Regulation of fatty acid delivery to metastases by tumor endothelium.

Tumor metastasis, the main cause of death in cancer patients, requires outgrowth of tumor cells after their dissemination and residence in microscopic niches. Nutrient sufficiency is a determinant of such outgrowth1. Fatty acids (FA) can be metabolized by cancer cells for their energetic and anabolic needs but impair the cytotoxicity of T cells in the tumor microenvironment (TME)2,3, thereby supporting metastatic progression. However, despite the important role of FA in metastatic outgrowth, the regulation of intratumoral FA is poorly understood. In this report, we show that tumor endothelium actively promotes tumor growth and restricts anti-tumor cytolysis by transferring FA into developing metastatic tumors. This process uses transendothelial fatty acid transport via endosome cargo trafficking in a mechanism that requires mTORC1 activity. Thus, tumor burden was significantly reduced upon endothelial-specific targeted deletion of Raptor, a unique component of the mTORC1 complex (RptorECKO). In vivo trafficking of a fluorescent palmitic acid analog to tumor cells and T cells was reduced in RptorECKO lung metastatic tumors, which correlated with improved markers of T cell cytotoxicity. Combination of anti-PD1 with RAD001/everolimus, at a low dose that selectively inhibits mTORC1 in endothelial cells4, impaired FA uptake in T cells and reduced metastatic disease, corresponding to improved anti-tumor immunity. These findings describe a novel mechanism of transendothelial fatty acid transfer into the TME during metastatic outgrowth and highlight a target for future development of therapeutic strategies.

Novel insights into genetic susceptibility for colorectal cancer from transcriptome-wide association and functional investigation.

BACKGROUND: Transcriptome-wide association studies have been successful in identifying candidate susceptibility genes for colorectal cancer (CRC). To strengthen susceptibility gene discovery, we conducted a large transcriptome-wide association study and an alternative splicing transcriptome-wide association study in CRC using improved genetic prediction models and performed in-depth functional investigations. METHODS: We analyzed RNA-sequencing data from normal colon tissues and genotype data from 423 European descendants to build genetic prediction models of gene expression and alternative splicing and evaluated model performance using independent RNA-sequencing data from normal colon tissues of the Genotype-Tissue Expression Project. We applied the verified models to genome-wide association studies (GWAS) summary statistics among 58 131 CRC cases and 67 347 controls of European ancestry to evaluate associations of genetically predicted gene expression and alternative splicing with CRC risk. We performed in vitro functional assays for 3 selected genes in multiple CRC cell lines. RESULTS: We identified 57 putative CRC susceptibility genes, which included the 48 genes from transcriptome-wide association studies and 15 genes from splicing transcriptome-wide association studies, at a Bonferroni-corrected P value less than .05. Of these, 16 genes were not previously implicated in CRC susceptibility, including a gene PDE7B (6q23.3) at locus previously not reported by CRC GWAS. Gene knockdown experiments confirmed the oncogenic roles for 2 unreported genes, TRPS1 and METRNL, and a recently reported gene, C14orf166. CONCLUSION: This study discovered new putative susceptibility genes of CRC and provided novel insights into the biological mechanisms underlying CRC development.

PIK3C3/VPS34 keeps body fats healthy.

Adipose tissue, or body fat, plays a critical role in the maintenance of health and the development of metabolic diseases. The pathological expansion of adipose tissue during obesity and the pathological reduction of adipose tissue during lipodystrophy can lead to a similar array of metabolic diseases that include diabetes, but mechanisms remain to be fully defined. In our recent studies, we explored the contribution of the lipid kinase PIK3C3/VPS34 to adipose tissue health and metabolic disease. We found that adipocyte-specific PIK3C3/VPS34 deficiency causes defects in the differentiation, survival and functional properties of adipocytes, resulting in reduced adipose tissue mass, altered blood lipid levels, fatty liver disease, diabetes, and defective body temperature control. These abnormalities mirror those observed in patients with lipodystrophy. These findings identify adipocyte PIK3C3/VPS34 as a potential target for therapeutic intervention in metabolic diseases.

Lipid kinase PIK3C3 maintains healthy brown and white adipose tissues to prevent metabolic diseases.

Adequate mass and function of adipose tissues (ATs) play essential roles in preventing metabolic perturbations. The pathological reduction of ATs in lipodystrophy leads to an array of metabolic diseases. Understanding the underlying mechanisms may benefit the development of effective therapies. Several cellular processes, including autophagy and vesicle trafficking, function collectively to maintain AT homeostasis. Here, we investigated the impact of adipocyte-specific deletion of the lipid kinase phosphatidylinositol 3-kinase catalytic subunit type 3 (PIK3C3) on AT homeostasis and systemic metabolism in mice. We report that PIK3C3 functions in all ATs and that its absence disturbs adipocyte autophagy and hinders adipocyte differentiation, survival, and function with differential effects on brown and white ATs. These abnormalities cause loss of white ATs, whitening followed by loss of brown ATs, and impaired "browning" of white ATs. Consequently, mice exhibit compromised thermogenic capacity and develop dyslipidemia, hepatic steatosis, insulin resistance, and type 2 diabetes. While these effects of PIK3C3 largely contrast previous findings with the autophagy-related (ATG) protein ATG7 in adipocytes, mice with a combined deficiency in both factors reveal a dominant role of the PIK3C3-deficient phenotype. We have also found that dietary lipid excess exacerbates AT pathologies caused by PIK3C3 deficiency. Surprisingly, glucose tolerance is spared in adipocyte-specific PIK3C3-deficient mice, a phenotype that is more evident during dietary lipid excess. These findings reveal a crucial yet complex role for PIK3C3 in ATs, with potential therapeutic implications.

PIK3C3/VPS34 helps school T cells in the thymus.

The development of a broad repertoire of T cells in the immune system requires interaction of T cell receptors expressed by immature T cells with peptide/major histocompatibility complexes (MHCs) displayed by specialized epithelial cells in the thymus, in a process called T cell positive selection. Thymic epithelial cells (TECs) display unique antigen processing machinery which shapes the collection of self-peptides that drive positive selection. In our recent studies, we explored the contribution of the lipid kinase PIK3C3/VPS34 to the generation of positively selecting peptides in TECs. We found that TEC-specific PIK3C3/VPS34 facilitates the positive selection of CD4 but not CD8 T lineage cells, in a mechanism independent of its role in canonical macroautophagy/autophagy. Instead, we propose that PIK3C3/VPS34 alters vesicle trafficking in TECs that modulates lysosomal protease activity which, in turn, controls the generation of MHC class II-presented peptides optimized for positive selection of CD4 T cells.

Thymic epithelial cells require lipid kinase Vps34 for CD4 but not CD8 T cell selection.

The generation of a functional, self-tolerant T cell receptor (TCR) repertoire depends on interactions between developing thymocytes and antigen-presenting thymic epithelial cells (TECs). Cortical TECs (cTECs) rely on unique antigen-processing machinery to generate self-peptides specialized for T cell positive selection. In our current study, we focus on the lipid kinase Vps34, which has been implicated in autophagy and endocytic vesicle trafficking. We show that loss of Vps34 in TECs causes profound defects in the positive selection of the CD4 T cell lineage but not the CD8 T cell lineage. Utilizing TCR sequencing, we show that T cell selection in conditional mutants causes altered repertoire properties including reduced clonal sharing. cTECs from mutant mice display an increased abundance of invariant chain intermediates bound to surface MHC class II molecules, indicating altered antigen processing. Collectively, these studies identify lipid kinase Vps34 as an important contributor to the repertoire of selecting ligands processed and presented by TECs to developing CD4 T cells.

Innate and Innate-like Effector Lymphocytes in Health and Disease.

Lymphocytes can be functionally partitioned into subsets belonging to the innate or adaptive arms of the immune system. Subsets of innate and innate-like lymphocytes may or may not express Ag-specific receptors of the adaptive immune system, yet they are poised to respond with innate-like speed to pathogenic insults but lack the capacity to develop classical immunological memory. These lymphocyte subsets display a number of common properties that permit them to integrate danger and stress signals dispatched by innate sensor cells to facilitate the generation of specialized effector immune responses tailored toward specific pathogens or other insults. In this review, we discuss the functions of distinct subsets of innate and innate-like lymphocytes. A better understanding of the mechanisms by which these cells are activated in different contexts, their interactions with other immune cells, and their role in health and disease may inform the development of new or improved immunotherapies.

Faraday Instability in Viscous Fluids Covered with Elastic Polymer Films.

Faraday instability has great application value in the fields of controlling polymer processing, micromolding colloidal lattices on structured suspensions, organizing particle layers, and conducting cell culture. To regulate Faraday instability, in this article, we attempt to introduce an elastic polymer film covering the surface of a viscous fluid layer and theoretically study the behaviors of the Faraday instability phenomenon and the effect of the elastic polymer film. Based on hydrodynamic theory, the Floquet theory is utilized to formulate its stability criterion, and the critical acceleration amplitude and critical wave number are calculated numerically. The results show that the critical acceleration amplitude for Faraday instability increases with three increasing bending stiffness of the elastic polymer film, and the critical wave number decreases with increasing bending stiffness. In addition, surface tension and viscosity also have important effects on the critical acceleration amplitude and critical wave number. The strategy of controlling Faraday instability by covering an elastic polymer film proposed in this paper has great application potential in new photonic devices, metamaterials, alternative energy, biology, and other fields.

Dendritic cell PIK3C3/VPS34 controls the pathogenicity of CNS autoimmunity independently of LC3-associated phagocytosis.

PIK3C3/VPS34 is a key player in macroautophagy/autophagy and MAP1LC3/LC3-associated phagocytosis (LAP), which play critical roles in dendritic cell (DC) function. In this study, we assessed the contribution of PIK3C3 to DC function during experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS). We found that Pik3c3-deficient DCs exhibit attenuated capacity to reactivate encephalitogenic T cells in the central nervous system, leading to reduced incidence and severity of EAE in DC-specific Pik3c3-deficient mice. Additionally, animals with a DC-specific deficiency in Rb1cc1/Fip200 but not Rubcn were protected against EAE, suggesting that the EAE phenotype of DC-specific Pik3c3-deficient mice is due to defective canonical autophagy rather than LAP. Collectively, our studies have revealed a critical role of PIK3C3 in DC function and the pathogenicity of these cells during EAE, with important implications for the development of immunotherapies for autoimmune diseases such as MS.Abbreviations: ATG: autophagy-related; CNS: central nervous system; DC: dendritic cell; DEG: differentially expressed gene; EAE: experimental autoimmune encephalomyelitis; LAP: LC3-associated phagocytosis; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MHC: major histocompatibility complex; MOG: myelin oligodendrocyte glycoprotein; MS: multiple sclerosis; PIK3C3/VPS34: phosphatidylinositol 3-kinase catalytic subunit type 3; ROS: reactive oxygen species.

Autophagy-related protein PIK3C3/VPS34 controls T cell metabolism and function.

The PIK3C3/VPS34 subunit of the class III phosphatidylinositol 3-kinase (PtdIns3K) complex is a key early player in macroautophagy/autophagy. In this study, we assessed the contribution of PIK3C3 to T cell metabolism and function. We found that Pik3c3-deficient T cells exhibited impaired cellular metabolism, and Pik3c3-deficient CD4+ T cells failed to differentiate into T helper 1 cells. These alterations were associated with reduced levels of active mitochondria upon T cell activation. In addition, conditional Pik3c3-deficient animals failed to mount autoreactive T cell responses and were resistant to experimental autoimmune encephalomyelitis (EAE). Interestingly, the deletion of Pik3c3 had little effect on the capacity of animals to clear tumor metastases. Collectively, our studies have revealed a critical role of PIK3C3 in T cell metabolism and the pathogenicity of these cells during EAE. Our findings also have important implications for the development of immunotherapies to treat multiple sclerosis and other inflammatory diseases by targeting PIK3C3.Abbreviations: CNS: central nervous system; DC: dendritic cell; DEG: differentially expressed gene; EAE: experimental autoimmune encephalomyelitis; ECAR: extracellular acidification rate; iNKT: invariant natural killer T; LAP: LC3-associated phagocytosis; LLC: Lewis lung carcinoma; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MDSC: myeloid-derived suppressor cell; MOG: myelin oligodendrocyte glycoprotein; NK: natural killer; OCR: oxygen consumption rate; PI: propidium iodide; PIK3C3/VPS34: phosphatidylinositol 3-kinase catalytic subunit type 3; RNA-seq: RNA-sequencing; TCR: T cell receptor; TMRE: tetramethylrhodamine ethyl ester perchlorate.

Pik3c3 deficiency in myeloid cells imparts partial resistance to experimental autoimmune encephalomyelitis associated with reduced IL-1ß production.

The PIK3C3/VPS34 subunit of the class III phosphatidylinositol 3-kinase (PtdIns3K) complex plays a role in both canonical and noncanonical autophagy, key processes that control immune-cell responsiveness to a variety of stimuli. Our previous studies found that PIK3C3 is a critical regulator that controls the development, homeostasis, and function of dendritic and T cells. In this study, we investigated the role of PIK3C3 in myeloid cell biology using myeloid cell-specific Pik3c3-deficient mice. We found that Pik3c3-deficient macrophages express increased surface levels of major histocompatibility complex (MHC) class I and class II molecules. In addition, myeloid cell-specific Pik3c3 ablation in mice caused a partial impairment in the homeostatic maintenance of macrophages expressing the apoptotic cell uptake receptor TIM-4. Pik3c3 deficiency caused phenotypic changes in myeloid cells that were dependent on the early machinery (initiation/nucleation) of the classical autophagy pathway. Consequently, myeloid cell-specific Pik3c3-deficient animals showed significantly reduced severity of experimental autoimmune encephalomyelitis (EAE), a primarily CD4+ T-cell-mediated mouse model of multiple sclerosis (MS). This disease protection was associated with reduced accumulation of myelin-specific CD4+ T cells in the central nervous system and decreased myeloid cell IL-1ß production. Further, administration of SAR405, a selective PIK3C3 inhibitor, delayed disease progression. Collectively, our studies establish PIK3C3 as an important regulator of macrophage functions and myeloid cell-mediated regulation of EAE. Our findings also have important implications for the development of small-molecule inhibitors of PIK3C3 as therapeutic modulators of MS and other autoimmune diseases.

Robust and Latch-Up-Immune LVTSCR Device with an Embedded PMOSFET for ESD Protection in a 28-nm CMOS Process.

Low-voltage-triggered silicon-controlled rectifier (LVTSCR) is expected to provide an electrostatic discharge (ESD) protection for a low-voltage integrated circuit. However, it is normally vulnerable to the latch-up effect due to its extremely low holding voltage. In this paper, a novel LVTSCR embedded with an extra p-type MOSFET called EP-LVTSCR has been proposed and verified in a 28-nm CMOS technology. The proposed device possesses a lower trigger voltage of ~ 6.2 V and a significantly higher holding voltage of ~ 5.5 V with only 23% degradation of the failure current under the transmission line pulse test. It is also shown that the EP-LVTSCR operates with a lower turn-on resistance of ~ 1.8 Ω as well as a reliable leakage current of ~ 1.8 nA measured at 3.63 V, making it suitable for ESD protections in 2.5 V/3.3 V CMOS processes. Moreover, the triggering mechanism and conduction characteristics of the proposed device were explored and demonstrated with TCAD simulation.

Phosphorylation of PLCγ1 by EphA2 Receptor Tyrosine Kinase Promotes Tumor Growth in Lung Cancer.

EphA2 receptor tyrosine kinase (RTK) is often expressed at high levels in cancer and has been shown to regulate tumor growth and metastasis across multiple tumor types, including non-small cell lung cancer. A number of signaling pathways downstream of EphA2 RTK have been identified; however, mechanisms of EphA2 proximal downstream signals are less well characterized. In this study, we used a yeast-two-hybrid screen to identify phospholipase C gamma 1 (PLCγ1) as a novel EphA2 interactor. EphA2 interacts with PLCγ1 and the kinase activity of EphA2 was required for phosphorylation of PLCγ1. In human lung cancer cells, genetic or pharmacologic inhibition of EphA2 decreased phosphorylation of PLCγ1 and loss of PLCγ1 inhibited tumor cell growth in vitro. Knockout of PLCγ1 by CRISPR-mediated genome editing also impaired tumor growth in a KrasG12D-p53-Lkb1 murine lung tumor model. Collectively, these data show that the EphA2-PLCγ1 signaling axis promotes tumor growth of lung cancer and provides rationale for disruption of this signaling axis as a potential therapeutic option. IMPLICATIONS: The EphA2-PLCG1 signaling axis promotes tumor growth of non-small cell lung cancer and can potentially be targeted as a therapeutic option.

Host deficiency in ephrin-A1 inhibits breast cancer metastasis.

Background: The conventional dogma of treating cancer by focusing on the elimination of tumor cells has been recently refined to include consideration of the tumor microenvironment, which includes host stromal cells. Ephrin-A1, a cell surface protein involved in adhesion and migration, has been shown to be tumor suppressive in the context of the cancer cell. However, its role in the host has not been fully investigated. Here, we examine how ephrin-A1 host deficiency affects cancer growth and metastasis in a murine model of breast cancer. Methods: 4T1 cells were orthotopically implanted into the mammary fat pads or injected into the tail veins of ephrin-A1 wild-type ( Efna1+/+), heterozygous ( Efna1+/-), or knockout ( Efna1-/-) mice. Tumor growth, lung metastasis, and tumor recurrence after surgical resection were measured. Flow cytometry and immunohistochemistry (IHC) were used to analyze various cell populations in primary tumors and tumor-bearing lungs. Results: While primary tumor growth did not differ between Efna1+/+, Efna1+/-, and Efna1-/- mice, lung metastasis and primary tumor recurrence were significantly decreased in knockout mice. Efna1-/- mice had reduced lung colonization of 4T1 cells compared to Efna1+/+ littermate controls as early as 24 hours after tail vein injection. Furthermore, established lung lesions in Efna1-/- mice had reduced proliferation compared to those in Efna1+/+ controls. Conclusions: Our studies demonstrate that host deficiency of ephrin-A1 does not impact primary tumor growth but does affect metastasis by providing a less favorable metastatic niche for cancer cell colonization and growth. Elucidating the mechanisms by which host ephrin-A1 impacts cancer relapse and metastasis may shed new light on novel therapeutic strategies.

Disruption of the Scaffolding Function of mLST8 Selectively Inhibits mTORC2 Assembly and Function and Suppresses mTORC2-Dependent Tumor Growth In Vivo.

mTOR is a serine/threonine kinase that acts in two distinct complexes, mTORC1 and mTORC2, and is dysregulated in many diseases including cancer. mLST8 is a shared component of both mTORC1 and mTORC2, yet little is known regarding how mLST8 contributes to assembly and activity of the mTOR complexes. Here we assessed mLST8 loss in a panel of normal and cancer cells and observed little to no impact on assembly or activity of mTORC1. However, mLST8 loss blocked mTOR association with mTORC2 cofactors RICTOR and SIN1, thus abrogating mTORC2 activity. Similarly, a single pair of mutations on mLST8 with a corresponding mutation on mTOR interfered with mTORC2 assembly and activity without affecting mTORC1. We also discovered a direct interaction between mLST8 and the NH2-terminal domain of the mTORC2 cofactor SIN1. In PTEN-null prostate cancer xenografts, mLST8 mutations disrupting the mTOR interaction motif inhibited AKT S473 phosphorylation and decreased tumor cell proliferation and tumor growth in vivo. Together, these data suggest that the scaffolding function of mLST8 is critical for assembly and activity of mTORC2, but not mTORC1, an observation that could enable therapeutic mTORC2-selective inhibition as a therapeutic strategy. SIGNIFICANCE: These findings show that mLST8 functions as a scaffold to maintain mTORC2 integrity and kinase activity, unveiling a new avenue for development of mTORC2-specific inhibitors.

EPHA2 Blockade Overcomes Acquired Resistance to EGFR Kinase Inhibitors in Lung Cancer.

Despite the success of treating EGFR-mutant lung cancer patients with EGFR tyrosine kinase inhibitors (TKI), all patients eventually acquire resistance to these therapies. Although various resistance mechanisms have been described, there are currently no FDA-approved therapies that target alternative mechanisms to treat lung tumors with acquired resistance to first-line EGFR TKI agents. Here we found that EPHA2 is overexpressed in EGFR TKI-resistant tumor cells. Loss of EPHA2 reduced the viability of erlotinib-resistant tumor cells harboring EGFR(T790M) mutations in vitro and inhibited tumor growth and progression in an inducible EGFR(L858R+T790M)-mutant lung cancer model in vivo. Targeting EPHA2 in erlotinib-resistant cells decreased S6K1-mediated phosphorylation of cell death agonist BAD, resulting in reduced tumor cell proliferation and increased apoptosis. Furthermore, pharmacologic inhibition of EPHA2 by the small-molecule inhibitor ALW-II-41-27 decreased both survival and proliferation of erlotinib-resistant tumor cells and inhibited tumor growth in vivo. ALW-II-41-27 was also effective in decreasing viability of cells with acquired resistance to the third-generation EGFR TKI AZD9291. Collectively, these data define a role for EPHA2 in the maintenance of cell survival of TKI-resistant, EGFR-mutant lung cancer and indicate that EPHA2 may serve as a useful therapeutic target in TKI-resistant tumors.

Regulation of endothelial cell proliferation and vascular assembly through distinct mTORC2 signaling pathways.

Mammalian target of rapamycin (mTOR) is a serine/threonine kinase that regulates a diverse array of cellular processes, including cell growth, survival, metabolism, and cytoskeleton dynamics. mTOR functions in two distinct complexes, mTORC1 and mTORC2, whose activities and substrate specificities are regulated by complex specific cofactors, including Raptor and Rictor, respectively. Little is known regarding the relative contribution of mTORC1 versus mTORC2 in vascular endothelial cells. Using mouse models of Raptor or Rictor gene targeting, we discovered that Rictor ablation inhibited vascular endothelial growth factor (VEGF)-induced endothelial cell proliferation and assembly in vitro and angiogenesis in vivo, whereas the loss of Raptor had only a modest effect on endothelial cells (ECs). Mechanistically, the loss of Rictor reduced the phosphorylation of AKT, protein kinase Cα (PKCα), and NDRG1 without affecting the mTORC1 pathway. In contrast, the loss of Raptor increased the phosphorylation of AKT despite inhibiting the phosphorylation of S6K1, a direct target of mTORC1. Reconstitution of Rictor-null cells with myristoylated AKT (Myr-AKT) rescued vascular assembly in Rictor-deficient endothelial cells, whereas PKCα rescued proliferation defects. Furthermore, tumor neovascularization in vivo was significantly decreased upon EC-specific Rictor deletion in mice. These data indicate that mTORC2 is a critical signaling node required for VEGF-mediated angiogenesis through the regulation of AKT and PKCα in vascular endothelial cells.

Eph receptor tyrosine kinases in cancer stem cells.

Eph receptor tyrosine kinases (RTKs) and their ligands, ephrins, play critical roles in development, tissue homeostasis, and cancer. Because Eph receptors are expressed in most adult stem cell niches and in many types of cancers, it has been long suspected that this family of RTKs may also regulate the function of cancer stem-like cells (CSCs). This review will focus on recent studies to elucidate the contribution of Eph/ephrin molecules in CSC self-renewal and tumorigenicity, as well as describe efforts to target these molecules in cancer. Because CSCs are often resistant to therapeutic intervention and have been shown to depend on Eph RTKs for self-renewal, targeting Eph receptors may hold promise for the treatment of drug-resistant cancers.

Elevated Slit2 Activity Impairs VEGF-Induced Angiogenesis and Tumor Neovascularization in EphA2-Deficient Endothelium.

UNLABELLED: Angiogenic remodeling during embryonic development and in adult tissue homeostasis is orchestrated by cooperative signaling between several distinct molecular pathways, which are often exploited by tumors. Indeed, tumors upregulate proangiogenic molecules while simultaneously suppressing angiostatic pathways to recruit blood vessels for growth, survival, and metastatic spread. Understanding how cancers exploit proangiogenic and antiangiogenic signals is a key step in developing new, molecularly targeted antiangiogenic therapies. While EphA2, a receptor tyrosine kinase (RTK), is required for VEGF-induced angiogenesis, the mechanism through which these pathways intersect remains unclear. Slit2 expression is elevated in EphA2-deficient endothelium, and here it is reported that inhibiting Slit activity rescues VEGF-induced angiogenesis in cell culture and in vivo, as well as VEGF-dependent tumor angiogenesis, in EphA2-deficient endothelial cells and animals. Moreover, blocking Slit activity or Slit2 expression in EphA2-deficient endothelial cells restores VEGF-induced activation of Src and Rac, both of which are required for VEGF-mediated angiogenesis. These data suggest that EphA2 suppression of Slit2 expression and Slit angiostatic activity enables VEGF-induced angiogenesis in vitro and in vivo, providing a plausible mechanism for impaired endothelial responses to VEGF in the absence of EphA2 function. IMPLICATIONS: Modulation of angiostatic factor Slit2 by EphA2 receptor regulates endothelial responses to VEGF-mediated angiogenesis and tumor neovascularization.

JNK signaling mediates EPHA2-dependent tumor cell proliferation, motility, and cancer stem cell-like properties in non-small cell lung cancer.

Recent genome-wide analyses in human lung cancer revealed that EPHA2 receptor tyrosine kinase is overexpressed in non-small cell lung cancer (NSCLC), and high levels of EPHA2 correlate with poor clinical outcome. However, the mechanistic basis for EPHA2-mediated tumor promotion in lung cancer remains poorly understood. Here, we show that the JNK/c-JUN signaling mediates EPHA2-dependent tumor cell proliferation and motility. A screen of phospho-kinase arrays revealed a decrease in phospho-c-JUN levels in EPHA2 knockdown cells. Knockdown of EPHA2 inhibited p-JNK and p-c-JUN levels in approximately 50% of NSCLC lines tested. Treatment of parental cells with SP600125, a c-JUN-NH2-kinase (JNK) inhibitor, recapitulated defects in EPHA2-deficient tumor cells, whereas constitutively activated JNK mutants were sufficient to rescue phenotypes. Knockdown of EPHA2 also inhibited tumor formation and progression in xenograft animal models in vivo. Furthermore, we investigated the role of EPHA2 in cancer stem-like cells (CSC). RNA interference-mediated depletion of EPHA2 in multiple NSCLC lines decreased the ALDH(+) cancer stem-like population and tumor spheroid formation in suspension. Depletion of EPHA2 in sorted ALDH(+) populations markedly inhibited tumorigenicity in nude mice. Furthermore, analysis of a human lung cancer tissue microarray revealed a significant, positive association between EPHA2 and ALDH expression, indicating an important role for EPHA2 in human lung CSCs. Collectively, these studies revealed a critical role of JNK signaling in EPHA2-dependent lung cancer cell proliferation and motility and a role for EPHA2 in CSC function, providing evidence for EPHA2 as a potential therapeutic target in NSCLC. Cancer Res; 74(9); 2444-54. ©2014 AACR.