Matrix Metalloproteinase 11 is a Potential Biomarker in Bladder Cancer Diagnosis and Prognosis

Figures & data

Table 1 Clinicopathological Characteristics in TCGA BLCA Cohort

Characteristics	Level of MMP11 Gene Expression		P-value
	Low (n=145)	High (n=262)
Sex			0.0671
Female	30	76
Male	115	186
Pathologic stage			<0.0001
I	2	0
II	70	60
III	41	98
IV	31	103
N/A	1	1
T stage			<0.0001
T0	1	0
T1	3	0
T2	59	60
T3	49	144
T4	15	43
TX	0	1
N/A	18	14
N stage			0.0086
N0	94	142
N1	10	36
N2	18	57
N3	1	7
NX	19	17
N/A	3	3
Recurrence^a			0.0358
No	100	151
Recurrence	36	89
N/A	9	22
Metastasis^b			0.0020
No	80	107
Metastasis	53	139
N/A	12	16
Histological grade			<0.0001
High grade	124	259
Low grade	20	1
N/A	1	2
Diagnosis subtype			<0.0001
Non-papillary	73	197
Papillary	70	62
N/A	2	3
Primary therapy outcome			0.0163^c
Complete remission	93	135
Partial remission	4	17
Progressive disease	20	49
Stable disease	9	20
N/A	19	41
Additional therapy outcome			0.0222^d
Complete response	73	101
Partial response	1	5
Progressive disease	27	71
Stable disease	6	8
N/A	38	77

Notes: ^a Recurrence was determined by new tumor event. ^b Metastasis was determined by N stage, M stage, and new distant metastatic tumor. ^c Complete remission vs Partial remission, Progressive disease and Stable disease. ^d Complete response vs Partial response, Progressive disease and Stable disease. P-value is calculated by Chi-square (χ²) test or Fisher’s exact test.

Figure 1 Promoter hypomethylation of MMP11 in bladder cancer. (A) Promoter hypomethylation in MMP11 gene in bladder cancer tissues. (B) Methylation status in each CpG site in MMP11 promoter in bladder cancer. (C) Compared to normal bladder tissues, a significant decrease of methylation was found at ⁻³⁴⁷CpG site (cg22418565) in MMP11 promoter in bladder cancer tissues. (D) Lower methylation level was found at ⁻³⁴⁷CpG site (cg22418565) in MMP11 promoter in bladder cancer tissues compared to their matched normal tissues. (E) Bisulfite sequencing assays showing hypomethylation in MMP11 promoter in bladder cancer cell T24 compared to normal bladder cell SV-HUC-1, in particular at ⁻³⁴⁷CpG, ⁻²⁶⁷CpG, and ⁻⁶⁶CpG sites. Upper left: MMP11 promoter sequence and CpG sites. Right: representative bisulfite sequencing results. Filled circles indicate methylated CpGs, while empty circles indicate unmethylated CpGs. Student’s t-test was performed in (A, B, C, E) (two tailed, unpaired) and (D) (two tailed, paired). Data represent means±SEM in (A, B, C, E). *P<0.05; **P<0.01; ****P<0.0001; ns, not statistically significant.

Figure 2 MMP11 is upregulated in bladder cancer in vivo and in vitro. (A, B) IHC staining in tissue microarray showing increased MMP11 protein level in bladder cancer tissues. The representative images of IHC and HE are shown in (A), while the quantification of MMP11 protein level is summarized in (B). (C) ELISA assays showing increased MMP11 protein levels in culture medium from bladder cancer cell lines T24, UMUC3, and 5637, compared to normal bladder cell SV-HUC-1. Scale bar in (A), 20 μm. Student’s t-test was performed (two-tailed, unpaired) and data represent means±SEM in (B) and (C). **P<0.01.

Figure 3 Big data analysis reveals MMP11 expression in bladder cancer. (A) GO term cellular component analysis of DEGs. (B) Top 10 most differentially expressed genes in yellow part in (A). (C) 14.9-fold increase of MMP11 expression was observed in bladder tumor tissues compared to adjacent normal tissues. (D) A higher upregulation of MMP11 expression (28.7-fold) is found in cancer tissues in bladder cancer patients compared to their matched normal tissues. (E) MMP11 expression level gradually increased along with tumor stage in bladder cancer. Student’s t-test was performed in (C) and (E) (two tailed, unpaired) and (D) (two tailed, paired). Data represent means±SEM in (C)and (E).

Figure 4 MMP11 overexpression is associated with tumor progression and poor patient survival in bladder cancer. MMP11 overexpression is associated with tumor progression and poor patient survival in bladder cancer. (A–C) High level of MMP11 was positively associated with advanced tumor stage, recurrence, metastasis, high disease grade, and non-papillary subtype. (D) MMP11 high expression was negatively correlated with therapy efficacy in bladder cancer. (E, F) Kaplan-Meier survival curve showing inverse association between MMP11 expression and overall survival and recurrence free survival in bladder cancer. Chi-square (χ²) test or Fisher’s exact test was performed in (A–D). Log rank test was performed in (E, F).