Han Chung Chong

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.

Matricellular Proteins: A Sticky Affair with Cancers

The multistep process of metastasis is a major hallmark of cancer progression involving the cointeraction and coevolution of the tumor and its microenvironment. In the tumor microenvironment, tumor cells and the surrounding stromal cells aberrantly secrete matricellular proteins, which are a family of nonstructural proteins in the extracellular matrix (ECM) that exert regulatory roles via a variety of molecular mechanisms. Matricellular proteins provide signals that support tumorigenic activities characteristic of the metastastic cascade such as epithelial-to-mesenchymal (EMT) transition, angiogenesis, tumor cell motility, proliferation, invasion, evasion from immune surveillance, and survival of anoikis. Herein, we review the current understanding of the following matricellular proteins and highlight their pivotal and multifacted roles in metastatic progression: angiopoietin-like protein 4 (ANGPTL4), CCN family members cysteine-rich angiogenic inducer 61 (Cyr61/CCN1) and CCN6, osteopontin (OPN), secreted protein acidic and rich in cysteine (SPARC), tenascin C (TNC), and thrombospondin-1 and -2 (TSP1, TSP2). Insights into the signaling mechanisms resulting from the interaction of these matricellular proteins and their respective molecular partner(s), as well as their subsequent contribution to tumor metastasis, are discussed. In addition, emerging evidences of their promising potential as therapeutic options and/or targets in the treatment of cancer are also highlighted.

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.

Angiopoietin-like 4 stimulates STAT3-mediated iNOS expression and enhances angiogenesis to accelerate wound healing in diabetic mice

Impaired wound healing is a major source of morbidity in diabetic patients. Poor outcome has, in part, been related to increased inflammation, poor angiogenesis, and deficiencies in extracellular matrix components. Despite the enormous impact of these chronic wounds, effective therapies are lacking. Here, we showed that the topical application of recombinant matricellular protein angiopoietin-like 4 (ANGPTL4) accelerated wound reepithelialization in diabetic mice, in part, by improving angiogenesis. ANGPTL4 expression is markedly elevated upon normal wound injury. In contrast, ANGPTL4 expression remains low throughout the healing period in diabetic wounds. Exogenous ANGPTL4 modulated several regulatory networks involved in cell migration, angiogenesis, and inflammation, as evidenced by an altered gene expression signature. ANGPTL4 influenced the expression profile of endothelial-specific CD31 in diabetic wounds, returning its profile to that observed in wild-type wounds. We showed ANGPTL4-induced nitric oxide production through an integrin/JAK/STAT3-mediated upregulation of inducible nitric oxide synthase (iNOS) expression in wound epithelia, thus revealing a hitherto unknown mechanism by which ANGPTL4 regulated angiogenesis via keratinocyte-to-endothelial-cell communication. These data show that the replacement of ANGPTL4 may be an effective adjunctive or new therapeutic avenue for treating poor healing wounds. The present finding also confirms that therapeutic angiogenesis remains an attractive treatment modality for diabetic wound healing.

Getting ‘Smad’ about obesity and diabetes

Recent findings on the role of transforming growth factor (TGF)-β/Smad3 signaling in the pathogenesis of obesity and type 2 diabetes have underscored its importance in metabolism and adiposity. Indeed, elevated TGF-β has been previously reported in human adipose tissue during morbid obesity and diabetic neuropathy. In this review, we discuss the pleiotropic effects of TGF-β/Smad3 signaling on metabolism and energy homeostasis, all of which has an important part in the etiology and progression of obesity-linked diabetes; these include adipocyte differentiation, white to brown fat phenotypic transition, glucose and lipid metabolism, pancreatic function, insulin signaling, adipocytokine secretion, inflammation and reactive oxygen species production. We summarize the recent in vivo findings on the role of TGF-β/Smad3 signaling in metabolism based on the studies using Smad3−/− mice. Based on the presence of a dual regulatory effect of Smad3 on peroxisome proliferator-activated receptor (PPAR)β/δ and PPARγ2 promoters, we propose a unifying mechanism by which this signaling pathway contributes to obesity and its associated diabetes. We also discuss how the inhibition of this signaling pathway has been implicated in the amelioration of many facets of metabolic syndromes, thereby offering novel therapeutic avenues for these metabolic conditions.

Early controlled release of peroxisome proliferator-activated receptor β/δ agonist GW501516 improves diabetic wound healing through redox modulation of wound microenvironment.

Diabetic wounds are imbued with an early excessive and protracted reactive oxygen species production. Despite the studies supporting PPARβ/δ as a valuable pharmacologic wound-healing target, the therapeutic potential of PPARβ/δ agonist GW501516 (GW) as a wound healing drug was never investigated. Using topical application of polymer-encapsulated GW, we revealed that different drug release profiles can significantly influence the therapeutic efficacy of GW and consequently diabetic wound closure. We showed that double-layer encapsulated GW microparticles (PLLA:PLGA:GW) provided an earlier and sustained dose of GW to the wound and reduced the oxidative wound microenvironment to accelerate healing, in contrast to single-layered PLLA:GW microparticles. The underlying mechanism involved an early GW-mediated activation of PPARβ/δ that stimulated GPx1 and catalase expression in fibroblasts. GPx1 and catalase scavenged excessive H2O2 accumulation in diabetic wound beds, prevented H2O2-induced ECM modification and facilitated keratinocyte migration. The microparticles with early and sustained rate of GW release had better therapeutic wound healing activity. The present study underscores the importance of drug release kinetics on the therapeutic efficacy of the drug and warrants investigations to better appreciate the full potential of controlled drug release.

Fabrication and Drug Release Study of Double-Layered Microparticles of Various Sizes

Double-layered microparticles, composed of poly(D,L-lactide-co-glycolide) (50:50) (PLGA) core and poly(L-lactide) (PLLA) shell, of controllable sizes ranging from several hundred microns to few microns were fabricated using a one-step solvent evaporation method. Metoclopramide monohydrochloride monohydrate (MCA), a hydrophilic drug, was selectively localized in the PLGA core. To achieve the double-layered particles of size approximately 2 µm, the process parameters were carefully manipulated to extend the phase separation time by increasing oil-to-water ratio and saturating the surrounding aqueous phase with solvent. Subsequently, the drug release profiles of the double-layered particles of various sizes were studied. Increased particle size resulted in faster degradation of polymers because of autocatalysis, accelerating the release rate of MCA. Interestingly, the effect of degradation rates, affected by particle sizes, on drug release was insignificant when the particle size was drastically reduced to 2-20 µm in the investigated double-layered particles. This understanding would provide critical insights into how the controllable formation and unique drug release profiles of double-layered particles of various sizes can be achieved.

Nox4-dependent ROS modulation by amino endoperoxides to induce apoptosis in cancer cells.

Tumor metastasis is the main cause of death in cancer patients. Anoikis resistance is one critical malefactor of metastatic cancer cells to resist current clinical chemotherapeutic treatments. Although endoperoxide-containing compounds have long been suggested as anticancer drugs, few have been clinically employed due to their instability, complex synthesis procedure or low tumor cell selectivity. Herein, we describe a one-pot strategy to synthesize novel amino endoperoxides and their derivatives with good yields and stabilities. In vitro cell-based assays revealed that 4 out of the 14 amino endoperoxides selectively induce metastatic breast carcinoma cells but not normal breast cells to undergo apoptosis, in a dose-dependent manner. Mechanistic studies showed that the most potent amino endoperoxide, 4-Me, is selective for cancer cells expressing a high level of Nox4. The anticancer effects are further shown to be associated with reduced O2−:H2O2 ratio and increased ·OH level in the cancerous cells. Animal study showed that 4-Me impairs orthotopic breast tumor growth as well as tumor cell metastasis to lymph nodes. Altogether, our study suggests that anticancer strategies that focus on redox-based apoptosis induction in tumors are clinically viable.