SerpinB3 upregulates the Cyclooxygenase-2 / β-Catenin positive loop in colorectal cancer

Abstract

Colorectal cancer is characterized by aberrant Cyclooxigenase-2 (COX-2) and β-Catenin pathways. Recently, the protease inhibitor SerpinB3 has been described overexpressed in more advanced stages of this tumor. Aim of the study was to explore the possible relationship between these molecules in this setting. We evaluated colorectal cancer specimens from 105 patients and a positive correlation between SerpinB3, COX-2 and β-Catenin expression was observed, with higher levels in tumor than in adjacent tissue. The highest levels were associated with pathologic parameters of poor prognosis, including vascular invasion, lymph node metastasis and perineural invasion. The molecular and protein profiles of COX-2 and β-Catenin were analyzed in cell lines with different expression of SerpinB3. In those with high expression of SerpinB3, COX-2 and β-Catenin were higher than in controls. Cells with high levels of SerpinB3 showed higher proliferation and invasion compared to controls. In conclusion, in colorectal cancer SerpinB3, COX-2 and β-Catenin are positively correlated and associated with more advanced tumor stage. The in vitro experimental results support a driving role of SerpinB3 in the upregulation of COX-2/ β-Catenin positive loop, associated with a more aggressive cellular phenotype.

Keywords: COX-2; SerpinB3; colorectal cancer; tumor stage; β-Catenin.

Figure 1. SerpinB3, COX-2 and β-Catenin mRNA expression levels in colorectal cancer tissue

Upper panel: Expression levels of SerpinB3, COX-2 and β-Catenin are significantly higher in tumor (T) samples than in the corresponding non-tumor tissue (N). In adenoma (A) samples the levels of the three molecules are similar to the adjacent tissue (N). Results are expressed as pg/μL. Boxes and whiskers represent the 25th to 75th and 5th to 95th percentiles, respectively; the median value is represented by the central line in each box and the range of values of all samples is represented by vertical bars. Statistical analysis was carried out using Mann Whitney test. Lower panels: Correlation of mRNA levels between SerpinB3, COX-2 and β-Catenin mRNA in tumor (T) specimens and in the corresponding non tumor samples (N). r_s = Sperman correlation coefficient.

Figure 2. Correlation between SerpinB3, COX-2, β-Catenin expression and tumor stage

A. Quantitative Real-Time PCR analysis of SerpinB3, COX-2 and β-Catenin expression levels in different tumor stages. Tumor (T) and non tumor (N) samples of early (I and II) and advanced (III and IV) tumor stages were analyzed using the Mann-Whitney test. Boxes and whiskers represent 25th to 75th and 5th to 95th percentiles, respectively; the median value is represented by the central line in each box and the range of values of all samples is represented by vertical bars. B. Immunohistochemical staining for SerpinB3, COX-2 and β-Catenin obtained in serial sections of a stage I and of a stage IV samples. In stage IV nuclear and cytoplasm positivity of SerpinB3 protein was detectable in a large number of cells, while in stage I only a small number of cells showed low level of expression. COX-2 was strongly positive in the cytoplasm and in the perinuclear area of stage IV tumor and the highest reactivity was found at the bottom of the crypts, decreasing along the longitudinal axis to the luminal region. A similar result was obtained for β-Catenin. Magnifications: 200 X, inserts 400X. In the right panel, graphical representation of the quantitative analysis of each staining has been provided. The average sum of intensities and stained area percentage of each patient was calculated using JmageJ software. Values are the mean ± SD (bar) of 10 different images analyzed [*p<0.001, Low vs High].

Figure 3. SerpinB3, COX-2 and β-Catenin expression in tumor specimens in relation with histological parameters of poor prognosis

Mann-Whitney Test analysis was applied to determine statistical differences between positive versus negative groups for Vascular Invasion, lymph node metastasis and perineural invasion. Boxes and whiskers represent the 25th to 75th and 5th to 95th percentiles, respectively. The central line in each box represents the median value, the range of values of all samples is represented by vertical bar. The level of significance was set at p < 0.05. Neg = negative and Pos = positive for the pathologic parameter.

Figure 4. Immunofluorescence analysis of SerpinB3, COX-2 and β-Catenin

Immunofluorescence staining of HTC15, HT29, HepG2 and HepG2/SerpinB3 cell lines after 48 hour of culture. Left panel: Double staining for SerpinB3 and β-Catenin proteins. Right panel: Double staining for SerpinB3 and COX-2 proteins. Proteins were visualized under a fluorescent microscope: TRIC (red, SerpinB3), FITC (green, β-Catenin and COX-2) and DAPI (blue, nuclei). Original magnification 400 X

Figure 5. Profile of expression of SerpinB3, COX-2 and β-Catenin in cell lines

A. In HTC15, HT29, HepG2 and HepG2/SerpinB3 cell lines SerpinB3, COX-2, and β-Catenin transcripts were quantified at 24, 48 and 72 hours after plating by real-time PCR. Results are expressed as mean +/− SE of three independent experiments. SerpinB3: HTC15 cell line 24hr versus 72hr *p = 0.0376 and 48hr versus 72hr *p = 0.0376; HT29 cell line 24hr versus 48hr **p = 0.0018, 24hr versus 72hr *p = 0.0181 and 48hr versus 72hr *p = 0.0134; HepG2/SerpinB3 cell line 24hr versus 48hr ***p < 0.0001, 24hr versus 72hr **p = 0.0014 and 48hr versus 72hr **p = 0.0028. COX-2: HT29 cell line 24hr versus 48hr ▪▪p = 0.0098, 24h versus 72h ▪p = 0.0316 and 48h versus 72h ▪p = 0.0103; HepG2 cell line 24h versus 72hr ▪p = 0.0180; HepG2/SerpinB3 cell line 24hr versus 48hr ▪▪▪p = 0.0002, 24hr versus 72hr ▪▪p = 0.0023. β-Catenin: HTC15 cell line 24hr versus 72hr ·p = 0.0421; HT29 cell line 24hr versus 48hr ··p = 0.0047, 24hr versus 72hr ··p = 0.0065 and 48hr versus 72hr ·p = 0.0201; HepG2 cell line 24hr versus 72hr ·p = 0.0300; HepG2/SerpinB3 cell line 24hr versus 48hr ···p = 0.0004 and 48hr versus 72hr ··p = 0.0024. B. SerpinB3 (45 KDa), COX-2 (60 KDa) and β-Catenin (92 KDa) proteins were analyzed by Western blot and C. Their levels were estimated by densitometric analysis (QuantityOne software, Biorad). β-Actin expression was used for sample normalization. Results are representative of three independent experiments.

Figure 6. The proposed mechanism the COX-2/ β-Catenin positive loop upregulation by SerpinB3

SerpinB3 determines an increase of β-Catenin. The accumulation of β-Catenin in the nucleus interacts with T-cell factor (TCF) and lymphoid enhancer binding protein (LEF) transcription factors to activate COX-2 gene. The COX-2 protein in turn increases PGE₂ levels and these two molecules strengthen the positive loop of β-Catenin/LEF1/TCF-mediated transcription.