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. 2008 Apr 30;3(4):e2054.
doi: 10.1371/journal.pone.0002054.

A minimal fragment of MUC1 mediates growth of cancer cells

Sanjeev Mahanta  1 , Shawn P Fessler, Jaehong Park, Cynthia Bamdad
Affiliations

A minimal fragment of MUC1 mediates growth of cancer cells

Sanjeev Mahanta et al. PLoS One. .

Abstract

The MUC1 protein is aberrantly expressed on many solid tumor cancers. In contrast to its apical clustering on healthy epithelial cells, it is uniformly distributed over cancer cells. However, a mechanistic link between aberrant expression and cancer has remained elusive. Herein, we report that a membrane-bound MUC1 cleavage product, that we call MUC1*, is the predominant form of the protein on cultured cancer cells and on cancerous tissues. Further, we demonstrate that transfection of a minimal fragment of MUC1, MUC1*(1110), containing a mere forty-five (45) amino acids of the extracellular domain, is sufficient to confer the oncogenic activities that were previously attributed to the full-length protein. By comparison of molecular weight and function, it appears that MUC1* and MUC1*(1110) are approximately equivalent. Evidence is presented that strongly supports a mechanism whereby dimerization of the extracellular domain of MUC1* activates the MAP kinase signaling cascade and stimulates cell growth. These findings suggest methods to manipulate this growth mechanism for therapeutic interventions in cancer treatments.

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Conflict of interest statement

Competing Interests: All authors are employees of a biotech company and participate in a stock option plan.

Figures

Figure 1
Figure 1. Schematic and antibody recognition of full-length MUC1 versus the membrane-bound cleavage product MUC1*.
A. Full-length MUC1 protein is comprised of a cytoplasmic tail (CT), a transmembrane domain (TM), a self-aggregation domain (SAD), and hundreds of tandem repeats (TRs). B. Cleavage product, MUC1*, consists of the cytoplasmic tail, transmembrane domain, and at least 45 amino acids of the extracellular domain (ECD). Although the exact site(s) of cleavage remain somewhat uncertain, to our knowledge, no cleavage sites have been reported that leave less than a 45 amino acid ECD. Binding sites for antibodies VUH5, Ab-5 and Anti-MUC1* are marked. C. Western analysis of whole cell lysates of MUC1-positive cultured cancer cells, Lanes 1–6, are compared to MUC1-negative cells, Lanes 7–9. A 6% gel (upper) was blotted with VU4H5 and a 12% gel (lower) was blotted with Anti-MUC1*. D. T47D, MUC1-positive cultured breast cancer cells, were immunoprecipitated with either Ab-5 or Anti-MUC1*. Samples of the whole cell lysate (WCL), Lane 1, or the immunoprecipitates, Lanes 2 and 3, were analyzed by western blot. Gels were probed with either VU4H5 (upper) or Anti-MUC1* (lower). E. Western analysis of three MUC1-positive breast cancer cell lines pre- and post- deglycosylation and blotted with Anti-MUC1* shows that the actual molecular weight of cleaved MUC1 is approximately 16–18 kDa. F. The table summarizes the reactivities of antibodies to either full-length MUC1 (Hi MW) or cleaved MUC1 (Lo MW).
Figure 2
Figure 2. MUC1* is the major species on cultured cancer cells and on human tumor tissue.
A. Fluorescent imaging of cultured breast cancer cells shows that the cleavage product MUC1* (red) is the predominant species and is uniformly distributed over the entire cell surface. Full-length MUC1 (green) is the minor species and is clustered. Cells were double stained with Anti-MUC1* (red) that binds to an epitope within the membrane proximal first 45 amino acids of the extracellular domain and VU4H5 (green) that binds to an epitope within the tandem repeat domain of the full-length protein. B–E. Pairs of contiguous sections of human tissue specimens from untreated patients were stained with either Anti-MUC1* (left) or VU4H5 (right). Images were photographed at 10× or 100× magnification. B. A cross section of a non-cancerous fallopian tube shows MUC1* surface staining at the luminal edge (lower left). Full-length MUC1 is cytoplasmic (lower right). C. Breast cancer tissue specimens. D. Lung cancer specimens. E. Colon cancer specimens. Arrows point to the most involved portions of the cancerous tissue where all cellular architecture has been lost and tissue is not stained by VU4H5.
Figure 3
Figure 3. MUC1* mediates growth factor receptor-like activity.
A. A clonogenic assay was performed on single cell clones of MUC1-negative 3Y1 cells that had been transfected with either an empty vector, full-length MUC1 or MUC1*1110. A photograph of Petri dishes containing cells plated at 1000 cells per 100 mm, grown for 9 days, then stained with crystal violet. Bar graph quantifies growth of each clone. Crystal violet absorbed by the cells on each plate was released and absorbance at 590 nm was measured on a spectrophotometer. Absorption measurements were normalized to the amount of crystal violet released from the empty vector clone. Nomenclature for our single cell clones is “parent cell name/construct/clone #”. B. Resistance to cell death induced by the chemotherapy drug AraC is tested in single cell clones of either empty vector, full-length MUC1 or MUC1* transfected into 3Y1 cells. C. Cell cycle analysis was performed on either empty vector, full-length MUC1 or MUC1* transfected into MUC1-negative cell line HCT116. The ratio of the percentage of cells in G2 to G1 phase is plotted as a function of serum concentration. The ratio of %G2∶%G1 for the empty vector transfectants has been normalized to 1. D. The growth of MUC1-positive breast cancer cells, ZR-75-30, is stimulated by the addition of bivalent (bv) Anti-MUC1* and inhibited by the addition of the monovalent (mv) Fab. The addition of bivalent antibody produces the bell-shaped growth curve that is characteristic of receptor dimerization. The growth of MUC1-negative HEK 293 cells was not impacted by either the bivalent or monovalent Anti-MUC1*. E. The stimulation of growth by the addition of bivalent Anti-MUC1* is tested using stably transfected siRNA to suppress MUC1 expression in breast cancer cell line T47D. Bivalent Anti-MUC1 stimulated growth in cells transfected with control siRNA, but did not significantly effect growth in the MUC1 suppressed cells. Western blot analysis (insert) shows that MUC1 siRNA suppressed MUC1 expression by about 90%. F. MUC1-positive breast cancer cells, T47Ds, are treated with either bivalent Anti-MUC1* or the monvalent Fab, then analyzed by Western for the presence of phosphorylated ERK1/2. Blots show the induction of ERK2 phosphorylation by bivalent Anti-MUC1* and inhibition of basal ERK1/2 phosphorylation after two days (2 d) treatment with the monovalent Fab.
Figure 4
Figure 4. NM23 is a MUC1* ligand that stimulates cell growth.
A. Lysate-supernatant mixtures from a panel of MUC1-positive breast cancer cells were tested for the presence of a ligand capable of dimerizing a MUC1*1110-ecd peptide (membrane proximal 45 amino acids of the extracellular domain: 1110–1155). Lysate-supernatant mixtures were incubated with nanoparticles bearing either a control peptide or a MUC1*1110-ecd peptide, histidine-tagged at the C-terminus. A nanoparticle solution color change from pink to a purple/blue indicates that a species in the mixture has simultaneously bound to 2 or more MUC1*1110-ecd peptides. B. Bivalent (bv) Anti-MUC1* (0.77 ug) is added to nanoparticles bearing either a control peptide (Row A) or MUC1*1110-ecd, peptides (Row B). Antibody binding to two peptides on separate nanoparticles causes nanoparticle aggregation and the solution color change from pink to purple/blue. In Row A, wells 3–5, this aggregation is inhibited by the addition monovalent Anti-MUC1* Fab (mv Fab). C. NM23 at 0.05, 0.1 or 0.2 uM, (Rows A, B and C respectively) is added to nanoparticles bearing MUC1*1110-ecd peptides (Columns 2–5). Added NM23 induces a color change, presumably as NM23 dimers bind to nanoparticle-immobilized MUC1*1110-ecd peptides. The addition of monovalent Anti-MUC1* (mv Fab), in Columns 3–5, inhibits the color change. D. Conditioned media from MUC1-positive breast cancer cells, T47Ds, and MUC1-negative rat fibroblasts, 3Y1s, was mixed with beads bearing the MUC1*1110-ecd peptide. Immunoprecipitated species were then analyzed by western, wherein the gel was blotted with an anti-NM23 antibody. The western blot shows that T47Ds secrete NM23 while 3Y1s do not. E. Surface plasmon resonance (SPR) was used to detect direct binding between NM23 (15 nM) and MUC1*1110-ecd peptides. The sensogram shows that 1044 RUs of NM23, injected at 15 nM, bound to a surface bearing MUC1*1110-ecd peptides (solid line) but no NM23 bound to a control surface bearing an irrelevant peptide (dashed line). F. Stimulation of T47D breast cancer cell growth by NM23 is dependent upon MUC1 expression. Exogenous NM23 added to T47D breast cancer cells stimulates growth and produces bell-shaped curve indicative of receptor dimerization. Growth is greatly reduced in cells stably expressing MUC1-specific siRNA, compared to cells expressing a control siRNA. Western blot quantifies the siRNA suppression of MUC1.
Figure 5
Figure 5. MUC1 has a self-aggregation domain (SAD) immediately N-terminal to MUC1*.
A. Degenerate peptides derived from the membrane-proximal extracellular domain of MUC1, as well as a portion of the tandem repeats, were separately attached to pools of nanoparticles. Aliquots of nanoparticles, each bearing a different peptide sequence, were mixed together to test for possible interaction between one peptide and another, or for self-interactions. Interactions among nanoparticle-immobilized peptides are seen as a color change from pink to purple/blue. The well in the upper left corner shows that the peptide MUC1SAD self-aggregates. No other peptides tested bound to each other. B. Wells 1–3 contain nanoparticles bearing histidine-tagged peptides of a single sequence. Well 1, containing MUC1*1110-ecd (N-1110-1154-C) does not self-aggregate as evidenced by the pink solution color. MUC1*SAD, (N-1084-1109-C), which is immediately adjacent to MUC1*1110-ecd, clearly self-aggregates as the solution color turned a deep purple/blue (Well 3). A third peptide MUC1ecd-EXT (N-1101-1148-C) comprised of 39 amino acids of MUC1*1110-ecd and extending nine additional amino acids into the self-aggregation domain (SAD), albeit to a lesser degree (Well 3).
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