ALK1 regulates the internalization of endoglin and the type III TGF-ß receptor.

Complex formation and endocytosis of transforming growth factor-ß (TGF-ß) receptors play important roles in signaling. However, their interdependence remained unexplored. Here, we demonstrate that ALK1, a TGF-ß type I receptor prevalent in endothelial cells, forms stable complexes at the cell surface with endoglin and with type III TGF-ß receptors (TßRIII). We show that ALK1 undergoes clathrin-mediated endocytosis (CME) faster than ALK5, type II TGF-ß receptor (TßRII), endoglin, or TßRIII. These complexes regulate the endocytosis of the TGF-ß receptors, with a major effect mediated by ALK1. Thus, ALK1 enhances the endocytosis of TßRIII and endoglin, while ALK5 and TßRII mildly enhance endoglin, but not TßRIII, internalization. Conversely, the slowly endocytosed endoglin has no effect on the endocytosis of either ALK1, ALK5, or TßRII, while TßRIII has a differential effect, slowing the internalization of ALK5 and TßRII, but not ALK1. Such effects may be relevant to signaling, as BMP9-mediated Smad1/5/8 phosphorylation is inhibited by CME blockade in endothelial cells. We propose a model that links TGF-ß receptor oligomerization and endocytosis, based on which endocytosis signals are exposed/functional in specific receptor complexes. This has broad implications for signaling, implying that complex formation among various receptors regulates their surface levels and signaling intensities.

Increased type III TGF-ß receptor shedding decreases tumorigenesis through induction of epithelial-to-mesenchymal transition.

The type III TGF-ß receptor (TßRIII) is a TGF-ß co-receptor that presents ligand to the type II TGF-ß receptor to initiate signaling. TßRIII also undergoes ectodomain shedding to release a soluble form (sTßRIII) that can bind ligand, sequestering it away from cell surface receptors. We have previously identified a TßRIII extracellular mutant that has enhanced ectodomain shedding ("super shedding (SS)"-TßRIII-SS). Here, we utilize TßRIII-SS to study the balance of cell surface and soluble TßRIII in the context of lung cancer. We demonstrate that expressing TßRIII-SS in lung cancer cell models induces epithelial-to-mesenchymal transition (EMT) and that these TßRIII-SS (EMT) cells are less migratory, invasive and adhesive and more resistant to gemcitabine. Moreover, TßRIII-SS (EMT) cells exhibit decreased tumorigenicity but increased growth rate in vitro and in vivo. These studies suggest that the balance of cell surface and soluble TßRIII may regulate a dichotomous role for TßRIII during cancer progression.

Mitochondrial dysregulation and glycolytic insufficiency functionally impair CD8 T cells infiltrating human renal cell carcinoma.

Cancer cells can inhibit effector T cells (Teff) through both immunomodulatory receptors and the impact of cancer metabolism on the tumor microenvironment. Indeed, Teff require high rates of glucose metabolism, and consumption of essential nutrients or generation of waste products by tumor cells may impede essential T cell metabolic pathways. Clear cell renal cell carcinoma (ccRCC) is characterized by loss of the tumor suppressor von Hippel-Lindau (VHL) and altered cancer cell metabolism. Here, we assessed how ccRCC influences the metabolism and activation of primary patient ccRCC tumor infiltrating lymphocytes (TIL). CD8 TIL were abundant in ccRCC, but they were phenotypically distinct and both functionally and metabolically impaired. ccRCC CD8 TIL were unable to efficiently uptake glucose or perform glycolysis and had small, fragmented mitochondria that were hyperpolarized and generated large amounts of ROS. Elevated ROS was associated with downregulated mitochondrial SOD2. CD8 T cells with hyperpolarized mitochondria were also visible in the blood of ccRCC patients. Importantly, provision of pyruvate to bypass glycolytic defects or scavengers to neutralize mitochondrial ROS could partially restore TIL activation. Thus, strategies to improve metabolic function of ccRCC CD8 TIL may promote the immune response to ccRCC.

Von Hippel-Lindau status influences phenotype of liver cancers arising from PTEN loss.

BACKGROUND: PTEN loss contributes to the development of liver diseases including hepatic steatosis and both hepatocellular carcinoma (HCC) and cholangiocarcinoma (CC). The factors that influence the penetrance of these conditions are unclear. We explored the influence of sustained hypoxia signaling through co-deletion of Pten and Vhl in a murine model. METHODS: We used a CreER-linked Keratin 18 mouse model to conditionally delete Pten, Vhl or both in somatic cells of adult mice, evaluating the resultant tumors by histology and gene expression microarray. Existing sets of gene expression data for human HCC and CC were examined for pathways related to those observed in the murine tumors, and a cohort of human CC samples was evaluated for relationships between HIF-1α expression and clinical outcomes. RESULTS: Both Pten deletion genotypes developed liver tumors, but with differing phenotypes. Pten deletion alone led to large hepatic tumors with widespread hepatosteatosis. Co-deletion of Pten and Vhl with the Keratin 18 promoter resulted in reduced steatosis and a reduced tumor burden that was characterized by a trabecular architecture similar to CC. Genes associated with hepatic steatosis were coordinately expressed in the human HCC dataset, while genes involved in hypoxia response were upregulated in tumors from the human CC dataset. HIF-1α expression and overall survival were examined in an independent cohort of human CC tumors with no statistical differences uncovered. CONCLUSION: Pten deletion in Keratin 18 expressing cells leads to aggressive tumor formation and widespread steatosis in mouse livers. Co-deletion of Vhl and Pten results in lower tumor burden with gene expression profiling suggesting a switch from a profile of lipid deposition to an expression profile more consistent with upregulation of the hypoxia response pathway. A relationship between tumor hypoxia signaling and altered hepatic steatotic response suggests that competing influences may alter tumor phenotypes.