Atypical protein kinase C activity is required for extracellular matrix degradation and invasion by Src-transformed cells.

Atypical protein kinase C (aPKC) isoforms have been shown to mediate Src-dependent signaling in response to growth factor stimulation. To determine if aPKC activity contributes to the transformed phenotype of cells expressing oncogenic Src, we have examined the activity and function of aPKCs in 3T3 cells expressing viral Src (v-Src). aPKC activity and tyrosine phosphorylation were found to be elevated in some but not all clones of mouse fibroblasts expressing v-Src. aPKC activity was inhibited either by addition of a membrane-permeable pseudosubstrate, by expression of a dominant-negative aPKC, or by RNAi-mediated knockdown of specific aPKC isoforms. aPKC activity contributes to morphological transformation and stress fiber disruption, and is required for migration of Src-transformed cells and for their ability to polarize at the edge of a monolayer. The lambda isoform of aPKC is specifically required for invasion through extracellular matrix in Boyden chamber assays and for degradation of the extracellular matrix in in situ zymography assays. Tyrosine phosphorylation of aPKClambda is required for its ability to promote cell invasion. The defect in invasion upon aPKC inhibition appears to result from a defect in the assembly and/or function of podosomes, invasive adhesions on the ventral surface of the cell that are sites of protease secretion. aPKC was also found to localize to podosomes of v-Src transformed cells, suggesting a direct role for aPKC in podosome assembly and/or function. We conclude that basal or elevated aPKC activity is required for the ability of Src-transformed cells to degrade and invade the extracellular matrix.

Dual role of SnoN in mammalian tumorigenesis.

SnoN is an important negative regulator of transforming growth factor beta signaling through its ability to interact with and repress the activity of Smad proteins. It was originally identified as an oncoprotein based on its ability to induce anchorage-independent growth in chicken embryo fibroblasts. However, the roles of SnoN in mammalian epithelial carcinogenesis have not been well defined. Here we show for the first time that SnoN plays an important but complex role in human cancer. SnoN expression is highly elevated in many human cancer cell lines, and this high level of SnoN promotes mitogenic transformation of breast and lung cancer cell lines in vitro and tumor growth in vivo, consistent with its proposed pro-oncogenic role. However, this high level of SnoN expression also inhibits epithelial-to-mesenchymal transdifferentiation. Breast and lung cancer cells expressing the shRNA for SnoN exhibited an increase in cell motility, actin stress fiber formation, metalloprotease activity, and extracellular matrix production as well as a reduction in adherens junction proteins. Supporting this observation, in an in vivo breast cancer metastasis model, reducing SnoN expression was found to moderately enhance metastasis of human breast cancer cells to bone and lung. Thus, SnoN plays both pro-tumorigenic and antitumorigenic roles at different stages of mammalian malignant progression. The growth-promoting activity of SnoN appears to require its ability to bind to and repress the Smad proteins, while the antitumorigenic activity can be mediated by both Smad-dependent and Smad-independent pathways and requires the activity of small GTPase RhoA. Our study has established the importance of SnoN in mammalian epithelial carcinogenesis and revealed a novel aspect of SnoN function in malignant progression.

The aryl hydrocarbon receptor signaling pathway is modified through interactions with a Kelch protein.

The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor with important roles in metabolic adaptation, dioxin toxicology, and vascular development. To understand the details of this signal transduction pathway, we have used the yeast two-hybrid system to identify proteins that physically interact with the AHR in a ligand-dependent manner. Using this strategy, we identified a novel modifier of the AHR signaling pathway that we named Ah-receptor associated protein 3 (ARA3). Coexpression of ARA3 with an AHR chimera in yeast and mammalian cells enhances signaling in response to agonists. The human full-length cDNA previously was described as influenza virus nonstructural protein-1 binding protein (NS1BP). This protein contains four apparent domains-a "broad complex/tramtrack/bric-a-brac" (BTB) domain, a "kelch" domain, a "BTB and C-terminal kelch" (BACK) domain, and an intervening region (IVR). The carboxyl terminus of the AHR "Per-ARNT-Sim" (periodicity/AHR nuclear translocator/simple-minded) domain and the BACK/IVR domains of ARA3 mediate the AHR-ARA3 interaction. The BACK/IVR domains of ARA3 also are sufficient to modify AHR signaling in yeast and mammalian cells. In an effort to provide a preliminary model of NS1BP activity in AHR signaling, we demonstrate that NS1BP regulates the concentration of functional AHR in mammalian cells.