Ming Jie Tan

Angiopoietin-like 4 Protein Elevates the Prosurvival Intracellular O2−:H2O2 Ratio and Confers Anoikis Resistance to Tumors

Cancer is a leading cause of death worldwide. Tumor cells exploit various signaling pathways to promote their growth and metastasis. To our knowledge, the role of angiopoietin-like 4 protein (ANGPTL4) in cancer remains undefined. Here, we found that elevated ANGPTL4 expression is widespread in tumors, and its suppression impairs tumor growth associated with enhanced apoptosis. Tumor-derived ANGPTL4 interacts with integrins to stimulate NADPH oxidase-dependent production of O(2)(-). A high ratio of O(2)(-):H(2)O(2) oxidizes/activates Src, triggering the PI3K/PKBα and ERK prosurvival pathways to confer anoikis resistance, thus promoting tumor growth. ANGPTL4 deficiency results in diminished O(2)(-) production and a reduced O(2)(-):H(2)O(2) ratio, creating a cellular environment conducive to apoptosis. ANGPTL4 is an important redox player in cancer and a potential therapeutic target.

ANGPTL4 modulates vascular junction integrity by integrin signaling and disruption of intercellular VE-cadherin and claudin-5 clusters

Vascular disruption induced by interactions between tumor-secreted permeability factors and adhesive proteins on endothelial cells facilitates metastasis. The role of tumor-secreted C-terminal fibrinogen-like domain of angiopoietin-like 4 (cANGPTL4) in vascular leakiness and metastasis is controversial because of the lack of understanding of how cANGPTL4 modulates vascular integrity. Here, we show that cANGPTL4 instigated the disruption of endothelial continuity by directly interacting with 3 novel binding partners, integrin α5β1, VE-cadherin, and claudin-5, in a temporally sequential manner, thus facilitating metastasis. We showed that cANGPTL4 binds and activates integrin α5β1-mediated Rac1/PAK signaling to weaken cell-cell contacts. cANGPTL4 subsequently associated with and declustered VE-cadherin and claudin-5, leading to endothelial disruption. Interfering with the formation of these cANGPTL4 complexes delayed vascular disruption. In vivo vascular permeability and metastatic assays performed using ANGPTL4-knockout and wild-type mice injected with either control or ANGPTL4-knockdown tumors confirmed that cANGPTL4 induced vascular leakiness and facilitated lung metastasis in mice. Thus, our findings elucidate how cANGPTL4 induces endothelial disruption. Our findings have direct implications for targeting cANGPTL4 to treat cancer and other vascular pathologies.

Angiopoietin-like 4 Interacts with Matrix Proteins to Modulate Wound Healing

A dynamic cell-matrix interaction is crucial for a rapid cellular response to changes in the environment. Appropriate cell behavior in response to the changing wound environment is required for efficient wound closure. However, the way in which wound keratinocytes modify the wound environment to coordinate with such cellular responses remains less studied. We demonstrated that angiopoietin-like 4 (ANGPTL4) produced by wound keratinocytes coordinates cell-matrix communication. ANGPTL4 interacts with vitronectin and fibronectin in the wound bed, delaying their proteolytic degradation by metalloproteinases. This interaction does not interfere with integrin-matrix protein recognition and directly affects cell-matrix communication by altering the availability of intact matrix proteins. These interactions stimulate integrin- focal adhesion kinase, 14-3-3, and PKC-mediated signaling pathways essential for effective wound healing. The deficiency of ANGPTL4 in mice delays wound re-epithelialization. Further analysis revealed that cell migration was impaired in the ANGPTL4-deficient keratinocytes. Altogether, the findings provide molecular insight into a novel control of wound healing via ANGPTL4-dependent regulation of cell-matrix communication. Given the known role of ANGPTL4 in glucose and lipid homeostasis, it is a prime therapeutic candidate for the treatment of diabetic wounds. It also underscores the importance of cell-matrix communication during angiogenesis and cancer metastasis.

Angiopoietin-Like 4 Interacts with Integrins β1 and β5 to Modulate Keratinocyte Migration

Adipose tissue secretes adipocytokines for energy homeostasis, but recent evidence indicates that some adipocytokines also have a profound local impact on wound healing. Upon skin injury, keratinocytes use various signaling molecules to promote reepithelialization for efficient wound closure. In this study, we identify a novel function of adipocytokine angiopoietin-like 4 (ANGPTL4) in keratinocytes during wound healing through the control of both integrin-mediated signaling and internalization. Using two different in vivo models based on topical immuno-neutralization of ANGPTL4 as well as ablation of the ANGPTL4 gene, we show that ANGPTL4-deficient mice exhibit delayed wound reepithelialization with impaired keratinocyte migration. Human keratinocytes in which endogenous ANGPTL4 expression was suppressed by either siRNA or a neutralizing antibody show impaired migration associated with diminished integrin-mediated signaling. Importantly, we identify integrins β1 and β5, but not β3, as novel binding partners of ANGPTL4. ANGPTL4-bound integrin β1 activated the FAK-Src-PAK1 signaling pathway, which is important for cell migration. The findings presented herein reveal an unpredicted role of ANGPTL4 during wound healing and demonstrate how ANGPTL4 stimulates intracellular signaling mechanisms to coordinate cellular behavior. Our findings provide insight into a novel cell migration control mechanism and underscore the physiological importance of the modulation of integrin activity in cancer metastasis.

Emerging roles of angiopoietin-like 4 in human cancer.

Angiopoietin-like 4 (ANGPTL4) is best known for its role as an adipokine involved in the regulation of lipid and glucose metabolism. The characterization of ANGPTL4 as an adipokine is largely due to our limited understanding of the interaction partners of ANGPTL4 and how ANGPTL4 initiates intracellular signaling. Recent findings have revealed a critical role for ANGPTL4 in cancer growth and progression, anoikis resistance, altered redox regulation, angiogenesis, and metastasis. Emerging evidence suggests that ANGPTL4 function may be drastically altered depending on the proteolytic processing and posttranslational modifications of ANGPTL4, which may clarify several conflicting roles of ANGPTL4 in different cancers. Although the N-terminal coiled-coil region of ANGPTL4 has been largely responsible for the endocrine regulatory role in lipid metabolism, insulin sensitivity, and glucose homeostasis, it has now emerged that the COOH-terminal fibrinogen-like domain of ANGPTL4 may be a key regulator in the multifaceted signaling during cancer development. New insights into the mechanistic action of this functional domain have opened a new chapter into the possible clinical application of ANGPTL4 as a promising candidate for clinical intervention in the fight against cancer. This review summarizes our current understanding of ANGPTL4 in cancer and highlights areas that warrant further investigation. A better understanding of the underlying cellular and molecular mechanisms of ANGPTL4 will reveal novel insights into other aspects of tumorigenesis and the potential therapeutic value of ANGPTL4. Mol Cancer Res; 10(6); 677–88. ©2012 AACR.

Smad3 Deficiency in Mice Protects Against Insulin Resistance and Obesity Induced by a High-Fat Diet

OBJECTIVE Obesity and associated pathologies are major global health problems. Transforming growth factor-β/Smad3 signaling has been implicated in various metabolic processes, including adipogenesis, insulin expression, and pancreatic β-cell function. However, the systemic effects of Smad3 deficiency on adiposity and insulin resistance in vivo remain elusive. This study investigated the effects of Smad3 deficiency on whole-body glucose and lipid homeostasis and its contribution to the development of obesity and type 2 diabetes. RESEARCH DESIGN AND METHODS We compared various metabolic profiles of Smad3-knockout and wild-type mice. We also determined the mechanism by which Smad3 deficiency affects the expression of genes involved in adipogenesis and metabolism. Mice were then challenged with a high-fat diet to study the impact of Smad3 deficiency on the development of obesity and insulin resistance. RESULTS Smad3-knockout mice exhibited diminished adiposity with improved glucose tolerance and insulin sensitivity. Chromatin immunoprecipitation assay revealed that Smad3 deficiency increased CCAAT/enhancer-binding protein β-C/EBP homologous protein 10 interaction and exerted a differential regulation on proliferator-activated receptor β/δ and proliferator-activated receptor γ expression in adipocytes. Focused gene expression profiling revealed an altered expression of genes involved in adipogenesis, lipid accumulation, and fatty acid β-oxidation, indicative of altered adipose physiology. Despite reduced physical activity with no modification in food intake, these mutant mice were resistant to obesity and insulin resistance induced by a high-fat diet. CONCLUSIONS Smad3 is a multifaceted regulator in adipose physiology and the pathogenesis of obesity and type 2 diabetes, suggesting that Smad3 may be a potential target for the treatment of obesity and its associated disorders.

Anti-cAngptl4 Ab-Conjugated N-TiO2/NaYF4:Yb,Tm Nanocomposite for Near Infrared-Triggered Drug Release and Enhanced Targeted Cancer Cell Ablation

Nanomedicine: NIR-active N-TiO(2) /NaYF(4) :Yb,Tm nanocomposites (NCs) were synthesized for the first time and its potential applications in drug release and targeted cancer cell ablation are explored. Upon 980 nm laser irradiation, the anti-cAngptl4 Ab-conjugated N-TiO(2) /NaYF(4) :Yb,Tm NCs shows a significant increase in apoptotic A-5RT3 cells when compared with that of the unconjugated NCs. The mechanisms for NIR-induced photocatalysis, drug release and targeted cancer cell killing are proposed.

Angiopoietin-like 4 regulates epidermal differentiation.

The nuclear hormone receptor PPARβ/δ is integral to efficient wound re-epithelialization and implicated in epidermal maturation. However, the mechanism underlying the latter process of epidermal differentiation remains unclear. We showed that ligand-activated PPARβ/δ indirectly stimulated keratinocyte differentiation, requiring de novo gene transcription and protein translation. Using organotypic skin cultures constructed from PPARβ/δ- and angiopoietin-like 4 (ANGPTL4)-knockdown human keratinocytes, we showed that the expression of ANGPTL4, a PPARβ/δ target gene, is essential for the receptor mediated epidermal differentiation. The pro-differentiation effect of PPARβ/δ agonist GW501516 was also abolished when keratinocytes were co-treated with PPARβ/δ antagonist GSK0660 and similarly in organotypic skin culture incubated with blocking ANGPTL4 monoclonal antibody targeted against the C-terminal fibrinogen-like domain. Our focused real-time PCR gene expression analysis comparing the skin biopsies from wildtype and ANGPTL4-knockout mice confirmed a consistent down-regulation of numerous genes involved in epidermal differentiation and proliferation in the ANGPTL4-knockout skin. We further showed that the deficiency of ANGPTL4 in human keratinocytes and mice skin have diminished expression of various protein kinase C isotypes and phosphorylated transcriptional factor activator protein-1, which are well-established for their roles in keratinocyte differentiation. Chromatin immunoprecipitation confirmed that ANGPTL4 stimulated the activation and binding of JUNB and c-JUN to the promoter region of human involucrin and transglutaminase type 1 genes, respectively. Taken together, we showed that PPARβ/δ regulates epidermal maturation via ANGPTL4-mediated signalling pathway.

TAK1 regulates SCF expression to modulate PKBα activity that protects keratinocytes from ROS-induced apoptosis.

Dysregulated reactive oxygen species (ROS) generation contributes to many human pathologies, including cancer and diabetes. During normal wound repair, inflammation-induced ROS production must be tightly controlled, but the mechanisms reining their generation remain unclear. Herein, we show that transforming growth factor β-activated kinase 1 (TAK1) directly regulates stem cell factor (SCF) expression, which activates the protein kinase B (PKB)α pro-survival pathway in a cell-autonomous manner to protect keratinocytes from ROS-mediated cell death. TAK1 is a pivotal inflammatory mediator whose expression was transiently elevated during wound healing, paralleling the ROS production profile. TAK1 deficiency in keratinocytes led to increased apoptosis in response to anoikis and TNF-α treatment and was associated with elevated ROS level as analyzed by FACS. Using organotypic skin co-culture and comparative growth factor array analysis, we revealed a cell-autonomous mechanism that involved the SCF/c-Kit/PKBα signaling cascade. Ectopic expression of TAK1 or treatment with exogenous recombinant SCF restored the increased ROS production and apoptotic cell death in TAK1-deficient keratinocytes. Conversely, normal keratinocytes treated with various inhibitors targeting the SCF/c-Kit/PKBα pathway exhibited increased ROS production and TNF-α- or anoikis-induced apoptosis. Our study reveals a novel anti-apoptotic role for SCF in keratinocytes and identifies TAK1 as a novel player uniting inflammation and ROS regulation in skin redox biology.

Regulation of Cell Proliferation and Migration by TAK1 via Transcriptional Control of von Hippel-Lindau Tumor Suppressor

Skin maintenance and healing after wounding requires complex epithelial-mesenchymal interactions purportedly mediated by growth factors and cytokines. We show here that, for wound healing, transforming growth factor-β-activated kinase 1 (TAK1) in keratinocytes activates von Hippel-Lindau tumor suppressor expression, which in turn represses the expression of platelet-derived growth factor-B (PDGF-B), integrin β1, and integrin β5 via inhibition of the Sp1-mediated signaling pathway in the keratinocytes. The reduced production of PDGF-B leads to a paracrine-decreased expression of hepatocyte growth factor in the underlying fibroblasts. This TAK1 regulation of the double paracrine PDGF/hepatocyte growth factor signaling can regulate keratinocyte cell proliferation and is required for proper wound healing. Strikingly, TAK1 deficiency enhances cell migration. TAK1-deficient keratinocytes displayed lamellipodia formation with distinct microspike protrusion, associated with an elevated expression of integrins β1 and β5 and sustained activation of cdc42, Rac1, and RhoA. Our findings provide evidence for a novel homeostatic control of keratinocyte proliferation and migration mediated via TAK1 regulation of von Hippel-Lindau tumor suppressor. Dysfunctional regulation of TAK1 may contribute to the pathology of non-healing chronic inflammatory wounds and psoriasis.