Roundabout Homolog Research Articles

Stromal Cell-SLIT3/Cardiomyocyte-ROBO1 Axis Regulates Pressure Overload-Induced Cardiac Hypertrophy.

Recently shown to regulate cardiac development, the secreted axon guidance molecule SLIT3 maintains its expression in the postnatal heart. Despite its known expression in the cardiovascular system after birth, SLIT3's relevance to cardiovascular function in the postnatal state remains unknown. As such, the objectives of this study were to determine the postnatal myocardial sources of SLIT3 and to evaluate its functional role in regulating the cardiac response to pressure overload stress. We performed in vitro studies on cardiomyocytes and myocardial tissue samples from patients and performed in vivo investigation with SLIT3 and ROBO1 (roundabout homolog 1) mutant mice undergoing transverse aortic constriction to establish the role of SLIT3-ROBO1 in adverse cardiac remodeling. We first found that SLIT3 transcription was increased in myocardial tissue obtained from patients with congenital heart defects that caused ventricular pressure overload. Immunostaining of hearts from WT (wild-type) and reporter mice revealed that SLIT3 is secreted by cardiac stromal cells, namely fibroblasts and vascular mural cells, within the heart. Conditioned media from cardiac fibroblasts and vascular mural cells both stimulated cardiomyocyte hypertrophy in vitro, an effect that was partially inhibited by an anti-SLIT3 antibody. Also, the N-terminal, but not the C-terminal, fragment of SLIT3 and the forced overexpression of SLIT3 stimulated cardiomyocyte hypertrophy and the transcription of hypertrophy-related genes. We next determined that ROBO1 was the most highly expressed roundabout receptor in cardiomyocytes and that ROBO1 mediated SLIT3's hypertrophic effects in vitro. In vivo, Tcf21+ fibroblast and Tbx18+ vascular mural cell-specific knockout of SLIT3 in mice resulted in decreased left ventricular hypertrophy and cardiac fibrosis after transverse aortic constriction. Furthermore, α-MHC+ cardiomyocyte-specific deletion of ROBO1 also preserved left ventricular function and abrogated hypertrophy, but not fibrosis, after transverse aortic constriction. Collectively, these results indicate a novel role for the SLIT3-ROBO1-signaling axis in regulating postnatal cardiomyocyte hypertrophy induced by pressure overload.

The tyrosine kinase inhibitor nintedanib enhances the efficacy of 90Y-labeled B5209B radioimmunotherapy targeting ROBO1 without increased toxicity in small-cell lung cancer xenograft mice.

Small cell lung cancer (SCLC) has a poor prognosis, and Roundabout homolog 1 (ROBO1) is frequently expressed in SCLC. ROBO1-targeted radioimmunotherapy (RIT) previously showed tumor shrinkage, but regrowth with fibroblast infiltration was observed. The fibroblasts would support tumor survival by secreting growth factors and cytokines. Inhibition of fibroblasts offers a candidate strategy for increasing RIT efficacy. Here, we evaluated the efficacy of combination therapy with 90Y-labeled anti-ROBO1 antibody B5209B (90Y-B5209B) and the tyrosine kinase inhibitor nintedanib in SCLC xenograft mice. Subcutaneous NCI-H69 SCLC xenograft mice were divided into four groups: saline, nintedanib alone, RIT alone, and a combination of RIT with nintedanib (combination). A single dose of 7.4 MBq of 90Y-B5209B was injected intravenously. Nintedanib was orally administered at a dose of 400 µg five times a week for 4 weeks. Tumor volumes and body weights were measured regularly. Tumor sections were stained with hematoxylin and eosin or Masson trichrome. All six tumors in the combination therapy group disappeared, and four tumors showed no regrowth. Although RIT alone induced similar tumor shrinkage, regrowth was observed. Prolonged survival in the combination therapy group was found compared with the other groups. Temporary body weight loss was observed in RIT and combination therapy. There is no difference in fibroblast infiltration between RIT alone and the combination. Nintedanib significantly enhanced the anti-tumor effects of RIT with the 90Y-B5209B without an increase in toxicity. These findings encourage further research into the potential clinical application of combining RIT with nintedanib.

Matrine suppresses liver cancer progression and the Warburg effect by regulating the circROBO1/miR-130a-5p/ROBO1 axis.

Matrine, an effective component extracted from the traditional Chinese herb, Sophora flavescens, has been indicated to exert antitumor activity in different types of cancer. However, the role and precise mechanism of matrine in the progression of liver cancer remains largely unclear. Cell viability, cell proliferation, cell apoptosis, and Warburg effect were estimated by cell counting kit-8 assay, colony formation assay, flow cytometry assay, and glucose uptake and lactate production assay, respectively. The candidate Circular RNAs (circRNAs) were screened by integrating the Gene Expression Omnibus database (GSE155949) analysis with the online program GEO2R. A quantitative real-time polymerase chain reaction was employed to test the expression of circRNA circROBO1, microRNA miR-130a-5p, and roundabout homolog 1 (ROBO1). The interaction of circROBO1/miR-130a-5p/ROBO1 axis was predicted and confirmed by bioinformatics analysis, a dual-luciferase reporter assay, and an RNA pull-down assay. A xenograft mouse model was employed to reveal the role of matrine in vivo. Matrine repressed liver cancer cell viability, proliferation, and Warburg effect, but increased cell apoptosis in vitro. CircROBO1 and ROBO1 were upregulated, but miR-130a-5p was downregulated in liver cancer tissues. Additionally, matrine could reduce the expression of circROBO1 and ROBO1, and increase the expression of miR-130a-5p. Mechanically, overexpression of circROBO1 partly recovered the effect of matrine on liver cancer cell viability, proliferation, apoptosis, and Warburg effect by regulating the miR-130a-5p/ROBO1 axis. Matrine impeded liver cancer development by mediating the circROBO1/miR-130a-5p/ROBO1 axis, which provided a theoretical basis for the application of matrine as an effective anticancer drug for liver cancer.

MicroRNA-29a-3p prevents Schistosoma japonicum-induced liver fibrosis by targeting Roundabout homolog 1 in hepatic stellate cells

BackgroundSchistosomiasis is a serious but neglected parasitic disease in humans that may lead to liver fibrosis and death. Activated hepatic stellate cells (HSCs) are the principal effectors that promote the accumulation of extracellular matrix (ECM) proteins during hepatic fibrosis. Aberrant microRNA-29 expression is involved in the development of fibrotic diseases. However, less is known about the role of miR-29 in Schistosoma japonicum (S. japonicum)-induced hepatic fibrosis.MethodsThe levels of microRNA-29a-3p (miR-29a-3p) and Roundabout homolog 1 (Robo1) were examined in liver tissues during S. japonicum infection. The possible involvement of the miR-29a-3p-Robo1 signaling pathway was determined. We used MIR29A conditional knock-in mice and mice injected with an miR-29a-3p agomir to investigate the role of miR-29a-3p in schistosomiasis-induced hepatic fibrosis. The functional contributions of miR-29a-3p-Robo1 signaling in liver fibrosis and HSC activation were investigated using primary mouse HSCs and the human HSC cell line LX-2.ResultsMiR-29a-3p was downregulated in humans and mice with schistosome-induced fibrosis, and Robo1 was upregulated in liver tissues. The miR-29a-3p targeted Robo1 and negatively regulated its expression. Additionally, the expression level of miR-29a-3p in schistosomiasis patients was highly correlated with the portal vein and spleen thickness diameter, which represent the severity of fibrosis. Furthermore, we demonstrated that efficient and sustained elevation of miR-29a-3p reversed schistosome-induced hepatic fibrosis. Notably, we showed that miR-29a-3p targeted Robo1 in HSCs to prevent the activation of HSCs during infection.ConclusionsOur results provide experimental and clinical evidence that the miR-29a-3p-Robo1 signaling pathway in HSCs plays an important role in the development of hepatic fibrosis. Therefore, our study highlights the potential of miR-29a-3p as a therapeutic intervention for schistosomiasis and other fibrotic diseases.Graphical

Binding and Efficacy of Anti-Robo4 CAR-T Cells against Solid Tumors.

Chimeric antigen receptor expression T (CAR-T) cell therapy has been shown be efficacious against relapsed/refractory B-cell malignant lymphoma and has attracted attention as an innovative cancer treatment. However, cells of solid tumors are less accessible to CAR-T cells; moreover, CAR-T function is decreased in the immunosuppressive state of the tumor microenvironment. Since most tumors induce angiogenesis, we constructed CAR-T cells targeting roundabout homolog 4 (Robo4), which is expressed at high levels in tumor vascular endothelial cells, by incorporating three anti-Robo4 single-chain variable fragments (scFv) that were identified using phage display. We found that binding affinities of the three CARs to mouse and human Robo4 reflected their scFv affinities. More importantly, when each CAR-T cell was assayed in vitro, antigen-specific cytotoxicity, cytokine-producing ability, and proliferation were correlated with binding affinity for Robo4. In vivo, all three T-cells inhibited tumor growth in a B16BL6 murine model, which also correlated with Robo4 binding affinities. However, growth inhibition of mouse Robo4-expressing tumors was observed only in the model with CAR-T cells with the lowest Robo4 affinity. Therefore, at high Robo4 expression, CAR-T in vitro and in vivo were no longer correlated, suggesting that clinical tumors will require Robo4 expression assays.

Pellino-2 in nonimmune cells: novel interaction partners and intracellular localization.

Pellino-2 is an E3 ubiquitin ligase that mediates intracellular signaling in innate immune pathways. Most studies of endogenous Pellino-2 have been performed in macrophages, but none in nonimmune cells. Using yeast two-hybrid screening and co-immunoprecipitation, we identified six novel interaction partners of Pellino-2, with various localizations: insulin receptor substrate 1, NIMA-related kinase 9, tumor necrosis factor receptor-associated factor 7, cyclin-F, roundabout homolog 1, and disheveled homolog 2. Pellino-2 showed cytoplasmic localization in a wide range of nonimmune cells under physiological potassium concentrations. Treatment with the potassium ionophore nigericin resulted in nuclear localization of Pellino-2, which was reversed by the potassium channel blocker tetraethylammonium. Live-cell imaging revealed intracellular migration of GFP-tagged Pellino-2. In summary, Pellino-2 interacts with proteins at different cellular locations, taking part in dynamic processes that change its intracellular localization influenced by potassium efflux.

Isolated congenital diaphragm hernia associated with homozygous SLIT3 gene variant in dizygous twins

Congenital diaphragmatic hernia (CDH) is a serious life-threatening birth defect characterized by abnormal development in the muscular or tendinous portion of the diaphragm during embryogenesis. Despite its high incidence, the etiology of CDH hasn't been fully understood. Genetic factors are important in pathogenesis; however, few single genes have been definitively implicated in human CDH. SLIT1, SLIT2, and SLIT3 (slit guidance ligand) are three human homologs of the drosophila Slit gene. They interact with roundabout (Robo) homolog receptors to affect cell migration, adhesion, cell motility, and angiogenesis and play important roles in cell signaling pathways including the guidance of axons. In this report, we presented dizygous twin babies with CDH related to the SLIT3 gene variant. Previous studies showed that Slit3 null mice had congenital diaphragmatic hernias on or near the ventral midline portion of the central tendon. This is the first report of homozygous SLIT3 variant associated with CDH in humans.

Roundabout homolog 1 inhibits proliferation via the YY1-ROBO1-CCNA2-CDK2 axis in human pancreatic cancer.

Pancreatic cancer (PC) is highly malignant and has a high mortality with a 5-year survival rate of less than 8%. As a member of the roundabout immunoglobulin superfamily of proteins, ROBO1 plays an important role in embryogenesis and organogenesis and also inhibits metastasis in PC. Our study was designed to explore whether ROBO1 has effects on the proliferation of PC and its specific mechanism. The expression of ROBO1 was higher in cancer tissues than in matched adjacent tissues by immunohistochemistry (IHC) and qRT-PCR. Low ROBO1 expression is associated with PC progression and poor prognosis. Overexpression of ROBO1 can inhibit the proliferation of PC cells in vitro, and the S phase fraction can also be induced. Further subcutaneous tumor formation in nude mice showed that ROBO1 overexpression can significantly inhibit tumor growth. YY1 was found to directly bind to the promoter region of ROBO1 to promote transcription by a luciferase reporter gene assay, a chromatin immunoprecipitation (ChIP) and an electrophoretic mobility shift assay (EMSA). Mechanistic studies showed that YY1 can inhibit the development of PC by directly regulating ROBO1 via the CCNA2/CDK2 axis. Taken together, our results suggest that ROBO1 may be involved in the development and progression of PC by regulating cell proliferation and shows that ROBO1 may be a novel and promising therapeutic target for PC.

Long non-coding RNA SNHG7 facilitates pancreatic cancer progression by regulating the miR-146b-5p/Robo1 axis.

Long non-coding RNA (lncRNA) small nucleolar RNA host gene 7 (SNHG7) plays a crucial role in the progression of pancreatic cancer (PC). SNHG7 is upregulated in PC; therefore, the purpose of the present study was to investigate the role and underlying mechanism of SNHG7 on PC progression. In the present study, the mRNA expression levels of SNHG7, microRNA(miR)-146b-5p and roundabout homolog 1 (Robo1) were measured via reverse transcription-quantitative PCR. Moreover, cell viability and apoptosis were assessed by MTT and flow cytometry assays, respectively. The ability of cells to migrate and invade was evaluated by Transwell assays. In addition, dual-luciferase reporter, RNA immunoprecipitation and RNA pull-down assays were conducted to assess the interaction between miR-146b-5p and SNHG7 or Robo1. The protein expression of Robo1 was measured via western blotting. Furthermore, mouse xenograft models were established to further investigate the effect of SNHG7 on PC progression in vivo. The results indicated that SNHG7 was highly expressed in PC tissues and cells. It was also found that SNHG7 was sponged by miR-146b-5p and that Robo1 was a target of miR-146b-5p. Moreover, it was demonstrated that SNHG7 knockdown inhibited cell proliferation, migration and invasion, as well as tumorigenesis and apoptosis of PC cells in vitro and in vivo by regulating miR-146b-5p. The results also suggested that miR-146b-5p overexpression inhibited the progression of PC cells by modulating Robo1. Furthermore, silencing of SNHG7 downregulated Robo1 expression by sponging miR-146b-5p. Collectively, the present results indicate that SNHG7 promotes PC progression by sponging miR-146b-5p and upregulating Robo1.

Roundabout homolog 1 inhibits proliferation via the YY1-ROBO1-CCNA2-CDK2 axis in human pancreatic cancer

Introduction: Pancreatic cancer (PC) is highly malignant and has a high mortality with a 5-year survival rate of less than 8%. As we know, ROBO1 plays an important role in embryogenesis and organogenesis and also inhibits metastasis in PC. Our study was designed to explore whether ROBO1 has effects on the proliferation of PC and its specific mechanism.

Roles of kininogen-1, basement membrane specific heparan sulfate proteoglycan core protein, and roundabout homolog 4 as potential urinary protein biomarkers in diabetic nephropathy.

Diabetic nephropathy, a major complication of diabetes mellitus (DM), is increasing worldwide and the large majority of patients have type 2 DM. Microalbuminuria has been used as a diagnostic marker of diabetic nephropathy. But owing to its insufficient sensitivity and specificity, other biomarkers are being sought. In addition, the pathophysiology of diabetic nephropathy is not fully understood and declines in renal function occur even without microalbuminuria. In this study, we investigated urinary proteins from three study groups (controls, and type 2 diabetic subjects with or without microalbuminuria). Non-targeted label-free Nano-LC QTOF analysis was conducted to discover underlying mechanisms and protein networks, and targeted label-free Nano-LC QTOF with SWATH was performed to qualify discovered protein candidates. Twenty-eight proteins were identified as candidates and functionally analyzed via String DB, gene ontology and pathway analysis. Four predictive mechanisms were analyzed: i) response to stimulus, ii) platelet activation, signaling and aggregation, iii) ECM-receptor interaction, and iv) angiogenesis. These mechanisms can provoke kidney dysfunction in type 2 diabetic patients via endothelial cell damage and glomerulus structural alteration. Based on these analyses, three proteins (kininogen-1, basement membrane-specific heparan sulfate proteoglycan core protein, and roundabout homolog 4) were proposed for further study as potential biomarkers. Our findings provide insights that may improve methods for both prevention and diagnosis of diabetic nephropathy.

Proteomic biomarkers of sleep apnea.

Obstructive sleep apnea (OSA) is characterized by recurrent partial to complete upper airway obstructions during sleep, leading to repetitive arousals and oxygen desaturations. Although many OSA biomarkers have been reported individually, only a small subset have been validated through both cross-sectional and intervention studies. We sought to profile serum protein biomarkers in OSA in unbiased high throughput assay. A highly multiplexed aptamer array (SomaScan) was used to profile 1300 proteins in serum samples from 713 individuals in the Stanford Sleep Cohort, a patient-based registry. Outcome measures derived from overnight polysomnography included Obstructive Apnea Hypopnea Index (OAHI), Central Apnea Index (CAI), 2% Oxygen Desaturation index, mean and minimum oxygen saturation indices during sleep. Additionally, a separate intervention-based cohort of 16 individuals was used to assess proteomic profiles pre- and post-intervention with positive airway pressure. OAHI was associated with 65 proteins, predominantly pathways of complement, coagulation, cytokine signaling, and hemostasis which were upregulated. CAI was associated with two proteins including Roundabout homolog 3 (ROBO3), a protein involved in bilateral synchronization of the pre-Bötzinger complex and cystatin F. Analysis of pre- and post intervention samples revealed IGFBP-3 protein to be increased while LEAP1 (Hepicidin) to be decreased with intervention. An OAHI machine learning classifier (OAHI >=15 vs OAHI<15) trained on SomaScan protein measures alone performed robustly, achieving 76% accuracy in a validation dataset. Multiplex protein assays offer diagnostic potential and provide new insights into the biological basis of sleep disordered breathing.

Abstract P5-04-14: Recombinant Slit2 suppresses breast cancer metastasis by activating anti-tumor macrophages

Abstract Metastasis is a major cause of mortality in breast cancer patients in part due to the lack of clinically established targeted therapies. Approximately 5%-20% of patients with Stage II, and ~50% of patients with stage III will recur distally and are likely to die from their disease. Metastatic, or stage IV breast cancers, have a 5-year relative survival rate of about 22%. The expression of a tumor suppressor protein, Slit2 has been shown to be downregulated in various types of tumors including breast cancer. The Slit2 acts through Roundabout Homolog1 (Robo1) receptor. Previously it has been shown that ectopic expression of Slit2 inhibits human MCF-7 breast cancer cell line xenograft tumor growth in mice. However, its role in breast cancer metastasis, tumor microenvironment (TME) and anti-tumor immunity has not been studied before. By using genetically engineered human breast cancer cells, spontaneous mammary tumor and pre-clinical mouse models, we evaluated the role of Slit2 in inhibiting breast cancer growth, metastasis by activating anti-tumor immune response. To study the role of Slit2 in breast cancer, we implanted Slit2 overexpressing human breast cancer cell line MDA-MB-231 (231-Slit2) or vector control cells (231-Vec) to the mammary fat-pads of NOD/SCID/gamma (NSG) mice and observed that 231-Slit2 had significantly reduced tumor growth and metastasis to the lungs compared to 231-Vec. To further confirm the anti-metastatic role of Slit2, we treated mouse mammary tumor virus- Polyoma Middle T antigen (MMTV-PyMT) mammary tumor model and MVT-1 orthotopic tumor bearing FVB/J wildtype mice with recombinant Slit2 (rSlit2) that resulted in significantly reduced tumor growth and metastasis to the lungs in both the models compared to PBS treated mice. The ex-vivo immunofluorescence and flow cytometry studies revealed that Slit2 treated tumors possess a very high number of tumor phagocytic macrophages compared to PBS. In-vitro analysis also showed that rSlit2 treated mouse macrophages (RAW264.7) has higher bacterial particle phagocytic ability. Further analysis of tumors elucidated that Slit2 treated tumors recruited higher number of CD4+ and CD8+ T-cells. In addition, The CD8+ cells were also positive for Granzyme-b showing higher number of effector T-cells in the Slit2 tumors compared to PBS. By using human breast cancer tissue microarray (TMA), we have found that Slit2 expression significantly correlates with better overall survival. These observations highlight the ability of Slit2 to enhance tumor phagocytic macrophages and anti-tumor CD8+/Granzyme-b+ T-cells, thereby restricting tumor growth and lung metastasis. These studies suggest that Slit2 could be used as a novel immunomodulatory therapeutic agent metastatic breast cancer. Citation Format: Dinesh Kumar Ahirwar, Ramesh Kumar Ganju, Sanjay Mishra, Konstantin Shilo, Kirti Kaul. Recombinant Slit2 suppresses breast cancer metastasis by activating anti-tumor macrophages [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P5-04-14.

Abstract 4561: Slit2 inhibits breast cancer growth and metastasis by activating anti-tumor immune response

Abstract Metastasis is a major cause of mortality in breast cancer patients in part due to the lack of clinically established targeted therapies. Approximately 5%-20% of patients with Stage II, and ~50% of patients with stage III will recur distally and are likely to die from their disease. Metastatic, or stage IV breast cancers, have a 5-year relative survival rate of about 22%. The expression of a tumor suppressor protein, Slit2 has been shown to be downregulated in various types of tumors including breast cancer. The Slit2 acts through Roundabout Homolog1 (Robo1) receptor. Previously it has been shown that ectopic expression of Slit2 inhibits human MCF-7 breast cancer cell line xenograft tumor growth in mice. However, its role in breast cancer metastasis, tumor microenvironment (TME) and anti-tumor immunity has not been studied before. By using genetically engineered human breast cancer cells, spontaneous mammary tumor and pre-clinical mouse models, we evaluated the role of Slit2 in inhibiting breast cancer growth, metastasis by activating anti-tumor immune response. To study the role of Slit2 in breast cancer, we implanted Slit2 overexpressing human breast cancer cell line MDA-MB-231 (231-Sli2) or vector control cells (231-Vec) to the mammary fat-pads of NOD/SCID/gamma (NSG) mice and observed that 231-Slit2 had significantly reduced tumor growth and metastasis to the lungs compared to 231-Vec. To further confirm the anti-metastatic role of Slit2, we treated mouse mammary tumor virus- Polyoma Middle T antigen (MMTV-PyMT) mammary tumor model and MVT-1 orthotopic tumor bearing FVB/J wildtype mice with recombinant Slit2 (rSlit2) that resulted in significantly reduced tumor growth and metastasis to the lungs in both the models compared to PBS treated mice. The ex-vivo immunofluorescence and flow cytometry studies revealed that Slit2 treated tumors possess a very high number of tumor phagocytic macrophages compared to PBS. In-vitro analysis also showed that rSlit2 treated mouse macrophages (RAW264.7) has higher bacterial particle phagocytic ability. Further analysis of tumors elucidated that Slit2 treated tumors recruited higher number of CD4+ and CD8+ T-cells. In addition, The CD8+ cells were also positive for Granzyme-b showing higher number of effector T-cells in the Slit2 tumors compared to PBS. By using human breast cancer tissue microarray (TMA), we have found that Slit2 expression significantly correlates with better overall survival. These observations highlight the ability of Slit2 to enhance tumor phagocytic macrophages and anti-tumor CD8+/Granzyme-b+ T-cells, thereby restricting tumor growth and lung metastasis. These studies suggest that Slit2 could be used as a novel immunomodulatory therapeutic agent. Citation Format: Dinesh K. Ahirwar, Nabanita Chatterjee, Sanjay Mishra, Kontestine Shilo, Ramesh Ganju. Slit2 inhibits breast cancer growth and metastasis by activating anti-tumor immune response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4561.

Cancer-selective nanoparticles for combinatorial siRNA delivery to primary human GBM in vitro and in vivo

Novel treatments for glioblastoma (GBM) are urgently needed, particularly those which can simultaneously target GBM cells’ ability to grow and migrate. Herein, we describe a synthetic, bioreducible, biodegradable polymer that can package and deliver hundreds of siRNA molecules into a single nanoparticle, facilitating combination therapy against multiple GBM-promoting targets. We demonstrate that siRNA delivery with these polymeric nanoparticles is cancer-selective, thereby avoiding potential side effects in healthy cells. We show that we can deliver siRNAs targeting several anti-GBM genes (Robo1, YAP1, NKCC1, EGFR, and survivin) simultaneously and within the same nanoparticles. Robo1 (roundabout homolog 1) siRNA delivery by biodegradable particles was found to trigger GBM cell death, as did non-viral delivery of NKCC1, EGFR, and survivin siRNA. Most importantly, combining several anti-GBM siRNAs into a nanoparticle formulation leads to high GBM cell death, reduces GBM migration in vitro, and reduces tumor burden over time following intratumoral administration. We show that certain genes, like survivin and EGFR, are important for GBM survival, while NKCC1, is more crucial for cancer cell migration. This represents a powerful platform technology with the potential to serve as a multimodal therapeutic for cancer.

ROBO1 Expression in Metastasizing Breast and Ovarian Cancer: SLIT2-induced Chemotaxis Requires Heparan Sulfates (Heparin).

The members of the slit homolog (SLIT) and roundabout homolog (ROBO) families have emerged as important signaling molecules in tumor metastasis. This study analyzed their role in regulating breast cancer (BC) cell motility and chemotaxis and assessed expression of ROBO1 in brain metastases (BMs) of breast, lung, and colon cancer, and in peritoneal metastases (PMs) of ovarian cancer. The BC cell line MDA-MB231 was subjected to scratch, motility, and chemotaxis assays using heparin and a purified recombinant N-terminal SLIT2 fragment. Protein expression was assessed in primary tumors and metastases by immunohistochemistry. Exposure to SLIT2 induced MDA-MB231 cell motility, but no significant chemotaxis without the presence of heparin. ROBO1 was expressed in 4/5 primary BC and in 18/21 BC-derived BM samples; 7/9 BM primary lung cancer samples also stained positive. In contrast, BMs from colorectal cancer were negative for ROBO1. Primary ovarian cancer and ovarian PM showed ROBO1 expression in 0/6 and in only 2/6 samples, respectively, whereas SLIT2 was observed in 1/6 primary cancer and in 6/6 PMs samples. SLIT2 can induce BC cell motility and chemotaxis, but the latter requires the presence of heparin. BM expression of ROBO1 is a common feature of some, but not all cancer types. SLIT2 expression appears to be a general feature of ovarian cancer-derived PMs.

Different Isoforms of the Neuronal Guidance Molecule Slit2 Directly Cause Chemoattraction or Chemorepulsion of Human Neutrophils.

The movement of neutrophils between blood and tissues appears to be regulated by chemoattractants and chemorepellents. Compared with neutrophil chemoattractants, relatively little is known about neutrophil chemorepellents. Slit proteins are endogenously cleaved into a variety of N- and C-terminal fragments, and these fragments are neuronal chemorepellents and inhibit chemoattraction of many cell types, including neutrophils. In this report, we show that the ∼140-kDa N-terminal Slit2 fragment (Slit2-N) is a chemoattractant and the ∼110-kDa N-terminal Slit2 fragment (Slit2-S) is a chemorepellent for human neutrophils. The effects of both Slit2 fragments were blocked by Abs to the Slit2 receptor Roundabout homolog 1 or the Slit2 coreceptor Syndecan-4. Slit2-N did not appear to activate Ras but increased phosphatidylinositol 3,4,5-triphosphate levels. Slit2-N-induced chemoattraction was unaffected by Ras inhibitors, reversed by PI3K inhibitors, and blocked by Cdc42 and Rac inhibitors. In contrast, Slit2-S activated Ras but did not increase phosphatidylinositol 3,4,5-triphosphate levels. Slit2-S-induced chemorepulsion was blocked by Ras and Rac inhibitors, not affected by PI3K inhibitors, and reversed by Cdc42 inhibitors. Slit2-N, but not Slit2-S, increased neutrophil adhesion, myosin L chain 2 phosphorylation, and polarized actin formation and single pseudopods at the leading edge of cells. Slit2-S induced multiple pseudopods. These data suggest that Slit2 isoforms use similar receptors but different intracellular signaling pathways and have different effects on the cytoskeleton and pseudopods to induce neutrophil chemoattraction or chemorepulsion.

Identification of differentially expressed genes in MG63 osteosarcoma cells with drug‑resistance by microarray analysis.

Osteosarcoma is the most common type of primary malignant bone tumor, with extremely poor prognosis in patients with metastatic disease and resistance to therapy, such as multidrug regimens. The mechanisms of drug resistance are quite complex and have not been fully elucidated; thus, novel therapeutic targets should be identified to alleviate drug resistance in osteosarcoma. In the present study, the transcriptomes of the human osteosarcoma cell line MG63 and vincristine (VCR)-resistant MG63 cells were compared by microarray analysis. A total of 1,300 genes (602 upregulated and 698 downregulated) were reported to be differentially expressed in MG63/VCR compared with MG63 cells. Bioinformatics analysis predicted that the differentially expressed genes were mainly enriched in the B cell receptor, UVA-induced mitogen-activated protein kinases and receptor tyrosine kinase 2/3 signaling pathways. In the present study, 10 of the dysregulated genes, including roundabout homolog 1, death-associated protein kinase 1 and A-kinase anchor protein 12 were further evaluated by reverse transcription-quantitative polymerase chain reaction. These results may aid the validation of candidate biomarkers for the treatment and prognosis of osteosarcoma, and provide novel insight into the molecular mechanisms underlying the drug resistance of osteosarcoma cells.

Thermodynamic analyses of amino acid residues at the interface of an antibody B2212A and its antigen roundabout homolog 1.

Artificial affinity maturation of antibodies is promising but often shows difficulties because the roles of each amino acid residue are not well known. To elucidate their roles in affinity against the antigen and thermal stability, interface residues in single-chain Fv of an antibody B2212A with its antigen roundabout homolog 1 were mutated and analyzed. Some amino acids played important roles in the affinity while others contributed to thermal stability.

Abstract 916: Slit2 inhibits breast cancer growth and metastasis by modulating tumor microenvironment

Abstract Breast cancer (BC) continues to be a major health issue particularly in developed countries. The Slit2 gene is hyper-methylated in BC and acts through Roundabout Homolog1 (Robo1) receptor. Slit2/Robo1 signaling has been shown to inhibit the migration of a variety of cells including BC cells. Recently, it has also been reported to improve chemotherapy outcome in intestinal cancer. However, Slit2/Robo1-mediated mechanisms that regulate tumor growth and metastasis are not well known. Using syngeneic mouse model, we report that the over-expression of Slit2 using Adeno-Slit2 transduction successfully restrains tumor growth and also restrict the metastasis to lung, compared to Adeno-null control. To explore the Slit2 mediated cellular events, we analyzed the stromal cells recruitment to the tumor and observed reduced number of tumor associated macrophages (TAMs) in the Adeno-Slit2 group compared to Adeno-null group. We further analyzed the underlying molecular mechanisms and found that the soluble Slit2 inhibits TGF-β1 mediated alternative activation of macrophages to TAMs. Treatment with TGF-β1 significantly enhances the markers of TAMs by upregulating β-catenin levels. Interestingly, we observed that Soluble Slit2 inhibits TGF-β1 mediated activation of TAMs by inhibiting downstream signaling molecules Smad2 and β-catenin. We also analyzed Slit2 expression in human breast cancer TMA and an inverse correlation of Slit2 expression was observed with the incidence of breast cancer, metastasis and macrophage recruitment. This study highlights the ability of Slit2 to prevent macrophage alternative activation to TAMs, thereby restrict tumor growth and lung metastasis. Anti-cancer activity of Adeno-Slit2 suggests therapeutic potential of Slit2 to treat advanced breast cancer. Citation Format: Dinesh K. Ahirwar, Mohd W. Nasser, Mohamad Elbaz, Kontestine Shilo, Ramesh Ganju. Slit2 inhibits breast cancer growth and metastasis by modulating tumor microenvironment. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 916.