Potent Inhibitor Of Angiogenesis Research Articles

Abstract 4480: SerRS is a key inhibitor of VEGFA through both transcriptional and translational controls

Abstract Vascular endothelial growth factor A (VEGFA) is the most important mediator of angiogenesis and a major target for cancer therapy. Recent studies from our lab revealed that seryl-tRNA synthetase (SerRS), by counteracting transcription factor c-Myc in the nucleus, is the crucial transcriptional repressor of VEGFA and a potent inhibitor of angiogenesis (1,2). Here we report that SerRS controls the expression of VEGFA not only in the nucleus but also in the cytoplasm through mediating its translational readthrough. Translational read-through of the VEGFA mRNA generates a novel isoform called VEGF-Ax that, compared with the non-readthrough isoform VEGF165, has a 22-amino acid C-terminal extension and much weakened angiogenic activity (3,4). Interestingly, serine was identified as the amino acid inserted in the place of the UGA stop codon during the read-through (3). In unpublished work, wWe findound that SerRS can bind to the 3’UTR region of the VEGFA mRNA. The binding anchors SerRS to charge serine onto tRNASec (the tRNA for the 21st amino acid selenocysteine that decodes the UGA stop codon), while preventing the charged serine from being modified into selenocysteine. SerRS binding also blocks the release factors (eRFs) from terminating translation at the stop codon. Therefore, SerRS is the key mediator of the translational readthrough that generates VEGF-Ax. Collectively, we have established that a member of the aminoacyl-tRNA synthetase family plays a critical role in regulating VEGF and angiogenesis through both transcriptional and translational controls.

Nutriproteomic Analysis of Hwangmaemok-Induced Antiangiogenic Effect Using Antibody-Arrayed Protein Chip Assay.

We investigated the antiangiogenic effects of Lindera obtusiloba Blume (Hwangmaemok, HMM), which is a plant in the Lauraceae family that is commonly used to treat colds and gastritis. Moreover, given that a recent study reported the inhibitory effects of HMM extract on cancer metastasis, we hypothesized that HMM extract might possess and antiangiogenic function. Thus, this study was conducted to investigate the effects of HMM extract on endothelial cell proliferation, migration, and neovascularization in chick chorioallantoic membrane (CAM), and investigated the molecular mechanism of antiangiogenesis using a ProteoChip-based proteomics technology. To examine the effects of HMM extracts on endothelial cell proliferation and migration, we conducted basic fibroblast growth factor (bFGF)-induced human umbilical vein endothelial cell (HUVEC) proliferation and migration. To assess the molecular mechanism of the antiangiogenic effects of HMM extract, a ProteoChip-based forwarded phase antibody array was employed to identify the differential expression of cell cycle proteins in HMM-treated HUVECs. HMM extract inhibited bFGF-induced HUVEC proliferation and migration in a dose-dependent manner and CAM angiogenesis. The ProteoChip-based antibody microarray data showed upregulation of Nibrin/NBS1 and downregulation of Plk-1 and Cyclin E, which are involved in cell division and controlling the cell cycle in bFGF-induced HUVECs. These data suggest that HMM may be a potent antitumor medicinal herb. The present study demonstrates that the antiangiogenic effect of HMM may be due to suppression of endothelial cell proliferation and migration. Taken together, these results emphasize the potential to use HMM extract as a potent angiogenesis inhibitor to treat cancer.

Potent Vasoconstrictor Kisspeptin-10 Induces Atherosclerotic Plaque Progression and Instability: Reversal by its Receptor GPR54 Antagonist.

BackgroundKisspeptin‐10 (KP‐10), a potent vasoconstrictor and inhibitor of angiogenesis, and its receptor, GPR54, have currently received much attention in relation to pre‐eclampsia. However, it still remains unknown whether KP‐10 could affect atherogenesis.Methods and ResultsWe evaluated the effects of KP‐10 on human umbilical vein endothelial cells, human monocyte‐derived macrophages, human aortic smooth muscle cells in vitro, and atherosclerotic lesions in apolipoprotein E–deficient (ApoE−/−) mice in vivo. KP‐10 significantly increased the adhesion of human monocytes to human umbilical vein endothelial cells, which was significantly inhibited by pretreatment with P234, a GPR54 antagonist. KP‐10 stimulated mRNA expression of tumor necrosis factor‐α, interleukin‐6, monocyte chemotactic protein‐1, intercellular adhesion molecule‐1, vascular adhesion molecule‐1, and E‐selectin in human umbilical vein endothelial cells. KP‐10 significantly enhanced oxidized low‐density lipoprotein–induced foam cell formation associated with upregulation of CD36 and acyl‐CoA:cholesterol acyltransferase‐1 in human monocyte‐derived macrophages. In human aortic smooth muscle cells, KP‐10 significantly suppressed angiotensin II–induced migration and proliferation, but enhanced apoptosis and activities of matrix metalloproteinase (MMP)‐2 and MMP‐9 by upregulation of extracellular signal‐regulated kinase 1 and 2, p38, Bcl‐2‐associated X protein, and caspase‐3. Four‐week‐infusion of KP‐10 into ApoE−/− mice significantly accelerated the development of aortic atherosclerotic lesions with increased monocyte/macrophage infiltration and vascular inflammation as well as decreased intraplaque vascular smooth muscle cells contents. Proatherosclerotic effects of endogenous and exogenous KP‐10 were completely canceled by P234 infusion in ApoE−/− mice.ConclusionsOur results suggest that KP‐10 may contribute to accelerate the progression and instability of atheromatous plaques, leading to plaque rupture. The GPR54 antagonist may be useful for prevention and treatment of atherosclerosis. Thus, the KP‐10/GPR54 system may serve as a novel therapeutic target for atherosclerotic diseases.

Design, synthesis and biological evaluation of new pyrazolyl-ureas and imidazopyrazolecarboxamides able to interfere with MAPK and PI3K upstream signaling involved in the angiogenesis

Taking into account the structure activity relationship information given by our previous studies, we designed and synthesized a small library of pyrazolylureas and imidazopyrazolecarboxamides fluorinated on urea moiety and differently decorated on pyrazole nucleus. All compounds were preliminary screened by Western blotting technique to evaluate their activity on MAPK and PI3K pathways by monitoring ERK1/2, p38MAPK and Akt phosphorylation, and also screened with a wound healing assay to assess their capacity in inhibiting endothelial cell migration, using human umbilical vein endothelial cells stimulated with VEGF. Pyrazoles and imidazopyrazoles did not show the same activity profile. SAR consideration showed that specific substituents and their position in pyrazole nucleus, as well as the type of substituent on the phenylurea moiety play a pivotal role in determining increase or decrease of kinases phosphorylation. On the other hand the loss of flexibility in imidazopyrazole derivatives is responsible for activity potentiation. Screening of the compound library for inhibition of endothelial cell migration, a function required for angiogenesis, showed significant activity for compound 3. This compound might interfere with cell migration by modulating the activity of different upstream target kinases. Therefore, compound 3 represents a potential inhibitor of angiogenesis. Furthermore, it may be used as a tool to identify unknown mediators of endothelial migration and thereby unveiling new therapeutic targets for controlling pathological angiogenesis in diseases such as cancers.

Therapeutic implication of ‘Iturin A’ for targeting MD-2/TLR4 complex to overcome angiogenesis and invasion

Tumor angiogenesis and invasion are deregulated biological processes that drive multistage transformation of tumors from a benign to a life-threatening malignant state activating multiple signaling pathways including MD-2/TLR4/NF-κB. Development of potential inhibitors of this signaling is emerging area for discovery of novel cancer therapeutics. In the current investigation, we identified Iturin A (A lipopeptide molecule from Bacillus megaterium) as a potent inhibitor of angiogenesis and cancer invasion by various in vitro and in vivo methods. Iturin A was found to suppress VEGF, a powerful inducer of angiogenesis and key player in tumor invasion, as confirmed by ELISA, western blot and real time PCR. Iturin A inhibited endothelial tube arrangement, blood capillary formation, endothelial sprouting and vascular growth inside the matrigel. In addition, Iturin A inhibited MMP-2/9 expression in MDA-MB-231 and HUVEC cells. Cancer invasion, migration and colony forming ability were significantly hampered by Iturin A. Expressions of MD-2/TLR4 and its downstream MyD88, IKK-α and NF-κB were also reduced in treated MDA-MB-231 and HUVEC cells. Western blot and immunofluorescence study showed that nuclear accumulation of NF-κB was hampered by Iturin A. MD-2 siRNA or plasmid further confirmed the efficacy of Iturin A by suppressing MD-2/TLR4 signaling pathway. The in silico docking study showed that the Iturin A interacted well with the MD-2 in MD-2/TLR4 receptor complex. Conclusively, inhibition of MD-2/TLR4 complex with Iturin A offered strategic advancement in cancer therapy.

Perindopril Induces TSP-1 Expression in Hypertensive Patients with Endothelial Dysfunction in Chronic Treatment.

Thrombospondin-1 (TSP-1) is a potent endogenous inhibitor of both physiological and pathological angiogenesis, widely studied as a target in drug development for treating cancer. Several studies performed in the cardiovascular field on TSP-1 are contradictory, the role of TSP-1 in the physiopathology of cardiovascular disorders (CVDs) being, for the moment, incompletely understood and may be due to the presence of several domains in its structure which can stimulate many cellular receptors. It has been reported to inhibit NO-mediated signaling and to act on the angiogenesis, tissue perfusion, endothelial cell proliferation, and homeostasis, so we aimed to quantify the effect Perindopril has on TSP-1 plasma levels in hypertensive patients with endothelial dysfunction in comparison with other antihypertensive drugs, such as beta blockers, calcium channel blockers, and diuretics, in a chronic treatment. As a conclusion, patients under treatment with Perindopril had increased plasma levels of TSP-1 compared with other hypertensive patients and with the control group. The results of this study confirms the pleiotropic properties of Perindopril: anti-proliferative, anti-inflammatory, with effects showed by quantifying a single biomarker: TSP-1.

Cryptotanshinone inhibits VEGF-induced angiogenesis by targeting the VEGFR2 signaling pathway

ObjectiveAnti-angiogenesis has been proposed as an important strategy for angiogenesis-related diseases. Cryptotanshinone (CPT), an active component of Salvia miltiorrhiza, may be a potential inhibitor of angiogenesis. However, the molecular mechanisms underlying its anti-angiogenic activities remain poorly understood. This study is to investigate the effects of CPT on VEGF-induced angiogenesis and VEGFR2 signaling pathway in human umbilical vein endothelial cells (HUVECs). MethodsHUVECs were treated with different concentration of CPT (5–20μmol/L) and the viability, endothelial cell migration, invasion, and tubular-like structure formation of HUVECs were detected by MTT, wound-healing migration, Transwell invasion and Matrigel tube formation assays, respectively. To assess the effect of CPT on VEGFR2 signaling pathway, VEGF-induced phosphorylation of VEGFR2 and its downstream molecules, including ERK1/2, p90RSK, Src and FAK were analyzed by Western blot. ResultsCPT significantly suppressed VEGF-induced cells proliferation, migration, invasion, and tubular-like structure formation in HUVECs in a dose- and time-dependent manner. Western blot results revealed that CPT significantly suppressed VEGF-induced phosphorylation of VEGFR2 and its key downstream protein kinases, including p-ERK1/2, p-p90RSK, pY416-Src and pY576/577-FAK, which are responsible for endothelial cell migration, proliferation, and survival. ConclusionOur study suggested that CPT potently inhibits VEGF-induced angiogenesis by suppressing VEGFR2 activation and its downstream Src/FAK and ERK1/2 signaling pathways in HUVECs, highlighting the therapeutic potential for the treatment of angiogenesis-related diseases.

A PlGF-1 Derived Peptide Inhibits Angiogenesis via HIF-1β/VEGF Pathway

Many diseases are associated with angiogenesis. Therefore, inhibition of angiogenesis is deemed as a treatment for those diseases. To date, many angiogenesis inhibitors are from large plasma proteins. In this study we identified a 21-aa peptide (named peptide ZY1) from human placenta growth factor-1 and it may serve as a potent angiogenesis inhibitor. Our study demonstrated ZY1 inhibited angiogenesis in vitro by suppressing proliferation, migration and tube formation of HUVECs. The in vivo inhibition activity of ZY1 was observed in chicken chorioallantoic membrane assays and tumor-bearing mouse models. Moreover, we found ZY1 inhibited angiogenesis by decreasing the expression of HIF-1β and subsequently reducing its downstream molecule VEGF. In conclusion, peptide ZY1 can inhibit angiogenesis and may serve as an anti-angiogenesis drug candidate.

In Vitro and In Vivo Activity of Lucitanib in FGFR1/2 Amplified or Mutated Cancer Models

The fibroblast growth factor receptor (FGFR) pathway has been implicated both as an escape mechanism from anti-angiogenic therapy and as a driver oncogene in different tumor types. Lucitanib is a small molecule inhibitor of vascular endothelial growth factor (VEGF) receptors 1 to 3 (VEGFR1 to 3), platelet derived growth factor α/β (PDGFRα/β) and FGFR1–3 tyrosine kinases and has demonstrated activity in a phase I/II clinical study, with objective RECIST responses in breast cancer patients with FGFR1 or FGF3/4/19 gene amplification, as well as in patients anticipated to benefit from anti-angiogenic agents. We report here the in vitro and in vivo antitumor activity of lucitanib in experimental models with or without FGFR1/2 amplification or mutations. In cell assays, lucitanib potently inhibited the growth of tumor cell lines with amplified FGFR1 or mutated/amplified FGFR2. In all xenograft models studied, lucitanib demonstrated marked tumor growth inhibition due to potent inhibition of angiogenesis. Notably, in two lung cancer models with FGFR1 amplification, the antitumor efficacy was higher, suggesting that the simultaneous inhibition of VEGF and FGF receptors in FGFR1 dependent tumors can be therapeutically advantageous. Similar antitumor activity was observed in FGFR2 wild-type and amplified or mutated xenograft models. Pharmacokinetic studies showed lucitanib plasma concentrations in the micro/sub-micromolar range demonstrated drug accumulation following repeated lucitanib administration.

Enhanced peripheral dopamine impairs post-ischemic healing by suppressing angiotensin receptor type 1 expression in endothelial cells and inhibiting angiogenesis.

Increased circulating catecholamines have been linked with cardiovascular anomalies as well as with peripheral vascular diseases. Although the roles of epinephrine and norepinephrine have received considerable attention, the role of the other catecholamine, dopamine, has been less studied. Since dopamine is a potent endogenous inhibitor of angiogenesis and as angiogenesis is essential for ischemic healing, we therefore studied the role played by dopamine during ischemic healing using dopamine D2 receptor knockout (KOD2) mice. Although concentration of dopamine and its rate-limiting enzyme, tyrosine hydroxylase, was considerably high in the muscle tissues of wild-type and KOD2 mice with unilateral hind limb ischemia (HLI), recovery was significantly faster in the KOD2 mice compared to the wild-type controls, thereby indicating that peripheral dopamine might have a role in this healing process. In addition, we observed significant differences in post-ischemic angiogenesis between these two groups. Our study further revealed that elevated dopamine independently suppressed activation of local tissue-based renin-angiotensin system (RAS), a critical growth factor system stimulating angiogenesis in ischemia. Angiotensin II (ATII) and its receptor, angiotensin receptor type 1 (AT1R), are the key players in RAS-mediated angiogenesis. Dopamine acting through its D2 receptors in endothelial cells inhibited ATII-mediated angiogenesis by suppressing the expression of AT1R in these cells. This study thus for the first time demonstrates the role played by dopamine in prolonging post-ischemic recovery. Therefore, pharmacological intervention inhibiting the action of dopamine holds promise as future therapeutic strategy for the treatment of HLI and other peripheral arterial diseases.

The noni anthraquinone damnacanthal is a multi-kinase inhibitor with potent anti-angiogenic effects

The natural bioactive compound damnacanthal inhibits several tyrosine kinases. Herein, we show that -in fact- damancanthal is a multi kinase inhibitor. A docking and molecular dynamics simulation approach allows getting further insight on the inhibitory effect of damnacanthal on three different kinases: vascular endothelial growth factor receptor-2, c-Met and focal adhesion kinase. Several of the kinases targeted and inhibited by damnacanthal are involved in angiogenesis. Ex vivo and in vivo experiments clearly demonstrate that, indeed, damnacanthal is a very potent inhibitor of angiogenesis. A number of in vitro assays contribute to determine the specific effects of damnacanthal on each of the steps of the angiogenic process, including inhibition of tubulogenesis, endothelial cell proliferation, survival, migration and production of extracellular matrix remodeling enzyme. Taken altogether, these results suggest that damancanthal could have potential interest for the treatment of cancer and other angiogenesis-dependent diseases.

Beta-adrenergic signaling promotes tumor angiogenesis and prostate cancer progression through HDAC2-mediated suppression of thrombospondin-1.

Chronic behavioral stress and beta-adrenergic signaling have been shown to promote cancer progression, whose underlying mechanisms are largely unclear, especially the involvement of epigenetic regulation. Histone deacetylase-2 (HDAC2), an epigenetic regulator, is critical for stress-induced cardiac hypertrophy. It is unknown whether it is necessary for beta-adrenergic signaling-promoted cancer progression. Using xenograft models, we showed that chronic behavioral stress and beta-adrenergic signaling promote angiogenesis and prostate cancer progression. HDAC2 was induced by beta-adrenergic signaling in vitro and in mouse xenografts. We next uncovered that HDAC2 is a direct target of cAMP response element-binding protein (CREB) that is activated by beta-adrenergic signaling. Notably, HDAC2 is necessary for beta-adrenergic signaling to induce angiogenesis. We further demonstrated that, upon CREB activation, HDAC2 represses thrombospondin-1 (TSP1), a potent angiogenesis inhibitor, through epigenetic regulation. Together, these data establish a novel pathway that HDAC2 and TSP1 act downstream of CREB activation in beta-adrenergic signaling to promote cancer progression.

Synthesis and biological evaluation of novel alkyl diamine linked bivalent β-carbolines as angiogenesis inhibitors

We have synthesized and evaluated a series of novel alkyl diamine linked bivalent β-carbolines as potent angiogenesis inhibitors. The results demonstrated that most bivalent β-carbolines exhibited significant antiproliferative effects against human umbilical vein cell lines EA.HY926. Compound 4m was found to be the most potent antiproliferative agent with IC50 value of 2.16 μM against EA.HY926 cell lines. Mechanism investigations revealed that compound 4m could significantly inhibit EA.HY926 cells migration and tube formation in a dose-dependent manner. Moreover, compound 4m also showed obvious angiogenesis inhibitory effects in CAM assay, and the antiangiogenetic potency was more potent than the reference drug Endostar. The bivalent β-carbolines might be served as candidates for the development of vascular targeting antitumor drugs.

In vitro study of 2,3-dehydrosilybin and its galloyl esters as potential inhibitors of angiogenesis.

2,3-Dehydrosilybin exhibits substantial anticancer and antiangiogenic effects, which can be potentially improved by semi-synthetic modification such as esterification with gallic acid. The aim of this study was to examine the potential antiangiogenic effect of 2,3-dehydrosilybin and its galloyl esters (3-O-galloyl-2,3-dehydrosilybin; 7-O-galloyl-2,3-dehydrosilybin; 20-O-galloyl-2,3-dehydrosilybin and 23-O-galloyl-2,3-dehydrosilybin) and to determine which molecular mechanism could be responsible for their activity. The effect on cell proliferation, tube formation, signal transduction pathways (PI3K/Akt and ERK) and the cell cycle was studied in human microvascular endothelial cells (HMEC). The results showed that all compounds decreased the growth of HMEC, but the strongest effect was observed for 20-O-galloyl-2,3-dehydrosilybin at 5 μmol/l. In addition, at 5 and 10 μmol/l, this was the only compound that significantly inhibited HMEC tube formation. Based on an assessment of Akt and ERK1/2 expression, we suggest that 20-O-galloyl-2,3-dehydrosilybin influences the angiogenic process through the Akt pathway.

Abstract 3264: Lenvatinib in combination with everolimus demonstrated enhanced antiangiogenesis and antitumor activity in human RCC xenograft models

Abstract Lenvatinib mesilate (lenvatinib) is an oral multiple receptor tyrosine kinase (RTK) inhibitor that selectively inhibits the kinase activities of vascular endothelial growth factor (VEGF) receptors VEGFR1 (FLT1), VEGFR2 (KDR), and VEGFR3 (FLT4), in addition to other proangiogenic and oncogenic pathway-related RTKs including fibroblast growth factor (FGF) receptors FGFR1, 2, 3, and 4; the platelet-derived growth factor (PDGF) receptor PDGFRα; KIT; and RET. Lenvatinib showed antitumor activity against various tumor types mainly through its potent inhibition of angiogenesis, and is currently marketed for the treatment of patients with radioactive iodine-refractory differentiated thyroid cancer. Recently, lenvatinib in combination with everolimus has shown longer progression free survival compared to lenvatinib or everolimus alone in renal cell carcinoma in Phase 2 study. The aim of this study is to elucidate the activity of the combination of lenvatinib and everolimus in preclinical human RCC xenograft models. We examined antitumor activity in two human RCC (A-498 and Caki-1) xenograft models orally treated with lenvatinib (10 mg/kg), everolimus (30 mg/kg), and the combination of lenvatinib and everolimus for 1 or 2 weeks. The antitumor proliferation and antiangiogenic effects were evaluated by immunohistochemistry (IHC) using anti Ki67 antibody and anti CD31 antibody, respectively. The induction of apoptosis was detected by TUNEL assay. To analyze the gene expression profile of tumor samples, microarray analysis were also conducted. The antitumor activity of the combination of lenvatinib and everolimus was greater than that of either agent administered alone in A-498 and Caki-1 xenograft models. The combination caused tumor regression and had no remarkable body weight loss. IHC analysis revealed decrease of microvessel density in lenvatinib and combination groups, and also decrease in the proportion of proliferative cells in everolimus treated and combination-treated group in A-498 model. In TUNEL assay, significant induction of apoptosis was observed only in the combination-treatment group. The analysis of gene expression profile in A-498 xenograft tumors also supported these results: lenvatinib alone upregulated hypoxia-related genes and everolimus decreased proliferation-related genes. The combination of these 2 drugs induced blends of the gene expression changes caused by each single treatment. Our results indicate that treatment of lenvatinib in combination with everolimus caused significant antitumor effect by combining the potent antiangiogenic activity of lenvatinib as well as direct antitumor activity of everolimus in A-498 model. These preclinical results provide one of the mechanisms to support the significant clinical benefit observed in RCC with the combination of lenvatinib and everolimus. Citation Format: Yusuke Adachi, Masahiro Matsuki, Atsumi Yamaguchi, Yoichi Ozawa, Kiyochi Okamoto, Kaoru Mitsuhashi, Takayuki Kimura, Taisuke Hoshi, Osamu Tohyama, Kenji Tai, Makoto Ogo, Yasuhiro Funahashi, Junji Matsui. Lenvatinib in combination with everolimus demonstrated enhanced antiangiogenesis and antitumor activity in human RCC xenograft models. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3264.

Abstract 3266: Antitumor activity of a combination of lenvatinib mesilate, ifosfamide, and etoposide against human pediatric osteosarcoma cell lines

Abstract Lenvatinib mesilate (lenvatinib) is an orally administered molecular targeted agent that selectively inhibits several receptor tyrosine kinases (RTKs) including vascular endothelial growth factor receptors (VEGFR1-3), fibroblast growth factor receptors (FGFR1-4), platelet-derived growth factor receptor (PDGFR) α, RET, and KIT. Lenvatinib has been marketed for the treatment of advanced or differentiated thyroid cancer refractory to radioactive iodine. Lenvatinib showed antitumor activity against various tumor types mainly through its potent inhibition of angiogenesis. In this study we evaluated the antitumor activities of lenvatinib in combination with ifosfamide and etoposide against human pediatric osteosarcoma cell lines. Five human pediatric osteosarcoma cell lines (143B, G-292 clone A141B1, HOS, HuO9, and Saos-2) were used. Antiproliferative activity of lenvatinb and etoposide was evaluated in WST-8-based colorimetric proliferation assay or in soft agar colony formation assay. To evaluate the in vivo antitumor activity, lenvatinib alone, the combination of etoposide and ifosfamide, and the combination of these 3 drugs were administered to mice bearing the human osteosarcoma xenografts. Etoposide inhibited cell proliferation with IC50 values of a range within 0.31 - 4.1 μmol/L. G-292 clone A141B1 (G-292) had the lowest sensitivity to etoposide. Four human osteosarcoma cell lines out of five were not sensitive to lenvatinib in proliferation assay (IC50 values >10 μmol/L), and only G-292 was susceptible to lenvatinib with IC50 values of 1.3 and 0.059 μmol/L in proliferation assay or soft agar colony formation assay, respectively. G-292 has been reported to have FGFR1 amplification and shows high transcript expression of FGFR1, suggesting that the antiproliferative activity of lenvatinib could be mediated by the FGFR1 blockade. In all xenograft models, tumor volumes with the triple combination treatment were significantly lower than that with vehicle treatment on Day 8. Antitumor effect of the triple combination was significantly more potent than that of lenvatinib alone and the ifosfamide-etoposide combination in 143B, G-292, and HOS, and more potent than that of lenvatinib alone in HuO9 and Saos-2 xenograft models. The triple combination of lenvatinib, ifosfamide, and etoposide was active against all examined human pediatric osteosarcoma xenografts in mice, and lenvatinib enhanced antitumor activity of the ifosfamide-etoposide combination in 3 out of 5 models. These results support further investigation of the combination of lenvatinib with ifosfamide and etoposide in osteosarcoma. In addition, direct antitumor activity of lenvatinib against the osteosarcoma cells harboring FGFR1 amplification warrants further investigation. Citation Format: Masahiro Matsuki, Kiyoshi Okamoto, Zoltan Dezso, Sergei I. Agoulnik, Yasuhiro Funahashi, Junji Matsui. Antitumor activity of a combination of lenvatinib mesilate, ifosfamide, and etoposide against human pediatric osteosarcoma cell lines. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3266.

Suramin blocks interaction between human FGF1 and FGFR2 D2 domain and reduces downstream signaling activity

The extracellular portion of the human fibroblast growth factor receptor2 D2 domain (FGFR2 D2) interacts with human fibroblast growth factor 1 (hFGF1) to activate a downstream signaling cascade that ultimately affects mitosis and differentiation. Suramin is an antiparasiticdrug and a potent inhibitor of FGF-induced angiogenesis. Suramin has been shown to bind to hFGF1, and might block the interaction between hFGF1 and FGFR2 D2. Here, we titrated hFGF1 with FGFR2 D2 and suramin to elucidate their interactions using the detection of NMR. The docking results of both hFGF1-FGFR2 D2 domain and hFGF1-suramin complex were superimposed. The results indicate that suramin blocks the interaction between hFGF1 and FGFR2 D2. We used the PyMOL software to show the hydrophobic interaction of hFGF1-suramin. In addition, we used a Water-soluble Tetrazolium salts assay (WST1) to assess hFGF1 bioactivity. The results will be useful for the development of new antimitogenic activity drugs.

Novel pyrazolopyridine derivatives as potential angiogenesis inhibitors: Synthesis, biological evaluation and transcriptome-based mechanistic analysis

Modified purine derivatives exemplified by pyrazolopyrimidines have emerged as highly selective inhibitors of several angiogenic receptor tyrosine kinases. Herein, we designed and synthesized a new series of substituted pyrazolopyridines and explored their ability to influence crucial pro-angiogenic attributes of endothelial cells. Four of the synthesized compounds, possessing analogous substitution pattern, were found able to inhibit at low micromolar concentrations endothelial cell proliferation, migration and differentiation, constitutively or in response to Vascular Endothelial Growth Factor (VEGF) and to attenuate VEGF-induced phosphorylation of VEGF receptor-2 and downstream kinases AKT and ERK1/2. Administration of effective compounds in mice delayed the growth of syngeneic Lewis lung carcinoma transplants and reduced tumor microvessel density, without causing toxicity. Genome-wide microarray and gene ontology analyses of treated endothelial cells revealed derivative 18c as the most efficient modulator of gene expression and “mitotic cell cycle/cell division” along with “cholesterol biosynthesis” as the most significantly altered biological processes.

Metabolic Symbiosis Enables Adaptive Resistance to Anti-angiogenic Therapy that Is Dependent on mTOR Signaling.

SummaryTherapeutic targeting of tumor angiogenesis with VEGF inhibitors results in demonstrable, but transitory efficacy in certain human tumors and mouse models of cancer, limited by unconventional forms of adaptive/evasive resistance. In one such mouse model, potent angiogenesis inhibitors elicit compartmental reorganization of cancer cells around remaining blood vessels. The glucose and lactate transporters GLUT1 and MCT4 are induced in distal hypoxic cells in a HIF1α-dependent fashion, indicative of glycolysis. Tumor cells proximal to blood vessels instead express the lactate transporter MCT1, and p-S6, the latter reflecting mTOR signaling. Normoxic cancer cells import and metabolize lactate, resulting in upregulation of mTOR signaling via glutamine metabolism enhanced by lactate catabolism. Thus, metabolic symbiosis is established in the face of angiogenesis inhibition, whereby hypoxic cancer cells import glucose and export lactate, while normoxic cells import and catabolize lactate. mTOR signaling inhibition disrupts this metabolic symbiosis, associated with upregulation of the glucose transporter GLUT2.

Mutations and altered expression of SERPINF1 in patients with familial otosclerosis.

Otosclerosis is a relatively common heterogenous condition, characterized by abnormal bone remodelling in the otic capsule leading to fixation of the stapedial footplate and an associated conductive hearing loss. Although familial linkage and candidate gene association studies have been performed in recent years, little progress has been made in identifying disease-causing genes. Here, we used whole-exome sequencing in four families exhibiting dominantly inherited otosclerosis to identify 23 candidate variants (reduced to 9 after segregation analysis) for further investigation in a secondary cohort of 84 familial cases. Multiple mutations were found in the SERPINF1 (Serpin Peptidase Inhibitor, Clade F) gene which encodes PEDF (pigment epithelium-derived factor), a potent inhibitor of angiogenesis and known regulator of bone density. Six rare heterozygous SERPINF1 variants were found in seven patients in our familial otosclerosis cohort; three are missense mutations predicted to be deleterious to protein function. The other three variants are all located in the 5′-untranslated region (UTR) of an alternative spliced transcript SERPINF1-012. RNA-seq analysis demonstrated that this is the major SERPINF1 transcript in human stapes bone. Analysis of stapes from two patients with the 5′-UTR mutations showed that they had reduced expression of SERPINF1-012. All three 5′-UTR mutations are predicted to occur within transcription factor binding sites and reporter gene assays confirmed that they affect gene expression levels. Furthermore, RT-qPCR analysis of stapes bone cDNA showed that SERPINF1-012 expression is reduced in otosclerosis patients with and without SERPINF1 mutations, suggesting that it may be a common pathogenic pathway in the disease.