Insufficient oxygen and nutrient supply often restrain solid tumor growth, and the hypoxia-inducible factors (HIF) 1␣ and HIF-2␣ are key transcription regulators of phenotypic adaptation to low oxygen levels. Moreover, mouse gene disruption studies have implicated HIF-2␣ in embryonic regulation of tyrosine hydroxylase, a hallmark gene of the sympathetic nervous system. Neuroblastoma tumors originate from immature sympathetic cells, and therefore we investigated the effect of hypoxia on the differentiation status of human neuroblastoma cells. Hypoxia stabilized HIF-1␣ and HIF-2␣ proteins and activated the expression of known hypoxia-induced genes, such as vascular endothelial growth factor and tyrosine hydroxylase. These changes in gene expression also occurred in hypoxic regions of experimental neuroblastoma xenografts grown in mice. In contrast, hypoxia decreased the expression of several neuronal͞neuroendocrine marker genes but induced genes expressed in neural crest sympathetic progenitors, for instance c-kit and Notch-1. Thus, hypoxia apparently causes dedifferentiation both in vitro and in vivo. These findings suggest a novel mechanism for selection of highly malignant tumor cells with stem-cell characteristics.
The tubules of the kidney display a remarkable capacity for self-renewal on damage. Whether this regeneration is mediated by dedifferentiating surviving cells or, as recently suggested, by stem cells has not been unequivocally settled. Herein, we demonstrate that aldehyde dehydrogenase (ALDH) activity may be used for isolation of cells with progenitor characteristics from adult human renal cortical tissue. Gene expression profiling of the isolated ALDH high and ALDH low cell fractions followed by immunohistochemical interrogation of renal tissues enabled us to delineate a tentative progenitor cell population scattered through the proximal tubules (PTs). These cells expressed CD24 and
The miR-17-92 microRNA cluster is often activated in cancer cells, but the identity of its targets remains elusive. Using SILAC and quantitative mass spectrometry, we examined the effects of activation of the miR-17-92 cluster on global protein expression in neuroblastoma cells. Our results reveal cooperation between individual miR-17-92 miRNAs and implicate miR-17-92 in multiple hallmarks of cancer, including proliferation and cell adhesion. Most importantly, we show that miR-17-92 is a potent inhibitor of TGFβ-signaling. By functioning both upstream and downstream of pSMAD2, miR-17-92 activation triggers downregulation of multiple key effectors along the TGFβ-signaling cascade as well as through direct inhibition of TGFβ-responsive genes.
Dysregulation of the von Hippel-Lindau/hypoxia-inducible transcription factor (HIF) signaling pathway promotes clear cell renal cell carcinoma (ccRCC) progression and metastasis. The protein kinase GAS6/AXL signaling pathway has recently been implicated as an essential mediator of metastasis and receptor tyrosine kinase crosstalk in cancer. Here we establish a molecular link between HIF stabilization and induction of AXL receptor expression in metastatic ccRCC. We found that HIF-1 and HIF-2 directly activate the expression of AXL by binding to the hypoxia-response element in the AXL proximal promoter. Importantly, genetic and therapeutic inactivation of AXL signaling in metastatic ccRCC cells reversed the invasive and metastatic phenotype in vivo. Furthermore, we define a pathway by which GAS6/AXL signaling uses lateral activation of the met proto-oncogene (MET) through SRC proto-oncogene nonreceptor tyrosine kinase to maximize cellular invasion. Clinically, AXL expression in primary tumors of ccRCC patients correlates with aggressive tumor behavior and patient lethality. These findings provide an alternative model for SRC and MET activation by growth arrest-specific 6 in ccRCC and identify AXL as a therapeutic target driving the aggressive phenotype in renal clear cell carcinoma.targeted therapy | kidney cancer | VHL | hepatocellular carcinoma K idney cancer is a leading cause of cancer-related deaths in the United States. Metastasis to distant organs including the lung, bone, liver, and brain is the primary cause of death in kidney cancer patients, as only 12% of patients with metastatic kidney cancer will survive past 5 y, in comparison with 92% of patients with a localized disease (1). Because kidney cancer is chemo-and radiation-resistant, targeted therapies are needed for the prevention and management of metastatic kidney cancer.The von Hippel-Lindau (VHL)-hypoxia-inducible transcription factor (HIF) pathway is a critical regulator of clear cell renal cell carcinoma (ccRCC) tumor initiation and metastasis. VHL is a classic tumor suppressor controlling tumor initiation in ∼90% of ccRCC tumors (2, 3). VHL is the substrate recognition component of an E3 ubiquitin ligase complex containing the elongins B and C (4, 5), Cullin-2 (6), and Rbx1 (7) that targets the hydroxylated, oxygen-sensitive α-subunits of HIFs (HIF-1, -2, and -3) for ubiquitination and degradation by the 26S proteasome (8, 9). Thus, the primary function ascribed to VHL is the regulation of HIF protein stability. In VHL-deficient tumors, HIF transcriptional activity is constitutively active and contributes to both ccRCC tumor initiation and metastasis (8-11). Although many downstream HIF targets controlling ccRCC tumor initiation have been defined, key targets involved in ccRCC metastasis remain to be identified.AXL, a member of the TAM family of receptor tyrosine kinases (RTKs), has recently been described as an essential mediator of cancer metastasis. Additionally, AXL has been reported to mediate RTK crosstalk and resistance to targeted kina...
AIBSTRACTThe RBTN2 LIM-domain protein, originally identified as an oncogenic protein in human T-cell leukemia, is essential for erythropoiesis. A possible role for RBTN2 in transcription during erythropoiesis has been investigated. Direct interaction of the RBTN2 protein was observed in vivo and in vitro with the GATAI or -2 zinc-finger transcription factors, as well as with the basic helix-loop-helix protein TALl. By using mammalian two-hybrid analysis, complexes involving RBTN2, TAL1, and GATA1, together with E47, the basic helix-loop-helix heterodimerization partner of TAL1, could be demonstrated. Thus, a molecular link exists between three proteins crucial for erythropoiesis, and the data suggest that variations in amounts of complexes involving RBTN2, TAL1, and GATAl could be important for erythroid differentiation.
Expression of antibodies inside cells has been used successfully to ablate protein function. This finding suggests that the technology should have an impact on disease treatment and in functional genomics where proteins of unknown function are predicted from genomic sequences. A major hindrance is the paucity of antibodies that function in eukaryotic cells, presumably because the antibodies fold incorrectly in the cytoplasm. To overcome this problem, we have developed an in vivo assay for functional intracellular antibodies using a two-hybrid approach. In this assay, antibody, as single-chain Fv (scFv) linked to a transcriptional transactivation domain, can interact with a target antigen, linked to a LexA-DNA binding domain, and thereby activate a reporter gene. We find that several characterized antibodies can bind their target antigen in eukaryotic cells in this two-hybrid format, and we have been able to isolate intracellular binders from among sets of scFv that can bind antigen in vitro. Furthermore, we show a model selection in which a single scFv was isolated from a mixture of half a million clones, indicating that this is a robust procedure that should facilitate capture of antibody specificities from complex mixtures. The approach can provide the basis for de novo selection of intracellular scFv from libraries, such as those made from spleen RNA after immunization with antigen, for intracellular analysis of protein function based only on genomic or cDNA sequences.intrabodies ͉ single-chain Fv ͉ therapy ͉ functional genomics
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