The Role of the MiR-181 Family in Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is the fourth-leading cause of cancer-related death worldwide. Due to the high mortality rate in HCC patients, discovering and developing novel systemic treatment options for HCC is a vital unmet medical need. Among the numerous molecular alterations in HCCs, microRNAs (miRNAs) have been increasingly recognised to play critical roles in hepatocarcinogenesis. We and others have recently revealed that members of the microRNA-181 (miR-181) family were up-regulated in some, though not all, human cirrhotic and HCC tissues-this up-regulation induced epithelial-mesenchymal transition (EMT) in hepatocytes and tumour cells, promoting HCC progression. MiR-181s play crucial roles in governing the fate and function of various cells, such as endothelial cells, immune cells, and tumour cells. Previous reviews have extensively covered these aspects in detail. This review aims to give some insights into miR-181s, their targets and roles in modulating signal transduction pathways, factors regulating miR-181 expression and function, and their roles in HCC.

Targeting the SphK1/S1P/PFKFB3 axis suppresses hepatocellular carcinoma progression by disrupting glycolytic energy supply that drives tumor angiogenesis.

BACKGROUND: Hepatocellular carcinoma (HCC) remains a leading life-threatening health challenge worldwide, with pressing needs for novel therapeutic strategies. Sphingosine kinase 1 (SphK1), a well-established pro-cancer enzyme, is aberrantly overexpressed in a multitude of malignancies, including HCC. Our previous research has shown that genetic ablation of Sphk1 mitigates HCC progression in mice. Therefore, the development of PF-543, a highly selective SphK1 inhibitor, opens a new avenue for HCC treatment. However, the anti-cancer efficacy of PF-543 has not yet been investigated in primary cancer models in vivo, thereby limiting its further translation. METHODS: Building upon the identification of the active form of SphK1 as a viable therapeutic target in human HCC specimens, we assessed the capacity of PF-543 in suppressing tumor progression using a diethylnitrosamine-induced mouse model of primary HCC. We further delineated its underlying mechanisms in both HCC and endothelial cells. Key findings were validated in Sphk1 knockout mice and lentiviral-mediated SphK1 knockdown cells. RESULTS: SphK1 activity was found to be elevated in human HCC tissues. Administration of PF-543 effectively abrogated hepatic SphK1 activity and significantly suppressed HCC progression in diethylnitrosamine-treated mice. The primary mechanism of action was through the inhibition of tumor neovascularization, as PF-543 disrupted endothelial cell angiogenesis even in a pro-angiogenic milieu. Mechanistically, PF-543 induced proteasomal degradation of the critical glycolytic enzyme 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3, thus restricting the energy supply essential for tumor angiogenesis. These effects of PF-543 could be reversed upon S1P supplementation in an S1P receptor-dependent manner. CONCLUSIONS: This study provides the first in vivo evidence supporting the potential of PF-543 as an effective anti-HCC agent. It also uncovers previously undescribed links between the pro-cancer, pro-angiogenic and pro-glycolytic roles of the SphK1/S1P/S1P receptor axis. Importantly, unlike conventional anti-HCC drugs that target individual pro-angiogenic drivers, PF-543 impairs the PFKFB3-dictated glycolytic energy engine that fuels tumor angiogenesis, representing a novel and potentially safer therapeutic strategy for HCC.

Novel miRNA-based drug CD5-2 reduces liver tumor growth in diethylnitrosamine-treated mice by normalizing tumor vasculature and altering immune infiltrate.

Introduction: Liver cancers exhibit abnormal (leaky) vasculature, hypoxia and an immunosuppressive microenvironment. Normalization of tumor vasculature is an emerging approach to treat many cancers. Blockmir CD5-2 is a novel oligonucleotide-based inhibitor of the miR-27a interaction with VE-Cadherin, the endothelial-specific cadherin. The combination of a vasoactive medication with inhibition of immune checkpoints such as programmed cell death protein 1 (PD1) has been shown to be effective in treating liver cancer in humans. We aimed to study the effect of CD5-2 combined with checkpoint inhibition (using an antibody against PD1) on liver tumor growth, vasculature and immune infiltrate in the diethylnitrosamine (DEN)-induced liver tumor mouse model. Methods: We first analyzed human miR-27a and VE-Cadherin expression data from The Cancer Genome Atlas for hepatocellular carcinoma. CD5-2 and/or anti-PD1 antibody were given to the DEN-treated mice from age 7-months until harvest at age 9-months. Tumor and non-tumor liver tissues were analyzed using histology, immunohistochemistry, immunofluorescence and scanning electron microscopy. Results: Human data showed high miR-27a and low VE-Cadherin were both significantly associated with poorer prognosis. Mice treated with CD5-2 plus anti-PD1 antibody had significantly smaller liver tumors (50% reduction) compared to mice treated with either agent alone, controls, or untreated mice. There was no difference in tumor number. Histologically, tumors in CD5-2-treated mice had less leaky vessels with higher VE-Cadherin expression and less tumor hypoxia compared to non-CD5-2-treated mice. Only tumors in the combination CD5-2 plus anti-PD1 antibody group exhibited a more favorable immune infiltrate (significantly higher CD3+ and CD8+ T cells and lower Ly6G+ neutrophils) compared to tumors from other groups. Discussion: CD5-2 normalized tumor vasculature and reduced hypoxia in DEN-induced liver tumors. CD5-2 plus anti-PD1 antibody reduced liver tumor size possibly by altering the immune infiltrate to a more immunosupportive one.

Liver-specific deletion of miR-181ab1 reduces liver tumour progression via upregulation of CBX7.

MiR-181 expression levels increased in hepatocellular carcinoma (HCC) compared to non-cancerous tissues. MiR-181 has been widely reported as a possible driver of tumourigenesis but also acts as a tumour suppressor. In addition, the miR-181 family regulates the development and function of immune and vascular cells, which play vital roles in the progression of tumours. More complicatedly, many genes have been identified as miR-181 targets to mediate the effects of miR-181. However, the role of miR-181 in the development of primary tumours remains largely unexplored. We aimed to examine the function of miR-181 and its vital mediators in the progression of diethylnitrosamine-induced primary liver cancers in mice. The size of liver tumours was significantly reduced by 90% in global (GKO) or liver-specific (LKO) 181ab1 knockout mice but not in hematopoietic and endothelial lineage-specific knockout mice, compared to WT mice. In addition, the number of tumours was significantly reduced by 50% in GKO mice. Whole-genome RNA-seq analysis and immunohistochemistry showed that epithelial-mesenchymal transition was partially reversed in GKO tumours compared to WT tumours. The expression of CBX7, a confirmed miR-181 target, was up-regulated in GKO compared to WT tumours. Stable CBX7 expression was achieved with an AAV/Transposase Hybrid-Vector System and up-regulated CBX7 expression inhibited liver tumour progression in WT mice. Hepatic CBX7 deletion restored the progression of LKO liver tumours. MiR-181a expression was the lowest and CBX7 expression the highest in iClust2 and 3 subclasses of human HCC compared to iClust1. Gene expression profiles of GKO tumours overlapped with low-proliferative peri-portal-type HCCs. Liver-specific loss of miR-181ab1 inhibited primary liver tumour progression via up-regulating CBX7 expression, but tumour induction requires both hepatic and non-hepatic miR-181. Also, miR-181ab1-deficient liver tumours may resemble low-proliferative periportal-type human HCC. miR-181 was increased with liver tumour growth. More miR-181, darker colour and higher shape. CBX7 was very low in pericentral hepatocytes, increased in early liver tumours, but reduced in advanced liver tumours. Its levels were maintained in miR-181 KO liver tumours. In tumours (T), brown (darker is more) represents miR-181, the blue circle (thicker is more) represents CBX7.

The Tumour Vasculature as a Target to Modulate Leucocyte Trafficking.

The effectiveness of immunotherapy against solid tumours is dependent on the appropriate leucocyte subsets trafficking and accumulating in the tumour microenvironment (TME) with recruitment occurring at the endothelium. Such recruitment involves interactions between the leucocytes and the endothelial cells (ECs) of the vessel and occurs through a series of steps including leucocyte capture, their rolling, adhesion, and intraluminal crawling, and finally leucocyte transendothelial migration across the endothelium. The tumour vasculature can curb the trafficking of leucocytes through influencing each step of the leucocyte recruitment process, ultimately producing an immunoresistant microenvironment. Modulation of the tumour vasculature by strategies such as vascular normalisation have proven to be efficient in facilitating leucocyte trafficking into tumours and enhancing immunotherapy. In this review, we discuss the underlying mechanisms of abnormal tumour vasculature and its impact on leucocyte trafficking, and potential strategies for overcoming the tumour vascular abnormalities to boost immunotherapy via increasing leucocyte recruitment.

The VE-Cadherin/ß-catenin signalling axis regulates immune cell infiltration into tumours.

Vascular normalisation, the process that reverses the structural and functional abnormalities seen in tumour-associated vessels, is also accompanied by changes in leucocyte trafficking. Our previous studies have shown the normalisation effects of the agent CD5-2 which acts to stabilise VE-Cadherin leading to increased penetration of CD8+ T cells but decreased infiltration of neutrophils (CD11b+Gr1hi) into tumour parenchyma. In the present study, we demonstrate that VE-Cadherin stabilisation through CD5-2 treatment of purified endothelial cells (ECs) results in a similar leucocyte-selective regulation of transmigration, suggesting the existence of an endothelial specific intrinsic mechanism. Further, we show by RNA sequencing (RNA-seq)-based transcriptomic analysis, that treatment of ECs with CD5-2 regulates chemokines known to be involved in leucocyte transmigration, including upregulation of CCL2 and CXCL10 that facilitate CD8+ T cell transmigration. Both in vitro and in vivo mechanistic studies revealed that the increased CCL2 expression was dependent on expression of VE-Cadherin and downstream activation of the AKT/GSK3ß/ß-catenin/TCF4 signalling pathway. CD5-2 treatment also contributed to the reorganisation of the cytoskeleton, inducing reorganisation of stress fibres to circumferential actin, which previously has been described as associated with the stabilisation of the endothelial barrier, and amplification of the transcellular migration of CD8+ T cells. Thus, we propose that promotion of endothelial junctional integrity during vascular normalisation not only inhibits vascular leak but also resets the endothelial dependent regulation of immune cell infiltration.

Regulation of hepatic insulin signaling and glucose homeostasis by sphingosine kinase 2.

Sphingolipid dysregulation is often associated with insulin resistance, while the enzymes controlling sphingolipid metabolism are emerging as therapeutic targets for improving insulin sensitivity. We report herein that sphingosine kinase 2 (SphK2), a key enzyme in sphingolipid catabolism, plays a critical role in the regulation of hepatic insulin signaling and glucose homeostasis both in vitro and in vivo. Hepatocyte-specific Sphk2 knockout mice exhibit pronounced insulin resistance and glucose intolerance. Likewise, SphK2-deficient hepatocytes are resistant to insulin-induced activation of the phosphoinositide 3-kinase (PI3K)-Akt-FoxO1 pathway and elevated hepatic glucose production. Mechanistically, SphK2 deficiency leads to the accumulation of sphingosine that, in turn, suppresses hepatic insulin signaling by inhibiting PI3K activation in hepatocytes. Either reexpressing functional SphK2 or pharmacologically inhibiting sphingosine production restores insulin sensitivity in SphK2-deficient hepatocytes. In conclusion, the current study provides both experimental findings and mechanistic data showing that SphK2 and sphingosine in the liver are critical regulators of insulin sensitivity and glucose homeostasis.

Targeting miR-27a/VE-cadherin interactions rescues cerebral cavernous malformations in mice.

Cerebral cavernous malformations (CCMs) are vascular lesions predominantly developing in the central nervous system (CNS), with no effective treatments other than surgery. Loss-of-function mutation in CCM1/krev interaction trapped 1 (KRIT1), CCM2, or CCM3/programmed cell death 10 (PDCD10) causes lesions that are characterized by abnormal vascular integrity. Vascular endothelial cadherin (VE-cadherin), a major regulator of endothelial cell (EC) junctional integrity is strongly disorganized in ECs lining the CCM lesions. We report here that microRNA-27a (miR-27a), a negative regulator of VE-cadherin, is elevated in ECs isolated from mouse brains developing early CCM lesions and in cultured ECs with CCM1 or CCM2 depletion. Furthermore, we show miR-27a acts downstream of kruppel-like factor (KLF)2 and KLF4, two known key transcription factors involved in CCM lesion development. Using CD5-2 (a target site blocker [TSB]) to prevent the miR-27a/VE-cadherin mRNA interaction, we present a potential therapy to increase VE-cadherin expression and thus rescue the abnormal vascular integrity. In CCM1- or CCM2-depleted ECs, CD5-2 reduces monolayer permeability, and in Ccm1 heterozygous mice, it restores dermal vessel barrier function. In a neonatal mouse model of CCM disease, CD5-2 normalizes vasculature and reduces vascular leakage in the lesions, inhibits the development of large lesions, and significantly reduces the size of established lesions in the hindbrain. Furthermore, CD5-2 limits the accumulation of inflammatory cells in the lesion area. Our work has established that VE-cadherin is a potential therapeutic target for normalization of the vasculature and highlights that targeting miR-27a/VE-cadherin interaction by CD5-2 is a potential novel therapy for the devastating disease, CCM.

Low fluid shear stress conditions contribute to activation of cerebral cavernous malformation signalling pathways.

Cerebral cavernous malformations (CCMs) are vascular malformations that cause hemorrhagic stroke. CCMs can arise from loss-of-function mutations in any one of CCM1 (KRIT1), CCM2 or CCM3 (PDCD10). Despite the mutation being in all endothelial cells the CCM lesions develop primarily in the regions with low fluid shear stress (FSS). Here we investigated the role of FSS in the signalling pathways associated with loss of function of CCM genes. We performed transcriptomic analysis on CCM1 or CCM2-silenced endothelial cells subjected to various FSS. The results showed 1382 genes were deregulated under low FSS, whereas only 29 genes were deregulated under high FSS. Key CCM downstream signalling pathways, including increased KLF2/4 expression, actin cytoskeleton reorganization, TGF-ß and toll-like receptor signalling pathways and also oxidative stress pathways, were all highly upregulated but only under low FSS. We also show that the key known phenotypes of CCM lesions such as disrupted endothelial cell junction, increased inflammatory response/oxidative stress and elevated RhoA-ROCK activity, are only exhibited in monolayers of CCM-silenced endothelial cells subjected to low FSS. Our data establishes that shear stress acts as a previously unappreciated but important regulator for CCM gene function and may determine the site of CCM lesion development.

Deletion of sphingosine kinase 1 inhibits liver tumorigenesis in diethylnitrosamine-treated mice.

Primary liver cancer is the 3rd leading cause of cancer deaths worldwide with very few effective treatments. Sphingosine kinase 1 (SphK1), a key regulator of sphingolipid metabolites, is over-expressed in human hepatocellular carcinoma (HCC) and our previous studies have shown that SphK1 is important in liver injury. We aimed to explore the role of SphK1 specifically in liver tumorigenesis using the SphK1 knockout (SphK1-/-) mouse. SphK1 deletion significantly reduced the number and the size of DEN-induced liver cancers in mice. Mechanistically, fewer proliferating but more apoptotic and senescent cells were detected in SphK1 deficient tumors compared to WT tumors. There was an increase in sphingosine rather than a decrease in sphingosine 1-phosphate (S1P) in SphK1 deficient tumors. Furthermore, the STAT3-S1PR pathway that has been reported previously to mediate the effect of SphK1 on colorectal cancers was not altered by SphK1 deletion in liver cancer. Instead, c-Myc protein expression was down-regulated by SphK1 deletion. In conclusion, this is the first in vivo evidence that SphK1 contributes to hepatocarcinogenesis. However, the downstream signaling pathways impacting on the development of HCC via SphK1 are organ specific providing further evidence that simply transferring known oncogenic molecular pathway targeting into HCC is not always valid.

Targeting Vascular Endothelial-Cadherin in Tumor-Associated Blood Vessels Promotes T-cell-Mediated Immunotherapy.

T-cell infiltration of solid tumors is associated with improved prognosis and favorable responses to immunotherapy. Mechanisms that enable tumor infiltration of CD8+ T cells have not been defined, nor have drugs that assist this process been discovered. Here we address these issues with a focus on VE-cadherin, a major endothelial cell-specific junctional protein that controls vascular integrity. A decrease in VE-cadherin expression is associated with tumor pathology. We developed an oligonucleotide-based inhibitor (CD5-2), which disrupted the interaction of VE-cadherin with its regulator miR-27a, resulting in increased VE-cadherin expression. Administration of CD5-2 in tumor-bearing mice enhanced expression of VE-cadherin in tumor endothelium, activating TIE-2 and tight junction pathways and normalizing vessel structure and function. CD5-2 administration also enhanced tumor-specific T-cell infiltration and spatially redistributed CD8+ T cells within the tumor parenchyma. Finally, CD5-2 treatment enhanced the efficacy of anti-PD-1 blocking antibody. Our work establishes a role for VE-cadherin in T-cell infiltration in tumors and offers a preclinical proof of concept for CD5-2 as a therapeutic modifier of cancer immunotherapy via effects on the tumor vasculature. Cancer Res; 77(16); 4434-47. ©2017 AACR.

Deletion of sphingosine kinase 1 ameliorates hepatic steatosis in diet-induced obese mice: Role of PPARγ.

Sphingolipid metabolites have emerged playing important roles in the pathogenesis of nonalcoholic fatty liver disease, whereas the underlying mechanism remains largely unknown. In the present study, we provide both in vitro and in vivo evidence showing a pathogenic role of sphingosine kinase 1 (SphK1) in hepatocellular steatosis. We found that levels of SphK1 expression were significantly increased in steatotic hepatocytes. Enforced overexpression of SphK1 or treatment with sphingosine 1-phosphate (S1P) markedly enhanced hepatic lipid accumulation. In contrast, the siRNA-mediated knockdown of SphK1 or S1P receptors, S1P2 and S1P3, profoundly inhibited lipid accumulation in hepatocytes. Moreover, Sphk1(-/-) mice exhibited a significant amelioration of hepatosteatosis under diet-induced obese (DIO) conditions, compared to wild-type littermates. In addition, DIO-induced up-regulation of PPARγ and its target genes were significantly reduced by SphK1 deficiency. Furthermore, treatment of hepatocytes with S1P induces a dose-dependent increase in PPARγ expression at the transcriptional level. Blockage of S1P receptors and the Akt-mTOR signaling profoundly inhibited S1P-induced PPARγ expression. Notably, down-regulation of PPARγ by using its siRNA significantly diminished the pro-steatotic effect of SphK1/S1P. Thus, the study demonstrates a new pathway connecting SphK1 and PPARγ involved in the pathogenesis of hepatocellular steatosis.

miR-181a mediates TGF-ß-induced hepatocyte EMT and is dysregulated in cirrhosis and hepatocellular cancer.

BACKGROUND & AIMS: Epithelial-mesenchymal transition (EMT) has been implicated in the processes of embryogenesis, tissue fibrosis and carcinogenesis. Transforming growth factor-ß (TGF-ß) has been identified as a key driver of EMT and plays a key role in the pathogenesis of cirrhosis and hepatocellular carcinoma (HCC). The aim was to identify microRNA (miR) expression in TGF-ß-induced hepatocyte EMT. METHODS: We treated a human hepatocyte cell line PH5CH8 with TGF-ß to induce an EMT-like change in phenotype and then identified dysregulated miRs using TaqMan Low Density Arrays. MiR expression was altered using miR-181a mimic and inhibitor in the same system and gene changes were identified using TaqMan gene arrays. MiR-181a gene expression was measured in human and mouse cirrhotic or HCC liver tissue samples. Gene changes were identified in rAAV-miR-181a-expressing mouse livers using TaqMan gene arrays. RESULTS: We identified miR-181a as a miR that was significantly up-regulated in response to TGF-ß treatment. Over-expression of a miR-181a mimic induced an in vitro EMT-like change with a phenotype similar to that seen with TGF-ß treatment alone and was reversed using a miR-181a inhibitor. MiR-181a was shown to be up-regulated in experimental and human cirrhotic and HCC tissue. Mouse livers expressing rAAV-miR-181a showed genetic changes associated with TGF-ß signalling and EMT. CONCLUSIONS: MiR-181a had a direct effect in inducing hepatocyte EMT and was able to replace TGF-ß-induced effects in vitro. MiR-181a was over-expressed in cirrhosis and HCC and is likely to play a role in disease pathogenesis.

Caveolae control the anti-inflammatory phenotype of senescent endothelial cells.

Senescent endothelial cells (EC) have been identified in cardiovascular disease, in angiogenic tumour associated vessels and in aged individuals. We have previously identified a novel anti-inflammatory senescent phenotype of EC. We show here that caveolae are critical in the induction of this anti-inflammatory senescent state. Senescent EC induced by either the overexpression of ARHGAP18/SENEX or by H2O2 showed significantly increased numbers of caveolae and associated proteins Caveolin-1, cavin-1 and cavin-2. Depletion of these proteins by RNA interference decreased senescence induced by ARHGAP18 and by H2O2. ARHGAP18 overexpression induced a predominantly anti-inflammatory senescent population and depletion of the caveolae-associated proteins resulted in the preferential reduction in this senescent population as measured by neutrophil adhesion and adhesion protein expression after TNFα treatment. In confirmation, EC isolated from the aortas of CAV-1(-/-) mice failed to induce this anti-inflammatory senescent cell population upon expression of ARHGAP18, whereas EC from wild-type mice showed a significant increase. NF-κB is one of the major transcription factors mediating the induction of E-selectin and VCAM-1 expression, adhesion molecules responsible for leucocyte attachment to EC. TNFα-induced activation of NF-κB was suppressed in ARHGAP18-induced senescent EC, and this inhibition was reversed by Caveolin-1 knock-down. Thus, out results demonstrate that an increase in caveolae and its component proteins in senescent ECs is associated with inhibition of the NF-kB signalling pathway and promotion of the anti-inflammatory senescent pathway.

ARHGAP18: an endogenous inhibitor of angiogenesis, limiting tip formation and stabilizing junctions.

The formation of the vascular network requires a tightly controlled balance of pro-angiogenic and stabilizing signals. Perturbation of this balance can result in dysregulated blood vessel morphogenesis and drive pathologies including cancer. Here, we have identified a novel gene, ARHGAP18, as an endogenous negative regulator of angiogenesis, limiting pro-angiogenic signaling and promoting vascular stability. Loss of ARHGAP18 promotes EC hypersprouting during zebrafish and murine retinal vessel development and enhances tumor vascularization and growth. Endogenous ARHGAP18 acts specifically on RhoC and relocalizes to the angiogenic and destabilized EC junctions in a ROCK dependent manner, where it is important in reaffirming stable EC junctions and suppressing tip cell behavior, at least partially through regulation of tip cell genes, Dll4, Flk-1 and Flt-4. These findings highlight ARHGAP18 as a specific RhoGAP to fine tune vascular morphogenesis, limiting tip cell formation and promoting junctional integrity to stabilize the angiogenic architecture.

Stress-induced premature senescence mediated by a novel gene, SENEX, results in an anti-inflammatory phenotype in endothelial cells.

Cellular senescence is a mechanism to inhibit the growth of mammalian cells after oncogenic activation, or in response to damage or stress. We describe here the identification of a novel gene, SENEX, that regulates stress induced premature senescence pathways in endothelial cells (ECs) involving p16(INK4a) and retinoblastoma protein activation. Endogenous levels of SENEX remain unchanged during replicative senescence but are regulated by H(2)O(2)-mediated stress. In contrast to that previously described for senescence in other cell types, the SENEX induced senescent ECs are profoundly anti-inflammatory. The cells are resistant to tumor necrosis factor (TNF)α-induced apoptosis, adhesion of neutrophils and mononuclear cells, and the surface (but not cytoplasmic) expression of endothelial leukocyte adhesion molecule 1 and vascular cell adhesion molecule 1. Furthermore they are resistant to thrombin induced vascular leak. Senescent ECs such as those lining atherosclerotic lesions may therefore function to limit the inflammatory response. SENEX is also essential for EC survival since depletion either ectopically by siRNA or by high- dose H(2)O(2) treatment causes apoptosis. Together, these findings expand our understanding of the role of senescence in the vasculature and identify SENEX as a fulcrum for driving the resultant phenotype of the endothelium after activation.

Sphingosine kinase-1 associates with integrin {alpha}V{beta}3 to mediate endothelial cell survival.

Sphingosine kinase (SK)-1 promotes endothelial cell (EC) survival through the cell junction molecule CD31 (platelet endothelial cell adhesion molecule-1). The integrin alpha(v)beta(3) is also essential for EC survival; inhibition of alpha(v)beta(3) ligation promotes apoptosis. Herein we demonstrate that under basal conditions, SK-1, alpha(v)beta(3), and CD31 exist as a heterotrimeric complex. Under conditions that affect EC survival such as loss of contact with the extracellular matrix or growth factor activation, more of this heterotrimeric complex forms. Overexpression studies demonstrate a requirement for SK-1 phosphorylation at serine 225 for increased heterotrimeric complex formation, activation of alpha(v)beta(3), and EC survival signals, including Bcl-X and nuclear factor-kappaB pathways. Moreover, beta(3) integrin depletion confirmed the requirement for this heterotrimeric complex in SK-1-mediated EC survival. Thus, with alpha(v)beta(3) integrin being identifiable primarily on angiogenic ECs and SK-1 being highly expressed in tumors, targeting SK-1 may affect multiple survival pathways, and its inhibition may be highly efficacious in controlling pathological EC survival.

Restoring endocrine response in breast cancer cells by inhibition of the sphingosine kinase-1 signaling pathway.

We previously demonstrated that sphingosine kinase-1 (SphK1) is an important mediator in the cytoplasmic signaling of estrogens, including Ca(2+) mobilization, ERK1/2 activation, and the epidermal growth factor receptor transactivation. Here we report for the first time that SphK1 activity is causally associated with endocrine resistance in MCF-7 human breast cancer cells. Enforced overexpression of human SphK1 in MCF-7 cells resulted in enhanced cell proliferation and resistance to tamoxifen-induced cell growth arrest and apoptosis. Tamoxifen-resistant (TamR) MCF-7 cells selected by prolonged exposure to 4-hydroxytamoxifen, exhibited higher levels in SphK1 expression and activity, compared with the control cells. Inhibition of SphK1 activity by either specific pharmaceutical inhibitors or the dominant-negative mutant SphK1(G82D) restored the antiproliferative and proapoptotic effects of tamoxifen in the TamR cells. Furthermore, silencing of SphK1, but not SphK2, expression by the specific small interference RNA also restored the tamoxifen responsiveness in the TamR cells. Thus, blockade of the SphK1 signaling pathway may reprogram cellular responsiveness to tamoxifen and abrogate antiestrogen resistance in human breast cancer cells.

JAM-C induces endothelial cell permeability through its association and regulation of {beta}3 integrins.

OBJECTIVE: The molecular mechanisms regulating vascular permeability are only now being elucidated. The junctional adhesion molecule (JAM) JAM-C has been linked to the induction of vascular permeability. We sought to understand the mechanism whereby JAM-C may disrupt junctional integrity in endothelial cells (ECs). METHODS AND RESULTS: We show here that JAM-C alters permeability through modulation of integrin activity. JAM-C overexpression results in an increase in JAM-C at junctions and an increase in permeability. Conversely, knockdown of JAM-C by siRNA results in a reduction in permeability. JAM-C associates with alphavbeta3 integrin and regulates its localization and activity. JAM-C also inhibits the activation state of the beta(1) integrin although it does not associate with this integrin. These changes induced on the integrins are mediated through regulation of the small GTPase, Rap1b but not Rap1a. Thrombin, a powerful inductor of vascular leak, causes localization of JAM-C into the junctions, whereas angiopoietin-1, an inhibitor of permeability, prevents JAM-C translocation. CONCLUSIONS: The regulation of EC junctional integrity involves the coordinated and dynamic modification of localization and activity of junctional stabilizers such as the integrin beta(3) and the destabilizer, JAM-C.