The Effects of Severe Hypoxia on Glycolytic Flux and Enzyme Activity in a Model of Solid Tumors.

Solid tumors contend with, and adapt to, a hostile micro-environment that includes limited availability of nutrient fuels and oxygen. The presence of hypoxia (O2 <5%) stabilizes the transcription factor Hif1 and results in numerous cellular adaptations including increased flux of glucose through glycolysis. Increasingly, more sophisticated analysis of tumor oxygenation has revealed large gradients of oxygen tension and significant regions under severe hypoxia (O2 ∼0.1%). The present investigation has demonstrated a significant increase in the glycolytic flux rate when tumor spheroids were exposed to 0.1% O2 . The severe hypoxia was associated with uniform pimonidazole adduct formation and elevated levels of Hif1α and c-Myc. This resulted in elevated expression of GLUT and MCT transporters, in addition to increased activity of PFK1 in comparison to that observed in normoxia. However, the protein expression and enzymatic capacity of HK2, G6PDH, PK, and LDH were all reduced by severe hypoxia. Clearly, the effects of exposure to severe hypoxia lead to a significantly abridged Hif1 response, yet one still able to elevate glycolytic flux and prevent loss of intermediates to anabolism. J. Cell. Biochem. 117: 1890-1901, 2016. © 2016 Wiley Periodicals, Inc.

Targeting p21-activated kinase 1 (PAK1) to induce apoptosis of tumor cells.

p21-activated kinases (PAKs) are serine/threonine protein kinases that serve as important mediators of Rac and Cdc42 GTPase function as well as pathways required for Ras-driven tumorigenesis. PAK1 has been implicated in signaling by growth factor receptors and morphogenetic processes that control cell polarity, invasion, and actin cytoskeleton organization. To better understand the role of PAK1 in tumorigenesis, PAK1 genomic copy number and expression were determined for a large panel of breast, lung, and head and neck tumors. PAK1 genomic amplification at 11q13 was prevalent in luminal breast cancer, and PAK1 protein expression was associated with lymph node metastasis. Breast cancer cells with PAK1 genomic amplification rapidly underwent apoptosis after inhibition of this kinase. Strong nuclear and cytoplasmic PAK1 expression was also prevalent in squamous nonsmall cell lung carcinomas (NSCLCs), and selective PAK1 inhibition was associated with delayed cell-cycle progression in vitro and in vivo. NSCLC cells were profiled using a library of pathway-targeted small-molecule inhibitors, and several synergistic combination therapies, including combination with antagonists of inhibitor of apoptosis proteins, were revealed for PAK1. Dual inhibition of PAK1 and X chromosome-linked inhibitor of apoptosis efficiently increased effector caspase activation and apoptosis of NSCLC cells. Together, our results provide evidence for dysregulation of PAK1 in breast and squamous NSCLCs and a role for PAK1 in cellular survival and proliferation in these indications.

CD31 angiogenesis and combined expression of HIF-1α and HIF-2α are prognostic in primary clear-cell renal cell carcinoma (CC-RCC), but HIFα transcriptional products are not: implications for antiangiogenic trials and HIFα biomarker studies in primary CC-RCC.

Hypoxia-inducible factors, HIF-1α and HIF-2α, are expressed in the majority of clear-cell renal cell carcinoma (CC-RCC). In vitro, HIFα isoforms regulate a differential set of genes, and their effects in vivo within CC-RCC tumours may affect outcome. The role of angiogenesis and HIFα transcriptional products, including those involved in cell metabolism and morphological dedifferentiation have not been extensively investigated and might have relevance to the development of antiangiogenic or anti-HIFα trials in primary CC-RCC, either before or after radical nephrectomy. We analysed 168 consecutive clear-cell renal tumours from 1983 to 1999 within tissue microarrays and assessed expression of HIF-1α and HIF-2α together with the protein expression of seven of their target genes (BNIP3, CA9, Cyclin D1, GLUT-1, LDH5, Oct-4 and VEGF). The expression of these factors was compared with patient overall survival and CD31 angiogenesis. We found that HIFα antigenicity deteriorated with the age of the paraffin block (P < 0.0001) and in tumours from 1983 to 1992 was deemed not to be reliable. Similar findings were found in aged archival osteosarcoma samples. This might have important implications for retrospective biomarker studies that rely on archival tissue material. HIF-1α(HIGH)/HIF-2α(LOW) tumours had a worse overall survival compared with HIF-1α(LOW)/HIF-2α(LOW) tumours (P = 0.04). Surprisingly, on multivariate analysis, high levels of CD31(+) angiogenesis was shown to be an independent prognostic marker of increased overall survival (P = 0.003). We propose that better differentiation of vascular endothelium may be a reflection of a greater production of vessel stabilization factors versus pro-angiogenic factors, and therefore a less aggressive phenotype.

New mechanism for Notch signaling to endothelium at a distance by Delta-like 4 incorporation into exosomes.

Notch signaling is an evolutionary conserved pathway that is mediated by cell-cell contact. It is involved in a variety of developmental processes and has an essential role in vascular development and angiogenesis. Delta-like 4 (Dll4) is a Notch ligand that is up-regulated during angiogenesis. It is expressed in endothelial cells and regulates the differentiation between tip cells and stalk cells of neovasculature. Here, we present evidence that Dll4 is incorporated into endothelial exosomes. It can also be incorporated into the exosomes of tumor cells that overexpress Dll4. These exosomes can transfer the Dll4 protein to other endothelial cells and incorporate it into their cell membrane, which results in an inhibition of Notch signaling and a loss of Notch receptor. Transfer of Dll4 was also shown in vivo from tumor cells to host endothelium. Addition of Dll4 exosomes confers a tip cell phenotype on the endothelial cell, which results in a high Dll4/Notch-receptor ratio, low Notch signaling, and filopodia formation. This was further evidenced by increased branching in a tube-formation assay and in vivo. This reversal in phenotype appears to enhance vessel formation and is a new form of signaling for Notch ligands that expands their signaling potential beyond cell-cell contact.

Expression of vascular Notch ligands Delta-like 4 and Jagged-1 in glioblastoma.

AIMS: The coordinated expression of the Notch ligands Delta-like 4 (Dll4) and Jagged (Jag)1 is believed to define appropriate endothelial sensitivity to vascular endothelial growth factor (VEGF). Preclinical data suggest that Dll4-Notch signalling may confer resistance to anti-VEGF therapy with bevacizumab, and Jag1 may antagonize Dll4-Notch. The aims of this study were to characterize the expression of Dll4 and Jag1 in primary glioblastomas. METHODS AND RESULTS: Immunohistochemistry was performed on 40 glioblastomas and normal brain using validated antibodies against Dll4 and Jag1. In-situ hybridization for Dll4 was performed on serial sections and compared with protein expression. Dll4 expression was localized to the cytoplasm and membrane of endothelial cells in all glioblastomas; it was weak or absent in normal brain. Jag1 expression was observed in the cytoplasm and membrane of glomeruloid and non-glomeruloid endothelial cells from 76% and 67% of glioblastomas, respectively. However, endothelial Jag1 expression was less intense and less prevalent than Dll4. There was no association between Dll4 and Jag1 expression. CONCLUSIONS: In summary, Dll4 and Jag1 are expressed in glioblastoma vasculature. These data may define subsets of glioblastoma that might be sensitive (Dll4(+) /Jag1(+) ) or resistant (Dll4(+) /Jag1(-) ) to bevacizumab. Our data also suggest that anti-Dll4 therapy should be evaluated experimentally in glioblastoma.

Gastrointestinal differentiation marker Cytokeratin 20 is regulated by homeobox gene CDX1.

CDX1 is a transcription factor that plays a key role in intestinal development and differentiation. However, the downstream targets of CDX1 are less well defined than those of its close homologue, CDX2. We report here the identification of downstream targets of CDX1 using microarray gene-expression analysis and other approaches. Keratin 20 (KRT20), a member of the intermediate filament and a well-known marker of intestinal differentiation, was initially identified as one of the genes likely to be directly regulated by CDX1. CDX1 and KRT20 mRNA expression were significantly correlated in a panel of 38 colorectal cancer cell lines. Deletion and mutation analysis of the KRT20 promoter showed that the minimum regulatory region for the control of KRT20 expression by CDX1 is within 246 bp upstream of the KRT20 transcription start site. ChIP analysis confirmed that CDX1 binds to the predicted CDX elements in this region of the KRT20 promoter in vivo. In addition, immunohistochemistry showed expression of CDX1 parallels that of KRT20 in the normal crypt, which further supports their close relationship. In summary, our observations strongly imply that KRT20 is directly regulated by CDX1, and therefore suggest a role for CDX1 in maintaining differentiation in intestinal epithelial cells. Because a key feature of the development of a cancer is an unbalanced program of proliferation and differentiation, dysregulation of CDX1 may be an advantage for the development of a colorectal carcinoma. This could, therefore, explain the relatively frequent down regulation of CDX1 in colorectal carcinomas by hypermethylation.

Expression of vascular notch ligand delta-like 4 and inflammatory markers in breast cancer.

Delta-like ligand 4 (Dll4) is a Notch ligand that is predominantly expressed in the endothelium. Evidence from xenografts suggests that inhibiting Dll4 may overcome resistance to antivascular endothelial growth factor therapy. The aims of this study were to characterize the expression of Dll4 in breast cancer and assess whether it is associated with inflammatory markers and prognosis. We examined 296 breast adenocarcinomas and 38 ductal carcinoma in situ tissues that were represented in tissue microarrays. Additional whole sections representing 10 breast adenocarcinomas, 10 normal breast tissues, and 16 angiosarcomas were included. Immunohistochemistry was then performed by using validated antibodies against Dll4, CD68, CD14, Dendritic Cell-Specific Intercellular adhesion molecule-3-Grabbing Non-integrin (DC-SIGN), CD123, neutrophil elastase, CD31, and carbonic anhydrase 9. Dll4 was selectively expressed by intratumoral endothelial cells in 73% to 100% of breast adenocarcinomas, 18% of in situ ductal carcinomas, and all lactating breast cases, but not normal nonlactating breast. High intensity of endothelial Dll4 expression was a statistically significant adverse prognostic factor in univariate (P = 0.002 and P = 0.01) and multivariate analyses (P = 0.03 and P = 0.04) of overall survival and relapse-free survival, respectively. Among the inflammatory markers, only CD68 and DC-SIGN were significant prognostic factors in univariate (but not multivariate) analyses of overall survival (P = 0.01 and 0.002, respectively). In summary, Dll4 was expressed by endothelium associated with breast cancer cells. In these retrospective subset analyses, endothelial Dll4 expression was a statistically significant multivariate prognostic factor.

Nuclear localization of factor inhibitor hypoxia-inducible factor in prostate cancer is associated with poor prognosis.

PURPOSE: We determined the role of factor inhibiting hypoxia-inducible factor-1 in prostate cancer specimens. MATERIALS AND METHODS: A tissue microarray of 152 prostate cancers was constructed and stained for factor inhibiting hypoxia-inducible factor-1, hypoxia-inducible factor-1α and 2α, and glucose transporter 1 as a prototypical downstream target of hypoxia-inducible factor-1α. Correlation analysis was done between these variables, and between factor inhibiting hypoxia-inducible factor-1, and clinical and pathological variables, including prostate specific antigen as a surrogate of recurrence. RESULTS: Factor inhibiting hypoxia-inducible factor-1 was expressed in the cytoplasm and/or the nucleus in 86.5% of tumors, including exclusive cytoplasmic expression in 51.3% and exclusive nuclear expression in 5.3%. Any nuclear and exclusive expression of factor inhibiting hypoxia-inducible factor was associated with poor prognosis on univariate analysis (p = 0.007 and 0.042, respectively). On multivariate analysis men with nuclear expression in tumors were twice as likely to experience recurrence (p = 0.034). CONCLUSIONS: Factor inhibiting hypoxia-inducible factor-1 is widely expressed in prostate tumors. Its differential subcellular expression suggests that regulation of its expression is an important factor in the activity of the hypoxia-inducible factor pathway. Its modulation may help treat hypoxia-inducible factor driven aggressive prostate cancer.

Expression of key hypoxia sensing prolyl-hydroxylases PHD1, -2 and -3 in pancreaticobiliary cancer.

AIMS: Tumour hypoxia is associated with an aggressive phenotype and resistance to chemotherapy and radiotherapy. The aim was to investigate whether key hypoxia sensing prolyl hydroxylases PHD1, PHD2 and PHD3 are dysregulated in pancreaticobiliary cancers, and to evaluate their potential clinical significance. METHODS AND RESULTS: Formalin-fixed human pancreatic tissue from 120 consecutive patients undergoing pancreatic resections between June 2001 and June 2006 was constructed into tissue microarrays. Expression of PHD1, PHD2 and PHD3 was analysed using immunohistochemistry and correlated with clinicopathological variables and disease-specific overall survival. PHD1, PHD2 and PHD3 were significantly overexpressed in pancreaticobiliary tumours compared with normal pancreatic ductal tissues (P = 0.03, P < 0.0001 and P < 0.0001, respectively). PHD3 expression in tumour tissue was associated with a trend towards worse overall disease-specific survival in ampullary adenocarcinomas (P = 0.035) and pancreatic adenocarcinomas (P = 0.084). Absence of PHD1 expression was significantly associated with perineural invasion in pancreatic adenocarcinomas (P = 0.02) and a trend towards significance was also seen for absence of PHD2 expression in pancreatic adenocarcinomas (P = 0.04). CONCLUSIONS: Our results provide the first clinical evidence that PHD1, PHD2 and PHD3 may be involved in pancreaticobiliary tumorigenesis.

Induction of the vascular endothelial growth factor pathway in the brain of adults with fatal falciparum malaria is a non-specific response to severe disease.

AIMS: Pathological or neuroprotective mechanisms in the brain in severe malaria may arise from microvascular obstruction with malaria-parasitized erythrocytes. This study aimed to investigate the role of hypoxia and induction of the vascular endothelial growth factor (VEGF) pathway in the neuropathophysiology of severe malaria. METHODS AND RESULTS: Immunohistochemistry was performed on post mortem brain tissue sections from 20 cases of severe malaria and examined for the expression of transcriptional regulators of VEGF [hypoxia-inducible factor-1 alpha (HIF-1alpha), HIF-2alpha], DEC-1, VEGF, VEGF receptors 1 and 2, and the activated, phosphorylated VEGF receptor 2 (pKDR). HIFs showed limited protein expression and/or translocation to cell nuclei in severe malaria, but DEC-1, which is more stable and regulated by HIF-1alpha, was observed. There was heterogeneous expression of VEGF and its receptors in severe malaria and non-malarial disease controls. pKDR expression on vessels was greater in malaria cases than in controls but did not correlate with parasite sequestration. VEGF uptake by malaria parasites was observed. CONCLUSIONS: VEGF and its receptor expression levels in severe malaria reflect a non-specific response to severe systemic disease. Potential manipulation of events at the vasculature by the parasite requires further investigation.

BHLHB2 controls Bdnf promoter 4 activity and neuronal excitability.

Brain-derived neurotrophic factor (BDNF), via activation of TrkB receptors, mediates vital physiological functions in the brain, ranging from neuronal survival to synaptic plasticity, and has been implicated in the pathophysiology of neurodegenerative disorders. Although transcriptional regulation of the BDNF gene (Bdnf) has been extensively studied, much remains to be understood. We discovered a sequence within Bdnf promoter 4 that binds the basic helix-loop-helix protein BHLHB2 and is a target for BHLHB2-mediated transcriptional repression. NMDA receptor activation de-repressed promoter 4-mediated transcription and correlated with reduced occupancy of the promoter by BHLHB2 in cultured hippocampal neurons. Bhlhb2 gene -/- mice showed increased hippocampal exon 4-specific Bdnf mRNA levels compared with +/+ littermates under basal and activity-dependent conditions. Bhlhb2 knock-out mice also showed increased status epilepticus susceptibility, suggesting that BHLHB2 alters neuronal excitability. Together, these results support a role for BHLHB2 as a new modulator of Bdnf transcription and neuronal excitability.

Nuclear and membrane expression of the angiogenesis regulator delta-like ligand 4 (DLL4) in normal and malignant human tissues.

AIMS: Delta-like ligand 4 (DLL4) is one of five known Notch ligands in mammals and interacts predominantly with Notch 1. DLL4 is induced by vascular endothelial growth factor (VEGF) and acts downstream of VEGF as a 'brake' on VEGF-induced vessel growth, forming an autoregulatory negative feedback loop inactivating VEGF. This action was believed to occur only in vascular development, raising hopes that DLL4 could be a specific drug target for controlling vessel growth in tumours and other pathological conditions. Our aim was to pursue this by raising a monoclonal antibody to the internal domain of DLL4 and assess its distribution in normal and malignant tissues in comparison with antibodies against the external domain of DLL4. METHODS AND RESULTS: The anti-DLL4 monoclonal antibody was raised using conventional mouse hybridoma techniques. The antibody has been fully characterized by Western blotting and transfectant immunostaining. It has also been comprehensively compared with other antibodies against both the internal and external domains of DLL4. The antigen is widely expressed on human tissues not only on endothelium but also on epithelium and stromal cells. Indeed, in our comprehensive survey only pulmonary alveoli failed to express DLL4. Of a wide range of malignancies, most also expressed DLL4 on tumour cells with a predominantly cytoplasmic pattern, although a number also displayed nuclear positivity. CONCLUSIONS: Contrary to previous beliefs, DLL4 is widely distributed in tissues other than vessels including many malignancies. Furthermore, the molecule is internalized on binding its receptor and often transported to the nucleus. These findings raise many interesting possibilities for further study of DLL4 and its potential as a therapeutic target.

Relation of a hypoxia metagene derived from head and neck cancer to prognosis of multiple cancers.

Affymetrix U133plus2 GeneChips were used to profile 59 head and neck squamous cell cancers. A hypoxia metagene was obtained by analysis of genes whose in vivo expression clustered with the expression of 10 well-known hypoxia-regulated genes (e.g., CA9, GLUT1, and VEGF). To minimize random aggregation, strongly correlated up-regulated genes appearing in >50% of clusters defined a signature comprising 99 genes, of which 27% were previously known to be hypoxia associated. The median RNA expression of the 99 genes in the signature was an independent prognostic factor for recurrence-free survival in a publicly available head and neck cancer data set, outdoing the original intrinsic classifier. In a published breast cancer series, the hypoxia signature was a significant prognostic factor for overall survival independent of clinicopathologic risk factors and a trained profile. The work highlights the validity and potential of using data from analysis of in vitro stress pathways for deriving a biological metagene/gene signature in vivo.

BNIP3 as a progression marker in primary human breast cancer; opposing functions in in situ versus invasive cancer.

PURPOSE: BNIP3 is involved in cell death and cell survival via autophagy. Its perinecrotic localization within ductal carcinoma in situ (DCIS) suggests an involvement in neoplastic cellular adaptation to low oxygen tension. This study has investigated the role of BNIP3 in normal and neoplastic breast. EXPERIMENTAL DESIGN: Whole sections from 11 normal breast and microarrayed tissue cores from 81 DCIS and 251 invasive carcinomas were stained for BNIP3 and hypoxia-inducible factor-1alpha. The pattern and level of BNIP3 expression were correlated with clinicopathologic variables and hypoxia-inducible factor-1alpha. RESULTS: BNIP3 expression was significantly up-regulated in the cytoplasm of DCIS and invasive carcinoma compared with normal breast (P = 0.0005 and P < 0.0001, respectively). Nuclear BNIP3 expression was associated with smaller tumor size (P = 0.04), low tumor grade (P = 0.005), and estrogen receptor positivity (P = 0.008) in invasive tumors. Nuclear BNIP3 expression was also associated with a longer disease-free survival among low-grade and estrogen receptor-positive tumors. (P = 0.03 and 0.04, respectively). Conversely, nuclear BNIP3 expression in DCIS was associated with a 3-fold increase in recurrence and a shorter disease-free survival (P = 0.03). CONCLUSIONS: Up-regulation of BNIP3 expression in DCIS and invasive carcinoma suggests a significant role in breast tumor progression. Its association with good survival outcome in invasive carcinoma but with an increased risk of recurrence and shorter disease-free survival in DCIS may suggest a pivotal switch from a cell death to survival function during the transition from preinvasive to invasive breast cancer.

Cytoplasmic location of factor-inhibiting hypoxia-inducible factor is associated with an enhanced hypoxic response and a shorter survival in invasive breast cancer.

INTRODUCTION: Hypoxia-inducible factor (HIF)-1alpha levels in invasive breast carcinoma have been shown to be an adverse prognostic indicator. Cellular HIF-1alpha activity is regulated by factor-inhibiting hypoxia-inducible factor 1 (FIH-1). In hypoxia, FIH-1 hydroxylation of Asn803 within the C-terminal transactivation domain does not occur and HIF-1alpha forms a fully active transcriptional complex. The present study investigates the role of FIH-1 in invasive breast carcinoma and its correlation with hypoxia. METHODS: Microarrayed tissue cores from 295 invasive carcinomas were stained for FIH-1, for HIF-1alpha and for carbonic anhydrase 9. FIH-1 expression was correlated with standard clinicopathological parameters and with the expression of the surrogate hypoxic markers HIF-1alpha and carbonic anhydrase 9. RESULTS: FIH-1 was positive in 239/295 (81%) tumours, 42/295 (14%) exclusively in the nucleus and 54/295 (18%) exclusively in the cytoplasm. Exclusive nuclear FIH-1 expression was significantly inversely associated with tumour grade (P = 0.02) and risk of recurrence (P = 0.04), whereas exclusive cytoplasmic FIH-1 was significantly positively associated with tumour grade (P = 0.004) and carbonic anhydrase 9 expression (P = 0.02). Patients with tumours that excluded FIH-1 from the nucleus had a significantly shorter survival compared with those with exclusive nuclear expression (P = 0.02). Cytoplasmic FIH-1 expression was also an independent poor prognostic factor for disease-free survival. CONCLUSION: FIH-1 is widely expressed in invasive breast carcinoma. As with other HIF regulators, its association between cellular compartmentalization and the hypoxic response and survival suggests that tumour regulation of FIH-1 is an additional important mechanism for HIF pathway activation.

Expression of terminally glycosylated calcitonin receptor-like receptor in uterine leiomyoma: endothelial phenotype and association with microvascular density.

PURPOSE: The role for the hypoxia-inducible angiogenic factor adrenomedullin (AM) in tumor growth and progression has been suggested. Calcitonin receptor-like receptor (CL) is a G protein-coupled receptor (GPCR) that mediates effects of AM, but little information is available on its expression and functional state in human tumors. The present study attempted to determine CL potential for antiangiogenic therapy of uterine leiomyoma. EXPERIMENTAL DESIGN AND RESULTS: GPCR CL is transported to the cell surface and recognized by AM only when terminally/mature glycosylated. The presence and localization of this form of the receptor in tumor and surrounding myometrial tissues obtained from leiomyoma-bearing uteri were examined using deglycosylation, immunoblotting, and immunofluorescence analysis. The mature CL glycoprotein was expressed in both tissues and localized exclusively in normal and tumor endothelium within leiomyoma-bearing uteri. The functionality of the receptor expressed in myometrial microvascular endothelial cells (MMVEC) was examined in vitro using receptor internalization and angiogenic assays. The mature CL glycoprotein expressed by primary MMVECs was functional because AM interacted with this GPCR and induced its internalization as well as angiogenic effects (proliferation and migration) in MMVECs in vitro. Finally, the levels of tissue-expressed mature CL glycoprotein as a functional form of this GPCR were analyzed by immunoblotting. The expression of this functional form of the receptor in vivo was significantly decreased (P = 0.01) in leiomyoma tissue, and this was concurrent with the decrease in microvascular density (measured by Chalkley counting) in tumor compared with surrounding myometrium (P = 0.031). CONCLUSIONS: Our findings suggest that GPCR CL mediates angiogenic effects of AM in myometrium and that further evaluation of the properties of the CL expressed in both normal and tumor endothelium in vivo may be essential before targeting this endothelial GPCR for antiangiogenic therapies.

Role of Delta-like 4 in Jagged1-induced tumour angiogenesis and tumour growth.

Delta-like 4 (DLL4) and Jagged1 (JAG1) are two key Notch ligands implicated in tumour angiogenesis. They were shown to have opposite effects on mouse retinal and adult regenerative angiogenesis. In tumours, both ligands are upregulated but their relative effects and interactions in tumour biology, particularly in tumour response to therapeutic intervention are unclear. Here we demonstrate that DLL4 and JAG1 displayed equal potency in stimulating Notch target genes in HMEC-1 endothelial cells but had opposing effects on sprouting angiogenesis in vitro. Mouse DLL4 or JAG1 expressed in glioblastoma cells decreased tumour cell proliferation in vitro but promoted tumour growth in vivo. mDLL4-expressing tumours showed fewer but larger vessels whereas mJAG1-tumours produced more vessels. In both tumour types pericyte coverage was decreased but the vessels were more perfused. Both ligands increased tumour resistance towards anti-VEGF therapy but the resistance was higher in mDLL4-tumours versus mJAG1-tumours. However, their sensitivity to the therapy was restored by blocking Notch signalling with dibenzazepine. Importantly, anti-DLL4 antibody blocked the effect of JAG1 on tumour growth and increased vessel branching in vivo. The mechanism behind the differential responsiveness was due to a positive feedback loop for DLL4-Notch signalling, rendering DLL4 more dominant in activating Notch signalling in the tumour microenvironment. We concluded that DLL4 and JAG1 promote tumour growth by modulating tumour angiogenesis via different mechanisms. JAG1 is not antagonistic but utilises DLL4 in tumour angiogenesis. The results suggest that anti-JAG1 therapy should be explored in conjunction with anti-DLL4 treatment in developing anti-Notch therapies in clinics.

Relation of erythropoietin and erythropoietin receptor expression to hypoxia and anemia in head and neck squamous cell carcinoma.

PURPOSE: The use of erythropoietin in head and neck squamous cell carcinoma (HNSCC) has been associated with poor survival. This study examines the protein and mRNA expression of erythropoietin and erythropoietin receptor in HNSCC and their relation to hypoxia, hemoglobin (Hb), and clinical outcome. EXPERIMENTAL DESIGN: The immunohistochemical expression of erythropoietin and erythropoietin receptor was assessed in 151 cases of HNSCC. Expression was compared with the hypoxia-dependent proteins hypoxia-inducible factor-1alpha (HIF-1alpha) and carbonic anhydrase-9 (CA-9) and correlated with clinical outcome. The mRNA expression of erythropoietin and erythropoietin receptor was measured in paired samples of HNSCC. RESULTS: Erythropoietin and erythropoietin receptor were expressed in 95% and 99% of tumors, respectively. Using a weighed expression score, there was a positive correlation between erythropoietin and erythropoietin receptor expression (r = 0.18, P = 0.03). HIF-1alpha (r = 0.38, P < 0.01) and CA-9 (r = 0.26, P = 0.002) correlated with erythropoietin expression, but there was no correlation with erythropoietin receptor. No correlation was found between Hb and erythropoietin (r = 0.07, P = 0.36) or erythropoietin receptor (r = -0.02, P = 0.8), and no survival difference between high and low erythropoietin or erythropoietin receptor expression (P = 0.59 and P = 0.98, respectively). The mRNA expression of erythropoietin (P = 0.03) but not erythropoietin receptor (P = 0.62) was significantly increased in 11 paired samples of HNSCC. CONCLUSION: In vivo, the HIF pathway regulates erythropoietin at the mRNA level but not erythropoietin receptor expression in HNSCC. Anemia does not seem to influence the hypoxic microenvironment of tumors sufficiently to alter the expression of erythropoietin. The effects of exogenous erythropoietin may be acting via receptors expressed on tumor cells in vivo, or on vascular cells, which also express the pathway.

The androgen receptor is significantly associated with vascular endothelial growth factor and hypoxia sensing via hypoxia-inducible factors HIF-1a, HIF-2a, and the prolyl hydroxylases in human prostate cancer.

PURPOSE: Hypoxia regulates key biological processes including angiogenesis via the transcription factor, hypoxia-inducible factor (HIF). In prostate cancer, angiogenesis is also influenced by androgens, and recent cell line studies suggest that this effect is partly mediated by HIF. The study aimed to assess whether a relationship exists in human prostate cancer between expression of the androgen receptor, HIFs, and the key angiogenesis factor, vascular endothelial growth factor (VEGF). EXPERIMENTAL DESIGN: A tissue microarray comprised of 149 radical prostatectomy specimens was constructed. Semiquantitative immunohistochemical analysis was used to assess the expression of the androgen receptor, VEGF and HIF-1a and 2a, and their regulatory prolyl hydroxylase enzymes (PHD1, PHD2, and PHD3). Statistical analysis compared these factors with each other and with prostate-specific antigen relapse. RESULTS: There was a significant correlation between HIF-1a and HIF-2a expression (P = 0.02), and with androgen receptor (P = 0.04 and P < 0.001, respectively) and VEGF expression (P = 0.05 and P < 0.001, respectively). VEGF was also significantly related to the androgen receptor (P = 0.05), whereas PHD2 was inversely related to HIF-2a expression. No significant association was shown between HIF-1a or HIF-2a and time to prostate-specific antigen recurrence (P = 0.20 and P = 0.94, respectively). CONCLUSIONS: These findings confirm the relationship between hypoxia and the androgen receptor in prostate cancer, and show for the first time, the role of HIF-2a in this disease process. They provide clinical evidence to support the recent cell line findings that androgens may regulate VEGF levels through the activation of HIF in androgen-sensitive tumors. Inhibition of both the HIF pathways may provide new therapeutic options in the management of this disease.

CITED4 inhibits hypoxia-activated transcription in cancer cells, and its cytoplasmic location in breast cancer is associated with elevated expression of tumor cell hypoxia-inducible factor 1alpha.

The interaction of hypoxia-inducible factor 1alpha and the CH1 domain of the transcriptional coactivator p300/CBP is necessary for the expression of hypoxia responsive genes and tumor angiogenesis. The transcription factor CITED2 binds p300/CBP at the CH1 domain and functions as a negative regulator of hypoxia signaling by competing with hypoxia-inducible factor 1alpha. CITED4, a recently identified member of the CITED family, binds p300/CBP via the CH1 domain and functions as a coactivator for transcription factor AP-2. Here, we show that CITED4 blocks the binding of hypoxia-inducible factor 1alpha to p300 in vitro and inhibits hypoxia-inducible factor-1alpha transactivation and hypoxia-mediated reporter gene activation. These studies suggest that CITED4 might function as an inhibitor of hypoxia-inducible factor 1alpha. To explore the function of CITED4 in breast cancer, we determined its expression in normal, in situ and invasive breast cancers. We also correlated its expression in 286 invasive breast tumors with clinicopathological, hypoxia markers and survival. In contrast to the nuclear localization of CITED4 in normal breast tissue, breast tumors were characterized by cytoplasmic and nuclear localization. Nuclear CITED4 expression was significantly inversely associated with tumor hypoxia-inducible factor 1alpha (P < 0.05), tumor size (P = 0.03), tumor grade (P = 0.0001), and Chalkley vessel count (P = 0.04). CITED4 showed no significant correlation with patient age (P = 0.45), estrogen receptor (P = 0.11), or epidermal growth factor receptor (P = 0.48). These results show that breast cancer development is characterized by either nuclear loss or cytoplasmic translocation of CITED4, with consequent loss of hypoxia-inducible factor-1alpha transcriptional antagonist activity. This may be an important mechanism by which tumors enhance hypoxia-inducible factor expression and result in an aggressive phenotype.