A Minimal Fragment of MUC1 Mediates Growth of Cancer Cells

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

MUC1* ligand, NM23-H1, is a novel growth factor that maintains human stem cells in a more naïve state.

We report that a single growth factor, NM23-H1, enables serial passaging of both human ES and iPS cells in the absence of feeder cells, their conditioned media or bFGF in a fully defined xeno-free media on a novel defined, xeno-free surface. Stem cells cultured in this system show a gene expression pattern indicative of a more "naïve" state than stem cells grown in bFGF-based media. NM23-H1 and MUC1* growth factor receptor cooperate to control stem cell self-replication. By manipulating the multimerization state of NM23-H1, we override the stem cell's inherent programming that turns off pluripotency and trick the cells into continuously replicating as pluripotent stem cells. Dimeric NM23-H1 binds to and dimerizes the extra cellular domain of the MUC1* transmembrane receptor which stimulates growth and promotes pluripotency. Inhibition of the NM23-H1/MUC1* interaction accelerates differentiation and causes a spike in miR-145 expression which signals a cell's exit from pluripotency

Human stem cells cultured in NM23-H1-MM over anti-MUC1* antibody surfaces express higher levels of naïve markers and lower levels of primed markers.

<p>RT-PCR was used to quantify expression of a subset of naïve markers that included Oct4, Nanog, Klf4 and Klf2, which should be high in the naïve state, and a subset of primed markers that included FoxA2, XIST, Otx2 and Lhx2, which are high in the primed state but low in the naïve state. Measurements were normalized to housekeeping gene GAPDH and expressed as fold change to H9 ES cells cultured in 4ng/ml bFGF over MEFs (control, n = 3). a) H9 ES cells cultured in NM23-H1-MM on anti-MUC1* antibody (MN-C3) surfaces, on average, showed increased expression of naïve markers and decreased expression of primed markers (n = 6). Conversely, H9 cells cultured in mTeSR over Matrigel showed decreased expression of naïve markers and increased expression of primed markers (n = 4). b) Individual measurements of the subset of naïve or primed markers are plotted as a function of passage number for NM23-H1-MM over anti-MUC1* antibody surfaces and c) for mTeSR over Matrigel. The trend toward the naïve state increased with successive passage in NM23-H1-MM but not with mTeSR. Large standard error for some experiments may be due to contamination of visually pluripotent stem cells with newly differentiating stem cells. For statistical analysis see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058601#pone.0058601.s010" target="_blank">Fig. S10</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058601#pone.0058601.s011" target="_blank">S11</a>.</p

ES and iPS cells cultured long-term in NM23-H1-MM on Anti-MUC1* surfaces express pluripotency markers and differentiate down all three germlines.

<p>a, b) H9 ES cells and iPS cells serially passaged on a monoclonal anti-MUC1* antibody surface in NM23-H1-MM stained positive for typical pluripotency markers. c, h) After more than 20 passages (H9, P20; iPS, P28), cells were allowed to differentiate by embryoid body method (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058601#pone.0058601.s013" target="_blank">Methods S1</a>). Staining with nuclear marker DAPI and antibodies against markers of the three germlines, endoderm - alpha feto protein (c, f), ectoderm–beta-III-tubulin (d, g), and mesoderm - smooth muscle actin (e, h) shows that the cells differentiate normally down all three germlines. (All images 4X). i–q) ES cells serially passaged (p10) on a monoclonal anti-MUC1* antibody surface in NM23-H1-MM were injected in the kidney capsule and in the testis of mice for teratoma formation analysis (Applied Stem Cell, Menlo Park, CA). Tumors were fixed, embedded in paraffin, cut into sections and stained (Hematoxylin and eosin) to detect embryonic germ cell layers (endoderm, mesoderm and ectoderm). Typical structures from each germ layer were detected. All images x200.</p