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Chemoresistance of triple negative breast cancer against paclitaxel (PAX) is one of the major issues for the patients under chemotherapy. However, the mechanism by which the breast cancer cells are resistant to PAX remains unclear. Here, we identified a circular RNA of angiomotin-like 1 (circAMOTL1) as an important player which may be responsible for the adverse resistance against PAX in breast cancer cells. The circAMOTL1 were overexpressed in MDA-MB-231 breast cancer cells via transfection of circAMOTL1 construct. Overexpression of circAMOTL1 caused significant increase of cell viability, reduction of apoptosis, and enhancement of invasion when MDA-MB-231 cells were exposed to PAX compared to those cells with vector control. Moreover, these resistant effects could be blocked by the application of circAMOTL1 siRNA. In these circAMOTL1 overexpressing cells, we found notable increase of both phosphorylated and total AKT protein, which suggested that AKT might be the downstream factor mediating the resistant effects. Consequently, the gene and protein expression of AKT related pro-apoptotic (BAX and BAK) and anti-apoptotic (BCL-2) factors were significantly changed by circAMOTL1 as well. These results suggest circAMOTL1 may play an important role in the PAX resistance of breast cancer cells via regulation of AKT pathway, facilitation of anti-apoptotic protein and inhibition of pro-apoptotic protein. While providing a new mechanism of PAX resistance in breast cancer cells, our findings may lay groundwork for a novel therapeutic target of the breast cancer treatment in the future.

Diabetic retinopathy (DR) is the most severe microvascular complication of diabetes and a major cause of visual impairment and blindness. However, the treatment for DR is still limited. Our study aimed to explore the role of circular RNA_0002570 in DR. First, we predicted the potential microRNA and mRNA that could bind to circ_0002570 and identified the miR-1243 and angiomotin gene; then, we used RT-PCR and Western blot to measure their expression. Next, we evaluated the abilities of proliferation, migration, and angiogenesis in vitro in human retinal microvascular endothelial cells (hRMECs) by CCK-8, transwell assay, and tube formation assay, respectively. To analyze the relationship among miR-1243, circ_0002570, and angiomotin, RNA pull-down and luciferase assay were performed. Our results showed that, in DR patients and high-glucose–induced hRMECs, miR-1243, circ_0002570, and angiomotin were all abnormally expressed. MiR-1243 could directly and competitively bind to both circ_0002570 and angiomotin mRNA to inhibit their expression. Moreover, circ_0002570 suppressed the abilities of proliferation, migration, and angiogenesis in hRMECs induced by high glucose, which was dependent on miR-1243-angiomotin axis. Furthermore, circ_0002570 could upregulate angiomotin by targeting miR-1243 to mediate the dysfunction of hRMECs induced by high glucose. In conclusion, circ_0002570 might serve as a potential target for diagnosis and treatment for DR.

// Hao Zeng 1 , Angelica Ortiz 2, 3, * , Peng-Fei Shen 1, * , Chien-Jui Cheng 4, 5 , Yu-Chen Lee 2 , Guoyu Yu 2 , Song-Chang Lin 2 , Chad J. Creighton 6 , Li-Yuan Yu-Lee 7 , Sue-Hwa Lin 2, 3, 8 1 Department of Urology, Institute of Urology, West China Hospital, Sichuan University, Chengdu, China 2 Department of Translational Molecular Pathology, The University of Texas M. D. Anderson Cancer Center, Houston, Texas, USA 3 The University of Texas Graduate School of Biomedical Sciences at Houston, Texas, USA 4 Department of Pathology, School of Medicine, College of Medicine, Taipei Medical University, Taipei, Taiwan 5 Department of Pathology, Taipei Medical University Hospital, Taipei, Taiwan 6 Dan L. Duncan Cancer Center, Baylor College of Medicine, Houston, Texas, USA 7 Department of Medicine, Baylor College of Medicine, Houston, Texas, USA 8 Department of Genitourinary Medical Oncology, The University of Texas M. D. Anderson Cancer Center, Houston, Texas, USA * These authors have contributed equally to this work Correspondence to: Hao Zeng, email: cdhx510@foxmail.com , kucaizeng@163.com Sue-Hwa Lin, email: slin@mdanderson.org Keywords: angiomotin, Hippo pathway, YAP, BMP4, proliferation Abbreviations: Angiomotin (AMOT), neurofibromatosis type 2 (NF2), prostate cancer (PCa) Received: October 11, 2016 Accepted: December 13, 2016 Published: December 29, 2016 ABSTRACT Angiomotin (AMOT) is a family of proteins found to be a component of the apical junctional complex of vertebrate epithelial cells and is recently found to play important roles in neurofibromatosis type 2 (NF-2). Whether AMOT plays a role in prostate cancer (PCa) is unknown. AMOT is expressed as two isoforms, AMOTp80 and AMOTp130, which has a 409 aa N-terminal domain that is absent in AMOTp80. Both AMOTp80 and AMOTp130 are expressed in LNCaP and C4-2B4, but at a low to undetectable level in PC3, DU145, and BPH1 cells. Further study showed that AMOTp130 and AMOTp80 have distinct functions in PCa cells. We found that AMOTp80, but not AMOT p130, functioned as a tumor promoter by enhancing PCa cell proliferation. Mechanistic studies showed that AMOTp80 signaled through the Hippo pathway by promoting nuclear translocation of YAP, resulting in an increased expression of YAP target protein BMP4. Moreover, inhibition of BMP receptor activity by LDN-193189 abrogates AMOTp80-mediated cell proliferation. Together, this study reveals a novel mechanism whereby the AMOTp80-Merlin-MST1-LATS-YAP-BMP4 pathway leads to AMOTp80-induced tumor cell proliferation.

// Albino Troilo 1,* , Erica K. Benson 1,* , Davide Esposito 1 , Rachel-Ann A. Garibsingh 1 , E. Premkumar Reddy 1 , Sathish Kumar Mungamuri 1 and Stuart A. Aaronson 1 1 Department of Oncological Sciences, Icahn School of Medicine at Mount Sinai, New York, NY, USA * These authors have contributed equally to this work Correspondence to: Stuart A. Aaronson, email: // Keywords : tankyrase inhibitors, TEAD, YAP, angiomotin, tumor cell proliferation Received : February 24, 2016 Accepted : March 26, 2016 Published : April 29, 2016 Abstract The evolutionarily conserved Hippo inhibitory pathway plays critical roles in tissue homeostasis and organ size control, while mutations affecting certain core components contribute to tumorigenesis. Here we demonstrate that proliferation of Hippo pathway mutant human tumor cells exhibiting high constitutive TEAD transcriptional activity was markedly inhibited by dominant negative TEAD4, which did not inhibit the growth of Hippo wild-type cells with low levels of regulatable TEAD-mediated transcription. The tankyrase inhibitor, XAV939, identified in a screen for inhibitors of TEAD transcriptional activity, phenocopied these effects independently of its other known functions by stabilizing angiomotin and sequestering YAP in the cytosol. We also identified one intrinsically XAV939 resistant Hippo mutant tumor line exhibiting lower and less durable angiomotin stabilization. Thus, angiomotin stabilization provides a new mechanism for targeting tumors with mutations in Hippo pathway core components as well as a biomarker for sensitivity to such therapy.

International audience; This study aimed to develop a PET imaging agent of angiomotin (AMOT) expression, a potential biomarker of functional tissue regeneration in post-ischaemic conditions. Methods: Hindlimb ischaemia was induced by ligature and resection of the right femoral artery in mice, and clinical score and limb perfusion were evaluated up to 30 days after surgery. AMOT expression was evaluated by histology and Western blot analysis. NODAGA-conjugates of AMOT ligand, sCD146, were designed, synthesised and radiolabelled with gallium-68. Ga-68-sCD146 microPET/CT imaging was performed from day 1 to day 30 after ischaemia. Ga-68-sCD146 specificity for AMOT was evaluated by autoradiography. Results: Immunohistochemistry showed a significant endothelial overexpression of AMOT from day 5 up to day 10 in the ischaemic hindlimb. Ga-68-sCD146 PET signal intensity correlated significantly with AMOT immunohistochemistry evaluation. Ga-68-sCD146 PET imaging showed a significant uptake in the ischaemic hindlimb from day 2 to day 15, peaking on day 5 (ipsi/contralateral ratio = 2.4 +/- 1.3, P = 0.0005) and significantly decreased after pharmacological blocking (62.57 +/- 11% decrease in PET signal P = 0.032). Finally, we observed a significant correlation between day 5 Ga-68-sCD146 PET signal intensity and clinical recovery (day 28) or hindlimb perfusion recovery (day 30). Conclusions: This work reports for the first time an early and sustained increase in AMOT expression after hindlimb ischaemia in mice. We therefore developed an AMOT-targeting imaging agent, Ga-68-sCD146, and showed its specific uptake up to 21 days after ischaemic hindlimb using microPET imaging. Correlation of early post-ischaemic PET signal with both delayed perfusion recovery and clinical outcome allows us to postulate that Ga-68-sCD146 represents a promising radiotracer for tissue angiogenesis assessment.

Renal cell carcinoma (RCC) is one of the common tumors in the urinary system without effective therapies. Angiomotin (Amot) can interact with Yes-associated protein (YAP) to either stimulate or inhibit YAP activity, playing a potential role in cell proliferation. However, the role of Amot in regulating the proliferation of renal epithelial and RCC cells is unknown. Here, we show that Amot is expressed predominantly in the nucleus of RCC cells and tissues, and in the cytoplasm and nucleus of renal epithelial cells and paracancerous tissues. Furthermore, Amot silencing inhibited proliferation of HK-2 and 786-O cells while Amot upregulation promoted proliferation of ACHN cells. Interestingly, the location of Amot and YAP in RCC clinical samples and cells was similar. Amot interacted with YAP in HK-2 and 786-O cells, particularly in the nucleus. Moreover, Amot silencing mitigated the levels of nuclear YAP in HK-2 and 786-O cells and reduced YAP-related CTGF and Cyr61 expression in 786-O cells. Amot upregulation slightly increased the nuclear YAP and YAP-related gene expression in ACHN cells. Finally, enhanced YAP expression restored proliferation of Amot-silencing 786-O cells. Together, these data indicate that Amot is crucial for the maintenance of nuclear YAP to promote renal epithelial and RCC proliferation.

Angiomotin (Amot) is one of several identified angiostatin receptors expressed by the endothelia of angiogenic tissues. We have shown that a DNA vaccine targeting Amot overcome immune tolerance and induce an antibody response that hampers the progression of incipient tumors. Following our observation of increased Amot expression on tumor endothelia concomitant with the progression from pre-neoplastic lesions to full-fledged carcinoma, we evaluated the effect of anti-Amot vaccination on clinically evident tumors. Electroporation of plasmid coding for the human Amot (pAmot) significantly delayed the progression both of autochthonous tumors in cancer prone BALB-neuT and PyMT genetically engineered mice and transplantable TUBO tumor in wild-type BALB/c mice. The intensity of the inhibition directly correlated with the titer of anti-Amot antibodies induced by the vaccine. Tumor inhibition was associated with an increase of vessels diameter with the formation of lacunar spaces, increase in vessel permeability, massive tumor perivascular necrosis and an effective epitope spreading that induces an immune response against other tumor associated antigens. Greater tumor vessel permeability also markedly enhances the antitumor effect of doxorubicin. These data provide a rationale for the development of novel anticancer treatments based on anti-Amot vaccination in conjunction with chemotherapy regimens. Electronic supplementary material The online version of this article (doi:10.1007/s10456-012-9263-3) contains supplementary material, which is available to authorized users.

The Yes-associated protein (YAP) is a transcription coactivator that plays a crucial role in organ size control by promoting cell proliferation and inhibiting apoptosis. The Hippo tumor suppressor pathway inhibits YAP through phosphorylation-induced cytoplasmic retention and degradation. Here we report a novel mechanism of YAP regulation by angiomotin (AMOT) family proteins via a direct interaction. Knockdown of AMOT family protein AMOTL2 in polarized Madin-Darby canine kidney (MDCK) cells leads to YAP activation, as indicated by decreased YAP tight junction localization, attenuated YAP phosphorylation, accumulation of nuclear YAP, and induction of YAP target gene expression. Transcriptional coactivator with PDZ-binding motif (TAZ), the YAP paralog, is also regulated by AMOT in a similar fashion. Furthermore, AMOTL2 knockdown results in loss of cell contact inhibition in a manner dependent on the functions of YAP and TAZ. Our results indicate a potential tumor-suppressing role of AMOT family proteins as components of the Hippo pathway, and demonstrate a novel mechanism of YAP and TAZ inhibition by AMOT-mediated tight junction localization. These observations provide a potential link between the Hippo pathway and cell contact inhibition.

The development of the embryonic vascular system into a highly ordered network requires precise control over the migration and branching of endothelial cells (ECs). We have previously identified angiomotin (Amot) as a receptor for the angiogenesis inhibitor angiostatin. Furthermore, DNA vaccination targeting Amot inhibits angiogenesis and tumor growth. However, little is known regarding the role of Amot in physiological angiogenesis. We therefore investigated the role of Amot in embryonic neovascularization during zebrafish and mouse embryogenesis. Here we report that knockdown of Amot in zebrafish reduced the number of filopodia of endothelial tip cells and severely impaired the migration of intersegmental vessels. We further show that 75% of Amot knockout mice die between embryonic day 11 (E11) and E11.5 and exhibit severe vascular insufficiency in the intersomitic region as well as dilated vessels in the brain. Furthermore, using ECs differentiated from embryonic stem (ES) cells, we demonstrate that Amot-deficient cells have intact response to vascular endothelial growth factor (VEGF) in regard to differentiation and proliferation. However, the chemotactic response to VEGF was abolished in Amot-deficient cells. We provide evidence that Amot is important for endothelial polarization during migration and that Amot controls Rac1 activity in endothelial and epithelial cells. Our data demonstrate a critical role for Amot during vascular patterning and endothelial polarization.

The modern era of angiogenesis research is generally considered to have begun with the proposal by Folkman 1971 that tumors release diffusible factors that promote the formation of new blood vessels. Although there has been interest in this topic for 30 yr, the last 6–7 yr have seen an explosion of interest in this field. This explosion is evidenced in the number of meetings on the topic, the number of press reports, and especially the number of publications. A search of the Medline database reveals that in 1990 approximately 200 papers were published on the topic of angiogenesis, whereas in the year 2000 more than 1,700 papers appeared on the same subject. To what can we attribute this rise? My own feeling is that while the previous two decades were devoted to proving that angiogenesis was a real biological phenomenon and to the isolation of angiogenesis-stimulating factors, the 1990s were devoted to the identification, purification, and cloning of potentially effective angiogenesis inhibitors. This allowed the establishment of clinical trials to test the therapeutic efficacy of these agents and has stimulated a torrent of activity among biologists in both academic and industrial settings to find out more about the mechanisms by which these inhibitors work. A large number of angiogenesis inhibitors have entered or will soon enter clinical trials. These have been identified and isolated using a variety of strategies. Some were chosen on the basis of their ability to interfere with key angiogenesis stimulators such as the vascular endothelial growth factor and its receptors (Presta et al. 1997; Fong et al. 1999) or key endothelial receptors for extracellular matrix molecules such as the αvβ3 integrin (Brooks et al. 1994). Others were isolated from tissues such as cartilage that are naturally devoid of blood vessels (Moses et al. 1999). Some were known cytokines such as interferon alpha (White et al. 1989) or interleukin 12 (Voest et al. 1995) that were subsequently found to have antiangiogenic properties. Still others were identified on the basis of their structural characteristics (D'Amato et al. 1994; Klauber et al. 1997) or on the basis of their ability to inhibit endothelial cell migration or proliferation (Ingber et al. 1990) or to function as a tumor suppressor (Good et al. 1990). Information regarding angiogenesis inhibitors in clinical trials can be found on the Cancer Trials web site of the National Cancer Institute (http://cancertrials.nci.nih.gov/news/angio/table.html). One of the first angiogenesis inhibitors described in the 1990s was angiostatin, a molecule originally collected from the urine of tumor-bearing mice (O'Reilly et al. 1994). Angiostatin is a proteolytically generated fragment of plasminogen consisting of the first four kringle domains of the parent molecule. Angiostatin is thought not to be produced inside the tumor cells, but rather to be generated in the circulation by tumor-derived proteolytic enzymes (Gately et al. 1996). The observation that angiostatin as well as other angiogenic inhibitors are derived from molecules involved in hemostasis has encouraged speculation that these molecules may have a role in normal wound healing, as well as in tumors and other pathological states (Browder et al. 2000). Angiostatin is currently in clinical trials to determine its utility as an antitumor drug. Despite the intense effort and research output on the topic of angiogenesis inhibitors, much remains to be learned. For many of these newly described inhibitors, we know little of their mechanism of action and less about the nature of the receptors that mediate their effects on endothelial cells. For this reason, the paper by Troyanovsky et al. 2001 is a welcome addition to our understanding of angiogenesis. This team has described angiomotin, a new, functional angiostatin-binding protein that mediates the inhibition of endothelial cell motility, a critical component of the angiogenic process (Zetter 1980). Because of the importance of cell migration in the angiogenesis process, the discovery of a cell-surface protein that mediates the effects of angiostatin on endothelial cell motility represents a significant step forward for this field. The data are convincing. Angiomotin was discovered using a yeast two-hybrid screen for proteins that interact with the first four kringle domains of plasminogen. It is described as a 72-kD cell surface–associated protein that is expressed in capillary endothelial cells as well as in actively angiogenic tissue such as placenta and solid tumors. Like other surface-associated proteins that can bind plasminogen and its derivatives, angiomotin does not appear to have a signal sequence, thus its association with the cell surface may be via protein–protein interaction. It is localized to the lamellipodia at the leading edge of migrating endothelial cells. Most importantly, transfection of control HeLa cells with angiomotin results in an increased response to migration-stimulating proteins. Furthermore, angiostatin treatment blocked cell migration and three-dimensional tube morphogenesis in cells transfected with angiomotin but not in vector-control cells. These results solidly implicate angiomotin in the angiostatin-mediated regulation of cell motility and capillary differentiation. Angiomotin is not the first angiostatin-binding protein discovered. ATP synthase was previously described as a high affinity angiostatin-binding protein (Moser et al. 1999) that bound preferentially to angiostatin relative to the parent molecule plasminogen. Antibodies to ATP synthase were reported to inhibit the antiproliferative activity of angiostatin on endothelial cells. Plasminogen itself binds to other cell-surface proteins such as annexin II and alpha enolase (Miles et al. 1991; Hajjar et al. 1994). Should we be concerned, then, that a potent antiangiogenic molecule should have more than one potentially important cell surface binding molecule? Probably not. The positive and negative regulation of blood vessel growth is a critical determinant of tissue repair and of normal tissue mass. Angiogenesis has essential roles in embryonic development, tissue hyperplasia, and a variety of diseases. Vascular endothelial growth factor (VEGF), a positive regulator of angiogenesis, has at least three receptors, and fibroblast growth factor, another positive regulator, has more receptors than VEGF. It would be no surprise if a potent angiogenesis inhibitor such as angiostatin were to have multiple receptors. These multiple receptors could act independently or in concert, as is the case for the high- and low-affinity receptors for fibroblast growth factor (Yayon et al. 1991). As always, a discovery of this type generates many new questions. Among those undoubtedly being asked in the Holmgren laboratory today are: (a) what is the binding affinity of angiostatin's interaction with angiomotin? (b) what is the nature of the binding interactions between angiomotin and plasminogen? (c) what signaling molecules are downstream of angiomotin? (d) does binding of angiostatin to angiomotin cause activation of the focal adhesion kinase previously shown to be modulated by angiostatin (Claesson-Welsh et al. 1998)? and (e) does angiomotin mediate other functions of angiostatin action such as endothelial proliferation in vitro or the inhibition of angiogenesis and tumor growth in vivo? There is considerable work left to be done. Who are the beneficiaries of this new discovery? Those who will try to add more flesh to the bones of this new finding, those who will attempt to identify or construct novel small molecules that regulate angiogenesis via the angiomotin pathway, those curious about angiogenesis, and, with any luck, those who may have or will later acquire a disease characterized by untoward angiogenesis.