Junko Sawada

Small GTPase R-Ras Regulates Integrity and Functionality of Tumor Blood Vessels

We show that R-Ras, a small GTPase of the Ras family, is essential for the establishment of mature, functional blood vessels in tumors. The genetic disruption of R-Ras severely impaired the maturation processes of tumor vessels in mice. Conversely, the gain of function of R-Ras improved vessel structure and blood perfusion and blocked plasma leakage by enhanced endothelial barrier function and pericyte association with nascent blood vessels. Thus, R-Ras promotes normalization of the tumor vasculature. These findings identify R-Ras as a critical regulator of vessel integrity and function during tumor vascularization.

Peptide-Directed Highly Selective Targeting of Pulmonary Arterial Hypertension

Pulmonary arterial hypertension (PAH) is a disorder of the pulmonary vasculature associated with elevated pulmonary vascular resistance. Despite recent advances in the treatment of PAH, with eight approved clinical therapies and additional therapies undergoing clinical trials, PAH remains a serious life-threatening condition. The lack of pulmonary vascular selectivity and associated systemic adverse effects of these therapies remain the main obstacles to successful treatment. Peptide-mediated drug delivery that specifically targets the vasculature of PAH lungs may offer a solution to the lack of drug selectivity. Herein, we show highly selective targeting of rat PAH lesions by a novel cyclic peptide, CARSKNKDC (CAR). Intravenous administration of CAR peptide resulted in intense accumulation of the peptide in monocrotaline-induced and SU5416/hypoxia-induced hypertensive lungs but not in healthy lungs or other organs of PAH rats. CAR homed to all layers of remodeled pulmonary arteries, ie, endothelium, neointima, medial smooth muscle, and adventitia, in the hypertensive lungs. CAR also homed to capillary vessels and accumulated in the interstitial space of the PAH lungs, manifesting its extravasation activity. These results demonstrated the remarkable ability of CAR to selectively target PAH lung vasculature and effectively penetrate and spread throughout the diseased lung tissue. These results suggest the clinical utility of CAR in the targeted delivery of therapeutic compounds and imaging probes to PAH lungs.

Control of mammary tumor differentiation by SKI-606 (bosutinib)

C-Src is infrequently mutated in human cancers but it mediates oncogenic signals of many activated growth factor receptors and thus remains a key target for cancer therapy. However, the broad function of Src in many cell types and processes requires evaluation of Src-targeted therapeutics within a normal developmental and immune-competent environment. In an effort to understand the appropriate clinical use of Src inhibitors, we tested an Src inhibitor, SKI-606 (bosutinib), in the MMTV-PyVmT transgenic mouse model of breast cancer. Tumor formation in this model is dependent on the presence of Src, but the necessity of Src kinase activity for tumor formation has not been determined. Furthermore, Src inhibitors have not been examined in an autochthonous tumor model that permits assessment of effects on different stages of tumor progression. Here we show that oral administration of SKI-606 inhibited the phosphorylation of Src in mammary tumors and caused a rapid decrease in the Ezh2 Polycomb group histone H3K27 methyltransferase and an increase in epithelial organization. SKI-606 prevented the appearance of palpable tumors in over 50% of the animals and stopped tumor growth in older animals with pre-existing tumors. These antitumor effects were accompanied by decreased cellular proliferation, altered tumor blood vessel organization and dramatically increased differentiation to lactational and epidermal cell fates. SKI-606 controls the development of mammary tumors by inducing differentiation.

R-Ras Inhibits VEGF-Induced p38MAPK Activation and HSP27 Phosphorylation in Endothelial Cells.

R-Ras is a Ras family small GTPase that is highly expressed in mature functional blood vessels in normal tissues. It inhibits pathological angiogenesis and promotes vessel maturation and stabilization. Previous studies suggest that R-Ras affects cellular signaling in endothelial cells, pericytes and smooth-muscle cells to regulate vessel formation and remodeling in adult tissues. R-Ras suppresses VEGF-induced endothelial permeability and vessel sprouting while promoting normalization of pathologically developing vessels in mice. It attenuates VEGF receptor-2 (VEGFR2) activation by inhibiting internalization of the receptor upon VEGF ligand binding, leading to significant reduction of VEGFR2 autophosphorylation. Here, we show that R-Ras strongly suppresses the VEGF-dependent activation of stress-activated protein kinase-2/p38 mitogen-activated protein kinase (SAPK2/p38MAPK) and the phosphorylation of downstream heat-shock protein 27 (HSP27), a regulator of actin cytoskeleton organization, in endothelial cells. The suppression of p38MAPK activation and HSP27 phosphorylation by R-Ras concurred with altered actin cytoskeleton architecture, reduced membrane protrusion and inhibition of endothelial cell migration toward VEGF. Silencing of endogenous R-Ras by RNA interference increased membrane protrusion and cell migration stimulated by VEGF, and these effects were offset by p38MAPK inhibitor SB203580. These results suggest that R-Ras regulates angiogenic activities of endothelial cells in part via inhibition of the p38MAPK-HSP27 axis of VEGF signaling.

Developmental origin of age-related coronary artery disease

AIM Age and injury cause structural and functional changes in coronary artery smooth muscle cells (caSMCs) that influence the pathogenesis of coronary artery disease. Although paracrine signalling is widely believed to drive phenotypic changes in caSMCs, here we show that developmental origin within the fetal epicardium can have a profound effect as well. METHODS AND RESULTS Fluorescent dye and transgene pulse-labelling techniques in mice revealed that the majority of caSMCs are derived from Wt1(+), Gata5-Cre(+) cells that migrate before E12.5, whereas a minority of cells are derived from a later-emigrating, Wt1(+), Gata5-Cre(-) population. We functionally evaluated the influence of early emigrating cells on coronary artery development and disease by Gata5-Cre excision of Rbpj, which prevents their contribution to coronary artery smooth muscle cells. Ablation of the Gata5-Cre(+) population resulted in coronary arteries consisting solely of Gata5-Cre(-) caSMCs. These coronary arteries appeared normal into early adulthood; however, by 5-8 months of age, they became progressively fibrotic, lost the adventitial outer elastin layer, were dysfunctional and leaky, and animals showed early mortality. CONCLUSION Taken together, these data reveal heterogeneity in the fetal epicardium that is linked to coronary artery integrity, and that distortion of the coronaries epicardial origin predisposes to adult onset disease.

R-Ras Protein Inhibits Autophosphorylation of Vascular Endothelial Growth Factor Receptor 2 in Endothelial Cells and Suppresses Receptor Activation in Tumor Vasculature

Background: Excessive stimulation of endothelial cells by VEGF is a major cause of aberrant angiogenesis and leaky blood vessels in various medical conditions. Results: The Small GTPase R-Ras inhibits VEGF-induced internalization and tyrosine phosphorylation of VEGF receptor 2. Conclusion: R-Ras suppresses chronic and acute endothelial cell stimulation by VEGF. Significance: Our study identifies a mechanism of attenuating VEGF signaling by a Ras homolog and offers mechanistic insights into the regulation of angiogenesis by this small GTPase. Abnormal angiogenesis is associated with a broad range of medical conditions, including cancer. The formation of neovasculature with functionally defective blood vessels significantly impacts tumor progression, metastasis, and the efficacy of anticancer therapies. Vascular endothelial growth factor (VEGF) potently induces vascular permeability and vessel growth in the tumor microenvironment, and its inhibition normalizes tumor vasculature. In contrast, the signaling of the small GTPase R-Ras inhibits excessive angiogenic growth and promotes the maturation of regenerating blood vessels. R-Ras signaling counteracts VEGF-induced vessel sprouting, permeability, and invasive activities of endothelial cells. In this study, we investigated the effect of R-Ras on VEGF receptor 2 (VEGFR2) activation by VEGF, the key mechanism for angiogenic stimulation. We show that tyrosine phosphorylation of VEGFR2 is significantly elevated in the tumor vasculature and dermal microvessels of VEGF-injected skin in R-Ras knockout mice. In cultured endothelial cells, R-Ras suppressed the internalization of VEGFR2, which is required for full activation of the receptor by VEGF. Consequently, R-Ras strongly suppressed autophosphorylation of the receptor at all five major tyrosine phosphorylation sites. Conversely, silencing of R-Ras resulted in increased VEGFR2 phosphorylation. This effect of R-Ras on VEGFR2 was, at least in part, dependent on vascular endothelial cadherin. These findings identify a novel function of R-Ras to control the response of endothelial cells to VEGF and suggest an underlying mechanism by which R-Ras regulates angiogenesis.

The Long Noncoding RNA SPRIGHTLY Regulates Cell Proliferation in Primary Human Melanocytes

The long noncoding RNA SPRIGHTLY (formerly SPRY4-IT1), which lies within the intronic region of the SPRY4 gene, is up-regulated in human melanoma cells compared to melanocytes. SPRIGHTLY regulates a number of cancer hallmarks, including proliferation, motility, and apoptosis. To better understand its oncogenic role, SPRIGHTLY was stably transfected into human melanocytes, which resulted in increased cellular proliferation, colony formation, invasion, and development of a multinucleated dendritic-like phenotype. RNA sequencing and mass spectrometric analysis of SPRIGHTLY-expressing cells revealed changes in the expression of genes involved in cell proliferation, apoptosis, chromosome organization, regulation of DNA damage responses, and cell cycle. The proliferation marker Ki67, minichromosome maintenance genes 2-5, antiapoptotic gene X-linked inhibitor of apoptosis, and baculoviral IAP repeat-containing 7 were all up-regulated in SPRIGHTLY-expressing melanocytes, whereas the proapoptotic tumor suppressor gene DPPIV/CD26 was down-regulated, followed by an increase in extracellular signal-regulated kinase 1/2 phosphorylation, suggesting an increase in mitogen-activated protein kinase activity. Because down-regulation of DPPIV is known to be associated with malignant transformation in melanocytes, SPRIGHTLY-mediated DPPIV down-regulation may play an important role in melanoma pathobiology. Together, these findings provide important insights into how SPRIGHTLY regulates cell proliferation and anchorage-independent colony formation in primary human melanocytes.

Normalization of tumor vasculature by R-Ras

Malfunction of blood vessels and abnormal vessel growth are associated with various medical conditions ranging from heart disease, cancer and metabolic problems, such as diabetes, to age-related conditions, such as macular degeneration. During normal development or regeneration of adult tissues, newly formed blood vessels undergo maturation. It is important to understand how this process occurs on a cellular and molecular level, because its failure leads to structural and functional deficiencies of blood vessels. Tumor blood vessels are highly permeable, tortuous, dilated and saccular and poorly covered by mural cells (pericytes).1 These properties, which are primarily attributed to impaired tumor vessel maturation, underlie inadequate blood circulation and poor oxygenation of tumors. Although these anomalies could potentially slow tumor growth, they also reduce the sensitivity of tumors to ionizing radiation and impede delivery of chemotherapeutic agents, allowing tumors to become resistant to cytotoxic therapies.2 Excessive vessel permeability associated with tumors also causes clinical complications, such as cerebral edema, in brain cancer patients. The ability to control vessel maturity in tumors, therefore, provides a potential therapeutic opportunity.

RGD-conjugated two-photon absorbing near-IR emitting fluorescent probes for tumor vascular imaging(Conference Presentation)

Observation of the activation and inhibition of angiogenesis processes is important in the progression of cancer. Application of targeting peptides, such as a small peptide that contains adjacent L-arginine (R), glycine (G) and L-aspartic acid (D) residues can afford high selectivity and deep penetration in vessel imaging. To facilitate deep tissue vasculature imaging, probes that can be excited via two-photon absorption (2PA) in the near-infrared (NIR) and subsequently emit in the NIR are essential. In this study, the enhancement of tissue image quality with RGD conjugates was investigated with new NIR-emitting pyranyl fluorophore derivatives in two-photon fluorescence microscopy. Linear and nonlinear photophysical properties of the new probes were comprehensively characterized; significantly the probes exhibited good 2PA over a broad spectral range from 700-1100 nm. Cell and tissue images were then acquired and examined, revealing deep penetration and high contrast with the new pyranyl RGD-conjugates up to 350 μm in tumor tissue.

Abstract B25: R-Ras is critical for endothelial lumen formation, blood perfusion, and normalization of regenerating vasculature

Formation of endothelial lumen is fundamental to angiogenesis and generation of functional vasculature. Tumors develop a large number of angiogenic blood vessels. However, a significant fraction of these vessels are poorly functional due to the impaired vessel maturation such as the improper formation of endothelial lumen. R-Ras is a small GTPase of the Ras family, which inhibits vessel sprouting and branching activities and redirects the vessel sprouts from these activities to the vessel maturation process. Through this effect, R-Ras normalizes pathologically regenerating blood vessels. Here we show that R-Ras regulates formation of endothelial lumen in the developing vessel sprouts. R-Ras deficient mice develop a large number of poorly perfused blood vessels in response to tissue ischemia. Notably, many of these vessels lack lumen assessed by the lack of PODXL staining of the endothelium. In 3D in vitro angiogenesis studies in fibrin gels, upregulation of R-Ras dramatically enhances formation of lumens in the endothelial sprouts. On the other hand, knockdown of the endogenous R-Ras abrogates lumen formation and endothelial cell differentiation into tubular structures resulting in abnormal extension of endothelial cells with numerous membrane protrusions. The stabilization of microtubules in endothelial cells is critical to the formation of endothelial lumen. Upregulation of R-Ras signaling in endothelial cells results in the formation of prominent microtubule cytoskeleton network that extends to the membrane periphery in 2D culture. This phenomenon is accompanied by the significant spreading of endothelial cells. We found that R-Ras stabilizes microtubules by increasing acetylation of alpha-tubulin. This effect of R-Ras is mediated by the PI3-kinase-Akt1/2 pathway, which phosphorylates GSK3-beta and inhibits the inhibitory activity of GSK3-beta on tubulin acetylation. GSK3-beta inhibition partially rescues tubulogenesis of R-Ras-silenced endothelial cells. These findings demonstrate a novel Akt-dependent mechanism of endothelial lumen formation and blood vessel maturation. The endothelial-specific upregulation of R-Ras in vivo leads to improved blood perfusion and normalization of VEGF-induced angiogenic vessels developing in Matrigel implants. The enhanced endothelial lumen formation and resulting improvement of vessel perfusion would significantly increase the delivery of chemotherapeutic drugs to tumors, which is otherwise hampered by the abnormal, poorly perfused vasculature. This R-Ras-dependent mechanism of endothelial lumen formation should be further exploited to potentially develop a strategy for improving drug delivery and enhancing treatment efficacy via normalization of the tumor vasculature. Citation Format: Fangfei Li, Junko Sawada, Masanobu Komatsu. R-Ras is critical for endothelial lumen formation, blood perfusion, and normalization of regenerating vasculature. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Angiogenesis and Vascular Normalization: Bench to Bedside to Biomarkers; Mar 5-8, 2015; Orlando, FL. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl):Abstract nr B25.