Inhibition of Sphingosine Phosphate Receptor 1 Signaling Enhances the Efficacy of VEGF Receptor Inhibition.

Inhibition of VEGFR signaling is an effective treatment for renal cell carcinoma, but resistance continues to be a major problem. Recently, the sphingosine phosphate (S1P) signaling pathway has been implicated in tumor growth, angiogenesis, and resistance to antiangiogenic therapy. S1P is a bioactive lipid that serves an essential role in developmental and pathologic angiogenesis via activation of the S1P receptor 1 (S1P1). S1P1 signaling counteracts VEGF signaling and is required for vascular stabilization. We used in vivo and in vitro angiogenesis models including a postnatal retinal angiogenesis model and a renal cell carcinoma murine tumor model to test whether simultaneous inhibition of S1P1 and VEGF leads to improved angiogenic inhibition. Here, we show that inhibition of S1P signaling reduces the endothelial cell barrier and leads to excessive angiogenic sprouting. Simultaneous inhibition of S1P and VEGF signaling further disrupts the tumor vascular beds, decreases tumor volume, and increases tumor cell death compared with monotherapies. These studies suggest that inhibition of angiogenesis at two stages of the multistep process may maximize the effects of antiangiogenic therapy. Together, these data suggest that combination of S1P1 and VEGFR-targeted therapy may be a useful therapeutic strategy for the treatment of renal cell carcinoma and other tumor types.

RhoB antibody alters retinal vascularization in models of murine retinopathy.

Neovascularization in cancer or retinopathy is driven by pathological changes that foster abnormal sprouting of endothelial cells. Mouse genetic studies indicate that the stress-induced small GTPase RhoB is dispensable for normal physiology but required for pathogenic angiogenesis. In diabetic retinopathy, retinopathy of prematurity (ROP) or age-related wet macular degeneration (AMD), progressive pathologic anatomic changes and ischemia foster neovascularization are characterized by abnormal sprouting of endothelial cells. This process is driven by the angiogenic growth factor VEGF, which induces and supports the formation of new blood vessels. While injectable biologics targeting VEGF have been used to treat these pathological conditions, many patients respond poorly, prompting interest in other types of mechanism-based therapy. Here we report the preclinical efficacy of a monoclonal antibody that specifically targets RhoB, a signaling molecule that is genetically dispensable for normal physiology but required for pathogenic retinal angiogenesis. In murine models of proliferative retinal angiogenesis or oxygen-induced retinopathy, administering a monoclonal RhoB antibody (7F7) was sufficient to block neoangiogenesis or avascular pathology, respectively. Our findings offer preclinical proof of concept for antibody targeting of RhoB to limit diabetic retinopathy, ROP or wet AMD and perhaps other diseases of neovasculogenesis such as hemangioma or hemangiosarcoma nonresponsive to existing therapies.

Stromal-Based Signatures for the Classification of Gastric Cancer.

Treatment of metastatic gastric cancer typically involves chemotherapy and monoclonal antibodies targeting HER2 (ERBB2) and VEGFR2 (KDR). However, reliable methods to identify patients who would benefit most from a combination of treatment modalities targeting the tumor stroma, including new immunotherapy approaches, are still lacking. Therefore, we integrated a mouse model of stromal activation and gastric cancer genomic information to identify gene expression signatures that may inform treatment strategies. We generated a mouse model in which VEGF-A is expressed via adenovirus, enabling a stromal response marked by immune infiltration and angiogenesis at the injection site, and identified distinct stromal gene expression signatures. With these data, we designed multiplexed IHC assays that were applied to human primary gastric tumors and classified each tumor to a dominant stromal phenotype representative of the vascular and immune diversity found in gastric cancer. We also refined the stromal gene signatures and explored their relation to the dominant patient phenotypes identified by recent large-scale studies of gastric cancer genomics (The Cancer Genome Atlas and Asian Cancer Research Group), revealing four distinct stromal phenotypes. Collectively, these findings suggest that a genomics-based systems approach focused on the tumor stroma can be used to discover putative predictive biomarkers of treatment response, especially to antiangiogenesis agents and immunotherapy, thus offering an opportunity to improve patient stratification. Cancer Res; 76(9); 2573-86. ©2016 AACR.

Akt1 and akt3 exert opposing roles in the regulation of vascular tumor growth.

Vascular tumors are endothelial cell neoplasms whose mechanisms of tumorigenesis are poorly understood. Moreover, current therapies, particularly those for malignant lesions, have little beneficial effect on clinical outcomes. In this study, we show that endothelial activation of the Akt1 kinase is sufficient to drive de novo tumor formation. Mechanistic investigations uncovered opposing functions for different Akt isoforms in this regulation, where Akt1 promotes and Akt3 inhibits vascular tumor growth. Akt3 exerted negative effects on tumor endothelial cell growth and migration by inhibiting activation of the translation regulatory kinase S6-Kinase (S6K) through modulation of Rictor expression. S6K in turn acted through a negative feedback loop to restrain Akt3 expression. Conversely, S6K signaling was increased in vascular tumor cells where Akt3 was silenced, and the growth of these tumor cells was inhibited by a novel S6K inhibitor. Overall, our findings offer a preclinical proof of concept for the therapeutic utility of treating vascular tumors, such as angiosarcomas, with S6K inhibitors.

RhoB controls coordination of adult angiogenesis and lymphangiogenesis following injury by regulating VEZF1-mediated transcription.

Mechanisms governing the distinct temporal dynamics that characterize post-natal angiogenesis and lymphangiogenesis elicited by cutaneous wounds and inflammation remain unclear. RhoB, a stress-induced small GTPase, modulates cellular responses to growth factors, genotoxic stress and neoplastic transformation. Here we show, using RhoB null mice, that loss of RhoB decreases pathological angiogenesis in the ischaemic retina and reduces angiogenesis in response to cutaneous wounding, but enhances lymphangiogenesis following both dermal wounding and inflammatory challenge. We link these unique and opposing roles of RhoB in blood versus lymphatic vasculatures to the RhoB-mediated differential regulation of sprouting and proliferation in primary human blood versus lymphatic endothelial cells. We demonstrate that nuclear RhoB-GTP controls expression of distinct gene sets in each endothelial lineage by regulating VEZF1-mediated transcription. Finally, we identify a small-molecule inhibitor of VEZF1-DNA interaction that recapitulates RhoB loss in ischaemic retinopathy. Our findings establish the first intra-endothelial molecular pathway governing the phased response of angiogenesis and lymphangiogenesis following injury.

Vascular tumors have increased p70 S6-kinase activation and are inhibited by topical rapamycin.

Vascular tumors are endothelial cell neoplasms whose cellular and molecular mechanisms, leading to tumor formation, are poorly understood, and current therapies have limited efficacy with significant side effects. We have investigated mechanistic (mammalian) target of rapamycin (mTOR) signaling in benign and malignant vascular tumors, and the effects of mTOR kinase inhibitor as a potential therapy for these lesions. Human vascular tumors (infantile hemangioma and angiosarcoma) were analyzed by immunohistochemical stains and western blot for the phosphorylation of p70 S6-kinase (S6K) and S6 ribosomal protein (S6), which are activated downstream of mTOR complex-1 (mTORC1). To assess the function of S6K, tumor cells with genetic knockdown of S6K were analyzed for cell proliferation and migration. The effects of topical rapamycin, an mTOR inhibitor, on mTORC1 and mTOR complex-2 (mTORC2) activities, as well as on tumor growth and migration, were determined. Vascular tumors showed increased activation of S6K and S6. Genetic knockdown of S6K resulted in reduced tumor cell proliferation and migration. Rapamycin fully inhibited mTORC1 and partially inhibited mTORC2 activities, including the phosphorylation of Akt (serine 473) and PKCα, in vascular tumor cells. Rapamycin significantly reduced vascular tumor growth in vitro and in vivo. As a potential localized therapy for cutaneous vascular tumors, topically applied rapamycin effectively reduced tumor growth with limited systemic drug absorption. These findings reveal the importance of mTOR signaling pathways in benign and malignant vascular tumors. The mTOR pathway is an important therapeutic target in vascular tumors, and topical mTOR inhibitors may provide an alternative and well-tolerated therapy for the treatment of cutaneous vascular lesions.

Angiopoietin-2: an attractive target for improved antiangiogenic tumor therapy.

Anti-VEGF pathway therapies primarily target immature blood vessels in tumors. However, emerging approaches to combine with targeted therapies impacting the later stages of remodeling and vessel maturation are expected to improve clinical efficacy by expanding the target vessel population. The angiopoietin/Tie ligand/receptor system is a prototypic regulator of vessel remodeling and maturation. Angiopoietin-2 (Ang2) appears to be a particularly attractive therapeutic target. In fact, the experimental proof-of-concept showing improved efficacy when VEGF and Ang2-targeting therapies are combined has been solidly established in preclinical models, and several Ang2-targeting drugs are in clinical trials. However, rational development of these second-generation combination therapies is hampered by a limited understanding of the biological complexity that is generated from agonistic and antagonistic Ang/Tie signaling. This review discusses recent mechanistic advances in angiopoietin signaling, particularly in light of the recent study published on REGN910 and summarizes the status quo of Ang2-targeting therapies. In light of the clarified partial agonist function of Ang2, we propose that clarity on the expression profile of the angiopoietin ligands and Tie1 and Tie2 receptors in subsets of cancer vessels and cancer cells will provide clearer hypotheses for more focused rational clinical trials to exploit this seminal pathway and improve current antiangiogenic therapies.

RhoB differentially controls Akt function in tumor cells and stromal endothelial cells during breast tumorigenesis.

Tumors are composed of cancer cells but also a larger number of diverse stromal cells in the tumor microenvironment. Stromal cells provide essential supports to tumor pathophysiology but the distinct characteristics of their signaling networks are not usually considered in developing drugs to target tumors. This oversight potentially confounds proof-of-concept studies and increases drug development risks. Here, we show in established murine and human models of breast cancer how differential regulation of Akt by the small GTPase RhoB in cancer cells or stromal endothelial cells determines their dormancy versus outgrowth when angiogenesis becomes critical. In cancer cells in vitro or in vivo, RhoB functions as a tumor suppressor that restricts EGF receptor (EGFR) cell surface occupancy as well as Akt signaling. However, after activation of the angiogenic switch, RhoB functions as a tumor promoter by sustaining endothelial Akt signaling, growth, and survival of stromal endothelial cells that mediate tumor neoangiogenesis. Altogether, the positive impact of RhoB on angiogenesis and progression supercedes its negative impact in cancer cells themselves. Our findings elucidate the dominant positive role of RhoB in cancer. More generally, they illustrate how differential gene function effects on signaling pathways in the tumor stromal component can complicate the challenge of developing therapeutics to target cancer pathophysiology.

Oxidative stress promotes myofibroblast differentiation and tumour spreading.

JunD regulates genes involved in antioxidant defence. We took advantage of the chronic oxidative stress resulting from junD deletion to examine the role of reactive oxygen species (ROS) in tumour development. In a model of mammary carcinogenesis, junD inactivation increased tumour incidence and revealed an associated reactive stroma. junD-inactivation in the stroma was sufficient to shorten tumour-free survival rate and enhance metastatic spread. ROS promoted conversion of fibroblasts into highly migrating myofibroblasts through accumulation of the hypoxia-inducible factor (HIF)-1alpha transcription factor and the CXCL12 chemokine. Accordingly, treatment with an antioxidant reduced the levels of HIF and CXCL12 and numerous myofibroblast features. CXCL12 accumulated in the stroma of HER2-human breast adenocarcinomas. Moreover, HER2 tumours exhibited a high proportion of myofibroblasts, which was significantly correlated to nodal metastases. Interestingly, this subset of tumours exhibited a significant nuclear exclusion of JunD and revealed an associated oxido-reduction signature, further demonstrating the relevance of our findings in human cancers. Collectively, our data uncover a new mechanism by which oxidative stress increases the migratory properties of stromal fibroblasts, which in turn potentiate tumour dissemination.

B-Raf(V600E) and thrombospondin-1 promote thyroid cancer progression.

Although B-Raf(V600E) is the most common somatic mutation in papillary thyroid carcinoma (PTC), how it induces tumor aggressiveness is not fully understood. Using gene set enrichment analysis and in vitro and in vivo functional studies, we identified and validated a B-Raf(V600E) gene set signature associated with tumor progression in PTCs. An independent cohort of B-Raf(V600E)-positive PTCs showed significantly higher expression levels of many extracellular matrix genes compared with controls. We performed extensive in vitro and in vivo validations on thrombospondin-1 (TSP-1), because it has been previously shown to be important in the regulation of tumor angiogenesis and metastasis and is present in abundance in tumor stroma. Knockdown of B-Raf(V600E) resulted in TSP-1 down-regulation and a reduction of adhesion and migration/invasion of human thyroid cancer cells. Knockdown of TSP-1 resulted in a similar phenotype. B-Raf(V600E) cells in which either B-Raf(V600E) or TSP-1 were knocked down were implanted orthotopically into the thyroids of immunocompromised mice, resulting in significant reduction in tumor size and fewer pulmonary metastases from the primary carcinoma as compared with the control cells. Treatment of orthotopic thyroid tumors, initiated 1 week after tumor cell implantation with PLX4720, an orally available selective inhibitor of B-Raf(V600E), caused a significant tumor growth delay and decreased distant metastases, without evidence of toxicity. In conclusion, B-Raf(V600E) plays an important role in PTC progression through genes (i.e., TSP-1) important in tumor invasion and metastasis. Testing of a patient's thyroid cancer for B-Raf(V600E) will yield important information about potential tumor aggressiveness and also allow for future use of targeted therapies with selective B-Raf(V600E) inhibitors, such as PLX4720.

JunD reduces tumor angiogenesis by protecting cells from oxidative stress.

Reactive oxygen species (ROS) are implicated in the pathophysiology of various diseases, including cancer. In this study, we show that JunD, a member of the AP-1 family of transcription factors, reduces tumor angiogenesis by limiting Ras-mediated production of ROS. Using junD-deficient cells, we demonstrate that JunD regulates genes involved in antioxidant defense, H2O2 production, and angiogenesis. The accumulation of H2O2 in junD-/- cells decreases the availability of FeII and reduces the activity of HIF prolyl hydroxylases (PHDs) that target hypoxia-inducible factors-alpha (HIFalpha) for degradation. Subsequently, HIF-alpha proteins accumulate and enhance the transcription of VEGF-A, a potent proangiogenic factor. Our study uncovers the mechanism by which JunD protects cells from oxidative stress and exerts an antiangiogenic effect. Furthermore, we provide new insights into the regulation of PHD activity, allowing immediate reactive adaptation to changes in O2 or iron levels in the cell.