ABSTRACT. Recent studies of a relatively rare hereditary cancer syndrome, von Hippel-Lindau (VHL) disease, have shed new light on the molecular pathogenesis of kidney cancer and, perhaps more important, on how mammalian cells sense and respond to changes in oxygen availability. This knowledge is already translating into new therapeutic targets for kidney cancer as well as for multiple conditions, such as myocardial infarction and stroke, in which ischemia plays a pathogenic role. This review summarizes the current knowledge of the molecular pathogenesis of von Hippel-Lindau disease and the role of the VHL gene product (pVHL) in kidney cancer and the mammalian oxygen sensing pathway. E-mail: william_kaelin@dfci.harvard.edu
Previous results using gene transfection methods have shown that the wild-type (WT) von Hippel-Lindau (VHL) gene can function as a potent tumor suppressor gene in vivo for renal cell carcinoma (RCC) cells in the absence of any suppressive effect on cell growth in monolayer cell culture under serum-rich conditions. Because we had previously found that the function of some oncogenes, such as mutant ras, can be influenced by three-dimensional growth as multicellular spheroids (J. Rak et aL, J. Cell Biol., 131: 1587-1598, 1995), we reasoned the same might be true for suppressor genes as well. We, therefore, decided to compare and study the effects of the WT VHL gene in monolayer versus three-dimensional culture systems of the RCC cell line 786-0, which contains an inactivated VHL gene. We found that the reintroduction of the WT VHL gene into mutant VHL RCC cells resulted in growth suppression in vitro, but only when the cells were grown as spheroid cultures. This decrease in cell proliferation was associated with several features of cell differentiation/morphogenesis, as shown by light and electron microscopy. Thus, in contrast to cultures of mutant VHL RCC cells, which formed very compact and cohesive spheroids, the WT VHL transfectants were loosely arranged and formed a network of tubular and trabecular structures within the spheroids. The morphological changes of the WT VHL spheroids were associated with the deposition of fibronectin in the extracellular space, a feature that was absent in the mutant and inactivated VHL gene-expressing spheroids. The results suggest the VHL gene may be involved in the maintenance of the epithelial phenotype of renal tubular cells, ie., it may act as a differentiation/morphogenetic factor. Moreover, this effect in tumors cells appears to be highly dependent on multicellular growth conditions that mimic the basic nature of solid tumors, such as RCC.
Mammalian target of rapamycin (mTOR) is a central regulator of protein synthesis whose activity is modulated by a variety of signals. Energy depletion and hypoxia result in mTOR inhibition. While energy depletion inhibits mTOR through a process involving the activation of AMP-activated protein kinase (AMPK) by LKB1 and subsequent phosphorylation of TSC2, the mechanism of mTOR inhibition by hypoxia is not known. Here we show that mTOR inhibition by hypoxia requires the TSC1/TSC2 tumor suppressor complex and the hypoxia-inducible gene REDD1/RTP801 . Disruption of the TSC1/TSC2 complex through loss of TSC1 or TSC2 blocks the effects of hypoxia on mTOR, as measured by changes in the mTOR targets S6K and 4E-BP1, and results in abnormal accumulation of Hypoxia-inducible factor (HIF). In contrast to energy depletion, mTOR inhibition by hypoxia does not require AMPK or LKB1. Down-regulation of mTOR activity by hypoxia requires de novo mRNA synthesis and correlates with increased expression of the hypoxia-inducible REDD1 gene. Disruption of REDD1 abrogates the hypoxia-induced inhibition of mTOR, and REDD1 overexpression is sufficient to down-regulate S6K phosphorylation in a TSC1/TSC2-dependent manner. Inhibition of mTOR function by hypoxia is likely to be important for tumor suppression as TSC2-deficient cells maintain abnormally high levels of cell proliferation under hypoxia.
It has previously been demonstrated that the simian virus 40 large T antigen and adenovirus E1A proteins can form complexes with the retinoblastoma susceptibility gene product (RB). We studied the ability of these proteins to bind to mutant RB proteins in vitro. A region of RB spanning residues 379 to 792 was found to be both necessary and sufficient for binding to T or E1A. Furthermore, this region of RB contains sufficient structural information to mimic wild-type RB in its ability to distinguish between wild-type T and the transformation-defective T mutant K1. The results of competition experiments with peptide analogs of the RB-binding sequence in T suggest that this region of RB makes direct contact with a short colinear region of T, i.e., residues 102 to 115, previously implicated in both transformation and RB binding.
Control of proliferation and differentiation by the retinoblastoma tumor suppressor protein (pRB) and related family members depends upon their interactions with key cellular substrates. Efforts to identify such cellular targets led to the isolation of a novel protein, EID-1 (for E1A-like inhibitor of differentiation 1). Here, we show that EID-1 is a potent inhibitor of differentiation and link this activity to its ability to inhibit p300 (and the highly related molecule, CREB-binding protein, or CBP) histone acetylation activity. EID-1 is rapidly degraded by the proteasome as cells exit the cell cycle. Ubiquitination of EID-1 requires an intact C-terminal region that is also necessary for stable binding to p300 and pRB, two proteins that bind to the ubiquitin ligase MDM2. A pRB variant that can bind to EID1, but not MDM2, stabilizes EID-1 in cells. Thus, EID-1 may act at a nodal point that couples cell cycle exit to the transcriptional activation of genes required for differentiation.
The von Hippel-Lindau tumor suppressor, pVHL, is a key player in one of the best characterized hypoxia signaling pathways, the VHL-hypoxia-inducible factor (VHL-HIF) pathway. To better understand the role of VHL in the hypoxia signaling pathways of tumor cells, we used serial analysis of gene expression (SAGE) to investigate hypoxia-regulated gene expression in renal carcinoma cells (786-0), with and without VHL. The gene expression profiles of the cancer cells were compared to SAGE profiles from normal renal proximal tubule cells grown under both normoxia and hypoxia. The data suggest that the role of VHL as a tumor suppressor may be more complex than previously thought. Further, the data reveal that renal carcinoma cells have evolved an alternative hypoxia signaling pathway(s) compared with normal renal cells. These alternative hypoxia pathways demonstrate VHL-dependent and VHL-independent regulation. The genes involved in such pathways include those with potential importance in the physiological and pathological regulation of tumor growth and angiogenesis. Some of the genes identified as showing overexpression in the cancer cells, particularly those encoding secreted or membrane-bound proteins, could be potential biomarkers for tumors or targets for rational therapeutics that are dependent on VHL status.
ABSTRACT Inactivation of pVHL tumor suppressor in clear cell Renal Cell Carcinoma (ccRCC) increases the abundance of Histone H3 lysine 27 acetylation (H3K27ac). We hypothesized that H3K27ac, a marker of transcriptional activation, drives the expression of critical oncogenes in ccRCC. Using H3K27ac ChIP-Seq; RNA-Seq; an in vivo positive selection screen; cell-based functional studies; and clinical validations; here, we report the identification of the SLC1A1/EAAT3 aspartate (Asp) and glutamate (Glu) transporter as a ccRCC oncogene. pVHL loss promotes SLC1A1 expression in a HIF-independent manner. Importantly, SLC1A1 inactivation depletes Asp/Glu-derived metabolites, impedes ccRCC growth both in vitro and in vivo , and sensitizes ccRCCs to metabolic therapeutics (e.g., glutaminase blockers). Finally, in human ccRCC biospecimens, higher SLC1A1 expression is associated with metastatic disease and clusters with elevated expression of other solute carriers, but not HIF/Hypoxia pathways. Altogether, our studies identify a HIF-independent metabolic hub in ccRCC and credential SLC1A1 as an actionable ccRCC oncogene. STATEMENT OF SIGNIFICANCE Targeting chronic HIF activation underlies many therapeutic strategies in ccRCC; but, unfortunately, is not curative. SLC1A1, instead, represents a HIF-independent ccRCC dependency, which is targetable alone and together with other antimetabolites, such as glutaminase inhibitors. These observations identify an actionable metabolic program that functions independent of HIF in ccRCC.
