Patients with a germline mutation in von Hippel-Lindau (VHL) develop renal cell cancers and hypervascular tumors of the brain, adrenal glands, and pancreas as well as erythrocytosis. These phenotypes are driven by aberrant expression of HIF2α, which induces expression of genes involved in cell proliferation, angiogenesis, and red blood cell production. Currently, there are no effective treatments available for VHL disease. Here, using an animal model of VHL, we report a marked improvement of VHL-associated phenotypes following treatment with HIF2α inhibitors. Inactivation of vhl in zebrafish led to constitutive activation of HIF2α orthologs and modeled several aspects of the human disease, including erythrocytosis, pathologic angiogenesis in the brain and retina, and aberrant kidney and liver proliferation. Treatment of vhl–/– mutant embryos with HIF2α-specific inhibitors downregulated Hif target gene expression in a dose-dependent manner, improved abnormal hematopoiesis, and substantially suppressed erythrocytosis and angiogenic sprouting. Moreover, pharmacologic inhibition of HIF2α reversed the compromised cardiac contractility of vhl–/– embryos and partially rescued early lethality. This study demonstrates that small-molecule targeting of HIF2α improves VHL-related phenotypes in a vertebrate animal model and supports further exploration of this strategy for treating VHL disease.
Identification of potential changes in the glycosylation of existing cancer biomarkers can result in a higher level of diagnostic sensitivity and specificity. Clusterin (Apolipoprotein J) has been implicated in renal cell carcinoma (RCC) and other types of malignancy as potential biomarker. In the present work, an automated multidimensional HPLC platform enabling high throughput affinity enrichment of clusterin from plasma samples was developed. Integrated with two dimensional gel electrophoresis, high purity clusterin in microgram quantities suitable for glycan characterization was isolated. The analytical platform was applied to study clusterin glycosylation in a small group of RCC patients before and after nephrectopy as a pilot study to evaluate the performance of the platform. A statistically significant decrease was observed in the levels of a bi-antennary digalactosyl disialylated (A2G2S(3)2) glycans while the levels of a core fucosylated bi-antennary digalactosyl disialylated glycan (FA2G2S(6)2) and a tri-antennary trigalactosyl disialylated glycan (A3G3S(6)2) were increased in the post-surgery plasma samples.
Multi-dimensional HPLC; clusterin; two dimensional gel electrophoresis (2-DE); UPLC-HILIC; N-glycan; renal cell carcinoma
To determine the role of hypoxia-inducible factor-2α (HIF2α) on the sensitivity of renal cell carcinoma (RCC) cell lines to ionizing radiation and to determine if the mTOR antagonist, rapamycin, could decrease HIF2α protein levels.
MATERIALS AND METHODS
Cell lines expressing stable short-hairpin RNAs (shRNAs) encoding HIF2α shRNAs or an empty vector were transfected with a hypoxia responsive element (HRE)-driven firefly luciferase reporter gene. Two separate paired cell lines were assayed for their response to increasing doses of ionizing radiation. Proliferation and cell cycle kinetics were compared for cell lines expressing HIF2α shRNAs and empty vectors. The effect of an mTOR antagonist, rapamycin on HIF1α and HIF2α proteins levels was also assessed.
We confirmed that the 786-O RCC lines with stably integrated shRNAs against HIF2α had decreased activation of a plasmid with a HRE-driven firefly luciferase reporter gene. Lines from two separate cell clones with decreased HIF2α levels showed a significant increase in radiation sensitivity and an increase in G2 cell cycle arrest. Rapamycin, while effective in decreasing HIF1α protein levels, did not affect HIF2α levels in either of the RCC cell lines.
These results show that decreasing levels of HIF2α leads to an increased sensitivity to ionizing radiation. This finding may explain in part, the known resistance of RCC to radiation therapy. Although mTOR antagonists are approved for the treatment of RCC, these agents do not decrease HIF2α levels and therefore might not be effective in enhancing the radio-sensitivity of these tumours.
renal cancer; radiation; hypoxia; hypoxia-inducible factor
Hypoxic and VHL-deficient cells use glutamine to generate citrate and lipids through reductive carboxylation (RC) of α-ketoglutarate. To gain insights into the role of HIF and the molecular mechanisms underlying RC, we took advantage of a panel of disease-associated VHL mutants and showed that HIF expression is necessary and sufficient for the induction of RC in human renal cell carcinoma (RCC) cells. HIF expression drastically reduced intracellular citrate levels. Feeding VHL-deficient RCC cells with acetate or citrate or knocking down PDK-1 and ACLY restored citrate levels and suppressed RC. These data suggest that HIF-induced low intracellular citrate levels promote the reductive flux by mass action to maintain lipogenesis. Using [1–13C] glutamine, we demonstrated in vivo RC activity in VHL-deficient tumors growing as xenografts in mice. Lastly, HIF rendered VHL-deficient cells sensitive to glutamine deprivation in vitro, and systemic administration of glutaminase inhibitors suppressed the growth of RCC cells as mice xenografts.
Cells transiently adapt to hypoxia by globally decreasing protein translation. However, specific proteins needed to respond to hypoxia evade this translational repression. The mechanisms of this phenomenon remain unclear. We screened for and identified small molecules that selectively decrease HIF-2a translation in an mTOR independent manner, by enhancing the binding of Iron Regulatory Protein 1 (IRP1) to a recently reported Iron-Responsive Element (IRE) within the 5’-untranslated region (UTR) of the HIF-2a message. Knocking down the expression of IRP1 by shRNA abolished the effect of the compounds. Hypoxia de-represses HIF-2a translation by disrupting the IRP1- HIF-2a IRE interaction. Thus, this chemical genetic analysis describes a molecular mechanism by which translation of the HIF-2a message is maintained during conditions of cellular hypoxia through inhibition of IRP-1 dependent repression. It also provides the chemical tools for studying this phenomenon.
Acetyl coenzyme A (AcCoA) is the central biosynthetic precursor for fatty acid synthesis and protein acetylation. In the conventional view of mammalian cell metabolism, AcCoA is primarily generated from glucose-derived pyruvate through the citrate shuttle and adenosine triphosphate citrate lyase (ACL) in the cytosol1-3. However, proliferating cells that exhibit aerobic glycolysis and those exposed to hypoxia convert glucose to lactate at near stoichiometric levels, directing glucose carbon away from the tricarboxylic acid cycle (TCA) and fatty acid synthesis4. Although glutamine is consumed at levels exceeding that required for nitrogen biosynthesis5, the regulation and utilization of glutamine metabolism in hypoxic cells is not well understood. Here we show that human cells employ reductive metabolism of alpha-ketoglutarate (αKG) to synthesize AcCoA for lipid synthesis. This isocitrate dehydrogenase 1 (IDH1) dependent pathway is active in most cell lines under normal culture conditions, but cells grown under hypoxia rely almost exclusively on the reductive carboxylation of glutamine-derived αKG for de novo lipogenesis. Furthermore, renal cell lines deficient in the von Hippel-Lindau (VHL) tumor suppressor protein preferentially utilize reductive glutamine metabolism for lipid biosynthesis even at normal oxygen levels. These results identify a critical role for oxygen in regulating carbon utilization in order to produce AcCoA and support lipid synthesis in mammalian cells.
The Birt-Hogg-Dube disease occurs as a result of germline mutations in the human Folliculin gene (FLCN), and is characterized by clinical features including fibrofolliculomas, lung cysts and multifocal renal neoplasia. Clinical and genetic evidence suggest that FLCN acts as a tumor suppressor gene. The human cell line UOK257, derived from the renal cell carcinoma of a patient with a germline mutation in the FLCN gene, harbors a truncated version of the FLCN protein. Reconstitution of the wild type FLCN protein into UOK257 cells delays cell cycle progression, due to a slower progression through the late S and G2/M-phases. Similarly, Flcn–/– mouse embryonic fibroblasts progress more rapidly through the cell cycle than wild type controls (Flcnflox/flox). The reintroduction of tumor-associated FLCN mutants (FLCN ΔF157, FLCN 1–469 or FLCN K508R) fails to delay cell cycle progression in UOK257 cells. Additionally, FLCN phosphorylation (on Serines 62 and 73) fluctuates throughout the cell cycle and peaks during the G2/M phase in cells treated with nocodazole. In keeping with this observation, the reintroduction of a FLCN phosphomimetic mutant into the UOK257 cell line results in faster progression through the cell cycle compared to those expressing the wild type FLCN protein. These findings suggest that the tumor suppression function of FLCN may be linked to its impact on the cell cycle and that FLCN phosphorylation is important for this activity. Additionally, these observations describe a novel in vitro assay for testing the functional significance of FLCN mutations and/or genetic polymorphisms.
Cancer epithelial cells often migrate away from the primary tumor to invade into the surrounding tissues. Their migration is commonly assumed to be directed by pre-existent spatial gradients of chemokines and growth factors in the target tissues. Unexpectedly however, we found that the guided migration of epithelial cells is possible in vitro in the absence of pre-existent chemical gradients. We observed that both normal and cancer epithelial cells can migrate persistently and reach the exit along the shortest path from microscopic mazes filled with uniform concentrations of
media. Using microscale engineering techniques and biophysical models, we uncovered a self-guidance strategy during which epithelial cells generate their own guiding cues under conditions of biochemical confinement. The self-guidance strategy depends on the balance between three interdependent processes: epidermal growth factor (EGF) uptake by the cells (U), the restricted transport of EGF through the structured microenvironment (T), and cell chemotaxis toward the resultant EGF gradients (C). The UTC self-guidance strategy can be perturbed by inhibition of signalling through EGF-receptors and appears to be independent from chemokine signalling. Better understanding of the UTC self-guidance strategy could eventually help devise new ways for modulating epithelial cell migration and delaying cancer cell invasion or accelerating wound healing.
Normal stem cells reside in functional niches critical for self-renewal and maintenance. Neural and hematopoietic stem cell niches, in particular, are characterized by restricted availability of oxygen and the resulting regulation by hypoxia inducible factors (HIFs). Glioblastoma multiforme (GBM) is the most common malignant brain tumor and also contains high degrees of hypoxia. Heterogeneity within the neoplastic compartment has been well characterized in GBM and may be derived from genetic and epigenetic sources that co-evolve during malignant progression. Recent experimental evidence has supported the importance of hypoxia in cancer stem cell (CSC) niches. We hypothesized that HIFs require epigenetic modifying proteins in order to promote tumor malignancy in GBM. Here we demonstrate that in GBM the histone methyltransferase Mixed-Lineage Leukemia 1 (MLL1) is induced by hypoxia and enhances hypoxic responses. Loss of MLL1 reduces expression of HIF transcripts and HIF2α protein. Targeting MLL1 by RNA interference inhibited expression of HIF2α and target genes, including VEGF. CSCs expressed higher levels of MLL1 than matched nonstem tumor cells and depletion of MLL1 reduced CSC self-renewal, growth, and tumorigenicity. These studies have uncovered a novel mechanism mediating tumor hypoxic responses linking microenvironmental regulation of epigenetic modifying proteins to cellular heterogeneity and provide rationale for the design of more sophisticated clinical approaches targeting epigenetic regulation.
hypoxia; MLL1; cancer stem cell; HIF2α; epigenetics
Liquid chromatography-selected reaction monitoring (LC-SRM) is a highly specific and sensitive mass spectrometry (MS) technique that is widely being applied to selectively qualify and validate candidate markers within complex biological samples. However, in order for LC-SRM methods to take on these attributes, target-specific optimization of sample processing is required, in order to reduce analyte complexity, prior to LC-SRM. In this study, we have developed a targeted platform consisting of protein immunoaffinity enrichment on magnetic beads and LC-SRM for measuring carbonic anhydrase 12 (CA12) protein in a renal cell carcinoma (RCC) cell line (PRC3), a candidate biomarker for RCC whose expression at the protein level has not been previously reported. Sample processing and LC-SRM assay were optimized for signature peptides selected as surrogate markers of CA12 protein. Using LC-SRM coupled with stable isotope dilution, we achieved limits of quantitation in the low fmol range sufficient for measuring clinically relevant biomarkers with good intra- and inter-assay accuracy and precision (≤17%). Our results show that using a quantitative immunoaffinity capture approach provides specific, accurate, and robust assays amenable to high-throughput verification of potential biomarkers.
immunoaffinity enrichment; selected reaction monitoring; enhanced signature peptide predictor; carbonic anhydrase 12; renal cell carcinoma; biomarker; Protein G magnetic beads
SIRT6 is a member of a highly conserved family of NAD+-dependent deacetylases with various roles in metabolism, stress resistance and lifespan. SIRT6 deficient mice develop normally but succumb to a lethal hypoglycemia early in life; however, the mechanism underlying this hypoglycemia remained unclear. Here, we demonstrate that SIRT6 functions as a histone H3K9 deacetylase to control the expression of multiple glycolytic genes. Specifically, SIRT6 appears to function as a co-repressor of the transcription factor Hif1α, a critical regulator of nutrient stress responses. Consistent with this notion, SIRT6 deficient cells exhibit increased Hif1α activity and show increased glucose uptake with up-regulation of glycolysis and diminished mitochondrial respiration. Our studies uncover a novel role for the chromatin factor SIRT6 as a master regulator of glucose homeostasis, and may provide the basis for novel therapeutic approaches against metabolic diseases, such as diabetes and obesity.
Hypoxia Inducible Factors 1 and 2 (HIF1 and HIF2) are heterodimeric transcription factors consisting of alpha regulatory subunits and a constitutively expressed beta subunit. The expression of alpha regulatory subunits is promoted by hypoxia, cancer-associated mutations and inflammatory cytokines. Thus, HIF1 and HIF2 provide a molecular link between cancer and inflammation. We have recently identified novel small molecules that selectively inhibit translation of the HIF2a message and thereby powerfully inhibit the expression of HIF2a target genes. We report here that Connectivity Map analysis links three of these compounds to the anti-inflammatory cytokine 15-deoxy-Δ12,14-Prostaglandin J2 (PGJ2). As with our identified compounds, PGJ2 inhibits translation of the HIF2a message in an mTOR independent manner by promoting the binding of Iron Regulatory Protein-1 (IRP1) to a non-canonical Iron Responsive Element (IRE) embedded within the 5′-UTR of the HIF2a message. The IRE is necessary and sufficient for mediating the effect. Mutation of the IRE sequence, or down regulation of IRP1 expression, blocks the effect of PGJ2 on HIF2a translation. This is the first report of an endogenous natural molecule regulating HIF2a translation and it suggests that part of the anti-inflammatory and putative anti-neoplastic effects of PGJ2 may be mediated through inhibition of HIF2a within tumor epithelial cells themselves and/or mesenchymal cells of the tumor microenvironment.
Renal Cancer; hypoxia; prostaglandin; angiogenesis; iron
The von Hippel-Lindau tumor suppressor protein (pVHL) binds to elongins B and C and posttranscriptionally regulates the accumulation of hypoxia-inducible mRNAs under normoxic (21% O2) conditions. Here we report that pVHL binds, via elongin C, to the human homolog of the Caenorhabditis elegans Cul2 protein. Coimmunoprecipitation and chromatographic copurification data suggest that pVHL-Cul2 complexes exist in native cells. pVHL mutants that were unable to bind to complexes containing elongin C and Cul2 were likewise unable to inhibit the accumulation of hypoxia-inducible mRNAs. A model for the regulation of hypoxia-inducible mRNAs by pVHL is presented based on the apparent similarity of elongin C and Cul2 to Skp1 and Cdc53, respectively. These latter proteins form complexes that target specific proteins for ubiquitin-dependent proteolysis.