CTGF is a therapeutic target for metastatic melanoma.
Metastatic melanoma remains a devastating disease with a 5-year survival rate of less than five percent. Despite recent advances in targeted therapies for melanoma, only a small percentage of melanoma patients experience durable remissions. Therefore, it is critical to identify new therapies for the treatment of advanced melanoma. Here, we define connective tissue growth factor (CTGF) as a therapeutic target for metastatic melanoma. Clinically, CTGF expression correlates with tumor progression and is strongly induced by hypoxia through HIF-1 and HIF-2-dependent mechanisms. Genetic inhibition of CTGF in human melanoma cells is sufficient to significantly reduce orthotopic tumor growth, as well as metastatic tumor growth in the lung of severe combined immunodeficient (SCID) mice. Mechanistically, inhibition of CTGF decreased invasion and migration associated with reduced matrix metalloproteinase-9 expression. Most importantly, the anti-CTGF antibody, FG-3019, had a profound inhibitory effect on the progression of established metastatic melanoma. These results offer the first preclinical validation of anti-CTGF therapy for the treatment of advanced melanoma and underscore the importance of tumor hypoxia in melanoma progression.Oncogene advance online publication, 25 February 2013; doi:10.1038/onc.2013.47.
View details for PubMedID 23435419
- Blood and bones Osteoblastic HIF signaling regulates erythropoiesis CELL CYCLE 2012; 11 (12): 2221-2222
The HIF Signaling Pathway in Osteoblasts Directly Modulates Erythropoiesis through the Production of EPO
2012; 149 (1): 63-74
Osteoblasts are an important component of the hematopoietic microenvironment in bone. However, the mechanisms by which osteoblasts control hematopoiesis remain unknown. We show that augmented HIF signaling in osteoprogenitors results in HSC niche expansion associated with selective expansion of the erythroid lineage. Increased red blood cell production occurred in an EPO-dependent manner with increased EPO expression in bone and suppressed EPO expression in the kidney. In contrast, inactivation of HIF in osteoprogenitors reduced EPO expression in bone. Importantly, augmented HIF activity in osteoprogenitors protected mice from stress-induced anemia. Pharmacologic or genetic inhibition of prolyl hydroxylases1/2/3 in osteoprogenitors elevated EPO expression in bone and increased hematocrit. These data reveal an unexpected role for osteoblasts in the production of EPO and modulation of erythropoiesis. Furthermore, these studies demonstrate a molecular role for osteoblastic PHD/VHL/HIF signaling that can be targeted to elevate both HSCs and erythroid progenitors in the local hematopoietic microenvironment.
View details for DOI 10.1016/j.cell.2012.01.051
View details for Web of Science ID 000302235400010
View details for PubMedID 22464323
A central role for hypoxic signaling in cartilage, bone, and hematopoiesis.
Current osteoporosis reports
2011; 9 (2): 46-52
Hypoxic signaling plays an essential role in maintaining oxygen homeostasis and cell survival. Hypoxia-inducible transcription factors HIF-1 and HIF-2 are central mediators of the cellular response to hypoxia by regulating the expression of genes controlling metabolic adaptation, oxygen delivery, and survival in response to oxygen deprivation. Recent studies have identified an important role for HIF-1 and HIF-2 in the regulation of skeletal development, bone formation, and regeneration, as well as joint formation and homeostasis. In addition, overexpression of HIF-1 and HIF-2 is clinically associated with osteosarcoma and osteoarthritis. Together, these findings implicate hypoxic signaling as a central regulator of bone biology and disease.
View details for DOI 10.1007/s11914-011-0047-2
View details for PubMedID 21360287
AXL Is an Essential Factor and Therapeutic Target for Metastatic Ovarian Cancer
2010; 70 (19): 7570-7579
The receptor tyrosine kinase AXL is thought to play a role in metastasis; however, the therapeutic efficacy of an AXL-targeting agent remains largely untested in metastatic disease. In this study, we defined AXL as a therapeutic target for metastatic ovarian cancer. AXL is primarily expressed in metastases and advanced-stage human ovarian tumors but not in normal ovarian epithelium. Genetic inhibition of AXL in human metastatic ovarian tumor cells is sufficient to prevent the initiation of metastatic disease in vivo. Mechanistically, inhibition of AXL signaling in animals with metastatic disease results in decreased invasion and matrix metalloproteinase activity. Most importantly, soluble human AXL receptors that imposed a specific blockade of the GAS6/AXL pathway had a profound inhibitory effect on progression of established metastatic ovarian cancer without normal tissue toxicity. These results offer the first genetic validation of GAS6/AXL targeting as an effective strategy for inhibition of metastatic tumor progression in vivo. Furthermore, this study defines the soluble AXL receptor as a therapeutic candidate agent for treatment of metastatic ovarian cancer, for which current therapies are ineffective.
View details for DOI 10.1158/0008-5472.CAN-10-1267
View details for Web of Science ID 000282647700021
View details for PubMedID 20858715
Regulation of the Histone Demethylase JMJD1A by Hypoxia-Inducible Factor 1 alpha Enhances Hypoxic Gene Expression and Tumor Growth
MOLECULAR AND CELLULAR BIOLOGY
2010; 30 (1): 344-353
The hypoxia-inducible transcription factors (HIFs) directly and indirectly mediate cellular adaptation to reduced oxygen tensions. Recent studies have shown that the histone demethylase genes JMJD1A, JMJD2B, and JARID1B are HIF targets, suggesting that HIFs indirectly influence gene expression at the level of histone methylation under hypoxia. In this study, we identify a subset of hypoxia-inducible genes that are dependent on JMJD1A in both renal cell and colon carcinoma cell lines. JMJD1A regulates the expression of adrenomedullin (ADM) and growth and differentiation factor 15 (GDF15) under hypoxia by decreasing promoter histone methylation. In addition, we demonstrate that loss of JMJD1A is sufficient to reduce tumor growth in vivo, demonstrating that histone demethylation plays a significant role in modulating growth within the tumor microenvironment. Thus, hypoxic regulation of JMJD1A acts as a signal amplifier to facilitate hypoxic gene expression, ultimately enhancing tumor growth.
View details for DOI 10.1128/MCB.00444-09
View details for Web of Science ID 000272569200028
View details for PubMedID 19858293
Hypoxia-Inducible Factor 2 Regulates Hepatic Lipid Metabolism
MOLECULAR AND CELLULAR BIOLOGY
2009; 29 (16): 4527-4538
In mammals, the liver integrates nutrient uptake and delivery of carbohydrates and lipids to peripheral tissues to control overall energy balance. Hepatocytes maintain metabolic homeostasis by coordinating gene expression programs in response to dietary and systemic signals. Hepatic tissue oxygenation is an important systemic signal that contributes to normal hepatocyte function as well as disease. Hypoxia-inducible factors 1 and 2 (HIF-1 and HIF-2, respectively) are oxygen-sensitive heterodimeric transcription factors, which act as key mediators of cellular adaptation to low oxygen. Previously, we have shown that HIF-2 plays an important role in both physiologic and pathophysiologic processes in the liver. HIF-2 is essential for normal fetal EPO production and erythropoiesis, while constitutive HIF-2 activity in the adult results in polycythemia and vascular tumorigenesis. Here we report a novel role for HIF-2 in regulating hepatic lipid metabolism. We found that constitutive activation of HIF-2 in the adult results in the development of severe hepatic steatosis associated with impaired fatty acid beta-oxidation, decreased lipogenic gene expression, and increased lipid storage capacity. These findings demonstrate that HIF-2 functions as an important regulator of hepatic lipid metabolism and identify HIF-2 as a potential target for the treatment of fatty liver disease.
View details for DOI 10.1128/MCB.00200-09
View details for Web of Science ID 000268310000016
View details for PubMedID 19528226
Bringing H2AX into the Angiogenesis Family
2009; 15 (6): 459-461
The cell's ability to sense and respond to DNA damage is critical to maintain homeostasis and prevent the development of cancer. Paradoxically, Economopoulou et al. recently reported that a DNA damage response protein, H2AX, promotes tumor growth and angiogenesis.
View details for DOI 10.1016/j.ccr.2009.05.004
View details for Web of Science ID 000266686500003
View details for PubMedID 19477424
Stable expression of HIF-1 alpha in tubular epithelial cells promotes interstitial fibrosis
AMERICAN JOURNAL OF PHYSIOLOGY-RENAL PHYSIOLOGY
2008; 295 (4): F1023-F1029
Chronic hypoxia accelerates renal fibrosis. The chief mediator of the hypoxic response is hypoxia-inducible factor 1 (HIF-1) and its oxygen-sensitive component HIF-1alpha. HIF-1 regulates a wide variety of genes, some of which are closely associated with tissue fibrosis. To determine the specific role of HIF-1 in renal fibrosis, we generated a knockout mouse in which tubular epithelial expression of von Hippel-Lindau tumor suppressor (VHL), which acts as a ubiquitin ligase to promote proteolysis of HIF-1alpha, was targeted. We investigated the effect of VHL deletion (i.e., stable expression of HIF-1alpha) histologically and used the anti-HIF-1alpha agent [3-(5'-hydroxymethyl-2'-furyl)-1-benzyl indazole] (YC-1) to test whether inhibition of HIF-1alpha could represent a novel approach to treating renal fibrosis. The area of renal fibrosis was significantly increased in a 5/6 renal ablation model of VHL-/- mice and in all VHL-/- mice at least 60 wk of age. Injection of YC-1 inhibited the progression of renal fibrosis in unilateral ureteral obstruction model mice. In conclusion, HIF-1alpha appears to be a critical contributor to the progression of renal fibrosis and could be a useful target for its treatment.
View details for DOI 10.1152/ajprenal.90209.2008
View details for Web of Science ID 000259913000019
View details for PubMedID 18667485
Hypoxia-inducible factor-2 regulates vascular tumorigenesis in mice
2008; 27 (40): 5354-5358
The von Hippel-Lindau tumor suppressor pVHL regulates the stability of hypoxia-inducible factors (HIF)-1 and -2, oxygen-sensitive basic helix-loop-helix transcription factors, which mediate the hypoxic induction of angiogenic growth factors such as vascular endothelial growth factor. Loss of pVHL function results in constitutive activation of HIF-1 and HIF-2 and is associated with the development of highly vascularized tumors in multiple organs. We have used a conditional gene-targeting approach to investigate the relative contributions of HIF-1 and HIF-2 to VHL-associated vascular tumorigenesis in a mouse model of liver hemangiomas. Here we demonstrate genetically that conditional inactivation of HIF-2alpha suppressed the development of VHL-associated liver hemangiomas and that angiogenic gene expression in hepatocytes is predominantly regulated by HIF-2 and not by HIF-1. These findings suggest that HIF-2 is the dominant HIF in the pathogenesis of VHL-associated vascular tumors and that pharmacologic targeting of HIF-2 may be an effective strategy for their treatment.
View details for DOI 10.1038/onc.2008.160
View details for Web of Science ID 000259096100008
View details for PubMedID 18490920
The role of hypoxia-inducible factors in tumorigenesis
CELL DEATH AND DIFFERENTIATION
2008; 15 (4): 678-685
Hypoxia-inducible factors (HIFs) are essential mediators of the cellular oxygen-signaling pathway. They are heterodimeric transcription factors consisting of an oxygen-sensitive alpha subunit (HIF-alpha) and a constitutive beta subunit (HIF-beta) that facilitate both oxygen delivery and adaptation to oxygen deprivation by regulating the expression of genes that control glucose uptake, metabolism, angiogenesis, erythropoiesis, cell proliferation, and apoptosis. In most experimental models, the HIF pathway is a positive regulator of tumor growth as its inhibition often results in tumor suppression. In clinical samples, HIF is found elevated and correlates with poor patient prognosis in a variety of cancers. In summary, HIF regulates multiple aspects of tumorigenesis, including angiogenesis, proliferation, metabolism, metastasis, differentiation, and response to radiation therapy, making it a critical regulator of the malignant phenotype.
View details for DOI 10.1038/cdd.2008.21
View details for Web of Science ID 000254041800010
View details for PubMedID 18259193
Regulation of iron homeostasis by the hypoxia-inducible transcription factors (HIFs)
JOURNAL OF CLINICAL INVESTIGATION
2007; 117 (7): 1926-1932
Iron is essential for many biological processes, including oxygen delivery, and its supply is tightly regulated. Hepcidin, a small peptide synthesized in the liver, is a key regulator of iron absorption and homeostasis in mammals. Hepcidin production is increased by iron overload and decreased by anemia and hypoxia; but the molecular mechanisms that govern the hepcidin response to these stimuli are not known. Here we establish that the von Hippel-Lindau/hypoxia-inducible transcription factor (VHL/HIF) pathway is an essential link between iron homeostasis and hepcidin regulation in vivo. Through coordinate downregulation of hepcidin and upregulation of erythropoietin and ferroportin, the VHL-HIF pathway mobilizes iron to support erythrocyte production.
View details for DOI 10.1172/JCI31370
View details for Web of Science ID 000247837700024
View details for PubMedID 17557118
Hypoxia-inducible factor-2 (HIF-2) regulates hepatic erythropoietin in vivo
JOURNAL OF CLINICAL INVESTIGATION
2007; 117 (4): 1068-1077
Erythropoiesis is critically dependent on erythropoietin (EPO), a glycoprotein hormone that is regulated by hypoxia-inducible factor (HIF). Hepatocytes are the primary source of extrarenal EPO in the adult and express HIF-1 and HIF-2, whose roles in the hypoxic induction of EPO remain controversial. In order to define the role of HIF-1 and HIF-2 in the regulation of hepatic EPO expression, we have generated mice with conditional inactivation of Hif-1alpha and/or Hif-2alpha (Epas1) in hepatocytes. We have previously shown that inactivation of the von Hippel-Lindau tumor suppressor pVHL, which targets both HIFs for proteasomal degradation, results in increased hepatic Epo production and polycythemia independent of Hif-1alpha. Here we show that conditional inactivation of Hif-2alpha in pVHL-deficient mice suppressed hepatic Epo and the development of polycythemia. Furthermore, we found that physiological Epo expression in infant livers required Hif-2alpha but not Hif-1alpha and that the hypoxic induction of liver Epo in anemic adults was Hif-2alpha dependent. Since other Hif target genes such phosphoglycerate kinase 1 (Pgk) were Hif-1alpha dependent, we provide genetic evidence that HIF-1 and HIF-2 have distinct roles in the regulation of hypoxia-inducible genes and that EPO is preferentially regulated by HIF-2 in the liver.
View details for DOI 10.1172/JCI30117
View details for Web of Science ID 000245451700029
View details for PubMedID 17404621
Rend cyst development in mice with conditional inactivation of the von Hippel-Lindau tumor suppressor
2006; 66 (5): 2576-2583
Inactivation of the von Hippel-Lindau tumor suppressor, pVHL, is associated with both hereditary and sporadic renal cysts and renal cell carcinoma, which are commonly thought to arise from the renal proximal tubule. pVHL regulates the protein stability of hypoxia-inducible factor (HIF)-alpha subunits and loss of pVHL function leads to HIF stabilization. The role of HIF in the development of VHL-associated renal lesions remains to be determined. To investigate the functional consequences of pVHL inactivation and the role of HIF signaling in renal epithelial cells, we used the phosphoenolpyruvate carboxykinase (PEPCK) promoter to generate transgenic mice in which Cre-recombinase is expressed in the renal proximal tubule and in hepatocytes. We found that conditional inactivation of VHL in PEPCK-Cre mutants resulted in renal cyst development that was associated with increased erythropoietin levels and polycythemia. Increased expression of the HIF target gene erythropoietin was limited to the liver, whereas expression of carbonic anhydrase 9 and multidrug resistance gene 1 was up-regulated in the renal cortex of mutant mice. Inactivation of the HIF-alpha binding partner, arylhydrocarbon receptor nuclear translocator (Arnt), but not Hif-1alpha, suppressed the development of renal cysts. Here, we present the first mouse model of VHL-associated renal disease that will provide a basis for further genetic studies to define the molecular events that are required for the progression of VHL-associated renal cysts to clear cell renal cell carcinoma.
View details for DOI 10.1158/0008-5472.CAN-05-3241
View details for Web of Science ID 000235826400014
View details for PubMedID 16510575
Inactivation of the arylhydrocarbon receptor nuclear translocator (Arnt) suppresses von Hippel-Lindau disease-associated vascular tumors in mice
MOLECULAR AND CELLULAR BIOLOGY
2005; 25 (8): 3163-3172
Patients with germ line mutations in the VHL tumor suppressor gene are predisposed to the development of highly vascularized tumors within multiple tissues. Loss of pVHL results in constitutive activation of the transcription factors HIF-1 and HIF-2, whose relative contributions to the pathogenesis of the VHL phenotype have yet to be defined. In order to examine the role of HIF in von Hippel-Lindau (VHL)-associated vascular tumorigenesis, we utilized Cre-loxP-mediated recombination to inactivate hypoxia-inducible factor-1alpha (Hif-1alpha) and arylhydrocarbon receptor nuclear translocator (Arnt) genes in a VHL mouse model of cavernous liver hemangiomas and polycythemia. Deletion of Hif-1alpha did not affect the development of vascular tumors and polycythemia, nor did it suppress the increased expression of vascular endothelial growth factor (Vegf) and erythropoietin (Epo). In contrast, phosphoglycerokinase (Pgk) expression was substantially decreased, providing evidence for target gene-dependent functional redundancy between different Hif transcription factors. Inactivation of Arnt completely suppressed the development of hemangiomas, polycythemia, and Hif-induced gene expression. Here, we demonstrate genetically that the development of VHL-associated vascular tumors in the liver depends on functional ARNT. Furthermore, we provide evidence that individual HIF transcription factors may play distinct roles in the development of specific VHL disease manifestations.
View details for DOI 10.1128/MCB.25.8.3163-3172.2005
View details for Web of Science ID 000228138500025
View details for PubMedID 15798202
Putative intestine-specific enhancers located in 5 ' sequence of the CDX1 gene regulate CDX1 expression in the intestine
AMERICAN JOURNAL OF PHYSIOLOGY-GASTROINTESTINAL AND LIVER PHYSIOLOGY
2004; 286 (5): G872-G880
CDX1 is a homeobox transcription factor that plays a critical role in intestinal epithelial cell growth and differentiation. CDX1 gene expression is tightly regulated in a temporal and cell-type specific manner. However, very little is known about the regulatory mechanisms that direct CDX1 gene expression in the intestine. To elucidate these mechanisms, we employed a series of transgenic mouse studies using the 5' flanking sequences of the human CDX1 gene. Transgenic mice containing nucleotides between -5667 and +68 relative to the transcription start site of the CDX1 gene demonstrated ectopic expression of the transgene in the brain and gastric smooth muscle. However, transgenic expression of the nucleotides -15601 to +68 of the CDX1 gene was restricted to the intestinal epithelium, which was identical to endogenous CDX1 gene expression. Taken together, the upstream sequences between -15601 and -5667 contain regulatory elements that direct transgene expression specifically to the intestinal epithelium. Furthermore, DNase I hypersensitivity assays revealed two active chromatin regions in the CDX1 gene (hypertensive sites 1 and 2) located at approximately -5.8 and -6.8 kb upstream of the CDX1 gene, respectively, which may function as potential intestine-specific enhancers.
View details for DOI 10.1152/ajpgi.00326.2003
View details for Web of Science ID 000220693200023
View details for PubMedID 14715525
An essential role of Th1 responses and interferon gamma in infection-mediated suppression of neoplastic growth
CANCER BIOLOGY & THERAPY
2003; 2 (6): 687-693
We had previously demonstrated that in mice acute toxoplasmosis leads to systemic inhibition of angiogenesis and, consequently, strong suppression of neoplastic growth. Here we investigated the role of Th1 cytokines, in particular interferon gamma (IFN-gamma), in this phenomenon. Besides toxoplasma, neoplastic growth was readily blocked during acute infection with other Th1 response-inducing pathogens such as Listeria monocytogenes and lymphocytic choriomeningitis virus (LCMV). In contrast, chronic infection with LCMV (when Th1 responses were strongly suppressed) and acute infection with Schistosoma mansoni (when Th2 responses predominated) afforded no anti-tumor protection. To corroborate the involvement of Th1 cytokines in infection-mediated suppression of neoplastic growth, we utilized mice deficient in interleukin-10 (IL10), a suppressor of Th1 responses. When challenged with B16 cells concomitantly with toxoplasma infection, both IL10-null and wild type mice exhibited resistance to neoplastic growth. However, tumors borne by IL10-null animals were even smaller than those borne by their wild type counterparts. This enhanced resistance correlated with dramatically elevated levels of circulating IFN-gamma, a principal Th1 cytokine. Furthermore, while interleukin-12 and tumor necrosis factor a were dispensable for tumor suppression, in animals deficient in IFN-gamma production or signaling, tumor growth and neovascularization were markedly enhanced. Interestingly, the enhancement was also apparent in uninfected animals suggesting that IFN-gamma and its anti-angiogenic effects underlie both infection-dependent and -independent tumor surveillance.
View details for Web of Science ID 000188846200017
View details for PubMedID 14688478
DNA methylation down-regulates CDX1 gene expression in colorectal cancer cell lines
JOURNAL OF BIOLOGICAL CHEMISTRY
2002; 277 (39): 35795-35800
CDX1 is a homeobox protein that inhibits proliferation of intestinal epithelial cells and regulates intestine-specific genes involved in differentiation. CDX1 expression is developmentally and spatially regulated, and its expression is aberrantly down-regulated in colorectal cancers and colon cancer-derived cell lines. However, very little is known about the molecular mechanism underlying the regulation of CDX1 gene expression. In this study, we characterized the CDX1 gene structure and identified that its gene promoter contained a typical CpG island with a CpG observed/expected ratio of 0.80, suggesting that the CDX1 gene is a target of aberrant methylation. Alterations of DNA methylation in the CDX1 gene promoter were investigated in a series of colorectal cancer cell lines. Combined Bisulfite Restriction Analysis (COBRA) and bisulfite sequencing analysis revealed that the CDX1 promoter is methylated in CDX1 non-expressing colorectal cancer cell lines but not in human normal colon tissue and T84 cells, which express CDX1. Treatment with 5'-aza-2'-deoxycytidine (5-azaC), a DNA methyltransferase inhibitor, induced CDX1 expression in the colorectal cancer cell lines. Furthermore, de novo methylation was determined by establishing stably transfected clones of the CDX1 promoter in SW480 cells and demethylation by 5-azaC-activated reporter gene expression. These results indicate that aberrant methylation of the CpG island in the CDX1 promoter is one of the mechanisms that mediate CDX1 down-regulation in colorectal cancer cell lines.
View details for DOI 10.1074/jbc.M205567200
View details for Web of Science ID 000178275100005
View details for PubMedID 12124393