William G. Kaelin

Binding of the von Hippel-Lindau tumor suppressor protein to Elongin B and C

Germ-line mutations of the von Hippel-Lindau tumor suppressor gene (VHL) predispose individuals to a variety of human tumors, and somatic mutations of this gene have been identified in sporadic renal cell carcinomas and cerebellar hemangioblastomas. Two transcriptional elongation factors, Elongin B and C, were shown to bind in vitro and in vivo to a short, colinear region of the VHL protein (pVHL) that is frequently mutated in human tumors. A peptide replica of this region inhibited binding of pVHL to Elongin B and C whereas a point-mutant derivative, corresponding to a naturally occurring VHL missense mutation, had no effect. These results suggest that the tumor suppression function of pVHL may be linked to its ability to bind to Elongin B and C.

HIF hydroxylation and the mammalian oxygen-sensing pathway

Hypoxia Hypoxia refers to below-normal levels of oxygen in air, blood, or tissue. Tissue hypoxia leads to cellular dysfunction and ultimately can lead to cell death. Causes of tissue hypoxia include (a) decreased blood oxygenation (such as occurs in certain pulmonary disorders), (b) altered oxygen release from hemoglobin (associated with some hemoglobinopathies), and (c) impaired blood delivery leading to localized anemia (i.e., ischemia) as a result of low cardiac output or vascular obstruction. In order to adapt to hypoxia, mammals use a number of physiological responses. These include (a) increased production of erythropoietin (EPO), which augments the production of red blood cells; (b) induction of tyrosine hydroxylase, which facilitates the control of ventilation through the carotid body; and (c) the stimulation of new blood vessels by upregulation of VEGF (1). At the cellular level, hypoxia induces a number of metabolic changes that allow for continued energy generation despite decreased oxygen availability.

Retinoblastoma protein and anaphase-promoting complex physically interact and functionally cooperate during cell-cycle exit

The retinoblastoma protein (pRB) negatively regulates the progression from G1 to S phase of the cell cycle, in part, by repressing E2F-dependent transcription. pRB also possesses E2F-independent functions that contribute to cell-cycle control — for example, during pRB-mediated cell-cycle arrest pRB associates with Skp2, the F-box protein of the Skp1–Cullin–F-box protein (SCF) E3 ubiquitin ligase complex, and promotes the stability of the cyclin-dependent kinase-inhibitor p27Kip1 through an unknown mechanism. Degradation of p27Kip1 is mediated by ubiquitin-dependent targeting of p27Kip1 by SCF –Skp2 (ref. 4). Here, we report a novel interaction between pRB and the anaphase-promoting complex/cyclosome (APC/C) that controls p27Kip1 stability by targeting Skp2 for ubiquitin-mediated degradation. Cdh1, an activator of APC/C, not only interacts with pRB but is also required for a pRB-induced cell-cycle arrest. The results reveal an unexpected physical convergence between the pRB tumour-suppressor protein and E3 ligase complexes, and raise the possibility that pRB may direct APC/C to additional targets during pRB-mediated cell-cycle exit.

HIF2α cooperates with RAS to promote lung tumorigenesis in mice

Members of the hypoxia-inducible factor (HIF) family of transcription factors regulate the cellular response to hypoxia. In non–small cell lung cancer (NSCLC), high HIF2α levels correlate with decreased overall survival, and inhibition of either the protein encoded by the canonical HIF target gene VEGF or VEGFR2 improves clinical outcomes. However, whether HIF2α is causal in imparting this poor prognosis is unknown. Here, we generated mice that conditionally express both a nondegradable variant of HIF2α and a mutant form of Kras (KrasG12D) that induces lung tumors. Mice expressing both Hif2a and KrasG12D in the lungs developed larger tumors and had an increased tumor burden and decreased survival compared with mice expressing only KrasG12D. Additionally, tumors expressing both KrasG12D and Hif2a were more invasive, demonstrated features of epithelial-mesenchymal transition (EMT), and exhibited increased angiogenesis associated with mobilization of circulating endothelial progenitor cells. These results implicate HIF2α causally in the pathogenesis of lung cancer in mice, demonstrate in vivo that HIF2α can promote expression of markers of EMT, and define HIF2α as a promoter of tumor growth and progression in a solid tumor other than renal cell carcinoma. They further suggest a possible causal relationship between HIF2α and prognosis in patients with NSCLC.

Synthetic peptides define critical contacts between elongin C, elongin B, and the von Hippel-Lindau protein

The von Hippel-Lindau tumor suppressor protein (pVHL) negatively regulates hypoxia-inducible mRNAs such as the mRNA encoding vascular endothelial growth factor (VEGF). This activity has been linked to its ability to form multimeric complexes that contain elongin C, elongin B, and Cul2. To understand this process in greater detail, we performed a series of in vitro binding assays using pVHL, elongin B, and elongin C variants as well as synthetic peptide competitors derived from pVHL or elongin C. A subdomain of elongin C (residues 17-50) was necessary and sufficient for detectable binding to elongin B. In contrast, elongin B residues required for binding to elongin C were not confined to a discrete colinear domain. We found that the pVHL (residues 157-171) is necessary and sufficient for binding to elongin C in vitro and is frequently mutated in families with VHL disease. These mutations preferentially involve residues that directly bind to elongin C and/or alter the conformation of pVHL such that binding to elongin C is at least partially diminished. These results are consistent with the view that diminished binding of pVHL to the elongins plays a causal role in VHL disease.

Choosing anticancer drug targets in the postgenomic era.

The human genome project will be completed in the early part of the next decade. For example, most of the approximately 105 human mRNAs have already been at least partially cloned and sequenced as cDNAs. These cDNAs, when completed, will provide valuable tools for studying the protein products of their respective mRNAs. The challenge in the postgenomic era will be to apply advances in molecular oncology to select from among this diverse set of proteins the most suitable candidates for anti-cancer drug discovery programs. Here, I outline some considerations that may be helpful in choosing rational drug targets for cancer therapy.

Alterations in G1/S Cell‐Cycle Control Contributing to Carcinogenesis

ABSTRACT: Mutations involving the retinoblastoma tumor‐suppressor gene (RB‐1) have been described in a variety of human neoplasms. In addition, many tumors that retain a wild‐type RB‐1 allele harbor mutations that indirectly impair the function of the RB‐1 gene product (pRB). pRB is a nuclear protein that regulates cell‐cycle progression and, in at least certain tissues, differentiation. The former has been linked to its ability to form complexes with members of the E2F transcription factor family. E2F DNA‐binding sites have been identified in the regulatory regions of a number of genes involved in cell‐cycle progression. pRB‐E2F complexes actively repress transcription when bound to these sites. All tumor‐derived pRB mutants have lost the ability to bind to E2F. Conversely, activation of E2F‐responsive genes is sufficient to overcome a pRB‐induced cell‐cycle block and, in certain cell types, can lead to transformation. Thus, E2F appears to be a physiologically relevant target of pRB action, and deregulation of E2F‐responsive genes is a common, and possibly universal, step in human carcinogenesis.

Good COP1 or bad COP1? In vivo veritas

The evolutionarily conserved protein COP1 has been shown to operate as an E3 ubiquitin ligase complex, and a number of putative substrates have been identified, including the c-JUN oncoprotein and p53 tumor suppressor protein. New work by Migliorini and colleagues described in the current issue of JCI demonstrates that COP1 acts as a tumor suppressor in vivo and does so, at least in part, by promoting the destruction of c-JUN. These findings challenge the view that COP1 regulates p53 stability and call into question the wisdom of developing COP1 inhibitors as potential anticancer agents.

Fluorescence-activated cell sorting of transfected cells

Publisher Summary This chapter discusses the fluorescence-activated cell sorting of transfected cells. Fluorescence-activated cell sorting (FACS) is a technique that can measure the fluorescence intensity of individual cells within a population. The cells within a population are labeled with a fluorescent label in such a way that the intensity of fluorescence of an individual cell provides a quantitative measure of the particular parameter of interest. By staining cells with fluorochromes of different emission wavelengths, multiple parameters can be measured simultaneously. FACS has a number of applications within cell cycle research. For example, cellular DNA content is commonly measured with a fluorescent DNA intercalating agent such as propidium iodide (PI). The study of putative cell cycle regulatory proteins often requires their activities within the cell to be either enhanced or inhibited, and the consequences observed in terms of, for example, changes in cell cycle distribution.

Bioluminescent imaging of ubiquitin ligase activity: measuring Cdk2 activity in vivo through changes in p27 turnover.

Optical imaging of reporter molecules such as firefly luciferase has become a popular method of tracking and visualizing cells in living animals. Many biological processes involve ubiquitin ligases, which target specific proteins for destruction under specific sets of conditions. Importantly, the motifs recognized by different ubiquitin ligases are often modular and can be used to target foreign proteins for destruction in cis. We recently fused the Cdk inhibitor p27, which is polyubiquitylated by a Skp2-containing ubiquitin ligase if phosphorylated by cdk2 to firefly luciferase. The resulting fusion protein, p27-Luc, was induced by cdk2 inhibitors in living cells grown in culture or in nude mice. This article describes protocols for validation of p27-Luc in cell culture using siRNA against cdk2 (or its partner cyclin A) and for imaging cells producing p27-Luc grown in transparent hollow fibers after treatment with cdk2 inhibitory drugs in vivo. These approaches should be generalizable to other ubiquitin-ligase substrate pairs.