Mesothelin Binding to CA125/MUC16 Promotes Pancreatic Cancer Cell Motility and Invasion

Mesothelin Binding to CA125/MUC16 Promotes Pancreatic Cancer Cell Motility and Invasion via MMP-7 Activation

Shih-Hsun Chen1,2, Wei-Chien Hung1,3, Pu Wang5, Colin Paul1,2,4, and Konstantinos Konstantopoulos1,2,3,4,*

From the 1Department of Chemical and Biomolecular Engineering, 2Physical Sciences-Oncology Center, 3Center for Cancer Nanotechnology Excellence, 4Institute for NanoBioTechnology, Johns Hopkins University, Baltimore, Maryland 21218. 5College of Life and Health Sciences, Northeastern University, Shenyang, P. R. China 110004

*To whom correspondence should be addressed:

Konstantinos Konstantopoulos, Ph.D.

Department of Chemical and Biomolecular Engineering

The Johns Hopkins University

3400 N. Charles Street

Baltimore, MD 21218

Tel: (410) 516-6290

Fax: (410) 516-5510

E-mail:

Figure-S1

Figure S1. Knockdown of MUC16 via shRNA in SW1990 and Pa03C pancreatic cancer cells. MUC16 mRNA (A), surface protein (B), and total protein levels (C) in scramble control or MUC16-KD SW1990 and Pa03C cells were assessed by qRT-PCR, flow cytometry, and immunoblotting, respectively. In (A), data represent the mean±S.E. of at least three independent experiments. *, p < 0.01 with respect to the corresponding scramble control cells. Flow cytometry histograms (B) and immunoblots (C) are representative of at least three independent experiments, all revealing similar results.

Figure-S2

Figure S2. MSLN induces MMP-7 expression in Pa03C cell in a time- and dose- dependent manner. (A) Pa03C cells were starved in serum-free medium overnight and incubated with MSLN (1 µg/ml) for the indicated periods of time. MMP-7 protein expression from Pa03C cell lysates was analyzed using Western blotting. (B) Pa03C 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 protein expression was then analyzed by Western blotting. β-actin served as an internal control.

Figure-S3

Figure S3. Diagram of microchannel migration assays. (A) Schematic of microchannel migration chamber. A chemotactic gradient is generated within the microchannels by diffusion of the chemoattractant from the chemoattractant inlet well. Pressure-balanced laminar flow on each side of the micro-channels minimizes convective flow within the channels. Feed lines are 50 µm high. (B) Cartoon of cells seeded at the entrance of the microchannels (WxH= 6x10 µm). Cells are induced to migrate through the microchannels in response to a chemotactic gradient.

Figure-S4

Figure S4. MSLN binding to MUC16 promotes pancreatic cancer cell motility in a wound healing assay. Scramble control and MUC16-KD SW1990 cells were seeded at 100% confluence and thin wounds were created and imaged at 0 h and 12 h of MSLN (1 µg/ml) incubation.

Figure-S5

Figure S5. MSLN induces MMP-7 synthesis via an ERK-dependent pathway in pancreatic cancer cells. SW1990 cells were incubated with MSLN (1 µg/ml) for 12 h in the presence or absence of the ERK inhibitor PD98029 (20 µM). The levels of phospho-ERK (Thr202/Tyr204) 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 ERK and β-actin. These Western blots are representative of three independent experiments, all revealing similar results.

Figure-S6

Figure S6. The effect of MSLN on cell proliferation in scramble control and MUC16-KD PC cells. Scramble control and MUC16-KD SW1990 (A) and Pa03C (B) cells were seeded in triplicate (104 cells/well) onto 96-well plates and starved in serum-free media overnight, followed by treatment with either MSLN (1 µg/ml) or vehicle control for 0, 6, 12, and 24 h. The cell number was then determined byWST-1assay.