HIF Inactivation of p53 in Ovarian Cancer Can Be Reversed by Topotecan, Restoring Cisplatin and Paclitaxel Sensitivity.

Ovarian cancer growth under hypoxic conditions results in hypoxia-inducible factor-1α (HIF1α) stabilization. HIF1α is an adverse prognostic factor that may contribute to worse outcomes via its capacity to bind to p53, potentially blocking p53-mediated apoptosis. We determined whether HIF1α-p53 binding occurred in hypoxic ovarian cancer cell lines, and if this blocked p53 transcriptional activity. Topotecan (TPT), used in the treatment of ovarian cancer, inhibits HIF1α translation via a topoisomerase-1 (TOPO1)-dependent mechanism. We examined if TPT knockdown of HIF1α restored p53 transcriptional function. TPT effects on HIF1α and p53-related transcriptional targets were assessed by PCR. Associations between TPT effects and TOPO1 expression levels were examined by Western blots and knockdown by siRNA. RNA-binding protein immunoprecipitation was used to assess if TOPO1 was resident on HIF1α mRNA. We determined if sublethal doses of TPT, used to knockdown HIF1α, reversed hypoxia-related cisplatin and paclitaxel resistance (XTT assay). Flow cytometry was used to assess HIF1α-mediated upregulation of ABCB1 and ABCB5 efflux pump expression. We found that HIF1α binding to, and inhibition of, p53 transcriptional activity in hypoxic ovarian cancer cells was associated with drug resistance. TPT-mediated downregulation of HIF1α in hypoxic cells required TOPO1 resident on HIF1α mRNA, restored p53 transcriptional activity, downregulated ABCB1/ABCB5 cell surface expression, and reversed hypoxia-related cisplatin and paclitaxel resistance. IMPLICATIONS: TPT-mediated reduction of HIF1α accumulation in hypoxic ovarian cancer cell lines restores p53 tumor-suppressor function, offering a novel approach to reverse chemoresistance. Further clinical investigation is warranted.

A novel antitumor activity of deguelin targeting the insulin-like growth factor (IGF) receptor pathway via up-regulation of IGF-binding protein-3 expression in breast cancer.

In this study, we investigated the antitumor effects of deguelin in several human breast cancer cells in vitro and in vivo. Deguelin inhibited cell viability and the anchorage-dependent and anchorage-independent colony formation of triple-negative (MDA-MB-231 and MDA-MB-468) and triple-positive (MCF-7) breast cancer cells, and it significantly reduced the growth of MCF-7 cell xenograft tumors. The induction of apoptosis, inhibition of insulin-like growth factor-1 receptor (IGF-1R) signaling activation, and up-regulation of IGF-binding protein-3 (IGFBP-3) expression may be associated with deguelin-mediated antitumor effects. Our findings suggest a potential therapeutic use for deguelin in patients with triple-negative breast cancer and for those with breast cancers who are sensitive to endocrine- and HER2-targeted therapies.

BMP9 induces EphrinB2 expression in endothelial cells through an Alk1-BMPRII/ActRII-ID1/ID3-dependent pathway: implications for hereditary hemorrhagic telangiectasia type II.

ALK1 (ACVRL1) is a member of the TGFß receptor family and is expressed predominantly by arterial endothelial cells (EC). Mutations in ACVRL1 are responsible for hereditary hemorrhagic telangiectasia type 2 (HHT2), a disease manifesting as fragile vessels, capillary overgrowth, and numerous arterio-venous malformations. Arterial EC also express EphrinB2, which has multiple roles in vascular development and angiogenesis and is known to be reduced in ACVRL1 knockout mice. Using an in vitro angiogenesis model we find that the Alk1 ligand BMP9 induces EphrinB2 in EC, and this is entirely dependent on expression of Alk1 and at least one of the co-receptors BMPRII or ActRII. BMP9 induces both ID1 and ID3, and both are necessary for full induction of EphrinB2. Loss of Alk1 or EphrinB2 results in increased arterial-venous anastomosis, while loss of Alk1 but not EphrinB2 results in increased VEGFR2 expression and enhanced capillary sprouting. Conversely, BMP9 blocks EC sprouting and this is dependent on Alk1, BMPRII/ActRII and ID1/ID3. Finally, notch signaling overcomes the loss of Alk1-restoring EphrinB2 expression in EC, and curbing excess sprouting. Thus, in an in vitro model of HHT2, loss of Alk1 blocks BMP9 signaling, resulting in reduced EphrinB2 expression, enhanced VEGFR2 expression, and misregulated EC sprouting and anastomosis.

Insulin-like growth factor binding protein-3 suppresses vascular endothelial growth factor expression and tumor angiogenesis in head and neck squamous cell carcinoma.

Angiogenesis, the process by which new blood vessels are recruited to existing ones, is essential for tumor development. Insulin-like growth factor (IGF) binding protein-3 (IGFBP-3), which modulates bioavailability of IGF, has been studied for its potential role in angiogenesis during tissue regeneration and cancer development. In this study, we assessed the role of IGFBP-3 in tumor angiogenesis in head and neck squamous cell carcinoma (HNSCC) and human umbilical vein endothelial cells (HUVECs) using adenoviral (Ad-BP3) and recombinant (rBP3) IGFBP-3. Using an in vivo orthotopic tongue tumor model, we confirmed that both Ad-BP3 and rBP3 suppress the growth of UMSCC38 HNSCC cells in vivo. Ad-BP3 inhibited vascularization in tongue tumors and chorio-allantoic membrane, and suppressed angiogenesis-stimulating activities in UMSCC38 cells. In HUVECs, Ad-BP3 decreased migration, invasion, and tube formation. rBP3 also suppressed production of vascular endothelial growth factor (VEGF) in HUVECs and UMSCC38 cells. IGFBP-3-GGG, a mutant IGFBP-3 with loss of IGF binding capacity, suppressed VEGF production. In addition, we found that IGFBP-3 suppressed VEGF expression, even in mouse embryonic fibroblasts from an IGF-1R-null mouse. Finally, we demonstrated that IGFBP-3-GGG inhibits tumor angiogenesis and growth to the same degree as wild-type IGFBP-3. Taken together, these results support the hypothesis that IGFBP-3 has anti-angiogenic activity in HNSCC, at least in part due to IGF-independent suppression of VEGF production from vascular endothelial cells and cancer cells.

Mouse and human strategies identify PTPN14 as a modifier of angiogenesis and hereditary haemorrhagic telangiectasia.

Hereditary haemorrhagic telangiectasia (HHT) [corrected] is a vascular dysplasia syndrome caused by mutations in transforming growth factor-ß/bone morphogenetic protein pathway genes, ENG and ACVRL1. HHT [corrected] shows considerable variation in clinical manifestations, suggesting environmental and/or genetic modifier effects. Strain-specific penetrance of the vascular phenotypes of Eng(+/-) and Tgfb1(-/-) mice provides further support for genetic modification of transforming growth factor-ß pathway deficits. We previously identified variant genomic loci, including Tgfbm2, which suppress prenatal vascular lethality of Tgfb1(-/-) mice. Here we show that human polymorphic variants of PTPN14 within the orthologous TGFBM2 locus influence clinical severity of HHT, [corrected] as assessed by development of pulmonary arteriovenous malformation. We also show that PTPN14, ACVRL1 and EFNB2, encoding EphrinB2, show interdependent expression in primary arterial endothelial cells in vitro. This suggests an involvement of PTPN14 in angiogenesis and/or arteriovenous fate, acting via EphrinB2 and ACVRL1/activin receptor-like kinase 1. These findings contribute to a deeper understanding of the molecular pathology of HHT [corrected] in particular and to angiogenesis in general.

Antiangiogenic antitumor activities of IGFBP-3 are mediated by IGF-independent suppression of Erk1/2 activation and Egr-1-mediated transcriptional events.

Most antiangiogenic therapies currently being evaluated in clinical trials target the vascular endothelial growth factor pathway; however, the tumor vasculature can acquire resistance to vascular endothelial growth factor-targeted therapy by shifting to other angiogenesis mechanisms. Insulin-like growth factor binding protein-3 (IGFBP-3) has been reported to suppress tumor growth and angiogenesis by both IGF-dependent and IGF-independent mechanisms; however, understanding of its IGF-independent mechanisms is limited. We observed that IGFBP-3 blocked tumor angiogenesis and growth in non-small cell lung cancer and head and neck squamous cell carcinoma. Conditioned media from an IGFBP-3-treated non-small cell lung cancer cell line displayed a significantly decreased capacity to induce HUVEC proliferation and aortic sprouting. In cancer cells, IGFBP-3 directly interacted with Erk1/2, leading to inactivation of Erk1/2 and Elk-1, and suppressed transcription of early growth response protein 1 and its target genes, basic fibroblast growth factor and platelet-derived growth factor. These data suggest that IGF-independent Erk1/2 inactivation and decreased IGFBP-3-induced Egr-1 expression block the autocrine and paracrine loops of angiogenic factors in vascular endothelial and cancer cells. Together, these findings provide a molecular framework of IGFBP-3's IGF-independent antiangiogenic antitumor activities. Future studies are needed for development of IGFBP-3 as a new line of antiangiogengic cancer drug.

Estradiol stimulates capillary formation by human endothelial progenitor cells: role of estrogen receptor-{alpha}/{beta}, heme oxygenase 1, and tyrosine kinase.

Endothelial progenitor cells (EPCs) repair damaged endothelium and promote capillary formation, processes involving receptor tyrosine kinases (RTKs) and heme oxygenase 1 (HO-1). Because estradiol augments vascular repair, we hypothesize that estradiol increases EPC proliferation and capillary formation via RTK activation and induction of HO-1. Physiological concentrations of estradiol (10 nmol/L) increased EPC-induced capillary sprout and lumen formation in matrigel/fibrin/collagen systems. Propyl-pyrazole-triol (PPT; 100 nmol/L; estrogen receptor [ER]-alpha agonist), but not diarylpropionitrile (ER-beta agonist), mimicked the stimulatory effects of estradiol on capillary formation, and methyl-piperidino-pyrazole (ER-alpha antagonist) abolished the effects of estradiol and PPT. Three different RTK activators (vascular endothelial growth factor, hepatocyte growth factor, and stromal derived growth factor 1) mimicked the capillary-stimulating effects of estradiol and PPT. SU5416 (RTK inhibitor) blocked the stimulatory effects of estradiol and PPT on capillary formation. Estradiol increased HO-1 expression by 2- to 3-fold, an effect blocked by SU5416, and PPT mimicked the effects of estradiol on HO-1. The ability of estradiol to enhance capillary formation, increase expression of HO-1, and augment phosphorylation of extracellular signal-regulated kinase 1/2, Akt, and vascular endothelial growth factor receptor 2 was mimicked by its cell-impermeable analog BSA estradiol. Actinomycin (transcription inhibitor) did not alter the effects of estradiol on RTK activity or vascular endothelial growth factor secretion. We conclude that estradiol via ER-alpha promotes EPC-mediated capillary formation by a mechanism that involves nongenomic activation of RTKs and HO-1 activation. Estradiol in particular and ER-alpha agonists in general may promote healing of injured vascular beds by promoting EPC activity leading to more rapid endothelial recovery and capillary formation after injury.

Integration of the beta-catenin-dependent Wnt pathway with integrin signaling through the adaptor molecule Grb2.

BACKGROUND: THE COMPLEXITY OF WNT SIGNALING LIKELY STEMS FROM TWO SOURCES: multiple pathways emanating from frizzled receptors in response to wnt binding, and modulation of those pathways and target gene responsiveness by context-dependent signals downstream of growth factor and matrix receptors. Both rac1 and c-jun have recently been implicated in wnt signaling, however their upstream activators have not been identified. METHODOLOGY/PRINCIPAL FINDINGS: Here we identify the adapter protein Grb2, which is itself an integrator of multiple signaling pathways, as a modifier of beta-catenin-dependent wnt signaling. Grb2 synergizes with wnt3A, constitutively active (CA) LRP6, Dvl2 or CA-beta-catenin to drive a LEF/TCF-responsive reporter, and dominant negative (DN) Grb2 or siRNA to Grb2 block wnt3A-mediated reporter activity. MMP9 is a target of beta-catenin-dependent wnt signaling, and an MMP9 promoter reporter is also responsive to signals downstream of Grb2. Both a jnk inhibitor and DN-c-jun block transcriptional activation downstream of Dvl2 and Grb2, as does DN-rac1. Integrin ligation by collagen also synergizes with wnt signaling as does overexpression of Focal Adhesion Kinase (FAK), and this is blocked by DN-Grb2. CONCLUSIONS/SIGNIFICANCE: These data suggest that integrin ligation and FAK activation synergize with wnt signaling through a Grb2-rac-jnk-c-jun pathway, providing a context-dependent mechanism for modulation of wnt signaling.

The unfolded protein response is necessary but not sufficient to compensate for defects in disulfide isomerization.

Pdi1p (protein-disulfide isomerase) is a folding assistant of the endoplasmic reticulum (ER) that catalyzes disulfide formation and the isomerization of incorrect disulfides. Its disulfide forming activity is its essential function in Saccharomyces cerevisiae. A truncation mutant (Pdi1a') that is competent in disulfide formation but deficient in catalyzing isomerization has only a small effect on growth, although the maturation of isomerase-requiring substrates (carboxypeptidase Y) is impaired (Xiao, R., Wilkinson, B., Solovyov, A., Winther, J. R., Holmgren, A., Lundstrom-Ljung, J., and Gilbert, H. F. (2004) J. Biol. Chem. 279, 49780-49786). We show here that there are multiple ways to compensate for defects in disulfide formation and isomerization in the ER. Genes of the unfolded protein response are induced, and deletions of the nonessential IRE1 or HAC1 genes are synthetically lethal. Diploid synthetic lethality analysis by microarray (dSLAM) using PDIa' and a temperature-sensitive mutant of PDIa' as query mutations reveals a group of 130 synthetically lethal genes. Only 10 of these correspond to genes clearly associated with the unfolded protein response. More than half are involved in vesicle traffic, not only out of and into the ER but anterograde and retrograde traffic from most cellular compartments. This suggests that defects in protein maturation in one intracellular compartment may be compensated for by adjusting vesicular traffic patterns throughout the cell.

Identification of insulin-like growth factor binding protein-3 as a farnesyl transferase inhibitor SCH66336-induced negative regulator of angiogenesis in head and neck squamous cell carcinoma.

The farnesyl transferase inhibitor (FTI) SCH66336 has been shown to have antitumor activities in head and neck squamous cell carcinoma (HNSCC) in vitro and in vivo. However, its mechanism of action has not been well defined. Here, we report that the insulin-like growth factor (IGF) binding protein (IGFBP)-3 mediates antitumor activities of SCH66336 in HNSCC by inhibiting angiogenesis. SCH66336 significantly suppressed HNSCC tumor growth and angiogenesis via mechanisms that are independent of H-Ras and RhoB. By inducing IGFBP-3 secretion from HNSCC cells, this compound suppresses angiogenic activities of endothelial cells, including vessel formation in chorioallantoic membranes of chick, endothelial cell sprouting from chick aorta, and capillary tube formation of human umbilical vascular endothelial cells (HUVEC). Knockdown of IGFBP-3 expression in HNSCC cells by RNA interference or depletion of IGFBP-3 in HUVECs by neutralizing antibody effectively blocked the effects of IGFBP-3 secreted from SCH66336-treated HNSCC cells on HUVECs. These findings suggest that IGFBP-3 could be a primary target for antitumor activities of FTIs and that IGFBP-3 is an effective therapeutic approach against angiogenesis in HNSCC.

Methylation of CpG islands in the rat 7-dehydrocholesterol reductase promoter suppresses transcriptional activation.

In mammals, 7-dehydrocholesterol reductase (Dhcr7) is the terminal enzyme in cholesterol biosynthesis. We previously reported that the Dhcr7 proximal promoter (-179 to +1), which contains CpG islands, is responsible for sterol-mediated expression of the rat gene. In the present study, we examined whether methylation of this region affects the transcriptional activity of the Dhcr7 gene. In vitro DNA methylation of the Dhcr7 promoter and luciferase-reporter assays showed that DNA methylation of the CpG islands suppressed transcription. Furthermore, treatment of the methylated Dhcr7 promoter with the demethylating agent, 5-aza-2'-deoxycytidine (5-Aza-CdR), reversed the suppression of promoter activity. These results indicate that methylation of the CpG islands is an important transcriptional regulatory mechanism in the Dhcr7 promoter.

Alterations in transcription clusters underlie development of bladder cancer along papillary and nonpapillary pathways.

Bladder cancer develops in the urothelial lining from intraurothelial preneoplasia via two pathways, papillary and nonpapillary, which correspond to nonaggressive and aggressive forms of the disease. Because these two forms of cancer may develop via distinct molecular events, we examined the gene expression patterns in the development of bladder cancer from preneoplasia along papillary and nonpapillary pathways. The expression profiles of 19 pairs of RNA samples from adjacent urothelium and tumors were analyzed using cDNA microarrays. For selected genes their expressions were verified on a cohort of 251 bladder cancer patients using tissue microarray and immunohistochemistry and were related to clinicopathological parameters including follow-up data. We identified alterations in seven gene clusters controlling proliferation, differentiation, and programmed cell death that were common for papillary and nonpapillary cancer. In contrast, genes controlling cellular and stromal interactions were altered in the nonpapillary cancer. The expression levels of only two genes from this group could be used to define an aggressive form of the disease. Tumors characterized by the low expression of e-cadherin and the high expression of DNA alpha-topoisomerase II had a high propensity for distant metastasis and were associated with poor survival.

A novel mutation of the human 7-dehydrocholesterol reductase gene reduces enzyme activity in patients with holoprosencephaly.

Defects in cholesterol biosynthesis genes are recognized as a leading cause for holoprosencephaly (HPE). Previous reports suggest that mutations of human 7-dehydrocholesterol reductase (Dhcr7), which catalyzes the final step of cholesterol biosynthesis, may cause HPE [Clin. Genet. 53 (1998) 155]. To determine whether Dhcr7 mutations are involved in HPE pathogenesis, we analyzed the sequence of exon 9, which contains both a catalytic domain and a mutational hot spot. We examined 36 prematurely terminated fetuses with HPE at their gestation ages in the range from 21 to 33 weeks by single strand conformation polymorphism analysis and DNA sequencing. A novel missense mutation was identified: G344D. Dhcr7 enzyme assays using overexpressed recombinant mutant proteins revealed altered enzyme activity. Mutant G344D harbored less than 50% of enzyme activity compared with the control. Two previously reported mutations, R404C and G410S, abolished enzyme activity. These results suggest that mutation of the Dhcr7 gene is involved in HPE pathogenesis.