MUC5B, a key secredted gel-forming mucin : embryogenesis, breast cancer and cystic fibrosis

Le mucus recouvre et protège les épithéliums des tractus respiratoire, gastro-intestinal et reproducteur. Les mucines sécrétées sont des molécules mosaïques de très grande taille moléculaire, fortement O-glycosylées et responsables des propriétés rhéologiques du gel de mucus. MUC5B est l’une des cinq mucines gélifiantes et est principalement sécrétée dans le tractus respiratoire. Chez l’Homme, MUC5B est impliquée dans le cancer mammaire et la mucoviscidose. MUC5B est exprimée dans plus de 80% des cancers mammaires alors que MUC5B n’est pas exprimée dans le tissu mammaire sain. Par ailleurs, MUC5B pourrait jouer un rôle majeur dans le poumon de patients atteints de mucoviscidose. Pour étudier la fonction de MUC5B, nous avons utilisé des protéines recombinantes composées de différents domaines de MUC5B et des modèles animaux. Nous avons montré que MUC5B favorise la prolifération cellulaire et l’invasion in vitro et favorise la croissance tumorale et la dissémination métastatique chez des souris immunodéprimées. MUC5B apparait donc comme une cible thérapeutique intéressante pour ralentir la prolifération cellulaire et la dissémination métastatique dans le cancer du sein. Nous avons également montré que l’expression de Muc5b et le nombre de cellules Muc5b positives sont plus élevés dans un modèle murin de mucoviscidose (Cftr–/–) que chez les souris frères-sœurs contrôles, ce qui suggère que les souris Cftr–/– sont prédisposées à former des bouchons bronchiques. Les souris Cftr–/– infectées expérimentalement par Pseudomonas aeruginosa développent des bouchons bronchiques AB-PAS positifs principalement composés de la mucine Muc5b et d’ADN. MUC5B semble être une cible thérapeutique intéressante pour diminuer la viscosité du mucus dans la mucoviscidose. Enfin, nous avons montré que la protéine Muc5b est exprimée très précocement (dès E8.5) au cours du développement embryonnaire murin. A E11.5, Muc5b est exprimée dans le bourgeon pulmonaire, dans le bourgeon formant la trachée et l’œsophage et dans l’estomac. De manière inattendue, Muc5b est exprimée au niveau baso-latéral des cellules épithéliales suggérant un rôle clé de MUC5B dans la morphogenèse.Respiratory, gastrointestinal and reproductive tracts are protected by a mucus layer. Secreted mucins are large, high molecular weight and heavily O-glycosylated mosaic proteins that are responsible for the rheological properties of mucus gel. MUC5B is one of the five secreted gel-forming mucins and is mainly secreted in the respiratory tract. In human pathology, MUC5B has been implicated in breast cancer and cystic fibrosis (CF). This ‘non mammary’ mucin is expressed in more than 80% of breast cancer and it has been suggested that MUC5B may represent the main mucin to be implicated in the lung disease of CF.To investigate the function of MUC5B, we used recombinant proteins of several MUC5B domains and animal models. We show that MUC5B promotes cell proliferation and invasion in vitro and tumor growth and metastasis using SCID mice suggesting that MUC5B may represent a therapeutic target to slow down the tumor growth and dissemination in breast cancer. We show also that Cftr–/– mice harbor more Muc5b positive Clara cells than do control littermates suggesting that CF mice are predisposed to develop mucus plugs. Experimental infection with Pseudomonas aeruginosa increased the number of Muc5b-positive cells and the formation of mucus plugs in bronchi or bronchioles of Cftr–/– mice. These plugs are mainly composed of Muc5b and DNA suggesting that MUC5B may be a valuable target for decreasing mucus viscosity in CF. Finally, Muc5b protein was detected very early in mouse embryogenesis (at day 8.5). At E11.5, Muc5b is expressed in pulmonary bud, in tracheo-esophagal groove and in stomach. Unexpectedly, Muc5b is expressed at the basal and lateral membrane of epithelial cells of the buds suggesting a key role in epithelial morphogenesis

HYPOTHESE D'UN AGENT INFECTIEUX DANS LA MALADIE DE CROHN

LILLE2-BU Santé-Recherche (593502101) / SudocSudocFranceF

MUC5B leads to aggressive behavior of breast cancer MCF7 cells.

The mucin MUC5B has a critical protective function in the normal lung, salivary glands, esophagus, and gallbladder, and has been reported to be aberrantly expressed in breast cancer, the second leading cause of cancer-related deaths among women worldwide. To understand better the implication of MUC5B in cancer pathogenesis, the luminal human breast cancer cell line MCF7 was transfected with a vector encoding a recombinant mini-mucin MUC5B and was then infected with a virus to deliver a short hairpin RNA to knock down the mini-mucin. The proliferative and invasive properties in Matrigel of MCF7 subclones and subpopulations were evaluated in vitro. A xenograft model was established by subcutaneous inoculation of MCF7 clones and subpopulations in SCID mice. Tumor growth was measured, and the tumors and metastases were assessed by histological and immunological analysis. The mini-mucin MUC5B promoted MCF7 cell proliferation and invasion in vitro. The xenograft experiments demonstrated that the mini-mucin promoted tumor growth and MCF7 cell dissemination. In conclusion, MUC5B expression is associated with aggressive behavior of MCF7 breast cancer cells. This study suggests that MUC5B may represent a good target for slowing tumor growth and metastasis

Non-C-mannosylable mucin CYS domains hindered proper folding and secretion of mucin

International audienceThe CYS domain occurs in multiple copies in many gel-forming mucins. It is believed that CYS domains can interact with each other in a reversible manner, suggesting a key role of the domain in gel formation. This domain always contains in its amino-terminal sequence the C-mannosylation motif WXXW, but whether the CYS domain is C-mannosylated is debated, and the putative role of C-mannosylation of the domain is unclear. We prepared recombinant CYS domains of the human mucin MUC5B with (WXXW→AXXW) and without a single amino acid mutation and mini-5B mucins made of a large Ser/Thr/Pro region flanked by two CYS domains with the WXXW motif or with the mutated AXXW motif on the first, second or both CYS domains. We found that the single CYS domain and the two CYS domains of mini-5B mucin must be C-mannosylable for the efficient maturation and secretion of the recombinant molecules; otherwise, they are retained in the cell and co-localized with a resident enzyme of the endoplasmic reticulum

Metastases following subcutaneous xenograft.

a<p>mice were injected with 10×10⁶ cells.</p>b<p>the presence of macrometastases was evaluated by histological analysis.</p>*<p><i>P</i> = 0.02. Mice injected with MCF7 clones/population expressing Mini5B were grouped and mice injected with clones/cell population not expressing Mini5B were grouped.</p

Abnormal expression of Muc5b in Cftr-null mice and in mammary tumors of MMTV-ras mice.

International audienceGel-forming mucins are large, high molecular weight, and heavily O-glycosylated proteins that are responsible for the rheological properties of mucus gel. Among them, the mucin MUC5B has been implicated in breast cancer and cystic fibrosis. We obtained a new polyclonal serum, named CP1, which was isolated from a rabbit immunized with a mouse Muc5b peptide. The immunoprofile of Muc5b was determined on paraffin-embedded and frozen mouse tissue sections and showed a similar expression pattern in mouse to that in the human. The "nonmammary" mucin Muc5b was detected in all mammary tumors analyzed from MMTV-ras mice, suggesting that the CP1 antibody is a valuable tool for investigating the involvement of this mucin in mammary cancer. We also found that uninfected Cftr( -/- ) mice harbored more Clara cells, which were Muc5b-positive, than did their wild-type control littermates. The number of Muc5b-positive cells increased in Cftr( -/- ) mice infected experimentally with Pseudomonas aeruginosa, and the mice developed mucus plugs in their bronchi and bronchioles with a high frequency of Muc5b content (87%, Cohen's kappa = 0.82; p < 0.0001). These findings suggest that mice genetically deficient in the Cftr gene are predisposed to develop mucus plugs and that MUC5B may provide a valuable target for decreasing mucus viscosity in cystic fibrosis

Impact of Mini5B expression on the dissemination of MCF7 cell xenografts in immunodeficient mice.

<p>(<b>A</b>) Immunofluorescence analysis was performed on paraffin-embedded sections of tumors from mice injected with the Ires-Luc clone, the Mini5B-Luc CYS shRNA clone, the Mini5B-Luc clone and the Mini5B-Luc empty shRNA clone and stained with anti-MUC5B antibody. Mini5B was expressed and secreted by tumoral cells in tumors of Mini5B-Luc group and Mini5B-Luc empty shRNA. (<b>B</b>) Serial sections of livers of mice injected with the Ires–Luc clone and stained with HE and anti-PCNA antibody, and serial sections of lungs of mice injected with the Mini5B–Luc clone and stained with HE and anti-PCNA antibody. Metastases in the liver and the lung were visualized. Metastatic cells were PCNA positive. (<b>C</b>) Histological analysis of a macrometastasis in lung of a mouse injected with the Mini5B–Luc clone using HE staining and E-cadherin immunostaining (serial sections). Mt; metastasis. Nuclei were counterstained with Hoechst 33258. Scale bar 50 µm.</p

Histological analysis of mice sacrificed during the experimentation.

<p>(<b>A</b>) HE staining was performed on paraffin-embedded sections of the liver, lungs, and lymph nodes of mice injected with Mini5B–Luc clones. Metastasis is showed in the magnified image. (<b>B</b>) Immunofluorescence analysis was performed on paraffin-embedded sections of liver from mice injected with Mini5B–Luc clones and stained with anti-PCNA antibody. Metastatic cells were PCNA positive. (<b>C</b>) Double immunofluorescence analysis using the anti-human E-cadherin and anti-MUC5B antibodies were performed on paraffin-embedded sections of thoracic lymph nodes from mice injected with Mini5B–Luc clones. Metastatic cells expressed both the epithelial marker E-cadherin and secreted MUC5B protein. Nuclei were counterstained with Hoechst 33258 or propidium iodide. Mt, metastasis. Scale bar 50 µm.</p

Clinical scores of xenografted SCID mice.

<p>The empty box and the box with the diagonal lines are for the Ires–Luc clone and the Mini5B KD groups, respectively. The black box and the box with vertical lines are for the Mini5B–Ires empty shRNA and the Mini5B–Ires–Luc groups, respectively.</p