Mesothelin binding to CA125/MUC16 promotes pancreatic cancer cell motility and invasion via MMP-7 activation

Abstract

Mesothelin (MSLN) and cancer antigen125/mucin 16 (CA125/MUC16) are potential biomarkers for pancreatic cancer (PC) that are co-overexpressed at the invading edges of PC tissues, and their expression correlates with poor survival rates. However, the role of MSLN-MUC16 molecular interaction in PC cell motility and invasion has yet to be elucidated. Using sophisticated bioengineering and molecular biology tools, we report that the binding of MSLN to MUC16 markedly enhances PC cell motility and invasion via the selective induction of matrix metalloproteinase (MMP)-7. MSLN-mediated MMP-7 upregulation in MUC16-expressing PC cells occurs via a p38 MAPK-dependent pathway. Depletion of MMP-7 or inhibition of p38 activity abolishes MSLN-mediated PC motility and invasion. These findings provide a novel perspective on the enhanced invasive potential associated with MSLN and MUC16 co-overexpression, and the mechanism underlying MMP-7 activation in PC invasion and metastasis.

Figures

Figure 1. MUC16 expression correlates with pancreatic cancer cell binding capacity to MSLN.

(A) hTERT-HPNE, HPDE, PANC-1, SW1990, and Pa03C cells were seeded in 96-well plates pre-coated with either mesothelin (MSLN) or BSA (control). After 30 min of incubation, the relative number of adherent cells to MSLN- versus BSA-coated wells was quantified by WST-1 assay. Data represent the mean ± S.E. of three independent experiments. *, p < 0.05 with respect to control. (B) Representative flow cytometric histograms of MUC16 surface expression on hTERT-HPNE, HPDE, PANC-1, SW1990, and Pa03C cells using indirect single-color immunofluorescence and an anti-MUC16 mAb or an isotype control. (C) MUC16-overexpressing SW1990 or Pa03C cells were pre-incubated with either a function-blocking anti-MUC16 mAb M11 (20 μg/ml) or an isotype control for 1 h at 37°C, and then used in the cell binding assay as described in (A). (D) Scramble control or MUC16-KD SW1990 and Pa03C cells were seeded in 96-well plates pre-coated with MSLN or BSA (control). After 30 min of incubation, adherent cells were quantified by WST-1 assay. Data represent the mean ± S.E. of three independent experiments. *, p < 0.05 with respect to MUC16-KD cells.

Figure 2. MSLN and MUC16 co-immunoprecipitate in pancreatic cancer cells.

Whole lysates from scramble control (A) or MUC16-KD (B) SW1990 and Pa03C cells were immunoprecipitated with either an anti-MUC16 or anti-MSLN or an isotype control antibody, and analyzed for MSLN or MUC16 reactivity by immunoblotting. Lysates from SW1990 and Pa03C cells were also analyzed as input controls.

Figure 3. MUC16 expression correlates with MSLN-mediated MMP-7 induction in pancreatic cancer cells.

hTERT-HPNE, HPDE, PANC-1, SW1990, and Pa03C cells were starved in serum-free medium and incubated in the presence or absence of MSLN (1 μg/ml) overnight. (A) MMP-7, (B) MMP-2, and (C) MMP-9 mRNA levels were then determined by qRT-PCR. GAPDH served as internal control. Data represent the mean ± S.E. of at least three independent experiments. *, p < 0.01 with respect to corresponding control. (D) MUC16, MSLN, and MMP-7 expression in pancreatic cancer tissue specimens was determined by immunohistochemical staining using an anti-MUC16, anti-MSLN, or anti-MMP-7 antibody, respectively. Strong immunostaining was detected in ∼ 90% of cancerous tissues, whereas no immunoreactivity was present in the ducts or acini of normal pancreas tissue.

Figure 4. MSLN-MUC16 interaction induces MMP-7 synthesis in pancreatic cancer cells.

(A) SW1990 cells were starved in serum-free medium overnight and incubated with MSLN (1 μg/ml) for the indicated periods of time. The mRNA levels of MMP-7 were quantified by qRT-PCR. GAPDH served as internal control. Data represent the mean ± S.E. of at least three independent experiments. *, p < 0.05 with respect to cells treated with MSLN (1 μg/ml) for 0 h. MMP-7 protein expression from SW1990 cell lysates was analyzed using Western blotting. β-actin served as an internal control. (B) SW1990 cells were starved in serum-free medium overnight and incubated with different concentrations of MSLN (0–2.0 μg/ml) for 12 h. MMP-7 mRNA and protein expression were then analyzed by qRT-PCR and Western blotting, respectively. Data from qRT-PCR represent the mean ± S.E. of three independent experiments. *, p < 0.05 with respect to untreated control cells. (C, D) Scramble control or MUC16-KD SW1990 and Pa03C cells were serum-starved overnight and then incubated for 12 h in the presence or absence of MSLN (1 μg/ml). MMP-7 mRNA (C) and protein (D) levels were quantified by qRT-PCR and immunoblotting, respectively. Data represent the mean ± S.E. of at least three independent experiments. *, p < 0.01 and §, p < 0.05 with respect to the corresponding scramble control cells. (E) Conditioned media from scramble control or MUC16-KD pancreatic cancer cells pre-treated with MSLN (1 μg/ml) for 12 h were collected and analyzed for MMP-7 secretion. Data are reported as the fold of MMP-7 levels detected in the conditioned medium of each cell line compared to that in the medium of untreated scramble control cells. Data represent the mean ± S.E. of three independent experiments. *, p < 0.05 with respect to corresponding untreated scramble control cells.

Figure 5. MSLN-MUC16 interaction promotes pancreatic cancer cell invasion via MMP-7 activation.

Scramble control or MUC16-KD SW1990 (A) or Pa03C (B) cells were incubated with MSLN (1 μg/ml) in the presence of either a neutralizing MMP-7 antibody (2 μg/ml) or an isotype control and then used in transwell invasion assays. Data are reported as normalized cell number that invaded through the transwell membrane relative to untreated scramble control (100%), and represent the mean ± S.E. of three independent experiments. **, p < 0.01; ***, p < 0.001 (one-way ANOVA followed by Tukey test). (C) SW1990 and Pa03C pancreatic cancer cells were incubated with either human recombinant active MMP-7 (2 μg/ml) or vehicle control, and then used in transwell invasion assays. Data are reported as normalized cell number that invaded through the membrane relative to vehicle control-treated cells (100%), and represent the mean ± S.E. of three independent experiments. *, p < 0.05 with respect to vehicle control cells (Student's t-test).

Figure 6. MSLN binding to MUC16 promotes pancreatic cancer cell motility via MMP-7 activation.

Representative time-lapse images of scramble control and MUC16-KD SW1990 (A) and Pa03C (C) pancreatic cancer cells migrating through channels of prescribed dimensions (WxHxL = 6 × 10 × 200 μm) using 10% FBS as chemoattractant. In all experiments, cells were incubated with or without MSLN (1 μg/ml) for 6 h before being seeded to the microchannel device. The average migration speed of individual scramble control and MUC16-KD SW1990 (B) and Pa03C (D) cells was determined over a 10 h period for > 30 cells from three independent experiments. *, p < 0.05 with respect to scramble control and MSLN-treated MUC16-KD cells. (E) The average migration speed of MSLN-stimulated SW1990 and Pa03C cells in the presence of either an MMP-7 neutralizing antibody (2 μg/ml) or an isotype control was quantified for > 30 cells from three independent experiments. *, p < 0.05 with respect to the control treatment. (F) The average migration speed of SW1990 and Pa03C cells in the presence or absence of human recombinant active MMP-7 (2 μg/ml) was quantified for > 30 cells from three independent experiments. *, p < 0.05 with respect to untreated control.

Figure 7. MSLN-MUC16 interaction promotes pancreatic cancer cell motility and invasion via MMP-7 induced by a p38 MAPK-dependent pathway.

(A) Scramble control and MUC16-KD SW1990 cells were serum-starved overnight, and then treated with MSLN (1 μg/ml) for 12 h. The levels of phospho-ERK1/2 (Thr202/Tyr204) and phospho-p38 MAPK (Thr180/Tyr182) from whole cell lysates were analyzed by immunoblotting using specific Abs. Equal loading in each lane was ensured by the similar intensities of total ERK1/2, p38 MAPK, and β-actin. (B) SW1990 cells were incubated with MSLN (1 μg/ml) for 12 h in the presence or absence of the p38 MAPK inhibitor SB203580 (20 μM). The levels of phospho-p38 MAPK (Thr180/Tyr182) and MMP-7 expression from cell lysates were analyzed by Western blotting using specific Abs. Equal loading in each lane is ensured by the similar intensities of total p38 MAPK and β-actin. These Western blots are representative of three independent experiments, all revealing similar results. The intensity of bands was quantified using the NIH ImageJ software and then normalized with respect to the value obtained for the untreated control. (C, D) SW1990 cells were stimulated with MSLN (1 μg/ml) in the presence or absence of SB203580 (20 μM) or an MMP-7 antisense oligonucleotide (50 nM), and then subjected to either transwell invasion or microchannel motility assays. (C) Data are reported as percentage of untreated control cells that invaded through the transwell membrane, and represent the mean ± S.E. of three independent experiments. (D) The average migration speed of individual cells was determined over a 10 h period for > 30 cells from three independent experiments for each of the indicated conditions. *, p < 0.01.

Figure 8. Proposed molecular pathway induced by MSLN-MUC16 binding, which results in MMP-7 induction in pancreatic cancer cells.

The binding of MSLN to MUC16 present on the pancreatic cancer cell surface activates a p38 MAPK-dependent pathway, which in turn upregulates MMP-7 synthesis, resulting in increased invasive and migratory potentials. In the absence of MUC16 expression by pancreatic cancer cells, MSLN is capable of upregulating MMP-7 expression via activation of an ERK-dependent pathway.