TGFß1-Endo180-dependent collagen deposition is dysregulated at the tumour-stromal interface in bone metastasis.

Cellular niches in adult tissue can harbour dysregulated microenvironments that become the driving force behind disease progression. The major environmental change when metastatic cells arrive in the bone is the destruction of mineralized type I collagen matrix. Once metastatic niches establish in bone, the invading tumour cells initiate a vicious cycle of osteolytic lesion formation via the dysregulation of paracrine signals and uncoupling of normal bone resorption and production. Here we report that the collagen receptor Endo180 (CD280, MRC2, uPARAP) participates in collagen deposition by primary human osteoblasts during de novo osteoid formation. This newly recognized function of Endo180 was suppressed in osteoblasts following heterotypic direct cell-cell contact in co-culture with prostate tumour cells. Reciprocal Endo180 up-regulation in osteolytic prostate tumour cells (PC3 and DU145) followed their direct contact with osteoblasts and promoted de novo collagen internalization, which is a previously characterized function of the constitutively recycling Endo180 receptor. The osteoblastic suppression and tumour cell-associated enhancement of Endo180 expression were equally sustained in these direct co-cultures. These findings are the first to demonstrate that increased tumour cell participation in collagen degradation and decreased collagen formation by osteoblasts in the osteolytic microenvironment are linked to the divergent regulation of a collagen-binding receptor. Immunohistochemical analysis of core biopsies from bone metastasis revealed higher levels of Endo180 expression in tumour cell foci than cells in the surrounding stroma. Additional experiments in prostate cell-osteoblast co-cultures indicate that divergent regulation of Endo180 is the result of dysregulated TGFß1 signalling. The findings of this study provide a rationale for targeting collagen remodelling by Endo180 in bone metastases and other collagen matrix pathologies.

Polyomavirus middle T-antigen is a transmembrane protein that binds signaling proteins in discrete subcellular membrane sites.

Murine polyomavirus middle T-antigen (MT) induces tumors by mimicking an activated growth factor receptor. An essential component of this action is a 22-amino-acid hydrophobic region close to the C terminus which locates MT to cell membranes. Here, we demonstrate that this sequence is a transmembrane domain (TMD) by showing that a hemagglutinin (HA) tag added to the MT C terminus is exposed on the outside of the cells, with the N terminus inside. To determine whether this MT TMD is inserted into the endoplasmic reticulum (ER) membrane, we added the ER retention signal KDEL to the MT C terminus (MTKDEL). This mutant protein locates only in the ER, demonstrating that MT does insert into membranes solely at this location. In addition, this ER-located MT failed to transform. Examination of the binding proteins associated with the MTKDEL protein demonstrated that it associates with PP2A and c-Src but fails to interact with ShcA, phosphatidylinositol 3-kinase (PI3K), and phospholipase C-γ1 (PLC-γ1), despite being tyrosine phosphorylated. Additional mutant and antibody studies show that MT binding to PP2A is probably required for MT to efficiently exit the ER and migrate to the plasma membrane though the TMD also plays a role in this relocation. Overall, these data, together with previous publications, illustrate that MT associates with signaling proteins at different sites in its maturation pathway. MT binds to PP2A in the cytoplasm, to c-Src at the endoplasmic reticulum, and to ShcA, PI3K, and PLC-γ1 at subsequent locations en route to the plasma membrane.