Maxine Tran

Contrasting Properties of Hypoxia-Inducible Factor 1 (HIF-1) and HIF-2 in von Hippel-Lindau-Associated Renal Cell Carcinoma

ABSTRACT Defective function of the von Hippel-Lindau (VHL) tumor suppressor ablates proteolytic regulation of hypoxia-inducible factor α subunits (HIF-1α and HIF-2α), leading to constitutive activation of hypoxia pathways in renal cell carcinoma (RCC). Here we report a comparative analysis of the functions of HIF-1α and HIF-2α in RCC and non-RCC cells. We demonstrate common patterns of HIF-α isoform transcriptional selectivity in VHL-defective RCC that show consistent and striking differences from patterns in other cell types. We also show that HIF-α isoforms display unexpected suppressive interactions in RCC cells, with enhanced expression of HIF-2α suppressing HIF-1α and vice-versa. In VHL-defective RCC cells, we demonstrate that the protumorigenic genes encoding cyclin D1, transforming growth factor alpha, and vascular endothelial growth factor respond specifically to HIF-2α and that the proapoptotic gene encoding BNip3 responds positively to HIF-1α and negatively to HIF-2α, indicating that HIF-1α and HIF-2α have contrasting properties in the biology of RCC. In keeping with this, HIF-α isoform-specific transcriptional selectivity was matched by differential effects on the growth of RCC as tumor xenografts, with HIF-1α retarding and HIF-2α enhancing tumor growth. These findings indicate that therapeutic approaches to targeting of the HIF system, at least in this setting, will need to take account of HIF isoform-specific functions.

New androgen receptor genomic targets show an interaction with the ETS1 transcription factor.

The androgen receptor (AR) initiates important developmental and oncogenic transcriptional pathways. The AR is known to bind as a homodimer to 15‐base pair bipartite palindromic androgen‐response elements; however, few direct AR gene targets are known. To identify AR promoter targets, we used chromatin immunoprecipitation with on‐chip detection of genomic fragments. We identified 1,532 potential AR‐binding sites, including previously known AR gene targets. Many of the new AR target genes show altered expression in prostate cancer. Analysis of sequences underlying AR‐binding sites showed that more than 50% of AR‐binding sites did not contain the established 15 bp AR‐binding element. Unbiased sequence analysis showed 6‐bp motifs, which were significantly enriched and were bound directly by the AR in vitro. Binding sequences for the avian erythroblastosis virus E26 homologue (ETS) transcription factor family were also highly enriched, and we uncovered an interaction between the AR and ETS1 at a subset of AR promoter targets.

Regulation of E-cadherin Expression by VHL and Hypoxia-Inducible Factor

Mutations in von Hippel-Lindau tumor suppressor gene (VHL) underlie the VHL hereditary cancer syndrome and also occur in most sporadic clear cell renal cell cancers (CCRCC). Currently, the mechanism(s) by which VHL loss of function promotes tumor development in the kidney are not fully elucidated. Here, we show that VHL inactivation in precancerous lesions in kidneys from patients with VHL disease correlates with marked down-regulation of the intercellular adhesion molecule E-cadherin. Moreover, in VHL-defective cell lines (RCC4 and RCC10) derived from sporadic CCRCC, reexpression of VHL was found to restore E-cadherin expression. The product of the VHL gene has multiple reported functions, the best characterized of which is its role as the recognition component of an ubiquitin E3 ligase complex responsible for mediating oxygen-dependent destruction of hypoxia-inducible factor-alpha (HIF-alpha) subunits. We show that HIF activation is necessary and sufficient to suppress E-cadherin in renal cancer cells. Given the fundamental role of E-cadherin in controlling epithelial behavior, our findings give insight into how VHL inactivation/HIF activation may lead to kidney cancer and also indicate a mechanism by which reduced oxygenation could alter E-cadherin expression in other cancers and influence normal homeostasis in other epithelia.

Inhibition of Hypoxia Inducible Factor Hydroxylases Protects Against Renal Ischemia-Reperfusion Injury

Acute renal failure resulting from hypoperfusion and hypoxia is a significant clinical problem. Hypoxia activates the heterodimeric transcription factor hypoxia inducible factor (HIF), leading to changes in gene expression that promote tissue adaptation and survival. To determine whether HIF may protect the kidney from ischemia-reperfusion injury, we subjected hif1a(+/-) and hif2a(+/-) mice to renal ischemia-reperfusion injury. Injury was substantially more severe in hif(+/-) than in littermate controls, consistent with a protective role for HIF. Because wild-type mice exhibited submaximal HIF accumulation in response to no-flow ischemia, we tested compounds that might augment the protective HIF response following ischemia-reperfusion in these animals. We found that l-mimosine and dimethyloxalylglycine, two small molecules that activate HIF by inhibiting HIF hydroxylases, protected mouse kidneys from ischemia-reperfusion injury. Therefore, pharmacological activation of HIF may offer an effective strategy to protect the kidney from ischemic injury.

Tumor cell plasticity in Ewing sarcoma, an alternative circulatory system stimulated by hypoxia.

A striking feature of Ewing sarcoma is the presence of blood lakes lined by tumor cells. The significance of these structures, if any, is unknown. Here, we report that the extent of blood lakes correlates with poor clinical outcomes, whereas variables of angiogenesis do not. We also show that Ewing sarcoma cells form vessel-like tubes in vitro and express genes associated with vasculogenic mimicry. In tumor models, we show that there is blood flow through the blood lakes, suggesting that these structures in Ewing sarcoma contribute to the circulation. Furthermore, we present evidence that reduced oxygen tension may be instrumental in tube formation by plastic tumor cells. The abundant presence of these vasculogenic structures, in contrast to other tumor types, makes Ewing sarcoma the ideal model system to study these phenomena. The results suggest that optimal tumor treatment may require targeting of these structures in combination with prevention of angiogenesis.

Recurrent chromosomal gains and heterogeneous driver mutations characterise papillary renal cancer evolution

Papillary renal cell carcinoma (pRCC) is an important subtype of kidney cancer with a problematic pathological classification and highly variable clinical behaviour. Here we sequence the genomes or exomes of 31 pRCCs, and in four tumours, multi-region sequencing is undertaken. We identify BAP1, SETD2, ARID2 and Nrf2 pathway genes (KEAP1, NHE2L2 and CUL3) as probable drivers, together with at least eight other possible drivers. However, only ~10% of tumours harbour detectable pathogenic changes in any one driver gene, and where present, the mutations are often predicted to be present within cancer sub-clones. We specifically detect parallel evolution of multiple SETD2 mutations within different sub-regions of the same tumour. By contrast, large copy number gains of chromosomes 7, 12, 16 and 17 are usually early, monoclonal changes in pRCC evolution. The predominance of large copy number variants as the major drivers for pRCC highlights an unusual mode of tumorigenesis that may challenge precision medicine approaches.

Renal Tubular HIF-2α Expression Requires VHL Inactivation and Causes Fibrosis and Cysts

The Hypoxia-inducible transcription Factor (HIF) represents an important adaptive mechanism under hypoxia, whereas sustained activation may also have deleterious effects. HIF activity is determined by the oxygen regulated α-subunits HIF-1α or HIF-2α. Both are regulated by oxygen dependent degradation, which is controlled by the tumor suppressor “von Hippel-Lindau” (VHL), the gatekeeper of renal tubular growth control. HIF appears to play a particular role for the kidney, where renal EPO production, organ preservation from ischemia-reperfusion injury and renal tumorigenesis are prominent examples. Whereas HIF-1α is inducible in physiological renal mouse, rat and human tubular epithelia, HIF-2α is never detected in these cells, in any species. In contrast, distinct early lesions of biallelic VHL inactivation in kidneys of the hereditary VHL syndrome show strong HIF-2α expression. Furthermore, knockout of VHL in the mouse tubular apparatus enables HIF-2α expression. Continuous transgenic expression of HIF-2α by the Ksp-Cadherin promotor leads to renal fibrosis and insufficiency, next to multiple renal cysts. In conclusion, VHL appears to specifically repress HIF-2α in renal epithelia. Unphysiological expression of HIF-2α in tubular epithelia has deleterious effects. Our data are compatible with dedifferentiation of renal epithelial cells by sustained HIF-2α expression. However, HIF-2α overexpression alone is insufficient to induce tumors. Thus, our data bear implications for renal tumorigenesis, epithelial differentiation and renal repair mechanisms.

Expression of Hypoxia-Inducible Factors in Normal Human Lung Development

Pulmonary vascular development is essential for proper lung development, and its disturbance can lead to neonatal morbidity and mortality, as exemplified in congenital diaphragmatic hernia. Hypoxia-inducible factors (HIFs) appear to be key molecules in physiologic angiogenesis and in certain forms of lung pathology, such as bronchopulmonary dysplasia. Little is known about the qualitative and quantitative expression of HIFs in normal human fetal lung development. Therefore, we investigated the expression of HIF-1α, HIF-2α, and HIF-3α, along with their upstream regulators and downstream targets, von Hippel-Lindau protein, vascular endothelial growth factor A (VEGF-A), and its receptor, VEGFR-2, in 20 normal human fetal lungs (13.5 weeks in gestation until term) and 5 adult lungs. Quantitative polymerase chain reaction demonstrated a positive correlation between HIF-2α and VEGF-A expression and gestational age. Although there appeared to be a decreasing trend in HIF-3α expression during pregnancy, it did not reach statistical significance. Immunohistochemistry for HIF-1α and HIF-2α revealed that HIF-1α is expressed in the epithelium, while HIF-2α is expressed in both interstitium and epithelium. Our data indicate that HIFs, most notably HIF-2α, appear to exert an important role in angiogenesis during human fetal lung development, especially in the last phases of pregnancy, preparing the fetus for extrauterine life. As such, our results form the baseline data for the evaluation and interpretation of abnormal pulmonary vascular development.

A glycolytic phenotype is associated with prostate cancer progression and aggressiveness: a role for Monocarboxylate Transporters as metabolic targets for therapy.

Metabolic adaptation is considered an emerging hallmark of cancer, whereby cancer cells exhibit high rates of glucose consumption with consequent lactate production. To ensure rapid efflux of lactate, most cancer cells express high levels of monocarboxylate transporters (MCTs), which therefore may constitute suitable therapeutic targets. The impact of MCT inhibition, along with the clinical impact of altered cellular metabolism during prostate cancer (PCa) initiation and progression, has not been described. Using a large cohort of human prostate tissues of different grades, in silico data, in vitro and ex vivo studies, we demonstrate the metabolic heterogeneity of PCa and its clinical relevance. We show an increased glycolytic phenotype in advanced stages of PCa and its correlation with poor prognosis. Finally, we present evidence supporting MCTs as suitable targets in PCa, affecting not only cancer cell proliferation and survival but also the expression of a number of hypoxia‐inducible factor target genes associated with poor prognosis. Herein, we suggest that patients with highly glycolytic tumours have poorer outcome, supporting the notion of targeting glycolytic tumour cells in prostate cancer through the use of MCT inhibitors.

Developmentally arrested structures preceding cerebellar tumors in von Hippel-Lindau disease.

There is increasing evidence that suggests that knockout of tumor-suppressor gene function causes developmental arrest and protraction of cellular differentiation. In the peripheral nervous system of patients with the tumor-suppressor gene disorder, von Hippel–Lindau disease, we have demonstrated developmentally arrested structural elements composed of hemangioblast progenitor cells. Some developmentally arrested structural elements progress to a frank tumor, hemangioblastoma. However, in von Hippel–Lindau disease, hemangioblastomas are frequently observed in the cerebellum, suggesting an origin in the central nervous system. We performed a structural and topographic analysis of cerebellar tissues obtained from von Hippel–Lindau disease patients to identify and characterize developmentally arrested structural elements in the central nervous system. We examined the entire cerebella of five tumor-free von Hippel–Lindau disease patients and of three non-von Hippel–Lindau disease controls. In all, 9 cerebellar developmentally arrested structural elements were detected and topographically mapped in 385 blocks of von Hippel–Lindau disease cerebella. No developmentally arrested structural elements were seen in 214 blocks from control cerebella. Developmentally arrested structural elements are composed of poorly differentiated cells that express hypoxia-inducible factor (HIF)2α, but not HIF1α or brachyury, and preferentially involve the molecular layer of the dorsum cerebelli. For the first time, we identify and characterize developmentally arrested structural elements in the central nervous system of von Hippel–Lindau patients. We provide evidence that developmentally arrested structural elements in the cerebellum are composed of developmentally arrested hemangioblast progenitor cells in the molecular layer of the dorsum cerebelli.