Human MUC4 mucin induces ultra-structural changes and tumorigenicity in pancreatic cancer cells

Abstract

MUC4 is a type-1 transmembrane glycoprotein and is overexpressed in many carcinomas. It is a heterodimeric protein of 930 kDa, composed of a mucin-type subunit, MUC4alpha, and a membrane-bound growth factor-like subunit, MUC4beta. MUC4 mRNA contains unique 5' and 3' coding sequences along with a large variable number of tandem repeat (VNTR) domain of 7-19 kb. A direct association of MUC4 overexpression has been established with the degree of invasiveness and poor prognosis of pancreatic cancer. To understand the precise role of MUC4 in pancreatic cancer, we engineered a MUC4 complementary DNA construct, mini-MUC4, whose deduced protein (320 kDa) is comparable with that of wild-type MUC4 (930 kDa) but represents only 10% of VNTR. Stable ectopic expression of mini-MUC4 in two human pancreatic cancer cell lines, Panc1 and MiaPaCa, showed that MUC4 minigene expression follows a biosynthesis and localisation pattern similar to the wild-type MUC4. Expression of MUC4 resulted in increased growth, motility, and invasiveness of the pancreatic cancer cells in vitro. Ultra-structural examination of MUC4-transfected cells showed the presence of increased number and size of mitochondria. The MUC4-expressing cells also demonstrated an enhanced tumorigenicity in an orthotopic xenograft nude mice model, further supporting a direct role of MUC4 in inducing the cancer properties. In conclusion, our results suggest that MUC4 promotes tumorigenicity and is directly involved in growth and survival of the cancer cells.

Figure 1

Construction of the mini-MUC4. (A) Schematic representation of cloning strategy for the generation of the mini-MUC4 construct. Fragments amplified by PCR were subcloned in PCR2.1 vector, sequenced, digested with specific restriction sites, and subcloned in the pBluescript vector. Finally, the 5′ region, JER64 fragment, and the 3′ fragments were subcloned in the pSecTag C vector. Each junction was sequenced to confirm the reading frame. (B) A schematic representation of mini-MUC4 gene. The sequence of the mini-MUC4 (320 kDa) is comparable with that of wild-type MUC4 (930 kDa), but with a repetitive domain representing only 10% the normal size.

Figure 2

Expression and subcellular localisation of mini-MUC4 in pancreatic adenocarcinoma cell lines. (A) A total of 10 μg protein from cell extracts were resolved by electrophoresis on a 2% SDS-agarose gel containing Tris–glycine (pH 8.8) buffer, transferred to PVDF membrane and incubated with anti-MUC4 monoclonal antibody. The membrane was then probed with HRPO-labelled goat anti-mouse Ig. Immunoblot of actin, obtained from 10% SDS-polyacrylamide gel, was used as an internal control. (B) Confocal photomicrograph demonstrating expression pattern of MUC4 in methanol-fixed human pancreatic tumor cells. The cells were grown at low density on coverslips for 24 h, and the acetone/methanol (1 : 1)-fixed cells were labelled using a FITC-conjugated anti-MUC4 monoclonal antibody and counterstained by propidium iodide. Magnification, × 630.

Figure 3

Growth kinetic patterns of the parental, vector control, and mini-MUC4-expressing cells. The cells were plated at a density of 10⁴ per well into six-well plates, grown for 6 days, and counted each day after seeding. The graph presents the result obtained in terms of the number of cells as a function of increasing duration. (A) The cells were maintained in medium containing 10% serum. (B) The cells were maintained in medium containing 1% serum.

Figure 4

Electronmicroscopy. Cells, cultured at 70% confluence, were fixed with 2.5% glutaraldehyde in 1 M Sorenson's phosphate buffer. The cell pellet was then subjected to overnight infiltration in Dureapam Acm. Epoxy Resin (Electron Microscopy Sciences), flat embedding, and polymerisation at 55°C for 48 h. Embedded cells were mounted on resin stubs and sectioned at 90 nm with an Ultracut E ultramicrotome. Sections were viewed and photographed with a Zeiss 600 EM10A TEM at 60 Kv. The pictures presented are representative for the experiment. Twenty cells for each cell line were observed in two independent experiments. The black arrows indicate dividing mitochondria in the MiaPaCa cell line transfected with mini-MUC4 construct and the red arrows indicate the golgi complex.

Figure 5

Determination of the mitochondrial mass. (A) The fluorescent dye NAO was used to monitor the mitochondrial mass in low MUC4-expressing (thin black curve) and high MUC4-expressing (thick black curve) Panc1 cell lines. These cell lines were compared with the parental Panc1 cells (thin grey curve) and the mock cells (discontinuous curve). The relative NAO intensity of the mini-MUC4-transfected cells (with a mean of 88.05 and 103.43 for the mini-MUC4 low and high expressing cell lines respectively) was higher than the control cell lines (with a mean of 67.3 and 63.25 for the parental and mock cell lines, respectively). (B) Western blot analysis of the low and high mini-MUC4 expressing cells compared with both Panc1 and Panc1 pSecTag control cell. As positive control, the HPAF/CD18 cells were used. (C) The NAO relative intensity was plotted depending on the mini-MUC4 expression. Mini-MUC4 expression was normalised with β-actin. One-hundred counts for MUC4 expression were empirically attributed to the mini-MUC4-expressing cells.

Figure 6

Analysis of the apoptotic and necrotic indices in mini-MUC4-overexpressing cells vs control cells. Panc1, either overexpressing mini-MUC4 or transfected with vector only, were serum starved 16 h to induce apoptosis. The percentage of cells undergoing apoptosis and necrosis was measured by annexin V and propidium iodide staining, respectively, followed by fluorescence-activated cell sorting analysis. (A) The bars represent the mean percentage of the apoptotic and necrotic cells (n=3, ^*P<0.05). (B) Western blot analysis to show the PARP activity in Panc1, Panc1-pSecTag C, and Panc1-mini-MUC4 cell lysates. A total of 30 μg protein from cell extracts were resolved by electrophoresis on a 10% SDS-polyacrylamide gel, transferred to PVDF membrane, and incubated with anti-PARP antibody. The membrane was then probed with HRP-labelled goat anti-mouse Ig. β-Actin was used as an internal control.

Figure 7

Expression of mini-MUC4 increases the motility and invasiveness of pancreatic cancer cells. (A) Cells were plated onto non-coated membranes for motility assays. The cells were incubated for 24 h, and those that did not migrate through the pores in the membrane were removed by scraping the membrane with a cotton swab and the remaining cells were stained. Cells that migrated through the pores were counted, and representative fields were photographed under bright field microscopy. Quantitation of cells invaded through the membrane in the above experiments is presented as a bar diagram. The mini-MUC4-expressing cells showed significant increase in cell motility as compared to control cells (^*P<0.001). (B) Matrigel invasion assay to measure the invasive potential of mini-MUC4-expressing cells. The invasion of mini-MUC4-expressing and control cells through extracellular matrix was investigated using Matrigel invasion chambers. The 10% FBS was used as a chemoattractant in lower chambers. Cells migrated through the Matrigel matrix were counted, and representative fields were photographed under bright field microscopy. Quantitation of cells invaded through the Matrigel matrix in the above experiments is presented as a bar diagram. The mini-MUC4-expressing cells showed higher invasive index than the control cells (^*P<0.05).