Efficient TGF-ß/SMAD signaling in human melanoma cells associated with high c-SKI/SnoN expression.

BACKGROUND: SKI and SnoN proteins have been shown to inhibit TGF-ß signaling, acting both as transcriptional co-repressors in the cell nucleus, and as sequestrators of SMAD proteins in the cytoplasm. TGF-ß, on the other hand, induces rapid, proteasome-mediated, degradation of both proteins. How elevated SKI and SnoN protein levels co-exist with active autocrine TGF-ß signaling in cancer cells is yet to be understood. RESULTS: In this study, we found elevated SKI and SnoN protein levels in a panel of melanoma cell lines, as compared to normal melanocytes. There was no correlation between SKI protein content and the capacity of melanoma cells to invade Matrigel™, to form subcutaneous tumors, or to metastasize to bone after intracardiac inoculation into nude mice. Nor did we find a correlation between SKI expression and histopathological staging of human melanoma. TGF-ß induced a rapid and dose-dependent degradation of SKI protein, associated with SMAD3/4 specific transcriptional response and induction of pro-metastatic target genes, partially prevented by pharmacologic blockade of proteasome activity. SKI knockdown in 1205Lu melanoma cells did not alter their invasive capacity or transcriptional responses to TGF-ß, and did not allow p21 expression in response to TGF-ß or reveal any growth inhibitory activity of TGF-ß. CONCLUSIONS: Despite high expression in melanoma cells, the role of SKI in melanoma remains elusive: SKI does not efficiently interfere with the pro-oncogenic activities of TGF-ß, unless stabilized by proteasome blockade. Its highly labile nature makes it an unlikely target for therapeutic intervention.

SnoN Antagonizes the Hippo Kinase Complex to Promote TAZ Signaling during Breast Carcinogenesis.

SnoN regulates multiple signaling pathways, including TGF-ß/Smad and p53, and displays both pro-oncogenic and anti-oncogenic activities in human cancer. We have observed previously that both its intracellular localization and expression levels are sensitive to cell density, suggesting that it may crosstalk with Hippo signaling. Here we report that, indeed, SnoN interacts with multiple components of the Hippo pathway to inhibit the binding of Lats2 to TAZ and the subsequent phosphorylation of TAZ, leading to TAZ stabilization. Consistently, SnoN enhances the transcriptional and oncogenic activities of TAZ, and reducing SnoN decreases TAZ expression as well as malignant progression of breast cancer cells. Interestingly, SnoN itself is downregulated by Lats2 that is activated by the Scribble basolateral polarity protein. Thus, SnoN is a critical component of the Hippo regulatory network that receives signals from the tissue architecture and polarity to coordinate the activity of intracellular signaling pathways.

Ski regulates Hippo and TAZ signaling to suppress breast cancer progression.

Ski, the transforming protein of the avian Sloan-Kettering retrovirus, inhibits transforming growth factor-ß (TGF-ß)/Smad signaling and displays both pro-oncogenic and anti-oncogenic activities in human cancer. Inhibition of TGF-ß signaling is likely responsible for the pro-oncogenic activity of Ski. We investigated the mechanism(s) underlying the tumor suppressor activity of Ski and found that Ski suppressed the activity of the Hippo signaling effectors TAZ and YAP to inhibit breast cancer progression. TAZ and YAP are transcriptional coactivators that can contribute to cancer by promoting proliferation, tumorigenesis, and cancer stem cell expansion. Hippo signaling activates the the Lats family of kinases, which phosphorylate TAZ and YAP, resulting in cytoplasmic retention and degradation and inhibition of their transcriptional activity. We showed that Ski interacted with multiple components of the Hippo pathway to facilitate activation of Lats2, resulting in increased phosphorylation and subsequent degradation of TAZ. Ski also promoted the degradation of a constitutively active TAZ mutant that is not phosphorylated by Lats, suggesting the existence of a Lats2-independent degradation pathway. Finally, we showed that Ski repressed the transcriptional activity of TAZ by binding to the TAZ partner TEAD and recruiting the transcriptional co-repressor NCoR1 to the TEAD-TAZ complex. Ski effectively reversed transformation and epithelial-to-mesenchyme transition in cultured breast cancer cells and metastasis in TAZ-expressing xenografted tumors. Thus, Ski inhibited the function of TAZ through multiple mechanisms in human cancer cells.

Metabolic profiling reveals PAFAH1B3 as a critical driver of breast cancer pathogenicity.

Many studies have identified metabolic pathways that underlie cellular transformation, but the metabolic drivers of cancer progression remain less well understood. The Hippo transducer pathway has been shown to confer malignant traits on breast cancer cells. In this study, we used metabolic mapping platforms to identify biochemical drivers of cellular transformation and malignant progression driven through RAS and the Hippo pathway in breast cancer and identified platelet-activating factor acetylhydrolase 1B3 (PAFAH1B3) as a key metabolic driver of breast cancer pathogenicity that is upregulated in primary human breast tumors and correlated with poor prognosis. Metabolomic profiling suggests that PAFAH1B3 inactivation attenuates cancer pathogenicity through enhancing tumor-suppressing signaling lipids. Our studies provide a map of altered metabolism that underlies breast cancer progression and put forth PAFAH1B3 as a critical metabolic node in breast cancer.

Transforming growth factor-beta suppresses the ability of Ski to inhibit tumor metastasis by inducing its degradation.

c-Ski is an important corepressor of transforming growth factor-beta (TGF-beta) signaling through its ability to bind to and repress the activity of the Smad proteins. It was initially identified as an oncogene that promotes anchorage-independent growth of chicken and quail embryo fibroblasts when overexpressed. Although increased Ski expression is detected in many human cancer cells, the roles of Ski in mammalian carcinogenesis have yet to be defined. Here, we report that reducing Ski expression in breast and lung cancer cells does not affect tumor growth but enhances tumor metastasis in vivo. Thus, in these cells, Ski plays an antitumorigenic role. We also showed that TGF-beta, a cytokine that is often highly expressed in metastatic tumors, induces Ski degradation through the ubiquitin-dependent proteasome in malignant human cancer cells. On TGF-beta treatment, the E3 ubiquitin ligase Arkadia mediates degradation of Ski in a Smad-dependent manner. Although Arkadia interacts with Ski in the absence of TGF-beta, binding of phosphorylated Smad2 or Smad3 to Ski is required to induce efficient degradation of Ski by Arkadia. Our results suggest that the ability of TGF-beta to induce degradation of Ski could be an additional mechanism contributing to its protumorigenic activity.

Overexpression of sphingosine kinase 1 is an oncogenic event in erythroleukemic progression.

The erythroleukemia developed by spi-1/PU.1-transgenic mice is a model of multistage oncogenic process. Isolation of tumor cells representing discrete stages of leukemic progression enables the dissection of some of the critical events required for malignant transformation. To elucidate the molecular mechanisms of multistage leukemogenesis, we developed a microarray transcriptome analysis of nontumorigenic (HS1) and tumorigenic (HS2) proerythroblasts from spi-1-transgenic mice. The data show that transcriptional up-regulation of the sphingosine kinase gene (SPHK1) is a recurrent event associated with the tumorigenic phenotype of these transgenic proerythroblasts. SPHK1 is an enzyme of the metabolism of sphingolipids, which are essential in several biologic processes, including cell proliferation and apoptosis. HS1 erythroleukemic cells engineered to overexpress the SPHK1 protein exhibited growth proliferative advantage, increased clonogenicity, and resistance to apoptosis in reduced serum level by a mechanism involving activation of the extracellular signal-related kinases 1/2 (ERK1/2) and phosphatidylinositol 3-kinase (PI3K)/AKT pathways. In addition, SPHK1-overexpressing HS1 cells acquired tumorigenicity when engrafted in vivo. Finally, enforced expression of a dominant-negative mutant of SPHK1 in HS2 tumorigenic cells or treatment with a pharmacologic inhibitor reduced both cell growth and apoptosis resistance. Altogether, these data suggest that overexpression of the sphingosine kinase may represent an oncogenic event during the multistep progression of an erythroleukemia.

Sorting nexin 4 and amphiphysin 2, a new partnership between endocytosis and intracellular trafficking.

Endocytosis is a regulated physiological process by which membrane receptors and their extracellular ligands are internalized. After internalization, they enter the endosomal trafficking pathway for sorting and processing. Amphiphysins consist of a family of proteins conserved throughout evolution that are crucial elements of the endocytosis machinery in mammalian cells. They act as adaptors for a series of proteins important for the endocytic process, such as dynamin. In order to improve our knowledge of amphiphysin function, we performed a two-hybrid screen with the N-terminal part of murine amphiphysin 2 (residues 1-304). One of the interacting clones corresponded to sorting nexin 4 (SNX4), a member of the SNX family of proteins which are suspected to regulate vesicular trafficking. This interaction was confirmed in vivo by co-immunoprecipitation. Immunofluorescence analysis revealed that amphiphysin 2 might bind reticulo-vesicular structures present throughout the cell body and be associated with SNX4 on these structures. In an endocytosis assay, overexpressed C-terminal or full-length SNX4 was able to inhibit transferrin receptor endocytosis as efficiently as the SH3 domain of amphiphysin 2. At lower levels of expression, SNX4 colocalized with transferrin-containing vesicles, some of which were also positive for amphiphysin 2. These results indicate that SNX4 may be part of the endocytic machinery or, alternatively, that SNX4 may associate with key elements of endocytosis such as amphiphysin 2 and sequester them when overexpressed. The presence of amphiphysin 2 on intracellular vesicles and its interplay with SNX4, which is likely to take part in intracellular trafficking, suggest that amphiphysin 2 is not only a regulator of the early steps of endocytosis. It could also play a role at the surface of the endocytic vesicle that has just been formed and of the future endosomes, in order to regulate intracellular trafficking.