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Oncogenic targets acting in both tumor cells and tumor stromal cells may offer special therapeutic appeal. Interrogation of the Oncomine database revealed that 52 of 53 human breast carcinomas showed substantial upregulation of WNT family ligand WNT7B. Immunolabeling of human mammary carcinoma showed that WNT7B immunoreactivity was associated with both tumor cells and with tumor-associated macrophages. In the MMTV-PymT mouse model of mammary carcinoma, we found tumor progression relied upon WNT7B produced by myeloid cells in the microenvironment. Wnt7b deletion in myeloid cells reduced the mass and volume of tumors due to a failure in the angiogenic switch. In the tumor overall, there was no change in expression of Wnt/β-catenin pathway target genes, but in vascular endothelial cells (VEC), expression of these genes was reduced, suggesting that VECs respond to Wnt/β-catenin signaling. Mechanistic investigations revealed that failure of the angiogenic switch could be attributed to reduced Vegfa mRNA and protein expression in VECs, a source of VEGFA mRNA in the tumor that was limiting in the absence of myeloid WNT7B. We also noted a dramatic reduction in lung metastasis associated with decreased macrophage-mediated tumor cell invasion. Together, these results illustrated the critical role of myeloid WNT7B in tumor progression, acting at the levels of angiogenesis, invasion, and metastasis. We suggest that therapeutic suppression of WNT7B signaling might be advantageous due to targeting multiple aspects of tumor progression. Cancer Res; 74(11); 2962–73. ©2014 AACR.

Macrophages are required for tissue homeostasis through their role in regulation of the immune response and the resolution of injury. Here we show, using the kidney as a model, that the Wnt pathway ligand Wnt7b is produced by macrophages to stimulate repair and regeneration. When macrophages are inducibly ablated from the injured kidney, the canonical Wnt pathway response in kidney epithelial cells is reduced. Furthermore, when Wnt7b is somatically deleted in macrophages, repair of injury is greatly diminished. Finally, injection of the Wnt pathway regulator Dkk2 enhances the repair process and suggests a therapeutic option. Because Wnt7b is known to stimulate epithelial responses during kidney development, these findings suggest that macrophages are able to rapidly invade an injured tissue and reestablish a developmental program that is beneficial for repair and regeneration.

Circulating erythropoietin (EPO) is mainly produced by the kidneys and mediates erythrogenesis in bone marrow and nonhematopoietic cell survival. EPO is also produced in other tissues where it functions as a paracrine. Moreover, the hypoxic induction of EPO is known to be mediated by HIF-1alpha and HIF-2alpha, but it remains obscure as to which of these two mediators mainly contributes to EPO expression. Thus, we designed in vivo experiments to evaluate the contributions made by HIF-1alpha and HIF-2alpha to EPO expression. In mice exposed to mild whole body hypoxia, HIF-1alpha and HIF-2alpha were both induced in all tissues examined. However, EPO mRNA was expressed in kidney and brain, but not in liver and lung. Likewise, chromatin immunoprecipitation (CHIP) analyses demonstrated that HIF-1alpha or HIF-2alpha binding to the EPO gene increased under hypoxic conditions only in kidney and brain. A comparison of CHIP data and EPO mRNA levels suggested that, during mild hypoxia, renal EPO transcription is induced equally by HIF-1alpha and HIF-2alpha, but that brain EPO is mainly induced during hypoxia by HIF-2alpha. Thus, HIF-1alpha and HIF-2alpha appear to contribute to EPO expression tissue specifically.

We have previously described the distribution pattern of inhibitory synapses on rat jaw-closing (JC) alpha- and gamma-motoneurons. In the present study, we investigated developmental changes in inhibitory synapses on JC motoneurons. We performed a quantitative ultrastructural analysis of putative inhibitory synaptic boutons on JC motoneuron somata by using postembedding immunogold labeling for GABA and glycine. In total, 206, 350, and 497 boutons contacting JC motoneuron somata were analyzed at postnatal days 2 (P2), 11 (P11) and 31 (P31), respectively. The size of the somata increased significantly during postnatal development. The size distribution was bimodal at P31. Mean length of the boutons and percentage of synaptic covering also increased during postnatal development, whereas bouton density did not differ significantly among the three age groups. Synaptic boutons on the somata of JC alpha-motoneurons could be classified into four types: boutons immunoreactive for 1) GABA only, 2) glycine only, 3) both GABA and glycine, and 4) neither GABA nor glycine. There was no developmental change in the proportion of putative inhibitory boutons to the total number of studied boutons. However, the glycine-only boutons increased significantly (15.1% to 27.3%), and the GABA-only boutons decreased significantly (17.7% to 2.6%) during the period from P11 to P31. Our ultrastructural data indicate that the inhibitory synaptic input to JC motoneurons is developmentally regulated and that there is a postnatal switch from GABA to glycine. The postnatal changes revealed in the present study could play an important role in the maturation of the oral motor system.

Qian et al. show that the receptor tyrosine kinase FLT1 is highly expressed in a subset of macrophages enriched in breast cancer metastatic sites. Inhibition of this kinase reduces metastasis to the lungs by blocking signaling via focal adhesion kinase 1 to an inflammatory state in the macrophages centered on signaling from the macrophage growth factor, colony stimulating factor-1., Although the link between inflammation and cancer initiation is well established, its role in metastatic diseases, the primary cause of cancer deaths, has been poorly explored. Our previous studies identified a population of metastasis-associated macrophages (MAMs) recruited to the lung that promote tumor cell seeding and growth. Here we show that FMS-like tyrosine kinase 1 (Flt1, also known as VEGFR1) labels a subset of macrophages in human breast cancers that are significantly enriched in metastatic sites. In mouse models of breast cancer pulmonary metastasis, MAMs uniquely express FLT1. Using several genetic models, we show that macrophage FLT1 signaling is critical for metastasis. FLT1 inhibition does not affect MAM recruitment to metastatic lesions but regulates a set of inflammatory response genes, including colony-stimulating factor 1 (CSF1), a central regulator of macrophage biology. Using a gain-of-function approach, we show that CSF1-mediated autocrine signaling in MAMs is downstream of FLT1 and can restore the tumor-promoting activity of FLT1-inhibited MAMs. Thus, CSF1 is epistatic to FLT1, establishing a link between FLT1 and inflammatory responses within breast tumor metastases. Importantly, FLT1 inhibition reduces tumor metastatic efficiency even after initial seeding, suggesting that these pathways represent therapeutic targets in metastatic disease.

Amphotericin B (AmB) is widely used for treating severe systemic fungal infections. However, long-term AmB treatment is invariably associated with adverse effects such as anemia. The erythropoietin (EPO) suppression by AmB has been proposed to contribute to the development of anemia. However, the mechanism whereby EPO is suppressed remains obscure. In this study, we investigated the possibility that AmB inhibits the transcription of the EPO gene by inactivating HIF-1, which is a known key transcription factor and regulator of EPO expression. EPO mRNA levels were markedly attenuated by AmB treatment both in rat kidneys and in Hep3B cells. AmB inactivated the transcriptional activity of HIF-1α, but did not affect the expression or localization of HIF-1 subunits. Moreover, AmB was found to specifically repress the C-terminal transactivation domain (CAD) of HIF-1α, and this repression by AmB required Asn803, a target site of the factor-inhibiting HIF-1 (FIH); moreover, this repressive effect was reversed by FIH inhibitors. Furthermore, AmB stimulated CAD-FIH interaction and inhibited the p300 recruitment by CAD. We propose that this mechanism underlies the unexplained anemia associated with AmB therapy.

During transplantation, donor organs or cells are subjected to hypoxia. Hypoxia-inducible factor-1 (HIF-1) is essential for cellular adaptation to hypoxia. Immunosuppressive agents should be used for preventing graft rejection, but of these, rapamycin and cyclosporine A have been reported to inhibit HIF-1. We examined whether or not another important immunosuppressant, FK506, inhibits HIF-1. In contrast to cyclosporine A, FK506 neither inhibits HIF-1alpha expression in 8 different cell lines, nor represses the transcriptional activity of HIF-1. Compared with cyclosporine A, FK506 significantly reduced the apoptotic cell death by hypoxia. FK506 could preserve HIF-1 activity in donor organs subjected to hypoxia.

Reactive oxygen species are involved in tumor promotion or apoptosis. In assaying prooxidant or antioxidant activities, cyanide has been commonly used as an inhibitor of mitochondrial oxidases, peroxidases, or Cu,Zn-superoxide dismutase, which have an influence on intracellular levels of reactive oxygen species. It has also been used to chemically mimic hypoxia. On the other hand, glycerol has been widely used as a stabilizer of various enzymes. In particular, glycerol is required to maintain the enzymatic activities of membrane-bound NAD(P)H oxidases extracted from surrounding phospholipids. Since both cyanide and glycerol are relatively inert, they have been used concomitantly regardless of any mutual interference. In this study, we demonstrate that a mixture of glycerol and cyanide reduced cytochrome c and nitroblue tetrazolium, both of which are superoxide anion indicators. The mixture also enhanced the production of superoxide anion in the presence of redox-cycling compounds. Superoxide production by the mixture was confirmed by electron spin resonance spectra. Moreover, the mixture induced lipid peroxidation and hemolysis in human erythrocytes. These results suggest that cyanide and glycerol should be used carefully in reaction systems used to measure superoxide production or antioxidant activity. However, sucrose and sodium azide in combination do not produce such artifacts and thus may be used as an alternative.

Hypoxia-inducible factor-1 (HIF-1), which is present at high levels in human tumors, plays crucial roles in tumor promotion by up-regulating its target genes, which are involved in anaerobic energy metabolism, angiogenesis, cell survival, cell invasion, and drug resistance. Therefore, it is apparent that the inhibition of HIF-1 activity may be a strategy for treating cancer. Recently, many efforts to develop new HIF-1-targeting agents have been made by both academic and pharmaceutical industry laboratories. The future success of these efforts will be a new class of HIF-1-targeting anticancer agents, which would improve the prognoses of many cancer patients. This review focuses on the potential of HIF-1 as a target molecule for anticancer therapy, and on possible strategies to inhibit HIF-1 activity. In addition, we introduce YC-1 as a new anti-HIF-1, anticancer agent. Although YC-1 was originally developed as a potential therapeutic agent for thrombosis and hypertension, recent studies demonstrated that YC-1 suppressed HIF-1 activity and vascular endothelial growth factor expression in cancer cells. Moreover, it halted tumor growth in immunodeficient mice without serious toxicity during the treatment period. Thus, we propose that YC-1 is a good lead compound for the development of new anti-HIF-1, anticancer agents.

YC-1 is a newly developed agent that inhibits platelet aggregation and vascular contraction. Although its effects are independent of nitric oxide (NO), it mimics some of the biological actions of NO. For example, it stimulates soluble guanylate cyclase (sGC) and increases intracellular cGMP concentration. Here, we tested the possibility that YC-1 inhibits hypoxia-inducible factor (HIF)-1-mediated hypoxic responses, as does NO. Hep3B cells were used during the course of this work to observe hypoxic induction of erythropoietin (EPO) and vascular endothelial growth factor (VEGF), and the effects of YC-1 were compared with those of a NO donor, sodium nitropurruside (SNP). In hypoxic cells, YC-1 blocked the induction of EPO and VEGF mRNAs, and inhibited the DNA-binding activity of HIF-1. It suppressed the hypoxic accumulation of HIF-1alpha, but not its mRNA level. It also reduced HIF-1alpha accumulation induced by cobalt and desferrioxamine. Treatment with antioxidants did not recover the HIF-1alpha suppressed by YC-1. We examined whether these effects of YC-1 are related to the sGC/cGMP signal transduction system. Two sGC inhibitors examined failed to block the effects of YC-1, and 8-bromo-cGMP did not mimic actions of YC-1. The effects of YC-1 on the hypoxic responses were comparable with those of SNP. These results suggest that YC-1 and SNP suppressed the hypoxic responses by post-translationally inhibiting HIF-1alpha accumulation. The YC-1 effect may be linked with the metal-related oxygen sensing pathway, and is not due to the stimulation of sGC. This observation implies that the inhibitory effects of YC-1 on hypoxic responses can be developed to suppress EPO-overproduction by tumor cells and tumor angiogenesis.

Macrophages are abundantly found in the tumor microenvironment and enhance malignancy. At distal metastatic sites, our previous studies identified a distinct population of metastasis associated macrophages (MAMs) that promotes tumor cell extravasation, seeding and persistent growth. These macrophages were derived from circulating inflammatory monocytes recruited by CCL2/CCR2 chemokine signaling and directly promote tumor cell extravasation and metastatic seeding in vivo through VEGF production. Our recent studies identified that MAMs express high levels of cell surface FMS-like tyrosine kinase 1 (FLT1, also known as VEGFR1) after their recruitment. Blockade of FLT1 signaling using specific inhibitory antibodies significantly inhibited the metastatic seeding and persistent growth. Using several genetic models of Flt1 deficiency, we show that macrophage specific FLT1 signaling is critical for breast tumor distal metastatic potential. FLT1 is not expressed by other hematopoietic cells and its inhibition did not affect the recruitment of MAMs, which indicated that specific FLT1 signaling in MAMs are important for their metastasis promoting functions. Indeed, we identified that FLT1 regulates a set of inflammatory response genes including Colony Stimulating Factor 1 (CSF1) a central regulator of macrophage biology. Using a genetic gain-of-function approach we show that CSF1 mediated autocrine signaling in MAMs is downstream of FLT1 and can restore the tumor-promoting activity in MAMs even when FLT1 has been inhibited. Together, our data established a link between inflammation and cancer metastasis and suggested the therapeutic potential of targeting these pathways in treating metastatic disease. Citation Format: Bin-Zhi Qian, Hui Zhang, Jiufeng Li, Eun-Jin Yeo, Neil O. Carragher, Anne R. Bresnick, Richard A. Lang, Jeffrey W. Pollard. Macrophage FLT1 mediated inflammatory response determines breast cancer distal metastasis. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Metastasis; 2015 Nov 30-Dec 3; Austin, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(7 Suppl):Abstract nr A13.

The Eyes Absent (EYA) proteins combine transactivation, tyrosine phosphatase, and threonine phosphatase activities in their function as part of a conserved regulatory cascade involved in embryonic organ development. EYA tyrosine phosphatase activity contributes to fly eye development, and vertebrate EYA is involved in promoting DNA damage repair subsequent to genotoxic stress. EYAs are known to be expressed at elevated levels in ovarian and breast cancers. Here, we show that the tyrosine phosphatase activity of the EYAs promotes tumor cell migration, invasion, and transformation. These cellular effects are accompanied by alterations of the actin cytoskeleton and increased levels of active Rac and Cdc42. The invasiveness conferred by EYA is reflected in vivo by inhibition of metastasis seen when EYA3 expression is silenced in the invasive breast cancer cell line MDA-MB-231. Together, our data directly associate the tyrosine phosphatase activity of the EYAs with the oncogenesis-associated cellular properties of motility and invasiveness.

HIF-1alpha is believed to promote tumor growth and metastasis, and many efforts have been made to develop new anticancer agents based on HIF-1alpha inhibition. YC-1 is a widely used HIF-1alpha inhibitor both in vitro and in vivo, and is being developed as a novel class of anticancer drug. However, little is known about the mechanism by which YC-1 degrades HIF-1alpha. As the first step for understanding the mechanism of action of YC-1, we here identified the HIF-1alpha domain responsible for YC-1-induced protein degradation. YC-1 blocked the HIF-1alpha induction by hypoxia, iron chelation, and proteasomal inhibition and also degraded ectopically expressed HIF-1alpha. In deletion analyses, C-terminal HIF-1alpha was found to be sensitively degraded by YC-1. Using a GFP-fusion method, the YC-1-induced degradation domain was identified as the aa. 720-780 region of HIF-1alpha. We next tested the possible involvement of HDAC7 or OS-9 in YC-1-induced HIF-1alpha degradation. However, their binding to HIF-1alpha was not affected by YC-1, suggesting that they are not involved in the YC-1 action. It is also suggested that YC-1 targets a novel pathway regulating HIF-1alpha stability.