Angiogenesis In Breast Cancer Research Articles

Yanghe Huayan decoction inhibits the capability of trans-endothelium and angiogenesis of HER2+ breast cancer via pAkt signaling.

Background: Yanghe Huayan Decoction (YHD), a traditional Chinese medicine, is one of the most common complementary medicine currently used in the treatment of breast cancer (BC). It has been recently linked to suppress precancerous lesion and tumor development. The current study sought to explore the role of YHD on trans-endothelium and angiogenesis of BC. Methods: HER2+ BC cells were treated with YHD, Trastuzumab, or the combination in vitro and in vivo to compare the effects of them on trans-endothelium and angiogenesis features. The present study also investigated the potential molecular mechanism of YHD in inhibiting angiogenesis of BC. Results: YHD significantly suppressed the invasion and angiogenesis of BC cells via elevated pAkt signaling. Administration of YHD in vivo also strikingly repressed angiogenesis in tumor grafts. Conclusion: YHD could partially inhibit and reverse tumorigenesis of BC. It also could inhibit Akt activation and angiogenesis in vitro and in vivo. Its effect was superior to trastuzumab. Thus it was suitable for prevention and treatment of BC.

MiR-126 Modulates Angiogenesis in Breast Cancer by Targeting VEGF-A -mRNA

Background:Breast cancer is most serious reasons of women death around worldwide result in increasing its morbidity and mortality. MicroRNAs are considered as significant regulators of cancer biological processes. The main aim of this study is restoration of miR-126 could lead to modulate breast cell line and impairs their proliferation by targeting vascular endothelial growth factor gene (VEGF-A).Methods:Breast cancer cell line (MCF7) was transfected by miR-126 lipofectamine and negative miR control for 24 hr. Cytotoxic effects of miR-126 lipofectamine were determined by cell viability assay. Cell proliferation and cell cycle were quantitatively measured using PicoGreen assay and DAPI stain-flow cytometer analysis. For further investigation, Taq-Man real time PCR assay was performed to detect relative VEGF-A and miRNA-126 level.Results:MiR-126 was overexpressed in treated breast cancer cell (MCF7) compared with control cells. miR-126 expression has been associated –with a decrease in cell proliferation and arrested MCF7 cells at G1 phase. The study found that vascular endothelial growth factor is regulated by miR-126. Hence, VEGF-A is considered as functional vital and direct target to miR-126 in breast cancer cell line (MCF7).Conclusions:This study provided that manipulated miR-126 level may suggest a novel therapeutic approach in breast cancer treatment. However, an animal models study is needed to address and prove predictive ability of miR-126 on breast cancer controlling.

CUL1 promotes breast cancer metastasis through regulating EZH2-induced the autocrine expression of the cytokines CXCL8 and IL11

CUL1 is an essential component of SCF (SKP1-CUL1-F-box protein) E3 ubiquitin ligase complex. Our previous study has showed that CUL1 is positively associated with poor overall and disease-specific survival of breast cancer patients. Here, we further explored its roles in breast cancer metastasis. Our data showed that CUL1 significantly promoted breast cancer cell migration, invasion, tube formation in vitro, as well as angiogenesis and metastasis in vivo. In mechanism, the human gene expression profiling was used to determine global transcriptional changes in MDA-MB-231 cells, and we identified autocrine expression of the cytokines CXCL8 and IL11 as the target genes of CUL1 in breast cancer cell migration, invasion, metastasis, and angiogenesis. CUL1 regulated EZH2 expression to promote the production of cytokines, and finally significantly aggravating the breast cancer cell metastasis and angiogenesis through the PI3K–AKT–mTOR signaling pathway. Combined with the previous report about CUL1, we proposed that CUL1 may serve as a promising therapeutic target for breast cancer metastasis.

C11, a novel fibroblast growth factor receptor 1 (FGFR1) inhibitor, suppresses breast cancer metastasis and angiogenesis.

The fibroblast growth factor receptors (FGFRs) are increasingly considered attractive targets for therapeutic cancer intervention due to their roles in tumor metastasis and angiogenesis. Here, we identified a new selective FGFR inhibitor, C11, and assessed its antitumor activities. C11 was a selective FGFR1 inhibitor with an IC50 of 19 nM among a panel of 20 tyrosine kinases. C11 inhibited cell proliferation in various tumors, particularly bladder cancer and breast cancer. C11 also inhibited breast cancer MDA-MB-231 cell migration and invasion via suppression of FGFR1 phosphorylation and its downstream signaling pathway. Suppression of matrix metalloproteinases 2/9 (MMP2/9) was associated with the anti-motility activity of C11. Furthermore, the anti-angiogenesis activity of C11 was verified in endothelial cells and chicken chorioallantoic membranes (CAMs). C11 inhibited the migration and tube formation of HMEC-1 endothelial cells and inhibited angiogenesis in a CAM assay. In sum, C11 is a novel selective FGFR1 inhibitor that exhibits potent activity against breast cancer metastasis and angiogenesis.

Deciphering cancer fibroblasts.

In this issue of JEM, Raz et al. (https://doi.org/10.1084/jem.20180818) identify a subset of bone marrow-derived cells that uniquely promotes breast cancer angiogenesis and tumor growth. The existence of functional heterogeneity among stromal populations motivates further fundamental and therapeutic inquiries.

Osterix promotes the migration and angiogenesis of breast cancer by upregulation of S100A4 expression.

As a key transcription factor required for bone formation, osterix (OSX) has been reported to be overexpressed in various cancers, however, its roles in breast cancer progression remain poorly understood. In this study, we demonstrated that OSX was highly expressed in metastatic breast cancer cells. Moreover, it could upregulate the expression of S100 calcium binding protein A4 (S100A4) and potentiate breast cancer cell migration and tumor angiogenesis in vitro and in vivo. Importantly, inhibition of S100A4 impaired OSX‐induced cell migration and capillary‐like tube formation. Restored S100A4 expression rescued OSX‐short hairpin RNA‐suppressed cell migration and capillary‐like tube formation. Moreover, the expression levels of OSX and S100A4 correlated significantly in human breast tumors. Our study suggested that OSX acts as an oncogenic driver in cell migration and tumor angiogenesis, and may serve as a potential therapeutic target for human breast cancer treatment.

Blocking Dimeric Pyruvate Kinase M2 Transformation and Nuclear Translocation Leading to Suppression of Aerobic Glycolysis Mediates Beta-Elemene Inhibition of Breast Cancer Metastasis

Pyruvate kinase M2 (PKM2) plays a central role in regulating aerobic glycolysis and is considered as a potential target for cancer therapy. However, its role in cancer metastasis is rarely known. Here, we found a tight relationship between PKM2 and breast cancer metastasis, which is demonstrated by the finding that beta-elemene (β-elemene), an approved drug for complementary cancer therapy, exerted distinct anti-metastatic activity dependent on PKM2. The results indicated thatβ-elemene inhibited breast cancer cell migration, invasion and angiogenesis. β-elemene also inhibited the process of glycolysis, and decreased the utilization of glucose and the production of pyruvate and lactate through suppressing pyruvate kinase activity by modulating the transformation of dimeric and tetrameric forms of PKM2, which was reversed in part by fructose-1,6-bisphosphate (FBP) or L-cysteine. Our further analysis revealed that β-elemene suppressed aerobic glycolysis through inhibiting the nuclear transportation of PKM2 and the expression of GLUT1, MCT1, MCT4 and LDHA by influencing the expression of importin α5. Additionally, β-elemene remarkably inhibited the breast cancer metastases in lung and liver and selectively influenced the glycolysis of the lung and liver metastatic foci in mouse models. Taken together, tetrameric transformation and nuclear translocation of PKM2 are essential for cancer metastasis, and β-elemene inhibits breast cancer metastasis via blocking aerobic glycolysis induced by dimeric PKM2 transformation and EGFR-importin α5 mediated PKM2 nuclear translocation, being a promising anti-metastatic agent. Funding Statement: The work was supported by National Natural Science Foundation of China (No. 81673648, 81673725, 81573859, 81603339), Natural Science Foundation of Higher School of Jiangsu Province (17KJA360003), and the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD). Declaration of Interests: The authors declare that they have no conflict of interest. Ethics Approval Statement: All animal studies were approved by the Animal Care and Use Committee of Nanjing University of Chinese Medicine. All mouse experimental procedures were performed in accordance with the Regulations for the Administration of Affairs Concerning Experimental Animals approved by the State Council of People’s Republic of China.

Vandetanib (ZD6474) induces antiangiogenesis through mTOR-HIF-1 alpha-VEGF signaling axis in breast cancer cells.

ObjectiveVandetanib, also known as ZD6474, has recently been proved to be a clinical drug for cancer by targeting vascular endothelial growth factor receptor 2 (VEGFR2), EGFR, and RET tyrosine kinases. We hypothesized that vandetanib will be a drug candidate for breast cancer treatment by targeting angiogenesis.Materials and methodsVandetanib was used to treat different breast cancer cell lines, and its effect on growth, apoptosis, and cell cycle was studied by MTT assay and flow cytometry. VEGF level in culture medium was measured by ELISA. Gene expression of mechanistic target of rapamycin (mTOR), hypoxia-inducible factor (HIF)-1 alpha, and VEGF at mRNA and protein level were analyzed by quantitative real-time-PCR and Western blot. The cellular behavior variations were investigated by using wound healing assay, transwell invasion assay, and tubular formation assay as well as experiments in vivo.ResultWe found that vandetanib can inhibit breast cancer cell line growth via apoptosis and cell cycle regulation. VEGF secretion decreases upon treatment. Vandetanib can reduce both mRNA and protein level of mTOR, HIF-1 alpha, and VEGF. Angiogenesis assays showed that vandetanib can inhibit wound healing, invasion, and tubular formation in culture. Furthermore, vandetanib inhibited the growth of breast tumor in vivo.ConclusionIn short, our study showed that vandetanib can control angiogenesis of breast cancer in culture via mTOR, HIF-1 alpha, and VEGF signaling pathway.

Boswellia frereana suppresses HGF-mediated breast cancer cell invasion and migration through inhibition of c-Met signalling

BackgroundHepatocyte growth factor (HGF) plays a pivotal role in breast cancer cell motility, invasion and angiogenesis. These pro-metastatic events are triggered through HGF coupling and activation of the c-Met receptor. Reports have demonstrated that HGF/c-Met signalling plays an important part in breast cancer progression and that their expression is linked to poor patient outcome. In the present study, we investigated the anti-metastatic potential of an extract from traditional Somalian frankincense, Boswellia frereana, on human breast cancer cells. In addition, we also examined the effect of this Boswellia frereana extract (BFE) upon HGF-mediated stimulation of the c-Met receptor.MethodsTwo triple negative human breast cancer cell lines, BT549 and MDA-MB-231, were utilised in the study to examine the effect of BFE on tumour cell proliferation, migration, matrix-adhesion, angiogenesis and invasion. Cell migration was investigated using a Cell IQ time-lapsed motion analysis system; while tumour cell–matrix adhesion, angiogenesis and invasion were assessed through Matrigel-based in vitro assays. Breast cancer cell growth and spheroid formation was examined through proliferation assay and 3D non-scaffold cell culture techniques. Western Blotting was employed to determine the phosphorylation status of the c-Met receptor tyrosine kinase following BFE treatment and subsequent HGF stimulation.ResultsFollowing HGF treatment, the breast cancer cells displayed a significant increase in migration, matrix adhesion, vessel/tubule formation, invasion and c-Met activation. HGF did not appear to have any bearing on the proliferation rate or spheroid formation of these breast cancer cells. The addition of the BFE extract quenched the HGF-enhanced migratory, angiogenic and invasive potential of these cells. Further study revealed that BFE inhibited c-Met receptor tyrosine kinase phosphorylation within these breast cancer cells.ConclusionsOur findings reveal that BFE was able to significantly suppress the influence of HGF in breast cancer cell motility and invasion in vitro, through the ability of BFE to reduce HGF/c-Met signalling events. Therefore, these results indicate that BFE could play a novel role in the treatment of breast cancer.

Implicit recognition of orthographic violations in Spanish. Eye-tracking and fMRI evidence

Qingdu granule (QDG), a traditional Chinese herbal prescription, had anti-tumor effect on breast cancer. However the underlying mechanism of QDG was unclear.The present study aimed to investigate whether QDG could inhibit angiogenesis of breast cancer via acting on nuclear factor of activated T-cells (NFAT) signaling pathway. This was implicated in human umbilical vein endothelial cells (HUVECs) in vitro and breast cancer xenograft model in vivo.The VEGF165 (15.58 ng/mL) induced human umbilical vein endothelial cells (HUVECs) were treated with serum samples containing tamoxifen (TAM), tacrolimus (FK506), or QDG with three dosages. The migration and canalization capacities of HUVECs were evaluated by transwell migration and tube formation assay. In 72 h-cultured HUVECs, The gene expression, protein amount, and nuclear translocation of NFATc3 were measured. The anti-tumor and anti-angiogenic effects of QDG in vivo were investigated in breast cancer xenograft model. The serum VEGF levels, microvessel density, and protein expressions (immunohistochemistry and western blot) of VEGF, VEGFR2 and NFATc3 were detected.The results showed that, QDG significantly inhibited HUVEC migration and tube formation. It downregulated NFATc3 gene expression, decreased NFATc3 protein amount, and reduced the ratio of NFATc3 nuclear translocation in HUVECs. In breast cancer xenograft model, QDG treatment significantly suppressed tumor growth, inhibited VEGF release, and decreased microvessel density. QDG reduced protein expressions of VEGF, VEGFR2 and NFATc3.The results suggested that QDG showed anti-angiogenic effects of breast cancer both in vitro and in vivo. The mechanism might be partially associated with inhibiting NFAT signaling pathway.

MALAT1 promotes angiogenesis of breast cancer.

Metastasis‑associated lung adenocarcinoma transcript1 (MALAT1) is a long non‑coding RNA (lncRNA) that has an oncogenic role in some types of cancers, uncluding breast cancer (BC). To investigate the role of MALAT1 in human BC progression, we detected MALAT1 expression levels based on tissue samples from 20BC cases and 20healthy controls and found MALAT1 expression levels to be significantly high (P<0.05). Then, we knocked down endogenous MALAT1 in MCF‑7 cells using MALAT1 short hairpin RNA (shRNA). The results revealed that MALAT1 knockdown could significantly inhibit proliferation, migration, and tube formation invitro. In addition, miR‑145 expression inversely changed in BC tissue cases. Furthermore, knockdown of endogenous MALAT1 significantly increased miR‑145 levels in MCF‑7 cells. This finding indicated an interaction between MALAT1 and miR‑145. In addition, knockdown of MALAT1 significantly reduced the expression of vascular endothelial growth factor in MCF‑7 cells. This outcome revealed that MALAT1 promoted angiogenesis in BC, which may be related to the expression of miR‑145.

Comparative inhibition of MCF-7 breast cancer cell growth, invasion and angiogenesis by Cannabis sativa L. sourced from sixteen different geographic locations

Molecular mechanism of Cannabis sativa L. on some of its claimed possible health benefits have not been thoroughly explored despite the global commercialization of Cannabis products. The scientific validation of C. sativa L. available in certain countries is required and demands to research its effect on cancer cell growth and progression. In the present study, several C. sativa L. samples were collected from various part of the world including Southern Africa and studied to determine their comparative cytotoxic and anti-angiogenic activity with exploring the possible mechanism of their action. Sixteen samples of C. sativa L. collected from various parts of the world and four extracts from each sample generated by 24-h successive maceration using hexane, dichloromethane (DCM), DCM: methanol (1:1) and methanol. Total flavonoid content (TFC) in each extract was quantified. Angiogenesis in Chick Chorioallantoic Membrane (CAM) was induced by vascular endothelial growth factor (VEGF) and l-arginine at the day 8th and the effect confirmed at the day 11th. Nitric oxide (NO) and matrix mettaloproteinase-1 (MMP-1) inhibition assays were performed to explore the possible mechanism of action. Cytotoxicity and VEGF inhibition assays were also performed in MCF-7 breast cancer cell line. The C. sativa L. of “Pondo” region, of the Eastern Cape, South Africa showed maximum inhibition of angiogenic index and inhibited the MMP-1 enzyme (whereas IC50 found <20 μg/ml) as compared to other samples. The flavonoid enriched extracts inhibited the NO whereas IC50 ranged from 80.83–90.15 μg/ml. The C. sativa L. sample of Eastern Cape South Africa and Denmark (Danish) were found more cytotoxic (IC50 ~ 25 μg/ml at 48 h) and able to reduce extracellular VEGF level significantly in MCF-7 cells as *p < 0.05 found statistically. The result collectively indicated that C. sativa L. from “Pondo” region of Eastern Cape, South Africa showed maximum inhibitory effect on MCF-7 cell growth and angiogenesis due to ability to inhibit NO, MMP-1 and VEGF meriting further studies.

Branched pentapeptides as potent inhibitors of the vascular endothelial growth factor 165 binding to Neuropilin-1: Design, synthesis and biological activity

The demonstrated involvement of VEGF165/NRP-1 complex in pathological angiogenesis has catalyzed interest in blocking this interaction to combat angiogenesis dependent diseases. It was shown before that Lys-Pro-Pro-Arg is a fairly strong inhibitor of the VEGF165/NRP-1 interaction. Our current findings suggest that the side chain elongation of the Lys1 by branching it with additional homoarginine (Har) residue, to obtain Lys(Har)-Pro-Pro-Arg, allows more effective inhibition. Moreover, increasing the flexibility of the middle part of molecule, in particular with simultaneous introduction of additional interacting elements at the second or third position, produced compounds up to 30-fold more active (IC50 = 0.2 μM) than the heptapeptide ATWLPPR (A7R) which is one of the first peptide known as an effective antagonist of the VEGF165 binding to NRP-1 and in vivo decreases breast cancer angiogenesis and growth. Herein, we present also the structure-activity study of Lys(Har)-Pro-Pro-Arg, discussing the design, synthesis, inhibitory activity, proteolytic stability and molecular modeling of the prepared derivatives. For two of the most active analogs the high proteolytic stability was also observed. These studies provide the next step for elucidating the optimal structure of the small peptidic inhibitors of VEGF165/NRP-1 interaction that could serve as research tools or be prospective drug candidates.

Specific Antibody Fragment Ligand Traps Blocking FGF1 Activity.

Fibroblast growth factor 1 (FGF1) and its receptors (FGFRs) regulate crucial biological processes such as cell proliferation and differentiation. Aberrant activation of FGFRs by their ligands can promote tumor growth and angiogenesis in many tumor types, including lung or breast cancer. The development of FGF1-targeting molecules with potential implications for the therapy of FGF1-driven tumors is recently being considered a promising approach in the treatment of cancer. In this study we have used phage display selection to find scFv antibody fragments selectively binding FGF1 and preventing it from binding to its receptor. Three identified scFv clones were expressed and characterized with regard to their binding to FGF1 and ability to interfere with FGF1-induced signaling cascades activation. In the next step the scFvs were cloned to scFv-Fc format, as dimeric Fc fusions prove beneficial in prospective therapeutic application. As expected, scFvs-Fc exhibited significantly increased affinity towards FGF1. We observed strong antiproliferative activity of the scFvs and scFvs-Fc in the in vitro cell models. Presented antibody fragments serve as novel FGF1 inhibitors and can be further utilized as powerful tools to use in the studies on the selective cancer therapy.

Tumor endothelial marker 8 promotes cancer progression and metastasis

Every year more than 8 million people suffer from cancer-related deaths worldwide [1]. Metastasis, the spread of cancer to distant sites, accounts for 90% of these deaths. A promising target for blocking tumor progression, without causing severe side effects [2], is Tumor Endothelial Marker 8 (TEM8), an integrin-like cell surface protein expressed predominantly in the tumor endothelium and in cancer cells [3, 4]. Here, we have investigated the role of TEM8 in cancer progression, angiogenesis and metastasis in invasive breast cancer, and validated the main findings and important results in colorectal cancer. We show that the loss of TEM8 in cancer cells results in inhibition of cancer progression, reduction in tumor angiogenesis and reduced metastatic burden in breast cancer mouse models. Furthermore, we show that TEM8 regulates cancer progression by affecting the expression levels of cell cycle-related genes. Taken together, our findings may have broad clinical and therapeutic potential for breast and colorectal primary tumor and metastasis treatment by targeting TEM8.

Abstract 2042: Hypoxia induces miR-153 through the IRE1α-XBP1 pathway to fine-tune the HIF1α/VEGFA axis in breast cancer angiogenesis

Abstract It is well documented that hypoxia activates the hypoxia-inducible factor (HIF1α)/vascular endothelial growth factor A (VEGFA) axis to promote angiogenesis in breast cancer. However, it is unclear how this axis is negatively regulated. A number of studies suggest that miR-153 inhibits tumor growth and metastasis by targeting SNAI1, ZEB2, MTDH, ADAM19, AKT1, HECTD3, and MCL1. We recently reported that miR-153 was induced by mifepristone and inhibited the breast cancer stem cells by down-regulating KLF5. In this study, we demonstrated that miR-153 directly inhibits expression of HIF1α by binding to the 3'UTR of HIF1A mRNA, as well as suppresses tube formation of primary human umbilical vein endothelial cells (HUVECs) and breast cancer angiogenesis by decreasing the secretion of VEGFA. Importantly, expression of miR-153 was induced by hypoxia-stimulated ER stress, which activates IRE1α and its downstream transcription factor XBP1. XBP1 directly binds to the promoter of the miR-153 host gene PTPRN and activates transcription. These results indicate that hypoxia induces miR-153 to fine tune the HIF1α/VEGFA axis in breast cancer angiogenesis and miR-153 could be used for breast cancer anti-angiogenesis therapy. Citation Format: Huichun Liang, Zhongmei Zhou, Ceshi Chen. Hypoxia induces miR-153 through the IRE1α-XBP1 pathway to fine-tune the HIF1α/VEGFA axis in breast cancer angiogenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2042.

FOXP3 inhibits angiogenesis by downregulating VEGF in breast cancer

Forkhead box P3 (FOXP3), an X-linked tumor suppressor gene, plays an important role in breast cancer. However, the biological functions of FOXP3 in breast cancer angiogenesis remain unclear. Here we found that the clinical expression of nuclear FOXP3 was inversely correlated with breast cancer angiogenesis. Moreover, the animal study demonstrated that FOXP3 significantly reduced the microvascular density of MDA-MB-231 tumors transplanted in mice. The cytological experiments showed that the supernatant from FOXP3-overexpressing cells exhibited a diminished ability to stimulate tube formation and sprouting in HUVECs in vitro. In addition, expression of vascular endothelial growth factor (VEGF) was downregulated by FOXP3 in breast cancer cell lines. Luciferase reporter assays and chromatin immunoprecipitation assays demonstrated that FOXP3 can directly interact with the VEGF promoter via specific forkhead-binding motifs to suppress its transcription. Importantly, the inhibitory effects of FOXP3 in the supernatant on tube formation and sprouting in HUVECs could be reversed by adding VEGF in vitro. Nuclear FOXP3 expression was inversely correlated with VEGF expression in clinical breast cancer tissues, and FOXP3 downregulation and VEGF upregulation were both correlated with reduced survival in breast cancer data sets in the Kaplan–Meier plotter. Taken together, our data demonstrate that FOXP3 suppresses breast cancer angiogenesis by downregulating VEGF expression.

Abstract 2045: Mechanistic insights into the role of cAMP-regulated EPAC1 in tumor-induced angiogenesis and metastatic potential in triple-negative breast cancer

Abstract Background: Breast cancer is the leading cause of cancer-related mortality in women. Triple negative breast cancer (TNBC) is one of most aggressive subtypes, which is often accompanied by metastasis, a feature that requires development of new blood vessels (neovasculature) to disseminate tumor cells throughout the body. We recently reported that down-regulation of exchange factor directly activated by Cyclic AMP (cAMP), also known as EPAC1, leads to a reduction in proliferation and inhibition of cell migration. Methods: Here, using gene knockdown by siRNA and a 3-dimensional in-vitro TNBC co-culture model with Human Vascular Endothelial Cells (HUVECs), we studied microvascular density and the role of EPAC1. Angiogenesis proteome profiling was employed to examine protein networks that were regulated by EPAC1 in the TNBC cell line, MDA-MB-231. Immunofluorescence along with Electric cell-substrate impedance sensing (ECIS) was used to study vascular permeability. To confirm these findings in a translational context, we employed a human tumor explant assay that predicts clinical response to therapy (CANscriptTM) and studied CD34+ nodes to determine how neovasculature associates to drug response. Results: We determined that downregulation of EPAC1 in the TNBC cell line, MDA-MB-231, leads to reduction of proteins involved in cell migration, metastasis, angiogenesis and adhesion. Interestingly, we determined that loss of EPAC1 leads to increased expression of negative regulators in these same pathways. Immunofluorescence imaging showed that EPAC1 downregulation in MDA-MB-231 cells leads to diminished expression of proteins such as Paxillin, MENA, MMP-9 and tubulin. indicated that EPAC1 role in vascular permeability. Finally, CANscriptTM demonstrated a link between response to therapy and microvascular density, which was reduced under drug pressure in patients that were predicted to respond to treatment. Conclusion: Our results suggest that EPAC1 is a driver of microvascular density in the tumor microenvironment, a feature that may play a key role in distant metastasis and therapy failure. Ex-vivo modeling of neovasculature may be a novel strategy to predict clinical response and distant metastasis. Citation Format: Muthu Dhandapani, Aaron Goldman, Naveen Kumar, Peeyush Prasad, Eshna Jash, Itender Singh, Kodaganur S. Gopinath, Krishnamurthy S, Prabhusankar P, Seema Sehrawat. Mechanistic insights into the role of cAMP-regulated EPAC1 in tumor-induced angiogenesis and metastatic potential in triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2045.

Abstract 2509: Blockage of angiotensin II type 1 receptor (AGTR1) inhibits epithelial-mesenchymal transition (EMT) and tumor growth in breast cancer

Abstract The angiotensin II type I receptor (AGTR1) has been implicated in diverse aspects of human disease, from the regulation of blood pressure and cardiovascular homeostasis. A recent study has reported pathogenic overexpression of the angiotensin II type 1 receptor (AGTR1) in breast cancers. We sought to investigate the effect of losartan, an angiotensin II receptor blocker on cell proliferation, epithelial-mesenchymal transition (EMT), angiogenesis and tumor growth in breast cancer. Overexpression of AGTR1 was associated with accelerated cell proliferation, concomitant with increased expression of survival factors including poly(ADP-ribose) polymerase (PARP) and X-linked inhibitor of apoptosis (XIAP), as well as extracellular signal-regulated kinase (ERK) activation. AGTR1-overexpressing MCF7 cells were more aggressive than their parent line, with significantly increased activity in migration and invasion assays. These observations were associated with changes in EMT markers, including reduced E-cadherin expression and increased p-Smad3, Smad4 and Snail levels. Treatment with the AGTR1 antagonist losartan attenuated these effects in vitro. To confirm the physiological relevance of our in vitro observations, we examined the effect of losartan on tumor growth using a mammary fat pad xenograft model with the AGTR1-overexpressing cells. Losartan administration resulted in a significant reduction in tumor growth and a significant decrease in the number of Ki-67-positive cells in the AGTR1-overexpressing xenograft tumors. Losartan administration notably upregulated E-cadherin and suppressed vimentin expression. Furthermore, microvessel density analysis revealed that the number of CD31-positive microvessels in both the peritumoral and intratumoral areas was significantly decreased in the losartan-treated groups. Our findings support the notion that AGTR1 is a potentially useful diagnostic marker and its inhibition may provide an effective therapeutic strategy for breast cancer treatment. Citation Format: Ji Young Kim, Eunhye Oh, Yoon-Jae Kim, Daeil Sung, Tae-Min Cho, Seojin Jang, Jae Hong Seo. Blockage of angiotensin II type 1 receptor (AGTR1) inhibits epithelial-mesenchymal transition (EMT) and tumor growth in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2509.

Tryptase promotes breast cancer angiogenesis through PAR-2 mediated endothelial progenitor cell activation.

Mast cells have been demonstrated to accumulate around and within solid tumors of numerous types, and express a number of pro-angiogenic compounds, including tryptase. They may serve an early role in angiogenesis within developing tumors. In the present study, the role and mechanism of tryptase in the activation of endothelial progenitor cells (EPCs) in breast cancer angiogenesis were evaluated. Human umbilical cord blood EPCs were isolated and cultured. MB-MDA-231 breast cancer cells were then pretreated with tryptase, and the conditioned medium was collected. The effects of tryptase on the migratory and angiogenesis abilities of EPCs were determined using wound-healing and tube formation assays, respectively. The effect of tryptase on the proliferation of EPCs was detected using a Cell Counting Kit-8 assay. Alterations in proteinase activated receptor (PAR)-2, phosphorylated (p)-protein kinase B (AKT), p-extracellular signal-regulated kinase (p-ERK) and vascular endothelial growth factor receptor (VEGFR)-2 expression were analyzed, in tryptase or conditioned medium-treated EPCs, by western blot analysis and reverse transcription-quantitative polymerase chain reaction. It was confirmed that the EPCs expressed PAR-2; and that tryptase treatment promoted the migration and tube formation of EPCs. Treatment with a PAR-2 agonist had a similar effect to tryptase, whereas treatment with a tryptase inhibitor, APC366, or a PAR-2 inhibitor, SAM 11, inhibited the effect of tryptase treatment. Tryptase and PAR-2 agonists did not affect the rate of EPC proliferation. MB-MDA-231 cells also expressed PAR-2. Treatment with tryptase or conditioned medium increased the expression of PAR-2, p-AKT, p-ERK and VEGFR-2 in EPCs. In conclusion, tryptase activated EPCs via PAR-2-mediated AKT and ERK signaling pathway activation, thereby enhancing angiogenesis in breast cancer.