By participating in the TGF-beta/Smad signaling pathway, LRP12 regulates the tumorigenicity of human fibrosarcoma-derived cell line SHAC.

It is generally accepted that tumorigenesis is a multi-step process that involves a series of genetic and/or epigenetic changes. In an effort to identify the nature of these changes, McCormick and Maher established a cell lineage in which normal human fibroblasts were transformed into malignant fibroblasts by acquiring a series of genetic changes. Using this cell lineage, differential display of mRNA from an infinite life span, nontumorigenic human fibroblast cell strain and one of its carcinogen-transformed, malignant derivatives led to the identification of a novel gene designated LRP12. LRP12 is expressed in normal fibroblasts, but is absent or only expressed at very low levels in human fibrosarcoma cells, suggesting it is a tumor suppressor gene. What is more, expression of LRP12 in human fibrosarcoma-derived cell line SHAC inhibits tumor formation in athymic mice. Structurally, LRP12 belongs to the low-density lipoprotein receptor (LDLR) family. Although members of this family are best known as endocytic receptors, recent studies demonstrate that the cytoplasmic tails of several LDLR family members interact with adaptor and scaffold proteins that are implicated in signal transduction. A yeast two-hybrid assay in this laboratory suggested that the cytoplasmic tail of LRP12 interacts with several signaling proteins, including a truncated form of the SMAD Anchor for Receptor Activation (SARA). The fact that SARA is a protein involved in TGF-beta signaling suggests that LRP12 participates in the TGF-beta signaling pathway. Using Western blotting analysis and luciferase assays, I determined that LRP12 expression increases TGF-beta-induced phosphorylation of Smad2 and Smad3. This function of LRP12 depends on the presence of its cytoplasmic tail, which I demonstrated to interact with the full-length SARA and the Smad7-Smurf2 complex. Using Wesern blotting analysis, I showed that expression of LRP12 significantly decreases the ubiquitination of TGF-beta receptor type I (TbetaRI) and activated Smad2. In contrast, LRP12 lacking the majority of the cytoplasmic tail cannot associate with SARA and Smurf2, nor does it increase TGF-beta-induced phosphorylation of Smad2. Using an in vitro cell migration assay and invasion assay, I further demonstrated that expression of LRP12 inhibits the ability of tumor cells to migrate and invade by enhancing the TGF-beta1 induction of PAI-1 expression. These data support the hypothesis that LRP12 is a tumor suppressor. Furthermore, they indicate that LRP12 regulates tumorigenicity by participating in the TGF-beta signaling pathway and by enhancing the TGF-beta1-induced PAI-1 expression.