months.

Relationship between MMPs expressions and clinicopathologic characteristics.
We first analyzed the expression profile of 22 MMPs in the above 293 OSC patients. As shown in Fig. 1, the mRNA expression levels of different MMPs varied among the patient samples. Generally, MMP-2, MMP-7, MMP-11 and MMP-14 were expressed at higher levels than the other MMPs in most of the patient samples.
Previous studies have shown that some MMP expression levels correlate with clinicopathologic characteristics, for example, MMP-1, MMP-9, MMP-12 and MMP-15 expressed higher in grade 3 than in grade 1 and 2 breast tumors 28 . To further explore whether the MMP expression levels correlate with clinicopathologic status in OSC, we systematically analyzed the correlations between them, using the Mann-Whitney U test. As shown in Table 2, upon this analysis, we found mRNA levels of MMP-3 and MMP-25 were significantly higher in stage III and IV than stage II cancers (p < 0.05), while the other MMPs did not show significant differences between www.nature.com/scientificreports www.nature.com/scientificreports/ different MMPs, the Spearman correlation analysis was used, and the results were shown in Table 3. Three different types of correlations were found between MMPs expression levels: strong correlations (|r| ≥ 0.8, also shown in  (Table 4), high expressions of MMP-2, MMP-14, MMP-19 and MMP-20 were found to associate with poor overall survival. To find out whether these MMPs could serve as independent variables, the derived four MMPs were again analyzed with stage, grade, and anatomic neoplasm subdivision in a multivariate model. Only high expression of MMP-19 and MMP-20 were significantly associated with poor overall survival independent of the above clinicopathologic characteristics after this analysis (Table 5). Thus, high expressions of MMP-19 or MMP-20 could serve as independent factors to predict poor prognosis in OSC patients.
High MMP-19 and -20 expressions induce drug resistance. Drug resistance 29 and metastasis or invasion 15,16 were the possible reasons of poor prognosis caused by high MMP expressions. We first test if drug resistance might be one of the reasons of the poor prognosis caused by high expressions of MMP-19 and MMP-20. MMP-19 and MMP-20 protein expression levels from six ovarian carcinoma cell lines (Ovcar5, Ovcar8, Cov362, Ov90, Ho8910 and Skov3) were assayed by western blotting. The MMP-19 and MMP-20 protein expression levels varied among these six cell lines, as indicated by optical density ratio of target protein to β-actin (Fig. 3a). Among these cell lines, MMP-19 protein level expressed highest in Ho8910 but lowest in Ovcar5, while MMP-20 expressed highest in Cov362 but lowest in Ovcar8. To further test the relationships of MMP-19 and MMP-20 protein levels and the anti-cancer drug sensitivities, both the MMP-19 and MMP-20 high expression and low expression cell lines were exposed to two different anti-cancer agents. One is A-1210477, which could direct target the anti-apoptotic protein MCL1 and induce apoptosis, and the other is Vincristine, which is a common chemotherapy drug used in many types of cancers, inducing cancer cell death through a microtubule polymerization mechanism. We observed that Ovcar8 (the lowest MMP-20 protein expression among the six cell lines), was more sensitive than Cov362 (highest MMP-20 expression among these lines) to both A-1210477 and Vincrinstine, as evaluated by the apoptotic pre-G1 cells (Fig. 3b,c) and also the annexin V and PI double staining assay (Fig. 3d,e). Moreover, less cell viabilities were observed in Ovcar8 than Cov362 cells after both A-1210477 and Vincrinstine treatments (Fig. 3f,g). Similarly, Ovcar5, which had lowest MMP-19 expression, was more sensitive to both A-1210477 and Vincristine than Ho8910, which had highest MMP-20 expression (Fig. 3h,i). Therefore, cell lines with higher MMP-19 or MMP-20 expression levels were associated with increased drug resistances to A-1210477 and Vincritine.
To further confirm the correlation between MMP-19 and MMP-20 high expressions and drug resistance, ovarian cancer cell lines Skov3 and Cov362 were performed with the MMP-19 and MMP-20 knockdown using siRNAs, followed by treatments of several anti-cancer drugs. The mRNA expressions of MMP-19 in Skov3 and Cov362 cells decreased about 50% and 74% respectively after MMP-19 siRNA treatments (Fig. 4a). MMP-20 siRNA also remarkablely decreased the MMP-20 protein levels in both cell lines (Fig. 4b). More importantly, knockdown of MMP-19 significantly increased sensitivities of Skov3 to A-1210477 (p < 0.05) and Carboplatin (p < 0.001), as well as the sensitivities of Cov362 to A-1210477 (p < 0.05) and Carboplatin (p < 0.05) ( Fig. 4ch). Similarly, knockdown of MMP-20 significantly increased sensitivities of Skov3 to A-1210477 (p < 0.01) and Paclitaxel (p < 0.05), as well as sensitivities of Cov362 to A-1210477 (p < 0.05) and Carboplatin (p < 0.01) (Fig. 4c-h). To rule out the possibility of non-specific target effect of siRNAs, another siRNA was performed in Cov362 cell lines followed by drug treatments. As shown in Fig. S1a- Table 2. Relationship between MMPs expression levels and clinicopathologic characteristics. a "mRNA" (messenger RNA); "MMP" (matrix metalloproteinase); "NS" (not significance). b The Mann-Whitney U test was used.

High MMP-19 and -20 expressions promote invasiveness. Previous studies have also shown that
MMPs are involved in cancer metastasis and invasion, so we also investigated the influence of MMP-19 and MMP-20 on cell invasion abilities using the Boyden chamber method. We observed that the MMP-19 and MMP-20 knockdown decreased Cov362 cell invasion abilities by 68% and 74%, respectively (Fig. 5a,c). Similar results were observed in ovarian cell line Skov3, that knockdown of MMP-19 and MMP-20 decreased the cell invasion abilities by 35% and 41%, respectively (Fig. 5b,c). In addition, another group of MMP-19 siRNA also decreased the Cov362 cell invasion ability by 52% (Fig. S2a,b), further confirmed the MMP-19 function in cell invasion ability. Therefore, the results in Figs 5 and S2 suggest high MMP-19 and -20 expressions related to high cancer cell invasion abilities.

Discussion
Several previous studies have investigated the potential prognostic impact of one or more MMPs in different cancer types 13,15,21 , however, most of these studies only involve one or several MMP family members. No systematic study between MMPs and cancer prognosis has been done. In this study, we systematically investigated the relationship between MMP expressions and the patient clinicopathologic characteristics, and outcomes using clinical and gene expression data from TCGA of the ovarian cancer patients.   Table 3. Correlation between mRNA levels of MMP family members in OSC patients. a "mRNA"(messenger RNA); "MMP" (matrix metalloproteinase); "NS" (not significance). b The Spearman correlation analysis was used.   Table 4. Relationship between MMP expression and overall survival in OSC. a "MMP" (matrix metalloproteinase); "CI" (confidence interval). b Data were analyzed using a univariate cox model. www.nature.com/scientificreports www.nature.com/scientificreports/ Through the analysis, we found the mRNA expression levels of certain MMP members were correlated with some clinic pathologic characteristics. For example, mRNA levels of MMP-3 and MMP-25 were significantly higher in stage III and IV tumors compared with stage II tumors ( Table 2). Both MMPs have previously been reported to be associated with cancer metastasis or cancer progression. Between them, MMP-3 was reported to be overexpressed in both chicken and human ovarian cancers cells 30 , which would lead to the invasion of ovarian cancer 31 , and the molecular mechanisms might involve the miR-200 down regulation. MMP-25 was one of the membrane type MMPs, which could promote cell growth and migration because of their presence on the cell surface 32 . Our work revealed that MMP-3 and MMP-25 high expression levels were correlated with higher OSC tumor stage, providing further evidence of these MMPs in OSC progression.
By the analysis of the relationships between expressions of the different MMPs, multiple statistically significant strong or medium correlations were found between MMP pairs, which would suggest these pairs of MMPs were co-regulated by similar pathways, or even that one MMP is under the regulation of the other. In the present study, strong correlations (Table 3) were found between MMP-2 and MMP-11 (P < 0.001, r = 0.820), and MMP-11 and MMP-13 (P < 0.001, r = 0.805), suggesting strong co-regulations between both pairs of MMPs in OSC patients. Previous studies have also found correlations of both MMP pairs in breast cancer 28 . Moreover, we found medium correlations in quite a few pairs of MMPs. For example, medium correlations were found between MMP-2 and www.nature.com/scientificreports www.nature.com/scientificreports/ MMP-14 (P < 0.001, r = 0.764). A previous study has also shown that MMP-14 could activate MMP2, both of which appeared to play important roles in regulating cell growth and proliferation by controlling matrix remodeling in aggressive ovarian cancer cells 33 . We also found some of the statistically significant medium correlations that have not been reported before, including the correlations between MMP-1/MMP-3, MMP-1/MMP-13, MMP-2/MMP-3, and MMP-8/MMP-12. The reason why these MMPs showed significant correlations in OSC patient samples needs further study.
In this study, we also found high expressions of MMP-2, MMP-14, MMP-19, and MMP-20 were associated with poor overall survival using univariate analysis, however, only the high expression of MMP-19 and MMP-20 predict poor prognosis in a multivariate model, suggesting MMP-19 and MMP-20 high expressions as independent predictive factors for poor prognosis in OSC patients. Previous studies have also found that MMP-19 was highly expressed in astroglial tumors and facilitate the invasion of gliomacells 13 , and MMP-20 might play an important role in the progression of esophageal cancer 14 . In addition, we found that MMP-2 and MMP-14 correlated with poor prognosis in OSC patients using univariate analysis, both of which had been reported to predict poor prognosis in several cancer types 15,19,21,22 .
To further explore the potential mechanisms of MMP-19 and MMP-20 involved in poor prognosis, MMP related anti-cancer drug resistance and invasion mechanisms were studied using ovarian cancer cells lines. We  , h), the percentage of pre-G1 cells were measured. Error bars, mean ± S.D. of three independent experiments. ns, not significant; *p < 0.05; **p < 0.01; ***p < 0.001.
found that ovarian cancer cell lines with higher MMP-19 and MMP-20 protein expressing levels were more resistant to anti-cancer drugs, such as A-1210477 and Vincristine (Fig. 3). While A-1210477 and Vincristine are not generally used in the treatment of OSC patient in clinic, we also included two other commonly used drugs to evaluate the relationship between drug resistance and the expressions of MMP-19 and MMP-20. One drug is Carboplatin, a DNA synthesis inhibitor, inhibits tumor growth by binding to DNA and interfering with DNA repair mechanisms, the other is Paclitaxel, a microtubule polymer stabilizer. MMP-19 and MMP-20 knockdown not only significantly increased the drug sensitivity to A-1210477 in both Skov3 and Cov362 cells, but also increased the drug sensitivity to Carboplatin in both cell lines, suggesting MMP-19 and MMP-20 high expression are related to at least some kind of anti-cancer drug resistances in OSC patients (Fig. 4). MMP-19 and MMP-20 knockdown did not increase Cov362 resistance to Paclitaxel (both p > 0.05, Fig. 4h), which might due to the fact that different molecular mechanisms are involved in the anti-cancer effects of these drugs.
Previous studies have also found high MMP expressions correlated with metastasis and invasion 13,15,16 . To further explore the mechanisms of OSC patients with poor prognosis and MMP-19 and MMP-20 high expression levels, we also conducted Boyden chamber assay to evaluate the ability of invasion after MMP-19 and MMP-20 knockdown. We found both MMP-19 and MMP-20 knockdown decreased the invasion ability in two OSC cell lines, Skov3 and Cov362 (Fig. 5). Taken together, our study suggested the potential mechanisms of poor prognosis of patients with MMP-19 and MMP-20 high expressions included both drug resistance and invasion caused by both MMPs.
In conclusion, our investigations of OSC patient samples from TCGA showed that high expression of MMP-19 and MMP-20 were independent predictors of poor outcome in patients with OSC. Moreover, through experiments using ovarian cancer cell lines, we found cell lines with high MMP-19 or MMP-20 expression levels were more resistant to several anti-cancer drugs. Further knockdown assay using MMP-19 and MMP-20 siRNAs confirmed the important roles of drug-resistance caused by MMP-19 and MMP-20 high expressions. In addition, we found MMP-19 and MMP-20 also increased cell invasion ability. All these in vitro studies provided potential mechanisms of the poor prognosis of OSC patient with high MMP-19 or MMP-20 RNA expressions.

Methods
Patients in TCGA. RNA-Seq expression and corresponding clinical data for OSC patients were downloaded from TCGA (http://cancergenome.nih.gov/). The methods of biospecimen procurement, RNA isolation, and RNA sequencing were previously described by the Cancer Genome Atlas Research Network 34 . RPKM (reads per kilobase per million mapped reads) was used as the expression value for statistical analysis.