Xin-an Lu

Cholesterol sequestration by nystatin enhances the uptake and activity of endostatin in endothelium via regulating distinct endocytic pathways

Specific internalization of endostatin into endothelial cells has been proved to be important for its biologic functions. However, the mechanism of endostatin internalization still remains elusive. In this study, we report for the first time that both caveolae/lipid rafts and clathrin-coated pits are involved in endostatin internalization. Inhibition of either the caveolae pathway or the clathrin pathway with the use of chemical inhibitors, small interfering RNAs, or dominant-negative mutants alters endostatin internalization in vitro. Intriguingly, cholesterol sequestration by nystatin, a polyene antifungal drug, significantly enhances endostatin uptake by endothelial cells through switching endostatin internalization predominantly to the clathrin-mediated pathway. Nystatin-enhanced internalization of endostatin also increases its inhibitory effects on endothelial cell tube formation and migration. More importantly, combined treatment with nystatin and endostatin selectively enhances endostatin uptake and biodistribution in tumor blood vessels and tumor tissues but not in normal tissues of tumor-bearing mice, ultimately resulting in elevated antiangiogenic and antitumor efficacies of endostatin in vivo. Taken together, our data show a novel mechanism of endostatin internalization and support the potential application of enhancing the uptake and therapeutic efficacy of endostatin via regulating distinct endocytic pathways with cholesterol-sequestering agents.

The CXCL12-CXCR4 Chemokine Pathway: A Novel Axis Regulates Lymphangiogenesis

Purpose: Lymphangiogenesis, the growth of lymphatic vessels, contributes to lymphatic metastasis. However, the precise mechanism underlying lymphangiogenesis remains poorly understood. This study aimed to examine chemokine/chemokine receptors that directly contribute to chemoattraction of activated lymphatic endothelial cells (LEC) and tumor lymphangiogenesis. Experimental Design: We used quantitative RT-PCR to analyze specifically expressed chemokine receptors in activated LECs upon stimulation of vascular endothelial growth factor-C (VEGF-C). Subsequently, we established in vitro and in vivo models to show lymphangiogenic functions of the chemokine axis. Effects of targeting the chemokine axis on tumor lymphangiogenesis and lymphatic metastasis were determined in an orthotopic breast cancer model. Results: VEGF-C specifically upregulates CXCR4 expression on lymphangiogenic endothelial cells. Moreover, hypoxia-inducible factor-1α (HIF-1α) mediates the CXCR4 expression induced by VEGF-C. Subsequent analyses identify the ligand CXCL12 as a chemoattractant for LECs. CXCL12 induces migration, tubule formation of LECs in vitro, and lymphangiogenesis in vivo. CXCL12 also stimulates the phosphorylation of intracellular signaling Akt and Erk, and their specific antagonists impede CXCL12-induced chemotaxis. In addition, its level is correlated with lymphatic vessel density in multiple cancer tissues microarray. Furthermore, the CXCL12–CXCR4 axis is independent of the VEGFR-3 pathway in promoting lymphangiogenesis. Intriguingly, combined treatment with anti-CXCL12 and anti-VEGF-C antibodies results in additive inhibiting effects on tumor lymphangiogenesis and lymphatic metastasis. Conclusions: These results show the role of the CXCL12–CXCR4 axis as a novel chemoattractant for LECs in promoting lymphangiogenesis, and support the potential application of combined targeting of both chemokines and lymphangiogenic factors in inhibiting lymphatic metastasis. Clin Cancer Res; 18(19); 5387–98. ©2012 AACR.

Endostatin Prevents Dietary-Induced Obesity by Inhibiting Adipogenesis and Angiogenesis

Endostatin is a well-known angiogenesis inhibitor. Although angiogenesis has been considered as a potential therapeutic target of obesity, the inhibitory effect of endostatin on adipogenesis and dietary-induced obesity has never been demonstrated. Adipogenesis plays a critical role in controlling adipocyte cell number, body weight, and metabolic profile in a homeostatic state. Here we reveal that endostatin inhibits adipogenesis and dietary-induced obesity. The antiadipogenic mechanism of endostatin lies in its interaction with Sam68 RNA-binding protein in the nuclei of preadipocytes. This interaction competitively impairs the binding of Sam68 to intron 5 of mammalian target of rapamycin (mTOR), causing an error in mTOR transcript. This consequently decreases the expression of mTOR, results in decreased activities of the mTOR complex 1 pathway, and leads to defects in adipogenesis. Moreover, our findings demonstrate that the antiangiogenic function of endostatin also contributes to its obesity-inhibitory activity. Through the combined functions on adipogenesis and angiogenesis, endostatin prevents dietary-induced obesity and its related metabolic disorders, including insulin resistance, glucose intolerance, and hepatic steatosis. Thus, our findings reveal that endostatin has a potential application for antiobesity therapy and the prevention of obesity-related metabolic syndromes.

The regulatory mechanism of a client kinase controlling its own release from Hsp90 chaperone machinery through phosphorylation

It is believed that the stability and activity of client proteins are passively regulated by the Hsp90 (heat-shock protein 90) chaperone machinery, which is known to be modulated by its intrinsic ATPase activity, co-chaperones and post-translational modifications. However, it is unclear whether client proteins themselves participate in regulation of the chaperoning process. The present study is the first example to show that a client kinase directly regulates Hsp90 activity, which is a novel level of regulation for the Hsp90 chaperone machinery. First, we prove that PKCγ (protein kinase Cγ) is a client protein of Hsp90α, and, that by interacting with PKCγ, Hsp90α prevents PKCγ degradation and facilitates its cytosol-to-membrane translocation and activation. A threonine residue set, Thr115/Thr425/Thr603, of Hsp90α is specifically phosphorylated by PKCγ, and, more interestingly, this threonine residue set serves as a ‘phosphorylation switch’ for Hsp90α binding or release of PKCγ. Moreover, phosphorylation of Hsp90α by PKCγ decreases the binding affinity of Hsp90α towards ATP and co-chaperones such as Cdc37 (cell-division cycle 37), thereby decreasing its chaperone activity. Further investigation demonstrated that the reciprocal regulation of Hsp90α and PKCγ plays a critical role in cancer cells, and that simultaneous inhibition of PKCγ and Hsp90α synergistically prevents cell migration and promotes apoptosis in cancer cells.

Endostatin Has ATPase Activity, Which Mediates Its Antiangiogenic and Antitumor Activities

Endostatin is an endogenous angiogenesis inhibitor with broad-spectrum antitumor activities. Although the molecular mechanisms of endostatin have been extensively explored, the intrinsic biochemical characteristics of endostatin are not completely understood. Here, we revealed for the first time that endostatin embedded novel ATPase activity. Moreover, mutagenesis study showed that the ATPase activity of endostatin mutants positively correlated with effects on endothelial cell activities and tumor growth. E-M, an endostatin mutant with higher ATPase activity than that of wild-type (WT) endostatin, significantly increased endostatin-mediated inhibitory effects on endothelial cell proliferation, migration, tube formation, and adhesion. In vivo study showed that E-M displayed enhanced antitumor effects compared with WT. On the other hand, K96A, K96R, and E176A, endostatin mutants with lower ATPase activities than that of WT, showed reduced or comparable effects on targeting both in vitro endothelial cell activities and in vivo tumor angiogenesis and tumor growth. Furthermore, endostatin and its mutants exhibited distinct abilities in regulations of gene expression (Id1, Id3), cell signaling (Erk, p38, and Src phosphorylation), and intracellular ATP levels. Collectively, our study demonstrates that endostatin has novel ATPase activity, which mediates its antiangiogenic and antitumor activities, suggesting that construction of endostatin analogues with high ATPase activity may provide a new direction for the development of more potent antiangiogenic drugs. Mol Cancer Ther; 14(5); 1192–201. ©2015 AACR.

Thr90 phosphorylation of Hsp90α by protein kinase A regulates its chaperone machinery.

Hsp90 (heat-shock protein 90) is one of the most important molecular chaperones in eukaryotes. Hsp90 facilitates the maturation, activation or degradation of its client proteins. It is now well accepted that both ATP binding and co-chaperone association are involved in regulating the Hsp90 chaperone machinery. However, other factors such as post-translational modifications are becoming increasingly recognized as being involved in this process. Recent studies have reported that phosphorylation of Hsp90 plays an unanticipated role in this process. In the present study, we systematically investigated the impact of phosphorylation of a single residue (Thr90) of Hsp90α (pThr90-Hsp90α) on its chaperone machinery. We demonstrate that protein kinase A specifically phosphorylates Hsp90α at Thr90, and that the pThr9090-Hsp90α level is significantly elevated in proliferating cells. Thr90 phosphorylation affects the binding affinity of Hsp90α to ATP. Subsequent examination of the interactions of Hsp90α with co-chaperones reveals that Thr90 phosphorylation specifically regulates the association of a subset of co-chaperones with Hsp90α. The Hsp90α T90E phosphor-mimic mutant exhibits increased association with Aha1 (activator of Hsp90 ATPase homologue 1), p23, PP5 (protein phosphatase 5) and CHIP (C-terminus of Hsp70-interacting protein), and decreased binding affinity with Hsp70, Cdc37 (cell division cycle 37) and Hop [Hsc70 (heat-shock cognate protein 70)/Hsp90-organizing protein], whereas its interaction with FKBP52 (FK506-binding protein 4) is only moderately affected. Moreover, we find that the ability of the T90E mutant to form complexes with its clients, such as Src, Akt or PKCγ (protein kinase Cγ), is dramatically impaired, suggesting that phosphorylation affects its chaperoning activity. Taken together, the results of the present study demonstrate that Thr90 phosphorylation is actively engaged in the regulation of the Hsp90α chaperone machinery and should be a generic determinant for the cycling of Hsp90α chaperone function.

PLCγ1–PKCγ Signaling-Mediated Hsp90α Plasma Membrane Translocation Facilitates Tumor Metastasis

The 90‐kDa heat shock protein (Hsp90α) has been identified on the surface of cancer cells, and is implicated in tumor invasion and metastasis, suggesting that it is a potentially important target for tumor therapy. However, the regulatory mechanism of Hsp90α plasma membrane translocation during tumor invasion remains poorly understood. Here, we show that Hsp90α plasma membrane expression is selectively upregulated upon epidermal growth factor (EGF) stimulation, which is a process independent of the extracellular matrix. Abrogation of EGF‐mediated activation of phospholipase (PLCγ1) by its siRNA or inhibitor prevents the accumulation of Hsp90α at cell protrusions. Inhibition of the downstream effectors of PLCγ1, including Ca2+ and protein kinase C (PKCγ), also blocks the membrane translocation of Hsp90α, while activation of PKCγ leads to increased levels of cell‐surface Hsp90α. Moreover, overexpression of PKCγ increases extracellular vesicle release, on which Hsp90α is present. Furthermore, activation or overexpression of PKCγ promotes tumor cell motility in vitro and tumor metastasis in vivo, whereas a specific neutralizing monoclonal antibody against Hsp90α inhibits such effects, demonstrating that PKCγ‐induced Hsp90α translocation is required for tumor metastasis. Taken together, our study provides a mechanistic basis for the role for the PLCγ1–PKCγ pathway in regulating Hsp90α plasma membrane translocation, which facilitates tumor cell motility and promotes tumor metastasis.

Genetically engineered endostatin-lidamycin fusion proteins effectively inhibit tumor growth and metastasis.

BackgroundEndostatin (ES) inhibits endothelial cell proliferation, migration, invasion, and tube formation. It also shows antiangiogenesis and antitumor activities in several animal models. Endostatin specifically targets tumor vasculature to block tumor growth. Lidamycin (LDM), which consists of an active enediyne chromophore (AE) and a non-covalently bound apo-protein (LDP), is a member of chromoprotein family of antitumor antibiotics with extremely potent cytotoxicity to cancer cells. Therefore, we reasoned that endostatin-lidamycin (ES-LDM) fusion proteins upon energizing with enediyne chromophore may obtain the combined capability targeting tumor vasculature and tumor cell by respective ES and LDM moiety.MethodsIn this study, we designed and obtained two new endostatin-based fusion proteins, endostatin-LDP (ES-LDP) and LDP-endostatin (LDP-ES). In vitro, the antiangiogenic effect of fusion proteins was determined by the wound healing assay and tube formation assay and the cytotoxicity of their enediyne-energized analogs was evaluated by CCK-8 assay. Tissue microarray was used to analyze the binding affinity of LDP, ES or ES-LDP with specimens of human lung tissue and lung tumor. The in vivo efficacy of the fusion proteins was evaluated with human lung carcinoma PG-BE1 xenograft and the experimental metastasis model of 4T1-luc breast cancer.ResultsES-LDP and LDP-ES disrupted the formation of endothelial tube structures and inhibited endothelial cell migration. Evidently, ES-LDP accumulated in the tumor and suppressed tumor growth and metastasis. ES-LDP and ES show higher binding capability than LDP to lung carcinoma; in addition, ES-LDP and ES share similar binding capability. Furthermore, the enediyne-energized fusion protein ES-LDP-AE demonstrated significant efficacy against lung carcinoma xenograft in athymic mice.ConclusionsThe ES-based fusion protein therapy provides some fundamental information for further drug development. Targeting both tumor vasculature and tumor cells by endostatin-based fusion proteins and their enediyne-energized analogs probably provides a promising modality in cancer therapy.

Endostatin sensitizes p53-deficient non-small-cell lung cancer to genotoxic chemotherapy by targeting DNA-dependent protein kinase catalytic subunit

Endostatin was discovered as an endogenous angiogenesis inhibitor with broad‐spectrum antitumour activities. Although clinical efficacy was observed when endostatin was combined with standard chemotherapy for non‐small cell lung cancer (NSCLC), as well as other cancer types, the specific mechanisms underlying the benefit of endostatin are not completely understood. Extensive investigations suggest that endostatin is a multifunctional protein possessing more than anti‐angiogenic activity. Here, we found that endostatin exerts a direct chemosensitizing effect on p53‐deficient tumour cells. Concomitant treatment with endostatin and genotoxic drugs resulted in therapeutic synergy in both cellular and animal models of p53‐deficient NSCLC. Mechanistically, endostatin specifically interacts with DNA‐dependent protein kinase catalytic subunit (DNA‐PKcs) in tumour cells and suppresses its DNA repair activity. Using isogenic NSCLC cells with different p53 statuses, we discovered that p53‐deficient tumour cells show chemoresistance to genotoxic drugs, creating a synthetic dependence on DNA‐PKcs‐mediated DNA repair. In this setting, endostatin exerted inhibitory effects on DNA‐PKcs activity, leading to accumulation of DNA lesions and promotion of the therapeutic effect of genotoxic chemotherapy. In contrast, p53‐proficient tumour cells were more sensitive to genotoxic drugs so that DNA‐PKcs could be cleaved by drug‐activated caspase‐3, making DNA‐PKcs inhibition less effective during this ongoing apoptotic process. Therefore, our data demonstrate a novel mechanism for endostatin as a DNA‐PKcs suppressor, and indicate that combination therapy of endostatin with genotoxic drugs could be a promising treatment strategy for cancer patients with p53‐deficient tumours. Copyright

The novel anti-CD19 chimeric antigen receptors with humanized scFv (single-chain variable fragment) trigger leukemia cell killing.

The molecular design of CARs (Chimeric Antigen Receptors), especially the scFv, has been a major part to use of CAR-T cells for targeted adoptive immunotherapy. To address this issue, we chose a vector backbone encoding a second-generation CAR based on efficacy of a murine scFv-based CAR. Next, we generated a panel of humanized scFvs and tested in vitro for their ability to direct CAR-T cells to specifically lyse, proliferate, and secrete cytokines in response to antigen-bearing targets. Furthermore, in a xenograft model of lymphoma, human T cells expressing humanized scFvs exhibited the same anti-tumor efficacy as those expressing murine scFv and prolonged survival compared with cells expressing control CAR. Therefore, we uncovered CARs expressing humanized scFv domain that contribute the similar enhanced antileukemic efficacy and survival in tumor bearing mice. These results provide the basis for the future clinical studies of CAR-T cells transduced with humanized scFv directed to CD19.