Reduced Tumor Angiogenesis Research Articles

Abstract B03: Integrin agonists reduce infiltration of tumor-associated macrophages to promote T-cell mediated anti-tumor immunity

Abstract Tumors associated macrophages (TAMs) mediate neovascularization and promote tumor growth. Integrin CD11b/CD18 is highly expressed on cells of myeloid and granulocytic lineage and mediates their adhesion, migration and tissue recruitment functions. CD11b+ TAMs are present at high numbers in several types of human cancers, including breast and non-small cell lung cancer, and are associated with reduced T-cell mediated anti-tumor immunity. This suggests that reducing infiltration of CD11b+ TAMs could have a therapeutic benefit. Recently, we have developed a novel small molecule CD11b agonist termed leukadherin-1 (LA1) that binds to and allosterically activates integrin CD11b on leukocytes. We also reported that CD11b activation is a more effective strategy for reducing CD11b+ cell tissue infiltration, as compared to integrin antagonists. LA1 binding transiently increases leukocyte adhesion to the vascular endothelium and reduces their transmigration in vivo. Here, we show that LA1 mediated CD11b activation reduced the number of CD11b+ TAMs in two different murine tumor models that was accompanied with diminished primary tumor growth. LA1 treated animals also had significantly reduced tumor angiogenesis and prolonged survival. These improved aspects were accompanied with reduction in vessel density as indicated by reduced intratumoral expression levels of CD31 and αSMA and increased numbers of CD8+ T cells indicating an anti-tumor immune profile response fostering tumor suppression. Our data provide a rationale for using allosteric CD11b agonists to target TAMs as a novel therapeutic approach for treating cancer. Citation Format: Samia Q. Khan, Mohd. H. Faridi, Shehryar J. Khaliqdina, Antonio J. Barbosa, Judith A. Varner, Vineet Gupta. Integrin agonists reduce infiltration of tumor-associated macrophages to promote T-cell mediated anti-tumor immunity. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr B03.

Abstract P3-06-01: Therapeutic targeting of RAGE in the tumor and tumor microenvironment inhibits breast progression and metastasis

Abstract Background: The Receptor for Advanced-Glycation End-products (RAGE) is highly expressed in various cancers and its expression is correlated with poorer outcomes in breast cancer. We have previously implicated RAGE in breast cancer, but whether RAGE drives breast cancer progression and metastasis either through tumor cell intrinsic effects, non-tumor cells of the tumor microenvironment, or both, is not fully understood. More importantly, studies are lacking that target RAGE therapeutically in cancer, and may therefore represent a novel treatment for breast cancer metastasis. Methods: Using multiple human and murine breast cancer models we dissected the tumor intrinsic versus tumor microenvironment role of RAGE in metastasis. RAGE was targeted in tumor cells using multiple shRNAs, in non-tumor cells by global gene knockout in mice, and both by therapeutically targeting with the novel RAGE inhibitor FPS-ZM1. In vivo orthotopic models included the NSG (NOD-SCID-gamma) xenograft mouse model (with MDA-MB-231 cells; herein 231), BALBc (4T-1 and 67NR), and C57BL6 wild-type and RAGE knockout (RAGE -/-) mice (with MMTV-PyMT spontaneous breast cancer derived AT-3 cells). Results: We first tested how RAGE impacts tumor cell intrinsic mechanisms using either RAGE shRNAs or FPS-ZM1 in 231, 4175 (231 isogenic highly metastatic cells) and 4T-1 cells. RAGE shRNA and FPS-ZM1 both decreased RAGE MAP-kinase signaling, transwell invasion and soft agar colony formation, without affecting proliferation. In vivo, RAGE shRNA knockdown in 231 cells did not affect tumor growth, but inhibited metastasis to lung and liver. RAGE shRNA knockdown in 4175 cells, decreased orthotopic tumor growth, and reduced tumor angiogenesis and tumor recruitment of leukocyte / macrophages. Furthermore, RAGE shRNA knockdown dramatically decreased metastasis of 4175 cells to lung and liver in a time and sized matched manner compared to shRNA controls. Similarly, RAGE knockdown in 4T-1 cells reduced cell invasion and colony formation, and inhibited lung metastasis from the orthotopic site in BALBc immunocompetent mice. To test the non-tumor cell microenvironment role of RAGE, we performed syngeneic studies with orthotopically injected AT-3 cells in RAGE +/+ and RAGE -/- C57BL6 mice. RAGE -/- mice displayed striking impairment of tumor cell growth compared to RAGE +/+ mice, along with decreased MAP-kinase signaling, tumor angiogenesis and inflammatory cell recruitment. Finally, to test the combined inhibition of RAGE in both tumor cell intrinsic and non-tumor cells of the microenvironment, we performed in vivo treatment of 4175 tumors with FPS-ZM1 (1mg/kg, twice per week). Compared to vehicle, FPS-ZM1 inhibited primary tumor growth, inhibited tumor angiogenesis and inflammatory cell recruitment, and most importantly prevented metastasis to lung and liver. Conclusion: These data clearly demonstrate a role for RAGE in breast cancer progression and metastasis through distinct effects in the tumor cell and non-tumor cells of the tumor microenvironment. Furthermore, our data from drug inhibitor studies highlight the combined targeting of RAGE in the tumor and tumor microenvironment, and as a viable therapeutic means for breast and other metastatic cancers. Citation Format: Kwak T, Drews-Elger K, Ergonul A, Braley A, Hwang GH, El-Ashry D, Slingerland JM, Lippman ME, Hudson BI. Therapeutic targeting of RAGE in the tumor and tumor microenvironment inhibits breast progression and metastasis [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P3-06-01.

Slug Is Associated With Tumor Metastasis and Angiogenesis in Ovarian Cancer.

Ovarian cancer is the most lethal gynecologic malignancy among women and usually initiated by the malignant transformation of epithelial cells. The progression of ovarian cancer involves a cascade of events, including tumor cell epithelial-mesenchymal transition (EMT), invasion, migration, and angiogenesis. Slug plays vital roles in the development of motile and invasive manner of cancer cells via EMT progression. The present work is devoted to investigate the effect of slug on the invasion and angiogenesis in ovarian cancer. The findings reveal that tumors with high expression of slug (44 of 60) represent higher tumor grade, lymph node metastasis, and worse prognosis than those with low expression (16 of 60; P < .05). We also identified a significant correlation between the slug and the microvessel density (MVD). Results of transwell migration assay showed that decreased slug induced by short hairpin RNA contributed to the repressed invasion and migration of SKOV3 cells. Additionally, the migration and tube formation capacity of human umbilical vein endothelial cells were markedly decreased in SKOV3-sh-conditioned medium compared to SKOV3 and SKOV3-NC. Furthermore, xenograft mouse models (SKOV3/SKOV3-sh cells injection into BALB/c nude mice) were developed to validate the effects of slug. The data confirmed that inhibited expression of slug extensively decreased the growth of tumor and MVD in vivo. Moreover, knockdown of slug can significantly reduce tumor angiogenesis of SKOV3 cells via ccn1/vascular endothelial growth factor. Thus, our present study demonstrates that slug is closely associated with tumor metastasis and angiogenesis in ovarian cancer.

ETS transcription factors Etv2 and Fli1b are required for tumor angiogenesis.

ETS transcription factor ETV2/Etsrp functions as a key regulator of embryonic vascular development in multiple vertebrates. However, its role in pathological vascular development has not been previously investigated. To analyze its role in tumor angiogenesis, we utilized a zebrafish xenotransplantation model. Using a photoconvertible kdrl:NLS-KikGR line, we demonstrated that all tumor vessels originate from the existing embryonic vasculature by the mechanism of angiogenesis. Xenotransplantation of mouse B16 melanoma cells resulted in a significant increase in expression of the ETS transcription factors etv2 and fli1b expression throughout the embryonic vasculature. etv2 null mutants which undergo significant recovery of embryonic angiogenesis during later developmental stages displayed a strong inhibition of tumor angiogenesis. We utilized highly specific and fully validated photoactivatable morpholinos to inhibit Etv2 function after embryonic vasculogenesis has completed. Inducible inhibition of Etv2 function resulted in a significant reduction of tumor angiogenesis and inhibition of tumor growth. Furthermore, inducible inhibition of Etv2 function in fli1b mutant embryos resulted in even stronger reduction in tumor angiogenesis and growth, demonstrating that Etv2 and Fli1b have a partially redundant requirement during tumor angiogenesis. These results demonstrate the requirement for Etv2 and Fli1b in tumor angiogenesis and suggest that inhibition of these ETS factors may present a novel strategy to inhibit tumor angiogenesis and reduce tumor growth.

Predictive model of thrombospondin-1 and vascular endothelial growth factor in breast tumor tissue.

Angiogenesis, the formation of new blood capillaries from pre-existing vessels, is a hallmark of cancer. Thus far, strategies for reducing tumor angiogenesis have focused on inhibiting pro-angiogenic factors, and less is known about the therapeutic effects of mimicking the actions of angiogenesis inhibitors. Thrombospondin-1 (TSP1) is an important endogenous inhibitor of angiogenesis that has been investigated as an anti-angiogenic agent. TSP1 impedes the growth of new blood vessels in many ways, including crosstalk with pro-angiogenic factors. Owing to the complexity of TSP1 signaling, a predictive systems biology model would provide quantitative understanding of the angiogenic balance in tumor tissue. Therefore, we have developed a molecular-detailed, mechanistic model of TSP1 and vascular endothelial growth factor (VEGF), a promoter of angiogenesis, in breast tumor tissue. The model predicts the distribution of the angiogenic factors in tumor tissue, revealing that TSP1 is primarily in an inactive, cleaved form owing to the action of proteases, rather than bound to its cellular receptors or to VEGF. The model also predicts the effects of enhancing TSP1’s interactions with its receptors and with VEGF. To provide additional predictions that can guide the development of new anti-angiogenic drugs, we simulate administration of exogenous TSP1 mimetics that bind specific targets. The model predicts that the CD47-binding TSP1 mimetic markedly decreases the ratio of receptor-bound VEGF to receptor-bound TSP1, in favor of anti-angiogenesis. Thus, we have established a model that provides a quantitative framework to study the response to TSP1 mimetics.

Targeting of RAGE-ligand signaling impairs breast cancer cell invasion and metastasis.

The receptor for advanced glycation end products (RAGE) is highly expressed in various cancers and is correlated with poorer outcome in breast and other cancers. Here we tested the role of targeting RAGE by multiple approaches in the tumor and tumor microenvironment, to inhibit the metastatic process. We first tested how RAGE impacts tumor cell-intrinsic mechanisms using either RAGE overexpression or knockdown with short hairpin RNAs (shRNAs). RAGE ectopic overexpression in breast cancer cells increased MEK-EMT (MEK-epithelial-to-mesenchymal transition) signaling, transwell invasion and soft agar colony formation, and in vivo promoted lung metastasis independent of tumor growth. RAGE knockdown with multiple independent shRNAs in breast cancer cells led to decreased transwell invasion and soft agar colony formation, without affecting proliferation. In vivo, targeting RAGE shRNA knockdown in human and mouse breast cancer cells, decreased orthotopic tumor growth, reduced tumor angiogenesis and recruitment of inflammatory cells, and markedly decreased metastasis to the lung and liver in multiple xenograft and syngeneic mouse models. To test the non-tumor cell microenvironment role of RAGE, we performed syngeneic studies with orthotopically injected breast cancer cells in wild-type and RAGE-knockout C57BL6 mice. RAGE-knockout mice displayed striking impairment of tumor cell growth compared with wild-type mice, along with decreased mitogen-activated protein kinase signaling, tumor angiogenesis and inflammatory cell recruitment. To test the combined inhibition of RAGE in both tumor cell-intrinsic and non-tumor cells of the microenvironment, we performed in vivo treatment of xenografted tumors with FPS-ZM1 (1 mg/kg, two times per week). Compared with vehicle, FPS-ZM1 inhibited primary tumor growth, inhibited tumor angiogenesis and inflammatory cell recruitment and, most importantly, prevented metastasis to the lung and liver. These data demonstrate that RAGE drives tumor progression and metastasis through distinct tumor cell-intrinsic and -extrinsic mechanisms, and may represent a novel and therapeutically viable approach for treating metastatic cancers.

SPARC overexpression combined with radiation retards angiogenesis by suppressing VEGF-A via miR‑410 in human neuroblastoma cells.

Neuroblastoma (NB) is the most common extracranial solid tumor in children and despite aggressive therapy survival rates remain low. One of the contributing factors for low survival rates is aggressive tumor angiogenesis, which is known to increase due to radiation, one of the standard therapies for neuroblastoma. Therefore, targeting tumor angiogenesis can be a viable add-on therapy for the treatment of neuroblastomas. In the present study, we demonstrate that overexpression of secreted protein acidic and rich in cysteine (SPARC) suppresses radiation induced angiogenesis in SK-N-BE(2) and NB1691 neuroblastoma cells. We observed that overexpression of SPARC in SK-N-BE(2) and NB1691 cells reduced radiation induced angiogenesis in an in vivo mouse dorsal skin model and an ex vivo chicken CAM (chorioallantoic-membrane) model and also reduced tumor size in subcutaneous mouse tumor models of NB. We also observed that SPARC overexpression reduces VEGF-A expression, in SK-N-BE(2) and NB1691 NB cells via miR-410, a VEGF-A targeting microRNA. SPARC overexpression alone or in combination with miR-410 and radiation was shown to be effective at reducing angiogenesis. Moreover, addition of miR-410 inhibitors reversed SPARC mediated inhibition of VEGF-A in NB1691 cells but not in SK-N-BE(2) NB cells. In conclusion, the present study demonstrates that the over-expression of SPARC in combination with radiation reduced tumor angiogenesis by downregulating VEGF-A via miR-410.

Spontaneous regression of adrenal metastasis from renal cell carcinoma after sunitinib withdrawal: Case report and literature review

The spontaneous regression of metastatic renal cell carcinoma is a rare phenomenon, with an estimated incidence of < 1%. We report a case of post-nephrectomy renal cell carcinoma adrenal metastasis, followed by the spontaneous regression of the metastasis after withdrawal of sunitinib. The patient was a 55-year-old male with clear cell type renal cell carcinoma who previously underwent a left laparoscopic radical nephrectomy. After 51 months of follow up, a recurrence in the left renal fossa was observed and subsequently excised. Four months after excision, an abdominal Computerized tomography (CT) identified an adrenal metastasis of 1.6 cm. The patient was treated with sunitinib. However, the treatment was discontinued because of gastrointestinal side effects and fatigue. Eleven months after the discontinuation of sunitinib treatment, a progression in the adrenal metastasis growth (5.7 cm) was observed, whereas 16 months after the discontinuation, a regression of the adrenal metastasis growth (3.4 cm) was observed. During subsequent follow-ups, a gradual reduction in the size of the adrenal metastasis (1.8 cm) was observed. After 44 months from the discontinuation of sunitinib treatment, the patient was still alive and followed up in the outpatient department. Sunitinib is a multi-targeted inhibitor of vascular endothelial growth factor (VEGF) receptors. This compound reduces tumor angiogenesis and has been approved worldwide for the treatment of advanced renal cell carcinoma. To our knowledge, this is the fourth case of the spontaneous regression of metastatic renal cell carcinoma after the discontinuation of sunitinib treatment.

Infra Red Dye and Endostar Loaded Poly Lactic Acid Nano Particles as a Novel Theranostic Nanomedicine for Breast Cancer.

Endostar, a novel recombinant human endostatin, has been proven to inhibit tumor angiogenesis and is utilized as an anticancer drug. While free drugs can display limited efficacy, nanoscaled anticancer drugs have been fabricated and proven to possess superior therapeutic effects. Poly(lactic acid) (PLA) is a FDA-approved biomaterial displaying excellent biocompatibility and low toxicity. In this study, Endostar-loaded PLA nanoparticles (EPNPs) were first prepared, and a near-infrared (NIR) dye, IRDye 800CW, was conjugated to the surface for detecting nanoparticle biodistribution through fluorescence molecular imaging (FMI) using an orthotopic breast tumor mouse model. The antitumor efficacy of EPNPs was examined using bioluminescence imaging (BLI) and immunohistology. To further improve the antitumor effects, we combined EPNPs with zoledronic acid monohydrate (ZA), which is known to decrease the tumor-associated macrophages (TAM) and inhibit tumor progression. We found that EPNPs decreased human umbilical vein endothelial cell (HUVEC) viability by inhibiting tumor growth gene expression more significantly than free Endostar in vitro. In vivo, EPNPs displayed better tumor growth inhibitory effects compared with free Endostar, and the combination of EPNPs with ZA exhibited more significant antitumor effects. As confirmed by CD31 and CD11b immunohistochemistry, the combination of EPNPs and ZA showed synergistic effects in reducing tumor angiogenesis and TAM accumulation in tumor regions. Taken together, this study presents a novel and effective form of nanoscaled Endostar for the treatment of breast cancer that displays synergistic antitumor effects in combination with ZA.

Brain metastasis in breast cancer: a comprehensive literature review.

This comprehensive review provides information on epidemiology, size, grade, cerebral localization, clinical symptoms, treatments, and factors associated with longer survival in 14,599 patients with brain metastasis from breast cancer; the molecular features of breast cancers most likely to develop brain metastases and the potential use of these predictive molecular alterations for patient management and future therapeutic targets are also addressed. The review covers the data from 106 articles representing this subject in the era of modern neuroimaging (past 35years). The incidence of brain metastasis from breast cancer (24% in this review) is increasing due to advances in both imaging technologies leading to earlier detection of the brain metastases and introduction of novel therapies resulting in longer survival from the primary breast cancer. The mean age at the time of breast cancer and brain metastasis diagnoses was 50.3 and 48.8years respectively. Axillary node metastasis was noted in 32.8% of the patients who developed brain metastasis. The median time intervals between the diagnosis of breast cancer to identification of brain metastasis and from identification of brain metastasis to death were 34 and 15months, respectively. The most common symptoms experienced in patients with brain metastasis consisted of headache (35%), vomiting (26%), nausea (23%), hemiparesis (22%), visual changes (13%) and seizures (12%). A majority of the patients had multiple metastases (54.2%). Cerebellum and frontal lobes were the most common sites of metastasis (33 and 16%, respectively). Of the primary tumors for which biomarkers were recorded, 37% were estrogen receptor (ER)+, 41% ER-, 36% progesterone receptor (PR)+, 34% PR-, 35% human epithelial growth factor receptor 2 (HER2)+, 41% HER2-, 27% triple negative and 18% triple positive (TP). Treatment in most patients consisted of a multimodality approach often with two or more of the following: whole brain radiation therapy (52%), chemotherapy (51%), stereotactic radiosurgery (20%), surgical resection (14%), trastuzumab (39%) for HER2 positive tumors, and hormonal therapy (34%) for ER and/or PR positive tumors. Factors that had an impact on prognosis included grade and size of the tumor, multiple metastases, presence of extra-cranial metastasis, triple negative or HER2+biomarker status, and high Karnovsky score. Novel therapies such as application of agents to reduce tumor angiogenesis or alter permeability of the blood brain barrier are being explored with preliminary results suggesting a potential to improve survival after brain metastasis. Other potential therapies based on genetic alterations in the tumor and the microenvironment in the brain are being investigated; these are briefly discussed.

Abstract B29: Anti-breast cancer effects of the Vav/Rac inhibitor Ehop-016 in the tumor microenvironment

Abstract Metastatic breast cancer still lacks effective treatment, and remains the primary cause of mortality. Therefore, new effective strategies to inhibit breast cancer metastasis are needed. The Rho family GTPase Rac is an ideal target for anti-metastatic cancer therapy, because Rac is a key molecular switch that can promote cancer cell migration/invasion and survival. Previously, we reported the design and development of EHop-016, a small molecule compound, which inhibits Rac activation by the oncogene Vav in metastatic cancer cells with an IC50 of 1 microM. Consequently, EHop-016 inhibits the activity of the Rac downstream effector p21-activated kinase (PAK) and cell migration in metastatic breast cancer cells; and significantly reduces tumor growth, metastasis, and angiogenesis in a mouse model of metastatic breast cancer. The reduced tumor angiogenesis in response to EHop-016 treatment may be attributed to the inhibition of the Vav/Rac activity of endothelial cells, as shown by the ability of EHop-016 to inhibit the Rac activation and capillary tube formation of endothelial cells, in vitro. Recent studies have also shown a pivotal role for tumor associated stromal cells in signaling to the cancer cells to leave the primary tumor to initiate metastasis. Because Ehop-016 acts as a specific inhibitor of the Vav/Rac interaction, we hypothesize that EHop-016 inhibits the Vav/Rac signaling in not only cancer cells, but also in fibroblasts and leukocytes. To determine whether EHop-016 treatment affects the tumor microenvironment (TME), athymic nude mice bearing mammary fatpad tumors were treated with vehicle or 30 mg/kg BW EHop-016 by IP, 3X a week, for 52 days. Immunofluorescence microscopy of mammary tumor tissue sections demonstrated a statistically significant 40% decrease in Ki-67 expression, a 60% decrease in capillary tubes as quantified by CD-31 staining, and a 33% decrease in F4/80 positive macrophages in the tumors from mice following EHop-016 treatment, compared to those treated with vehicle. To investigate the effect of EHop-016 on the cytokine milieu, plasma recovered from the mice in this study were subjected to a cytokine array for common inflammatory mediators. EHop-016 treatment demonstrated a statistically significant decrease in mouse plasma levels of the major inflammatory mediator interleukin-6 (IL-6) that has been implicated with cancer progression in the TME, as well as a trend in reduced monocyte chemoattractant protein and tumor necrosis factor alpha. A PCR array analysis of the mammary tumors from this study also demonstrated a decrease in IL-6 levels in tumor tissue. Reduced macrophages and IL-6 signaling in the TME, in response to EHop-016 treatment, is expected to decrease the cytokine-mediated malignant signaling cross talk between cancer cells and stromal cells. Therefore, inhibition of Vav/Rac signaling in the TME may additionally enhance the anti-metastatic effects of EHop-016. This study was supported by National Institute on Minority Health and Health Disparities of the National Institutes of Health (NIMHHD/NIH) U54MD008149 and the Puerto Rico Science and Technology Trust to SD; Title V PPOHA P031M10505 and Title V Cooperative P031S130068 from U.S. Department of Education, and RCMI 8G12MD007583 to UCC; and UPR RCM NIH/NIMHHD grants 5U54CA096297 andR25GM061838 to THB. Citation Format: Linette Castillo-Pichardo, Cristina Del Valle, Jessica Morales, Valance Washington, Krizia Rohena-Rivera, Magaly Martinez, Luis Cubano, Suranganie Dharmawardhane. Anti-breast cancer effects of the Vav/Rac inhibitor Ehop-016 in the tumor microenvironment. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research; Oct 17-20, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(2_Suppl):Abstract nr B29.

Theaflavin-3, 3'-digallate decreases human ovarian carcinoma OVCAR-3 cell-induced angiogenesis via Akt and Notch-1 pathways, not via MAPK pathways.

Theaflavin-3, 3′-digallate (TF3) is a black tea poly-phenol produced from polymerization and oxidization of the green tea ployphenols epicatechin gallate and (−)-epigallocatechin-3-gallate (EGCG) during fermentation of fresh tea leaves. TF3 has been reported to have anticancer properties. However, the effect of TF3 on tumor angiogenesis and the underlying mechanisms are not clear. In the present study, TF3 was verified to inhibit tumor angiogenesis. Compared with EGCG, TF3 was more potent. TF3 inhibited human ovarian carcinoma OVCAR-3 cell-induced angiogenesis in human umbilical vein endothelial cell model and in chick chorioallantoic membrane model. TF3 reduced tumor angiogenesis by downregulating HIF-1α and VEGF. One of the mechanisms was TF3 inactivated Akt/mTOR/p70S6K/4E-BP1 pathway and Akt/c-Myc pathway. Besides, TF3 suppressed the cleavage of Notch-1, subsequently decreased the expression of c-Myc, HIF-1α and VEGF, and finally the impaired cancer cells induced angiogenesis. Nevertheless, TF3 did not have any influence on the MAPK pathways. Taken together, these findings suggest that TF3 might serve as a potential anti-angiogenic agent for cancer treatment.

Antitumor activity of the multikinase inhibitor regorafenib in patient-derived xenograft models of gastric cancer.

BackgroundUnresectable gastric cancer is associated with poor outcomes, with few treatment options available after failure of cytotoxic chemotherapy. Clinical trials of targeted therapies have generally shown no survival benefit in gastric cancer, with the exceptions of the antibodies ramucirumab (anti-VEGFR2) and trastuzumab (anti-HER2/neu). Given the efficacy of the multikinase inhibitor regorafenib in other gastrointestinal tumors, we investigated its potential in gastric cancer.MethodsThe antitumor activity of oral regorafenib was assessed in eight murine patient-derived gastric cancer xenograft models. Dose–response experiments assessed the efficacy and tolerability of oral regorafenib 5, 10, and 15 mg/kg/day in two models, with 10 mg/kg/day selected for further investigation in all eight models. Tumor weight and volume was monitored during treatment; tumor cell proliferation, angiogenesis, apoptosis, and intracellular signaling were assessed using immunohistochemistry and Western blotting of total tumor lysates at the end of treatment.ResultsRegorafenib showed dose-dependent inhibition of tumor growth and was well tolerated, with no significant decreases in bodyweight or evident toxicity. Regorafenib 10 mg/kg/day significantly inhibited tumor growth in all eight models (72 to 96 %; all p < 0.01), resulting in reduced tumor weight versus vehicle controls. Regorafenib reduced tumor angiogenesis 3- to 11-fold versus controls in all models (all p < 0.05), reduced tumor proliferation 2- to 5-fold in six of the eight models (all p < 0.05), and induced apoptosis in seven models.ConclusionRegorafenib was effective in patient-derived models of gastric cancer of different histological subtypes, with inhibition of tumor growth, angiogenesis, and tumor-cell proliferation observed in almost all models. These findings are consistent with the observed activity of regorafenib in preclinical models of other gastrointestinal tumors, and support further clinical investigation in gastric cancer.Electronic supplementary materialThe online version of this article (doi:10.1186/s13046-015-0243-5) contains supplementary material, which is available to authorized users.

Endothelial Robo4 suppresses breast cancer growth and metastasis through regulation of tumor angiogenesis

Targeting tumor angiogenesis is a promising alternative strategy for improvement of breast cancer therapy. Robo4 (roundabout homolog 4) signaling has been shown to protect endothelial integrity during sepsis shock and arthritis, and inhibit Vascular Endothelial Growth Factor (VEGF) signaling during pathological angiogenesis of retinopathy, which indicates that Robo4 might be a potential target for angiogenesis in breast cancer. In this study, we used immune competent Robo4 knockout mouse model to show that endothelial Robo4 is important for suppressing breast cancer growth and metastasis. And this effect does not involve the function of Robo4 on hematopoietic stem cells. Robo4 inhibits breast cancer growth and metastasis by regulating tumor angiogenesis, endothelial leakage and tight junction protein zonula occludens protein-1 (ZO-1) downregulation. Treatment with SecinH3, a small molecule drug which deactivates ARF6 downstream of Robo4, can enhance Robo4 signaling and thus inhibit breast cancer growth and metastasis. SecinH3 mediated its effect by reducing tumor angiogenesis rather than directly affecting cancer cell proliferation. In conclusion, endothelial Robo4 signaling is important for suppressing breast cancer growth and metastasis, and it can be targeted (enhanced) by administrating a small molecular drug.

Abstract 4410: An ICAM-1-targeted, Lcn2 siRNA-encapsulating liposome as a potent anti-angiogenic agent for triple-negative breast cancer

Abstract Introduction: The significant growth and metastasis of triple negative breast cancer (TNBC) tumors, coupled with limited treatment options, have made TNBC a clinically challenging disease. We have previously shown that the intercellular adhesion molecule-1 (ICAM-1) is highly overexpressed in human TNBC tissues and cell lines, suggesting that ICAM-1 could be a potential TNBC molecular target. Lipocalin 2 (Lcn2), a 25-kDa protein belonging to the lipocalin protein superfamily. We have recently reported that Lcn2 is a regulator of angiogenesis. We have also found that down-regulation of TNBC-secreted Lcn2 is associated with reduced tumor angiogenesis in preclinical studies of breast cancer, making it a promising TNBC anti-angiogenic target. Here, we describe a novel liposomal drug delivery system that integrates TNBC targeting via ICAM-1, with Lcn2 siRNA silencing, to synergistically inhibit TNBC angiogenesis in vitro and in vivo, representing a novel anti-angiogenic therapeutic approach for the treatment of TNBC. Materials and Methods: ICAM-1 antibody, or non-targeted IgG conjugated, Lcn2 siRNA encapsulating liposomes (ICAM-Lcn2-LPs or IgG-Lcn2-LPs (non-targeting control)) were prepared by the extrusion method. Lcn2 siRNA silencing in TNBC cells was determined by qRT-PCR, and the anti-angiogenic functions of ICAM-Lcn2-LP were evaluated via a series of in vitro angiogenic assays including: endothelial cell proliferation, endothelial cell migration, and endothelial cell tube formation. These in vitro anti-angiogenic results were further confirmed in vivo using the chick chorioallantoic membrane (CAM) assay. Results: We have engineered a liposomal siRNA delivery system to target triple negative breast cancer (TNBC) via a novel molecular target, ICAM-1, and selectively silenced a TNBC-overexpressing angiogenic mediator, Lcn2 using siRNA. This ICAM-1 targeted, Lcn2 siRNA delivery liposome binds human TNBC MDA-MB-231 cells 3.1-fold greater than non-neoplastic MCF-10A cells. Potent Lcn2 knockdown by ICAM-Lcn2-LP led to a significant reduction in VEGF secretion by MDA-MB-231 cells, which led to reduced TNBC-induced angiogenesis both in vitro and in vivo. Conclusions: We have successfully engineered an immunoliposome that synergistically couples TNBC-targeting with Lcn2 siRNA silencing. Synthesized ICAM-Lcn2-LPs can significantly suppress in vitro and in vivo angiogenic activities of TNBC cells by regulating VEGF secretion. The modular design introduced here provides a basis for future TNBC-targeted therapeutics. Acknowledgements: This research is supported by NIH(NCI 1DP2CA174495) and the Breast Cancer Research Foundation. Citation Format: Peng Guo, Jiang Yang, Marsha Moses, Debra Auguste. An ICAM-1-targeted, Lcn2 siRNA-encapsulating liposome as a potent anti-angiogenic agent for triple-negative breast cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4410. doi:10.1158/1538-7445.AM2015-4410

Abstract 1379: IKKβ is a potential anti-angiogenic therapeutic target in KRAS-induced lung cancer

Abstract Activating mutations in KRAS are prevalent in cancer, but therapies targeted to oncogenic RAS have been ineffective to date. An alternative route for blocking RAS-driven oncogenic pathways is to target downstream effectors of RAS involved in promoting the oncogenic phenotype. One of the critical characteristics required for tumors to grow and progress is the ability of tumor cells to drive angiogenesis. Interestingly, oncogenic RAS promotes angiogenesis by upregulating the proangiogenic IL-8 cytokine, an NF-kB target gene; and recent studies have shown that oncogenic RAS also activates the NF-κB transcription factor pathway and that KRAS-induced lung tumorigenesis is suppressed by expression of a degradation-resistant form of the IκBα inhibitor or by genetic or pharmacological inhibition of IKKβ or deletion of the RELA/p65 subunit of NF-κB. Therefore, we hypothesized that IKKβ inhibition would reduce KRAS-induced angiogenesis and tumor progression. To test this hypothesis, we used genetic and pharmacological approaches to inhibit IKKβ in K-Ras mutant lung cancer cell lines. Treatment of KRAS-positive A549 e H358 lung cancer cells with the highly specific IKKβ inhibitor Compound A (CmpdA) or siRNA-mediated knockdown of IKKβ in these cells reduced expression and secretion of the proangiogenic IL-8 cytokine. We found that IKKβ targeting also reduced expression and secretion of VEGF, a growth factor involved in promoting angiogenesis, which is also regulated by NF-κB. Moreover, conditioned media from A549 and H358 cells with siRNA-mediated IKKβ knockdown reduced endothelial cell (HUVECS) migration. In order to ascertain whether IKKβ inhibition can directly affect endothelial cell function, we treated HUVECs with CmpdA, which also resulted in reduced HUVEC migration. To evaluate how IKKβ affects endothelial cell function in vivo, we used a mouse model of neonatal retinopathy, where pathological retinal angiogenesis is induced by transient exposure of neonatal mice to hyperoxic conditions. Angiogenesis was significantly reduced in this model when neonatal mice were treated with 3 doses of 10 mg/Kg CmpdA. Finally, IKKβ knockdown in A549 cells also reduced expression of MMP-2 and MMP9 metalloproteases and reduced A549 cell invasion. Taken together, these results suggest that IKKβ inhibition therapy may reduce tumor angiogenesis, as well as invasive properties of KRAS-induced lung tumors, thereby having the potential to result in a sustained therapeutic effect, particularly if combined with other therapeutic approaches. Citation Format: Tatiana C. Lobo, Leila Magalhães, Laura Cardeal, Ricardo Giordano, Albert Baldwin, Daniela Bassères. IKKβ is a potential anti-angiogenic therapeutic target in KRAS-induced lung cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1379. doi:10.1158/1538-7445.AM2015-1379

Abstract 5224: Non-canonical activity of threonyl-tRNA synthetase promotes angiogenesis and invasion in a mouse model of ovarian cancer

Abstract Despite clear connections between the tumor microenvironment and ovarian cancer invasion, the underlying molecular mechanisms remain elusive. We have recently shown that the protein synthesis regulator threonyl-tRNA synthetase (TARS) has a unique extracellular angiogenic activity separate from its canonical function. Secreted TARS promotes endothelial cell migration and tube formation, and a selective TARS inhibitor, BC194, disrupts normal vessel development in zebra fish and chick embryo models. TARS expression also correlates with tumor stage and angiogenesis in human ovarian cancer. The objective of this study was to validate the biological and clinical relationship between TARS and ovarian tumor progression using a syngenic mouse model of epithelial ovarian cancer. Tumors were initiated by intraperitoneal injection of ID8 mouse ovarian cancer cells, and tumors formed at 4-6 weeks were scored for invasiveness and analyzed by immunostaining for TARS expression and microvascular density. To test the hypothesis that overexpression of TARS promotes invasion, cells were stably transfected with a TARS expression plasmid prior to injection. To test the hypothesis that TARS inhibition reduces tumor invasion, animals harboring ID8 tumors were treated with the high-affinity TARS inhibitor BC194. We found that TARS levels were elevated in ovarian tumors as compared with normal ovarian tissue. Overexpression of TARS in ID8 cells also resulted in enhanced invasiveness and microvascular density of the resulting tumors (p = 0.026). Preliminary results also indicated that inhibition of TARS by BC194 treatment reduced tumor angiogenesis and growth (p = 0.005) without observed toxicities in the animals. Overall, these results show that modifying TARS expression or activity can affect in vivo ovarian tumor angiogenesis and progression. These results encourage further study of TARS as a regulator of the tumor microenvironment and as a possible target for diagnosis and treatment of ovarian cancer. Citation Format: Peibin Wo, Theresa Wellman, Alan Howe, Christopher Francklyn, Karen M. Lounsbury. Non-canonical activity of threonyl-tRNA synthetase promotes angiogenesis and invasion in a mouse model of ovarian cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5224. doi:10.1158/1538-7445.AM2015-5224

Effect of NF- κ B inhibitor PDTC on VEGF and endostatin expression of mice with Lewis lung cancer.

To investigate the effects of NF- κ B inhibitor pyrrolidine dithiocarbamate hydrochloride (PDTC) on vascular endothelial growth factor (VEGF) and endostatin expression in mice with Lewis lung cance; and its mechanism. Mice survival rate and anti-tumor effects were observed in different concentrations of NF- κ B inhibitor PDTC after the Lewis lung cancer mice model was established. VEGF and endostatin expressions were detected by immunohistochemical assay. Lewis lung cancer was be inhibited by 0.5 mg/kg, 1.5 mg/kg and 3.0 mg/kg of NF- κ B inhibitor PDTC (P<0.05). Microvessel density (MVD) in 0.5 mg/kg, 1.5 mg/kg and 3.0 mg/kg NF- κ B inhibitor PDTC groups were significantly lower than the control group (P<0.05). Immunohistochemical assay results showed that VEGF and endostatin expressions in the 0.5 mg/kg, 1.5 mg/kg and 3.0 mg/kg NF-κ B inhibitor PDTC groups were significantly lower than the control group (P<0.05). Western blot results also showed that NF- κ B inhibitor PDTC could inhibit VEGF and endostatin expressions in tumor tissues. NF- κ B inhibitor PDTC can inhibit tumor formation and reduce tumor angiogenesis in mice with Lewis lung cancer; and its mechanism maybe associated to VEGF and endostatin down-regulation.

Epidermal growth factor receptor inhibition reduces angiogenesis via hypoxia-inducible factor-1α and Notch1 in head neck squamous cell carcinoma.

Angiogenesis, a marker of cancer development, affects response to radiotherapy sensibility. This preclinical study aims to understand the receptor tyrosine kinase-mediated angiogenesis in head neck squamous cell carcinoma (HNSCC). The receptor tyrosine kinase activity in a transgenic mouse model of HNSCC was assessed. The anti-tumorigenetic and anti-angiogenetic effects of cetuximab-induced epidermal growth factor receptor (EGFR) inhibition were investigated in xenograft and transgenic mouse models of HNSCC. The signaling transduction of Notch1 and hypoxia-inducible factor-1α (HIF-1α) was also analyzed. EGFR was overexpressed and activated in the Tgfbr1/Pten deletion (2cKO) mouse model of HNSCC. Cetuximab significantly delayed tumor onset by reducing tumor angiogenesis. This drug exerted similar effects on heterotopic xenograft tumors. In the human HNSCC tissue array, increased EGFR expression correlated with increased HIF-1α and micro vessel density. Cetuximab inhibited tumor-induced angiogenesis in vitro and in vivo by significantly downregulating HIF-1α and Notch1. EGFR is involved in the tumor angiogenesis of HNSCC via the HIF-1α and Notch1 pathways. Therefore, targeting EGFR by suppressing hypoxia- and Notch-induced angiogenesis may benefit HNSCC therapy.

The Role of Dietary Fat throughout the Prostate Cancer Trajectory

Prostate cancer is the second most common cancer diagnosed world-wide; however, patients demonstrate exceptionally high survival rates. Many lifestyle factors, including obesity and diet, are considered risk factors for advanced prostate cancer. Dietary fat is a fundamental contributor to obesity and may be specifically important for prostate cancer patients. Prostate cancer treatment can result in changes in body composition, affecting quality of life for survivors by increasing the risk of co-morbidities, like cardiovascular disease and diabetes. We aim to examine dietary fat throughout the prostate cancer treatment trajectory, including risk, cancer development and survivorship. Focusing on one specific nutrient throughout the prostate cancer trajectory provides a unique perspective of dietary fat in prostate cancer and the mechanisms that may exacerbate prostate cancer risk, progression and recurrence. Through this approach, we noted that high intake of dietary fat, especially, high intake of animal and saturated fats, may be associated with increased prostate cancer risk. In contrast, a low-fat diet, specifically low in saturated fat, may be beneficial for prostate cancer survivors by reducing tumor angiogenesis and cancer recurrence. The insulin-like growth factor (IGF)/Akt signaling pathway appears to be the key pathway moderating dietary fat intake and prostate cancer development and progression.